Bioorganic Synthesis - An Introduction (PDFDrive)

Bioorganic
Synthesis

BIOORGANIC
SYNTHESIS
AN
INTRODUCTION
Gary W. Morrow
1

1
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Library of Congress Cataloging-in-Publication Data

Names: Morrow, Gary W., 1951–
Title: Bioorganic synthesis : an introduction / Gary W. Morrow.
Description: New York, NY : Oxford University Press, [2016] | Includes index.
Identifiers: LCCN 2016003452 | ISBN 9780199860531
Subjects: LCSH: Organic compounds—Synthesis. | Biosynthesis. | Chemistry, Organic.
Classification: LCC QD262 .M744 2016 | DDC 572/.45—dc23 LC record available
at http://lccn.loc.gov/2016003452
9 8 7 6 5 4 3 2 1
Printed by Sheridan Books, Inc., United States of America

Contents
xiii Acknowledgments
xv Introduction
xv The Unique Role of Carbon
xvii Distinguishing Primary Versus Secondary Metabolism
xvii Secondary Metabolites and Natural Products
xviii Natural Products in Organic Chemistry and Medicine
xix The Organic Chemistry of Biosynthesis
xx Goals and Structure of This Book
1 1. Brief Organic Review

1 Review of Functional Groups, Stereochemistry, and Conformational Analysis
5 Prochiral Relationships: One Step from Chirality
6 Prochiral π-Systems: “Two-Faced” Reaction Centers
7 Diastereotopic Atoms and Groups: One Step from a Diasteroeomer
9 Monosubstituted Cyclohexanes: Favoring Equatorial Positions
10 Disubstituted Cyclohexanes: Equivalent and Nonequivalent Combinations
11 Bicyclic Systems: Joining of Rings
12 Heterocyclic Ring Systems: One Atom Makes All the Difference
15 Bond Making and Breaking: Have Pair, Will Share; Need Two from You
15 Brønsted Acid–Base Reactions: Proton Donors Gladly Accepted
17 Acidity Trends: Why that Proton Is or Isn’t Acidic
20 Carbocations: Three Bonds to Carbon Can Be a Plus
20 Radicals: Odd and Reactive
22 Elimination Reactions: Introducing the Carbon–Carbon π-Bond
24 Carbocations: Rearrangements and Fates
25 Electrophilic Additions: π-Bonds as Nucleophilic Agents
26 Nucleophilic Substitutions and Alkylations: Make or Break for C–X Bonds
27 Nucleophilic Carbonyl Addition Reactions: C=O π-Bond under Attack
27 Imine Formation: Making the Essential C=N Linkage
v

30 Nucleophilic 1,4-(Conjugate) Addition Reactions: Remote Attack on

Contents vi Conjugated Carbonyls
36 Nucleophilic Acyl Substitution Reactions: Turning One Acyl
Compound into Another
37 Looking Ahead
37 Study Problems
42 2.Bioorganic Reactions
42 Enzymes: The Catalysts of Biological Organic Chemistry
44 Cofactors: Enzyme Assistants in Bioorganic Reactions
45 NADH/NADPH: Nature’s Version of Sodium Borohydride
for Carbonyl Reduction
47 NAD+/NADP+: Nature’s Version of PCC for Alcohol Oxidation
48 FAD: Another Hydride Acceptor for Dehydrogenations
49 Monooxygenases: Special Delivery of One O atom from O2
53 Dioxygenases: Delivering Both O Atoms from O2
54 Other Oxidations: Hydroquinone and Catechol Oxidations
55 Amine Oxidations: From Imines to Carbonyl Compounds and Beyond
57 PLP: Transamination and Decarboxylation of Amino Acids
59 Other Important Decarboxylations: β-Keto Acids,
o- and p-Hydroxybenzoic Acids
60 Thiamine Diphosphate (TPP) and Lipoic Acid: Decarboxylation
and Acyl Transfer
63 Biotin: The CO2 Carrier, Transport, and Transfer Agent
65 SAM: A C1 Fragment for Methyl Groups
65 DMAPP: An Allylic C5 Fragment for Structure Building
68 Other Essential Structural Fragments: Putting it All Together
70 Looking Ahead
71 Study Problems
75 3. Biosynthesis of Carbohydrates and Amino Acids

75 What Makes a Carbohydrate?
77 Cyclic Hemiacetals and Anomers
80 C-2 Epimers and Enediols—Simple Conversion of One
Carbohydrate into Another
81 Other Important Monosaccharides: Deoxy and Amino Sugars
83 The Significance of the Anomeric Carbon: Glycoside Formation
87 UDP-Sugars and Glycoside Formation: SN2 Chemistry at Work
90 Organic Reactions in Carbohydrate Chemistry: Overview
of Glucose Metabolism
92 Glycolysis: A 10-Step Program

97 What Happens to the Pyruvic Acid from Glycolysis
vii Contents
99 The Citric Acid Cycle: Another 10-Step Program
105 The Pentose Phosphate Pathway: Seven Alternative Steps
to Some Familiar Intermediates
109 The Big Picture
112 Amino Acids: More Important Primary Metabolite Building
Blocks for Biosynthesis
114 Biosynthesis of Serine: A Good Place to Start
120 Peptides and Proteins: A Very Brief Review
124 Putting Proteins and Carbohydrates Together: Glycoproteins
Versus Protein Glycosylation
127 Looking Ahead
127 Study Problems
131 4. The Terpenoid Pathway: Products from Mevalonic

Acid and Deoxyxylulose Phosphate
132 Classification of Terpenes: How Many Isoprene Units?
135 The Mevalonic Acid Route to DMAPP and IPP
136 The Deoxyxylulose Phosphate Route to IPP and DMAPP
139 Hemiterpenes: Just One Isoprene Unit
140 Monoterpenes (C10) and Isoprene Linkage: Heads, IPP Wins;
Tails, DMAPP Loses
141 Geranyl PP to Neryl PP via Linalyl PP: The Importance
of Alkene Stereochemistry
143 Some Acyclic Monoterpenes and Their Uses
144 Mono-and Bicyclic Monoterpenes via Cationic Cyclizations
and Wagner–Meerwein Shifts
147 What’s that Smell? Limonene Derivatives as Flavor
and Fragrance Compounds
149 Irregular Monoterpenes: If Not Head-to-Tail, then How?
151 Iridoids: From Catnip to Alkaloids
152 Sesquiterpenes (C15): Linking of Different Starter Units
154 Some FPP Cyclizations in Sesquiterpene Biosynthesis
156 Trichodiene and the Trichothecenes: How to Trace a Rearrangement Pathway
159 Diterpenes (C20): Taking it to the Next Level of Molecular
Complexity and Diversity
159 Cyclic Diterpenes: From Baseball and Plant Hormones to Anticancer Drugs
162 Sesterterpenes (C25): Less Common, More Complex
164 Triterpenes and Steroids: Another Case of Irregular Linkage of Terpene Units
165 Oxidosqualene and Steroid Biosynthesis: Cyclization
to Lanosterol and Beyond

170 Conversion of Lanosterol (C30) to Cholesterol (C27): Where Did

Contents viii the Carbons Go?
172 Conversions of Cholesterol: Production of the Sex Hormones
175 Dehydrocholesterol, Sunshine, and Vitamin D3 Biosynthesis
176 Tetraterpenes and Carotenoids: Tail-to-Tail Linkage of C20 Units
180 Looking Ahead
180 Study Problems
184 5. The Acetate Pathway: Biosynthesis of Polyketides and Related Compounds

185 Fatty Acids: Multiples of Two Carbons, Saturated or Unsaturated
190 Saturated Fatty Acid Biosynthesis: It All Starts with Acetyl-CoA
194 Branched Fatty Acids: Different Routes and Different Results
195 Mono-and Polyunsaturated Fatty Acids: Putting in the “Essential”
Double Bonds
196 Aerobic Versus Anaerobic Routes to Desaturation
200 Further Desaturation of Fatty Acids: Triple Bonds and Rings
202 Prostaglandins, Thromboxanes, and Leukotrienes: The Power
of Oxygenated FAs
208 Polyketide Biosynthesis: More Starter Units and Extender Units,
but with a Twist
211 Aromatic Polyketide Natural Products: Phenols and Related Structures
213 Isotopic Labeling Studies: Biosynthetic Insights via 13C NMR
219 Further Modification of Polyketides: Alkylations, Oxidations,
Reductions, and Decarboxylations
221 Other Oxidative Modifications of Aromatic Rings: Expansion
or Cleavage Processes
223 Oxidative Coupling of Phenols: Formation of Aryl–Aryl Bonds
227 The Use of Other Starter Groups: From Cancer Drugs and Antibiotics
to Poison Ivy
234 More on Polyketide Synthase (PKS) Systems: Increasing Product Diversity
240 Modular Type I PKS Complexes and Macrolide Antibiotics:
Erythromycin Biosynthesis
245 Genetic Manipulation of Modular PKS Systems: Rational Drug Modification
249 Some Final PKS Products of Medicinal Importance
252 Looking Ahead
252 Study Problems
258 6. The Shikimate Pathway: Biosynthesis of Phenolic Products

from Shikimic Acid
259 What Is Shikimic Acid?
260 Shikimic, Chorismic, and Prephenic Acids at the Heart of the Pathway
261 The Claisen Rearrangement: Allyl Vinyl Ethers in a Chair

263 Conversion of Chorismic Acid to Prephenic Acid
ix Contents
263 Conversion of Prephenic Acid to Phenylalanine or Tyrosine
265 More Uses for Chorismic Acid
268 Shikimic Acid Pathway Products from Phenylalanine
and Tyrosine: An Overview
269 Phenylpropanoids: A Large Family of Phenyl C3 Compounds
270 Phenylpropanoids: Reduction of Acids to Phenyl C3 Aldehydes and Alcohols
270 Reduction of Phenyl C3 Alcohols to Phenylpropenes
272 Lignans and Lignin: Oxidative Phenolic Coupling with a Twist
272 Coniferyl Alcohol Oxidative Coupling: Allyl C-Radical + Allyl C-Radical
273 Coniferyl Alcohol Oxidative Coupling: Ortho C-Radical + Allyl C-Radical
274 Coniferyl Alcohol Oxidative Coupling: O-Radical + Allyl C-Radical
274 Lignin: A Plant Polymer and Major Source of Carbon
276 Podophyllotoxin Biosynthesis: Aryltetralin Lignans from
the American Mayapple
278 Cleavage of Cinnamic Acids to Phenyl C1 Compounds: Different
Routes, Similar Outcomes
279 Coumarins: Sweet-Smelling Benzopyrones
281 Mixed Products: Combining the Shikimate, Polyketide,
and Terpenoid Pathways
282 Kavalactones: Natural Sedatives from the South Pacific
284 Flavonoids: Structurally Diverse Plant Polyphenolics
285 The Chalcone-to-Flavanone-to-Flavone Sequence: Formation of Apigenin
286 The Flavanone-to-Dihydroflavonol-to-Anthocyanin Sequence:
Formation of Pelargonidin
288 The Flavanone-to-Isoflavanone-to-Isoflavone Sequence:
Formation of Genistein
289 Isoflavanoid Structural Modifications: Production of Antimicrobial
Phytoalexins
292 Rotenoids: Fish Poisons from Isoflavones
293 Looking Ahead
294 Study Problems
300 7. Biosynthesis of Alkaloids and Related Compounds

302 Alkaloid Structure: The Importance of N-Heterocycles
302 Alkaloids Not Derived from Amino Acids: Amination Reactions,
Poisons, and Venoms
304 Amino Acids and Mannich Reactions: Important Keys
to Alkaloid Biosynthesis
306 Alkaloids from Ornithine: Tropanes via the Mannich Reaction in Action
310 Pyrrolizidine Alkaloids: Poison Plants and Insect Defense

312 Piperidine-Type Alkaloids Derived from Lysine

Contents x 315 Quinolizidine Alkaloids: Livestock Poisons from Cadaverine
317 Alkaloids from Phenylalanine: From Neurotransmitters
to Decongestants and Narcotics
318 Alkaloids from Tyrosine: The Pictet–Spengler Reaction
in Alkaloid Biosynthesis
319 (S)-Reticuline: A Versatile Pictet–Spengler-Derived
Benzyltetrahydroisoquinoline
320 Oxidative Coupling in Alkaloid Biosynthesis: Biosynthesis
of Corytuberine and Morphine
324 The Morphine Rule
327 Alkaloids from Tryptophan: Adventures in Indole Alkaloid
Structural Complexity
331 Pictet–Spengler-Type Reactions of Tryptamine: β-Carbolines
and Indole Terpene Alkaloids
338 Alkaloids from Nicotinic Acid: Toxic Addictive Derivatives
of a Common Nutrient
341 Alkaloids from Anthranilic Acid: From Tryptophan to Quinolines
and Acridines
341 Alkaloids from Histidine: From Simple Amides to Glaucoma Drugs
343 Purine Alkaloids: Addictive Stimulants in our Coffee, Tea, and Chocolate
344 Cyclic and Macrocyclic Peptides: From Sweeteners to Antibiotics and Beyond
349 Penicillins, Cephalosporins, and Carbapenums: The Essential
β-Lactam Antibiotics
354 A Final Look Ahead
356 Study Problems
361 8. Organic Synthesis in the Laboratory

362 Why We Synthesize Organic Compounds
364 Synthetic Challenges: Total Synthesis
366 Synthetic Challenges: Semisynthesis
369 Synthetic Challenges: Biomimetic Synthesis
372 Synthetic Challenges: Structural Revision or Confirmation
373 Synthetic Challenges: Formal Synthesis
377 Synthetic Challenges: Stereoselective Synthesis of Optically Pure Compounds
378 Resolution of Enantiomers to Obtain Optically Pure Compounds
380 Use of Chiral Pool Compounds for Synthesis of Optically
Pure Natural Products
381 Use of Chiral Reagents for Synthesis of Optically Pure Compounds
384 Use of Chiral Substrate Control for Stereoselective Synthesis
386 Use of Chiral Auxiliaries for Synthesis of Optically Pure Compounds
390 Use of Chiral Catalysis for Synthesis of Optically Pure Compounds

399 Use of Enzymes for Synthesis of Optically Pure Compounds: Biocatalysis
xiâ•…Contents
405 Some Final Thoughts
406 Study Problems
411 Suggested Further Readings

417 Index

Acknowledgments
A book like this is the result of the input, support, and effort of so many individuals
that properly thanking them all would require yet another chapter. Nevertheless,
I would like to acknowledge a global host of scientists in various fields whose re-
search efforts over many decades provided the foundation for all that is found in
this book. Among the many essential reference materials listed at the end of the
text, I especially wanted to acknowledge the important works of Paul Dewick, John
Mann, and Richard Silverman; their volumes proved to be inspirational and indis-
pensible guides for me throughout the preparation of my own. I would also like to
thank all those at Oxford University Press who assisted in the development of this
book at various stages, especially Jeremy Lewis who encouraged the initial ideas and
then waited patiently over a number of years for the work to finally be completed.
I am also deeply indebted to the University of Dayton for providing a sabbatical
leave during which several portions of the book were completed. Most importantly,
I wanted to thank my wife Barbara Smith for her abundant patience, encourage-
ment, and support throughout the writing process.
GWM
May 2016
xiii

Introduction
Nearly twenty years ago while on a sabbatical at Ohio State University, I decided to sit
in on an advanced graduate level course that dealt with a topic I knew almost nothing
about, namely the biosynthesis of organic compounds. Though I was (and still am)
a traditional organic chemist, I nevertheless found many of the topics in that course
to be fascinating, especially the remarkable similarities between the mechanisms of
long-established organic reactions and those of cellular bioorganic processes. In the
years that followed, I gradually accumulated additional research materials with the
thought of eventually developing an undergraduate-level course in bioorganic syn-
thesis for students who I believed would have an interest in this topical area. I sub-
sequently taught such a course for a number of years to many interested students,
majors in chemistry, biology, premedicine, and bioengineering, and their positive
response to the course and the great fun I had in teaching it were my prime motiva-
tion for converting most of the lecture materials into this textbook. To help set the
stage for further understanding of what the book is all about, a brief look at several
important introductory points would seem to be in order, so here goes.
THE UNIQUE ROLE OF CARBON
It all begins with carbon, the essential element of all organic and biological chem-
istry. As illustrated in Fig. I.1, the carbon cycle is basically a description of the
processes involved in the storage and transport of carbon throughout the planet.
Though carbon is of relatively low abundance in the earth’s crust (~0.03% by weight)
compared to elements such as silicon (~45%) and oxygen (~29%), its importance
cannot be overstated since it is the fundamental element essential for the existence
of life. Understanding how carbon is distributed and stored throughout the environ-
ment and how it is converted from one form to another is a good place to start in
gaining a deeper understanding of the unique relationship between carbon and life.
CARBON STORAGE AND THE ROLE OF METABOLISM
The group of processes that converts carbon from one form into another in living
systems is called metabolism. Anabolic metabolism uses energy derived from
xv

Introduction xvi
atmospheric
carbon dioxide
combustion respiration decomposer

respiration
acidification
fossil fuel ocean organic compounds death dead organic

carbon fixation respiration photosynthesis
(in animals) matter
sea shell,
sedimentary
rock
fossilization feeding death
organic compounds
(in plants)
FIGURE I.1
The carbon cycle, illustrating the various fates of carbon in the biosphere.
sunlight or other sources to synthesize complex organic molecules from simpler

ones. Autotrophs (mainly plants) are organisms that carry out anabolic processes
such as photosynthesis, for which the fundamental source of energy is sunlight and
the source of carbon is atmospheric carbon dioxide (carbon fixation). In terms of
the carbon cycle, the most important autotrophs are trees and phytoplankton, which
fix carbon by carrying out photosynthesis, converting carbon dioxide and water
into glucose (C6H12O6), one of the basic organic materials from which most other
organic compounds essential for living organisms are derived. Overall, this is a re-
ductive process, converting carbon from an oxidized form (CO2) to a reduced form
(glucose) via the familiar photosynthesis equation: 6CO2 + 6H2O + light energy →
C6H12O6 + 6O2.
When atmospheric carbon dioxide dissolves in the oceans, it is used in pho-
tosynthesis, but may also be converted to bicarbonate (HCO3–) and carbonate
(CO32–); marine organisms utilize calcium ion for the formation of shell material
(CaCO3). The accumulation of seabed shell deposits over millions of years ulti-
mately leads to significant carbon storage in the form of sedimentary rock deposits
such as chalk, limestone, and dolomite. Considerable amounts of carbon are also
stored within fossil fuels such as petroleum and natural gas. These are believed to
have formed over time by decomposition of large quantities of marine organisms
in oxygen-depleted waters in combination with high heat and pressure. Coal, an-
other significant carbon source, dates from Earth’s carboniferous period and was
presumably formed by similar processes over the ages through decomposition of
terrestrial plant material.

For organisms which cannot fix carbon to make their own organic compounds
xvii Introduction
(heterotrophs), the consumption of other organisms or their parts (such as seeds or
fruits) is necessary to obtain both carbon and the energy required for the anabolic
processes used to transform it. The fermentation or decay of dead organic matter by
fungi or bacteria also transfers carbon: these are examples of catabolism, degrada-
tive metabolic processes which involve the breaking down of more complex organic
materials (derived from anabolic processes) into simpler ones and the principal path
by which most carbon leaves the biosphere. Aerobic (with oxygen) catabolism (res-
piration) releases carbon back to the environment mainly by oxidative degradation
of organic compounds to give carbon dioxide and water in what is essentially the
reverse of the photosynthesis equation (with energy released in forms other than
light). Anaerobic respiration (without oxygen) may also occur, releasing significant
amounts of carbon back into the environment in the form of methane (as in marsh
gas or cattle flatulence). Additional release of carbon dioxide into the atmosphere
also occurs through the burning of fossil fuels and other combustion processes and
by acidification of carbonate sources. With the subsequent conversion of atmo-
spheric CO2 back to glucose by photosynthesis, the carbon cycle, in which living
organisms play a key role, continues.
DISTINGUISHING PRIMARY VERSUS

SECONDARY METABOLISM
From a chemistry standpoint, both autotrophs and heterotrophs vary a great deal in
their ability and capacity to synthesize and transform complex organic compounds
in a set of anabolic processes collectively referred to as biosynthesis. As noted above,
plants are efficient at constructing glucose via photosynthesis using simple inorganic
materials (CO2 and H2O) derived from the environment. By contrast, most other or-
ganisms, from animals to simple microorganisms, must rely primarily on degrada-
tion of plant or animal material in the diet in order to obtain both the energy and the
raw materials necessary to carry out biosynthesis. But in all cases, the chemical reac-
tion mechanisms and pathways employed by living cells for synthesizing or mod-
ifying the basic organic compounds essential for life, such as carbohydrates, fats,
proteins, or nucleic acids, are essentially identical in all known organisms, owing to
their common evolutionary ancestry. Collectively, this set of processes constitutes
the core of biochemistry and is known as primary metabolism. The compounds
derived from such processes, all of which are essential for life, are generally referred
to as primary metabolites (Fig. I.2).
SECONDARY METABOLITES AND NATURAL PRODUCTS
Alternatively, secondary metabolism in organisms employs metabolic pathways

for the production of certain complex organic compounds which are unrelated to

Introduction xviii
H
H2N C C OH
NH2 OH OH
CH 2
H O
N H
N
H OH
H
HN OH
N N OH H
H
tryptophan adenine glucopyranose
FIGURE I.2
Three examples of primary metabolites: an amino acid, a DNA base, and a carbohydrate.
OH
O
O
H H OCH3 O OCH3
H O
O N S CH3 O
CH3 O
N
O COOK H CO O
3
Cl H3CO OCH3
penicillin G
griseofulvin OCH3
podophyllotoxin
FIGURE I.3
Three examples of secondary metabolites: an antibiotic, an antifungal, and an anticancer agent.
and therefore unnecessary for an organism’s growth, development, or reproduction.

Such secondary metabolites are usually unique to a specific organism or group
of organisms and may be thought of as fundamental expressions of the organism’s
identity, even though these compounds may be produced only under specific condi-
tions. Such organic natural products have functions that are frequently obscure;
that is, the benefits to the producing organism may be entirely unknown, though
in some cases they have been found to be associated with some specific ecological
activity, such as acting as chemical agents for the direction of sexual attraction or
social behavior in insects or for the suppression of pathogens or predators in plants.
Most importantly, since many secondary metabolites and their derivatives have
been found to possess powerful antibacterial (e.g., penicillin G), antifungal (e.g.,
griseofulvin), or anticancer (e.g., podophyllotoxin) activity, such natural prod-
ucts are of extraordinary interest and significance in medicine and human health
(Fig. I.3).
NATURAL PRODUCTS IN ORGANIC

CHEMISTRY AND MEDICINE
Humans have been using organic compounds derived from plant sources for the
treatment of various health conditions for well over 4000 years, but it was not until
the 1800s that many of the active principals from various plants were actually

isolated and studied. This important work helped to establish a scientific basis for
xix Introduction
the often-powerful action of organic compounds as medicines, toxins, or halluci-
nogens, moving them for the first time out of the realms of superstition, magic, or
folklore. In fact, up until the early 1900s, the study of organic chemistry was es-
sentially the study of natural products, and many of the now familiar techniques for
isolation and characterization of organic compounds were originally developed as
part of such studies.
In modern times, natural products still play an essential role in medicine and
drug discovery. Of the nearly 120 plant-derived organic compounds in use as me-
dicinal agents around the world today, almost three-fourths were derived from stud-
ies of the active principals isolated from plants used in traditional folk medicine.
Although the pharmaceutical industry had begun in the 1990s to move away from
drug discovery modeled on bioactive natural product structures in favor of rapid
biological screening of enormous libraries of organic compounds that could be pro-
duced in small quantities via automated or combinatorial synthetic methods, it had
become apparent by the middle of the first decade of the 21st century that this newer
approach to drug discovery had met with only limited success, leading to a renewed
interest in natural products chemistry as a complementary source of lead com-
pounds. Today, screening assays continue to uncover new organic compounds from
nature that in some cases reveal previously unknown modes of biological action.
Others may someday hold the key to treating conditions we are currently unable
to adequately address with known medicinal agents. Such “bioprospecting” con-
tinues to expand into areas involving marine organisms and microbes—relatively
untapped sources of new organics as compared to plant sources—making it increas-
ingly important for scientists from a variety of different fields to have a better under-
standing of the fundamental organic chemistry used by organisms to produce such
important bioactive compounds.
THE ORGANIC CHEMISTRY OF SECONDARY

METABOLITE BIOSYNTHESIS
Most students are probably aware of the role that cholesterol-reducing drugs such as
lovastatin (Mevacor) have played in the control of heart disease in recent years, but
even chemistry majors might be surprised to learn that many statin drugs are or-
ganic compounds isolated from nature and that cells use an enzymatic Diels–Alder
reaction to form the two 6-membered rings at the structural core of this life-saving
compound (Fig. I.4).
Familiar organic reactions such as this one constitute the fundamental core of
methods used by all organisms to construct the complex organic compounds associ-
ated with life on our planet. For this reason, development of a book focusing exclu-
sively on such organic reactions and their mechanisms, with special attention given
to compounds of medicinal and pharmacological interest, seemed a reasonable way

HO O
Introduction xx Enzyme-S O Enzyme-S O O

O
H
O
Diels–Alder H
cyclase
many steps
lovastatin (Mevacor)
FIGURE I.4
Diels–Alder cyclization in the biosynthesis of lovastatin.
to introduce undergraduate students to the elegantly simple organic chemistry used

in various biosynthetic pathways.
GOALS AND STRUCTURE OF THIS BOOK
My hope is that Bioorganic Synthesis: An Introduction will prove useful in support-

ing various advanced undergraduate courses for chemistry, biochemistry, biology,
premedicine, bioengineering, and related majors whose students could benefit from
a deeper understanding of the chemical logic of reactions carried out in organisms
and the origins and uses of the biologically active compounds they often produce.
Importantly, the book assumes no prior background in biochemistry, though stu-
dents who have had an introductory biochemistry course should find the material
enlightening, and all students with the necessary organic chemistry prerequisites
should find the book supportive of their previous studies as it guides them through
the development of deductive skills to identify the relationship between specific or-
ganic compound structural features and the biosynthetic mechanisms and pathways
involved in their production.
The topics and sequence differ somewhat from the many excellent available
monographs on biosynthesis and natural products chemistry by focusing primarily
on the reaction mechanisms involved in the production of representative and phar-
macologically significant compounds from each of the major biosynthetic pathways
known to us. Most importantly, the book is designed specifically for undergraduate
students, rather than as an exhaustive reference work for graduate students or pro-
fessional researchers. Most organic chemistry instructors at the undergraduate level
will find their own background in organic chemistry more than sufficient for the
development of a meaningful course based on the text.
The book consists of eight chapters, beginning with a brief review of relevant
topics from introductory organic chemistry. This is followed by a detailed presen-
tation in Chapter 2 of an essential set of organic and biochemical reactions used
throughout the book as well as a brief introduction to the function and chemistry of
the most important coenzymes.

Chapter 3 covers basic carbohydrate chemistry and the biosynthesis of amino
xxi Introduction
acids for those students who may have had only a brief introduction to these topics
from their previous organic studies. This material is essential for a proper under-
standing of the role these important compounds play as building blocks in natural
product biosynthesis via glycoside and polypeptide formation and other processes.
The next four chapters are each devoted to a particular biosynthetic pathway.
The terpenoid pathway in Chapter 4 is the starting point for biosynthesis stud-
ies, since carbocations are the principal intermediates in the mechanisms of this
pathway and so should be most familiar to students from an introductory organic
chemistry sequence. The chapter covers biosynthesis of all the important classes of
terpenoids, with a detailed description of the biosynthesis and biological relevance
of the steroids.
This is followed by the acetate pathway presented in Chapter 5. This chapter
begins with examination of the biosynthesis of saturated and unsaturated fatty acids
and prostaglandins and their biological relevance, a discussion which provides the
basis for subsequent understanding of the biosynthetic origins of acetate-derived
polyketide natural products through cyclizations and transformations of intermedi-
ates via familiar aldol, Claisen, and related carbonyl condensation reactions.
Chapter 6 introduces the shikimate pathway. The biosynthesis of aromatic amino
acids and other shikimate-derived metabolites such as coumarins, lignans, and
products derived from the intersection of shikimate metabolites and the acetate
pathway are also examined. This material is followed by an introduction to alkaloids
and related nitrogenous compounds in Chapter 7.
Finally, Chapter 8 considers some of the obstacles faced by synthetic and medici-
nal chemists who tackle the challenge of recreating intricate molecules like lovatstin
in the laboratory from scratch. We will be left to marvel at just how difficult and
intellectually challenging it can be to replicate many of the molecular construction
projects that our cellular friends seem to achieve with such ease. But we will also
see how knowledge of biosynthetic pathways can also lead to new ways of thinking
about chemical problems and the development of new methods for organic syn-
thesis. And we’ll find that occasionally we can even employ the direct assistance of
simple organisms and their enzymes in the laboratory to help us accomplish goals in
synthetic and medicinal organic chemistry.
In the end, it is my sincere hope that both students and instructors of organic
chemistry will find the book to be a useful introductory guide to further exploration
of the exciting world of biological organic chemistry.

1 Brief Organic Review
As soon as we touch the complex processes that go on in a living thing, be it plant or animal,
we are at once forced to use the methods of this science [chemistry].
—John Jacob Abel (co-founder of The Journal of Biological Chemistry, 1905)
REVIEW OF FUNCTIONAL
GROUPS, STEREOCHEMISTRY,
AND CONFORMATIONAL ANALYSIS
In addition to simple hydrocarbon structures (alkanes, alkenes, alkynes, and aro-

matic systems) and alkyl groups (methyl, ethyl, propyl, isopropyl, etc.), this text
assumes a familiarity with the most common functional groups associated with or-
ganic chemical structures and their basic reactivity patterns. Table 1.1 summarizes
the names and structures of some of the more important functional groups we will
be dealing with throughout the remainder of the book. It is important to remember
that functional groups containing O or N with nonbonding electrons have an affin-
ity for both protic and Lewis acids and are important participators in H-bonding.
Groups containing a carbonyl (C=O) function are especially important, as these
bonds are strongly polarized (δ+C=Oδ–), the C atom being electron deficient and the
O atom electron excessive; this strong polarization is mainly responsible for the fa-
miliar reactivity patterns associated with carbonyl compounds.
1

Table 1.1 Common Organic Functional Groups

Bioorganic Synthesis 2
Name Structure Name Structure
O
alcohol ROH monophosphate RO P O (ROP)
O
O O
phenol ArOH
diphosphate RO P O P O (ROPP)
O O
n
ether R-O-R, O
O
acid R C OH
R-NH2, R 2NH, R 3N, O
amine n n ester R C OR'
N N
H R O
thiol RSH n
lactone
O
O
sulfide R-S-R thioester R C SR'
O
disulfide R-S-S-R amide R C NH2 , NHR , NR2
O
O n
aldehyde lactam N
R C H H(R)
O O NH(R)
n
imine
ketone R C R' (Schiff's base) R C H(R)
STEREOCHEMISTRY REVIEW: THE SHAPES

OF THINGS TO COME
Figure 1.1 depicts the standard classification of isomers in organic chemical struc-
tures. Recall that constitutional isomers are compounds with the same molecular
formula but different atom connectivity, such as 1-butanol versus 2-butanol (Fig. 1.2).
Stereoisomers, on the other hand, are compounds with the same formula and the
same atom connectivity, differing from one another only in the three-dimensional
orientation of their atoms in space. These are divided into two groups: enantiomers
and diastereomers.
Enantiomers are nonsuperimposable mirror image molecules whose asymme-
try is usually the result of a tetrahedral carbon atom with four different atoms or
groups attached to it, as in the 2-butanol enantiomers. Such chiral molecules rotate
the plane of polarized light either (+) or (−) and so are said to be optically active.
Achiral molecules, such as 1-butanol, do not rotate the plane of polarized light and
so are optically inactive.
A standard formalism for representation of a chiral center is to use bond line
drawings with two of the four atoms or groups lying in the plane of the paper, a third
projecting outward (wedge bond), and the fourth projecting inward (dashed bond).

Isomers
Constitutional Isomers Stereoisomers
Enantiomers Diastereomers
non-superimposable non-superimposable
mirror-image stereoisomers non-mirror-image stereoisomers
FIGURE 1.1
Classification of isomers in organic chemical structures.
R S S
=
OH vs.
OH HO H H OH H OH
constitutional isomers: 1- vs. 2-butanol enantiomers: (R)- vs. (S)-2-butanol
HO H H OH
S CO2H S CO2H
S R
vs. H NH2
H NH2 E vs. Z
diastereomers: (2S,3S)- vs. (2S,3R)-3-phenylserine and (E)- vs. (Z)-3-methyl-2-pentene
FIGURE 1.2
Comparison of constitutional isomers, enantiomers, and diastereomers.
Note that an enantiomeric pair may be drawn formally as reflected mirror images,
though it is useful to remember that an enantiomer may be generated simply by
interchanging any two of the four atoms or groups attached to the chiral carbon
(compare (R)-versus (S)-2-butanol in Fig. 1.2). The stereochemical prefixes S and R
are added to the chemical names of enantiomers to distinguish one from the other
and are assigned on the basis of the priorities established for the four atoms or
groups attached to the chiral carbon atom, using the Cahn–Ingold–Prelog (CIP)
rules. When the molecule is oriented so that it may be viewed looking down the
bond between the chiral carbon and the lowest-ranked atom or group, the three re-
maining atoms or groups are arranged much as in a three-spoked wheel. If the order
of decreasing priority of these three traces a counter-clockwise path, the absolute
configuration of the molecule is assigned as S. If the path is clockwise, the configu-
ration is assigned as R. Recall that R versus S and (+) versus (−) are not necessar-
ily related: some R compounds are (+) while others are (−). Optical rotation, both
magnitude and sign, must be measured and cannot be predicted simply on the basis

of absolute configuration assignment. Compounds with more than one stereocenter

Bioorganic Synthesis 4 will have more than two stereoisomers. In general, n stereocenters give 2n stereo-
isomers. Thus, 3-phenylserine (Fig. 1.2) has two stereocenters and a total of four
stereoisomers, but while (2R,3R) and (2S,3S) 3-phenylserine are enantiomers (non-
superimposable mirror image stereoisomers), the (2S,3S) and (2S,3R) stereoisomers
are related as diastereomers (nonsuperimposable, nonmirror image stereoisomers).
Unlike enantiomers, diastereomers have different chemical and physical properties.
Recall that the relationship between cis and trans stereoisomers is also diastereo-
meric and most commonly occurs among both alkenes and disubstituted cycloalkane
systems. For alkenes, the cis or trans stereochemical designation is unambiguous
only for disubstituted alkenes, while the E,Z designation is more general and may
be applied to all stereochemically different alkenes. The E,Z designation, as in
(E)-versus (Z)-3-methyl-2-pentene (Fig. 1.2) assigns priorities to the two atoms or
groups attached to each end of the double bond (one higher priority, one lower pri-
ority for each atom or group attached to C). These priorities are assigned one end
at a time, using the same CIP rules as for chiral carbons. The Z designation results
when the higher priority atoms or groups are on the same side of the double bond;
E when they are on opposite sides.
Certain compounds with multiple stereocenters may nevertheless be achiral
(optically inactive) if the molecule possesses an internal plane of symmetry. Such
achiral molecules are known as meso compounds and can often be recognized as
such by finding a mirror plane that reflects one half of the molecule in the other half,
as shown for 2,3-dichlorobutane isomers and 1,2-dimethylcyclopentane isomers
(Fig. 1.3). A meso compound and its mirror image are superimposable and therefore
identical, even though two (or more) stereocenters are involved.
CH 3 CH 3 CH 3 CH 3
Cl H H Cl H Cl Cl H
vs.
H Cl Cl H H Cl Cl H
CH 3 CH 3 CH 3 CH 3
(2R,3R) (2S,3S) (2R,3S) (2R,3S)
enantiomers of identical:
2,3-dichlorobutane meso-2,3-dichlorobutane
vs.
(1S,2S) (1R,2R) (1R,2S) (1R,2S)

enantiomers of identical and meso:
trans-1,2-dimethylcyclopentane cis-1,2-dimethylcyclopentane
FIGURE 1.3
Comparison of enantiomers, diastereomers, and meso compounds.

1 CH3
H 1 CH3 CH3
Cl 2
Cl H
H 2 Cl Cl H
2
H 3 Cl H 3 Cl
CH3 3 H Cl
1 H Cl
4 CH3 4 CH3 CH3
4 CH3
staggered eclipsed vertical Fischer Projection
FIGURE 1.4
Conversion of a staggered conformation into a Fischer projection.
A convenient way to represent the configuration of certain acyclic compounds

with one or more chirality centers without drawing wedge and dashed bond structures
is through the use of Fischer projections. The process for generating such structures
from more conventional representations is illustrated for (2R,3R)-2,3-dichlorobutane
(Fig. 1.4). Beginning with a staggered conformer, the molecule is then rotated about
the C2–C3 bond to generate an eclipsed conformer which, when viewed from above,
is redrawn as a vertical chain with correct use of wedge and dashed bonds, with the
lowest numbered atom of the compound (from the IUPAC name, not from CIP pri-
orities) at the top of the chain. Finally, the wedge and dashed bonds are converted
to simple lines. When drawn this way, it is understood that in a Fischer projection
horizontal bonds project outward while vertical bonds project inward, with chiral-
ity centers at the points of intersection. Such projections are especially useful in the
representation of carbohydrate structures, as will be seen in Chapter 3.
PROCHIRAL RELATIONSHIPS: ONE STEP

FROM CHIRALITY
By prochiral carbon we mean an achiral carbon atom in a molecule which, through

a chemical transformation, may be converted into a chiral carbon. One such case in-
volves prochiral carbon with so-called enantiotopic atoms (or groups). For example,
if replacement of one of two identical atoms (or groups) at an achiral carbon by an
arbitrary atom X produces a chiral enantiomer as product, such an achiral carbon is
said to be prochiral and the atom replaced is said to have been enantiotopic (Fig. 1.5).
Furthermore, assuming that X has a CIP priority higher than that of the atom
replaced but lower than that of the other two groups, if the enantiomer produced
has the R configuration, then the enantiotopic atom replaced by X is called the pro-R
atom; replacement of the other enantiotopic atom would produce the S enantiomer
and so would be the pro-S atom. In the example shown, HR and HS are identical in
all respects (chemical and physical properties), but will differ in their reactions with
chiral reagents. Thus, the chiral enzyme that catalyzes the oxidation (dehydrogena-
tion) of ethanol to acetaldehyde in the liver is selective for removal of the pro-R
hydrogen atom of ethanol (Fig. 1.6).

HS HR HS X
X
HR is the pro-R hydrogen
OH R OH
HS HR X HR
X
HS is the pro-S hydrogen
OH S OH
FIGURE 1.5
Enantiotopic hydrogen atoms on the prochiral carbon of an alcohol.
HS
HS HR liver alcohol
OH dehydrogenase O
FIGURE 1.6
Enantiotopic proton selectivity in an enzymatic alcohol dehydrogenation reaction.
PROCHIRAL Π-S YSTEMS: “TWO-FACED”

REACTION CENTERS
In appropriately substituted systems, an sp2 carbon may also be prochiral. In such cases,
we find that in reactions in which an achiral sp2 carbon is converted to a chiral sp3 carbon
by addition of a new atom or group, different enantiomers will be formed depending on
which “face” of the planar π system is approached when the new bond is formed. These
two prochiral faces are distinguished from one another by the stereochemical terms Si
and Re which are readily assigned to a prochiral sp2 center by application of the CIP
rules to the three different atoms or groups around the sp2 carbon, as shown in Fig. 1.7.
Thus, we see that reaction of achiral 2-butanone with phenylmagnesium bromide pro-
duces chiral 2-phenyl-2-butanol by nucleophilic attack from the front or the back of the
planar ketone π-system. While achiral reactants (such as PhMgBr) cannot distinguish
between Si and Re faces (and so produce racemic mixtures), chiral reagents or catalysts
such as enzymes can distinguish between them and may result in formation of enantio-
merically pure products from such prochiral π-systems.
Note that attack at a Si face does not necessarily indicate subsequent generation of the
S enantiomer of the resulting chiral product and vice versa. The Si and Re stereochemi-
cal assignments are used only to distinguish between the two prochiral π-system faces.
One example of addition to a prochiral alkene carbon to produce an enantiomer-
ically pure product is the enzyme-catalyzed hydration of the π-bond of fumaric acid
to give only (S)-malic acid (Fig. 1.8). Note that in addition of the elements H–O–H
across the π-bond, the O atom approaches only the Si face of the prochiral carbon.
Since the fumarase enzyme binding the substrate is chiral, it can distinguish between
the two faces, blocking one and facilitating the addition of water to the other, pro-
ducing a single enantiomer product.

3
O direction O
in going R
S from highest
2 1 priority to 1 2
("sinister" "Si " face lowest (CIP) ("rectus"
"Re" face
CCW, to the left) CW, to the right)
O Ph OH HO Ph
1. PhMgBr, THF
+
2. H3O+
prochiral attack on attack on

carbon "Si" face "Re" face
FIGURE 1.7
Assignment of Si and Re faces at a prochiral carbonyl center.
prochiral carbon addition to "Si" face only

H O O
H 2O
HO HO
OH OH
fumarase
O H enzyme O HO H
fumaric acid (S)-malic acid
FIGURE 1.8
Face selectivity of fumarase enzyme in the hydration of fumaric acid to (S)-malic acid.
DIASTEREOTOPIC ATOMS AND GROUPS: ONE

STEP FROM A DIASTEROEOMER
If replacement of one of two identical atoms (or groups) at an achiral carbon by an

arbitrary atom X produces a diastereomer as product, such atoms on the prochiral
carbon are said to be diastereotopic (Fig. 1.9).
Unlike enantiotopic atoms, diastereotopic atoms are not in identical chemical
environments and so are distinguishable by both chiral and achiral reagents. For
example, the enzyme phenylalanine ammonia lyase is selective for the diastereotopic
HS proton in its catalysis of the elimination of ammonia to yield trans-cinnamic acid
(Fig. 1.10).
Finally, we note that when substitution occurs at a chiral carbon, several possible
stereochemical outcomes are possible, depending on the mechanistic details and
reactive intermediates involved. These outcomes are summarized in Fig. 1.11, using
examples in which a new atom X replaces atom D at a chirality center. Thus, we may
refer to a reaction at chiral carbon as proceeding with retention of configuration, in-
version of configuration, or racemization (complete or partial) at the chirality center
(Fig. 1.11).

X HR HS X
HS HR
CO 2H CO2H CO2H
X
or
H NH2 H NH2 H NH2
phenylalanine diastereomers
FIGURE 1.9
Diastereotopic protons on the prochiral carbon of phenylalanine.
HR
HS HR
CO2H
CO 2H phenylalanine
H NH2 ammonia lyase

H
(anti elimination
phenylalanine of H S -NH 2) trans-cinnamic acid
FIGURE 1.10
Diastereotopic proton selectivity in an enzyme-catalyzed antielimination reaction.
A A
B retention B
X + D X + D
C C
A A
B inversion B
X + D X + D
C C
A A A
B racemization B B
X + D X + X + D
C C C
FIGURE 1.11
Possible stereochemical outcomes at a chiral reaction center.
RING SYSTEM CONFORMATIONS: FROM LYING

FLAT TO SITTING IN A CHAIR
Cyclopropanes are the only truly flat cycloalkane rings. These rings possess signifi-
cant angle strain due to their 60o bond angles versus 109.5o for an ideal sp3 tetra-
hedral angle, so the C–C bonds have poor sp3–sp3 orbital overlap. Along any C–C
bond, adjacent C–H bonds are completely eclipsed leading to a total ring strain
(angle strain plus torsional strain) of about 27 kcal/mol. (Fig. 1.12).
Cyclobutanes are slightly puckered rather than flat with a total ring strain of
about 26 kcal/mol, though the strain is distributed over four carbons rather than
three as in cyclopropane (Fig. 1.13). The vicinal C–H bonds are no longer com-
pletely eclipsed, leading to less torsional strain.
The cyclopentane ring adopts a folded “envelope” conformation that rapidly
equilibrates with only ~6 kcal/mol of ring strain. While the base of the envelope

H
H H
H
H
H H H H
H H
FIGURE 1.12
Strained planar geometry of cyclopropane ring systems.
H H H
H H
H
H
H H H
H H H
H H
H
FIGURE 1.13
Puckered cyclobutane ring conformation.
H H
H H H H
H H H
H
H H
H H
H H H H H H
FIGURE 1.14
Envelope conformation of the cyclopentane ring system.
has some torsional strain, the vicinal C–H bonds in the “flap” have minimal strain
(Fig. 1.14).
The cyclohexane ring has essentially no ring strain due to its rapidly intercon-
verting “chair” conformations. Interconversion between chair conformers is some-
times called a “ring flip” and is important because this conformational equilibrium
interchanges all axial and equatorial positions on the ring. Ring flipping involves
tipping one end of the ring up into a higher energy “half-chair” conformation
(~7 kcal/mol higher than chair) which can relax to a slightly lower energy “twist-
boat” conformation (about 1.5 kcal/mol lower in energy than the boat) and so on
(Fig. 1.15).
MONOSUBSTITUTED CYCLOHEXANES: FAVORING
EQUATORIAL POSITIONS
The more stable of two chair conformers for a monosubstituted cyclohexane

is one in which the substituent occupies an equatorial rather than an axial po-
sition. Axial substituents introduce steric strain via “1,3-diaxial” interactions.
This strain is relieved via a ring flip to the lower energy equatorial conformer
(Fig. 1.16).

H D
D D H D H H
H D H D
D H
H D H D H D
H D H D
half-chair half-chair
H D
D D H H
D H
D 7.0 kcal/mol
HH D
Energy
twist boat
5.5 kcal/mol
H D D
H D H
H D
D H
D H
D H H
D
H D
D H D H D
H chair
chair
FIGURE 1.15
Chair-chair interconversion (“ring flipping”) of cyclohexane conformers.
H Xax
H H
H H H Xeq
H
H H
1,3-diaxial H
interactions
FIGURE 1.16
Equatorial preference of monosubstituted cyclohexanes and 1,3-diaxial interactions.
DISUBSTITUTED CYCLOHEXANES: EQUIVALENT
AND NONEQUIVALENT COMBINATIONS
For 1,4-dimethylcyclohexane, the trans stereoisomer is more stable than cis, even
though the methyl groups are clearly too far apart to crowd one another. Both
methyls become equatorial after a ring flip. The cis stereoisomer has one axial
methyl regardless of conformation. Similar stabilities are observed with the 1,2-
dimethylcyclohexanes, with the trans stereoisomer more stable in the diequatorial
conformer than either conformer of the cis (Fig. 1.17).
For 1,3-dimethylcyclohexane, the cis stereoisomer is more stable than trans; one
conformer of cis has both methyl groups equatorial (Fig. 1.18).
In some cases, factors other than steric crowding may influence conformational
stability. For example, the diaxial conformation of cis-1,3-cyclohexanediol is more
stable in nonpolar environments due to intramolecular H-bonding (Fig. 1.19).


trans-1,4
cis-1,4
cis-1,2
trans-1,2
FIGURE 1.17
Conformational preferences in 1,4-and 1,2-disubstituted cyclohexanes.
cis-1,3
trans-1,3
FIGURE 1.18
Conformational preferences in 1,3-disubstituted cyclohexanes.
OH
HO OH
O OH
OH H favored in polar media
favored in
non-polar media
FIGURE 1.19
Intramolecular H-bonding in cis-1,3-cyclohexanediol.
BICYCLIC SYSTEMS: JOINING OF RINGS
Of the fused bicyclic ring systems, the 6-5 and 6-6 ring junctions are the most fre-
quently observed. The 6-5 or hydrindane ring fusion (bicyclo[4.3.0]nonane) has
both cis and trans ring junctures, with the methyl-substituted trans junction a
common feature of steroid structures (Fig. 1.20).

H H
H H
trans cis
H H
FIGURE 1.20
Cis and trans hydrindane (6-5) ring systems.
H H
H H
trans cis H
H
H
H H
FIGURE 1.21
Cis and trans decalin (6-6) ring systems.
The cis and trans decalin ring systems (bicyclo[4.4.0]decanes) are especially note-
worthy. The trans junction is conformationally immobile, while the cis-fused ring
system exists as equilibrating chair forms (Fig. 1.21). Both are also important struc-
tural components of the tetracyclic steroids.
HETEROCYCLIC RING SYSTEMS: ONE ATOM

MAKES ALL THE DIFFERENCE
Many of the medicinal agents important to natural products chemistry contain one
or more heterocyclic rings. Examples include the powerful analgesic N-heterocycle
morphine (from poppy seeds), O-heterocycles such as catechin, a flavonoid compound
with antioxidant and antiHIV activities, and lipoic acid, an important S-heterocyclic
cofactor in many enzymatic systems as well as an over-the-counter dietary supplement
(Fig. 1.22).
A brief review of the most important heterocyclic ring systems and their common
names is found in Table 1.2. While all the names used here are not rigorously cor-
rect in terms of IUPAC nomenclature, we will use them when convenient to refer to

HO

OH
O HO O CO2H
OH
N
CH 3 S S
OH
HO
morphine OH catechin lipoic acid
FIGURE 1.22
Some natural products containing N-, O-, and S-heterocycles.
Table 1.2 Some Common Heterocyclic Ring Systems
O
O O O S
oxirane oxetane furan tetrahydrofuran thiophene
O O O O O
benzofuran pyran tetrahydropyran chromene chroman
NH N N N N
H H H H
aziridine pyrrole pyrrolidine imidazole indole
N N
N N
O S H N H
oxazol e thiazole piperidine pyridine dihydropyridine
N N
N N
N N N
N N N H
pyrimidine pyrazine quinoline isoquinoline purine
portions of more complex organic structures which may contain one or more such
heterocyclic components.
NUCLEOPHILES AND ELECTROPHILES: GIVE

AND TAKE IN BOND MAKING
Since one of our main topical focuses will be organic reaction mechanisms in a bio-
logical context, a brief review of the basics of how organic mechanisms are depicted

HO: Br: HS: H 2 O: :NH 3 H 2 S:

Bioorganic Synthesis 14 anionic nucleophiles neutral nucleophiles
FIGURE 1.23
Examples of charged and uncharged nucleophilic species.
SN 2
Nu: + R X Nu R + :X
δ δ
FIGURE 1.24
Nucleophilic attack on an electrophilic atom in a polarized covalent bond.
is in order. Recall that an electron pair that initiates the “attack” in an organic reac-
tion usually comes from a nonbonding pair on an atom. The atom can be nega-
tively charged (like a hydroxide ion) or neutral (like NH3). For simplicity’s sake,
only one nonbonding pair is shown on the nucleophilic atoms in Fig. 1.23. Electrons
from a two-electron σ-bond or a π-bond can also participate in some cases (we’ll
see specific examples of this later on). When an attacking base forms a new cova-
lent bond in the resulting organic reaction product, the base is called a nucleophile
(“nucleus-loving”).
Bases and nucleophiles are always electron-rich: they have at least one pair of
electrons to share (or a readily available pair from a two-electron bond). The terms
“nucleophile” and “base” are often used interchangeably. Species that are attacked by
nucleophiles are called electrophiles (“electron-loving”). Electrophiles are always
electron-poor or electron-deficient. This characteristic of electrophiles can be due
to any one of several factors: i) presence of a full positive charge on an atom with
an empty orbital (i.e., a cation or formal positive charge); ii) electrically neutral but
electron-deficient due to an empty orbital (lack of an octet, such as in boron or alu-
minum compounds); 3) partially positive due to bonding to a more electronegative
atom (part of a “polar” covalent bond). Polarized bonds are common electrophilic
components in many organic reactions, as in the familiar SN2 reaction (Fig. 1.24).
The reaction of the nucleophile (Nu:–) with an alkyl halide (R–X) is an example of
nucleophilic attack by an electron-rich atom (nonbonding electron pair, negatively
charged) on an electron-deficient atom (R is partially positive due to its covalent
bond to the more electronegative halogen atom X).
Understanding and use of curved arrow notation (or arrow pushing formalism)
is fundamental to the depiction of the mechanistic details of organic and biological
reactions. The better you understand this notation, the greater your understand-
ing of the mechanism will be, which will help you develop the intuition needed to
predict reaction products. The simplest use of the arrow-pushing formalism is to
show the “movement” of electron pairs in the resonance forms of molecules or ions
(Fig. 1.25). Note the use of the double-headed arrow rather than equilibrium arrows
between resonance forms. This is especially useful in evaluating where electron pairs
and negative or positive charges may reside in structures.


O O
OR OR
FIGURE 1.25
Curved arrows showing electron pair movement in resonance forms.
BOND MAKING AND BREAKING: HAVE PAIR,

WILL SHARE; NEED TWO FROM YOU
In almost all organic chemistry reactions, two-electron covalent bonds are made
and broken. To represent these bond making/breaking processes, we use the curved
arrows to show how the electrons are moving or “flowing” in the mechanism. We
represent two-electron covalent bond formation resulting from an electron pair
“attacking” an electron-deficient atom by using a curved arrow pointing from the
electron pair toward the electron-deficient atom (never the other way around). Such
curved arrows are always meant to represent the movement of one electron pair.
They are never used to represent anything else. A simple example would be the reac-
tion of ammonia with hydrogen ion to give ammonium ion (Fig. 1.26).
Bond breaking often occurs in conjunction with bond making. In addition to the
use of a curved arrow to show where a bond is forming, arrow pushing will also be
used to show where an electron pair is going when a bond breaks. In this case, the
arrow will be pointing away from the two-electron bond that is breaking and toward
the atom on which the electron pair will ultimately reside, as in the reaction of a base
B:– with an acid H–A shown in Fig. 1.27.
In some instances, a curved arrow will be used to show the formation of a two-
electron bond that becomes one of the bonds in a multiple bond (double or triple
bond). In such cases, the arrow will be pointing away from a two-electron bond
that is breaking and toward the area between two other atoms where the new two-
electron bond will form. In the E2 elimination reaction depicted in Fig. 1.28, a base
B:– attacks and breaks a C–H bond.
In concert with C–H bond breaking, a B–H bond forms and the electron pair from
the broken C–H bond moves between the two carbon atoms, pushing the electron
pair of the C–Br bond onto Br, thereby breaking the C–Br bond and forming bromide
ion. Note that three electron pairs are moving when going from left to right in the
equation, so three curved arrows are needed to show how and where the electrons
have moved. In any correctly written arrow-pushing mechanism (with charges cor-
rectly assigned), the mass and net charge on both sides of the equation will balance.
BRØNSTED ACID–B ASE REACTIONS: PROTON

DONORS GLADLY ACCEPTED
Acids that act as proton (H+) donors and bases that act as proton acceptors are
called Brønsted acids or bases. These are among the most important processes in

H
H
H N: + H H N H
H H
lone pair on N becomes a shared pair in NH4+
FIGURE 1.26
Curved arrow depiction of a two-electron bond-making process.
B: + H A B H + :A
shared pair in H-A becomes a lone pair on A -
FIGURE 1.27
Curved arrow depiction of simultaneous bond making and bond breaking.
new lone pair

B:
H H
H
H + B H + :Br
H Br H H
three electron pairs are moving;
lone pair on B, shared pair new 2 e- bond new 2 e- bond
in C-H bond, shared pair in C-Br bond;
three electron pairs have moved
so, three curved arrows are needed
FIGURE 1.28
Curved arrow depiction of electron pair movement in an E2 elimination reaction.
O O
RC O + H3O + RCOH + H 2O
pK a = –1.7 pK a = 5
FIGURE 1.29
Acid–base equilibrium position favored in the direction of the weaker acid.
our studies of organic reaction mechanism, and the following trend is an important
one to remember for all such equilibria: acid-base reactions always proceed from the
stronger acid/base to the weaker acid/base. Thus, for the reaction shown in Fig. 1.29,
the equilibrium clearly lies to the right (on the side of the carboxylic acid and water),
not to the left (carboxylate ion and hydronium ion).
To predict the position of an acid–base equilibrium, it is necessary to know the
relative strengths of a variety of acids and bases, keeping in mind that the larger the
pKa, the weaker the acid. Table 1.3 provides a brief list of some useful pKa values for
our purposes. While exact numbers can be difficult to keep in mind, it is often useful
to have a few simple approximations memorized. The following “skip five” sequence

Table 1.3 Some Common pKa Values (acidic proton underlined)

Acid pKa Acid pKa
NH2
H3 O+ –1.7 RNHCNH 2 12.5
RCO2H 4–6 H2O 15.7
HN NH
6.9 ROH 16–18
O O O
RCCH2CR 9 RCCH3 19–20
O
ArOH, RNH3+, RSH 10 RSCCH3 21
O O O
RCCH2COR 11 ROCCH3 25
of pKa values, while only approximate, is easily memorized and is frequently useful
in evaluating or comparing the acidity of many simple carbon compounds: car-
boxylic acids ~5; phenols, thiols, and ammonium ions, all ~10; water and alcohols,
~16; ketones and thioesters, ~20; esters and terminal alkynes, ~25. The sequence
can be extended to include much weaker acids: ammonia and amines ~35; allylic
C–H (RCH=CH–CH3), benzylic C–H (Ph–CH3), aryl C–H (Ph–H), and vinyl C–H
(R–CH=CH2) all ~45; and alkyl C–H (R–CH3) ~50.
ACIDITY TRENDS: WHY THAT PROTON

IS OR ISN’T ACIDIC
By referring to the pKa values from Table 1.3 or other sources, simple trends in acid-
ity can be seen, which can be useful when evaluating or comparing the relative acid-
ity of various species.
i. Within a series of X–H bonds where X does not change, the greater the positive
charge on X, the greater the acidity of the X–H bond. Compare the O–H bond
pKa in H3O+ (−1.7) versus H2O (15.7) or the N–H bond pKa in NH4+ (~10)
versus and NH3 (35). The positively charged X “wants” the electrons in the
X–H bond more than neutral X does.
ii. Within a series of X–H bonds where X does not change, the greater the
resonance delocalization of negative charge on X after loss of H+, the greater the
acidity of the X–H bond. A developing negative charge on X has the ability to
spread out from X via resonance, thereby weakening the X–H bond. Compare

RCH 2OH H+ + RCH 2O

Bioorganic Synthesis 18 pK a = 16 (localized negative charge)
O O O
RCOH H+ + RC O RC=O
pK a = 5 (delocalized negative charge)
FIGURE 1.30
Charge delocalization effect on O–H bond acidity.
the O–H bond pKa in an alcohol R–OH (16) versus a carboxylic acid RCO2H
(5) (Fig. 1.30). Likewise, the lower acidity of alkyl C–H bonds in alkanes, R–
CH3 (~50) versus allylic C–H bonds in alkenes, RCH=CH–CH3 (~45) is due to
resonance stabilization of the resulting allylic versus alkyl carbanions.
iii. Within a series of X–H bonds where X has the same charge and is in the same
row of the periodic table, the more electronegative X is, the greater the acidity
of the X–H bond (compare H3O+ (−1.7) versus NH4+ (~10); H2O (16) versus
NH3 (35) versus CH4 (50). The more electronegative X is, the more it “wants”
the electrons in the X–H bond.
iv. Within a series of X–H bonds where X has the same charge and is in the same
column of the periodic table, the greater the size of X, the greater the acidity of
the X–H bond. Compare the pKa values for alcohols, RO–H (16) versus thiols,
RS–H (10) or H–F (5) versus H–Cl (−6). The size of X increases going down a
column, so the distance of H from the nucleus of X also increases, thus making
the X–H bond longer and weaker.
v. Within a series of X–H bonds where X remains constant but changes hybridization,
the X–H bond acidity increases as the % “s-character” of the hybridization of
X increases (sp (50% s-character) > sp2 (33% s-character) > sp3 (25% s-character)).
In this way we can understand the relative pKa values of terminal alkyne C–H
bonds (sp), RC ≡ C–H (25) vs alkene vinylic C–H bonds (sp2), RCH=CH2 (45)
versus alkane C–H bonds (sp3), RCH3 (50). Since the energy of hybrid orbitals
follows the order sp < sp2 < sp3, anionic X will follow the same stability trend; the
greater the % “s-character” of the anion’s hybrid orbitals, the lower its energy.
These pKa values are also useful in evaluating the relative strength of various
bases. Rather than using a separate pKb value for basicity, we need only compare the
pKa values for their respective conjugate acids, keeping in mind that the stronger the
conjugate acid, the weaker the base and vice versa. For instance, in comparing
the relative basicity of imidazole versus methylamine, we need only consider the
relative acidity of their conjugate acids (Fig. 1.31). That imidazole is a weaker base
than methylamine immediately becomes clear simply by comparing the pKa values
of their respective conjugate acids.
Similarly, we can quickly answer the question regarding the relative basicity of
a carboxyate ion versus water (Fig. 1.29). Again, such a process must proceed from

H H

N N
+ H+
N N
H
pK a = 6.9 imidazole
weaker base
stronger
conjugate acid
CH3NH3 CH3NH2 + H+
pK a = 10 methylamine
weaker stronger base
conjugate acid
FIGURE 1.31
Comparison of conjugate acid pKa values for evaluation of relative basicities.
CH3 CH3
H+
CH3 + O
O protic H H H
O
acid
H H
CH3 CH 3
BF3
CH3 + O
O Lewis BF3 H BF3
O
acid
H H
FIGURE 1.32
Comparison of catalytic action of a protic acid vs. a Lewis acid.
the stronger base to the weaker base, and the acid pKa values clearly reveal that water
must indeed be a weaker base than a carboxylate ion.
While many acid–base reactions involve proton transfer from an acid to a base
(Brønsted acid–base reactions), many others do not, and a broader definition of
acid–base chemistry is often more useful. In Lewis acid–base theory, any species
which can donate a nonbonding electron pair to an acid can be considered a Lewis
base, while any species that can accept an electron pair from a base can be consid-
ered a Lewis acid. This much broader definition of acids and bases means that Lewis
acids include many species other than H+ which may accept an electron pair from
a base, the only requirement being that a Lewis acid must have a low-lying, empty
orbital into which an electron pair may come to form a bond. Thus, while H+ is for-
mally a Lewis acid since its 1s orbital is empty, so is the boron atom in BF3, which
has an empty p orbital perpendicular to its trigonal planar, sp2 hybridized frame-
work. For many proton-catalyzed reactions, Lewis acid catalysis works just as well
(Fig. 1.32). Metal ions such as Fe3+, Zn2+, or Mg2+ can be especially important Lewis
acid catalysts in certain biological systems, where they can facilitate acid catalysis at
physiological pH.

Bioorganic Synthesis 20 Y
X H :B H B
X Y
R R
H O H O
B B:
FIGURE 1.33
Simultaneous enzymatic acid–base catalysis.
It is important to note that in biological systems, some enzymes can catalyze

acid–base reactions that would be impossible to carry out via conventional solution
chemistry. In particular, simultaneous acid and base enzyme catalysis can occur as
illustrated in Fig. 1.33. In solution chemistry, having both acid and base together in
the same system would lead to simple neutralization.
CARBOCATIONS: THREE BONDS TO CARBON

CAN BE A PLUS
As reactive intermediates, carbocations are among the most important spe-

cies we will encounter in biosynthetic processes. Generally speaking, their ease
of formation and stability will follow the usual simple order of 3o (tertiary)
> 2o (secondary) > 1o (primary), but it will also be useful to expand this series
somewhat to take into account some additional stabilizing features such as the
presence of adjacent heteroatoms with lone pairs or conjugation with π-bonds
(Fig. 1.34). Such categorizations, while only approximate, serve as useful guides
for anticipating the relative ease with which carbocations are likely to form at
various carbon centers.
We can quickly summarize some of the most important means for the formation of
carbocations in both laboratory and biosynthetic processes by considering four cases
(Fig. 1.35). The first is simple loss of a leaving group (such as a phosphate or diphos-
phate ester) which may include such processes as acid-catalyzed alcohol dehydration.
Next is electrophilic additions to π-bonds which may include processes such as simple
alkene protonation, reaction of alkenes with carbocations, or other processes such as
electrophilic aromatic substitution. The third case derives from the importance of ep-
oxides in biosynthesis, which frequently provide a reliable source of carbocations via
acid-catalyzed ring opening reactions. Finally, we consider alkyl radicals which are fre-
quently formed by hydrogen atom abstraction or radical addition to π-bonds and may
undergo subsequent one-electron oxidation to afford the corresponding carbocations.
RADICALS: ODD AND REACTIVE
In terms of organic reaction mechanisms, radicals are especially important reac-

tive intermediates in biosynthetic processes, so a brief review of their depiction in


R R R
R R R
NR2 OH O
R R
>
R
iminium protonated acylium
ion ketone 3o benzylic 3o allylic 3o 2o benzylic 2o allylic ion
very stable stable
R R
>
R R
2o 1o benzylic 1o allylic 1o phenyl 2o vinyl
moderately stable relatively unstable
FIGURE 1.34
Classification and comparison of stability of various types of carbocations.
HOH
- H2O
R R loss of a leaving group
R R
E+ E electrophilic addition to a π bond

R R
O H+
OH acid-catalyzed epoxide ring opening
R R
- e- oxidation of a radical
R R R R
FIGURE 1.35
Typical processes involved in carbocation formation.
mechanistic terms is necessary. Unlike more common two-electron bond-making

and breaking processes involving cations or anions where electrons are moving in
pairs, radicals are electrically neutral, one-electron species and so cannot be ap-
propriately represented mechanistically via the use of the standard curved arrow
formalism. Instead, we use “barbed” arrows to show the movement of only one elec-
tron. Compare the mechanistic depictions of a two-electron versus a one-electron
process in Fig. 1.36. In the two-electron process, an electron pair from the styrene
π-bond attacks a carbocation leading to a new two-electron σ-bond and a benzylic
carbocation. One curved arrow shows how one electron pair has moved.
By contrast, in the one-electron radical process, a new two-electron σ-bond is
formed as well as a benzylic radical, but only one of the two electrons in the new
σ-bond is provided by the π-bond; the other is provided by the initial radical spe-
cies. Depiction of both σ-bond and radical formation requires a total of three barbed
arrows, one leading away from the radical toward the π-bond and another leading

two electron bond,

both from π-bond
R+ R
carbocation carbocation
on left on right
two electron bond,

only one from π-bond
R R
radical radical
on left on right
FIGURE 1.36
Comparison of curved arrow formalisms for a carbocation vs. a radical process.
proton
R H + :B R: + H B
removal
H atom
R H + B R + H B
abstraction
FIGURE 1.37
Mechanistic depiction of proton removal vs. H atom abstraction.
away from the π-bond toward the radical to show how electrons have paired to form
the new σ-bond, with a third arrow showing how the remaining electron moves to
reside on the benzylic carbon. Note that just as charge must balance on both sides
in the cationic process, total electron count must balance in the radical process.
Finally, note also that radical reactions frequently involve hydrogen atom abstrac-
tion as opposed to proton removal in more familiar acid–base reactions (Fig. 1.37).
The facility with which such radical C–H bond-breaking processes will occur de-
pends mainly on homolytic bond dissociation energies or BDEs (C–H → C• + H•)
as opposed to proton acidity (and pKa) which at least partially reflects the het-
erolytic bond dissociation energy associated with simple ionization processes
(C–H → C:– + H+). Resonance stabilization of the resulting radical species is also
an important consideration regarding the ease with which H atoms are abstracted
in such processes.
ELIMINATION REACTIONS: INTRODUCING
THE CARBON–C ARBON Π-B OND
While we will see a number of new and different ways to introduce carbon–carbon
double bonds into molecules, many familiar elimination processes will still be of
significance in biosynthetic schemes. The familiar E1 and E2 reactions will play an
important role in this context as well as the somewhat less familiar E1cB process
(Fig. 1.38).


B:
H H
E1 + B-H
X
+ X:
B:
H
E2
+ X: + B-H
X
B:
H
E1cB + X:
X X
+ B-H
FIGURE 1.38
The three main types of 1,2-elimination processes: E1, E2, and E1cB.
OH O
OH O R' R
O
E1cB
R' R + HO
R' R
H OH O
:B
R' R
FIGURE 1.39
E1cB mechanism in the dehydration step of an aldol condensation.
Recall that the E1 reaction is a first-order process with carbocation formation as

the rate-limiting first step. Subsequent loss of a proton from the carbocation leads
to π-bond formation in the second faster step. The E1 reaction is usually observed
when loss of a leaving group can readily lead to a stable carbocation (typically ter-
tiary, allylic, or benzylic). This stands in contrast to the single-step E2 reaction, a
second order, concerted anti elimination process with a specific anticoplanar confor-
mational requirement. This process usually involves strong bases and will be useful
when carbocation formation is not favorable. Finally, the E1cB process involves
initial proton removal by a suitable base to form an anionic intermediate as the con-
jugate base (cB) of the substrate. Subsequent expulsion of the leaving group by the
anionic lone pair leads to π-bond formation (Fig. 1.39) This process is most often seen
when electron withdrawing groups present on the proton-bearing carbon make the
proton more acidic. Such groups also help to stabilize the resulting carbanion nega-
tive charge, often via resonance. One example of such an E1cB process will be seen
later in the base-induced dehydration of an aldol addition product to yield the cor-
responding α,β-unsaturated carbonyl derivative. Interestingly, in certain biological

examples this reaction will be seen to occur as a stereospecific syn elimination rather
Bioorganic Synthesis 24 than the usual anti orientation seen in most organic elimination reactions.
CARBOCATIONS: REARRANGEMENTS AND FATES
Certain carbocations may undergo internal structural rearrangement in order to

increase their stability. These rearrangements are usually achieved by so-called
1,2-shifts or 1,2-migrations of hydrogen, alkyl, or aryl groups and are known
as Wagner–Meerwein shifts. 1,3-hydride shifts, though not formally Wagner-
Meerwein shifts, are also sometimes observed in biosynthetic processes (Fig. 1.40).
The ultimate fate of carbocations, regardless of how they are initially formed, may
be loosely summarized as follows: i) loss of a β-proton to a base, resulting in forma-
tion of a π-bond; ii) trapping by a nucleophile to form a new σ−bond; and iii) loss of
γ−proton to a base, resulting in cyclization to a cyclopropane ring. This latter pro-
cess, though not common in laboratory organic chemistry, is occasionally observed
in certain biological systems. Generally speaking, Wagner–Meerwein shifts occur
only when the 1,2-shift process will lower the energy of the carbocation by increas-
ing its degree of substitution, that is, from 1o → 2o/3o or 2o → 3o.
A related and unusual carbocation rearrangement is observed when cyclopro-
pylmethyl carbocation is generated under solvolysis conditions by loss of a leaving
group. A mixture of three products is obtained as shown, but interestingly, the same
mixture of alcohol products is obtained regardless of whether cyclopropylmethyl,
cyclobutyl, or homoallyl substrates are used (Fig. 1.41). This implies a common non-
classical carbocation intermediate or rapidly equilibrating carbocations that arise
from any one of the three starting structures. This cyclopropylmethyl-cyclobutyl-
homoallyl carbocation system is one that has been much studied and is closely
H R
H R - H-B R
R R H :B R
1,2-H shift loss of a β-proton to form a π-bond

R' Nu
R' :Nu
R R R R
R R
1,2-alkyl shift nucleophilic trapping
H
H - H-B
R H
R :B R R
R R
1,3-H shift loss of a γ-proton to form a cyclopropane
carbocation rearrangements carbocation fates
FIGURE 1.40
Examples of the principal rearrangements and fates of carbocation intermediates.

heat

X H2O
or
heat
+ +
OH
H2O OH
X OH
or 48% 47% 5%
heat
X H 2O H2O - H+
cyclopropylmethyl-cyclobutyl-homoallyl cation
FIGURE 1.41
The cyclopropylmethyl-cyclobutyl-homoallyl carbocation system.
related to a key rearrangement that is just one of a large number carbocation rear-
rangements and 1,2-shift processes that are of particular mechanistic importance in
the biosynthesis of steroids and related structures (Chapter 4).
ELECTROPHILIC ADDITIONS: Π-B ONDS

AS NUCLEOPHILIC AGENTS
In carbon–carbon double bonds, the electrons in the π-bond are far more accessible
than those in the σ-bond and reaction of π-bonds with electrophilic agents (E+) is an
essential bond-making step in many organic reactions. Acid-catalyzed hydration of
an alkene to give an alcohol is an illustrative example (Fig. 1.42), with H+ acting as
the electrophile. The familiar Markovnikov orientation of addition is observed here,
since initial addition of H+ to the π-bond occurs in such a way as to give the more
stable tertiary carbocation. Nucleophilic attack by water and proton loss complete
the process.
Other familiar electrophilic agents that react with π-bonds include the halogens
(Cl2, Br2, I2), the active oxygen from peroxyacids (RCO3H) and many strong Lewis
acids (such as BH3 in alkene hydroboration reactions). In biosynthetic processes,
carbocations themselves are among the most important electrophiles that react with
alkenes to make new C–C bonds, as shown in the acid-catalyzed reaction of linalool,
an alcohol found in many flowers and spice plants, to give α-terpineol, a fragrant
and structurally related alcohol isolated from sources such as pine oil (Fig. 1.43).
Interestingly, α-terpineol has been shown to suppress the production of some proin-
flammatory mediators and may impair the growth of certain human melanoma cells.

CH3 CH3 H 3C OH2 H3C OH
H+ H2O H H
H
+ H+
FIGURE 1.42
Typical reaction of an alkene with an electrophilic agent such as H+.
H
CH 3 CH 3
HO CH 3 O CH 3
H
H+
+ H 2O
linalool
CH 3 CH 3
- H+
H2 O
OH
α-terpineol
FIGURE 1.43
C–C bond formation via intramolecular trapping of an electrophilic carbocation.
NUCLEOPHILIC SUBSTITUTIONS AND

ALKYLATIONS: MAKE OR BREAK FOR C–X BONDS
The two principal types of nucleophilic substitutions are the familiar SN1 and SN2
reactions. Recall that the SN1 reaction (substitution, nucleophilic, 1st order) is a
two-step process with bond breaking via an initial carbocation formation as the
rate-limiting step, followed by nucleophilic bond making in the fast second step
(Fig. 1.44). Because of the trigonal planar geometry of the carbocation intermediate,
any SN1 process occurring at a chirality center leads to racemization.
Conversely, the SN2 reaction (substitution, nucleophilic, 2nd order) occurs in
a single step with concerted bondmaking and bond-breaking events (Fig. 1.45)
and with no reactive intermediates involved (no carbocations or radicals).
Nucleophilic attack occurs at the backside of the carbon bearing the leaving group,
and inversion of configuration occurs when such substitution takes place at a chi-
rality center.
In the reaction shown in Fig. 1.45, hydrosulfide ion (HS–) has formed a bond to
an alkyl group (the 1-phenylethyl group). Reactions that deliver an alkyl group to a
nucleophilic atom are often referred to as alkylation reactions. In biological systems,

H Br H

CH 3 heat
S CH 3
1) (slow)
+ Br
SN 1 H H OH
i) H2O
CH 3 CH 3 (fast)
2)
ii) –H+
racemic
FIGURE 1.44
Stereochemical outcome of SN1 at a chirality center.
H Br HS H
HS
S CH 3 R CH 3
S N2
+ Br
FIGURE 1.45
Stereochemical outcome of SN2 at a chirality center.
we see alkylation reactions of both the SN1 and the SN2 type, depending on the alkyl
group involved.
NUCLEOPHILIC CARBONYL ADDITION REACTIONS:

C=O Π-B OND UNDER ATTACK
The electrophilic character of the carbon atom in the highly polarized carbonyl
(C=O) group of aldehydes and ketones makes these compounds very susceptible to
attack by many different nucleophiles, forming a variety of potentially useful deriva-
tives (Fig. 1.46). Additions of anionic nucleophiles are usually followed by proton-
ation of the resulting alkoxide intermediate to give a neutral product. For example, if
the nucleophile is hydride ion (H:–) or a carbanion (R:–), protonation leads to a stable
alcohol. For less reactive neutral nucleophiles such as alcohols (ROH), activation of
the carbonyl group by protonation (acid catalysis) may be required to facilitate addi-
tion of the nucleophile; in the case or addition of ROH via the mechanism shown in
Fig. 1.46, the product would be the familiar (though unstable) hemiacetal product.
For purposes of review, we focus on some key carbonyl addition reactions that will be
of particular importance in biological transformations.
IMINE FORMATION: MAKING THE ESSENTIAL

C=N LINKAGE
In the next chapter we will see some specific examples of imine formation (also
called Schiff ’s bases) that recur as fundamental components of many of the

O OH
O
H+
Nu: + R H(R') R H(R')
R H(R') Nu Nu
anionic
nucleophile
or
neutral H OH OH
O O
nucleophile - H+
H + Nu: + R H(R') R H(R')
R H(R') R H(R') Nu Nu
H
H
FIGURE 1.46
Anionic vs. neutral nucleophiles in addition reactions with carbonyl groups.
biosynthetic processes treated throughout the remainder of the book. The favor-
able equilibrium for this reaction of aldehydes or ketones with primary amines is
driven in part by the nucleophilicity of the amine nitrogen as well as the thermo-
dynamic stability of the resulting C=N double bond after loss of water. The general
mechanism for such condensations is depicted in Fig. 1.47, which shows initial
carbinolamine formation resulting from addition of the amine N–H bond across
the carbonyl C=O bond. This is followed by acid-catalyzed dehydration to give the
corresponding imine linkage.
Note also that imines may undergo a tautomerization process similar to keto–
enol tautomerism (Fig. 1.47), leading to the structurally related enamines. This tau-
tomerization will be an important one in several mechanistic analyses. Note that
these processes are reversible. Thus both imines and enamines are subject to acid-
catalyzed hydrolysis, reverting to their constituent amines and carbonyls by addition
of excess water.
ACETAL FORMATION: PROTECTING
ALDEHYDES OR KETONES
Both aldehydes and ketones react with alcohols (usually primary alcohols, always
two equivalents) via an acid-catalyzed nucleophilic addition sequence that yields
the corresponding acetal derivatives. The reaction is more favorable for aldehydes
than for ketones and proceeds mechanistically through an intermediate hemiacetal
for which the equilibrium is generally unfavorable (Fig. 1.48). The equilibrium is
driven forward by dehydration of the hemiacetal to a resonance-stabilized carboca-
tion which is subsequently trapped by a second equivalent of alcohol and proton loss
to give the acetal product. In acetal form, the reactive carbonyl function has been
converted to a much less reactive ether-type linkage. Like imines and enamines, ac-
etals are subject to acid-catalyzed hydrolysis, reverting to their constituent aldehyde/
ketone and alcohol components by addition of excess water.

O OH
O
+ H2 N R"
R H(R') R H(R')
i) R H(R') N N
H R" H R"
H carbinolamine
BH B: BH
OH
R H(R') R H(R')
ii) R H(R')
N N N
H R" R" + H2O
H R"
imine
(Schiff's base)
B:
H
BH
H(R') H(R')
R R
N HN
R"
BH R"
B: enamine
imine
(Schiff's base)
FIGURE 1.47
Conversion of aldehydes or ketones to imines (Schiff ’s bases) and enamines.
BH
BH B: OH OH
O
+ HO R"
R H(R') R H(R')
i) R H(R') O O
H R" R"
a hemiacetal
BH B:
OH R H(R')
R H(R')
+ H2O
ii) R H(R') O
O O
R" R"
R"
B: BH R"
H R" O
R H(R') O
iii) HO R"
R H(R')
O R H(R') O
R" O R"
R"
an acetal
FIGURE 1.48
Conversion of aldehydes or ketones to acetals via a hemiacetal intermediate.

O
H
1CH 5 H
2 HO O
H OH HO H
4 HO 1
3 H
HO H 3 2 OH
5
H 4 OH H OH
H in "furanose"
OH
hemiacetal form O
CH 2OH
1CH
D-glucose H OH 2
6 H OH
4 5 H O
HO HO 3 H
HO OH
3 H 2 OH 1 H 4 OH
H H 6
H 5 OH
in "pyranose"
hemiacetal form CH 2OH
D-glucose
FIGURE 1.49
Formation of furanose and pyranose cyclic hemiacetal forms of glucose.
While the equilibrium for hemiacetal formation from bimolecular reactions of

alcohols and aldehydes or ketone carbonyls is generally unfavorable, the equilibrium
for the corresponding intramolecular reaction is favorable; this means that cyclic
hemiacetal derivatives (5-or 6-membered rings are best) may often be isolated from
hydroxy aldehydes or hydroxy ketones with their alcohol oxygen as the number 5 or
number 6 atom relative to the carbonyl carbon (Fig. 1.49). Note the oxygen hetero-
cycle nomenclature employed to describe the two different cyclic hemiacetal forms
possible for D-glucose (the pyranose form is favored over the furanose form). Such
cyclic hemiacetal structures play an essential role in the chemistry of carbohydrates,
a topic we will address in some detail in Chapter 3.
NUCLEOPHILIC 1,4-( CONJUGATE) ADDITION

REACTIONS: REMOTE ATTACK ON
CONJUGATED CARBONYLS
An especially important component of our review of carbonyl addition reactions fo-

cuses on the unique nucleophilic addition chemistry associated with α,β-unsaturated
carbonyl compounds. Up to this point, we have been concerned with the direct
attack at the carbonyl carbon (C=O) by nucleophiles. For simple aldehydes and ke-
tones, this is the only mode of attack possible, since the carbonyl carbon is the sole
electrophilic center in the polarized C=O bond. However, when we consider the
dipolar resonance contributors possible for carbonyl compounds in which the C=O
bond is in conjugation with a C=C bond, we find two sites available for nucleophilic
attack: i) the carbonyl carbon (as usual), and ii) the β-carbon of the C=C π-bond
(Fig 1.50). Both carbons are electrophilic centers in such molecules; when direct nu-
cleophilic addition across the C=O π-bond occurs, we refer to this as the 1,2-addition

O O

O
β
α
electrophilic
carbons
O 1OH
1O
2 H+ 2
Nu: 4 4 1,2-addition
Nu 3 Nu
3
1O Nu O Nu 1 OH
2 H+ 2
Nu: 4 4 1,4-addition
3 3
enolate ion keto-enol

tautomerization
Nu O Nu O
FIGURE 1.50
1,2-vs. 1,4-addition to α,β-unsaturated carbonyl compounds.
mode; when nucleophilic addition occurs at the β-carbon of the C=C π-bond, we
refer to this as the 1,4-addition mode, also called conjugate addition. These modes
of addition are not limited to aldehydes and ketones but rather may include other
α,β-unsaturated carbonyl compounds such as esters, thioesters, or amides.
Note that 1,2-addition of an anionic nucleophile initially leads to an alkoxide ion
which is then protonated to give the corresponding alcohol product, whereas in the
case of 1,4-addition, an enolate ion is formed after initial nucleophilic addition. If
subsequent protonation occurs on oxygen, this leads to an enol product which rap-
idly tautomerizes to the keto form as shown. Alternatively, protonation of the eno-
late may occur on carbon, leading directly to the keto form of the product. Generally
speaking, strongly basic nucleophiles tend to add to α,β-unsaturated carbonyl sys-
tems in a 1,2-fashion, while weakly basic nucleophiles tend to favor the 1,4-mode
of addition (there are exceptions). For our purposes, we mainly need to be able to
recognize when an opportunity for 1,4-(conjugate) addition exists or to recognize
when such an addition has taken place within the context of a biosynthetic scheme.
NUCLEOPHILIC CARBONYL CONDENSATION

REACTIONS: MAKING C–C BONDS VIA ENOLATE IONS
Recall that aldehydes, ketones, and thioesters all have pKa values of ~20, making
them sufficiently acidic to be deprotonated by suitable bases. The resulting enolate
ions are nucleophilic and can attack the carbonyl groups of aldehydes or ketones to

B: BH O
O O
H
R R R
pK a ~ 20
BH
OH O O O O
R' R R' R R'
aldol addition product
OH O BH OH O
R' O
+ H2O
R' R R' R
R
H
:B aldol condensation
product
FIGURE 1.51
Aldol addition and aldol condensation reactions.
form alkoxide intermediates which after protonation, afford β-hydroxyaldehydes,

ketones, or thioesters, depending on the source of the enolate ion itself (Fig. 1.51).
This process is the familiar aldol addition reaction. Such aldol addition products
can readily lead to the corresponding α,β-unsaturated carbonyl derivatives via a
simple dehydration. In the presence of a suitable base, this reaction is again initiated
by deprotonation to form an enolate ion, which then expels hydroxide ion to intro-
duce the double bond. When aldol addition is followed by subsequent dehydration,
the overall process is known as aldol condensation.
We will see many examples of such processes occurring in both a bimolecular
and an intramolecular fashion, the latter serving as a particularly useful method for
formation of 5-and 6-membered rings. While a synthetic rather than a biosynthetic
process, the Robinson annulation provides an excellent example of the use of both
the conjugate addition of an enolate ion to an α,β-unsaturated carbonyl system and
a subsequent intramolecular aldol condensation to form a fused 6-6 bicyclic ring
system (Fig 1.52).
An important reaction closely related to the aldol condensation is the
Knoevenagel condensation, which specifically involves enolate ion formation from
relatively acidic 1,3-dicarbonyl compounds such as 1,3-diketones, β-ketoesters,
β-cyanoesters, or 1,3-diesters, and their subsequent condensation with aldehydes
(or in some cases, ketones) to give α,β-unsaturated carbonyl derivatives. The greater
acidity of β-dicarbonyl compounds (pKa ~9-13) relative to simple aldehydes or ke-
tones (~17–20) means that mild bases can be used to form the β-dicarbonyl enolate
ion without initiating a competing self-condensation of the less acidic aldehydes or

O O

O CH 3
CH 3 CH 3
:B
H OH
O O
O O H B
enolate formation conjugate addition
O O
O O CH 3 CH 3
CH 3 CH 3
O O
O O
O O
HO
H :B intramolecular H B
enolate formation
H :B
Robinson annulation B H
aldol condensation
product
FIGURE 1.52
Conjugate addition and intramolecular aldol condensations in the Robinson annulation reaction.
O O
O O O
B:
R R'
R R' + R" H(R'") + H2O
H H "R H(R'")
R, R' = alkyl, alkoxy,
B: CN in any combination. Knoevenagel product
HB O O
O O O
R R'
R R' + R" H(R'") H OH
B:
H "R HB
H(R'")
FIGURE 1.53
Reaction of 1,3-dicarbonyls with aldehydes or ketones in the Knoevenagel condensation.
ketones involved (Fig. 1.53). While often more complex mechanistically than the
simple condensation shown in Fig. 1.53, we will nevertheless see a number of situa-
tions in which it will be useful to invoke a Knoevenagel-type process for depiction of
C=C bond formation in certain biosynthetic schemes, and as a particularly powerful
component of phenolic ring formation in the polyketide pathway that is the subject
of Chapter 5.
Since enolate ions are nucleophilic, we should also expect them to participate
in alkylations as well as carbonyl addition reactions, and we will see some spe-
cific examples of such alkylations throughout our study of biosynthetic pathways.
One related process of particular note in this context is the Mannich reaction
(Fig. 1.54) which will be of particular significance in alkaloid biosynthesis
(Chapter 7). Often referred to as an enolate aminoalkylation process, the reac-
tion consists of condensation of ammonia or primary or secondary amines with a
nonenolizable aldehyde to produce the corresponding iminium ion (in the case of

B: BH O
O O
O
enolate ion H
formation R R R
R
R' H
BH
O R' H N
-H2O H R"
iminium ion + H2N R"
formation R' H N Mannich base
H R"
FIGURE 1.54
General mechanistic scheme for the Mannich reaction.
B: BH
O O O
H
SR SR SR
pK a ~ 20
O O O
O O - R"OH
R' SR R' OR"
R' SR OR"
HB
Claisen condensation
product
FIGURE 1.55
Reaction of a thioester enolate ion with an ester in a Claisen condensation.
ammonia or primary amines, this product is simply a protonated imine). Attack

on the electrophilic carbon of the iminium ion by an enolate carbanion leads to
C–C bond formation producing a β-amino-carbonyl compound known as the
Mannich base.
When the enolate ion of an ester or thioester reacts with the carbonyl group of
another ester or thioester rather than one from an aldehyde or ketone, the alkoxide
initially formed from such nucleophilic attack is not stable and will collapse with
expulsion of RO– or RS– from the tetrahedral intermediate, leading to formation of
a β-ketoester or β-ketothioester in a process known as the Claisen condensation.
In the example shown in Fig. 1.55, a thioester enolate ion reacts with a typical ester
carbonyl with resultant loss of alkoxide from the tetrahedral intermediate and for-
mation of a β-ketothioester as product. Such chemistry is the basis of the biosyn-
thesis of fatty acids and related products, which is also explored in Chapter5.
Intramolecular versions of the Claisen condensation are also important ring-
forming reactions in biosynthetic processes and are sometimes referred to as
Dieckmann condensations. Using the intramolecular case, we can also make some
additional useful observations about enolate ion reaction pathways. For example, we
can see that deprotonation of certain α-substituted esters may lead to formation of


O O
O
OR OR
OR
:B
H
OR
OR OR
O E-enolate
O O
O O
O
OR OR
OR
:B
H
O O O
OR Z-enolate
OR OR
FIGURE 1.56
Isomeric E and Z enolates from deprotonation of a 1,6-diester.
O O
E-enolate O OR
O
OR OR O
OR
O
OR OR
OR
OR
O
O
O O
O
OR
OR
O
cyclic β-ketoester lactone
FIGURE 1.57
C vs. O nucleophilic attack and ring closure from an E-enolate.
enolate ions possessing either E or Z stereochemistry, as shown in the deprotonation

of a diester of glutaric acid in Fig. 1.56.
Keeping in mind that enolate ions are ambident nucleophiles, we see that in the
intramolecular case, the E-enolate may adopt a conformation that favors attack on
the proximate ester carbonyl via either its nucleophilic carbanion or its nucleo-
philic oxyanion form: thus, C-atom nucleophilic attack leads to the expected cyclic
β-ketoester (Dieckmann product) while O-atom nucleophilic attack leads to the
corresponding lactone structure (Fig. 1.57). Note that lactone formation from
the corresponding Z-enolate would be geometrically constrained and strongly
disfavored.

O (H-Nu:) O(H) O (H-X:)
RC X + Nu: RC X RC Nu + X:
Nu
tetrahedral
intermediate
FIGURE 1.58
General mechanistic scheme for nucleophilic acyl substitution reactions.
As we look more closely at the various pathways utilized in biosynthetic pro-

cesses, we will find numerous examples of the use of all of these important reactions
as key C–C bond forming processes for the production of an incredible array of
widely different organic structures.
NUCLEOPHILIC ACYL SUBSTITUTION REACTIONS:

TURNING ONE ACYL COMPOUND INTO ANOTHER
The Claisen condensation may be thought of as a special case of the more general
process we know as nucleophilic acyl substitution. In such processes, a nucleophile
(anionic or neutral) attacks the carbonyl carbon of a carboxylic acid derivative, lead-
ing to a new carboxylic acid derivative via substitution after collapse of the usual
tetrahedral intermediate (Fig. 1.58). While we may for simplicity’s sake occasionally
skip the depiction of this intermediate in abbreviated mechanistic analyses, it is im-
portant to remember that direct nucleophilic displacement (as in the SN2 reaction)
does not take place in acyl systems and that a tetrahedral intermediate is always
involved.
In general organic chemistry terms, the reactivity order of carboxylic acid deriva-
tives follows the familiar order of acid chloride > acid anhydride > thioester > ester >
amide. In biological systems, the most reactive acyl derivative available is the acyl
phosphate (RCOOP), derived from phosphorylation of a carboxylic acid by adenos-
ine triphosphate (ATP), a topic taken up in Chapter 2.
The acyl phosphate is actually a mixed anhydride of a carboxylic acid and phos-
phoric acid and so is quite reactive toward nucleophilic acyl substitution. Thus,
acyl phosphates may be converted to any of the less reactive derivatives by nucleo-
philic acyl substitution with thiols, alcohols, or amines (Fig. 1.59). The next most
reactive derivative, the thioester, may be converted to both esters and amides,
while esters may be converted only to amides. All derivatives will yield the cor-
responding carboxylic acid by hydrolysis, with the acyl phosphate being hydro-
lyzed most readily and amides being the most resistant to hydrolysis. While the
thioester is an uncommon derivative in laboratory organic chemistry compared
to alcohol-derived esters, it is a crucial acyl derivative in biosynthesis owing to its
greater reactivity toward nucleophilic substitution relative to simple esters. Equally


RC OH
ATP
ADP
O
RC OP
acyl phosphate
R'SH
relative reactivity towards Nu:
- HOP
O
RC SR' R'OH - HOP O HOP
thioester H2O R'SH
RC OH +
R'OH
HNR'2 - HOP R'2NH
O
R'OH
RC OR'
- R'SH ester
HNR'2 - R'OH O
HNR'2 RC NR'2
amide
- R'SH
FIGURE 1.59
Relative reactivities and reaction pathways for interconversion of acyl compounds.
significant is the greater acidity of thioester α-hydrogens (pKa ~20) versus those
of simple esters (pKa ~25), making their enolate ions more accessible and there-
fore of particular importance as sources of nucleophilic carbon in biosynthetic
processes.
LOOKING AHEAD
Now that we have reviewed some essential organic chemistry concepts (and intro-
duced a few new ones), we are in a position to move on to Chapter 2 where we will
become familiar with some specific kinds of organic reactions and reagents that
are used by biological systems. Let’s see how cells achieve the same kinds of trans-
formations or synthesis goals that we ordinarily tackle in our own laboratory-
based organic chemistry experiments. The similarities are often surprising.
STUDY PROBLEMS
1. 6-
aminopenicillanic acid (6-
APA) and 2,6-
dimethoxybenzoyl chloride are
used to commercially prepare the antibiotic methicillin. Give the structure of
methicillin and indicate what type of functional group is formed by the reac-
tion shown.

OCH 3
O
H H
Cl
H 2N S
CH 3 OCH 3
methicillin
N CH 3
O
CO2H
2. Use of pKb values for comparing relative base strengths has largely been sup-
planted by comparison of pKa values for the corresponding conjugate acids of
the bases under consideration. Taking note of the relationship: pKb + pKa(conj.
acid) = 14, rank the following compounds in order of increasing basicity (weak-
est base first):
I. guanidine, pKa(conj. acid) = 13.6 II. ethyl amine, Kb = 6.5 x 10–4
III. aniline, pKa(conj. acid) = 4.6 IV. ammonia, pKb = 4.76
3. Arrange the indicated carbon–hydrogen bonds in order of increasing homolytic
bond dissociation energy (weakest bond first). Justify your order using resonance
structures.
A B
H
H H
H
H3C
H3C H
CH3
4. In each of the following, label the designated hydrogen atoms as being either pro-
R or pro-S. Are these hydrogens enantiotopic or diasterotopic? Explain.
H H H H
H2N CO 2H
H H CO2H
CO2H
HO2C N H H H NH2 H
H
H H
succinic acid proline lysine
5. Choose the most acidic hydrogen for the following compound and then explain
your choice using appropriate resonance forms.
O
H 3C CH 2 C CH 2 CH 3
A. B. C. D.

6. Assign Si or Re to the faces of the prochiral center(s) in each of the following:

H CO2H H H
O
H H
7. A simple bond-line structure for the compound (+)-juvabione is shown.
OCH3
O
O
a) What does the (+) in front of the name of the compound indicate?
b) Using wedge and hatched bonds at its two stereogenic centers, draw a stereo-
chemically correct structure of (+)-juvabione, given that the natural product
has the R,R configuration.
c) Draw the structure of the alcohol product resulting from hydride addition to
the Re face of juvabione by NADH/H+.
d) Draw correct structures for the enantiomer and the two diastereomers of
(+)-juvabione.
8. Japonilure is the chemical sex attractant (pheromone) secreted by the female Japanese
beetle (Popillia japonica) a destructive garden pest of over 200 species of plants in
America, where natural predators are lacking. Draw a stereochemically correct
structure for Japonilure, given that the natural product has the R,Z configuration as
indicated.
Z
O O CH=CH(CH2) 7CH3
R
Japonilure

9. Provide reagents and mechanisms for conversion of each of the following sub-
Bioorganic Synthesis 40 strates into 2-ethylcyclohexanone.
10. What is the correct order of acidity for the numbered hydrogens in the
structure shown?
1
O OH
2 a) 1 > 2 > 3 > 4 b) 4 > 3 > 2 > 1

OH
3 4
c) 1 > 3 > 2 > 4 d) 1 > 4 > 3 > 2
CH 3
O O
11. Which of the following statements describes this first step of the reaction?
OH O
O
HO
2
H2 O
a) The enol form of acetone is formed by proton removal by base.

b) The carbonyl oxygen of acetone is protonated by hydroxide ion.
c) Nucleophilic attack by hydroxide ion on the carbonyl carbon gives an alkoxide.
d) A methyl proton is removed by hydroxide ion to form acetone enolate ion.

12. When 2-methylpropanal is reacted with methyl vinyl ketone in the presence

of aqueous base, some 4,4-dimethyl-2-cyclohexen-1-one is formed. Outline in
clear, mechanistic detail the sequence of reactions involved in this transformation.
HO -
O +
H 2O
O
13. Which of the following is also known as a Schiff ’s base?

a) an imine b) a cyanohydrin c) a hydrate d) sodium hydroxide
14. Which reagent can be used to convert butanol into butanal?
a) LiAlH4 b) Na2Cr2O7 c) O3 d) PCC
15. Which of the following conditions can drive the equilibrium of acid-catalyzed
acetal formation from reaction of an aldehyde and two moles of an alcohol?
a) addition of excess H2O b) removal of H2O as it is formed c) addition of
excess alcohol d) b and d
16. When (S)-3-bromo-1-pentene is heated in water, which of the following is
produced?
a) (S)-1-penten-3-ol b) (R)-1-penten-3-ol c) (E)-2-penten-1-ol d) (Z)-2-penten-
1-ol e) all of these

2 Bioorganic Reactions
Few scientists acquainted with the chemistry of biological systems at the molecular level can
avoid being inspired. Evolution has produced chemical compounds exquisitely organized to
accomplish the most complicated and delicate of tasks.
—Donald J. Cram (Nobel Prize in Chemistry, 1987)
ENZYMES: THE CATALYSTS OF BIOLOGICAL

ORGANIC CHEMISTRY
It is not essential to have a background in enzymology or biochemistry to gain at

least an introductory-level understanding of many biosynthetic processes, so this
book does not deal with enzymology or enzyme structure or function in any signifi-
cant way, even though much of the chemistry we will be examining depends almost
entirely on enzyme catalysis. Nevertheless, we will refer to enzyme catalysis and the
names of specific enzymes throughout the text as we examine biosynthetic processes
and reactions in significant detail.
So what exactly are enzymes? Simply put, enzymes are naturally occurring pro-
teins that catalyze various biochemical reactions in living systems. As we will see,
many of the reactions they catalyze are familiar organic reactions, but have spe-
cific purposes and target structures. Generally speaking, enzymes catalyze organic
reactions by lowering transition state energies or raising ground state energies of
reactants in much the same way as nonenzymatic catalysts in laboratory chemical
reactions, though in the case of enzyme catalysis, rate enhancements of as much
as 1023 have been reported, far exceeding rate enhancements currently achievable
42

by conventional chemical means. Understanding the interaction of enzymes and

substrates (reactants) to form an enzyme–substrate complex (E–S complex) is
fundamental to having some appreciation for how enzymes carry out their work.
While overly simplistic, the “lock-and-key” model of enzyme–substrate interac-
tion provides an intuitive context for understanding the remarkable substrate
specificity of enzyme-mediated reactions. Thus, so-called enzyme active sites or
binding sites (the “lock”) will only accept certain specific substrate structures (the
“key”), with shape, conformation, intermolecular forces, and other factors deter-
mining the lock-and-key fit. Enzymes not only catalyze specific kinds of reac-
tions, they can act specifically on certain compounds or classes of compounds
or functional groups, often showing remarkable selectivity and stereospecificity,
especially in the recognition and/or introduction of chirality centers in organic
molecules.
In terms of nomenclature, enzyme names always end with an ase suffix and are
typically named in accordance with the substrate they act upon and/or the kind of
reaction process they catalyze. For example, an enzyme that catalyzes an oxidation
process in which only one of the two atoms of O2 is delivered to a substrate may be
named as a “monooxygenase.” A more specific example involves “toluene dioxygen-
ase,” an enzyme that mediates delivery of both oxygen atoms from O2 to the substrate
toluene (Fig. 2.1), with H+ and the enzyme cofactor NADH providing the diol H
atoms. Note that an enantiomerically pure chiral product is produced in this process
from an achiral substrate (toluene) and an achiral reactant (O2). In conventional
organic chemistry, such a result would be impossible, since achiral reactants cannot
produce optically active products. But the chirality of the enzyme catalyst in this
case leads to a chiral binding site for the substrate, with delivery of the two oxygen
atoms to only one of the two faces of the toluene π-bond.
Enzymes may be broadly grouped into six main classes, each with a number of
subclasses:
• Oxidoreductases: include the subclasses oxidases (for oxidation), reductases (for

reductions), and dehydrogenases (for π-bond formation via loss of the elements
of H2).
CH3 CH3
H
OH
toluene dioxygenase
+ NAD+
NADH, H +, O 2
OH
H
(1S,2R)-3-methyl-3,5-
cyclohexadiene-1,2-diol
FIGURE 2.1
Enzyme-mediated dioxygenation of toluene.

• Transferases: include the subclasses kinases (for phosphate transfer), trans-

Bioorganic Synthesis 44 aminases (for amino group transfer), and transketolases and transaldolases (for
ketone or aldehyde group transfer).
• Hydrolases: include the subclasses lipases (for ester hydrolysis), proteases (for
amide hydrolysis), and nucleases (for phosphate hydrolysis).
• Lyases: include decarboxylases (for decarboxylation reactions) and dehydrases
(for dehydration reactions).
• Isomerases: include epimerases (for chiral center isomerizations), racemases (for
chiral center racemizations), and cis–trans isomerases (for interconversion of E/Z
isomers).
• Ligases: include synthetases (for new bond formations), carboxylases (for addi-
tions of CO2), and cyclases (for formation of cyclic compounds).
COFACTORS: ENZYME ASSISTANTS
IN BIOORGANIC REACTIONS
Most biosynthetic steps are catalyzed by specific, individual enzymes that perform
familiar transformations such as alkylation, hydrolysis, oxidation, reduction, acyla-
tion, hydroxylation, elimination, decarboxylation, and other such processes. While
very different enzymes may carry out similar reactions in various organisms, most
employ a set of common organic or inorganic “assistants” known as cofactors. These
are molecules or ions which are necessary for enzyme activity, broadly divided into
two groups: the organic cofactors (nonprotein organic compounds) and metals ions
(such as Fe2+, Mg2+, Zn2+, etc.). Certain enzymes may require multiple cofactors as
a combination of organic cofactors and/or metal ions. When loosely bound to an
enzyme host, cofactors are sometimes called coenzymes, while tightly bound co-
factors are often referred to as prosthetic groups. Regardless of their role, we will
refer to them mainly as cofactors for simplicity’s sake. In the sections to follow, we
will treat the cofactors of most significance to biosynthetic processes in some detail
along with some specific types of bioorganic transformations of importance to our
further studies.
Often called the “molecular currency unit” for energy transfer in living cells,
adenosine-5’-triphosphate (ATP) is used by enzymes and structural proteins for
a wide variety of cellular processes. Each ATP molecule has three linked phosphate
groups and is produced from either adenosine diphosphate (ADP) or adenosine
monophosphate (AMP) and inorganic phosphate (Pi) by the action of the enzyme
ATP synthase. The energy derived from the degradation of nutrients is almost
always coupled to the stoichiometric production of ATP which, when degraded to
lower energy phosphate derivatives (ADP or AMP) by chemical reaction, releases
the energy stored in its phosphate bonds to drive other energy-demanding reaction
processes. Our interest in ATP is related to one of its main roles in biosynthesis,

NH2

= OPP
= OPPP
N N
O O O O
O O O O O O N enzyme P P
N
R-OH + P P P R-O OH
HO O O O Mn2+ or
O an alkyl diphosphate
H H Mg2+ (R-OPP)
H H
OH OH + NH2
adenosine triphosphate (ATP)
= OP N N
SN 2
Nu: + R-OH Nu R + OH O O N N
unfavorable P
HO O O
SN 2 H H
Nu: + R-OPP + OPP H H
Nu R
OH OH
favorable
adenosine monophosphate (AMP)
FIGURE 2.2
Conversion of alcohols to diphosphates via reaction with adenosine triphosphate (ATP).
namely the conversion of the –OH group of alcohols (R–OH) or carboxylic acids

(R(CO)–OH) into their corresponding mono-or diphosphate derivatives (Fig. 2.2).
Recall that the –OH group of alcohols is a very poor leaving group for nucleo-
philic substitution (such as SN1 or SN2), so to facilitate such reactions, the –OH
group must often be converted into a better leaving group. ATP, which is essentially
a substituted organic anhydride of phosphoric acid, reacts exothermically (ΔG ~−31
kJ/mol)) with alcohols to produce phosphate or diphosphate esters in much the
same way that alcohols are readily converted to acetate esters by reaction with acetic
anhydride. Once an alcohol is converted to its mono-or diphosphate ester deriva-
tive (R–OP or R–OPP), the substrate is activated toward subsequent nucleophilic
displacement or carbocation formation with loss of monophosphate or diphosphate,
depending on which derivative was produced initially. This overall process is analo-
gous to the familiar conversion of alcohols into tosylate or mesylate esters for subse-
quent nucleophilic substitution reactions (Fig. 2.2).
Similarly, nucleophilic acyl substitution of carboxylic acids (R(C=O)–OH) in
biosynthesis may be greatly facilitated by conversion of the acid to an acyl phosphate
derivative (R(C=O)–OP) by reaction with ATP, just as the conversion of carbox-
ylic acids to their acid chloride derivatives (R(C=O)–Cl) facilitates such reactions in
conventional organic synthesis.
NADH/N ADPH: NATURE’S VERSION OF SODIUM

BOROHYDRIDE FOR CARBONYL REDUCTION
Redox processes are probably the most common reactions in all of biosynthesis.
Living systems carry out hydride-type reductions of carbonyls using either the re-
duced form of nicotinamide adenine dinucleotide (NADH) or its phosphate

derivative (NADPH) as hydride donors. In these enzyme cofactors, we focus on the

Bioorganic Synthesis 46 dihydropyridine portion of the molecule that serves as the essential cellular source
of hydride ion for the reduction of carbonyl groups to alcohols. Note that unlike
laboratory NaBH4 reductions of prochiral ketones which lead to chiral but racemic
alcohols, the NADH reduction shown (enzyme-mediated) leads to generation of a
single enantiomer product by stereospecific delivery of the pro-R hydrogen from
NADH to the Si face of the β-ketothioester (Fig. 2.3). Such 1,2-hydride additions may
also be carried out on thioester carbonyls (RCO–SR’), leading to the correspond-
ing aldehydes with liberation of a thiol, R’–SH. Further reduction of the aldehyde
to the primary alcohol may also follow in some instances. Coupled with subsequent
protonation, the delivery of hydride by NADH/NADPH to carbonyls is a reductive
two-electron transfer reaction (H:– + H+ = 2e– + 2H+). This is the main function of
NADH/NADPH, though they may also be used in other cellular processes such as
posttranslational modification of proteins. The enzymes involved in NADH/NADPH
metabolism are considered important targets for drug discovery because of their sig-
nificance in such cellular processes.
In addition to reductive 1,2-hydride additions to carbonyls, conjugate (1,4-)
additions of hydride may also take place in certain α,β-unsaturated carbonyl
systems. For example, one step in the biosynthesis of fatty acids involves an enzyme-
mediated reduction of the double bond of an α,β-unsaturated thioester via sequen-
tial conjugate addition of hydride from NADPH followed by protonation to give the
H H O
NH2
N O O NH2
N
P P N
N N O O
OH OH O
O H H
R OH H H
OH OH
NADH (R = OH)
NADPH (R = OP)
= R'
BH B:
O O HR OH O
S-Enz S-Enz
H R at
HS HR O + HS O
Si face
NH2 NH2
dihydropyridine pyridinium ion
N N
ring of NADH, NADPH of NAD +, NADP +
R' R'
FIGURE 2.3
Carbonyl reduction with NADH or NADPH as hydride source.


NADP-H H-B
O O
enoyl
S-Enz reductase S-Enz
O NADP + :B
keto- H OH
S-Enz enol
S-Enz
FIGURE 2.4
Conjugate reduction of an α,β-unsaturated carbonyl compound using NADPH.
corresponding saturated thioester (Fig. 2.4). We will examine this process in greater
detail in Chapter 5.
NAD +/N ADP +: NATURE’S VERSION OF PCC

FOR ALCOHOL OXIDATION
Unlike laboratory oxidation and reduction processes which usually lead to spent
reagents that often end up as waste products, cellular systems often employ cofac-
tors for reagents that are readily recycled from their oxidized or reduced forms for
reuse. Representing models of efficiency for such chemical processes, we often see
the oxidized form of a cofactor used by other enzymes to carry out an oxidation, the
reverse of the kind of reduction reaction that originally produced the cofactor in oxi-
dized form. This leads to an oxidized organic product with simultaneous regenera-
tion of the cofactor in its reduced form. Such is the case with NAD+/NADP+, which
are the oxidized forms of NADH/NADPH. In oxidized form, these cofactors may
act as hydride acceptors, and in their reactions with primary and secondary alco-
hols they produce the corresponding aldehyde or ketone products—the same result
obtained from oxidation of such alcohols with the laboratory oxidizer, pyridinium
chlorochromate (PCC). While often referred to as alcohol oxidations, conversion of
alcohols to aldehydes or ketones by NAD+/NADH+ are formally dehydrogenation
reactions (Fig. 2.5). Note the stereospecific delivery of the prochiral hydride (HR)
from the alcohol to the Re face of NAD+/NADP+, yielding the corresponding al-
dehydes and NADH/NADPH. Aldehydes (RCH=O) can also be dehydrogenated
by NADP+ via their hydrated form to give carboxylic acids (RCO2H). Primary or
secondary amines (RCH2NH2 or RCHNHR’) can also be dehydrogenated to the cor-
responding imines (RCH=NH or RC=NR’) in this fashion.
Nature takes advantage of a carefully balanced system in these processes. In
oxidized form, the pyridinium ring of NAD+/NADP+ is aromatic but not electri-
cally neutral, while in reduced form, the dihydropyridine ring of NADH/NADPH is

H O H HR O
HR HS
alcohol HS
H NH 2 dehydrogenase NH 2
R O +
HR at R O
N N
Re face
B: BH R'
R'
NADH/NADPH
NAD+/ NADP +
FIGURE 2.5
Alcohol dehydrogenation using NAD+/NADP+ as hydride acceptor.
E H H O E+ H H O
NH2 NH2
N N
R' R'
aromatic but neutral but
not neutral not aromatic
FIGURE 2.6
Redox gain or loss of hydride in NAD+ NADH interconversion.
electrically neutral but not aromatic (Fig. 2.6). Trapped between the stability associ-
ated with electroneutrality and the stability associated with aromaticity, the system
is an ideal one for a reversible organic redox process.
FAD: ANOTHER HYDRIDE ACCEPTOR

FOR DEHYDROGENATIONS
For reactions that involve dehydrogenation of a carbon chain to give an alkene func-
tion, enzymes may use the cofactor flavin adenine dinucleotide (FAD). The various
components of this polyfunctional coenzyme include a diphosphate bridge between
an AMP unit and a ribose phosphate unit bound via a C–N linkage to the flavin unit
that constitutes the heart of FAD’s redox functionality (Fig. 2.7). Removal of the
AMP unit leads to a structurally simpler and closely related cofactor, flavin mono-
nucleotide (FMN), also called riboflavin-5’-phosphate, which functions like FAD
in conjunction with certain enzymes. FMN in turn is derived from phosphorylation
of riboflavin or vitamin B2 at the primary –OH group of the ribose unit. Any oxi-
doreductase enzyme that uses FAD as an electron carrier is called a flavoprotein.
In conjunction with such enzymes, FAD can act as a hydride acceptor in a pro-
cess that is formally a 1,4-addition of hydride across the conjugated –N=C–C=N–π-
system of the flavin core. As in the NADH/NAD+ system, this reversible redox couple
delivers two electrons to a conjugated π-system, in this instance by hydride attack at
one end and protonation at the other, giving the reduced form of the cofactor, FADH2
(Fig. 2.8). While in some instances FADH2 is used as a hydride donor for specific

ribose-OP unit

NH2
N
O O O O N
OH OH
P P
O O O N N
O
H H
OH
O N N H H
OH OH
HN
N AMP unit
O
flavin unit flavin adenine dinucleotide (FAD)
FIGURE 2.7
Flavin, ribose, and AMP components of flavin adenine dinucleotide (FAD).
R B: R
BH H
O N N O N N
oxidation FADH 2
FAD HN
HN reduction
N N
H
H R O R' H
O
H
R'
H H :B H R HB
FIGURE 2.8
FAD/FADH2 as hydride acceptor/donor in redox processes.
O acylCoA O
dehydrogenase
R SCoA R SCoA
FAD FADH 2
FIGURE 2.9
FAD dehydrogenation of a fatty acid thioester.
reductions in which the reverse process takes place, the principal path for regenera-
tion of FAD involves oxidation via FADH2 + O2 → FAD + H2O2, with hydrogen per-
oxide subsequently reduced to water by the action of other agents.
One example of the action of FAD in dehydrogenation is found in the β-oxidation
of fatty acids which begins with dehydrogenation of a saturated fatty acid thioseter
to the corresponding α,β−unsaturated derivative (Fig. 2.9).
MONOOXYGENASES: SPECIAL DELIVERY
OF ONE O ATOM FROM O 2
An important process to consider in the biosynthetic transformation of organic

compounds is the simple oxidative transformation of a C–H bond into a C–O–H

N N O
N N
FeII O2 Fe
N N N N
CYP-SH S
HO2C CO2 H HO2C CO2H

Heme
Heme iron–oxo complex
FIGURE 2.10
Formation of the heme iron–oxo complex involved in cytochrome P-450 (CYP) oxidations.
bond. Such processes convert lipid soluble compounds into more water soluble
ones. The delivery of oxygen in this manner may occur for alkane C–H bonds (lead-
ing to alcohols), alkene C–H bonds (leading from enols to carbonyls) or aromatic
C–H bonds (leading to phenols). While simple in conception, such processes are
often complex, vary widely from a mechanistic point of view, and may only be par-
tially understood. Nevertheless, since such transformations are fundamental to bio-
synthetic processes, some simplified representations will be useful. Many of these
processes involve an important family of enzymes known as cytochrome P-450
monooxygenases (P is for Pigment, 450 for the wavelength of absorption, in nm)
or simply CYP enzymes of which more than 500 are currently known. At their core
is a porphyrin-bound Fe3+ that is the key to their function activating molecular
oxygen via a heme iron–oxo complex (Fig. 2.10). Initial binding to the heme iron is
via a cysteine thiol of the enzyme.
While of great importance, the key intermediates in many of these processes have
proven to be among the most elusive in all of mechanistic bioorganic chemistry. For
simplicity’s sake, a simple diagram representing what are believed to be some of the
key steps will suffice for our purposes (Fig. 2.11). The overall oxidation is: R–H +
NADPH + H+ + O2 → R–OH + NADP+ + H2O. Thus, NADPH supplies the electrons
required, with the second oxygen atom from O2 being passed to water. A simple ex-
ample of a CYP-mediated hydroxylation is the stereospecific conversion of camphor
to the corresponding exo alcohol by cytochrome P450cam (Fig. 2.11).
Another monoxygenase system of importance is found in the group of amino
acid hydroxylases that use the cofactor tetrahydrobiopterin (BH4) and a nonheme
iron for catalysis. Like some CYP enzymes, this system converts aromatic C–H
bonds to phenols and is used in the conversion of phenylalanine to tyrosine as well
as in the biosynthesis of neurotransmitters such as serotonin, melatonin, dopa-
mine, norepinephrine and epinephrine (adrenaline), and many other compounds of
note. In the reaction, molecular oxygen is incorporated into BH4 to give BH4OOH
(a hydroperoxide) which delivers its active oxygen to a substrate and is thus reduced
to BH4OH (hydroxy BH4) in the process, in much the same way that a peroxyacid
(RCO3H) delivers an active oxygen to an alkene substrate (giving an epoxide) while
being reduced to the corresponding carboxylic acid (RCO2H) (Fig. 2.12).

O O

O O
FeIII + e- FeII O2 FeIII + e- FeIII
+ H+ S S S
S
+ 2H + - H 2 O
OH O
R H
R OH R + FeIV FeV
S S
heme
iron–oxo complex
O O
P450cam
example: OH (exo)
O2, NADPH
camphor H (endo)
FIGURE 2.11
Simplified P-450 mechanism and example of R–H to R–OH oxidation.
O OH
O OH O OH O OH
H H H
N N N
HN HN HN
OH OH OH
H2N N N H2N N N H2N N N
BH4 H
O O
O OH OH
O OH O H
OHH O N
N HN
HN
+
OH
OH H2N N N
H2N N N
hydroxy BH4 hydroperoxy BH4
FIGURE 2.12
Simplified mechanistic scheme for tetrahydrobiopterin (BH4) oxidation of alkenes to epoxides.
For example, in the conversion of phenylalanine to tyrosine (Fig. 2.13), an epoxide

known as an arene oxide is produced. This epoxide then undergoes an acid-catalyzed
ring-opening reaction to yield a carbocation which then undergoes a special kind
of 1,2-hydride shift known as the NIH shift, named after the National Institutes of
Health laboratories where the process was discovered. Tautomerization of the result-
ing keto form to the aromatic phenol follows.
Given the energy cost associated with loss of aromaticity in arene oxides, such
intermediates might seem unlikely, though isolation of an arene oxide from CYP-
catalyzed oxidation of naphthalene lends support. NIH-type shifts in aromatic hy-
droxylations are also observed in certain CYP-catalyzed oxidations, as is epoxide
formation from alkenes.

H H CO2H
CO2H phenylalanine BH CO2H
hydroxylase O H
H2N H H2N H H2 N H
O2, BH4 H H O
phenylalanine an arene oxide B:
1,2-H shift
"NIH Shift"
H
CO2H CO2H
H
H2N H keto-enol H2N H
HO O
tyrosine
FIGURE 2.13
Arene oxide intermediate and NIH shift in conversion of phenylalanine to tyrosine.
O
O
cyclohexanone O
oxygenase
FAD
O2, NADPH
flavin-OOH
H B :B
H
O
O O flavin
HO O
+ HO-flavin
FIGURE 2.14
Flavin-dependent biological Baeyer–Villiger oxidation of 2-methylcyclohexanone.
A final example of some importance in biosynthesis is found in the so-called

Baeyer–Villiger monooxygenases, a group of flavin-dependent enzymes which
catalyze the conversion of ketones to the corresponding esters in much the same
way that ketones are converted to esters via peroxyacids in the laboratory version
of the Baeyer–Villiger reaction. Studies have shown that the enzymatic version fol-
lows the same migratory aptitude for groups attached to the carbonyl (3o > 2o > 1o >
methyl) and shows the same stereospecificity, leading to retention of configuration
in chiral systems. For example, oxidation of (S)-2-methylcyclohexanone with O2/
NADPH in the presence of flavin-dependent cyclohexanone oxygenase yields only
the (S)-lactone product arising from exclusive migration of the chiral 3o carbon
(Fig. 2.14). The oxidized form of the flavin coenzyme is represented here as a
hydroperoxide similar in structure to the tetrahydrobiopterin hydroperoxide we
previously encountered. Nucleophilic addition of flavin-O OH to the ketone car-
bonyl (in much the same way that a peroxyacid would add) gives a hemiacetal-like

intermediate that cleaves via a stereospecific 1,2-carbon-to-oxygen migration,

expelling the reduced flavin-OH and releasing the chiral lactone product.
DIOXYGENASES: DELIVERING BOTH O ATOMS FROM O 2
Enzymes that catalyze the incorporation of both oxygen atoms from O2 into a single sub-
strate are called intramolecular dioxygenases, while those which lead to oxygen atom
incorporation into two separate substrates are known as intermolecular dioxygenases.
Both types require a nonheme form of Fe. Of the intramolecular types, the catechol
dioxygenases are among the most important because of their role in environmental
degradation of aromatic compounds, both natural and man-made, by oxidative cleav-
age of the catechol ring. While these processes are far more complex mechanistically
than is depicted here, the overall results may be usefully summarized by considering
the three different cleavage patterns that distinguish one enzyme system from another,
namely i) proximal extradiol; ii) intradiol; and iii) distal extradiol cleavages (Fig. 2.15).
Dioxygenase-mediated cleavage of nonhydroxylated aromatic rings follows a similar
pattern, leading to dialdehydes, diketones, or ketoaldehydes, depending on ring sub-
stitution, in a manner analogous to the cleavage of cycloalkene π-bonds via ozonolysis.
Intermolecular dioxygenases usually require α-ketoglutarate (glutaric acid in
ionized form), which assists the enzyme by being oxidized along with the prin-
cipal substrate. Thus, such enzymes are frequently referred to as α-ketoglutarate-
dependent dioxygenases. In this process, a ligand (L) bound nonheme Fe2+ forms
a complex with O2 and α-ketoglutarate, leading to transfer of one O atom to Fe
and one O atom to the ketoacid with simultaneous decarboxylation, converting
O R
R
O
O
O2 OH CO2H
i) proximal extradiol
OH
cleavage OH
R R R
i) OH
OH
O2 O CO2H
ii)
ii) intradiol cleavage O CO2H
OH
R OH
iii) R
OH OH
O2
iii) distal extradiol OH CO2H
cleavage O H
O
O
FIGURE 2.15
Different modes of oxidative ring cleavage catalyzed by catechol dioxygenases.

FIGURE 2.16
Simplified mechanism for α-ketoglutarate-dependent oxidations.
α-ketoglutarate to succinate (succinic acid in ionized form). The remaining iron–

oxo complex subsequently carries out an oxidation of the R–H to R–OH type in a
fashion similar to the P-450-type oxidations we previously encountered. A simpli-
fied representation of a possible mechanism for the overall process is shown here
and should be sufficient to gain a reasonable sense of how such processes may work
(Fig. 2.16).
OTHER OXIDATIONS: HYDROQUINONE
AND CATECHOL OXIDATIONS
Certain O2 oxidations involve hydrogen removal from compounds rather than

oxygen incorporation and are catalyzed by oxidase enzymes. Passing the hydro-
gen atoms on to water or hydrogen peroxide, these enzyme systems are especially
important in the oxidation of various structures containing hydroquinone or cat-
echol moieties. Mechanisms are complex and not always well understood, but may
be generally formulated as 2e–/2H+ redox processes (Fig. 2.17). Thus, oxidation of
hydroquinone (1,4-benzenediol) yields p-benzoquinone and vice versa, providing
an important and useful electron shuttle mechanism in biological systems. Catechol
benzenediol) is similarly oxidized to the highly reactive o-benzoquinone.
(1,2-
Catechol is often released when plant tissues are injured, and in the presence of
the abundant plant enzyme catechol oxidase, it undergoes rapid oxidation to
o-benzoquinone which then quickly converts to brown-colored polymeric prod-
ucts responsible for most of the dark color of “bruised” fruits like apples or bananas
(sometimes called “fruit browning”). The reactivity of o-benzoquinone is also re-
lated to its toxicity toward bacteria, which can help to protect plants from infections.
Closely related to these oxidations is an important process in biosynthesis known
as oxidative phenolic coupling. A number of different enzyme systems have been
found to participate in these processes, including oxidases, peroxidases, and P-450-
type enzymes employing NADPH and O2 cofactors. The key reactive intermediates
in these processes are phenoxy radicals (Fig. 2.18), which can participate in inter-
molecular radical–radical coupling reactions, forming C–O or C–C bonds in the
process.

OH O

- 2 e-, - 2 H +
hydroquinone + 2 e-, + 2 H + p-benzoquinone
- H+ OH O
+ H+
O O O O
- e- - H+ - e-
+ e- + H+ + e-
OH OH O O
OH catechol O
oxidase
1/ + H2O
2 O2
OH O
catechol o-benzoquinone
FIGURE 2.17
Oxidation of hydroquinone and catechol to p-and o-benzoquinones.
OH O O O
- H+
- e-
phenoxy ortho para

radical radical radical
FIGURE 2.18
Resonance forms of the phenoxy radical.
Different resonance forms of the radicals involved may connect in different com-
binations, leading to ortho- or para-phenoxyphenol ethers or ortho–ortho, ortho–
para, and para–para C–C coupled diphenolic products (Fig. 2.19). Both C–O and
C–C bond-forming processes are important here, but the C–C phenolic coupling
reactions are of special significance, as they constitute one of the principal means
by which aromatic rings are joined one to another in various biosynthetic processes,
occurring in both inter-and intramolecular fashions in a variety of contexts in dif-
ferent pathways.
AMINE OXIDATIONS: FROM IMINES TO CARBONYL

COMPOUNDS AND BEYOND
Just as 1o and 2o alcohols may be oxidized (actually dehydrogenated) to give alde-

hydes and ketones, so 1o and 2o amines can be oxidized to imines which then may
be hydrolyzed to give aldehydes or ketones. The oxidation of compounds containing

OH
O
O
ortho para
OH O radical radical
O
O C–O C–O p-coupling
H coupling coupling O
o-coupling H O
phenoxy
radical
O O
OH O
O ortho para H
H radical radical
C–C C–C H
H coupling
OH O coupling
ortho
o,o-coupling radical
OH
HO O OH
O
para ortho
H radical radical
C–C C–C
H coupling coupling
p,p-coupling OH O para o,p-coupling
radical
FIGURE 2.19
Various possible C–O and C–C coupling modes for phenoxy radical resonance forms.
R NH2 FAD, O2
H2O R O
H H + NH3
monoamine
oxidase H
FAD
H2 O
FADH -
R NH2 R NH
FADH - FADH2 H
H
FAD
O2 H2O2
FIGURE 2.20
Monoamine oxidase-mediated amine to imine to aldehyde transformations.
a single amino group is catalyzed by the action of enzymes known as monoamine

oxidases (MAOs). A number of different types are known, and a variety of mech-
anisms have been proposed to explain their action, with a simplified mechanism
shown (Fig. 2.20) in which FAD acts as a hydride acceptor, converting the amine to
an iminium ion. Loss of a proton to FADH– yields the imine and FADH2 (which is
reoxidized to FAD by O2 with loss of H2O2). Hydrolysis of the imine yields the cor-
responding carbonyl compound and ammonia.
A similar oxidation of compounds containing two amino groups is catalyzed
by the so-called diamine oxidase enzymes, although in this process only one of
the two amino groups is oxidized. For example, oxidation of the foul-smelling
diaminobutane (common name: putrescine) leads to the corresponding
1,4-

FAD, O 2

NH2 H2O O
H2N H2N + NH3
diamine
oxidase H
+ H+ :Nu
O imine formation
H2N
N - H+ N N Nu
H
H H
FIGURE 2.21
Diamine oxidase-mediated oxidation followed by cyclization and nucleophilic trapping.
4-aminobutanal. This transformation is an important one in the biosynthesis of

many alkaloids containing a pyrrolidine ring, as intramolecular Schiff ’s base forma-
tion from the aminoaldehyde leads to a cyclic imine that may be further elaborated
by addition of various nucleophiles (Fig. 2.21).
PLP: TRANSAMINATION AND DECARBOXYLATION

OF AMINO ACIDS
Another mechanism for conversion of amino groups into carbonyl groups (and vice
versa) but which is especially important in amino acid chemistry is transamination.
This process uses another essential enzyme cofactor, pyridoxal-5’-phosphate (PLP).
For simplicity’s sake, we show PLP in aldehyde form condensing with an amino acid
to form the corresponding imine (Schiff ’s base) as a first step (Fig. 2.22), though this
step actually involves PLP as an enzyme-bound imine which undergoes an imine ex-
change with the amino acid to form a new imine, with release of the enzyme’s amino
group. Protonation of the ring nitrogen of PLP enhances the acidity of the amino
acid imine α-proton by providing an extended set of conjugated π-bonds over which
the electron pair from the α-C–H bond may be delocalized, leading all the way to the
protonated PLP ring nitrogen giving, after deprotonation, the “quinonoid” form of
the pyridine ring, so-called due to the resemblance of the upper portion of its ring to
the conjugated π-system of p-benzoquinone. Rearomatization of the pyridine ring via
concurrent protonation at the PLP exocyclic π-bond gives an α-ketoacid imine which
upon hydrolysis releases the corresponding α-ketoacid and pyridoxamine phosphate.
As one might expect, most of the important amino acids have a familiar correspond-
ing α-ketoacid (alanine → pyruvic acid; glutamic acid → α-ketoglutaric acid; phe-
nylalanine → phenylpyruvic acid, etc.) and many of these play an important role as
readily available intermediates for various biosynthetic processes.
An alternative pathway for the PLP imine intermediate can be followed by car-
boxyl group deprotonation of the amino acid rather than removal of an α-proton.
The ensuing loss of CO2 as well as a proton provides the means for conversion of
amino acids to their corresponding primary amines via the resulting PLP
α-

FIGURE 2.22
Mechanism for transamination of α-aminoacids by the action of pyridoxal phosphate (PLP).
O
R
O
H O H
H N H :B
OH R CO2H decarboxylase
PO OH
- H2O PO
+ H2N H
N α-amino N
H acid
PLP imine H
-CO2
pyridoxal phosphate, PLP
(ring N-protonated form) :B HB
H
R H R H
H
R H H N H N
imine
PLP + NH2 hydroylsis OH OH
PO PO
primary
amine
N N
PLP imine PLP
H ("quinonoid" form) H
FIGURE 2.23
Mechanism for decarboxylation of α-amino acids by the action of pyridoxal phosphate (PLP).
quinonoid form. Again, rearomatization of the pyridine ring via concurrent pro-
tonation at the PLP exocyclic π-bond gives an imine which upon hydrolysis releases
the primary amine with concurrent regeneration of PLP (Fig. 2.23). This remark-
ably versatile cooenzyme also facilitates racemization reactions of amino acids (in
conjunction with racemase enzymes) via the PLP imine as well as some specific
retroaldol and beta-elimination reactions which we will encounter later on as we
move forward.

OTHER IMPORTANT DECARBOXYLATIONS: β-K ETO

ACIDS, O- AND P-H YDROXYBENZOIC ACIDS
Recall that the β-ketoester products derived from Claisen condensation reactions
are versatile intermediates in laboratory organic synthesis, in part due to the ease
with which these β-dicarbonyl compounds can be deprotonated by simple bases
(pKa ~ 11) to yield nucleophilic enolate ions which may then be alkylated by simple
SN2 chemistry. Just as important is the chemistry that results from hydrolysis of
their ester function. Unlike β-ketoesters, which are quite stable, the ester hydrolysis
products (β-ketoacids) are thermally unstable, undergoing a spontaneous decar-
boxylation via a cyclic 6-membered ring transition state to yield the corresponding
ketones. This reaction is also an important one in biosynthetic processes with many
examples of β-ketoacid decarboxylation providing a simple means of shedding a
single carbon atom from a structure. Such β-ketoacids need not be derived from
Claisen condensations and the cyclic transition state shown may not necessarily be
involved in biosynthetic examples (Fig. 2.24).
Closely related to the decarboxylation of β-keto acids is the decarboxylation of
ortho- or para-hydroxybenzoic acids. While these processes are only rarely encoun-
tered in laboratory organic synthesis, they are quite common in biosynthetic pro-
cesses. Phenols may be considered stable aromatic enol forms of one of two different
unstable and nonaromatic keto forms: the conjugated 2,4-cyclohexadienone form
or the nonconjugated 2,5-cyclohexadienone form. It is worth noting that cyclo-
hexadienones of these types, regardless of their origin, will always be unstable and
will quickly aromatize to the corresponding phenol, provided that there is at least
one H atom at the 6-position of a 2,4-cyclohexadienone and/or one H atom at the
4-position of a 2,5-cyclohexadienone (Fig. 2.25).
With an ortho-carboxyl group present, the 2,4-cyclohexadiene form is essentially
a β-ketoacid and may decarboxylate via the familiar cyclic transition state to yield the
phenol. Similarly, phenolic systems with a carboxyl group in the para position can
decarboxylate to regain aromaticity via the nonconjugated 2,5-cyclohexadienone
form (Fig. 2.26). In biosynthesis, the enzymes involved presumably provide a means
O O O O O
Claisen R ester R
2 R
OR' condensation OR' hydrolysis OH
β-ketoester R β-ketoacid R
OH H
O O O
- CO2
R R R
O
ketone R enol R R
FIGURE 2.24
Spontaneous decarboxylation of β-ketoacids to give ketones.

O OH O
R
R R
1 H 1
2 2
3 4 5 3
4
R R H R
2,4-cyclohexadieneone phenol 2,5-cyclohexadieneone
FIGURE 2.25
Keto-enol tautomerization of 2,4-and 2,5-cyclohexadienones to phenols.
H
OH O O
CO2H
O
H
OH
aromatic
o-keto form
enol form
- CO2
HB
OH O
H :B
CO2H H O
aromatic O
enol form p-keto form
FIGURE 2.26
Decarboxylation of o- and p-hydroxybenzoic acids.
for stabilizing the unfavorable keto forms, thereby facilitating such decarboxyl-
ations. Note that in each case, aromaticity can be restored either via loss of a proton
ortho or para to the ketone function (essentially via 1,2-or 1,4-enolization) or via
loss of a proton and CO2 from one or the other of these positions (essentially decar-
boxylative 1,2-or 1,4-enolization processes).
THIAMINE DIPHOSPHATE (TPP) AND LIPOIC

ACID: DECARBOXYLATION AND ACYL TRANSFER
We have seen how PLP facilitates the decarboxylation of amino acids to their corre-
sponding primary amines and also how it participates in the transamination process
that converts amino acids to their corresponding α-ketoacids. These keto acids are
also available from other pathway sources and their decarboxylation is an important
component of various metabolic processes, such as in the decarboxylation of pyru-
vic acid to ethanal (which then undergoes NADH reduction to ethanol) during yeast
fermentation. However, unlike β-ketoacids, decarboxylation of α-ketoacids is not
spontaneous and requires the action of a cofactor known as thiamine diphosphate

(TPP). How TPP works is related in part to the acidity of the proton on the posi-

tively charged thiazole ring of its structure (Fig. 2.27).
When its thiazole proton is removed (pKa ~18) by a base, the resulting anion is
called TPP ylide. Recall that ylides are dipolar reactive intermediates with a nega-
tive charge on carbon and a positive charge on an adjacent heteroatom (N in this
case). Thus, TPP ylide (shown in abbreviated form using R and R’ groups attached
to the core thiazole ring) is similar to the phosphorous ylide of the Wittig reaction
in which an ylide acts as a nucleophile toward the carbonyl group of ketones or
aldehydes. Similarly, the nucleophilic TPP ylide attacks the ketone carbonyl of an
α-ketoacid to initiate the subsequent decarboxylation of the resulting tetrahedral
intermediate (Fig. 2.28). After loss of CO2, a resonance-stabilized carbanion is pro-
duced which is first protonated on carbon then deprotonated at its –OH group to
release an aldehyde with concurrent regeneration of TPP ylide.
The most important function of TPP in biosynthesis is found in its ability to work
in conjunction with two other coenzymes, lipoic acid (LA) and the thiol-contain-
ing cofactor, coenzyme A (HSCoA or CoASH). This triad of cofactors couples the
R'
N OPP
S
N N
R NH2 H
pKa ~18
thiamine diphosphate (TPP)
FIGURE 2.27
Acidic proton on the thiazole ring component of thiamine diphosphate (TPP).
HB R'
R'
R'
O
OH S
S N
N S R R OH
R N
R O O
H :B H
TPP ylide R
TPP α-ketoacid
(nucleophilic) O :B
- CO2
R' R' R'
H
S S S
R O N N N
R R R
H B: BH OH OH
R O R R
H
FIGURE 2.28
Mechanism of action of TPP ylide in the decarboxylation of α-ketoacids.

disulfide bond
Bioorganic Synthesis 62 H2N Enz S S S S

S S H =
N
Enz R-Enz
CO2H
lipoic acid enzyme-bound LA O
NH2
N
N
thiol O O N N
HS O O O
N N P P O
H H H H
HO O O O O
H H
coenzyme A O OH
HS CoA O P O-
O-
FIGURE 2.29
Structures of the enzyme cofactors lipoic acid (LA) and coenzyme A (CoASH).
decarboxylation of pyruvic acid to its conversion to the important biological acyl

group carrier, acetyl-CoA. To sort all this out, we begin with the structures of both
LA and CoASH, focusing on the key components involved in their reactions with
TPP (Fig. 2.29). Of particular note are the thiol component of CoASH and the disul-
fide linkage of lipoic acid (LA). For the purposes of mechanistic analysis, both struc-
tures are usually abbreviated to include only those portions actually transformed
during a biosynthetic sequence. In the reactions considered here, LA functions in
enzyme-bound form, but usually only the disulfide-containing ring is involved in
chemical transformations. The generic abbreviations HSCoA or CoASH are also
frequently used as simple structural abbreviations for coenzyme A, since they place
all the emphasis on the thiol (HS) that is the most important functional group in its
chemical transformations.
In its reaction with pyruvic acid (shown here in nonionized form for simplicity’s
sake, in Fig. 2.30), TPP ylide leads to the same decarboxylated carbanion intermedi-
ate we saw in Fig. 2.28, but its fate will be different in this case.
Rather than attacking a proton source as before, the carbanion formed after de-
carboxylation attacks a sulfur atom in the disulfide linkage of enzyme-bound LA,
leading to formation of a hemithioacetal intermediate, deprotonation of which
produces an acetylthioester derivative with concurrent regeneration of TPP ylide
(Fig. 2.31). Finally, the acetylthioester undergoes nucleophilic attack by the thiol
sulphur atom of HSCoA, leading to a simple thiol exchange reaction, producing
a new thioester, acetyl-CoA, and the dithiol form of enzyme-bound LA, which is
subsequently recycled to its cyclic disulfide form by FAD oxidation. This complex
sequence of events, mediated by a multi-enzyme complex called pyruvate dehydro-
genase complex, is not only important because of the key role acetyl-CoA plays in
biosynthesis, it is especially important in the basic biochemistry of living systems, as
it links glucose metabolism (glycolysis, the main source of pyruvic acid) to the citric

HB

R' R' R'
R' O
S - CO2
OH N S S
N N
S H3C R OH R R
N
R O O
H OH OH
H3C H3C H3C
TPP ylide pyruvic acid
O :B
FIGURE 2.30
Carbanion intermediate from decarboxylation of pyruvic acid by TPP ylide.
R' R' R' O
N S S S + H3C S SH
N N
R R R
H :B
OH O TPP ylide R-Enz
H3C HB H3C S SH
S S CoASH
R-Enz
R-Enz
B: H O
O
S S HB
SH SH H3C
+ H3C SCoA S SH
R-Enz CoAS
R-Enz FADH2 FAD acetyl-CoA
R-Enz
FIGURE 2.31
Action of enzyme-bound lipoic acid in formation of acetyl-CoA and regeneration of TPP ylide.
acid cycle (or Krebs cycle), a process that combines acetyl-CoA and oxaloacetic acid
(the ketoacid of aspartic acid) as part of the many biochemical reactions involved in
cellular respiration.
As we will see, acetyl-CoA has many other important uses in biosynthesis, func-
tioning as an acyl transfer agent and participating as a two-carbon fragment for
the building up of complex structures via a variety of familiar transformations, not
only as an electrophilic carbonyl compound, but also as a nucleophile via its enolate
form. Some of these transformations, summarized in Fig. 2.32, take advantage of
the higher reactivity of thioesters relative to simple esters in both nucleophilic acyl
substitution reactions (acyl transfer) and the ease with which their α-protons may be
removed to form enolate ions (pKa ~20 for thioesters vs. pKa ~25 for esters).
BIOTIN: THE CO 2 CARRIER, TRANSPORT,

AND TRANSFER AGENT
The product derived from α-carboxylation of acetyl-CoA is called malonyl-CoA,

an especially important derivative of acetyl-CoA for generation of its thioester
enolate ion in certain Claisen condensation reactions. The incorporation of CO2
in this fashion requires a cofactor called biotin which acts as a CO2 carrier, trans-
port, and transfer agent in several biosynthetic sequences, working in conjunction

O O
H2O Nu:
H3C OH + CoASH H3C SCoA
H3C Nu + CoAS
hydrolysis acetyl-CoA acyl transfer
B:
R' O O
O
O
R R R' H2C SCoA R-X R
SCoA
OH aldol addition α-alkylation SCoA
O O O
O O
O CO2
R OR'
R SCoA Claisen α-carboxylation HO SCoA
H2C SCoA
condensation
FIGURE 2.32
Some important reactions in biosynthesis involving acetyl-CoA as a key intermediate.
O O
HN NH HN NH
Enz-NH2
=R O
H H H H
CO2H
NH-Enz
S S
biotin
O O
O O
HO O- HO OP
bicarbonate
ATP ADP - H+ O N NH
O O H H
- Pi
R
B: HN NH N NH S
H H H H N-carboxybiotin
R R
S S
FIGURE 2.33
Structure of biotin and mechanism of formation of N-carboxybiotin.
with specific carboxylase enzymes (Fig. 2.33). Biotin is first converted to an enzyme-
bound form which is then deprotonated to give an enolate-like anion which attacks
a carboxyphosphate derived from phosphorylation of bicarbonate (HCO3–) with
ATP. This results in formation of N-carboxybiotin, formally the carrier of CO2 for
subsequent carboxylation reactions.
A number of different mechanisms have been proposed and studied regarding
how N-carboxybiotin delivers CO2 to nucleophilic agents. We will consider what is
regarded by some as the most likely mechanism for carboxylation of acetyl-CoA eno-
late ion, namely initial release of CO2, with concurrent deprotonation of acetyl-CoA by
the biotin enolate ion. This regenerates biotin (enzyme-bound) with the acetyl-CoA
enolate ion and CO2 in close proximity, resulting in nucleophilic carboxylation to give
malonyl-CoA as shown in Fig. 2.34. Later on we’ll see how malonyl-CoA functions as


CoAS CH2
H
O O O O
O O
O N NH N NH CoAS CH2
- biotin
H H CoAS O
H H O
R R C malonyl-CoA
S O
S
N-carboxybiotin
FIGURE 2.34
N-carboxybiotin and carboxylation of acetyl-CoA to give malonyl-CoA.
a source of the enolate ion of acetyl-CoA via simple decarboxylation, forming an essen-
tial component in the biosynthesis of fatty acids and polyketide products in Chapter 5.
SAM: A C 1 FRAGMENT FOR METHYL GROUPS
CO2 can be considered a one-carbon fragment for the building up of larger struc-
tures from smaller ones by nucleophilic carboxylations; SN2-type alkylations can
also accomplish this by simple nucleophilic methylation using the methyl donor
S-adenosylmethionine or SAM (Fig. 2.35). This important alkylating agent may
be thought of as the biological equivalent of methyl iodide for such reactions, as it
takes advantage of the leaving group ability of a positively charged sulphur atom.
Generally speaking, CH3–O–, CH3–N– or CH3–S–groups found in natural product
structures usually originate via SAM alkylation of the heteroatom involved. For sim-
plicity’s sake, when SAM methylation is depicted mechanistically, we will employ
the abbreviated representation shown.
Certain methyl groups attached to carbon chains or rings may also be derived
from SAM alkylation in systems where carbon is available in a suitable nucleophilic
form, such as in enolate ions or phenolate ions (Fig. 2.36). Such ions may alkylate
either on carbon (C-alkylation) or oxygen (O-alkylation) with site selectivity mainly
controlled by the enzyme mediating the process. The conversion of tyramine to
N-methyltyramine via SAM represents a typical N-methylation process. Both com-
pounds are naturally occurring phenethylamine alkaloids (Chapter 7) isolated from
a number of different sources and able to act as CNS stimulants.
DMAPP: AN ALLYLIC C 5 FRAGMENT

FOR STRUCTURE BUILDING
We’ve seen CO2 and SAM as C1 fragments and acetyl-CoA as a C2 fragment for
the building up of more complex organic structures from simpler ones. Another
important alkylating agent that will be employed in a variety of biosynthetic trans-
formations is dimethylallyl diphosphate or DMAPP. Unlike SAM, which is limited

NH2
Bioorganic Synthesis 66 N
N
Nu: H3C
(C, N, O, S) N N
S O
H H ~ CH3 I
=
H H methyl iodide
OH OH
H2N
S-adenosylmethionine
CO2H (SAM)
NH2
N
N
N N
Nu CH3 + S O
H H
H H
OH OH
H2N
S-adenosylhomocysteine
CO2H
(SAH)
Nu: H3C SAM Nu CH3 + SAH
FIGURE 2.35
Structure and function of S-adenosylmethionine (SAM) for biosynthetic methylations.
B: H-O H3C SAM OCH3 BH
O-alkylation + SAH
BH
B: H-O O OH
H3C SAM
CH3 CH3
H keto-enol
C-alkylation + SAH
tautomerization
H CH3
N H3C SAM B: BH N
H H
H CH3
N-alkylation N
HO H HO
tyramine (- SAH) N-methyltyramine
FIGURE 2.36
O-, C-, and N-alkylations using SAM as a methyl group donor.
to SN2-type alkylations due to the inherent instability of the CH3+ that would be
required for SN1-type processes, DMAPP has its leaving group attached to a pri-
mary allylic carbon, so this important five-carbon fragment may be delivered via
either SN2 nucleophilic substitutions or via SN1 substitutions, owing to the resonance

stabilization of the allylic carbocation resulting from initial loss of the diphosphate

leaving group (Fig. 2.37). In both cases, DMAPP may be thought of as the biological
equivalent of allyl bromide, the laboratory reagent most commonly used for allylic
alkylation reactions. Alternatively, alkyl diphosphates (R–OPP) may be thought of
as biological equivalents of the more familiar tosylate or mesylate ester derivatives of
alcohols which, like the diphosphate ester, transform a normally poor leaving group
(–OH) into an excellent leaving group for nucleophilic substitution processes.
An example of one way DMAPP is used in biosynthesis is the late-stage ortho-
alkylation of the phenolic ring of the compound shown in Fig. 2.38 (a pterocarpene),
producing the alkylated isoflavonoid derivative erypoegin H, a potent natural an-
tibiotic isolated from the roots of the ornamental plant Erythrina poeppigiana and
found to be active against vancomycin-resistant strains of bacteria.
We will also see that DMAPP and its closely related partner, isopentenyl di-
phosphate (IPP) constitute the basic so-called isoprene building blocks of the bio-
synthetic pathway that produces the large and structurally diverse group of natural
products known as the terpenes. This terpenoid pathway produces many com-
pounds of great significance to human health. Retinal, a key molecule involved in
O O O O
or
P P ~
O O O OPP =
Br
dimethylallyl diphosphate (DMAPP)
SN2
OPP :Nu Nu + OPP
Nu
SN1
or + OPP
OPP :Nu
Nu
FIGURE 2.37
Structure of dimethylallyl diphosphate (DMAPP) and its use in SN2 and SN1 alkylations.
O O
OH ortho-alkylation OH
B:
O then HO O
HO keto-to-enol
erypoegin H
OPP
FIGURE 2.38
Ortho-alkylation of a phenolic ring with DMAPP to produce the antibiotic erypoegin H.

enzyme
OPP OPP
isoprene dimethylallyl PP isopentenyl PP
(DMAPP) (IPP)
OH
O
OH
an "isoprene unit"
HO
O HO
PhCO2 AcO O
retinal 10-deacetyl baccatin III
FIGURE 2.39
Isoprene units highlighted in the structures of two important natural products.
human vision and taxol, a powerful clinical chemotherapy drug derived from the
intermediate compound 10-deacetylbaccatin III, are just two examples; their five-
carbon isoprene structural units are highlighted in Fig. 2.39. In Chapter 4, we will
also see how this pathway assembles all the steroid structures, from cholesterol to
the sex hormones.
OTHER ESSENTIAL STRUCTURAL

FRAGMENTS: PUTTING IT ALL TOGETHER
Two key amino acids, ornithine and lysine, often provide basic C4N and C5N frag-
ments that are essential components in biosynthetic pathways leading to different
types of alkaloids that we will examine later in Chapter 7. The pyrrolizidine alka-
loids, which include compounds such as retronecine (Fig. 2.40) are examples of
frequently toxic C4N-containing alkaloids produced by plants as a defense against
herbivores. Swainsonine, a C5N-
containing alkaloid toxin found in locoweed,
causes significant annual economic losses in grazing livestock but has also shown
potential as an anticancer agent in a number of studies.
Many structures containing PhC1, PhC2, PhC3, as well as PhC2N fragments are
derived from two important amino acids: phenylalanine (Phe) and tyrosine (Tyr)
(Fig. 2.41). Starting with either structure, decarboxylation can provide PhC2N frag-
ments which may then be further modified by transamination or amine oxidations
to give PhC2 fragments for further elaboration. If Phe or Tyr undergo 1,2-elimination
of ammonia, a PhC3 fragment is produced which can also undergo various subse-
quent biosynthetic transformations. 1,2-elimination of NH3 followed by oxidative
C=C bond cleavage can also provide access to PhC1 fragments such as benzalde-
hydes or benzoic acids which may then be modified further. We can recognize the
tyrosine origin of the PhC3 units of etoposide, an important chemotherapy agent
in the treatment of lymphoma, lung, and testicular cancer. This nonalkaloid lignan

HO H OH

CO2H
H2N N or
N
NH2 N
ornithine
C4N retronecine
OH
H OH
H2N CO2H
N or OH
N N
NH2
lysine swainsonine
C5N
FIGURE 2.40
Lysine and ornithine as sources of C4N and C5N fragments in biosynthesis.
- CO2
N
etc. X X
CO2H
PhC2N PhC2
NH2
X
X = H: phenylalanine - NH3
X = OH: tyrosine
etc.
O X X
PhC3 PhC1
O
OH
O OH
H3CO
H3CO O
O NH2
H3CO
O
HO O O OCH3
OCH3 etoposide mescaline
FIGURE 2.41
Phenylalanine and tyrosine as PhC2N, PhC2, PhC3, and PhC1 structural fragments.
is derived from a compound isolated from a common forest plant, the American
Mayapple (Podophyllum peltatum), which produces it via the shikimic acid biosyn-
thetic pathway we will examine in detail in Chapter 6. We can also see the tyrosine
origins of the PhC2N component of mescaline, a notorious psychedelic alkaloid iso-
lated from peyote cactus which, though used in religious rites and other contexts by
native American populations for several millennia, is nevertheless illegal in most
countries. Note the SAM-derived methyl groups on the aromatic ring oxygens in
both these compounds. SAM is also the origin of the single C atom bridging the
two ortho-oxygen atoms on the second aromatic ring of etoposide. This so-called
methylenedioxy group is a common function in many natural product structures,
the formation of which we will address later on.
A final example of an essential fragment in biosynthesis finds its origin in the
amino acid tryptophan (Trp) to give the so-called indole-C2N fragment (Fig. 2.42)

CO2H
NH2 N
N N
H
tryptophan indole.C2N
HO2C
CH3
N N
N
O
N
H
O strychnine
lysergic acid
FIGURE 2.42
Tryptophan as an indole.C2N fragment source in biosynthesis.
which forms portions of the structures of a number of familiar alkaloids including

lysergic acid, a member of the ergot alkaloid family whose diethyl amide deriva-
tive is the potent hallucinogen LSD (lysergic acid diethylamide). Strychnine, a
highly toxic terpene alkaloid which contains the indole-C2N fragment from Trp as
well as isoprene units from the terpenoid pathway, has been known for thousands
of years, most famously as the poison that may have found its way into the wine
goblet of the young Alexander the Great in 323 BC, taking his life at the tender
age of 32.
LOOKING AHEAD
New and remarkably complex natural products continue to be discovered almost

on a daily basis, some of which not only show important beneficial effects for
human health, but may even provide new perspectives for historical analysis. For
example, a recent theory suggests that rather than being killed by strychnine poi-
soning, Alexander the Great might actually have been poisoned by drinking water
from the river Styx, which is now known to be contaminated with a highly toxic
compound called calicheamicin (Fig. 2.43). Discovered in 1981, this nonalkaloid
natural product is produced by certain soil bacteria utilizing the polyketide bio-
synthetic pathway (Chapter 5) with acetyl-CoA as the principal building block.
Because of its highly specific toxicity to DNA in virtually all kinds of cells, many
derivatives of calicheamicin have now been prepared, examined, and used in care-
fully targeted cancer chemotherapy regimens. Two of these derivatives, calicheami-
cin γ1 and esperamicin, are considered to be the most powerful anticancer agents
known to science.


HO H
N OCH3
H3C S
CH3 O S S
H3C O
I O H3C
S O NH O
HO O
O OCH3 OH O
H3C H
O OCH3 N O
HO
H3CO H3CO
OH
calicheamicin
FIGURE 2.43
Structure of calicheamicin, another poison possibly responsible for the death of Alexander
the Great.
You may recognize a number of the components of calecheamicin’s structure

as being carbohydrate (sugar) molecules. Clearly, we will need a good grasp of
carbohydrate structure and chemistry, one of the topics of our next chapter, before
moving further into the study of biosynthetic pathways. We have already seen the
significance of familiar amino acids such as phenylalanine, tyrosine, and trypto-
phan as well as some less familiar nonpeptidic amino acids such a ornithine as
basic biosynthetic building blocks, so our next chapter will also include a brief
overview of amino acid structure and biosynthesis. Once we understand how
amino acids and carbohydrates are constructed, how and where they are often
added to the core structures of more complex molecules, and how they are used to
assemble some of the key starting materials for biosynthesis, we will then be in a
position to systematically build up our knowledge of how specific natural products
are produced by a set of fundamental pathways in living systems. In doing so, we
will eventually be able to dissect even a remarkably complex compound like cali-
cheamicin to see what kinds of reactions and components are likely to have been
used by organisms to assemble such a marvel of molecular architecture and how
such knowledge can be used to solve problems in structure identification and to
even generate new ideas for the assembly of complex organic compounds of our
own making.
STUDY PROBLEMS
1. Provide the missing structures expected for A and B, then provide a reasonable
mechanism for the NAD+ oxidation that would produce the thioester.
R' SH O
NAD + thiol NAD +
R OH R S R'
alcohol thioester
A B

2. Propose a reasonable sequence of three different biological oxidation reactions

Bioorganic Synthesis 72 that could transform A into B. Show the intermediate product from each step
and provide mechanistic details of the third oxidation step.
O
H3C OH
H3 C
1) 2) 3)
O
O
CH3
CH3
A B
3. Pummerer’s Ketone 2 is formed by oxidation of para-cresol 1 using potassium

ferricyanide under basic conditions and has also been formed by biological
oxidation of 1 in the presence of a specific enzyme. Propose reasonable mecha-
nisms for the sequence of steps leading to 2. To begin, you may assume that 1
initially oxidizes to the usual phenoxy radical intermediate.
OH O
H 3C O
H 3C H 3C
1 2
4. Give a brief and concise description of the role that each of the following enzyme
cofactors plays in biosynthetic processes.
a. DMAPP
b. NADH/NAD+
c. SAM
d. PLP
e. TPP
f. FAD/FADH2
g. ATP
h. Biotin
5. In animals, phenylalanine is oxidized to tyrosine via a monooxygenase-mediated
process. If deuterium-labeled phenylalanine (as shown) undergoes this oxida-
tion, what happens to the deuterium? Provide a mechanistic analysis (keep in
mind that a C–D bond is stronger than a C–H bond).
CO2H
CO2H
NH2
NH2 HO
D D? if so, where?
6. Rifamycin B, a potent inhibitor of Escherichia coli RNA polymerase, contains a number

of different functional groups, some of which are labeled in the structure shown.

What is the likely biosynthetic origin (precursor molecule) of methyl

group A?
group B?
group C?
Name the functional group involving the atoms directly attached to (and
including) carbon 1.
For the H atoms labeled X, Y, and Z, what is their correct order of increasing
acidity (least acidic first)?
Z
3
O HO
B O Y
OH O
OH OH
A H3CO
H 3C NH 2
C
O
O OCH2CO2H X
O
rifamycin B 1
7. Compound A is a substituted pyrrole intermediate used in porphyrin biosyn-

thesis. Propose a detailed mechanism to account for the formation of A from
5-aminolevulinic acid as shown (hint: the mechanism requires no co-factors
or reagents other than water and a proton. You need only use imine formation,
imine-enamine tautomerism, and other intramolecular steps including a final
dehydration).
CO2H
CO2H CO2H CO2H
O O
N
H
NH2 NH2 NH2
A
5-aminolevulinic acid

8. Frontalin is the sex attractant of the southern pine beetle (Dendroctonus fron-
Bioorganic Synthesis 74 talis), a remarkably destructive pest of pine forests of the American south. The
attractant is biosynthesized from 6-methyl-6-heptene-2-one by the action of a
monooxygenase-mediated epoxidation followed by a simple acid-catalyzed cy-
clization. Propose a mechanism for the epoxide cyclization leading to frontalin.
O
O O
O2, NADPH
then cyclization
frontalin
9. Fenchone undergoes a monooxygenase-

mediated oxidation and hydrolysis
to give a mixture of hydroxyacids as shown.Provide a reasonable mechanism
to account for the formation of the two products shown from fenchone and
flavin–OOH.
O
OH
flavin–OOH OH
then hydrolysis +
O
OH O
OH
10. When the antibiotic emycin F is treated with aqueous acid, a molecular rear-
rangement occurs, producing a constitutional isomer known as emycin E. Write
a reasonable mechanism that accounts for this acid-catalyzed transformation.
OH O
O
O
O H+ HO
H2O
HO
HO emycin E
emycin F
11. The enzyme-catalyzed reaction of TPP ylide with 2 moles of pyruvic acid leads
to the product shown plus CO2. Propose a reasonable mechanism for this
transformation.
R' O O
enzyme CO2H
C OH C
2 H3C C H3 C C OH + CO2
S +
N
R O CH3

3 Biosynthesis
of Carbohydrates
and Amino Acids
I called it ignose, not knowing which carbohydrate it was. This name was turned down by
my editor. “God-nose” was not more successful, so in the end “hexuronic acid” was agreed
upon. Today the substance is called “ascorbic acid” and I will use this name.
—Albert Szent-Gyorgyi (Nobel Prize in Medicine, 1937)
We have already seen that some of the basic building blocks used in the biosynthesis
of natural products are amino acids such as phenylalanine, tyrosine, and others.
These and other crucial construction materials such as the acyl group in acetyl-CoA
are all ultimately derived from carbohydrates. In this chapter, we will present an
abbreviated overview of the components of carbohydrate structure and metabolism
sufficient for our purposes going forward, with a schematic flowchart showing how
carbohydrates and amino acids are modified, combined, and branched off in vari-
ous ways to yield the distinct set of biosynthetic pathways that will form the core of
the remainder of the text. We will finish the chapter with a brief, general review of
amino acid nomenclature and structure with emphasis on the key amino acids that
will be used throughout the remainder of the text.
WHAT MAKES A CARBOHYDRATE?
We know that plants make glucose (C6H12O6) by photosynthesis using light, water
(H2O), and carbon dioxide (CO2). Another way of looking at the formula for glucose
is C6(H2O)6, that is, six carbon atoms and six water molecules. Thus, glucose was
originally referred to as a hydrated form of carbon—a carbohydrate. But this is a
75

very general term since there are many different types of carbohydrate compounds.
Bioorganic Synthesis 76 One way to broadly classify carbohydrates is to identify them as either mono-(one),
di-(two), oligo-(a few) or poly-(many) saccharides. For example, glucose (C6H12O6)
cannot be broken down into simpler carbohydrates by simple hydrolysis, so it is clas-
sified as a monosaccharide, that is, a single, discrete carbohydrate compound. On
the other hand, the carbohydrate sucrose (C12H22O11) is classified as a disaccharide
since when it is subjected to aqueous hydrolysis, it yields two different monosaccha-
ride carbohydrates, namely glucose (C6H12O6) and fructose (C6H12O6). Noting that
glucose and fructose are different compounds but with the same molecular formula,
they must be related to one another either as stereoisomers or as constitutional iso-
mers, so further refinement of classification is needed.
Structurally speaking, most monosaccharide carbohydrates are simply poly-
hydroxyaldehydes (aldoses) or polyhydroxyketones (ketoses) which can be further
classified using a combination of aldo-or keto-prefixes along with suffixes such as
triose, tetrose, pentose, or hexose to designate the number of carbon atoms. Thus,
a ketopentose is a five-carbon ketone carbohydrate, while an aldotetrose is a four-
carbon aldehyde carbohydrate. In the hydrolysis of the disaccharide sucrose above,
the resulting monosaccharide carbohydrates produced (Fig. 3.1) turn out to be an
aldohexose (glucose) and a ketohexose (fructose).
Note that carbohydrates are further classified as D or L sugars according to
whether the last (lowermost) chiral –OH group in a Fischer projection of the mono-
saccharide is pointing to the right (D) or to the left (L). D-sugars are by far the pre-
dominant naturally occurring forms. Remember that a D or L designation is related
to configuration at a specific chirality center (like R or S) and not to the direction of
optical rotation as referred to by the familiar lower case d (+) or l (−).
The smallest carbohydrate possible is (+)-D-glyceraldehyde, the reduced form of
which is glycerine, the triol component from which triacylglycerol esters (triglycer-
ides) are formed from condensation with fatty acids (more on this later). Beyond this
simple D-aldotriose, we see in Fig. 3.2 the names and configurational relationships
of most of the important aldotetrose, aldopentose, and aldohexose carbohydrates.
CHO CH2OH
H OH O
HO H HO H
H3O+
C12H22O11 H OH +
H OH
sucrose H OH H OH
CH2OH CH2OH
glucose fructose
a D-aldohexose a D-ketohexose
FIGURE 3.1
Fischer projections of D-glucose and D-fructose obtained from sucrose hydrolysis.

CHO
77 Biosynthesis of Carbohydrates and Amino Acids

H OH
CH2OH
Glyceraldehyde
CHO CHO
H OH HO H
H OH H OH
CH2OH CH2OH
Erythrose Threose
CHO CHO CHO CHO
H OH HO H H OH HO H
H OH H OH HO H HO H
H OH H OH H OH H OH
CH2OH CH2OH CH2OH CH2OH
Ribose Arabinose Xylose Lyxose
CHO CHO CHO CHO CHO CHO CHO CHO

H OH HO H H OH HO H H OH HO H H OH HO H
H OH H OH HO H HO H H OH H OH HO H HO H
H OH H OH H OH H OH HO H HO H HO H HO H
H OH H OH H OH H OH H OH H OH H OH H OH
CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH
Allose Altrose Glucose Mannose Gulose Idose Galactose Talose
FIGURE 3.2
The configurational relationships of the aldose carbohydrates.
CYCLIC HEMIACETALS AND ANOMERS
We noted previously (Chapter 1, Fig. 1.49) that aldehydes and ketones containing

one or more –OH groups elsewhere in the molecule can undergo intramolecular
hemiacetal formation and that the equilibrium favors the cyclic hemiacetal if the
ring is 5-or 6-membered. This is especially important in carbohydrates; most any
sugar molecule with a hydroxyl O atom four or five atoms distant from the carbonyl
carbon will prefer to exist in cyclic hemiacetal form. The resulting 5-membered ring
hemicacetals are called furanose forms (from the O-heterocycle furan), while the
6-membered rings are called pyranose forms (from the O-heterocycle pyran); when
multiple hydroxyl groups are located relative to the carbonyl in such a way as to
allow formation of either type, the predominant form will depend on the relative
isomeric relationships of the hydroxyls to one another on the resulting ring system.
The relationship of open-chain versus furanose and pyranose forms for D-glucose in
aqueous solution is illustrated in Fig. 3.3.
Note that in open-chain form, glucose has only four chirality centers, but in cyclic
form, an additional chirality center is present due to formation of the hemiacetal func-
tional group. Since the carbonyl carbon can present either of its two prochiral faces
to the attacking nucleophilic –OH group, the resulting hemiacetal is produced as a
mixture of stereoisomers at the hemiacetal carbon; this unique chiral carbon in the

O
anomeric 1CH
anomeric
H carbon
5 H H 2 OH H carbon
HO O 5 OH
HO
4 HO H 1 HO 3 H HO
HO
O
5 4 HO H 1
H 3 H 4 OH
2 OH
H 3 2 H
H OH H OH
H OH
α-anomer of furanose form: CH2OH β-anomer of furanose form:
α-D-glucofuranose β-D-glucofuranose
(trace) D-glucose (~0.01%) (trace)
O
anomeric 1CH
H OH H OH anomeric
4 H 6 carbon H 2 OH 4 6 carbon
5 O 5 H O
HO HO 3 H HO
HO H HO OH
3 H 2 OH 1 H 4 OH 3 H 2 OH 1
H OH 6 H H
H 5 OH
α-anomer of pyranose form: β-anomer of pyranose form:
CH2OH
α-D-glucopyranose β-D-glucopyranose
(32%) (68%)
FIGURE 3.3
Interconversion and equilibrium composition of glucose forms in aqueous solution: open chain vs.
furanose and pyranose α and β anomers.
cyclic carbohydrate is known as the anomeric carbon and the resulting stereoisomers
are a special class of diastereomers called anomers. The anomeric carbon is always an
easy one to spot in cyclic carbohydrates; it will be the only carbon atom bonded to two
oxygens; all other carbons will be bonded to only one oxygen. In chair representations
of glucose, the so-called α-anomer will have the anomeric hydroxyl group in an axial
position on the ring (a for axial as well as a for alpha is an easy way to remember this)
when all other groups on the ring are equatorial, while the β-anomer will have the ano-
meric hydroxyl in an equatorial position on the ring when all other groups are equato-
rial. In the case of glucose, the predominant equilibrium form in aqueous solution is
the pyranose form, with the β-anomer (68%) favored over the α-anomer (32%); the fu-
ranose and open-chain polyhydroxyaldehyde forms are present in only trace amounts.
The process of interconversion of anomers to their equilibrium composition in solution
is known as mutarotation and occurs for all aldopentoses and aldohexoses.
Technically, α and β anomeric assignments are based on comparison of the R/S
configurational assignments at the anomeric carbon and the corresponding Fischer
projection’s lowermost chiral carbon. If the two assignments are the same, the anomer
is β; if different, the anomer is designated α. Most D-sugars have the R configuration at
the lowermost chiral carbon, and an anomeric equatorial –OH will correspond to the
R configuration for most aldopyranose forms drawn in their most stable chair confor-
mation; thus, the equatorial anomer will usually be β for D-aldopyranose sugars, while
the axial anomer will be α. But keep in mind that the axial and equatorial positions of
chair conformations are interconvertible via chair-chair “ring flipping,” so the above
analysis only holds for single conformations of the most stable chair form where only

H H

R OH R OH
HO OH OH
HO
H H O
H OH H
OH O OH
OH
CH2OH
O H H
HO H R OH R OH
HO O HO O
H OH H S H R OH
H OH H
H R OH α-anomer β-anomer
OH OH OH
CH2OH OH
D-fructose
OH
OH
H S H R
α-anomer β-anomer O OH
O OH
H R OH H R OH
HO HO
OH H OH H
FIGURE 3.4
Chair representations of α and β-D-fructopyranose.
the position of the pyranose anomeric –OH is changeable relative to all the others. To
see how this applies, consider the pyranose forms of fructose, by far the most signifi-
cant ketohexose carbohydrate, as shown in Fig. 3.4.
As we can see, the β-anomer (R,R) may have the anomeric –OH either axial or
equatorial, depending on which chair form is represented. The more stable form
of most pyranose carbohydrates will correspond to the chair form that maximizes
the number of equatorial substituents versus axial substituents. That’s an easy call
with D-glucopyranose, since the conformation that has all groups equatorial (in-
cluding the anomeric –OH) equatorial is the β-anomer. It’s a closer call in the case
of D-fructopyranose; for the β-anomer, the conformation with the anomeric –OH
group axial has two axial and three equatorial substituents; with the anomeric –OH
equatorial, there are three axial and two equatorial substituents, so the former is
probably slightly more stable. It is exactly the opposite case for the α-anomer. Thus,
unlike D-glucopyranose, the most stable chair form of the β-anomer will have the
anomeric –OH axial rather than equatorial, while the α-anomer will have its ano-
meric –OH equatorial in the more stable chair form.
The above discussion is less important for furanose forms since they lack the ob-
vious axial and equatorial chair positions characteristic of pyranose forms. Haworth
projections can be useful for representation of both furanose and pyranose forms,
as shown for D-fructose and D-glucose in Fig. 3.5, though their use is more common
for furanose forms. For D-sugars, converting a Fischer projection into a Haworth
projection is straightforward. With the ring O-atom projecting toward the back of
the structure and the anomeric carbon on the right side, any atom or group pointing

CH2OH
Bioorganic Synthesis 80 O
OH
HO H HOH2C OH HOH2C H
O
H OH CH2OH
H OH vs. O OH
H CH2OH H
H OH HO H
CH2OH OH H
D-fructose Haworth projection of Envelope conformation of

β-D-fructofuranose β-D-fructofuranose
O
CH
CH2OH H OH
H OH
H O OH HO
HO H H HO
OH H vs. HO OH
H OH OH H H OH
H OH H OH H H
CH2OH Haworth projection of Chair conformation of

D-glucose β-D-glucopyranose β-D-glucopyranose
FIGURE 3.5
Haworth vs. conformational representations of β-D-fructofuranose and β-D-glucopyranose.
to the right in the open-chain Fischer projection points down in the corresponding
Haworth projection; if pointing to the left in the Fischer projection, it points up in
the Haworth projection.
Significantly, the terminal –CH2OH group of D-carbohydrates (located at the
bottom of the Fischer projection) is always pointing up in the Haworth projection
(unless this group’s –OH is used to form the hemiacetal). Using the terminal –CH2OH
group as a reference point (Fig. 3.5), for most Haworth projections of D-furanose or
D-pyranose forms, the α-anomer will have the anomeric –OH group pointing down
(trans to the terminal –CH2OH), while the β-anomer will have the anomeric –OH
pointing up (cis to the terminal –CH2OH). Note how the all-trans relationship of
the ring substituents in β-D-glucopyranose is emphasized by the Haworth projec-
tion compared to the chair representation. The latter is more correct from a con-
formational point of view, while the former helps to clarify the stereochemical
relationships.
C-2 EPIMERS AND ENEDIOLS—S IMPLE CONVERSION

OF ONE CARBOHYDRATE INTO ANOTHER
We know that protons attached to the α-carbon of carbonyl compounds are es-
pecially acidic and may be removed by bases to form enolate ions. When such
α-protons are located at a chirality center, proton removal followed by reproton-
ation of the resulting enolate leads to racemization of the chirality center, since the
intermediate enolate is only prochiral and so will produce one enantiomer from
protonation at its Si face and the other from protonation at its Re face. If this process
occurs in the open-chain form of aldose sugars, the chirality center at C-2 becomes
scrambled, resulting in the formation of diastereomeric sugars. In systems such as


O
1CH
HO H 2 OH
HO H
H OH
H OH
CH2OH
D-glucose
O O
1CH O H O H 1CH
H 2 OH H OH HO 2 H
HO H OH OH
HO H HO H
HO H HO H
H OH vs. H OH
H OH H OH
H OH H OH
H OH H OH
CH2OH CH2OH
D-glucose CH2OH enolate ion CH2OH
D-mannose
FIGURE 3.6
Base-catalyzed conversion of D-glucose into C-2 epimers: D-glucose and D-mannose.
this, the process is referred to as epimerization, and in the case of aldose carbohy-
drates, the two disastereomers formed are related to one another as so-called C-2
epimers as illustrated in Fig. 3.6. Thus, under base-catalyzed conditions, D-glucose
is epimerized to a mixture of D-glucose and D-mannose.
If the intermediate enolate ion protonates at oxygen instead of carbon, the result-
ing enol form of the carbonyl group has an –OH group at each end of the double
bond, making it a so-called enediol. These unusual enol forms, when reverting back
to carbonyl form, can produce either an aldehyde or a ketone, depending only on
which of the two enediol carbons acts as the proton acceptor (Fig. 3.7).
In practice, treatment of glucose with aqueous base leads to a mixture that con-
sists of about 65% glucose, 32% fructose, and 3% mannose. A mixture with the same
composition will also be produced starting from mannose, since mannose and glu-
cose both form the same enediol intermediate under these conditions. Similarly,
other pairs of D-aldohexoses may be epimerized and/or converted to give the three
remaining D-ketose sugars: psicose, sorbose, and tagatose, while two D-aldopentose
pairs each yield the two possible corresponding D-ketopentose sugars. Thus, either
ribose or arabinose may yield the ketopentose ribulose, while either xylose or lyxose
will give the ketopentose xylulose as in Fig. 3.8.
OTHER IMPORTANT MONOSACCHARIDES:

DEOXY AND AMINO SUGARS
Not all carbohydrates are formulated as shown for the aldose sugars. Two impor-
tant examples are D-2-deoxyribose, a deoxy sugar, and D-glucosamine, an amino

HO H O H
OH
HO H
H OH
H OH
CH2OH
HO
O H OH
H O H HO H
CH CH2OH
OH
CHOH OH O H O
HO H
HO H HO H HO H HO H
vs.
H OH H OH H OH H OH
H OH H OH H OH H OH
CH2OH CH2OH enediol form CH2OH CH2OH
D-glucose + D-mannose D-fructose
FIGURE 3.7
Base-catalyzed epimerization of D-glucose vs. conversion to D-fructose via the enediol.
CHO CHO CH2OH

H OH HO H O
HO-(aq) HO-(aq)
H OH H OH H OH
H OH H OH H OH
CH2OH CH2OH CH2OH
Ribose Arabinose Ribulose
CHO CH2OH
CHO
HO H O
H OH HO-(aq)
HO-(aq)
HO H HO H
HO H
H OH H OH
H OH
CH2OH CH2OH CH2OH
Xylose Lyxose Xylulose
FIGURE 3.8
Base-catalyzed interconversion of D-aldopentose and D-ketopentose carbohydrates.
sugar (Fig. 3.9). The prefix “deoxy” is related to the missing –OH group at C-2 in
what would otherwise be the structure of D-ribose. This simple deoxy designation
is widely used in natural products chemistry, providing a consistent and useful no-
menclature that uses familiar names (and structures) of known compounds rather
than deriving yet another new common name. Similarly, D-glucosamine, also
known as D-2-deoxy-2-aminoglucose, relates the name and structure of a known
compound to a new one.
Deoxyribose may be familiar as an essential component in DNA (deoxyribo-
nucleic acid) and as one of the features that distinguish DNA from RNA (ribo-
nucleic acid, which employs ribose rather than 2-deoxyribose). Glucosamine is a

H H

O H
O OH
HO H
H H
2 HO 2
OH OH OH
CHO α-D-2-deoxyribopyranose (40%) OH
H
H 2 H H
O H
HO O
H OH H
H 2 OH
HO 2
H OH
OH H OH
CH2OH β-D-2-deoxyribopyranose (35%)
D-2-deoxyribose HO H HO
O OH
O
H H
H 2 OH H 2
H
OH OH
α-D-2-deoxyribofuranose (13%) β-D-2-deoxyribofuranose (12%)
CHO
2 H OH
H NH2 H OH
HO H H O H O
HO HO
H OH HO H HO OH
H 2 H 2
H OH NH2 NH2
H OH H H
CH2OH
α-D-2-deoxy-2-amino- β-D-2-deoxy-2-amino-
D-glucosamine
glucopyranose (63%) glucopyranose (37%)
FIGURE 3.9
D-2-deoxyribose, D-glucosamine, and their cyclic hemiacetal forms.
common dietary supplement (usually as the hydrochloride salt or sulphate ester)

used by many who suffer from osteoarthritis. More importantly, glucosamine is the
biosynthetic precursor of all other nitrogen-containing sugars. For dietary purposes,
glucosamine is obtained commercially from the hydrolysis of a crustacean exoskel-
eton material known as chitin, a biopolymer in which glucosamine, as its N-acetyl
derivative, is the principal component.
THE SIGNIFICANCE OF THE ANOMERIC

CARBON: GLYCOSIDE FORMATION
Recall that any hemiacetal may be considered an intermediate in the formation of a

corresponding acetal under acid catalyzed conditions if the protonated –OH group
of the hemiacetal is lost as water and the resulting resonance-stabilized carbocation
is trapped by an available nucleophilic alcohol –OH group. Such acetals, when de-
rived from the cyclic hemiacetals of carbohydrates, are known as O-glycosides. The
nucleophilic alcohol component, ROH, may be any simple alcohol such as methanol
or ethanol or a more complex alkanol or cycloalkanol. The general process is illus-
trated in Fig. 3.10.
This is an example of a very general and extremely important process: namely,
the linking of carbohydrate structures to other molecules by nucleophilic trap-
ping at the anomeric carbon of the carbohydrate. This is how disaccharides and

H OH
H OH H OH
HO
HO H+ HO HO
HO HO HO
H HO H + H2O
H H HO H
OH OH H
H OH
OH H O H H
α-D-glucopyranose H
means undefined
H OH stereochemistry H OH H OH
HO - H+ HO H
HO
HO HO HO
HO OR HO OR HO
H
H
OH H
OH OH H HO-R
H H H H
resonance stabilized
a mixture of α- and β-
carbocation
glucopyranosides
FIGURE 3.10
Acid-catalyzed formation of a glucopyranoside mixture from glucose and an alcohol, ROH.
OH a α-(1,4)-glycoside linkage
O
HO
HO H OH
OH
OH O O
OH - H2O
HO OH
O 4
HO O OH
HO 1 OH HO maltose (from starch) H
OH HO OH
H OH
H OH a β-(1,4)-glycoside linkage
- H2O
2 x D-glucopyranose OH
O
HO
HO O O
OH HO OH
H OH
cellobiose (from cellulose) H
FIGURE 3.11
Structures of disaccharides derived from glucose: maltose and cellobiose.
polysaccharides are formed from monosaccharides. Since the resulting products

are acetals, we can also now see how and why di-and polysaccharides are hydro-
lyzed to monosaccharides: the reverse process is simply acetal hydrolysis.
Unfortunately, the simple scheme in Fig. 3.10 is not one that is stereospecific.
Glycosides, like their hemiacetal precursors, can be formed as either α or β ano-
mers, and under such conditions we would also expect the carbohydrate to exist as a
mixture of pyranose and furanose forms, thus leading to complex mixtures. For lab-
oratory synthesis of glycosides, there are a number of ways to overcome these limi-
tations, though we will not address those methods here. We will be more interested
in seeing how biological systems overcome these limitations to produce different
glycosides stereospecifically. But before we look into how nature manages this pro-
cess, let’s learn a bit more about some important examples of glycoside structures.
Figure 3.11 illustrates in general terms how two glucose molecules come together
by linking the –OH group at C-4 of one glucose to C-1 (the anomeric carbon) of a

OH

O
a β-(1,4)-glycoside linkage HO
OH OH HO 1 H
OH glucose OH α
O
4 HO O
HO 1 O O O β
H HO 2
galactose OH HO OH
H OH H
glucose OH
H fructose OH H
lactose (milk sugar)
sucrose (table sugar)
FIGURE 3.12
Structures of the disaccharides lactose and sucrose.
second glucose. Since the C-4 –OH can attack at either the top face or the bottom
face at C-1, both α and β glycoside linkages are possible. When the bond to C-1 is
axial, the disaccharide formed is maltose, a product obtained by hydrolysis of starch
and whose glycoside bond is designated an α-(1,4)-glycoside linkage, making malt-
ose an α-glycoside. Alternatively, if the resulting glycoside bond at C-1 is equatorial,
the disaccharide product is cellobiose, a hydrolysis product obtained from cellulose.
Here the glycoside bond is designated as a β-(1,4)-glycoside linkage, making cel-
lobiose a β-glycoside.
Other O-glycosides of note are the disaccharides lactose and sucrose (Fig. 3.12).
These disaccharides both contain a single glucose component joined to a second
monosaccharide that is different in each case. Thus, lactose, also known as milk
sugar, is composed of a galactose unit linked at its anomeric carbon (C-1) to the
C-4 hydroxyl of a glucose unit via a β-(1,4) linkage. In humans, a deficiency in the
enzyme lactase, which facilitates the hydrolysis and breakdown of lactose, leads
to a condition known as lactose intolerance, a genetic trait that leads to gastric
upset when milk products are ingested, though the condition is easily treated
with over-the-counter lactase formulations. Sucrose, better known as table sugar,
is composed of glucose and fructose, but it differs from other disaccharides in
that the glycoside linkage between the two individual sugar components involves
the anomeric carbon of both units. Thus, the unique glycoside linkage of sucrose
is designated a 1α, 2β glycoside linkage, since it is α with respect to the glucose
anomeric carbon (at C-1) and β with respect to the fructose anomeric carbon
(at C-2).
Polysaccharides are examples of high molecular weight carbohydrate biopoly-
mers. Unlike some disaccharides, most polysaccharides contain only a single type
of carbohydrate as a repeating unit. Two important examples are amylose, one of
two glucose-derived components of starch, and cellulose, the main structural ma-
terial of many plants and also a polymer of glucose (cotton is essentially pure cel-
lulose). These two very different biomaterials (one is a key food source while the

α-(1,4)-glycoside linkages
OH
O
O
HO H OH
OH
O O
HO H OH
OH OH 4
4 amylose O O
O
HO HO 1 H
HO 1 OH
OH
OH O
glucose H
(many)
OH β-(1,4)-glycoside linkages
OH
O
O OH
HO O O
OH HO O O
H OH HO O
cellolose H OH
H
FIGURE 3.13
Structures of the glucose polysaccharides amylose and cellulose.
other is an indigestible yet valuable source of tough, durable fibers) differ mainly
in the type of O-glycoside bond linking the individual glucose units. Thus, amylose
utilizes α-(1,4)-glycoside linkages while cellulose utilizes β-(1,4)-glycoside linkages
(Fig. 3.13). That is a relatively small difference, chemically speaking, for two such
very dissimilar materials. The polysaccharide chitin, mentioned earlier as a compo-
nent of crustacean exoskeletons, also uses β-(1,4)-glycoside linkages, but the repeat-
ing unit is N-acetylglucosamine rather than glucose.
Other glycosides in which a carbohydrate is linked to various types of organic
alcohols or amines are quite abundant in nature, and many are important biological
or medicinal compounds. When the carbohydrate component of such compounds
is released from the glycoside linkage by hydrolysis, the residual organic structure
is known as the aglycone of the original glycoside structure. For example, salicin is
a component of willow bark, whose use in the treatment of fever, pain, and inflam-
mation dates to as far back as 400 BC. When ingested, hydrolysis of the glycoside
linkage in the stomach affords the aglycone, salicyl alcohol (Fig. 3.14). Metabolic
oxidation of this benzylic alcohol affords salicylic acid, the active principal in acetyl
salicylic acid (aspirin).
The O-glycoside daunorubicin, a powerful cancer chemotherapy agent isolated
from Streptomyces peucetius, contains a unique amino sugar called daunosamine
(Fig. 3.15) which is essential for the bioactivity of the compound. In other glyco-
sides, the sugar anomeric carbon may be linked to N-or C-atoms of the organic
aglycone. Adenosine, a familiar nucleic acid component of RNA, is an N-glycoside
of β-D-ribfuranose and the heterocyclic base adenine. Mangiferin, an antioxidant
found principally in mangoes but also widely distributed in other higher plants, is a

OH

glucose +
OH OH
O CO2H
HO H3O + oxidation
HO O HO HO
OH
H
salicin salicyl alcohol salicylic acid
FIGURE 3.14
The O-glycoside salicin, its aglycone salicyl alcohol, and the oxidation product salicylic acid.
NH2
O OH O
N
N
OH N
N OH
HO HO O OH
O O
OCH3 O OH O HO
H H HO OH
O H H OH
H OH O
H 2N OH OH
OH daunorubicin adenosine mangiferin
FIGURE 3.15
An O-glycoside (daunorubicin), an N-glycoside (adenosine), and a C-glycoside (mangiferin).
glucose C-glycoside connected to a xanthone-based aglycone and is a constituent of

some folk medicines.
A large and important class of antibiotics, the aminoglycosides, are so-called not
because they are necessarily N-glycosides, but rather because they contain one or
more amino sugars. Examples include streptomycin, the first antibiotic found to be
effective against tuberculosis, and neomycin, widely used in many over-the-counter
topical antibiotic ointments and creams. By contrast, the relatively rare thiogly-
cosides are specifically S-glycosides, such as the white mustard seed component
sinalbin, a well-known example found in many wild plants. All these are shown in
Fig. 3.16 using conventional organic stereochemical structural representations
rather than conformational or Haworth diagrams.
In many glycosidic natural products, we will be mainly interested in how the
aglycone portion is biosynthesized, since the carbohydrate component is usually
added to the aglycone by glycosylation at a late stage. But before moving on, let’s
take a look at how glycoside linkages are usually formed biosynthetically.
UDP-S UGARS AND GLYCOSIDE FORMATION: S N 2

CHEMISTRY AT WORK
As mentioned earlier, acid-catalyzed reaction of alcohols with carbohydrate hemi-

acetals to give glycoside (acetal) derivatives can be difficult to control in terms of
pyranose versus furanose and α versus β anomeric forms. Nature takes a different
approach to glycoside formation by making use of carbohydrate derivatives known

NH2
Bioorganic Synthesis 88 H2N NH H2N NH H2 N NH2 OH

O
OH
O HN NH HO O OH O
O O
O NH2 O S N
H HO O
HO O OH HO
S
O OH O
O HO
O O OH
OH H2N
H3CHN HO OH
OH HO NH2 OH
HO
OH OH
streptomycin neomycin sinalbin
FIGURE 3.16
The aminoglycoside antibiotics streptomycin and neomycin and the S-glycoside sinalbin.
OH
O
O uracil
HO NH OH
HO H
OH O O
O
α-D-glucopyranose O P O P O N O HO
O HO H
O- O- β-D-ribofuranose OH
O-UDP
OH OH UDP-glucose
uridine diphosophate (UDP)
FIGURE 3.17
Structural components of UDP-glucose.
as uridine diphosphosugars or simply UDP-sugars. The most important of these

is UDP-glucose (Fig. 3.17). This seemingly complex structure is relatively easy to
dissect into its various components: an α-D-glucopyranose linked by a diphosphate
bridge to the C-5 hydroxyl group of D-ribofuranose which in turn is connected via a
β-N-glycoside linkage to a molecule of uracil (uracil + β-D-ribofuranose = uridine,
an RNA base).
The diphosphate ester linkage is employed here to activate the anomeric O-atom
of the glucose component toward nucleophilic displacement in much the same way
that an alcohol –OH group can be turned into a good leaving group by conversion
to a tosylate or mesylate ester. When a nucleophile (Nu–H) attacks at the activated
anomeric center, an SN2 reaction displaces the UDP component as a leaving group,
leading to inversion of configuration at the anomeric carbon and formation of a
β-glycoside derivative (Fig. 3.18). Many O-, N-, and C-glycosides found in nature
are β-anomers for this reason.
When α-linkages are required, a nucleophilic functional group in an enzyme
mediating the process may form a β-linkage by initial nucleophilic SN2 attack;
this group may then be displaced by an external nucleophile (Nu–H) in a second
SN2 process (Fig. 3.19). Inversion followed by inversion leads to a net retention of

OH

OH
O H-Nu
HO SN 2 O
HO H HO
Nu + UDP-OH
OH α inversion HO
O-UDP OH β
H
a β-D-glucopyranoside
FIGURE 3.18
Conversion of UDP-glucose to a β-D-glucopyranoside via SN2 reaction with a nucleophile, Nu–H.
OH R2N OH
O
HO SN2 O
HO H HO R2N
α inversion HO
HO + UDP-OH
HO β
O-UDP
H
H B SN2
inversion Nu- H :B
OH R2N
O
HO
HO H
α
HO
Nu H B
FIGURE 3.19
α-D-glycoside formation via enzyme-mediated sequential SN2 reactions.
configuration and formation of an α-linkage at the anomeric center of the resulting

glycoside.
In some instances, certain important UDP-carbohydrates are biosynthesized
from other UDP-carbohydrates. For example, the UDP derivative of the aldopen-
tose xylose, an important building block component of plant cell wall structure,
is synthesized from UDP-glucose using some of the biological transformations in-
troduced in Chapter 2. The steps involved are NAD+ oxidation of the C-6 hydroxyl
group of UDP-glucose to give the corresponding UDP-glucaronic acid. Further
oxidation at C-4 gives a β-keto acid which decarboxylates to yield the correspond-
ing ketone. The sequence is completed by NADH reduction of the ketone to give the
pyranose form of UDP-xylose. Hydrolysis of the phosphate linkage will yield free
xylose, as shown in Fig. 3.20. The name xylose is derived from xylos, the Greek word
for wood, from which xylose was first isolated.
Another example is found in the synthesis of the O-methyl sugar cladinose, one
of two unusual carbohydrate O-glycoside components connected to the core struc-
ture of erythromycin, a macrolide antibiotic whose aglycone biosynthesis we will
explore further in Chapter 5. The sequence of reactions involved in cladinose syn-
thesis (Fig. 3.21) again begins with glucose, but linked not to a uridine diphosphate
(UDP) group, but rather to thymidine diphosphate group (TDP, a β-N-glycoside
of thymine and 2-deoxyribose). TDP-glucose is first oxidized at C-4 by NAD+; the

OH
HO2C O HO2C
O 2 NAD+
HO O NAD + O
HO H HO
HO H HO H
HO HO HO
O-UDP O-UDP O-UDP
UDP-glucose UDP-glucuronic acid
- CO2
O O
HO H3O + O NADH O
HO
HO HO H H
HO
HO OH HO HO
D-xylopyranose O-UDP O-UDP
UDP-xylose
CHO
H OH HO
O
HO H HO H OH
H OH
CH2OH H OH
D-xylose D-xylofuranose
FIGURE 3.20
Biosynthesis of UDP-xylose from UDP-glucose.
resulting β-hydroxyketone then undergoes a base-induced dehydration to give

the corresponding α,β-unsaturated ketone (similar to dehydration of aldol prod-
ucts); conjugate (1,4) reduction by NADH and keto-enol tautomerization gives the
α-methylketone TDP-6-deoxy-D-xylo-4-hexulose. Subsequent dehydration (loss
of the C-2 hydroxyl) gives a second α,β-unsaturated ketone which then undergoes
epimerization at C-5 (α to the ketone carbonyl) followed by conjugate addition of
hydride from NADH with concurrent methylation of the intermediate enolate by
S-adenosylmethione (SAM). NADH reduction of the ketone, SAM methylation of
the C-3 hydroxyl, and hydrolysis completes the sequence to give cladinose.
Now that we have a reasonable grasp of carbohydrate nomenclature and struc-
ture, let’s take a bit of time to look at the chemistry involved in the breaking down
of glucose into various organic fragments, some of which constitute the essential
building blocks for the biosynthesis of all other primary and secondary metabolite
structures.
ORGANIC REACTIONS IN CARBOHYDRATE

CHEMISTRY: OVERVIEW OF GLUCOSE METABOLISM
Glucose is broken down as a fuel source for energy production in living systems in
two important ways, both of which will be of interest to us, though mainly from the
point of view of the mechanistic aspects of the organic chemistry of the processes as
well as the use of the products produced by these pathways. The more important of
these two processes is known as glycolysis, but we will also be interested in a second

H+
OH O O :H
+
HO OH H HO
HO
OH NAD+ H
- H2O
O at C-4 O
HO O :B
HO HO
O-TDP O-TDP O-TDP
TDP-glucose
conjugate
NADH reduction
O OH
O O
B: HO CH3 HO CH3
HO CH3 HO CH3
- H2O H enol-keto
epimerize
O O
O at C-5 O HO HO
H: H+ O-TDP O-TDP
O-TDP O-TDP
TDP-6-deoxy-D-xylo-4-hexulose
S
CH3 O OH
O
H3C H3C
HO CH3 CH3 CH3
NADH i. NADH H3CO
HO
conjugate O ii. SAM O
addition O
iii. H2O
then SAM
O-TDP O-TDP OH
cladinose
FIGURE 3.21
Biosynthesis of cladinose, a carbohydrate component of the antibiotic erythromycin.

route to glucose metabolism known as the pentose phosphate pathway. To keep

Bioorganic Synthesis 92 our studies focused on the organic chemistry of these processes and the uses of their
products, we will defer to specialists in biochemistry for detailed treatments of their
enzymology, pathway regulation, and thermochemical aspects. To begin to sort all
this out, let’s take it one pathway at a time, starting with glycolysis.
GLYCOLYSIS: A 10-S TEP PROGRAM
While glycolysis ultimately converts glucose to pyruvic acid, the overall transfor-
mation occurs through a complex series of individual enzyme-mediated reactions
which we can now examine one by one. The first step in glycolysis is essentially
an esterification reaction that converts α-glucopyranose to glucose-6-phosphate
(Fig. 3.22) by reaction with ATP. A basic site of the meditating enzyme hexokinase
is involved in the deprotonation of the alcohol function, while Mg+2 (not shown)
coordinates with ATP to facilitate the phosphorylation. We will continue to use our
previous abbreviations of ROPPP for triphosphates, ROPP for diphosphates, and
ROP for monophosphate derivatives of alcohols for simplicity. This reaction is very
similar to the conversion of an alcohol to an acetate ester by reaction with acetic
anhydride.
The next step involves conversion of glucose-
6-
phosphate to fructose-
6-
phosphate. This phosphoglucose isomerase-catalyzed process is basically a sequence
that involves ring opening of the pyranose form to the open-chain aldohexose form,
followed by keto-enol tautomerization from aldohexose to enediol to the ketohexose
form, giving fructose-6-phosphate which then cyclizes to its α-furanose hemiacetal
form (Fig. 3.23).
The third step, catalyzed by phosphofructokinase, is similar to the first in that it
again involves phosphorylation of a primary alcohol by ATP, in this case convert-
ing fructose-6-phosphate to fructose-1,6-diphosphate, as shown in Fig. 3.24. This
represents the standard formulation of this step, though some studies indicate that
the actual product is the β-anomer rather than the α-anomer, though this distinc-
tion has little bearing on the very important chemistry that follows, since either
ADP P
OH
OH OH OP
O O
HO HO
HO H HO H
HO ATP HO
ADP
OH OH
α-glucopyranose α-glucose-6-P
FIGURE 3.22
Glycolysis Step 1: Conversion of α-glucopyranose to α-glucose-6-phosphate.

OP OP OP

O OH OH
HO HO HO
HO H HO H H
HO
HO HO HO
OH O OH
α-glucose-6-P open-chain aldose form enediol form
PO OH OP OP
O OH
H OH O HO
HO OH HO
H OH HO
OH O
OH H OH
α-fructose-6-P cyclized α-furanose hemiacetal form open-chain ketose form
FIGURE 3.23
Glycolysis Step 2: Isomerization of α-glucose-6-phosphate to α-fructose-6-phosphate.
ADP P
OH
OH
PO OH PO OP
O O
H OH H OH
H OH H OH
OH H ATP ADP OH H
α-fructose-6-P α-fructose-1,6-PP
FIGURE 3.24
Glycolysis Step 3: Phosphorylation of α-fructose-6-phosphate to α-fructose-1,6-diphosphate.
anomeric form may be considered to be in equilibrium with the open-chain ketose

form which participates in the next step.
For the fourth step in glycolysis, the open-chain ketohexose form of fructose
1,6-diphosphate undergoes what is essentially a retroaldol addition reaction cata-
lyzed by fructose diphosphate aldolase. This key step is the only one in which a C–C
bond is broken in the glycolysis pathway and therefore may be considered the most
important step in the breakdown of glucose from a structural degradation perspec-
tive, since all previous and subsequent steps mainly involve simple condensations,
isomerizations, or oxidations. The retroaldol addition reaction involves initial con-
version of the fructose 1,6-diphosphate carbonyl group into an imine link using
an enzyme side chain amino group, thereby holding the carbohydrate chain in a
favorable position relative to other basic and acidic enzyme functions that facili-
tate the subsequent cleavage reaction, as shown in Fig. 3.25. The retroaldol process
releases the first three-carbon carbonyl product, glyceraldehyde-3-phosphate, as
well as an enzyme-bound three-carbon enamine component which undergoes sub-
sequent isomerization and hydrolysis of the resulting imine linkage to regenerate

H CH2OP
NH2 CH2OP
N
PO OP O
O HO H :B
H OH HO H -H2O
H O H
H OH H OH imine
formation H OH
OH H H OH
CH2OP
α-fructose-1,6-PP CH2OP
fructose-1,6-PP retroaldol
H CH2OP H CH2OP
N C N C
CH2OH HO C H H-B
imine enamine
H2O
hydrolysis
+
H O
CH2OP C
NH2 + O C H C OH
CH2OH CH2OP
dihydroxyacetone-P glyceraldehyde-3-P
FIGURE 3.25
Glycolysis Step 4: Retro aldol cleavage of fructose-1,6-diphosphate to glyceraldehyde-3-phosphate
and dihydroxyacetone phosphate.
H O
CH2OP CH2OH H C OH C
keto-enol enol-keto
O C rotate C O C OH H C OH
CH2OH CH2OP CH2OP CH2OP
dihydroxyacetone-P enediol intermediate glyceraldehyde-3-P
FIGURE 3.26
Glycolysis Step 5: Conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate.
the enzymatic amino group and release the second three-carbon carbonyl product,
dihydroxyacetone phosphate.
The fifth step in the sequence, catalyzed by triose phosphate isomerase, converts
dihydroxyacetone phosphate to glyceraldehyde 3-phosphate via the correspond-
ing intermediate enediol, similar to the process seen in step 2. Thus, at this point
in glycolysis, we note that one molecule of glucose has been broken down into two
molecules of glyceraldehyde 3-phosphate (Fig. 3.26).
In the sixth step, glyceraldehyde-3-phosphate undergoes nucleophilic attack
by a glyceraldehyde phosphate dehydrogenase thiol group to give a hemithioac-
etal intermediate which then undergoes oxidation by NAD+ to give to the cor-
responding thioester (Fig. 3.27). Recall that thioesters are more reactive than


H O OH O OPO(OH)2
C HS
H C S C S
H C OH
H C OH H C OH
CH2OP
CH2OP NAD+ NADH/H + CH2OP
glyceraldehyde-3-P hemithioacetal thioester
Pi
O H B
OP OP
C
O C S
H C OH
H C OH
CH2OP
CH2OP
1,3-diphosphoglycerate
FIGURE 3.27
Glycolysis Step 6: Oxidation and phosphorylation of glyceraldehyde-3-phosphate.
O
O O P OH HO-ADP O
C OH O
OH C
H C OH H C OH + HO P O-ADP
= ATP
CH2OP HO
CH2OP
1,3-diphosphoglycerate 3-phosphoglycerate
FIGURE 3.28
Glycolysis Step 7: Conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate and ATP.
simple esters toward nucleophilic acyl substitution. Thus, we see inorganic phos-
phate ion (PO43– or Pi) acting as a nucleophile to attack the thioester carbonyl,
leading to a tetrahedral intermediate that expels the enzymatic thiol component,
giving 1,3-diphosphoglycerate: a phosphate ester at C-3 and an acylphosphate
at C-1. The acylphosphate linkage is actually a mixed anhydride of a carboxylic
acid and phosphoric acid, and as we saw earlier in Chapter 2, such mixed an-
hydrides are similar in reactivity to acid chlorides in terms of nucleophilic acyl
substitutions reactions.
This enhanced reactivity of the acyl phosphate is then exploited in the seventh step
of glycolysis for the transfer of the phosphate group from 1,3-diphosphoglycerate
to ADP via a simple nucleophilic substitution catalyzed by phosphoglycerate kinase.
This step generates 3-phosphoglycerate as well as giving back ATP in the process
(Fig. 3.28). This important step in glycolysis is an example of substrate-level phos-
phorylation, as it directly produces energy for the cell by generating ATP through
transfer of phosphate from a phosphorylated substrate intermediate directly to
ADP (again, note that all acids in these schemes are shown in nonionized form for
simplicity’s sake).

O OH
O P N C O P N
OH N OH
C H C OP C
H C O H CH2O H C OP
P H
CH2OP :B CH2OH :B
B
3-phosphoglycerate 2-phosphoglycerate
FIGURE 3.29
Glycolysis Step 8: Isomerization of 3-phosphoglycerate to 2-phosphoglycerate.
O OH
O C O OH
OH
C C
C OP - H2O
H C OP C OP
CH2-OH
B: CH2OH CH2
H B
2-phosphoglycerate phosphoenolpyruvate
FIGURE 3.30
Glycolysis Step 9: Dehydration of 2-phosphoglycerate to phosphoenolpyruvate.
The eighth step in glycolysis is essentially an isomerization reaction catalyzed by phos

phoglycerate mutase that converts 3-phosphoglycerate to 2-phosphoglycerate. This trans-
formation actually proceeds via formation of the intermediate 2,3-diphosphoglycerate
from which the C-3 phosphate group is removed. This is accomplished by a phosphory-
lated enzyme function which initially gives up its phosphate group to the C-2 hydroxyl
group and then recovers it from the phosphate ester at C-3 to regenerate the active
enzyme, as shown in Fig. 3.29.
The ninth step is an enolase-mediated dehydration reaction somewhat analogous
to the dehydration of β-hydroxycarbonyl compounds from aldol-type processes to
give α,β-unsaturated carbonyl compounds. The acidic α-proton of 2-phosphoglycer-
ate is removed by a basic enzyme side chain (Fig. 3.30) to give the corresponding
enolate ion in what is formally an E1cB-type mechanism. Loss of HO– to a proton
donor gives H2O and the α,β-unsaturated product, phosphoenolpyruvate. This is
essentially a phosphate ester of the enol form of pyruvic acid, as the name implies.
In the tenth and final step of glycolysis, the phosphate group of phosphoenol-
pyruvate is transferred to ADP by pyruvate kinase-catalyzed nucleophilic substitu-
tion, thereby regenerating ATP. The resulting enol form then tautomerizes to the
keto form as expected, yielding the final product of glycolysis, pyruvic acid, as
shown in Fig. 3.31. The reactivity of the enol phosphate ester here is greater than
that of a simple alcohol phosphate due to the final driving force of tautomerization
from enol to keto form; note also that this is another example of substrate-level
phosphorylation.
If we add up all the balanced equations from each step, we find the overall process
of glycolysis boils down to: C6H12O6 + 2 NAD+ + 2 ADP + 2 Pi ¾→ 2 C3H4O3 + 2
NADH + 2 H+ + 2 ATP + 2 H2O.


O OH O OH O OH
C C C HO
OH HO-ADP enol-keto
C O C OH C O + HO P O-ADP
P OH
CH2 CH2 CH3 O
O = ATP
enol form pyruvic acid
phosphoenolpyruvate
FIGURE 3.31
Glycolysis Step 10: Conversion of phosphoenolpyruvate to pyruvic acid and ATP.
oxidative cleavage of glucose to pyruvic acid with reduction of NAD+
glucose (C6H12O6) + 2 NAD+ + 2 ADP + 2Pi → 2 pyruvic acid (C3H4O3) + 2 NADH + 2 H+ + 2 ATP + 2 H2O
dehydrative formation of the anhydride ATP

from ADP and Pi
FIGURE 3.32
Overall transformations of glycolysis separated into redox and anhydride formation components.
Another way of viewing this chemically is to consider the overall glycolysis process
as being one that essentially couples the oxidative breakdown of 1 mole of glucose into
2 moles of pyruvic acid (with concurrent reduction of 2 NAD+ → 2 NADH + 2 H+)
with the dehydrative conversion of 2 moles of ADP and 2 moles of Pi (inorganic phos-
phate) into 2 moles of the more reactive “anhydride” ATP (with concurrent removal of
2 moles of H2O). The two balanced processes are linked on each side of the equation
in Fig. 3.32.
If we assume that ample glucose, ADP, and Pi are available, the limiting reagent
in the process will be the cofactor NAD+. This means that for glycolysis to continue,
more NAD+ will be required. In principle, this could be accomplished under aerobic
conditions by regeneration of NAD+ from NADH via direct reaction with molecular
oxygen (2 NADH + 2 H+ + O2 → 2 NAD+ + 2 H2O); in practice, this is accomplished
using protons and a complex, multistep sequence known as the electron transport
chain that regenerates NAD+ from NADH and eventually passes the hydrogen atoms
and associated electrons along to O2 which is discharged in reduced form as H2O.
The details are very complicated, so for now let’s just briefly consider how NAD+ is
regenerated under conditions of limited O2 supply or under anaerobic conditions.
WHAT HAPPENS TO THE PYRUVIC ACID FROM GLYCOLYSIS
The whole point of the oxidative breakdown of glucose is to supply energy in the
form of ATP to power other cellular processes. In some situations, an oxygen supply
may be limited for a period of time; one example is when the energy demands of
vigorous exercise exceed the ability to supply oxygen to muscle cells for regenerat-
ing the NAD+ needed to keep glycolysis going. There is an alternative way to recycle

O OH O OH
C C
C O HO C H
CH3 CH3
pyruvic acid NADH/H + NAD + (S)-lactic acid
FIGURE 3.33
Stereospecific reduction of pyruvic acid to (S)-lactic acid and regeneration of NAD+ from
NADH/H+.
NADH back to NAD+ when O2 availability as an electron acceptor (via the electron
transport chain) is limited. What is needed is another electron acceptor and this
constitutes another use for the pyruvic acid formed from glycolysis. Direct NADH/
H+ reduction of the ketone function of pyruvic acid to the corresponding alcohol
produces lactic acid (Fig. 3.33), but also recycles NADH back to its oxidized form,
NAD+. In this way, ATP and energy production from glycolysis can continue for a
while—at least until lactic acid build-up leads to the eventual aches and pains associ-
ated with muscle exhaustion. Note the reduction is stereospecific, with the hydride
from NADH being delivered to the Re face of pyruvic acid, giving only the S enan-
tiomer of lactic acid.
Of course, lactic acid has fates of its own, mainly to be transported to the liver
where it is oxidized back to pyruvic acid to serve as a building block for the local
production of more glucose by a pathway called gluconeogenesis (“new glucose
formation”). While this nonphotosynthetic process is not precisely the reverse of
glycolysis, it is fair to say that the two processes share many of the same steps and
intermediates, so we need not examine it in further detail for our current purposes.
Under anaerobic conditions, some microorganisms like yeast can break down
glucose by glycolysis, then convert the resulting pyruvic acid to ethanol and CO2
in the familiar process of fermentation that forms the basis for production of bev-
erage alcohol in beer and wine. The conversion of pyruvic acid is driven by TPP
ylide, as shown in Fig. 3.34. After decarboxylation and protonation, acetaldehyde
is released which is then reduced by NADH/H+ to give ethanol. Retention of the
by-product CO2 in beverage fermentation is responsible for the carbonation of
beer and sparkling wines, while removal of the residual CO2 by degassing leads
to “still” wines. When yeasts are used in baking, CO2 production causes dough
to expand or “rise” while the volatile ethanol produced is driven off during the
baking process.
As we saw previously in Chapter 2, another fate of pyruvic acid closely related to
the nonoxidative decarboxylation process shown above is the oxidative decarboxyl-
ation and conversion of pyruvic acid to acetyl-CoA by the action of TPP ylide and
enzyme-bound lipoic acid. The process shown in Fig. 3.35 is nonoxidative, since
the ketone carbonyl carbon in pyruvic acid ends up in the same oxidation state in
the product acetaldehyde, while in the conversion to acetyl-CoA, its oxidation state
increases to that of a thioester carbonyl carbon.


FIGURE 3.34
Decarboxylation of pyruvic acid by TPP ylide, generation of acetaldehyde and its subsequent
reduction to ethanol by NADH/H+ in anaerobic fermentation.
R' R' R' O
S C
N S S + H3C S SH
N N
R R R
H :B
C OH C O TPP ylide R-Enz
H3C HB H3C
S SH
S S CoASH
R-Enz
R-Enz
enzyme-bound lipoic acid B: H O
O
S HB
SH SH
S + C H3C C SH
H3C SCoA S
CoAS
R-Enz FADH2 FAD R-Enz acetyl-CoA
R-Enz
FIGURE 3.35
Formation of acetyl-CoA via decarboxylated TTP-bound intermediate from pyruvic acid.
THE CITRIC ACID CYCLE: ANOTHER 10-S TEP PROGRAM
While production of acetyl-CoA represents one of the more important uses of pyru-
vic acid, another essential sequence that utilizes both pyruvic acid and acetyl-CoA
is the so-called citric acid cycle. This indispensable closed loop of reactions is used
by all aerobic organisms for the oxidative conversion of the acetate in acetyl-CoA
(derived not only from metabolism of carbohydrates, but also from fats or proteins)
into CO2. The cycle also generates important precursors for the biosynthesis of spe-
cific amino acids as well as some of the NADH required by numerous other bio-
chemical processes. The pivotal nature of the cycle in terms of cellular metabolism
indicates that it was surely one of the earliest biochemical mechanisms associated
with the evolution of living systems.
To gain an appreciation for the subsequent workings of the citric acid cycle,
we will begin by looking briefly at how pyruvic acid and acetyl-CoA can come to-
gether to set things into motion. This starts by a carboxylation reaction of pyruvic

O
Bioorganic Synthesis 100 C
HO2C CH2 :B
H
pyruvic acid O
O
C
HO2C CH2 C
O O HO2C CH2CO2H
O
C - biotin C oxaloacetic acid
O N NH O
H H
R
S
N-carboxybiotin
FIGURE 3.36
N-carboxybiotin and carboxylation of pyruvic acid enolate ion to give oxaloacetic acid.
acid catalyzed by pyruvate carboxylase (for the purposes of depicting the chem-
istry and intermediate products of the citric acid cycle, we will stick with show-
ing all carboxylic acids and their names in nonionized form). Here, the universal
carboxyl carrier N-carboxybiotin releases CO2 to be trapped by the ketone enolate
ion of pyruvic acid, leading to the carboxylated product known as oxaloacetic acid
(Fig. 3.36). This important intermediate is the starting point for the process of glu-
coneogenesis referred to earlier and as we will see, is also produced by the citric
acid cycle itself, making this process a closed loop of reactions, since this starting
reactant is also the final product of the process. Once we have seen how these start-
ing materials set the process in motion, we can examine the overall cycle in a single
diagram. It should be pointed out that seven of the ten intermediates involved in
the cycle, including oxaloacetic acid, are also available from other metabolic pro-
cesses such as the degradation of amino acids and may enter the cycle at any point.
Next comes the crucial step in which acetyl-CoA reacts with oxaloacetate. This
reaction is essentially a citrate synthase-catalyzed aldol addition process in which the
enolate ion of acetyl-CoA attacks the ketone carbonyl of oxaloacetic acid, yielding the
intermediate product (S)-citryl-CoA which is then hydrolyzed to liberate citric
acid, the product from which the cycle takes its name, and HSCoA (Fig. 3.37). Note
that while (S)-citryl-CoA is chiral, its hydrolysis product, citric acid, is an achiral
compound.
While we are not emphasizing the role of the enzymes here, this step reminds us
of the important specificity of such enzyme-mediated processes. Since oxaloacetic
acid is a β-dicarbonyl compound, we would ordinarily expect its carbonyl α-pro-
tons to be much more acidic than those of acetyl-CoA, which is a simple thioester.
This means that this addition reaction could never take place under ordinary base-
catalyzed conditions, since the oxaloacetic acid enolate ion would form much more
readily. Here, the enzyme holds both reactants in a specific spatial relationship that


:B B-H
O O O
C H
CoAS CH2 C C
CoAS CH2 HO2C CH2CO2H
acetyl-CoA oxaloacetic acid
aldol addition
O
C
H2O CoAS CH2
OH OH
HO2CH2C
C C
HO2C CH2CO2H HO2C CH2CO2H
CoASH
citric acid (S)-citryl-CoA
FIGURE 3.37
Aldol addition reaction of acetyl-CoA and oxaloacetate to produce citrate.
brings the thioester into intimate contact with the basic site needed for its deprot-
onation while simultaneously providing the acidic site necessary to protonate the
oxygen of the oxaloacetic acid ketone carbonyl as the nucleophilic attack proceeds.
Now that we have initiated the citric acid cycle by the introduction of oxaloacetic
acid and acetyl-CoA, we can take a look at the overall process to see how it consumes
acetyl-CoA, discharging its carbons as CO2 and its hydrogens to NADH and FADH2
as shown in Fig. 3.38. It is convenient to think of the cycle as a sort of engine that
uses acetyl-CoA for fuel, while discharging CO2 and water. As long as there is fuel
available, the engine will keep running, though this engine can also run on any of
the intermediate products for fuel, most of which are available through the degrada-
tion of amino acids and other sources.
Referring to Fig. 3.39, we can see that step 2 in the process is simply a dehydra-
tion reaction, as often follows an aldol-type addition process. This reaction, cata-
lyzed by the enzyme aconitase, is a stereospecific anti elimination, producing the
α,β-unsaturated dicarbonyl product, cis-aconitic acid. This is followed by step 3,
which is a nucleophilic conjugate addition of water at C-2 of the α,β-unsaturated di-
carbonyl catalyzed by the action of the same enzyme as in step 2. This anti addition
is also stereospecific, with the OH of water being delivered to the Re face at C-2 and
the proton at the Re face of C-3 in cis-aconitic acid, leading to the isomeric product
(2R,3S)-isocitric acid. Thus, the stereospecificity of both the elimination and the
addition processes further emphasizes the careful regio-and stereochemical control
exerted by the mediating enzyme.
Step 4 is the simple isocitrate dehydrogenase-catalyzed NAD+ oxidation of the
secondary alcohol function at C-2 of isocitric acid to give the corresponding ketone,
oxalosuccinic acid, while also producing the first of three equivalents of NADH
from the cycle (Fig. 3.40). This β-ketoacid product then undergoes the expected

HO2C H
C
O acetyl-CoA/H2O
HO2CH2C OH 2
1
C C C
HO2C CH2CO2H HO2C CH2CO2H HO2C CH2CO2H
H2O
oxaloacetic acid CoASH citric acid cis-aconitic acid
H2O
NADH/H+ 3
10
NAD +
H
OH O HO2C H
C
HO2C C CH2CO2H C
CoAS CH 2CH 2 CO 2H HO C
H HO2C CH2CO2H
(S)-malic acid succinyl-CoA (2R,3S)-isocitric acid
CoASH
NAD +
CoASH
H2O GTP NAD + 4
9 Pi 6
7 CO 2 NADH/H+
GDP NADH/H + O
O
HO2C H 8 C H
C 5
C C HO2CCH2CH2CO2H HO2C
HO2C CH2CH2CO2H C
H CO2H HO2C CH2CO2H
FADH2 FAD succinic CO2
fumaric acid acid α-ketoglutaric acid oxalosuccinic acid
FIGURE 3.38
The 10 steps of the citric acid cycle.


HO2C H H B H
HO2C C H B HO2C C H
C OH
anti- anti-
H C C CH2CO2H HO C
B: elimination H2O addition CH2CO2H
HO2C CH2CO2H HO2C HO2C
citric acid cis-aconitic acid (2R,3S)-isocitric acid
FIGURE 3.39
Stereospecific dehydration of citric acid and hydration of cis-aconitic acid to give isocitric acid.
H
H O O O
HO2C OH
C H C C C
HO HO2C O HO2C C CH HO2C CH2
C C
HO2C CH2CO2H H HO2CH2C
NAD+ NADH/H+ HO2CH2C CO2 HO2CH2C
(2R,3S)-isocitric acid oxalosuccinic acid enol α-ketoglutaric acid
FIGURE 3.40
Oxidation of isocitric acid to oxalosuccinic acid and decarboxylation to α−ketoglutaric acid.
decarboxylation in step 5. While enzyme-assisted, this decarboxylation would prob-

ably occur spontaneously as it does for most β-keto acids; in this instance, the decar-
boxylation generates the first of the two equivalents of CO2 produced in each turn of
the cycle. We also note that the product, α-ketoglutaric acid, is a common biomol-
ecule that could also be introduced into the cycle from other sources; it could also
be diverted to the production of the amino acid glutamic acid via transamination.
The next two steps in the cycle are somewhat more complex and require some
additional clarification. Step 6, catalyzed by a multienzyme complex, represents the
second decarboxylative step in the cycle, and while not as simple as the decarbox-
ylation in the previous step, is actually just another example of the oxidative decar-
boxylation of α-ketoacids to acylCoA derivatives facilitated by TPP ylide, CoASH,
and enzyme-bound lipoic acid, as seen previously in the conversion of pyruvic acid
to acetyl-CoA (see Figs. 3.35 and 3.36). Thus, this sequence converts α-ketoglutaric
acid to succinyl-CoA with concurrent loss of CO2. The role of NAD+ in this step is
to regenerate FAD from the FADH2 produced during oxidative regeneration of the
lipoic acid cyclic disulfide ring (Fig. 3.41).
Step 7 in this sequence, catalyzed by succinyl-CoA synthetase, is very similar to the
seventh and eighth steps of the glycolysis pathway (see Figs. 3.28 and 3.29) which in-
volved conversion of a thioester to an intermediate acylphosphate by nucleophilic acyl
substitution by inorganic phosphate (Pi) which in turn transferred the reactive phos-
phate group to ADP, converting it to ATP and releasing the corresponding carbox-
ylic acid. Here, the thioester is succinyl-CoA rather than an enzyme-bound thioes-
ter, and the resulting intermediate acylphosphate undergoes nucleophilic attack by
GDP (guanosine diphosphate) rather than by ADP, producing GTP and succinic acid
(Fig. 3.42). While this is also an example of substrate-level phosphorylation as seen pre-
viously in glycolysis, there is no obvious reason why GDP is used here rather than ADP.
In the final steps of the citric acid cycle (Fig. 3.43), we see first the oxidative de-
hydrogenation of succinic acid to fumaric acid by the action of FAD and catalyzed

R'

R' HB R'
S O
S - CO2 S
C OH N N
N R R
H2C C OH
R C O C OH
H2C O C H
TPP ylide H2 C H2C HB
CO2H
S S
CH2 O :B CH2
α-ketooglutaric acid HO2C
HO2C
R-Enz
enzyme-bound lipoic acid
TPP ylide R'

B: O
H O
HB CoASH C
C H2C S SH S
N
CoAS S SH R
CH2 CH2 H
HO2C R-Enz C O :B
H2C H2C
R-Enz SH
CO2H S
CH2
HO2C
R-Enz
O
H2 SH S
C C S
HO2C C SCoA + SH and FADH2 FAD
H2
FAD FADH2 R-Enz NAD+ NADH/H+
succinyl-CoA
R-Enz
FIGURE 3.41
Formation of succinyl-CoA via decarboxylated TTP-bound intermediate from α-ketoglutaric acid.
H B O
O OPO(OH)2 OP
O O P OH HO-GDP
C SCoA Pi
O C SCoA C CH2CO2H
OH
CH2CH2CO2H CH2CH2CO2H CH2CH2CO2H CH2CO2H
GDP GTP
succinyl-CoA CoASH succinic acid
FIGURE 3.42
Conversion of succinyl-CoA to succinic acid coupled to conversion of GDP to GTP.
+
H B H
H
CH2CO2H C CO2H H2O H C CO2H HO2C CH2CO2H
HO2C C H C
CH2CO2H FAD FADH HO2C C
2 H
OH NAD+ NADH/H+ O
H2 O
succinic acid fumaric acid (S)-malic acid oxaloacetic acid
FIGURE 3.43
Oxidation of succinic to fumaric acid, hydration to (S)-malic acid, and final oxidation to
oxaloacetic acid.
by succinate dehydrogenase in step 8. Once again, this is a stereospecific anti elimi-

nation, removing the elements of H2. This is followed by a stereospecific conjugate
addition of water to the α,β-unsaturated dicarboxylic acid catalyzed by fumarase in
step 9 to produce (S)-malic acid in a process quite similar to the anti addition of
water in step 4 that produced isocitric acid. We complete one full turn of the cycle
in malate dehydrogenase-catalyzed step 10 by regenerating the oxaloacetic acid we

originally started with in step 1 by NAD+ oxidation of the secondary alcohol func-

tion of (S)-malic acid to the corresponding ketone.
So, one turn of the citric acid cycle accomplishes the degradation of a single acetyl-
CoA into 2 CO2 while also producing one each of HSCoA, GTP, and FADH2, along
with 3 NADH and 3 H+. Now that we have a better appreciation for glycolysis and one
of the important fates of its principal product, pyruvic acid, let’s turn to another path-
way for metabolism of glucose that also provides some useful intermediate products.
THE PENTOSE PHOSPHATE PATHWAY: SEVEN

ALTERNATIVE STEPS TO SOME FAMILIAR
INTERMEDIATES
While not as significant as glycolysis for the degradation of glucose, the pentose
phosphate pathway is nevertheless still important for a number of different reasons.
In animals, liver and mammary gland tissues use this pathway to metabolize a sig-
nificant fraction of the glucose they process, though other tissues, such as skeletal
or heart muscle, rely almost completely on glycolysis. The main reasons for use of
the pentose phosphate cycle are to generate ribose-5-phosphate needed for the pro-
duction of ribonucleic acids, to produce erythrose-4-phosphate for the production
of aromatic amino acids in plants, and to convert NADP+ to NADPH, a reducing
agent whose function and usage are similar to those of NADH. For our purposes, we
mainly want to gain an appreciation for the structures and nomenclature associated
with the intermediates produced as well as an understanding of the stoichiometry,
which at first glance appears unusual, as can be seen in the overall equation for me-
tabolism of one mole of glucose via this pathway:
glucose-6-P + 2 NADP+ + H2O → 2/3 fructose-6-P + 1/3 glyceraldehyde-3-P

+ CO2 + 2 NADPH + 2 H
We can think of this as a sort of partial degradation pathway which produces some
CO2 but also produces intermediate products that may be diverted to other uses as
well as products that may ultimately be passed along to glycolysis for further degra-
dation. An overview of the entire pathway is shown in Fig. 3.44.
The first step in the pathway is the glucose-6-phosphate dehydrogenase-catalyzed
NADP+ oxidation of the β-anomeric hydroxyl of glucose-6-phosphate at C-1 to
give the corresponding 6-phosphogluconolactone which is hydrolyzed in the
second step to give the open-chain form, 6-phosphogluconic acid. The third step,
(Fig. 3.45), is catalyzed by phosphogluconate dehydrogenase and involves the regio-
specific oxidation of the C-3 hydroxyl by NADP+, giving an intermediate β-ketoacid
which then decarboxylates as expected to give ribulose-5-phosphate. Note that
steps 1 and 3 are responsible for the production of the two equivalents of NADPH
produced per equivalent of glucose metabolized by this pathway.

CO2H

OH OH
H OH
HO HO
OP 1 OP 2 HO H
O O H OH
HO HO H2O
NADP + NADPH/H+ H OH
OH at C-1 O
CH2OP
glucose-6-P 6-phosphoglucono-
lactone 6-phosphogluconic acid
CH2OH NADP +
CH2OH
O CH2OH 3
O CO 2
O
HO H
HO H NADPH/H +
H OH HO H
H OH CH2OH
H OH H OH
H OH O
CH2OP CH2OP
H OH (2) xylulose-5-P H OH
fructose-6-P 6 5 4
CH2OP (1:1) + (2:1) H OH
+
sedopheptulose-7-P CHO
CHO CH2OP
+ H OH ribulose-5-P
H OH CHO H OH
H OH H OH H OH
CH2OP CH2OP CH2OP
erythrose-4-P glyceraldehyde-3-P (1) ribose-5-P
CH2OH
O
7 CHO
HO H
(1) xylulose-5-P + H OH
H OH
CH2OP
H OH
glyceraldehyde-3-P
CH2OP
fructose-6-P
FIGURE 3.44
The seven steps of the pentose phosphate pathway.
CO2H CO2H
H OH α CH2OH
H OH CO2
HO H β O O
H OH H OH H OH
H OH NADP + NADPH/H+ H OH
H OH
CH2OP CH2OP CH2OP
6-phosphogluconic acid a β-keto acid ribulose-5-P
FIGURE 3.45
Step 3 of the pentose phosphate pathway.
The fourth step in the sequence involves a series of isomerizations of ribulose-5-

phosphate, each involving the generation of isomeric products derived from two dif-
ferent enediol forms of ribose-5-phosphate, namely the corresponding 1,2-enediol
and 2,3-enediol forms, as shown in Fig. 3.46. These isomerizations, each catalyzed
by a specific epimerase enzyme, lead to the corresponding carbohydrates xylulose-5-
phosphate and ribose-5-phosphate in a relative ratio of 2:1, respectively, for book-
keeping purposes (or, 3 moles of ribulose-3-phosphate will be isomerized to 2 moles
of xylulose-3-phosphate and 1 mole of ribose-3-phosphate). The actual amount and


CH2OH CH2OH 1CH2OH H OH CHO
O OH 2 O OH H OH
HO H OH H 3 OH H OH H OH
H OH H OH H OH H OH H OH
CH2OP CH2OP CH2OP CH2OP CH2OP
xylulose-5-P 2,3-enediol ribulose-5-P 1,2-enediol ribose-5-P
(0.67 mole) (1.00 mole) (0.33 mole)
FIGURE 3.46
Step 4 of the pentose phosphate pathway: isomerizations of ribulose-5-P.
fate of the products will depend in part on local demands for ribose-5-phosphate
production in connection with ribonucleic acid synthesis.
Excess ribose-5-phosphate can then be converted to other products by the reac-
tions in the subsequent steps. In the fifth step, catalyzed by a transketolase enzyme,
TPP ylide adds to the ketone carbonyl of half of the xylulose-5-phosphate from step 4
in a manner similar to that seen in the TPP reaction with α-ketoacids (Fig. 3.47, i));
the resulting intermediate undergoes a retroaldol reaction which leads to formation of
glyceraldehyde-3-phosphate; the TPP-bound two-carbon fragment originally from
xylulose-5-P then acts as a nucleophile, attacking the aldehyde carbonyl of ribose-
5-phosphate, leading to an intermediate that undergoes deprotonation, followed by
expulsion of TPP ylide and simultaneous production of the ketoheptose product,
sedoheptulose-7-phosphate (Fig. 3.47, ii)). Thus we have the reaction of one equiv-
alent of ribose-5-P and one equivalent of xylulose-5-P producing one equivalent of
glyceraldehyde-3-P and sedoheptulose-7-P. In this process, we see an aldotriose pro-
duced from a ketopentose by removal of a two-carbon fragment which is then added
to an aldopentose to produce a ketoheptose. Also note that half of the xylulose-5-P
produced in step 4 remains available for use in step 7, but for now we have to carry out
step 6 to produce a product that will ultimately react with this remaining xylulose-5-P.
In the sixth step of the sequence, a transaldolase enzyme amino group condenses
with the ketone carbonyl of sedoheptulose-7-phosphate, producing an iminium ion
derivative (a protonated imine in this case) which then undergoes a retroaldol cleav-
age (nearly identical to that seen in the fourth step of the glycolysis pathway). This
produces a three-carbon enamine intermediate as well as the important product
erythrose-4-phosphate (Fig. 3.48, i)) which will ultimately play a key role in the
plant biosynthesis of aromatic amino acids and other natural products from the shi-
kimic acid pathway (Chapter 6); the erythrose-4-P will also serve as a key reactant
in the seventh and final step of the pentose phosphate pathway by combining with
unused xylulose-5-P produced in the fourth step.
In the next part of step 6 (Fig. 3.48, ii), the three-carbon enamine intermedi-
ate acts as a nucleophile, attacking the aldehyde carbonyl of glyceraldehyde-3-
phosphate (from step 5) in an aldol-type addition process. This step knits together
the two three-carbon fragments to produce a six-carbon carbohydrate imine de-
rivative which upon hydrolysis yields fructose-6-phosphate. For further metabolic
degradation, this may be passed along to the glycolysis pathway, along with the

R'
R'
CH2OH R'
S :B
H B
O S CHO
N
N R H S
HO H OH H OH + N
C O R
i) R H OH
HOH2C CH2OP C
TPP ylide H OH
CH2OP H OH glyceraldehyde-3-P HOH2C
xylulose-5-P
CH2OP
R'
:B
CH2OH
S R'
R' H B N
HC=O H O
R
S O
H OH C HO H
HOH2C S
ii) OH + N
N C H OH HO H H OH R
R CH2OH H OH H OH H OH TPP ylide
CH2OP H OH H OH
ribose-5-P H OH CH2OP
CH2OP sedoheptulose-7-P
FIGURE 3.47
Step 5 of the pentose phosphate pathway: production of glyceraldehyde-3-P and sedoheptulose-7-P.

CH2OH

CH2OH CH2OH H
enamine N
O H2N H
N
HO H HO H
HO H +
H OH - H 2O :B retroaldol CHO
i) H O H
H OH imine H OH
formation H OH
H OH H OH
H OH
CH2OP CH2OP
CH2OP erythrose-4-P
sedoheptulose-7-P
CH2OH CH2OH CH2OH

H
enamine
H
N O +H N
N 2
aldol HO H H2O HO H
HO H H B
imine H OH
HC=O H OH
ii) hydrolysis
H OH H OH H OH
CH2OP CH2OP CH2OP
glyceraldehyde-3-P fructose-6-P
FIGURE 3.48
Step 6 of the pentose phosphate pathway: production of erythrose-4-P and fructose-6-P.
fructose-6-P and glyceraldehyde-3-P that are the products in the seventh and final
step, which we can now examine.
As mentioned above, the pathway is completed in the seventh and final step in
which the remaining unused xylulose-5-phosphate from step 4 reacts with TPP
ylide in a sequence that is identical mechanistically to that in step 5 (Fig. 3.49).
Thus, the transketolase-mediated retroaldol process again releases the first final
product of the sequence, glyceraldehyde-3-phosphate, along with the usual TPP-
bound nucleophilic two-carbon fragment. This fragment then attacks the aldehyde
carbonyl of the erythrose-3-phosphate derived from the previous step. After knit-
ting together these two-carbon and four-carbon fragments, a subsequent deprot-
onation and elimination of TPP ylide yields the second final product of the step,
fructose-6-phosphate. Again, these two products are familiar ones which now may
enter the glycolysis pathway for final degradation to pyruvic acid.
THE BIG PICTURE
As we have seen, the breakdown of glucose via glycolysis ultimately leads to the
production of pyruvic acid, and our interest in the process stemmed in part from the
various fates of pyruvic acid itself which we have looked at in some detail. To gain a
better perspective of where all of this is leading, a general overview of the relation-
ships of the various pathways we have examined is presented in Fig. 3.50. While this
may look hopelessly complex at first glance, if we look more closely we can see how
this flow chart actually organizes and simplifies a lot of information while pointing
the way to where we will be going later in the text in Chapters 4–7.

R'
R'
CH2OH R'
S :B
H B
O S CHO
N
N R H S
HO H OH H OH + N
C O R
i) R H OH
HOH2C CH2OP C
TPP ylide H OH
CH2OP H OH glyceral- HOH2C
xylulose-5-P dehyde-3-P
CH2OP
R'
:B CH2OH
S
R' H B N O
HC=O H
R
S O HO H
H OH HOH2C C
ii) OH H OH + TPP ylide
N C H OH HO H
H OH
R CH2OH CH2OP H OH
CH2OP
erythrose-4-P H OH
fructose-6-P
CH2OP
FIGURE 3.49
Step 7 of the pentose phosphate pathway: final production of glyceraldehyde-3-P and fructose-6-P.

CO2 + H2O

photosynthesis
gluconeogenesis
glucose, C6H12O6
pentose phosphate
glycolysis pathway CHO
CHO
CO2H H OH
H OH
C OP + H OH ribonucleotides
H OH
CH2 H OH
phosphoenol CH2OP
pyruvic acid CH2OP
erythrose-
4-phosphate ribose-
4-phosphate
CO2H
CO2H
aromatic amino acids,
HO H shikimate lignans, cinnamic acids,
CH3 pathway and other aromatic
HO OH natural products (Ch. 6)
lactic acid
OH
shikimic acid
aliphatic aromatic peptides, proteins, penicillins, and
CO2H alkaloid natural products (Ch. 7)
amino acids amino acids
C O
O
CH3 acetate fatty acids, prostaglandins, and
CH3CSCoA pathway polyketide natural products (Ch. 5)
pyruvic acid acetyl-CoA
fermentation terpenoid steroids, carotenoids, and other
CH3 CH2 OH + CO2 terpene natural products (Ch. 4)
pathway
citric acid cycle
CO2 + H2O + ATP
O2
FIGURE 3.50
Breakdown of glucose and use of its components in various biosynthetic pathways.
As we saw, reduction of the ketone carbonyl of pyruvic acid gives lactic acid, which
can then be used to resynthesize glucose in gluconeogenesis, a process very nearly
the reverse of glycolysis in terms of the chemistry of its individual steps. Alternatively,
under anaerobic conditions, such as in yeasts, pyruvic acid underwent reduction and
decarboxylation, producing ethanol and CO2 in the familiar process of fermentation.
We then saw how acetyl-CoA (prepared from pyruvic acid by the action of TPP ylide
and enzyme-bound lipoic acid) moved into the citric acid cycle where its two carbons
were subsequently degraded to CO2. Later on we’ll see how acetyl-CoA is used as
one of the fundamental building blocks for the biosynthesis of terpene natural prod-
ucts (The Terpenoid Pathway, Chapter 4) as well as fatty acids, prostaglandins and a
wide variety of polyketide natural products (The Acetate Pathway, Chapter 5). The
alternative route for glucose breakdown, the pentose phosphate pathway, was also
significant for a number of reasons, but mainly for its production of ribose-5-phos-
phate, needed for the synthesis of ribonucleotides and erythrose-4-phosphate used
by plants for the production of shikimic acid, from which they then produce the
aromatic amino acids phenylalanine and tyrosine, two fundamental building blocks
of the so-called shikimate natural products (The Shikimic Acid Pathway, Chapter
6). The aromatic amino acids, when combined with aliphatic amino acids derived
from pyruvic acid and intermediates from the citric acid cycle, lead not only to famil-
iar peptides, proteins, and important modified peptides such as the penicillins, but

also to the vast array of nitrogen-containing natural products known as the alkaloids
Bioorganic Synthesis 112 (Biosynthesis of Alkaloids and Related Compounds, Chapter 7).
Since amino acids will clearly play a prominent role in much of what comes later
in our text, we will devote the remainder of this chapter to an overview of their
structure and nomenclature, followed by an abbreviated look at how a number of
them are produced from some of the carbohydrate-derived intermediate products
produced in the pathways we have just studied. The biosynthesis of a number of
others, especially the aromatic amino acids, will be deferred to later chapters as
topics of special significance in terms of their relationship to specific pathways or
classes of natural products.
AMINO ACIDS: MORE IMPORTANT PRIMARY

METABOLITE BUILDING BLOCKS FOR BIOSYNTHESIS
Amino acids are, of course, among the most important components of all biological
systems, since they combine to make peptides and proteins, the biopolymers that con-
stitute hair, nails, connective tissue, tendons, antibodies, and so on. More importantly,
all enzymes are specialized proteins with very specific catalytic properties essential to
the functioning and integrated operation of all cellular processes. Clearly, life goes no-
where without amino acids. But our limited focus here will be on their organic chemis-
try: their structure and functions as individual amino acids, both from the standpoint
of their biosynthesis as well as their later use as important raw materials for the con-
struction of a variety of important nonpeptide natural products in subsequent chapters.
The acid–base properties of amino acids, their analysis, purification, and catabolism,
along with the intimate and seemingly infinite details of peptide, protein, and enzyme
structure and function are all core components of the study of biochemistry which,
except for a very brief review, we must leave to more advanced texts in the field.
To begin our brief tour, we turn to Fig. 3.51 which lists the names, abbrevia-
tions, and structures of the 20 standard α-amino acids, in alphabetical order. The
names of the essential amino acids, those which humans cannot biosynthesize and
therefore must obtain in the diet, are indicated with an asterisk (*). This is by no
means an exhaustive list of all amino acids (well over 100 have been isolated from
various sources), but rather a list of the so-called proteinogenic amino acids, those
which are genetically coded for and produced by cellular machinery for peptide and
protein synthesis. Some lists also include pyrrolysine and selenocysteine, though the
former is rather rare and the latter is not truly proteinogenic.
With the exception of glycine, all the α-amino acids are produced in nature as
the corresponding (S)-enantiomers at the amino-bearing carbon (note that (2S,3R)-
threonine and (2S,3S)-isoleucine have a second chirality center). When drawn in
Fischer projection form with their carboxyl group at the top of the vertical chain, the
α-amino group at the chirality center of the standard amino acids will be found point-
ing to the left, leading to their being designated as L-amino acids in the same way that

O NH O O O

H2N HO
OH H2N N OH OH OH
H
NH2 NH2 O NH2 O NH2
alanine arginine asparagine aspartic acid
Ala Arg Asn Asp
O O O O O O
HS OH HO OH H2 N OH H2N
OH
NH2 NH2 NH2
cysteine glutamic acid glutamine glycine
Cys Glu Gln Gly
O O O O
N H2N
OH OH OH OH
HN NH2 NH2 NH2 NH2
histidine* isoleucine* leucine* lysine*
His Ile Leu Lys
O O O O
S
OH OH OH HO OH
NH2 NH2 NH NH2
methionine* phenylalanine* proline serine

Met Phe Pro Ser
OH O O O O
OH OH OH OH
NH2 HN NH2 NH2 NH2
HO
threonine* tryptophan* tyrosine valine*
Thr Trp Tyr Val
FIGURE 3.51
Names, abbreviations, and structures of the 20 standard α-amino acids (*essential).
D-carbohydrates are designated as such when their lowermost chiral carbon hydroxyl
group points to the right in a Fischer projection (Fig. 3.52). We will ordinarily repre-
sent these structures in “amino acid” form as shown here, rather than in their more ap-
propriate “zwitterionic” form, for simplicity’s sake, when used in mechanistic schemes.
To consider the biosynthesis of amino acids, we can use the flow chart in Fig. 3.53
which makes it clear that all trace their origin back to one or more of the basic car-
bohydrate intermediates that we encountered in glycolysis, the citric acid cycle, or
the pentose phosphate pathway. Using this chart as a starting point, we will spend
a bit of time looking at some of the reactions involved in the biosynthesis of several
of the amino acids, though as mentioned above, we will defer detailed treatment of
the aromatic amino acids (His, Trp, Phe, and Tyr) and some of the others to later
chapters in the text.
As can be gleaned from the flow chart, all nonessential amino acids are synthe-
sized from common intermediates from glycolysis and the citric acid cycle: pyru-
vic acid, 3-phosphoglycerate, oxaloacetic acid, and α-ketoglutaric acid. Similarly,

CO2-
CO2H R CO2H
H2N H vs. H OH H2N H vs. H 3N H
R CH2OH R R
L-amino acid D-carbohydrate amino acid form zwitterion form
FIGURE 3.52
Stereoisomeric forms and zwitterionic forms of α-amino acids.
pentose phosphate
glucose-6-P ribose-5-P His*
pathway
glycolysis
3-phosphoglycerate Ser Cys, Gly
glycolysis
Trp*
phosphoenolpyruvate + erythrose-4-P
Phe* Tyr
glycolysis
pyruvic acid Ala, Val*, Leu*
citric acid
cycle
Asn, Lys*
oxaloacetic acid Asp Met * Cys
Thr* Ile*
+
α-ketoglutaric acid Glu Gln, Arg, Pro
FIGURE 3.53
Flow chart for amino acid biosynthesis from carbohydrate metabolism components (*essential).
some of the essential amino acids come from pyruvic acid or phosphoenolpyruvate,
while others may come from the pentose phosphate pathway products erythrose-4-
phosphate or ribose-5-phosphate, and still others come from subsequent transfor-
mations of other amino acids. For the essential amino acids, biosynthesis is limited
to plants or microorganisms and may occur by different pathways in different spe-
cies, in stark contrast to the pathways associated with carbohydrate or lipid metabo-
lism, which are nearly universal. It is presumed that the loss of these pathways in
mammals is a result of various evolutionary mutations over time.
BIOSYNTHESIS OF SERINE: A GOOD PLACE TO START
We can start by taking a look at how 3-phosphoglycerate is converted to serine,

from which cysteine and glycine are both subsequently derived. The process begins
with NAD+ oxidation of the secondary alcohol of 3-phosphoglycerate to give the
corresponding ketone (Fig. 3.54). This α-ketoacid then undergoes a PLP-mediated
transamination reaction with glutamic acid acting as the amine donor, giving


OH O OH O O
C C transamination H2O
H C OH C O PO OH HO OH
CH2OP NAD+ NH2 Pi NH2
NADH/H+ CH2OP glu α-keto-
3-phosphoglycerate glutaric acid serine
FIGURE 3.54
Biosynthesis of serine from 3-phosphoglycerate.
α-ketoacid imine BH
R CO2H R CO2H
H R CO2H
H NH2 B:
O H
H N H N
α-keto imine
OH
PO acid formation OH OH
+ PO
- H2O PO
N transaminase
N N
H
pyridoxamine phosphate, PMP H PLP H
(quinonoid form)
(ring N-protonated form)
α-amino acid R CO2H

imine
H O H
H N
OH R CO2H
PO H2O OH
+ PO
H2N H imine hydroysis
N
N
H α-amino acid
pyridoxal phosphate, PLP PLP imine H
(ring N-protonated form)
FIGURE 3.55
Mechanism for transamination of α-ketoacids by the action of pyridoxamine phosphate (PMP).
α-ketoglutaric acid and the intermediate product, 3-phosphoserine. Simple hydro-

lysis of the phosphate ester to the alcohol and inorganic phosphate leads directly to
serine.
We’ll take this as an opportunity to briefly revisit the mechanism for transami-
nation that was discussed previously in Chapter 2. In Fig. 3.55, we see the general
scheme for converting an α-ketoacid to an α-amino acid by reaction with pyridox-
amine phosphate (PMP) which in turn is converted to pyridoxal phosphate (PLP).
To see how the overall process works to deliver the amino group of glutamic acid to
3-phosphoserine in Fig. 3.54, work the process in Fig. 3.55 in the reverse direction
using PLP and glutamic acid as the amino acid (or see Fig. 2.22); this will produce
α-ketoglutaric acid and PMP. Subsequent reaction of PMP with the ketoacid from
Fig. 3.54 will then produce 3-phosphoserine.
This basic transamination scheme is one we will see used for the synthesis of
several other amino acids and is also an important and useful process for a va-
riety of other transformations in biosynthetic schemes to come later. One way
to gain a further appreciation for the complexity and versatility of PLP-mediated

transformations is to take a detailed look at the 10-step conversion of serine into

Bioorganic Synthesis 116 cysteine, as shown in Fig. 3.56. This remarkably complex process stands in stark
contrast to the relative simplicity of the formation of serine itself, and while we
will not look at all subsequent biosynthetic schemes in such detail, this one should
give us a good sense of how to push the arrows in other PLP processes and to de-
velop the intuition needed to predict likely mechanistic details in similar or related
transformations.
In step 1, imine formation between PLP and serine takes places, followed by a de-
protonation to give the usual quinonoid form of the PLP imine in step 2. A rearoma-
tization of the PLP ring with loss of H2O from the serine chain in step 3 is followed
by nucleophilic addition of the thiol S atom of homocysteine (derived from methio-
nine), then PLP ring aromatization and protonation to yield an imine in steps 4 and 5.
Temporary loss of PLP from the imine to produce the key intermediate cystathionine
CO2H CO2H BH
HO HO CO2H
HO
NH2 serine N H :B
H O imine N
+ 2
formation OH
OH PO OH
PO -H2O PO
1 N
N N
H
H H 3
NH2 NH2 HB homocysteine

CO2H HO2C S H :B
CO2H
HO2C S HO2C S
N NH2 CO2H
N 5 4
OH N
OH PO
PO
OH
N PO
PLP imine N H
H N
H H
H
6 N N
OP OP
HO O
PLP PLP
NH2 H
7 8 N
N
CO2H CO2H
HO2C S CO2H HO2C S
HO2C S
H2 N H H :B NH2
cystathionine :B NH2 9
H
H
N
N
OP CO2H
HO HS 10 OP
succinyl-CoA O
+ NH2
N
HN HB
cysteine CO2H
HO2C
HO2C S
NH2
FIGURE 3.56
Ten-step PLP-mediated conversion of serine to cysteine by way of intermediate cystathionine.

in step 6 is then followed by PLP imine formation at the second amino group of cys-

tathionine in step 7. Sequential deprotonations in steps 8 and 9 are followed by a
final elimination and protonation in step 10 that produces cysteine as well as a PLP-
bound intermediate which is subsequently transformed in a number of steps into
succinyl-CoA. While perhaps intimidating at first, the sequence makes good chemi-
cal sense and nicely illustrates a number of commonly encountered transformations
that are made possible via PLP intermediates.
The same PLP imine derivative of serine from step 1 in the cysteine biosynthesis
is also involved in conversion of serine to the achiral amino acid glycine, further il-
lustrating the versatility of PLP-mediated processes (Fig. 3.57). The reaction begins
with a simple elimination reaction that produces formaldehyde and the usual PLP-
imine quinonoid form. PLP ring rearomatization and subsequent hydrolysis gives
glycine directly.
As shown in Fig. 3.58, transamination plays a direct role in the conversion of
pyruvic acid to alanine, oxaloacetic acid to aspartic acid, and α-ketoglutaric acid to
B:
H CO2H BH CO2H
O CO2H
N N
N
OH OH O
PO PO OH imine
PO hydrolysis H2N
OH
N N glycine
H2C=O N PLP
H H
serine-PLP imine H
FIGURE 3.57
PLP-mediated conversion of serine to glycine.
O O
OH OH
O NH2
pyruvic acid alanine
O O
HO transamination HO
OH OH
O O O NH2
oxaloacetic acid aspartic acid
O O O O
HO OH HO OH
O NH2
α-ketoglutaric acid glutamic acid
FIGURE 3.58
Transaminations for the direct biosynthesis of alanine, aspartic acid, and glutamic acid.

glutamic acid, three other nonessential amino acids for which PLP plays a pivotal
Bioorganic Synthesis 118 role in their biosynthesis.
Both aspartic acid and glutamic acid are subsequently converted to the cor-
responding amides asparagine and glutamine by acyl phosphate substitution
reactions using ammonia as the nucleophile, as shown in Fig. 3.59. The acyl
phosphates are derived from ATP but differ from one another in that the acyl
phosphate intermediate from aspartic acid is connected to adenosine by a mono-
phosphate bridge, while the glutamic acid phosphate is an acyl monophosphate.
The amide function of glutamine is as an amino group donor in numerous other
biosynthetic processes and provides the nitrogen used here as well as serving as
a general storehouse for NH3 derived from other processes; glutamine synthetase,
the enzyme responsible for glutamic acid amidation, clearly plays a key role in
nitrogen metabolism regulation.
In addition to alanine, pyruvic acid is also a direct precursor of valine and leucine
and provides two of the six carbons of isoleucine. For both valine and isoleucine,
the usual TPP-bound intermediate from pyruvic acid decarboxylation serves as a
nucleophilic 2-carbon fragment. In the case of valine, the electrophile is a second
equivalent of pyruvic acid while for isoleucine, this role is played by a degradation
product of threonine, α-ketobutyric acid (Fig. 3.60). The key step in both cases is
an α-ketol-type 1,2-alkyl migration, a common base-catalyzed rearrangement reac-
tion of α-hydroxyketones. Reduction to the respective 1,2-diols followed by dehy-
dration gives enols which tautomerize to the corresponding α-ketoacids which are
then converted to the α-amino acids by the usual transamination process.
Interestingly, the same α-ketoacid intermediate that undergoes transamination
to valine may also participate in an aldol condensation via the enolate of acetyl-
CoA. Hydrolysis and dehydration gives an α,β-unsaturated 1,4-dicarboxylic acid
which then undergoes conjugate addition of water to give a β-hydroxy-1,4-dicar-
boxylic acid. NAD+ oxidation of the hydroxyl group gives a β-keto-1,4-dicarboxylic
acid which decarboxylates to the α-ketoacid. A final transamination then affords
leucine. Much of the chemistry involved in biosynthesis of the remaining amino
acids is more specialized and will be encountered later in Chapters 6 and 7 when we
O AMP O O
HO O H2N
OH OH OH + AMP
O NH2 O NH2 O NH2
ATP PPi
aspartic acid NH3 asparagine
O O O O O O
HO OH PO OH H2N OH + Pi
NH2 NH2 NH2

ATP ADP NH3
glutamic acid glutamine
FIGURE 3.59
Conversion of aspartic and glutamic acids to asparagine and glutamine.

HB
R' :B
O :B
BH CO2H S H
S O N H O
R
OH O CO2H
N R
OH R
R R = CH3: pyruvic acid TPP ylide α -ketol
R CO2H
R = CH3CH2: α−ketobutyric acid rearrangement
BH
R
OH O
CO2H trans- O HO HO
amination R enol to keto
R CO2H CO2H
NH2 CO2H H R
NADH NAD +
valine O H2O
(R = CH3) :B
isoleucine (R = CH3) CH2CSCoA
(R = CH3CH2) (aldol, then
H2O, - HSCoA)
H2O
BH OH
CO2H OH
CO2H
HO2C HO2C CO2H
HO2C H H2O NAD +
:B BH
NADH/H+
trans- O
CO2H O
amination
CO2H
NH2
HO2C CO2H
leucine CO2
a β-ketoacid
FIGURE 3.60
Conversion of pyruvic acid and α-ketobutyric acid to valine, leucine, and isoleucine.


look at both the shikimic acid pathway and the fascinating chemistry involved in the
biosynthesis of some of the major plant alkaloids.
PEPTIDES AND PROTEINS: A VERY BRIEF REVIEW
While we need not labor over peptides and proteins for the purposes of this text, it
will be helpful to at least briefly review how amino acids bond with one another to
produce these important biomolecules, not only to better appreciate the essential
connection between protein shape and enzyme catalytic action, but to also recog-
nize that peptides and proteins may bond with other organic structures such as car-
bohydrates to produce biologically significant “hybrid” biopolymer structures.
Like saccharides, peptides may be classified according to the number of mono-
mer units linked together to form them by using the usual prefixes (di-, tri-,
tetra-, etc.). Thus, a tripeptide is made up of three amino acids linked together by
peptide bonds which, of course, are simply amide linkages derived from the amino
group of one amino acid and the carboxyl group of another. However, unlike many
polysaccharides such as cellulose or glycogen, polypeptides are not usually made up
of a single monomer as the repeating unit, making peptide structures and proper-
ties more complex and dependent on the specific sequence of amino acids pres-
ent. For example, using the three simple amino acids alanine (Ala), glycine (Gly),
and phenylalanine (Phe), a total of six distinctly different tripeptides may be as-
sembled: Ala-Gly-Phe, Ala-Phe-Gly, Gly-Ala-Phe, Gly-Phe-Ala, Phe-Ala-Gly, and
Phe-Gly-Ala (Fig. 3.61). Taking into account the possibility of using any one of the
three amino acids more than once in a sequence leads to even more possible tripep-
tide structures. In all peptides, there will be one amino acid whose amino group is
O
O O
O N
OH H2N + OH H
+ OH NH2 amide
NH2
resonance
Ala Gly Phe
O
peptide (amide) bonds N
H
C-terminal AA
O O O
H O O O
N H H
N OH N H2N N
H N N OH
NH2 O OH H
H O
N-terminal AA NH2 O
Ala-Gly-Phe Ala-Phe-Gly Gly-Ala-Phe
O O O O O O
H H H
H2N N N N
N OH N OH N OH
H H H
O NH2 O NH2 O
Gly-Phe-Ala Phe-Ala-Gly Phe-Gly-Ala
FIGURE 3.61
Amino acid sequences of six different tripeptides, each derived from Gly, Ala, and Phe.

unbound, known as the N-terminal amino acid and another whose carboxyl group

is unbound, known as the C-terminal amino acid. Peptide amino acid sequences
are usually read and drawn from left-to-right, with the N-terminal AA at the left
end and the C-terminal AA at the right end. It is also useful to recall that since pep-
tide bonds are actually amide linkages, amide resonance plays an important role in
the 3-D structure of most polypeptides and proteins due to the restricted rotation
around peptide C–N bonds, all of which have a substantial degree of π-bond charac-
ter. The resulting staggered or “zigzag” structure of peptide chains, as represented in
the tripeptides shown, has a specific geometry that places the N–H and C=O bonds
of peptide linkages in a “trans” orientation relative to one another, again the result of
the partial π-bond character of amide C–N bonds.
Peptides and proteins are generally linear polymers, though some cyclic peptides
are of particular interest and will be seen in later chapters. Various polypeptides can
have a variety of different functions, acting as enzymatic catalysts, neurotransmit-
ters, hormones, cellular structural components, or even sometimes toxins. This wide
variation in biological function is always dependent on the overall 3-D shape of
the protein, which in turn is determined exclusively by the original amino acid se-
quence, often referred to as the primary structure of the protein, a structural desig-
nation which may also include any disulfide linkages (or disulfide bridges) which
may be present. Such linkages can form when cysteine thiol groups located at vari-
ous positions within a peptide chain become bonded to one another (or to thiol
groups in another chain) by the oxidative transformation given by RSH + RSH →
RS–SR. Since the reaction is reversible, disulfide linkages may be broken reductively
to regenerate the original thiol groups. As shown in Fig. 3.62, the connections pos-
sible are nicely illustrated by the peptide hormone insulin which consists of two
separate peptide chains A (with 21 amino acids) and B (with 30 amino acids) linked
together by disulfide bridges at two different points. Note also that the A-chain con-
tains an internal disulfide link between two cysteine residues fairly close together
N-terminus 21 AA's
N-terminus (A-chain)
(A-chain)
C-terminus
C-terminus (A-chain)
(A-chain)
S SH SH
S
reduction C-terminus
C-terminus HS
S HS (B-chain)
S (B-chain) oxidation
HS SH
S S
N-terminus 30 AA's
N-terminus
human insulin hormone (B-chain)
(B-chain)
FIGURE 3.62
Insulin molecule composed of two polypeptide chains A and B, cross-linked by disulfide bridges.

in the chain. We thus see that in order to correctly determine the primary structure
Bioorganic Synthesis 122 (amino acid sequence) of any peptide that contains disulfide linkages, it will first
be necessary to reductively break the disulfide linkages; only then can the actual
sequence of the chain be established. It is for this reason that disulfide linkages are
often included as components of primary peptide structure, since these are true co-
valent bonding interactions, though these links may also be considered as part of the
tertiary structure of proteins, as we will see shortly.
Further refinement of protein shape is determined by secondary structure, a
result of H-bonding interactions between peptide bond N–H and C=O functions
fairly close to one another within the chain. The zigzag structure of peptide chains,
as represented earlier in Fig. 3.61, minimizes steric interactions between amino
acid side chains but also allows for extensive H-bonding interactions. The two
principal types of secondary structure in proteins that result from such H-bonding
are the α-helix and the β-pleated sheet, either of which will be important in de-
termining the final overall shape of a given protein, which in turn will determine
its biological properties and function. The α-helix illustrated in Fig. 3.63 shows
the preference for a “right-handed” twist in the helical arrangement in proceeding
from the N-terminus to the C-terminus (the mirror image of such a helix would
be “left-handed”) for proteins derived from naturally occurring L-amino acids
(unnatural D-amino acid-derived proteins would prefer a left-handed helix). For
natural proteins, this “telephone cord” helical arrangement maximizes H-bonding
interactions between the amino acid N–H and C=O functions; note also that the
amino acid side chains (R groups) always project outward from the backbone of
the helix (Fig. 3.63).
As shown in Fig. 3.64, the β-pleated sheet form of secondary protein structure
can involve C=O and N–H hydrogen bonding interactions in either a parallel or an
antiparallel fashion and may involve intermolecular interactions between separate
peptide chains or the formation of pleated sheet regions within a large polypeptide
(and therefore a protein) which has folded back upon itself.
H
C
N R
O H R
R
C N C H
OH O N
N C
H R
R C O R H
N
O C
N
H
O R
C
N
R O
FIGURE 3.63
Peptide chain secondary structure with α-helix and right-hand twist.


O R
R
N R R
R
H R
O N R
O C R R R
H R R R
N R R
R O N R
H C R
H R
R N
O R O R
C H R
parallel β-pleated sheet R
N R
H R
R
R
O R
R R
N R
R
H O N R
C R R R
O H R R R
O C R R
N R R
N R
H H R
R N R
R O
O C H R
R
N antiparallel β-pleated sheet R
R
H
FIGURE 3.64
Comparison of C=O and N–H hydrogen bonding interactions in parallel vs. anti parallel β-pleated
sheets.
Some proteins are fibrous in nature and may be elastic, due to the spring-like
behavior of helical structure H-bonding, or inelastic pleated sheets which have
H-bonding essentially perpendicular to the directionality of the chain backbone.
However, most proteins are termed globular in shape; such a spherical 3-D confor-
mation is due to multiple levels of structural interaction.
The third level of protein structure, known as tertiary structure, is associated
with the unique overall 3-D shape of a protein. This shape results from the character-
istic folding of a given protein due to noncovalent interactions between amino acid
side chain groups often far removed from one another in the amino acid sequence.
These interactions may include: i) salt bridges which are pH-dependent attractive
interactions between positively and negatively charged ionic functions contained
within certain amino acid side chains; ii) H-bonding interactions between neutral
hydrophilic side chains containing C=O, N–H, O–H, or S–H groups; and iii) weak
van der Waals interactions between hydrophobic hydrocarbon side chain groups
(Fig. 3.65). Disulfide bridges are also sometimes included in tertiary structure since
they involve interactions between amino acid side chains that significantly affect
overall protein shape, though these are covalent rather than noncovalent interac-
tions and are therefore much more robust.
Quaternary structure represents an additional level of protein complexity that
can arise in certain cases when two or more separate chains interact to form an over-
all aggregate protein unit with specific functionality that is present only when this
higher order of assembly is employed. For all proteins, their unique nooks, crannies,


O H2C X O C
H2C X
H 2C C H H N H
O
O C X CH2
X CH2 H
H3N CH2 HN
H
a salt bridge some hydrophilic H-bonding interactions (X = O, N, S)
some hydrophobic hydrocarbon side-chains for van der Waals interactions
helical
pleated
regions
region
side-chain
interactions
FIGURE 3.65
Examples of different amino acid side chain interactions within an overall protein structure.
and surfaces can allow binding or interaction with other molecules, giving enzymes
their characteristic catalytic activity as a result of the subtle interplay of side chain
inter-and intramolecular forces that ultimately produce the three-dimensional
shape of these biopolymers.
The foregoing represents a bare minimum of some of the essentials of an ex-
traordinarily complex area of study. For now, we’ll leave the treatment of the organic
mechanisms associated with the biosynthesis of peptides to a later chapter dealing
with some specific examples of peptide-based natural products and turn finally to
some concluding examples of how carbohydrates, amino acids, peptides and pro-
teins can further interact with one another in biologically significant ways.
PUTTING PROTEINS AND CARBOHYDRATES

TOGETHER: GLYCOPROTEINS VERSUS PROTEIN
GLYCOSYLATION
We know that carbohydrates can have electrophilic components (an aldehyde car-
bonyl or an activated anomeric leaving group) while amino acids, peptides, and
proteins have many potential nucleophilic components (either N-terminal amino
groups or side chain nitrogens or oxygens), so it should not be surprising that these
two separate groups of biomolecules can interact with one another to form hybrid

structures in some instances. We finish off this chapter by taking a brief look at some

examples of these complex and important biopolymers.
When linkages between carbohydrates and proteins are formed in an enzyme-
controlled process, a hybrid structure known as a glycoprotein is formed. The link-
age is usually one in which a nucleophilic atom from a protein is attached to the
anomeric center of the carbohydrate component via a UDP-glucose-type nucleo-
philic substitution. The final carbohydrate component is usually an oligosaccharide
and can play a critical role in protein folding and function. These interesting struc-
tures are normally divided into three groups: 1) N-linked, in which the carbohydrate
component is linked to the nitrogen of the amide side chain of an asparagine residue
in the protein; 2) O-linked, in which the carbohydrate is linked to the hydroxyl
side chain of a serine or threonine residue in the protein; and 3) C-linked, in which
the carbohydrate is linked to an indole ring carbon of a protein tryptophan residue
(C-linked glycoproteins are rare compared to N-and O-linked). The basic compo-
nents of the three are outlined in Fig. 3.66.
In membranes, glycoproteins can play an important role in interactions between
cells. They also appear as structural features in connective tissues and are especially
important components of the immune system. One interesting example is the group
of glycoproteins involved in designation of blood types, as shown in Fig. 3.67. The
disaccharide shown is connected to a carbohydrate hydroxyl component of a glyco-
protein, with A, B, and O blood types being distinguished by the substitution on the
C-3 oxygen of the galactose component. Connection at this position to an N-acetyl
galactosamine (Type A), a galactose (Type B), or simple termination in a –OH group
(Type O) distinguishes the three blood group antigens.
XO
XO O
OH
XO HO
HO O OX
O O protein
α-(1,6)- O O NH
mannose HO O HO O O H
β-(1,4)- N
NHAc HO N
mannose asp
HO β-(1,4)- NHAc H O
XO glucosamine β-(1,N)-
O
XO OX glucosamine
Core of N-linked glycoproteins
α-(1,3)-
(X = H or additional
mannose
monosaccharide)
OH
OH OX
HO
HO O
O HO O
XO protein
NHAc NH α-(1,C)-
H N
mannose HN
α-(1,O)- O N H
galactosamine HN protein
R O
trp
Core of most O-linked glycoproteins
(X = H or additional monosaccharide; Core of C-linked glycoproteins
R = H, serine, or R = CH3, threonine)
FIGURE 3.66
Core components of N-, O-, and C-linked glycoproteins.

OH OH
β-(1,O)-
X O galactose Type A: R = glycoprotein; X = α-(1,3)-N-acetyl galactosamine
O O
O R Type B: R = glycoprotein; X = α-(1,3)-galactose
O OH
Type O: R = glycoprotein; X = H
OH α-(1,2)-fucose
OH
FIGURE 3.67
Glycoproteins and core structural components of type A, B, and O blood group antigens.
O protein N HN HN
amino group HN
CH H N CH CH CH2
2 CH2
HC OH HC OH C OH C O ketone to C OH
- H2O imine to enol to enediol
HO CH HO CH HO CH HO CH H-O C
HC OH HC OH enamine HC OH ketone HC OH HC OH
HC OH HC OH HC OH HC OH HC OH
R R R R R
carbohydrate Amadori Rearrangement Amadori Product loss of
Hydroxyacetone H 2N
Hydroxyacetaldehyde retro-aldols CH3
CH3
Acetoin, Acetaldehyde CH2
C O C O
Biacetyl, Glyoxal,
ketone to R OH
H 2S NH3 Pyruvaldehyde enediol O C enol to
HO C
H 2S O C
furans, thiophenes C OH HC OH
ketone
pyrroles NH3 HC OH
HC OH HC OH
Pyrazines HC OH
aldehydes, CO2 amino R R
Pyridines
α-aminoketones acids a dehydro- R
Oxazoles a reductone
NH3, H2S Strecker reductone
Thiazoles
Degradation
FIGURE 3.68
Maillard reaction of carbohydrates and proteins to produce food flavor and fragrance compounds.
The relatively recent interest in glycoproteins was partly inspired by early 20th
century studies by Louis Camille Maillard (1878–1936) of the reactions between car-
bohydrates and proteins that occur during the cooking of foods. The Maillard reac-
tion, as it is now known, is actually a complex series of reactions between protein
N-terminal or side chain amino groups and reducing sugars that results in a form of
“browning” that produces a vast array of different flavor and fragrance compounds
associated with baking, frying, brewing, and other high-temperature cooking pro-
cesses (Fig. 3.68).
Unlike glycoprotein formation, the Maillard reaction is an example of nonenzy-
matic protein glycosylation or glycation, a process that begins with simple imine
(Schiff ’s base) formation followed by an Amadori rearrangement which is akin to
enediol formation but involves an α-hydroxyimine rather than an α-hydroxy alde-
hyde or ketone. The resulting Amadori compounds may then break down further
via loss of the amino function as shown in Fig. 3.68 to form so-called reductones

and dehydroreductones which can then undergo a variety of further rearrange-

ments, retroaldol reactions, condensation reactions with simple amino acids and
various cyclizations to produce more complex flavor and fragrance products that
may be characteristic of the type of food or beverage involved, the cooking process
or other factors such as temperature and pH.
In biological systems, nonenzymatic Maillard-type processes have been impli-
cated in protein–carbohydrate reactions that result in the formation of so-called
advanced glycation end-products or AGEs. It turns out that many cells in the
body bear a receptor (commonly referred to as RAGE) for AGEs that, upon binding
such products, is believed to contribute to various age-and diabetes-related chronic
inflammatory conditions including atherosclerosis, asthma, and arthritis, some
of which may result from loss or modification of normal protein function due to
Maillard nonenzymatic glycation processes. Investigation of the health-related im-
plications of the biological Maillard reaction is bound to be an area of active research
interest in the years ahead.
LOOKING AHEAD
For now, we have accumulated enough organic review and foundational material
in bioorganic reactions, carbohydrates, and amino acids to take the next step in our
journey through the organic chemistry of the major biosynthetic pathways, starting
with the vast array of significant terpenoid natural products covered in the chapter
that follows.
STUDY PROBLEMS
1. Two D-
aldopentose carbohydrates give optically inactive (achiral) products
when treated with sodium borohydride in ethanol. Which ones and why?
2. Most aldose monosaccharides and disaccharides will react with certain mild oxi-
dizing agents in aqueous solution and are thus known as “reducing sugars.”
a) At which carbon would such oxidations most likely take place in β-D-
glucopyranose? In maltose?
b) Neither methyl-α-D-glucopyranoside nor sucrose are reducing sugars. Explain
why not.
3. Draw Haworth and conformation diagrams for each of the following:
a) methyl-β-D-lyxofuranoside
b) L-cellobiose
c) β-D-arabinofuranosyl-α-L-arabinofuranoside.
4. A proposed mechanism for conversion of UDP-1-glucose to UDP-1-galactose
involves a 3-keto-UDP-1-glucose derivative. Suggest an overall mechanism for
the conversion.

O O-UDP

HO
UDP-1-glucose UDP-1-galactose
HO OH
O
5. Provide a likely mechanism for the laboratory reactions shown:
KOH, CH3OH
a)
HO O HO
O
OH
OH
O
HO
HO O
O
b) H+
O OH
HO OH
O
OH
NaNO2/HCl CO2H
c) HO2C CO2H
H
O
NH2 O
6. While achiral, citric acid is a prochiral compound, since a specific chemical

change applied to one of the “wings” attached to the central carbon of the struc-
ture would produce an R enantiomer while the same change applied to other
wing would produce the S enantiomer.
a) Label the pro-S and pro-R wings of citric acid.
b) When citric acid is prepared by reaction of oxaloacetic acid with 13C-labeled
acetyl-CoA (labeled at its carbonyl carbon), subsequent reactions of the citric
acid cycle convert it to only one of the two 13C-labeled α-ketoglutaric acid
structures shown. If the enzyme aconitase which catalyzes the anti elimination
dehydration step is completely selective for the pro-R wing of citric acid, which
of the two labeled α-ketoglutaric acid structures A or B would be produced?
HO CO2H
HO2C CO2H
citric acid
i) anti elimination (dehydration)

HO CO2H ii) anti addition (hydration) *CO2H HO2C *CO2H
HO2C
HO2C *CO2H B
iii) NAD+ oxidation A vs.
O O
13C-labeled citric acid
iv) decarboxylation 13C-labeled α-ketoglutaric acid

c) The methylene carbon of the pro-R wing of citric acid has two H atoms;

one is pro-R and one is pro-S. When the pro-R wing of citric acid undergoes
anti elimination of water to produce cis-aconitic acid, which H atom was
removed in the elimination, the pro-R or the pro-S proton? Explain.
7. Cleavage of the aminoacid A shown employs a pyridoxal-5’-phosphate (PLP)
dependent enzyme that catalyzes the hydrolytic cleavage of the β,γ-carbon-
carbon bond in the amino acid to give anthranilic acid B and L-alanine C.
Propose a chemical mechanism for this transformation using your knowledge
of other PLP dependent reactions.
O OH O
O O
PLP OH
NH2 + H2O OH
enzyme NH2
NH2 NH2
A B C
8. Glucosamine 6-phosphate is normally produced by enzyme-mediated NH3

transfer from glutamine to fructose-
6-
phosphate. Propose a reasonable
mechanism.
PO H OP
CH2OH
O NH3 H O
H OH HO
-H2O HO OH
H OH H
NH2
HO H H H
9. In a polypeptide with an α-helical structure, the α-helix would be considered a

part of which of the polypeptide’s structures?
a) primary structure
b) secondary structure
c) tertiary structure
d) quaternary structure
10. The planar peptide linkage in proteins and peptides has restricted rotation about
the C–N bond. The reason for this restricted rotation is mainly due to which of
the following?
a) the presence of disulfide linkages in the polypeptide
b) the presence of H-bonding to the carbonyl oxygens
c) the presence of amide resonance which gives peptide bonds some partial
π-bond character
d) the presence of bulky amino acid side chains which sterically interact with
one another
11. The major form of D-glucose present in aqueous solution is:
a) the open-chain hydroxyaldehyde form

b) α-D-glucopyranose
Bioorganic Synthesis 130 c) β-D-glucopyranose
d) β-D-glucofuranose
12. Explain the principal structural difference between amylose (from starch) and
cellulose. Use appropriate structural diagrams to illustrate your explanation.
13. Acid-catalyzed reaction of D-maltose with excess methanol yields a mixture of
products. Draw the structures of those products.
14. Side chains of proteins or peptides are frequently involved in H-bonding or
ionic bonding with other side chains. What level of protein structure do these
interactions involve?
a) primary structure
b) tertiary structure
c) secondary structure
d) quaternary structure

4 The Terpenoid Pathway

Products from Mevalonic Acid
and Deoxyxylulose Phosphate
But behind all this there looms a vast new problem, in comparison to which the one already
solved seems quite small: the problem, what kind of chemical processes in the plant organ-
ism cause the formation of essential oils. How can we explain their infinite variety?
—Otto Wallach (Nobel Prize in Chemistry, 1910)
It was Otto Wallach (1847–1931) who first coined the term “terpene” and made the
observation that many plant-derived essential oils had chemical structures whose
composition was based on multiples of a basic five-carbon unit. His work with tur-
pentine and the organic products derived from it was consistent with earlier studies
of natural rubber which had shown that its thermal decomposition released “iso-
prene” (2-methyl-1,3-butadiene) as the principal product (Fig. 4.1). This led eventu-
ally to the formulation of the so-called biogenetic isoprene rule of Leopold Ruzicka
(1887–1976) in 1953 which stated that “the carbon skeleton of the terpenes is com-
posed of isoprene units linked in regular or irregular arrangement.”
As it turns out, biosynthetic pathways to terpenes are found in nearly all organ-
isms, producing a remarkable variety of different structural types, as we will soon
see. In fact, something in excess of over 25,000 different terpenes with a wide va-
riety of biological functions have been isolated from the plant kingdom over the
years. Interestingly, while many terpenes are simple achiral compounds, others are
chiral as can be seen in the case of α-pinene in Fig. 4.1. But unlike the naturally oc-
curring L-amino acids and D-carbohydrates, different organisms may produce the
same terpene product but in different enantiomeric forms. For example, limonene
is formed by more than 300 plants, with the (+)-(R) enantiomer being the most
131


2 or
isoprene
(-)-α−pinene (+)-α−pinene
FIGURE 4.1
Isoprene units in α-pinene, a component of “terpentine” obtained from pine tree resin distillation.
strong scent vs. strong scent

of oranges of pine
(+)-(R)-limonene (-)-(S )-limonene
FIGURE 4.2
Enantiomers of limonene, the principal component of orange oil.
widespread form as the major constituent of citrus peel essential oils (orange oil).
As the most abundant of all terpenes, its pleasant citrus fragrance and flavor have
led to its worldwide use in the food and fragrance industries and also as a botanical
insecticide. A number of plants produce both enantiomers of limonene, while others
produce only the (−)-(S)-enantiomer which possesses a strong pine smell reminis-
cent of turpentine. This obviously speaks to the chirality and enantioselectivity of
our own olfactory receptor sites which can readily distinguish between the two en-
antiomers, thus signaling a different odor response in each case (Fig. 4.2).
As with the carbohydrates, any detailed examination of a large and widely vari-
able family of compounds such as the terpenes must begin with a systematic classi-
fication of the principal structural types that are encountered in nature. From there,
we may then move forward to begin to understand how members of each group are
constructed, modified, and elaborated into a vast array of organic natural products
of extraordinary structural complexity, diversity, and biological significance.
CLASSIFICATION OF TERPENES: HOW
MANY ISOPRENE UNITS?
Terpenes are usually classified into one of seven basic families according to the
number of individual isoprene units employed in their basic construction (Table 4.1).
Polymeric terpenes such as natural rubber or gutta-percha are of variable chain
length and so do not truly represent a distinct terpene family.
Within these families, the vast majority of compounds are found to be so-called
“regular” terpenes, those in which the isoprene units are linked in a “head-to-tail”
fashion (Fig. 4.3). As the name implies, the far less abundant “irregular” terpenes

Table 4.1 General Classifications for Terpenes

Terpene family name # of isoprene units # of carbon atoms # of rings possible
Hemiterpenes 1 5 0
Monoterpenes 2 10 0,1,2
Sesquiterpenes 3 15 0,1,2,3
Diterpenes 4 20 0,1,2,3,4
Sesterterpenes 5 25 0,1,2,3,4,5
Triterpenes 6 30 0,1,2,3,4,5,6
Tetraterpenes 8 40 Various
Rubber/gutta-percha >100 >500 —
are those which have their isoprene units linked in some fashion other than head-
to-tail. Beyond this, terpenes may be further subdivided into alicyclic, cyclic, bicy-
clic, tricyclic, and so on, according to the number of rings that may be present in a
given compound from a particular family. The term “terpenoid” is sometimes used
interchangeably with terpene, though the latter usually implies a hydrocarbon struc-
ture while the former ordinarily refers to modified terpenes containing one or more
oxygen-containing functional groups such as alcohols, aldehydes, esters, or ethers.
Among the examples of monoterpenes shown in Fig. 4.3 are menthol and cam-
phor, familiar and highly fragrant terpenes derived from mint oils with mild an-
esthetic properties that make them both common ingredients in throat lozenges,
cooling gels, and vaporizer additives. The sesquiterpene examples include farnesol,
a natural antibacterial agent and pesticide, and zingiberene, a fragrant component
of ginger oil. The diterpene alcohol vitamin A (retinol) is essential to the eye for
low-light and color vision in its aldehyde form (retinal) and is also a cellular growth
factor as the corresponding carboxylic acid (retinoic acid). Taxadiene is the im-
portant terpene precursor to the terpenoid anticancer drug taxol, widely used in
the treatment of breast, ovarian, lung, head, and neck cancers as well as the AIDS-
related Kaposi’s sarcoma. The pentacyclic diterpene cafestol, an aroma compound
found in Arabica coffee beans, has also been shown to possess some anticancer ac-
tivity and may also have the ability to inhibit the progress of Parkinson’s symptoms.
Some examples from the remaining families of terpenes are shown in Fig. 4.4. Of
these, the sesterterpenes such as the plant toxin ophiobolin A are relatively rare,
being produced mainly by fungi and marine organisms. Conversely, the triterpenes
are widely distributed in nature, with squalene serving as the essential terpene pre-
cursor to all the steroids, including cholesterol and the sex hormones (note that
there is a single nonhead-to-tail isoprene linkage in squalene). The tetraterpenes,
also known as carotenoids, include a vast array of biologically significant pigments
such as β-carotene and related structures, but unlike the triterpenes, these com-
pounds are not biosynthesized in animals and so must be obtained in the diet. The
polyterpenes are represented by the two isoprene polymers, natural rubber (from
rubber trees) and gutta-percha (dental rubber), differing from one another only in


Hemiterpenes:
C5 - (one isoprene unit) OH OH OH
isoprene dimethylallyl alcohol isopentenyl isovaleric acid
(prenol) alcohol
"head"
"tail"
Monoterpenes:
C10 - (two isoprene units)
OH
alicyclic:
OH
geraniol linalool
mono- or bicyclic: OH
OH
camphor
α−terpineol menthol
Sesquiterpenes:
C15 - (three isoprene units)
alicyclic: OH
farnesol
H
mono- or bicyclic: H
H
zingiberene caryophyllene
Diterpenes:
C20- (four isoprene units)
OH
monocyclic:
vitamin A
OH
OH
polycyclic: H
O
H cafestol
taxadiene
FIGURE 4.3
Head-to-tail isoprene linkage and some hemi-, mono-, sesqui-, and diterpene natural products.
the geometry of their isoprene linkages, that is, cis-1,4 for latex versus trans-1,4 for
gutta-percha. Both are natural forms of polyisoprene with similar physical proper-
ties; gutta-percha is less common but notable as a “bioinert” material compared to
natural rubber, which can elicit a latex allergy response in some individuals. It is for
this reason that gutta-percha is used for certain kinds of dental fillings and in some
surgical equipment.
Now that we have a better idea of what terpenes look like and how they are clas-
sified, we can now take a closer look at exactly how these important compounds


O
O
H
Sesterterpenes:
H
C25 - (five isoprene units)
H O
OH H
ophiobolin A
Triterpenes:
C30 - (six isoprene units)
squalene
Tetraterpenes:
C40 - (eight isoprene units)
β-carotene
Polyterpenes:
(>100 isoprene units)
H H H H H
natural rubber (cis-1,4)
H H H H H
gutta-percha (trans-1,4)
FIGURE 4.4
Examples of some sester-, tri-, tetra-, and polyterpene natural products.
are constructed. In fact, we will see that products of the terpene pathway are not
actually constructed from individual units of isoprene, but rather from two closely
related building blocks, dimethylallyl diphosphate (DMAPP) and isopentenyl di-
phosphate (IPP), both of which are produced by two distinctly different routes in
different organisms.
THE MEVALONIC ACID ROUTE TO DMAPP AND IPP
The mevalonic acid route begins with intermediates we have encountered before, as
shown in Fig. 4.5. The process begins in step 1 with a Claisen condensation between
malonyl-CoA and acetyl-CoA to produce acetoacetyl-CoA. This is followed by step 2
in which acetoacetyl-CoA, converted to an enzyme-bound thioester, undergoes attack


malonylSCoA
O O BH SCoA
O O O O
O SCoA 1 HSEnz
SCoA SEnz
SCoA acetoacetylSCoA
CO2, CoASH CoASH
2
O
acetylSCoA
O OH O H2 O O OH O
3
HO SCoA EnzS SCoA
NADPH/ β-hydroxymethylglutarylCoA HSEnz
H+ (HMG-CoA)
O OH OH O OH O O OH
4
HO SCoA HO H NADPH/H+ HO OH
H
CoASH mevaldic acid (3R)-mevalonic acid
(MVA)
2 ATP
5
AMP, ADP
BH :B
O OP
OPP OPP 6
isomerase H
HS HR HS O OPP
dimethylallyl diphosphate isopentenyl diphosphate CO2, -OP
(DMAPP) (IPP)
FIGURE 4.5
Biosynthesis of the terpene precursors IPP and DMAPP via mevalonic acid.
by acetyl-CoA enolate at the ketone carbonyl in a simple aldol addition process. After
hydrolysis and loss of EnzSH, the resulting β-hydroxymethylglutarylCoA then un-
dergoes NADPH reduction of the thioester to a hemithioacetal in step 3 which suf-
fers loss of HSCoA to yield the corresponding aldehyde, mevaldic acid. A second
NADPH reduction of the aldehyde to the primary alcohol in step 4 yields the impor-
tant intermediate product, (3R)-mevalonic acid (MVA) from which this particular
route to DMAPP and IPP takes its name. In step 5, the tertiary and primary alcohols
are converted to mono-and diphosphate derivatives, respectively, by the action of
2 ATP. A final decarboxylation-driven elimination of the monophosphate as a leaving
group in step 6 produces isopentenyl diphosphate (IPP) which is then converted to
dimethylallyl diphosphate (DMAPP) by the action of an isomerase enzyme via what
is believed to be an intermediate carbocation. Note the stereospecificity of this inter-
conversion in which only the pro-R proton of IPP is lost in the formation of DMAPP.
THE DEOXYXYLULOSE PHOSPHATE ROUTE

TO IPP AND DMAPP
Unlike the mevalonic acid route, which Ruzicka first described in the early 1950s and
which operates in animals, plants, bacteria, algae, fungi, and protozoa, the deoxyx-
ylulose phosphate route to IPP and DMAPP, discovered only in the early 1990s,

HB

R' R' R'
R'
O
- CO2
OH S S S
N N N
S H3C R OH R R
N
R O O
H OH OH
H3C H3C H3C
O :B B
H
HC=O
H OH
CH2OP
R'
CH3 glyceraldehyde-3-P
:B
R' O S
N H
HO H R
S O
N + H OH H3C
R HO H
CH2OP
TPP ylide 1-deoxyxylulose-5- H OH
phosphate CH2OP
FIGURE 4.6
Biosynthesis of 1-deoxyxylulose-5-phosphate from pyruvic acid, glyceraldehyde-3-P, and
TPP ylide.
has been found to operate widely in bacteria and in some algae, plants, and proto-
zoa but not in animals or fungi, though some organisms have been found to utilize
both pathways for terpene biosynthesis. Also unlike the mevalonate route, whose
details have been extensively studied over many years, a number of the late steps as-
sociated with the deoxyxylulose phosphate route remain obscure or are not entirely
understood. What is known for certain is that the process begins with biosynthesis
of 1-deoxyxylulose-5-phosphate from TPP ylide, pyruvic acid, and glyceraldehyde-
3-phosphate, as shown in Fig. 4.6.
As shown, the nucleophilic TPP-bound dipolar intermediate from decarboxyl-
ation of pyruvic acid attacks the aldehyde carbonyl of glyceraldehyde-3-P derived
from glycolysis. The resulting tetrahedral intermediate is then deprotonated with sub-
sequent expulsion of TPP ylide and generation of 1-deoxyxylulose-5-phosphate in
a process similar to that seen earlier in the generation of sedoheptulose-7-phosphate
from ribose-5-phosphate in the pentose phosphate pathway (see Fig. 3.47).
For the subsequent conversion of 1-
deoxyxylulose-
5-
phosphate to IPP and
DMAPP, much is known of the early steps, as shown in Fig. 4.7. An α-ketol rear-
rangement similar to one seen previously (see Fig. 3.59) in the conversion of pyru-
vic acid to valine begins the sequence (the starred carbons emphasize the 1,2-carbon
migration that occurs in this step). NADPH reduction of the resulting aldehyde gives
2C-methylerythritol-4-phosphate. The details of the next transformation involve
nucleophilic displacement of diphosphate (PPi) from cytidinetriphosphate (CTP)
by an oxygen of the C4-phosphate group of 2C-methylerythritol-4-phosphate to
form a CMP derivative. Conversion of the C2 tertiary –OH to a monophosphate
by reaction with ATP is then followed by an intramolecular nucleophilic displace-
ment of CMP by an oxygen from the C-2 monophosphate group to yield an unusual
cyclic derivative, 2C-methylerythritol-2,3-cyclodiphosphate. Beyond this key

:B CH3 B
H O CH3 CH=O CH2OH
* O
α-ketol OH
H O H * = H 3C OH NADPH H3C OH
rearrangement H
H * OH H * OH H OH H OH
CH2OP CH2OP CH2OP CH2OP

2C-methylerythrose-4-P 2C-methylerythritol-4-P
1-deoxyxylulose-5-
phosphate
CTP
PPi
HO O
OH HO O O O
O P O
P P OH O-P-O-Cytidine
O O O-P-O-Cytidine
HO OH OH HO
O HO P P OH
HO O HO
HO H O O O
B: vs. :B CMP
HO H HO H
2C-methylerythritol-2,4- ATP ADP
cyclodiphosphate
HO OPP
OPP ? IPP
or OH +
OPP
HO
OPP OPP
O H DMAPP
FIGURE 4.7
Known and speculative steps in the conversion of 1-deoxyxylulose-5-P to IPP and DMAPP.

intermediate, not much is known regarding the remaining chemistry involved in its

conversion to IPP and DMAPP. Some speculation centers on two alternative elimi-
nation pathways to yield the terminal diphosphate group, one involving formation
of an enol, the other an epoxide, as shown. In either case, both oxygen atoms must
eventually be lost, perhaps giving rise to the final products via reductive transforma-
tion of an allylic cation, though it is not clear if the products are formed separately or
if IPP is formed first and then isomerized to DMAPP as in the mevalonic acid route.
Now that we have seen how the two basic building blocks of the terpene biosyn-
thetic pathway are formed, we next take a systematic look at how IPP and DMAPP
are combined with one another and subsequently modified in various ways to create
each of the families of the vast array of abundant terpene and steroid natural prod-
ucts that are found in nature, starting with the hemiterpenes.
HEMITERPENES: JUST ONE ISOPRENE UNIT
While isopentenyl alcohol would appear to be a simple hydrolysis product from IPP,
dimethylallyl alcohol probably arises from addition of water to the intermediate car-
bocation after loss of diphosphate from DMAPP. Isoprene is similarly produced by
isoprene synthase-mediated proton loss from the same carbocation (Fig. 4.8).
Aside from its commercial production, an enormous amount of isoprene, the
monomer of natural rubber, is emitted into the atmosphere by many species of trees
and shrubs including oaks and poplars, among others, in a process that is believed
to help moderate heat stress in these plants. Global annual emissions of isoprene
from natural sources rival those of methane, accounting for approximately one-
third of the hydrocarbon content of the atmosphere. When combined with water
and oxygen, isoprene may be converted into a variety of haze-producing derivatives,
some of which are partly responsible for such phenomena as the so-called smoke of
the Smoky Mountains portion of the Appalachian chain. Interestingly, the average
human may produce as much as 17 mg of isoprene daily.
In the United States, the annual market for synthetic isoprene is about two billion
pounds, of which roughly 60% is used in the manufacture of synthetic rubber for
H2O
OPP OH
IPP - PPi isopentenyl
alcohol
H 2O OH
- H+ dimethylallyl alcohol
- PPi (prenol)
OPP
DMAPP - H+
isoprene
FIGURE 4.8
Hemiterpenes from IPP and DMAPP.

automobile tires, with the remainder used in adhesives or specialty chemicals. While
Bioorganic Synthesis 140 most of this isoprene currently comes from petroleum feedstock, the production
of biological isoprene or bioprene via fermentation technologies is rapidly matur-
ing and may eventually supplant petroleum sources for the manufacture of polyiso-
prene; fermentation may also eventually prove to be a viable source of hydrocarbon
building blocks for the manufacture of isoprene-based biofuels.
MONOTERPENES (C 10 ) AND ISOPRENE LINKAGE:

HEADS, IPP WINS; TAILS, DMAPP LOSES
As was mentioned earlier, when isoprene units are linked together, it is not actually
isoprene itself that is involved, but rather DMAPP and IPP units which are linked
together in a specific way, that is, in a head-to-tail fashion. To see how that works, it
helps to recognize that these two components are complementary to one another in
their chemical behavior, with DMAPP serving as an electrophilic component while
IPP serves as a nucleophilic component (Fig. 4.9). Thus, the diphosphate group is
lost as a leaving group from the “tail” of DMAPP, yielding an electrophilic allylic
carbocation which then undergoes nucleophilic attack by the electron pair from the
π-bond at the “head” of the IPP component. The resulting C–C bond-forming reac-
tion produces a tertiary carbocation that is discharged by simple loss of a proton to
yield a new derivative called geranyl diphosphate or geranyl PP or just GPP. This
process is completely analogous to the simple acid-catalyzed cationic dimerization
of isobutylene to yield diisobutylene.
FIGURE 4.9
Linkage of IPP and DMAPP to produce geranyl PP compared to isobutylene dimerization.


OPP would expect a rate
CF3-GPP comparable to DMAPP
F 3C OPP
for a concerted
CF3-DMAPP H H displacement mechanism
:B
- PPi IPP Rel. Rate

OPP GPP 1
- H+
DMAPP stabilized by resonance
- PPi
IPP
F 3C OPP F3C F3C CF3-GPP 1.0 x 10–6
- H+
CF3-DMAPP
destabilized by resonance,
due to inductive effect
FIGURE 4.10
Evidence for carbocation intermediate in DMAPP/IPP linkage via prenyl transferase.
Note that in the linkage of DMAPP and IPP units catalyzed by enzymes called
prenyl transferases, final proton loss to discharge the intermediate cation is specific
for removal of the HR proton and is analogous to the proton loss in isomerization
of IPP to DMAPP. Note also the (E) configuration for the double bond produced in
the GPP product.
The importance of carbocation intermediates in the organic chemistry of ter-
penes cannot be overemphasized, and the linkage of DMAPP and IPP is just one
example, though we might reasonably ask for evidence supporting such an interme-
diate here, since a direct nucleophilic displacement process to produce GPP might
seem just as reasonable (Fig. 4.10). As it turns out, if trifluoromethyl-substituted
DMAPP is used, the rate of conversion to GPP is over a million times slower. This
result makes sense only if a carbocationic intermediate is involved, since no effect
on the rate of GPP production would be expected from a concerted nucelophilic
displacement mechanism. The validity of such an experiment rests in part on the
similarity in size of a CF3 group to a CH3 group which would lead to similar enzyme
binding rates for the two substrates, that is, both would be expected to “fit” in the
active site. But the electronic properties of the two substrates would be vastly dif-
ferent; the rate of formation of a CF3-substituted allylic carbocation should be very
slow due to a highly unfavorable inductive effect.
GERANYL PP TO NERYL PP VIA LINALYL PP: THE

IMPORTANCE OF ALKENE STEREOCHEMISTRY
We noted that DMAPP can readily ionize to an allylic carbocation via loss of di-
phosphate as a leaving group. As we will see, GPP can do the same thing, and
for this reason GPP is an important precursor to a variety of other terpene prod-
ucts. However, the (E) geometry of its double bond limits what can happen to the

GPP-derived carbocation, since such allylic carbocations tend to preserve their ge-
Bioorganic Synthesis 142 ometry. At first glance, this might seem unreasonable, since the allylic carbocation
has a resonance form that would appear capable of isomerization via a simple rota-
tion around a C–C single bond. But we must remember that the actual carbocation
is a resonance hybrid of the two structures we ordinarily draw, meaning that there is
actually some double bond character between all three carbons involved, preventing
a simple C–C bond rotation, as shown in Fig. 4.11.
Note, however, that if after initial loss of –OPP, the tertiary carbon of GPP cation
recaptures the –OPP as a nucleophile, the structurally different product linalyl di-
phosphate (LPP) is produced (Fig. 4.12). Since this tertiary diphosphate is free to
rotate about the C–C bond adjacent to the tertiary center, subsequent ionization
of the less sterically congested conformer leads to an allylic carbocation which, if
(E) geometry no free rotation here
OPP - OPP
= (E) geometry is
maintained in
resonance hybrid
GPP
FIGURE 4.11
Retention of double-bond geometry in allylic carbocation from GPP.
(E) geometry
OPP
OPP
OPP - OPP
GPP GPP cation LPP
(Z) geometry
OPP
OPP
OPP
NPP LPP/NPP cation LPP
FIGURE 4.12
Isomerization of geranyl PP (GPP) to neryl PP (NPP) via linalyl PP (LPP).

trapped again by –OPP at its primary carbon, affords an isomer of GPP with (Z) ste-

reochemistry at its double bond. This isomer is known as neryl diphosphate (NPP).
The significance of this isomerization of GPP to NPP by way of LPP will be evident
shortly when we examine the various transformations possible for each of these de-
rivatives via their corresponding carbocations.
SOME ACYCLIC MONOTERPENES AND THEIR USES
From GPP, a number of different products are possible, as shown in Fig. 4.13. Many
of these are significant flavor or fragrance compounds found in volatile aroma plant
extracts known as essential oils. Few are found in pure form, but rather appear as
complex mixtures which can be challenging to separate. Geraniol is a component
of many different essential oils such as geranium oil, rose oil, and lemon oil, among
others. Citronellol is obtained from geraniol by reduction of the allylic alcohol
double bond and is an example from this group with a chirality center. Both enan-
tiomeric forms are found in nature; the (+) isomer (shown) is a component of lemon
grass oil and is more common, while the (−) isomer is found in rose oil.
O OH OH O
geranial geraniol (+)-citronellol (+)-citronellal

(lemon oil) (geranium oil) (lemon grass oil) (citronella oil)
O
OPP
O
O
GPP neral
(–)-linalyl acetate β-myrcene
(lemon oil)
(lavendar oil) (wild thyme oil)
OH
OPP
OPP OH
(–)-linalool LPP NPP nerol

(basil oil) (rose oil)
FIGURE 4.13
Representative structural relationships among some acyclic monoterpenes derived from GPP.

Oxidation of citronellol produces the corresponding aldehyde citronellal, a com-

Bioorganic Synthesis 144 ponent of citronella oil which has been shown to have significant mosquito repel-
lent activity, hence the use of citronella candles for summer evenings out of doors.
Oxidation of geraniol itself leads to the α,β-unsaturated geranial (also known as
(E)-citral), a component of lemon, orange, and many other essential oils which is
widely used in the flavor and perfumery industries due to its intense lemon fragrance.
Isomerization of GPP to LPP and NPP ultimately leads to similar derivatives. Loss of
diphosphate and a proton from LPP can give different triene hydrocarbons, one of
which is β-myrcene, a fragrant component from wild thyme and hops. The alcohol
corresponding to LPP is known as linalool which, like citronellol, possesses a chi-
rality center. The (+) form predominates in many plants producing it, such as basil
and thyme, while a near racemic mixture is isolated from lemon and grapefruit. The
corresponding linalyl acetate ester is found in lavender oil, among others. Nerol, the
alcohol derived directly from NPP, is found in hops, lemon grass, and neroli oil from
the bitter orange tree. It has a rose fragrance similar to that of geraniol. Its aldehyde
derivative is neral (also known as (Z)-citral), another important flavor and fragrance
compound with a sweet lemon aroma that is less intense than geranial.
MONO- AND BICYCLIC MONOTERPENES VIA CATIONIC

CYCLIZATIONS AND WAGNER–M EERWEIN SHIFTS
As we have seen above, much of the chemistry of the terpenes is dominated by

typical carbocation reactions such as nucleophilic additions, eliminations, or rear-
rangements. Furthermore, we have seen that bimolecular C–C bond formation be-
tween carbocations and alkenes, as in the linking of IPP and DMAP units, is also
commonly encountered. Not surprisingly, ring-forming unimolecular C–C bond-
forming reactions are also quite common for appropriate substrates and rings sizes.
Such simple transformations can lead to a remarkably diverse array of monocyclic
and bicyclic monoterpenoid structures.
Unlike the GPP cation which is geometrically constrained, intramolecular C–C
bond formation by cationic cyclization is readily achievable via the NPP/LPP cation
with its (Z)-configuration, as shown in Fig. 4.14. This cascade of reactions illustrates
some typical transformations possible in the series, though the compounds shown
are by no means an exhaustive catalogue of the many products that can be produced
from this same starting material.
Initial cyclization of the NPP/LPP cation leads to α-terpinyl cation which may
then be quenched by the usual sequences of carbocation quenching reactions.
For many of these compounds, different enantiomers may be produced by differ-
ent sources, so for our purposes the more commonly encountered form is usually
shown. Simple proton loss from α-terpinyl cation affords limonene, a terpene from
citrus fruits with a strong lemon smell that makes it useful as a food additive, as a
fragrance for household products, and as a hydrocarbon from renewable sources


LPP/NPP
- H+ - H+
LPP/NPP cation
(–)-β-phellandrene α-terpinene
- H+ 1,3-H 1,2-H H2O
shift shift
- H+
phellandryl cation 4−terpinenyl cation
- H+ H2O OH
- H+
(+)-α-phellandrene α-terpinyl cation (–)-4-terpineol
OH
(+)-limonene (+)-α-terpineol
FIGURE 4.14
Cationic cyclization and some representative examples of monocyclic monoterpenoids.
that is sometimes used to replace petroleum-based solvents. An unusual 1,3-hydride

shift in α-terpinyl cation may seem unlikely at first, but note that the resulting carbo-
cation is allylic and therefore more stable. Proton loss from different resonance con-
tributors affords α- and β-phellandrenes, both useful fragrance compounds with
peppery-minty aromas. Nucleophilic trapping of α-terpinyl cation by water leads
directly to α-terpineol, which has a heavy floral aroma similar to lilac. Products aris-
ing from a 1,2-H shift of α-terpinyl cation followed by either nucleophilic trapping of
water or simple proton loss are 4-terpineol, with a fragrance similar to α-terpineol,
and α-terpenine with an odor that has been described as “woody.”
Further intramolecular cyclizations of α-terpinyl cation are also possible, lead-
ing to various bicyclic monoterpene derivatives, as shown in Fig. 4.15 using a more
conformationally useful representation of the carbocation.
Initial cyclization occurs by trapping of the isopropyl cation side chain by the
ring π-bond, leading to pinyl cation. For each of the two representations of the pinyl
cation shown, two pathways a and b are possible: path a involves alternate 1,2-C
shift possibilities leading to either fenchyl cation or bornyl cation, while path b
involves alternate modes of β-proton loss for pinyl cation, leading to α-or β-pinene
products. Like isoprene, α-pinene is a significant source of atmospheric hydrocar-
bon, being emitted in substantial quantities by pine trees. Both enantiomeric forms
are produced in nature, with the (−)-form predominant from European pines, and
the (+)-form predominant in North America. The isomeric β-pinene is found in
rosemary, parsley, rose, and hop; both pinenes are major constituents of turpentine.
Nucleophilic trapping of fenchyl cation by water affords the terpenoid alcohol
fenchol, which has a bittersweet, lime-like flavor and is found in lime oil, grape,
nutmeg, and rosemary. Fenchol undergoes oxidation to fenchone which possesses
a distinct camphor-like aroma and is one of the components of absinthe, a potent,

H
b a b
b H
b
- H+ a α-terpinyl cation - H+
(+)-β-pinene pinyl cation pinyl cation (–)-α-pinene
a a
1,2-C shift 1,2-C shift
H2O H2O O
O OH
OH fenchyl cation bornyl cation
(–)-fenchone (+)-fenchol (+)-borneol (+)-camphor
FIGURE 4.15
1,2-C shifts in α-terpinyl cation and some representative bicyclic monoterpenoids.


H H H
- H+
H vs. P450
allylic
oxidation
C-H to C-OH
4−terpinenyl cation thujyl cation (–)-α-thujene (+)-sabinene
H H H H
NADPH
conjugate NAD+
O + O O HO
reduction CHOH to C=O
(–)-α-thujone (+)-β-thujone (–)-sabinone (+)-sabinol
FIGURE 4.16
Cyclization of 4-terpinenyl cation to thujyl cation and some resulting bicyclic monoterpenoids.
light-green alcoholic spirit made from distilled wormwood and anise (also known
as the “Green Fury”) which only recently has returned to popularity after being out-
lawed in most countries for nearly 100 years. Camphor itself is a ketone similar
to fenchone and is derived from oxidation of borneol, the alcohol obtained from
nucleophilic trapping of bornyl cation by water. Camphor, available from many
sources, can comprise as much as 20% by weight of the dried leaves of rosemary and
has many medicinal and culinary uses similar to those of menthol, as mentioned
earlier.
The 4-terpinenyl cation (Fig. 4.14) can also undergo a further intramolecu-
lar cyclization as shown in Fig. 4.16. The initial cyclization is reminiscent of the
homoallyl-cyclopropyl carbinyl cation system we discussed briefly in Chapter 1 and
leads to the so-called thujyl cation. Two fates for thujyl cation involve simple proton
loss to afford either α-thujene, a pungent flavor component of the herb summer
savory, or sabinene, a component of carrot seed oil and black pepper among other
sources. A P450-type allylic oxidation of sabinene leads to sabinol which after NAD+
oxidation to sabinone finally undergoes conjugate addition of hydride to the α,β-
unsaturated carbonyl system to produce α- or β-thujone, both of which are readily
epimerized due to the acidic proton at the chirality center α-to the ketone carbonyl.
As another terpene component of absinthe, thujone was partly responsible for the
formerly illegal status of this spirit due to its supposed dangerous psychedelic prop-
erties. While that role has now largely been discredited, thujone levels in absinthe
are still controlled by law.
WHAT’S THAT SMELL? LIMONENE DERIVATIVES

AS FLAVOR AND FRAGRANCE COMPOUNDS
As shown in Fig. 4.17, limonene undergoes oxidation at two different allylic positions
to give the corresponding alcohol derivatives trans-carveol, a component of both

Bioorganic Synthesis 148 HO
P450 P450
allylic allylic OH
oxidation oxidation
a
(–)-trans- (–)-trans-
(a) C-H to C-H to (b)
carveol isopiperitenol
C-OH C-OH
NAD + CHOH to C=O NAD + CHOH to C=O

b
(–)-limonene
O
(–)-carvone (–)-isopiperitenone
FIGURE 4.17
Biosynthesis of some oxygenated terpenoid derivatives of (−)-limonene.
spearmint and caraway seed oil among others, and also trans-isopiperitenol; both
undergo NAD+ oxidations to the corresponding ketones carvone and isopiperiten-
one as shown. The (−)-enantiomer of carvone (shown) has a strong spearmint odor,
while the (+)-enantiomer has the aroma of caraway seeds; these are frequently in-
voked as prime examples of the ability of the olfactory senses to distinguish between
enantiomeric forms. Isopiperitenone has a herbal fragrance described as sweet or
fruity. All these are useful as flavor or fragrance additives in various commercial
applications.
Isopiperitenone undergoes a series of further transformations that ultimately
lead to the widely distributed peppermint oil component menthol (Fig. 4.18). Initial
conjugate reduction of the α,β-unsaturated ketone function yields cis-isopulegone
which then undergoes an allylic isomerization via its acidic tertiary α-hydrogen to
yield the α,β-unsaturated ketone isomer, pulegone. Conjugate addition of hydride
may lead to the trans-stereoisomer, menthone, while alternate face reduction (or
epimerization of menthone at the tertiary center alpha to the carbonyl) produces
the corresponding cis-isomer, isomenthone. Final addition of hydride to the Si face
of the menthone carbonyl gives menthol, while addition to the Re face gives neo-
menthol. Similar face-selective reductions of isomenthone yield isomenthol and
neoisomenthol. Production of the different stereoisomers is controlled by different
enzymes in different plants at each step, but of all the stereoisomers, (−)-menthol
is the most abundant and it is also the most important, owing to its mild analgesic,
anesthetic, and cooling properties as well as its pleasing aroma.
There are other mono-and bicyclic monoterpenes structures far too numer-
ous to deal with in a brief overview, and new ones are isolated on a regular basis.


NADPH isomeriz- NADPH
conjugate ation conjugate
O reduction O O reduction O
H
B: H-B
(–)-isopiperitenone (+)-cis-isopulegone (+)-pulegone (+)-isomenthone
conjugate C=O to
NADPH NADPH CHOH
reduction
NADPH or
or
C=O to
OH OH CHOH O OH OH
(+)-neomenthol (–)-menthol (–)-menthone (+)-isomenthol (+)-neoisomenthol
FIGURE 4.18
Biosynthetic transformations in the conversion of isopiperitenone to menthol and its
disastereomers.
Nevertheless, we have seen some representative groups which have given us a fairly
good idea of some of the basic principles involved in their synthesis and subsequent
modifications. But before we move on from the monoterpenes to the C15 sesquiter-
penes, let’s take a brief look at some rather unusual monoterpene structures in which
the individual C5 units are not connected in a head-to-tail fashion.
IRREGULAR MONOTERPENES: IF NOT

HEAD-T O-TAIL, THEN HOW?
As the name suggests, the irregular monoterpenes are compounds which violate the
biogenetic isoprene rule. The four structural groups shown in Fig. 4.19 are chosen to
illustrate basic differences among some of the irregular monoterpenes, with the con-
nectivity between C5 units clearly not head-to-tail, but nevertheless variable from
group to group. In the first three, that is, the artemisyl, chrysanthemyl, and lavan-
dulyl groups, the individual isoprene units are intact, while in the fourth santolinyl
group, one of the isoprene units appears to have been disconnected and reassembled
by a skeletal rearrangement. Neither GPP nor LPP appear to be involved in the bio-
synthesis of these monoterpenes; overall there are relatively few members in each
family with the exception of the chrysanthemyl group whose members appear as
various esters derived from the corresponding parent carboxylic acids. We’ll take
a brief look at how one of these systems is assembled by an alternate method for
the linking of isoprene units and which we will encounter again later in the chapter
when we look at the earliest stages of steroid biosynthesis and the assembly of the
carotenoid terpenes.
The generally accepted route to the chrysanthemyl family involves linkage of two
DMAPP units rather than one DMAPP and one IPP unit as is the case for regular
terpenes (Fig. 4.20). Esterification of the resulting chrysanthemic acid or pyrethric

artemisyl chrysanthemyl lavandulyl santolinyl

Examples:
OH
HO2C
O OH H
artemesia ketone chrysanthemic acid lavandulol santolina alcohol
O
HO2C H
OH OH
yomogi alcohol pyrethric acid CO2CH3 β-cyclolavandulal lyratol
FIGURE 4.19
Four families of irregular monoterpenes and some representative members.
:B
H
PPO - OPP
DMAPP OPP OPP

DMAPP
OH
O
HO2C R
ester formation
- H2O chrysanthemic acid (R= CH3) OPP
pyrethric acid (R = CO2CH3)
O O
CO2CH3
or
O O
O pyrethrin I O pyrethrin II
FIGURE 4.20
Biosynthesis of chrysanthemic and pyrethric acids and conversion to pyrethrins I and II.
acid with the cyclopentyl alcohol (Z)-pyrethrelone leads to pyrethrins I and II, two
members of a group of six potent, structurally related insect neurotoxins that are
widely used as natural insecticides.
A good deal of experimental work has been done to try to determine the pathways
involved in biosynthesis of the other systems. Since the chrysanthemyl, artemisyl, and
santolinyl are the most commonly encountered systems, it has been proposed that the
chrysanthemyl PP may be a precursor to the others via ionization to a cyclopropyl-
methyl carbocation which would be expected to rearrange by different paths to yield
the santolinyl or artemisyl cation systems, both of which would readily produce some of
the known products of these families by simple additions of water, and so on (Fig. 4.21).


H :B
H b
a
OPP OPP
lavandulyl
b
products
santolinyl
OPP
chrysanthemyl
artemisyl
FIGURE 4.21
Possible relationships among the chrysanthemyl, santolinly, and artemisyl systems.
Similarly, the chrysanthemyl PP precursor may follow an alternate pathway of depro-

tonation rather than cyclization which would afford the lavandulyl system.
IRIDOIDS: FROM CATNIP TO ALKALOIDS
Iridoids are a class of monoterpene-derived compounds characterized by a cyclo-

pentane ring fused to a six-membered cyclic ether. They have their biosynthetic
origin in geraniol and are found in a wide variety of plants, usually appearing as
glycosides bound to a glucose molecule. Most iridoids are produced as a defense
against infection by microorganisms or as a bitter-tasting deterrent to herbivores
and can exhibit a wide range of biological activities including cardiovascular, anal-
gesic, anti-inflammatory, antimutagenic, antitumor, and antiviral activities among
others. Figure 4.22 traces a typical sequence for iridoids starting from geraniol in
the biosynthesis of nepetalactone, from the catnip plant. Roughly 50% of all cats
react to nepetalactone, suggesting a genetic susceptibility to its aroma; it is also an
active repellent for insects such as cockroaches and mosquitoes. As can be seen,
oxidation of geraniol to 8-hydroxygeraniol is followed by oxidation of the two pri-
mary alcohols to the corresponding dialdehyde, 8-oxogeranial. This is the key inter-
mediate in formation of the cyclopentane ring system, formulated as occurring via
intramolecular conjugate addition by the aldehyde enolate moiety initially derived
from conjugate hydride addition to the β-methyl-α,β-unsaturated aldehyde end of
the dial system as shown. Intramolecular trapping of the resulting enol form of the
cyclic irododial by the proximate aldehyde function leads to a hemiacetal which is
then oxidized to give nepetalactone.
A small number of iridoids are also found in the insect world. Figure 4.23 shows
the structures of chrysomelidial from leaf beetle larvae Chrysomelidae, anisomor-
phal from the defensive glands of the large walking stick Anisomorpha buprestoide,
and dolichodial from ants of the genera Dolichoderus, along with two important


H:
OH OH O
P450 2 NAD+
allylic CHOH
oxidation to C=O
OH O
geraniol 8-hydroxygeraniol 8-oxogeranial
H
NADPH conjugate
addition
H
H
H H intramolecular O
H O OH
conjugate
oxidize O
lactol to O addition of
O OH
H lactone H H enolate ion O
nepetalactone hemiacetal irododial (enol) H

H+
FIGURE 4.22
Biosynthesis of the iridoid nepetalactone from geraniol by way of irododial.
O H H O
O
O O O
H H H
chrysomelidial anisomorphal dolichodial
HO O H
H O-Glucose O-Glucose
O O
H H
H3CO2C H3CO2C
loganin secologanin
FIGURE 4.23
Three insect-derived iridoids and plant-derived iridoid glucosides loganin and secologanin.
plant-derived glucoside iridoids, loganin and secologanin, the latter of which plays
an important role in the biosynthesis of terpene indole alkaloids which we will ad-
dress later on in Chapter 7.
SESQUITERPENES (C 15 ): LINKING OF DIFFERENT

STARTER UNITS
Given that geranyl diphosphate (GPP) is a primary allylic diphosphate ester, it is able
to participate as a substrate for the same kind of head-to-tail linking with IPP units


GPP OPP
-OPP H H
IPP
E E OPP
OPP
(E,E)-farnesyl diphosphate (FPP) nerolidyl diphosphate (NLPP)
E Z
OPP
(E,Z)-farnesyl diphosphate (FPP) OPP
OH
OH
farnesol (E)-nerolidol
(E,E)-α-farnesene
FIGURE 4.24
Linkage of GPP and IPP to give FPP, NLPP, and some sesquiterpene products.
that we previously saw for DMAPP. The result of this linkage is a C15 sesquiterpene
diphosphate, (E,E)-farnesyl diphosphate or FPP. Like the cationic isomerizations
associated with GPP, we find that FPP undergoes ionization and allylic isomeriza-
tion to give the corresponding tertiary derivative, nerolidyl diphosphate (NLPP),
which after bond rotation may then undergo allylic isomerization to give the stereo-
isomeric derivative, (E,Z)-FPP as shown in Fig. 4.24.
Farnesol, the alcohol derivative of (E,E)-FPP, is present in citronella, lemongrass,
and many other essential oils and is widely used in perfumery as a fixative to en-
hance the scent and moderate the volatility of other fragrance compounds. It has
also been shown to induce apoptosis (cell suicide) in a number of different cancer
cell types and to inhibit the initiation of cancer cell development in some animal
models. Nerolidol is a fragrant tertiary alcohol from NLPP that is found in ginger,
jasmine, lavender, and lemongrass; its ability to penetrate the skin has led to testing
for potential use in transdermal drug delivery. As would be expected, elimination
of diphosphate and a proton from FPP leads to the common acyclic sesquiterpene
(E,E)-α-farnesene, which is partly responsible for the scent of gardenia and green
apples. The biosynthesis of this compound has been extensively studied due to its
key role in a condition of stored fruit deterioration known as scalding which has
been found to involve oxidative transformations of α-farnesene in the skins of af-
fected fruits.

SOME FPP CYCLIZATIONS IN SESQUITERPENE

Bioorganic Synthesis 154 BIOSYNTHESIS
In the formation of cyclic sesquiterpenes, various enzymes in different organisms

may establish specific folding patterns for FPP that lead to specific modes of cycliza-
tion after initial cation formation via loss of diphosphate, as seen in Fig. 4.25.
The cyclization of farnesyl cation leads initially to germacryl cation which may
then lose a proton to produce germacrene A or B. For germacrene B, sequential
oxidation at the indicated allylic position produces germacrone, a bioactive sesqui-
terpene ketone from Rhizoma curcuma which has been shown to inhibit prolifera-
tion of breast cancer cell lines via an apoptosis pathway. Alternatively, germacrene
A undergoes a more complex sequence of enzyme-controlled, site-specific oxidative
transformations, ultimately leading to the sesquiterpene lactone parthenolide, the
active principal found in the herbal remedy plant feverfew (Tanacetum parthenium)
enzyme
OPP folding
(E,E)-FPP PPO
-OPP
Ha
- Ha+
Hb
- Hb+ farnesyl cation
(+)-germacrene A
P450 C-H to P450

allylic allylic O
C-OH
oxidation oxidation
then NADP +
germacrene B germacrone
P450
2 NADP + allylic
OH oxidation
CH2OH to
CH=O C-H to
C-OH
to CO2H OH
O OH O OH
lactone
formation H2O
O2
mono-
O oxygenase
O O
O O
parthenolide
FIGURE 4.25
Cyclization of farnesyl cation to germacrene A or B and conversion to germacrone or parthenolide.

used for reduction of fevers (hence the name of the plant) and arthritis; it has also

been shown to be particularly effective for the treatment of migraine headaches.
As might be expected, the stereochemistry of nerolidyl PP allows folding patterns
and cyclization modes that are not possible via farnesyl PP. A significant example of
cyclization of nerolidyl cation is shown in Fig. 4.26, resulting in the formation of bis-
abolyl cation, a key intermediate which has been studied extensively due to its role
in the biosynthesis of a variety of different bicyclic and tricyclic sesquiterpenoids.
Simple proton loss from bisabolyl cation as shown gives (Z)-alpha-bisabolene,
one of the main constituents of opopanax oil. Alternatively, a 1,3-hydride shift (fa-
vorable due to the formation of a resonance-stabilized allylic carbocation) affords
a second opportunity for cationic cyclization leading to a 6,6-bicyclic ring system
which loses a proton to afford amorpha-4,11-diene, the terpene precursor to the
globally important antimalarial drug artemisinin, a sesquiterpenoid lactone iso-
lated from the plant Artemisia annua, long used as a medicinal herb in Chinese folk
remedies.
In Fig. 4.27, we can see an alternative 1,2-hydride shift for bisabolyl cation which
leads to another tertiary carbocation; this cation subsequently cyclizes to give the
spirocyclic acoryl cation which then may either lose a proton (via pathway a) to
afford the spiro[6.5]decane derivative, α-acoradiene, a flour beetle aggregation
enzyme
(E)-NLPP
folding
OPP -OPP
nerolidyl cation
Ha
H
1,3-hydride
shift
H
H Ha
Hb
- H+
Ha
- Hb +
H
bisabolyl cation
amorpha-4,11-diene (Z)-α-bisabolene
FIGURE 4.26
Nerolidyl to bisabolyl cation and its conversion to mono-and bicyclic sesquiterpenes.


H 1,2-
hydride
shift a
b - H+
bisabolyl a acoryl
cation
H cation α-acoradiene
b
- H+ - H+
β-cedrene cedryl cation α-cedrene
FIGURE 4.27
Bisabolyl cation and its conversion to other bi-and tricyclic sesquiterpenes.
pheromone, or it may undergo intramolecular cyclization (pathway b) to give the

tricyclic cedryl cation. Loss of an available β-proton yields either α-cedrene or
β-cedrene, two of several fragrant sesquiterpene cedar oil components found in pencil
cedar which are responsible for the familiar smell of our favorite writing instrument.
TRICHODIENE AND THE TRICHOTHECENES: HOW

TO TRACE A REARRANGEMENT PATHWAY
Another very important aspect of the chemistry of bisabolyl cation is its role in the
biosynthesis of trichodiene, the fundamental precursor to an especially important
family of mold toxins known collectively as the trichothecenes, a large family of
related toxins including deoxynivalenol (DON) and T-2 toxin which are pro-
duced by various mold species including Fusarium and Acremonium among others
(Fig. 4.28). The T-2 mycotoxin in particular is a significantly toxic trichothecene
that has been implicated in a number of historic outbreaks of toxin-related illness
and death as the result of the ingestion of moldy grains, including a massive case of
toxin poisoning in post-WWII Russia and the famous Plague of Athens in 430 B.C.
The unusual structure of trichodiene relative to bisabolyl cation indicates that
converting one into the other is a cationic process involving a number of differ-
ent specific hydride and methyl shifts. This provides us with an opportunity to see
how organic chemists have used isotopic labeling studies to determine the likely
sequence of events that produce various substitution patterns in such complex
rearrangements.
The process outlined in Fig. 4.29 begins with the use of 13C-labeled DMAPP as
shown. Note that providing labeled DMAPP precursors to an organism in “feed-
ing studies” would also lead to formation of some 13C-labeled IPP; therefore we
would expect the eventual formation of 13
C-labeled NLPP and thence bisab-
olyl cation with enhanced C content at specific positions within the structure.
13


H O
HO
O
deoxynivalenol O
H
OH
OH
H
H O
O
bisabolyl HO
cation trichodiene O
T-2 toxin O
OH
AcO OAc
FIGURE 4.28
Rearrangement of bisabolyl cation to trichodiene and conversion to trichothecene toxins.
OPP
13C-labeled DMAPP
OPP -OPP
OPP *NLPP *nerolidyl cation

13C-labeled IPP cyclize
H
- H+ =
H+
*bisabolyl cation
H cyclize
1,2-CH3 shift
*cuprenyl
cation
- H+ 1,2-CH3 shift
13C-labeled trichodiene H
FIGURE 4.29
Methyl migrations in 13C-labeled bisabolyl cation to give 13C-labeled trichodiene.
By using 13C NMR to determine where the labeled carbon atoms eventually reside
in the final product trichodiene, one may infer the sequence of events involved in
the various hydride and methyl migrations that must have taken place during the
rearrangement. Thus, rearrangement of labeled bisabolyl cation may be formu-
lated as undergoing initial loss of a proton followed by protonation of the remote
alkene function shown to give a carbocation that can cyclize to generate the
5-membered ring, thereby producing labeled cuprenyl cation, a known interme-
diate in the process (a more traditional alternative formulation of this sequence

involves initial cyclization of bisabolyl cation to produce the five-membered ring,

Bioorganic Synthesis 158 followed by a 1,5-hydride shift to produce cuprenyl cation). From here, a sequence
of 1,2-methyl shifts produces the final carbocation which then undergoes loss of a
proton to produce trichodiene with enhanced 13C-content at the indicated posi-
tions. While broken down here into a series of individual steps to aid in visualiza-
tion of the overall sequence, it should be recognized that this is probably a con-
certed process, with cyclizations, methyl migrations, and proton loss occurring
simultaneously. Labeling studies such as this one have proven to be invaluable in
helping to determine the complex mechanistic sequences likely to be involved in
the biosynthetic conversion of many different intermediates into specific products
in diverse enzyme-mediated processes, and we will be seeing other uses of this
technique throughout the remaining chapters.
FPP
OPP
- OPP FPP cation H H IPP
OPP
geranyl geranyl diphosphate (GGPP)
NADPH, H+ reduction
H2O
phytol
OPP
phytyl diphosphate - OPP (in chlorophyll)
- OPP OH
HO2C
phytyl cation
electrophilic 1,4-dihydroxy
CO2 OH
aromatic
naphthoic acid
substitution
(from shikimate
OH
pathway)
SAM
OH oxidize
(hydroquinone
O to quinone)
vitamin K1
(phylloquinone) H3C
O
FIGURE 4.30
Reaction of FPP and IPP to give GGPP, precursor to C20 diterpenes and vitamin K1.

What has been shown here may be thought of as only the tip of the iceberg with

regard to the number of different possible sesquiterpene compounds produced in
nature, with many more being discovered on a regular basis and all sharing their
origin in farnesyl diphosphate. The isolation, characterization, and elucidation of
biosynthetic pathways associated with these compounds represent some of the most
significant achievements of the organic chemistry community over the last 50 years.
And the wealth of information obtained in these studies finds applicability not only
in many other related fields, but also serves as the foundation upon which further
understanding of more complex terpene structures is built. So, while we still have far
to go in exploring the vast world of terpenoid organic structures, we are now armed
with a depth of knowledge that will make what remains much easier to grasp. So
now it’s time to move on.
DITERPENES (C 20 ): TAKING IT TO THE NEXT LEVEL

OF MOLECULAR COMPLEXITY AND DIVERSITY
Just as we used GPP and IPP as the building blocks to produce the FPP from which
the sesquiterpenes were all derived, the same sequence may be applied to the uti-
lization of FPP and IPP in combination to produce the basic starting material for
the production of all diterpenes, geranyl geranyl diphosphate or GGPP, as shown
in Fig. 4.30. Thus, in spite of its name, this C20 derivative is not produced from di-
merization of two C10 geranyl PP units. Among the important acyclic diterpene
derivatives is a reduced form of GGPP known as phytyl PP which is used in con-
junction with the shikimic acid pathway product 1,4-dihydroxynaphthoic acid
in the biosynthesis of the mixed pathway product vitamin K1, an essential human
blood-clotting factor. The hydrolysis product of phytyl PP is phytol, an alcohol con-
stituent incorporated into the structure of chlorophyll that is also used in the manu-
facture of synthetic vitamin E.
CYCLIC DITERPENES: FROM BASEBALL AND PLANT

HORMONES TO ANTICANCER DRUGS
Cyclization of GGPP is involved in the biosynthesis of abietic acid, the most abundant
of a group of related compounds found in the rosin of coniferous trees, a substance
which acts to help seal wounds in such trees and has long been used as a watertight
caulking for ships and also as an agent to improve the grip on baseballs and baseball
bats or the tackiness of the bow used with stringed instruments (Fig. 4.31). Anyone
who has gotten pine tree resin on their hands is familiar with its uniquely dry and tacky
sensation.
Gibberellic acid, a potent diterpenoid plant hormone first identified in Japan as
a byproduct of the plant pathogen Gibberella fujikuroi has been found to regulate

Bioorganic Synthesis 160 OPP

enzyme
OPP folding H
GGPP :B
B-H
OPP
H H H H
-OPP
H H H
1,2-methyl shift (+)-copalyl PP
- H+
H H methyl H
H group
H H oxidation H
CO2H (–)-abietic acid
FIGURE 4.31
Cyclization of GGPP and transformations to produce abietic acid.
OPP
H H H
-OPP methyl CH to C-OH
H H H
group oxidation
(–)-copalyl PP oxidation ent-kaurene
H pinacol-
pinacolone-type
rearrangement?
H H
CH to C-OH
O O-H oxidation? OH
H OH H H
HO2C HO2C OH HO2C
H :B
GA12 aldehyde H+ ent-7α-hydroxykaurenoic acid
O
multiple OH OH
oxidations,
lactone H H
cyclization
O dehydrogenation O
HO HO
H CO2H H CO2H
O O
gibberelllic acid (GA3)
FIGURE 4.32
Cyclization of (−)-copalyl PP and subsequent transformations to produce gibberellic acid.
stem and root growth as well as seed germination and dormancy. Its proposed bio-
synthesis from (−)-copalyl PP is shown in Fig. 4.32. It begins with a cyclization and
carbon migration to produce the intermediate compound ent-kaurene.
1,2-
Oxidation of one geminal dimethyl group to the carboxylic acid and C–H to CH–
OH oxidation leads to ent-7α-hydroxykaurenoic acid which is then oxidatively

converted to the key ring-contracted intermediate, GA12-aldehyde. The ring con-

traction mechanism shown is speculative, being formulated as a pinacol-pinacolone-
type rearrangement from a vicinal diol (pinacol) intermediate, though alternative
radical-type mechanisms are also possible. Additional oxidations, a decarboxyl-
ation, lactonization, and dehydrogenation with introduction of a ring double bond
finally completes this complex sequence of biotransformations to give gibberellic
acid or GA3.
A final example of diterpene cyclizations is a very important one, as it outlines
the basic biosynthetic pathway to the important cancer chemotherapy agent taxol,
mentioned earlier as a compound widely used in the treatment of breast, ovarian,
lung, head, and neck cancers as well as AIDS-related Kaposi’s sarcoma. Originally
isolated from the bark of the Pacific yew tree, Taxus brevifolia, intense interest
in taxol in the late 1970s stemmed in part from the discovery that the compound
manifested a previously unknown mechanism of anticancer action that involved
stabilization of cell microtubules in a way that interfered with their normal break-
down during the process of cell division. While its effectiveness was clearly dem-
onstrated, a major drawback to clinical use of taxol was its relative scarcity, after it
was determined that more than 350,000 Pacific yew trees would have to be stripped
annually to yield sufficient amounts of taxol for projected needs. This drawback was
initially addressed by semisynthetic routes to taxol, but more recently, plant cell
fermentation technology has been developed that now supplies most of the taxol
produced worldwide. Thus, the biosynthetic pathway to this compound, shown
in abbreviated form in Fig. 4.33, is not only interesting from an organic chemis-
try standpoint but also represents a highly valuable commercial process for taxol
production.
Initial carbocationic cyclization of GGPP, initiated by loss of –OPP, yields a ter-
tiary carbocation that loses a proton to give a cyclic diene; this diene then captures
the same proton in an enzyme-mediated 1,5-proton transfer process to initiate a
second carbocationic cyclization; subsequent proton loss from the resulting cation
yields 4,11-taxadiene, the basic diterpene precursor to taxol. Allylic oxidation
of 4,11-taxadiene at C-5 with rearrangement yields an intermediate allylic alco-
hol which is esterified by the action of acetyl-CoA to produce the intermediate
product 4,11-taxadien-5α,10β-diol monoacetate. The remaining steps involve a
complex series of oxidations and esterifications to produce the terpenoid alcohol
precursor 10-deacetylbaccatin III; sequential steps for construction and instal-
lation at the C-5 hydroxyl of an ester side chain derived from β-phenylalanine
and phenylisoserine finally yields taxol. As mentioned earlier, semi-synthetic
routes to taxol developed in the 1990s helped to relieve potential stress on the
Pacific yew population by synthesizing and installing the required ester side chain
onto 10-deacetylbaccatin III harvested from the more abundant European yew.
So, while the plant cell fermentation technology referred to earlier continues to
provide taxol, efforts are still underway to find other economical and ecologically

Bioorganic Synthesis 162 - H+

H+
-OPP
OPP
GGPP H+ (same proton lost

in previous step)
P450 - H+
allylic oxdiation
with rearrangement
then acetyl-CoA
OAc H
4,11-taxadien-5α-yl acetate 4,11-taxadiene
P450 C-H to CH-OH

oxidation
O-R'
OH
O
10-deacetylbaccatin III: R, R' = H
- or -
R-O
taxol: O
R' = Ac
many
steps HO R = Ph NH O
Ph O
AcO O Ph
OAc
O OH
4,11-taxadien-5α,10β-diol monoacetate
FIGURE 4.33
Cyclization of GGPP to yield 4,11-taxadiene and its subsequent conversion to taxol.
favorable natural sources of these compounds from plant waste materials like nut
shells or seed hulls.
SESTERTERPENES (C 25 ): LESS COMMON,

MORE COMPLEX
The C25 group of terpenes is derived from initial linkage of IPP and GGPP units to
produce geranylfarnesyl PP (GFPP) as shown in Fig. 4.34. Representative members
of this somewhat rare family of natural products, derived mainly either from fungi,
as with ophiobolin F, or from marine organisms, as with cheilanthadiol, are shown.
The ophiobolin F cationic cyclization is initiated by loss of –OPP, with a GFPP
folding pattern enforced by the mediating enzyme which presumably holds the re-
acting π-bonds in the necessary alignment to facilitate the resulting cascade of C–C
bond and ring formation. The intermediate tertiary carbocation formed then under-
goes a concerted, stereospecific 1,5-hydride shift leading to further cyclization and
discharge of the final stage carbocation by nucleophilic attack by water. Several other
members of the ophiobolin group are produced by further oxidative transforma-
tions of this precursor, including ophiobolin A, a phytotoxic calmodulin inhibitor


GGPP
OPP
-OPP GGPP cation H H IPP
OPP
fold GFPP fold
OPP
OPP
1,5-hydride
shift
H+
H
H H
H2O OPP
H2O H 2O
H OH
H
H
H H
H
OH
OH ophiobolin F cheilanthadiol
FIGURE 4.34
Formation of GFPP and cyclization to some representative sesterterpenoid products.
that has also shown antitumor and antibacterial activity. An alternate folding pattern
and cyclization mode for GFPP is shown in the biosynthetic sequence leading to
cheilanthadiol. Note that cyclization here is initiated by π-bond protonation rather
than initial loss of –OPP. Again, following the cyclization cascade, the final carboca-
tion is discharged by water which presumably also hydrolyzes the diphosphate link-
age to produce the diol product as shown.

TRITERPENES AND STEROIDS: ANOTHER CASE

Bioorganic Synthesis 164 OF IRREGULAR LINKAGE OF TERPENE UNITS
For the generation of C30 triterpenes and triterpenoids, we might expect head-to-tail
linkage of a C25 GFPP unit and a C5 IPP unit for the usual terpene chain extension
process. Instead, two C15 units of FPP are linked together in what is essentially a
“tail-to-tail” fashion, mediated by an enzyme known as squalene synthase, leading to
production of the fundamental C30 triterpene hydrocarbon, squalene, from which
all other triterpenes, steroids, sterols, and related compounds are ultimately derived
(Fig. 4.35). Squalene was originally isolated from shark liver oil (hence its name,
which comes from that of the shark, Squalus spp.), though many plant oils such as
olive, palm, and wheat-germ oil also contain significant amounts of squalene.
Upon careful consideration of the structure of squalene, we have to wonder ex-
actly how this tail-to-tail linkage of FPP units actually takes place mechanistically.
After all, instead of pairing a nucleophilic carbon with an electrophilic carbon to
form a C–C bond in the usual fashion, this process somehow involves the joining of
two electrophilic carbons (each with a leaving group, OPP), to make a single C–C
bond. Fig. 4.36 provides the standard sequence of transformations believed to be
involved in this unusual C–C bond-forming process.
The sequence of events here is quite similar to the nonstandard linkage of
DMAPP units seen in the formation of the irregular monoterpenes (see Figs. 4.20
and 4.21). Thus, ionization of the first FPP unit to its carbocation is followed by
attack from the π-electrons of the second FPP unit, leading to C–C bond formation
and an enzyme-stabilized carbocation. Discharge of this carbocation by subsequent
deprotonation-cyclopropane ring closure leads to the formation of presqualene
diphosphate, a key intermediate in this process which can be isolated and char-
acterized in the absence of NADPH. Loss of –OPP from presqualene PP leads to a
PPO
OPP +
FPP FPP
squalene
cholesterol other C30 triterpenes

(C27)
sex hormones, vitamin D, other steroids
FIGURE 4.35
Linkage of two FPP units to form squalene for biosynthesis of other triterpenes and steroids.


B: R B
R R
R
PPO PPO H
PPO :B
FPP(1) FPP(2) FPP(1)
cation
R=
R R R
R R R
PPO
cyclobutyl cation cyclopropylcarbinyl presqualene PP

cation
R
R
NADPH R
R
-H
squalene
homoallyl cation
(also allylic)
FIGURE 4.36
Tail-to-tail mechanism for squalene formation from two FPP units.
cyclopropylcarbinyl cation which, as we have seen previously, can lead to prod-

ucts derived from the cyclopropylcarbinyl-cyclobutyl-homoallyl cation system. In this
instance, the homoallyl carbocation component of this system is also allylic and
therefore especially stable; discharge of this carbocation by addition of hydride ion
from NADPH completes the sequence, affording squalene, shown with the linked
terminal carbons from each FPP unit highlighted for emphasis.
OXIDOSQUALENE AND STEROID BIOSYNTHESIS:

CYCLIZATION TO LANOSTEROL AND BEYOND
The significance of squalene cannot be overstated as it is the basic template upon

which all steroidal compounds are ultimately built. The fundamental biosynthetic
transformation that sets all this into motion is the initial conversion of squalene to
the epoxide derivative (S)-2,3-oxidosqualene (or (S)-2,3-epoxysqualene), catalyzed
by squalene monooxygenase (Fig. 4.37).
The cationic cyclization of 2,3-oxidosqualene is of enormous significance, and
different organisms deal with this process in different ways. For instance in some
plants, 2,3-oxidosqualene is converted to an important plant sterol known as
cycloartenol, while in animals and fungi it is initially converted to lanosterol, the
essential precursor to all other steroids produced by this path. Both compounds are
derived from a common protosteryl carbocation intermediate which arises from
a “chair-boat-chair” cyclization conformation, as shown in Fig. 4.38, and the di-
vergence of the fates of this cation are ultimately enzyme-dependent. The same set
of 1,2-hydride and methyl migrations takes place in both systems but ultimately

O2, NADPH
squalene
H
monooxygenase
squalene O
(S)-2,3-oxidosqualene
cyclization
H H
H
H H
HO HO
cholesterol H lanosterol
FIGURE 4.37
Conversion of squalene to (S)-2,3-oxidosqualene, precursor to lanosterol and cholesterol.
terminates in carbocation discharge by cyclopropane ring formation in plants while

proton loss to yield an alkene is the final act in animals and fungi.
The sequence of stereospecific 1,2-migrations of hydride and methyl in the
conversion of oxidosqualene to both products is probably concerted and involves
precise orbital alignment in which each migrating group is anti to the one preced-
ing it, as shown in Fig. 4.39 for the migrations involved in formation of lanosterol.
Note the cis relationship between the last remaining hydrogen atom (ultimately lost
as a proton) and the methyl group immediately adjacent to it: at this point, 1,2-
migrations cease since the methyl group is not anti to the H-atom and π-bond for-
mation results instead, leading to lanosterol.
In animals, lanosterol is the most significant product since it is ultimately con-
verted to cholesterol and all the other essential steroid structures, as we will see
shortly. In plants however, there is enormous variety in the various ways that tri-
terpenoid cyclizations can take place and in the products ultimately produced.
For instance, different enzymes can enforce alternate folding patterns for 2,3-
oxidosqualene cyclization, such as the “chair-
chair-
chair-
boat” folding pattern
employed to yield dammarenyl cation (Fig. 4.40). This cation can then undergo a
variety of different fates including a sequence of 1,2-shifts of hydride and carbon
similar to those seen for protosteryl cation to give euphol; alternatively, simple cap-
ture of water by the cation gives the related product dammerenediol.
In some instances oxidosqualene cyclization can be interrupted and then ex-
tended, leading to unusual structural features. This is illustrated in Fig. 4.41 in the
presumed biosynthetic origin of malabaricol, the main triterpene constituent of
yellow pigments from the woody plant Ailanthus malabarica. Initial cyclization
to the indicated tricyclic carbocation via the indicated folding pattern is then ter-
minated by water trapping, after which a second epoxidation occurs, followed by


CH3
H3C
CH3
B-H CH3 CH3
O
=
H CH3 CH3
CH3
O (S)-2,3-oxidosqualene
cationic cyclizations
CH3 CH3
i H3C
ii :B H3C
B: H i
CH2 H ii i CH3 H CH3 H
CH3 CH3 CH3 H
CH3 1,2-H and
HO HO
methyl CH3
H3C CH3 H migrations H3C
H H CH3 H
1,2-H shift, protosteryl cation
path ii:
path i: cyclopropane proton loss
ring closure
CH3 H CH3 CH3 H

H CH3
CH3
HO HO
CH3 CH3
H3C CH3 H H3C
H CH3 H
H
H H
H
HO HO
H cycloartenol H lanosterol
FIGURE 4.38
Cyclization of 2,3-oxidosqualene to protosteryl cation and final paths to cycloartenol and
lanosterol.
another acid-catalyzed intramolecular nucleophilic ring opening of the epoxide

which produces a five-membered cyclic ether. A final oxidation of alcohol to ketone
produces the final product.
Before moving on to the complex chemistry involved in the conversion of la-
nosterol to cholesterol and beyond, we take a brief look at a small sampling of the
remarkable structural variety possible in some of the medicinally significant plant-
derived sterols shown in Fig. 4.42. Note that these compounds are highly modified
structures relative to their precursor, cycloartenol; like cholesterol and other ste-
roids, they contain fewer than the 30 carbons characteristic of standard triterpenes.
Stigmasterol, a plant sterol believed to be beneficial in the prevention of certain
cancers, is found in the oils of soybean, calabar bean, various nuts and seeds, and in
a variety of medicinal herbs such as American ginseng. Diosgenin, the aglycone of a
glycosidic sterol extracted from the tubers of wild yam, is of considerable economic
significance as an abundant natural source of raw material used for the commercial
semisynthesis of medicinal steroids such as cortisone, pregnenolone, progesterone,
and other pharmaceutical steroids. Another useful example is β-sitosterol, a widely
distributed phytosterol found in corn oil, soybean, saw palmetto, and avocado

CH3
CH3
B: H3C H3C
CH3 H
CH3 CH3 H CH3 CH3 H
HO CH3
H3C CH3 HO
CH3
H CH3 H H3C CH3 H
protosteryl cation H lanosterol
syn :B
CH3
H CH3 H CH3 H
CH3
CH3 H CH3 H
FIGURE 4.39
The anti relationship of migrating atoms or groups in conversion of protosteryl cation to lanosterol.


H2O
H b
H
B-H H
O
HO
2,3-oxidosqualene dammarenyl carbocation
(chair-chair-chair-boat)
b
a H2O, - H+
1,2-H and CH3
shifts, - H+ OH
H H
H
HO HO
euphol dammarenediol
FIGURE 4.40
Chair-chair-chair-boat folding for oxidosqualene leading to dammarenediol or euphol formation.
H+ O
H+
H H OH
O
i) H2O
H ii) O2, NADPH H H
HO HO
H H
OH OH
H O H O
H NAD+ H
H H
O HO
H H
malabaricol
FIGURE 4.41
Alternative 2,3-oxidosqualene cyclization and subsequent biosynthetic steps to malabaricol.
which is often used to help treat elevated cholesterol levels by inhibiting cholesterol
absorption in the intestine. The compounds oubain and digoxin are examples of
the so-called cardiac glycosides (digoxigenin is the aglycone of digoxin), powerful
compounds that act as inhibitors of the sodium pump (Na+/K+ ATPase pump) in-
volved in heart electrical activity. While similar in action, digoxin, isolated from the
foxglove plant, has replaced oubain for treatment of various heart conditions such
as atrial fibrillation and flutter and congestive heart failure uncontrolled by other

O
H H
H H O
H
H
H H H H H H
HO HO HO
stigmasterol (soybean) diosgenin (mexican yam) β-sitosterol (corn oil)
O
O O
O
OH
HO HO
HO H
H
HO
O H
H OH OH
HO O HO
OH H
OH digoxigenin (digoxin aglycone)
ouabain (cardiac poison)
O O
OH
OH OH
H H
HO O O
H OH
O O O O
H H H
OH
digoxin (foxglove, Digitalis Spp)
FIGURE 4.42
Examples of some significant plant sterols and cardiac aglycones and glycosides.
drugs. Oubain, derived from the seeds of the African plant Strophanthus gratus,
was originally used in concentrated form by native tribesmen as an effective arrow
poison for bringing down large game animals, presumably by inducing cardiac or
respiratory failure.
CONVERSION OF LANOSTEROL (C 30 ) TO CHOLESTEROL

(C 27 ): WHERE DID THE CARBONS GO?
Conversion of lanosterol to cholesterol is a complex, 19-step process of double-

bond reductions, isomerizations, oxidations, reductions, decarboxylations, and
eliminations occurring in four different regions of the tetracyclic ring system. The
specifics of the process are summarized briefly in Fig. 4.43. As can be seen, the overall
conversion of the lanosterol skeleton involves removal of a double bond (Region 1),
removal of the C-14 methyl group (Region 2), sequential removal of the geminal
dimethyl groups (Region 3) and a double-bond rearrangement (Region 4). While
complex overall, many steps are fairly straightforward and some represent typical
processes used for methyl group removal from other structures.
Step 1 is a typical reduction of the CH=CH to CH2–CH2 type using NADPH/
H and this is the only step in Region 1 of the tetracyclic structure. In Region 2,
+


H H Step 1
H
NADPH/H+
19 steps H H
HO HO Region 1 (Step 1 only)
H lanosterol cholesterol
H Steps 2–4 H
NADPH/H+, O2
2. CH3 to CH2OH, :B
Steps 7–9 3. CH2OH to CH=O H
NADPH/H+, O2 4. CH=O to O-CH=O OCH=O
7. CH3 to CH2OH, HO (Baeyer-Villiger-type)
HO
H - HCO2-
H 8. CH2OH to CH=O Step 5
CO2H
9. CH=O to CO2H
Step 10 NADP+ H
H Step 6
NADPH/H+
- CO2
H
Region 2 (Steps 2–6)
O
H O
H H
CO2H
β-keto acid H+
Step 11 NADPH/H+
Step 17
H
Steps 12–14 double bond
H isomerization
NADPH/H+, O2 H H
12. CH3 to CH2OH, HO B:
HO H NADPH, O2
H 13. CH2OH to CH=O Step 18 dehydrogenation
H H CO2H
14. CH=O to CO2H
NADP+ (CHOH to C=O) Step 15
then - CO2 H
via β-keto acid Step 19
H H
NADPH/H+
Step 16
HO NADPH/H+ cholesterol
H O
H
Region 3 (Steps 7–16) Region 4 (Steps 17–19)
FIGURE 4.43
Conversion of lanosterol to cholesterol: 19 steps in four different regions.
steps 2–3 involve sequential C–H to C–OH oxidations of the C-14 methyl group of
lanosterol, leading to the aldehyde. For simplicity’s sake, step 4 is formulated here
as Baeyer–Villiger-type oxidation to give a formate ester followed by a 1,2-elimina-
tion of formate to yield the alkene, though a more complex radical-type mechanism
has been shown to be more likely for this step. A final CH=CH to CH2–CH2 reduc-
tion via NADPH/H+ in step 6 gives the saturated cyclopentane ring with the indi-
cated trans ring juncture. Moving to Region 3, we again see sequential oxidations
of one of the geminal dimethyl groups, ending in formation of the corresponding
carboxylic acid in steps 7–9. An NADP+ oxidation of the cyclohexanol hydroxyl
group in step 10 leads to the corresponding ketone after which the resulting in-
termediate β-keto acid undergoes decarboxylation as expected to give the mono-
methyl-substituted ketone. In step 11, the ketone function is reduced back to the
alcohol oxidation state by NADPH/H+, and the same oxidation sequence seen in
steps 7–9 is repeated on the remaining methyl group in steps 12–14. Re-oxidation
of the cyclohexanol hydroxyl in step 15 leads once more to a β-keto acid and sub-
sequent decarboxylation. Final NADPH/H+ reduction of the ketone function again
affords the cyclohexanol component. To complete the conversion, the cyclohexene

double bond in Region 4 of the tetracycle must undergo a change in position within
Bioorganic Synthesis 172 the ring. This is accomplished by a simple isomerization in step 17, formulated
as a simple protonation–deprotonation sequence. This is followed by an oxidative
dehydrogenation to give a conjugated diene system in step 18 (the intermediate
formed in this step, known as 7-dehydrocholesterol, will play a role later on in
the formation of vitamin D). The final double bond reduction in step 19 completes
the process, affording cholesterol.
CONVERSIONS OF CHOLESTEROL: PRODUCTION
OF THE SEX HORMONES
Cholesterol is the precursor to many other steroid structures, chief among these
are the male and female sex hormones. As shown in Fig. 4.44, this process begins
by removal of a six-carbon fragment from the acyclic portion of the structure.
This is accomplished by sequential C–H to C–OH oxidations leading to a vicinal
diol. Oxidative glycol cleavage produces the steroidal prohormone pregnenelone.
Oxidation of the cyclohexanol hydroxyl to the corresponding ketone, accompanied
by double bond isomerization, leads to progesterone, a hormone involved in the
menstrual cycle and pregnancy in humans and which has a number of medicinal
uses, including supplementation of declining natural levels of the hormone due
to aging (the yam sterol diosgenin (see Fig. 4.42) is used for the manufacture of
semisynthetic progesterone). The derivative 17-α-hydroxyprogesterone, formed
by a typical P450-type oxidation process, provides one of two possible pathways
for biosynthesis of the important sex hormone precursor androstenedione. The
pathway shown involves oxidative cleavage of the methyl ketone side chain of
17-α-hydroxyprogesterone, formulated here as another example of a Baeyer–Villiger
type oxidation followed by loss of AcOH to give the cyclopentanone ring system of
androstenedione as shown (the other pathway to androstenedione begins with preg-
nenolone instead of progesterone).
Androstenedione was taken as a supplement (known as “andro” prior to and
during the time of the major league baseball doping scandals) to boost testosterone
levels and to increase muscle mass, though these claims have not been substantiated
over the years, and its use has now been banned in most sports. Its in vivo conver-
sion to testosterone requires only NADPH reduction of the cyclopentanone func-
tion to the corresponding alcohol, while its conversion to the female sex hormones
estrone and estradiol involves a complex sequence of C-10 methyl group oxidation
and elimination that, for simplicity’s sake, is formulated here as being essentially
the same process used in removal of the C-14 methyl group of lanosterol (Fig. 4.43,
Region 2), with elimination of formate being followed here by aromatization to the
phenolic ring of estrone. NADPH reduction of the cyclopentanone function of es-
trone gives the corresponding alcohol, estradiol. Taken together, estrone, estradiol,


B: H
O O-H A+
(hydride
acceptor)
H H
H NADPH H
H H O2 H H
HO cholesterol HO
oxidative glycol
cleavage
O O O
H H H
H H H
isomerize NADP+
H H H H H H
O O HO
progesterone H+ pregnenolone
B: H
NADPH O2
O O O O
OH OH
H NADPH H - AcOH H
O2
H H H H H H
O O O
17-α-hydroxyprogesterone androstenedione
C-10 methyl oxidation
NADPH O2
O
O
H O
O HO
O H
B: H NADPH H NADPH
O H
O2 O2 H H
H H
H H
O
O
O
then keto-enol
1,2-elimination aromatization C-10 methyl oxidation, elimination, and aromatization (as above)
O OH
OH OH
H H
NADPH NADPH OH
H H H H NADPH
O2
HO H H O
estrone estradiol estriol testosterone
FIGURE 4.44
Pathways involved in conversion of cholesterol to various steroidal hormones.
and estriol form the fundamental group of natural estrogenic female hormones
which have been used in both contraceptive formulations and in postmenopausal
hormone replacement therapy for the prevention of osteoporosis.
Another group of steroid hormones of some significance is the corticosteroids
(Fig. 4.45), so called due to their origin from the adrenal cortex in animals. They
are ordinarily categorized into two main groups: the glucocorticoids and the
mineralocorticoids, the former deriving their name from the role they play in
glucose metabolism and the latter from their association with the management of
sodium, potassium, and water levels in certain tissues. Glucocorticoids are usually
typified by the presence of the primary/tertiary α-dihydroxyketone function at the

HO

O O
OH OH
H H
NADPH NADPH
progesterone
O2 H H O2 H H
O O
NADPH O2 17-α-hydroxyprogesterone 11-deoxycortisol
NADPH O2
HO HO HO
O O O
O OH HO OH
H H H
NAD+
H H H H H H
O O O
11-deoxycorticosterone cortisone hydrocortisone
NADPH O2
HO
HO O HO O
O HO O
HO HO HO
H H H
NADPH NADPH
O2 H H O2 H H
H H
O O O
corticosterone 18-hydroxycorticosterone aldosterone
FIGURE 4.45
Conversion of progesterone into the corticosteroid hormones.
cyclopentane ring side chain, as seen in hydrocortisone (though corticosterone is

an exception to this), while mineralocorticoids are usually limited to only a primary
α-hydroxyketone function at this position, as in aldosterone. Of particular interest
in this group is the corticosteroid known as cortisone. Proclaimed a “wonder drug”
following its discovery in 1949 due to its dramatic anti-inflammatory effects in the
treatment of patients suffering from debilitating rheumatoid arthritis, cortisone re-
mains a widely used treatment for many different ailments, as it is remarkable in its
ability to reduce pain and inflammation at sites of injury. Because of its suppression
of the immune response, it is particularly effective in the reduction of swelling of the
joints, hence its usefulness in the treatment of arthritic conditions. It is also used in
therapies to help suppress organ rejection following transplants.
Since the long-term use of corticosteroids such as cortisone can have many un-
desirable side effects, considerable effort has been made over the years to develop
synthetic analogues of cortisone designed to enhance anti-inflammatory properties
at the expense of less desirable ones; many such steroidal drugs have been developed
(Fig. 4.46) and are commonly prescribed for a variety of conditions, though most are
still unsuitable for long-term use, hence the enduring interest in the development
and marketing of synthetic NSAIDs (nonsteroidal anti-inflammatory drugs) such
as ibuprofen, naproxen, and celecoxib (celebrex).

HO

HO HO O
O O
HO OH
O OH O OH H
H H
H H
H H H H O
O O
cortisone prednisone methylprednisolone
HO Cl HO
O O O
HO HO O
OH HO O
H H H O
O
F H F H F H
O O O
betamethasone clobetasol propionate triamcinolone acetonide
O SO2NH2
OH
ibuprofen
N N
O
O F3C
HO
celecoxib
naproxen
FIGURE 4.46
Some important steroidal and nonsteroidal anti-inflammatory drugs.
DEHYDROCHOLESTEROL, SUNSHINE,
AND VITAMIN D 3 BIOSYNTHESIS
Before we leave our examination of steroidal structures, recall that we previously

noted that in the double bond isomerization to complete the conversion of lanos-
terol to cholesterol, the system passes through an intermediate cyclohexadiene inter-
mediate (Fig. 4.43, Region 4). This intermediate, known as 7-dehydrocholesterol,
also serves as the essential precursor to vitamin D3 (the so-called “sunshine vita-
min”) which plays a key role in bone health through regulation of absorption and
metabolism of calcium and phosphorous. In this process, 7-dehydrocholesterol is
photochemically converted to vitamin D3 (or cholecalciferol) upon exposure of the
skin to UV wavelengths between 270 and 300 nm in sunlight (Fig. 4.47).
The process involves a photochemical conrotatory cyclohexadiene ring-opening
reaction to produce the corresponding hexatrienyl derivative precholecalciferol.
This triene then undergoes a sigmatropic rearrangement or 1,7-hydride shift to
produce the sterically crowded s-cis conformer of the isomerized hexatriene which
relaxes to the more stable s-trans conformer of cholecalciferol (vitamin D3). Our
ability to synthesize this compound means that it is not a vitamin in the strictest
sense, since these are usually considered to be compounds that must be obtained
in the diet, but because deficient levels may lead to diseases such as rickets, vita-
min D is commonly added to foods and is synthesized commercially by the same

Bioorganic Synthesis 176 H

hν
H
H H conrotatory
ring opening HO
HO
7-dehydrocholesterol precholecalciferol
R1 1,7-hydride shift
calcidiol
(R1 = OH,R2 = H) H
H
HO
calcitriol cholecalciferol cholecalciferol
(R1 = R2 = OH)
HO R2 (s-trans conformer) (s-cis conformer)
(R1 = R2 = H)
FIGURE 4.47
Photochemical conversion of cholesterol to cholecalciferol (vitamin D3), calcidiol, and calcitriol.
photochemical process. The vitamin D group includes two other components, cal-
cidiol and calcitriol, both of which are derived from cholecalciferol by sequential
hydroxylations occurring in the liver and the kidneys, with calcitriol being the most
hormonally active component of the group.
TETRATERPENES AND CAROTENOIDS:

TAIL-T O-TAIL LINKAGE OF C 20 UNITS
You have probably noted that we are skipping over the possible C35 group of terpenes
containing seven isoprene units. These are known as sesquarterpenes and are even
less common than the sesterterpenes (C25), being produced mainly in certain bacteria.
Of far more interest are the C40 tetraterpenes, compounds which are produced by the
initial tail-to-tail linkage of two C20 GGPP units in a fashion analogous to that used in
the biosynthesis of squalene, but without the final NADPH reduction step. In plants or
fungi, this process produces the basic precursor to tetraterpenes known as Z-phytoene
(Fig. 4.48); in bacteria, the double bond isomerizes from Z to E.
From Z-phytoene, central bond isomerization and a sequence of four desatu-
ration reactions ultimately leads to formation of lycopene, as shown in Fig. 4.49.
This bright red pigment is found most commonly in tomatoes but is also present in
many other red fruits and vegetables. The eleven conjugated double bonds in lyco-
pene lead to an array of π-MOs with a HOMO–LUMO gap in the visible region of
the spectrum, hence its deep red color. Though unproven, an association between
lower levels of certain cancers and increased levels of lycopene consumption has
been observed, due perhaps to its known antioxidant properties. Its lack of toxicity
and bright red color make lycopene one of the most commonly used food colorings.


+
OPP PPO
GGPP GGPP
Z-phytoene
R R
B: B
R R
R
PPO PPO H
PPO :B
GGPP(1) GGPP(2) GGPP(1)
cation
R=
R R R
R R R
PPO
- H+
R R R R
Z-phytoene
FIGURE 4.48
Linkage of GGPP units to form Z-phytoene for biosynthesis of tetraterpenes and carotenoids.
Z-phytoene
isomerize Z to E
Ha Hb
E
Ha Hb E-phytoene
desaturation - 4H2
Hb
Ha lycopene
FIGURE 4.49
Z to E isomerization and stereospecific desaturation of phytoene to produce lycopene.
Phytoene and lycopene are the principal acyclic tetraterpenes of significance.

Both acyclic and cyclic tetraterpenes are also known as carotenoids, plant pigments
of special significance to human health and which must be obtained in the diet (ani-
mals do not biosynthesize tetraterpenes). The carotenoids are divided into two main
groups, the hydrocarbon carotenes and the oxygenated xanthophylls, and typically
result from cationic cyclization of one or both ends of the lycopene framework (end
group cyclization). In the case of the xanthophylls, further oxidative transformations
usually take place following initial cyclization. The combination of processes leads

lycopene
H
H+
H+
H
end group cyclization - H+ - H+ end group cyclization
C-H to C-OH
oxidation
β-carotene
both both
ends ends
α-carotene α vs. γ γ-carotene
H H
OH OH
H
or
HO lutein HO zeaxanthin
OH monooxygenase
O epoxidations
O
HO
violaxanthin
FIGURE 4.50
End group cyclization of lycopene and formation of α-, β-, and γ-carotenes and subsequent
oxidative transformations to zeaxanthin, lutein, and violaxanthin.
to a wide variety of structurally related compounds; as seen in Fig. 4.50, cationic

cyclization at both ends of the lycopene framework followed by simple proton loss
leads to the widely distributed natural product β-carotene, a carotenoid responsible
for the orange color of carrots and many other vegetables and fruits.
Oxidative transformations at one or both ends of the β-carotene ring system produce
a number of important carotenoids. For example, both zeazanthin and lutein are found
in many green vegetables such as spinach, peas, broccoli, romaine lettuce, and zucchini
(note the double bond isomerization and epimerization at only one of the two end rings
in lutein). These carotenoid alcohols are included in many ocular vitamin formulations
since high levels of these have been associated with decreased incidence of age-related
macular degeneration, possibly by protection of eye tissues from degenerative exposure
to blue light, of which these compounds are strong absorbers. Further epoxidation of
zeaxanthin also produces the common orange plant pigment violaxanthin.
The relationship between carotenoids and vision is also seen in the production
and use of the essential terpenoid vitamin A which can be obtained in the diet but

FIGURE 4.51
Cleavage of β-carotene to trans-retinal and retinol (vitamin A) as part of the visual cycle.
can also result from oxidative cleavage of the β-carotene central C=C bond in a pro-
cess somewhat analogous to alkene ozonolysis, catalyzed by intestinal monoxygen-
ase enzymes. This oxidative transformation leads initially to two molecules of the
key vision-related aldehyde, all-trans-retinal or vitamin A aldehyde. Subsequent
reduction by NADH then leads directly to all-trans-retinol (vitamin A). These im-
portant compounds are part of the visual cycle, as shown in Fig. 4.51.
Esterification of trans-retinol with lecithin yields an ester which can undergo an
SN2’-type nucleophilic substitution (nucleophilic attack at a π-bond of an allylic or
a conjugated polyene system with a leaving group, leading to products with allylic
isomerization) by an enzyme basic site, followed by a single bond rotation and a
second SN2’ attack by nucleophilic water which displaces the enzyme base while
producing the isomerized cis-retinol. An NAD+ oxidation to cis-retinal is then fol-
lowed by condensation with a lysine amino group within an opsin protein. The re-
sulting imine linkage produces rhodopsin, the essential pigment associated with

transmission of color-related visual signals in the retina of the eye. In this system,
Bioorganic Synthesis 180 cis-rhodopsin captures a photon of light, leading to an electronically excited state
that returns to the ground state with isomerization of the 11-position double bond
(i.e., from 11-cis-rhodpsin to 11-trans-rhodopsin). This light-induced change in the
overall molecular geometry of rhodopsin initiates a nerve impulse interpreted by
the brain as a visual signal. Subsequent hydrolysis of the imine linkage releases the
opsin protein and regenerates all-trans-retinal, which now can be cycled back into
the system for subsequent visual events.
LOOKING AHEAD
We have not seen the last of DMAPP or terpenoid compounds by any means, as we
will find that they can intersect with compounds from other pathways to produce a
variety of mixed products. As we move on to the next chapter to further expand our
knowledge of biosynthetic pathways, we will see some recurring themes from our
study of terpenes: fundamental building blocks, repeating units, acyclic, monocyclic
and polycyclic structures, alternate folding patterns, and pathways that can produce
a seemingly endless variety of biologically significant organic structures, all with a
logic behind them based on organic chemistry that we can understand, appreciate,
and build upon.
STUDY PROBLEMS
1. From among all eight possible diastereomers of the basic structural system asso-
ciated with menthol, neomenthol, isomenthol, and neoisomenthol, the menthol
enantiomers would be expected to be the most stable. Explain using appropriate
stereochemical diagrams.
HO
2. Eucalyptol, a simple bicyclic monoterpenoid ether, makes up about 90% of ge-

neric eucalyptus oil, but is also found in bay leaves, sweet basil, rosemary, and
sage among other aromatic plants. Propose a biosynthesis from GPP.
eucalyptol

3. When heated, β‐pinene undergoes a retro-[2+2] cycloaddition reaction to give

myrcene which may then be reacted with acrolein (propenal) to give a valuable
commercial fragrance compound as a mixture of regioisomers (one major, one
minor). Draw curved arrow mechanisms for both the formation of myrcene and
also for the expected structure of the fragrance compound.
Δ H
?
retro [2+2] Diels-Alder
myrcene [4+2]
β-pinene
4. The boll weevil (Anthonomus grandis) has been among the most destructive
of pests for the cotton-growing industry over the years. Many methods have
been employed in attempts to eradicate or control the insect’s spread, includ-
ing the monitoring of populations using traps baited with its sex pheromone
which is a mixture of four components, grandlure I–IV (grandlure I is also
known as grandisol). It has been proposed that all four components of the
pheromone mixture may be derived biosynthetically from geraniol. Propose a
sequence of plausible reactions for the biosynthesis of these compounds from
geraniol.
OH
OH O O
OH
?
H
geraniol grandlure I grandlure II grandlure III grandlure IV
(grandisol)
5. For a plant employing the DXP pathway for biosynthesis of geraniol, predict
where 13C labeling in geraniol would occur if the plant was provided with pyru-
vic acid labeled at the 2-position. Where would the labeled positions in geraniol
occur from pyruvic acid labeled at the 3-position?
OH OH
O
pyruvic acid geraniol
6. The enzyme aristolochene synthase catalyzes the cyclization of FPP to a large

group of fungal toxins. The intermediate carbocation can undergo a variety of

simple shifts and deprotonations to give different products. Show how the two
Bioorganic Synthesis 182 products shown are formed via a series of 1,2-shifts and a final proton loss.
aristolochene
FPP
synthase
7. Mevalonic acid labeled with 13C and 2H has been used to demonstrate the se-
quence of cyclizations and 1,2-H shifts involved in biosynthesis of the hydrocar-
bon precursors to various natural products.
a) Starting with the GGPP folding pattern provided, clearly demonstrate a likely
sequence of cation-
initiated cyclizations and accompanying 1,2-
H shifts
which would lead to the hydrocarbon precursor of Fusicoccin, a fungal me-
tabolite responsible for plant wilting. Use only loss of OPP or a protonation to
generate initial carbocations.
OPP
fusicoccin core
b) Star (*) the carbons in the hydrocarbon core to indicate where labeled carbons
would appear if acetate, 13C-labeled at the methyl group, was introduced into
the organism producing fusicoccin (assume generation of 13C-labeled acetyl-
CoA and a mevalonic acid pathway).
8. Propose a folding pattern and mechanism for cyclization of GGPP to produce
casbene.
casbene

9. Vitamin E is an important group of structurally related phenolic antioxidants

of which δ-tocopherol is the simplest member, possessing a monomethylated
aryl ring (β- and γ-tocopherols are para- and ortho-dimethyl-substituted iso-
mers, respectively, while α-tocopherol has a trimethylated aryl ring). Propose a
biosynthetic sequence to produce δ-
tocopherol from phytol PP and the
shikimate-derived product, homogentisic acid (in the first step, both alkylation
and decarboxylation occur). Indicate the reactions and cofactors most likely in-
volved in producing the remaining tocopherol isomers (note that α-tocopherol
is derived from γ-tocopherol).
OH
HO
HO2C
OH O
homogentisic acid δ-tocopherol
10. Propose a mechanism for the acid-catalyzed rearrangement of epoxidized de-

rivatives of zeaxanthin to produce capsanthin and capsorubin, food colorings
isolated from paprika oleoresin used as coloring agents for orange juice as well
as various cheeses and spice mixtures.
H+
O OH
rearrangement
HO
HO
capsanthin
one end
epoxidation
only
zeazanthin
epoxidation both ends

OH
O
OH
O
H+
O rearrangement
HO both ends
O
HO capsorubin

5 The Acetate Pathway

Biosynthesis of Polyketides
and Related Compounds
The group –CH2CO–(which the author proposes to call the “ketide” group) can be made to
yield by means of the simplest reactions a very large number of interesting compounds, all
belonging to classes which are largely represented amongst the compounds obtained from
plants.
—John Norman Collie (1907 lecture)
Within a few minutes these ideas reduced to order what had previously seemed an unre-
lated jumble of structures of many natural products. It was an immensely satisfying and
emotional moment.
—Arthur J. Birch (1952 lecture)
We saw in the previous chapter how Otto Wallach’s early proposal regarding the
structural origin of terpenoid natural products was later refined by the insightful
work of Leopold Rudzicka, leading to the biogenetic isoprene rule and all that it
implies. In a nearly parallel fashion, we find in our present chapter a second, unre-
lated class of naturally occurring compounds whose characteristic structural fea-
tures prompted an initial innovative hypothesis by J. N. Collie near the turn of the
20th century. Collie proposed that certain natural compounds might arise from pre-
cursors containing repeated “ketide” (–CH2CO–) units which could then undergo
subsequent condensations and other reactions typical of carbonyl compounds to
produce some of the observed structures. Unfortunately, Collie’s work was more or
less ignored and largely forgotten for nearly a half century, only to be reimagined
and expanded in the middle of the century by A. J. Birch, another pioneer whose
184

proposals met with considerable initial resistance. But unlike his predecessor, Birch

ultimately prevailed by providing experimental results that supported a comprehen-
sive theory of the biochemical origin of the group of compounds now universally
known as “polyketide” natural products.
This structurally diverse family includes some of the most useful medicinal
agents now known to us, with many members possessing powerful antibacterial,
antifungal, anticancer, immunosuppressant, and even cholesterol-lowering biologi-
cal properties. As we see in Fig. 5.1, such structures range from the relatively simple
to the exceedingly complex and may include large macrocyclic lactone rings (mac-
rolides) such as erythromycin, polycyclic ethers such as monensin A, polycyclic
structures which may be partly or mostly aromatic as in tetracycline, griseofulvin,
or daunorubicin, or nonaromatic polycyclics such as tacrolimus and lovastatin.
Some also contain noncyclic linear components that may be saturated, oxygenated,
or unsaturated, as seen in different regions of amphotericin B which, like erythro-
mycin, daunorubicin, and many other polyketides, also possesses an aglycone core
which has been glycosylated with a carbohydrate component at a specific position.
But in spite of this range of structures, many polyketide compounds share some
common features that ultimately become more evident upon closer inspection; six-
membered rings (either aromatic or nonaromatic) and multiple oxygens which tend
to appear in a repeating 1,3-relationship to one another on both acyclic, cyclic, and
aromatic structures. These features will turn out to be important clues associated
with Collie’s and Birch’s original work on understanding how polyketide natural
products are actually formed.
But before we delve into the complex organic chemistry associated with
polyketide biosynthesis, we must first spend some time examining a closely related
process—fatty acid biosynthesis—whose mechanistic details and dependence on
enzyme catalysis were understood well after Collie’s time but also ultimately pro-
vided the inspiration for most of Birch’s subsequent insights and investigations. As
we will see, fatty acid biosynthesis is revealed as the original template upon which
nature built the polyketide pathway, with only a few minor modifications or varia-
tions ultimately responsible for the vast structural differences observed in the array
of different products produced by each pathway.
FATTY ACIDS: MULTIPLES OF TWO CARBONS,

SATURATED OR UNSATURATED
Fats and oils, like steroids, are among the group of natural products referred to
as lipids, compounds with substantial hydrocarbon character and limited water
solubility. Two structurally similar types are the glycerophospholipids which are
important cell membrane constituents and the triacylglycerols (or triglycerides)
which constitute most of the familiar fats and oils obtained from plant or animal
sources. Both of these types of lipids are esters derived from glycerol (glycerine or

OH OH
OCH3 O OCH3 O OH
HO O OH OH OH OH O O
O
H3CO O OH
Cl O O
griseofulvin amphoterecin B OH
(antifungal) (antifungal) H2N
HO
O O O OH O
HO N OH O N
H HO
OH OH O OH
NH2 O O
O OCH3 O OH O
OH O O
OH O OH O O O
tetracycline erythromycin O OH daunorubicin
(antibiotic) (antibiotic) (anticancer) HO H2N
H OH O
OH
O
O H O
O O HO
O H OH HO O
O O
H O H OCH3 HO O OCH3
O OH N
HO
OCH3 O O OCH3
lovastatin monensin A tacrolimus
(cholesterol-lowering) OH
(veterinary antibiotic) (immunosuppressant)
FIGURE 5.1
Some important examples of structurally diverse medicinal polyketide natural products.

1,2,3-propanetriol) which is linked to fatty acids—even-numbered, unbranched

long-chain carboxylic acids containing between 12 and 20 carbon atoms, either with
saturated chains (no double bonds) or unsaturated chains (one or more double
bonds, almost always Z in configuration). A list of the most common fatty acids is
given in Table 5.1.
Glycerophospholipids such as phosphatidyl cholines are based on a phosphate
ester linkage between an amino alcohol such as choline, ethanolamine, or serine and
phosphatidic acid, a monophosphate ester of glycerol which has been further esteri-
fied by two fatty acids, one of which is usually saturated and the other unsaturated.
Triacylglycerols such as tristearin contain ester linkages between glycerol and three
fatty acids, though the three need not be the same and can be a mixture of both satu-
rated and unsaturated fatty acids (Fig. 5.2). Most fats or oils are not homogenous in
composition but rather are complex mixtures of variously configured triacylglycerols.
One important difference between animal and plant-derived fats is that animal fats
are usually saturated fats which are often solids at room temperature, due in part to
the ready aggregation or packing of their saturated chains. Conversely, plant-derived
fats are usually oils at room temperature; these fats contain one or more nonconju-
gated double bonds in some or all of the chains; these are referred to as monounsatu-
rated or polyunsaturated fats, depending on the number of double bonds present in
the unsaturated chains. The nonlinear geometry of the Z-double bond in unsaturated
fats tends to inhibit close aggregation or packing of the hydrocarbon tails and is the
main reason for their lower melting points relative to saturated fats.
Most of us are familiar with the rather notorious trans fats which, like dietary
saturated fats, have been shown to raise serum cholesterol levels (and the attendant
Table 5.1 Names and Structures of Some Common Saturated and Unsaturated

Fatty Acids
Name C-atoms π-bonds (Z) Structure
Lauric 12 0 CH3(CH2)10CO2H
Myristic 14 0 CH3(CH2)12CO2H
Palmitic 16 0 CH3(CH2)14CO2H
Stearic 18 0 CH3(CH2)16CO2H
Arachidic 20 0 CH3(CH2)18CO2H
Oleic 18 1: at C-9 CH3(CH2)7CH=CH(CH2)7CO2H
Linoleic 18 2: at C-9,12 CH3(CH2)4CH=CHCH2CH=
CH(CH2)7CO2H
α-Linolenic 18 3: at C-9,12,15 CH3CH2CH=CHCH2CH=CHCH2CH=
CH(CH2)7CO2H
γ-Linolenic 18 3: at C-6,9,12 CH3(CH2)4CH=CHCH2CH=CHCH2CH=
CH(CH2)4CO2H
Arachidonic 20 4: at C-5,8,11,14 CH3(CH2)4CH=CHCH2CH=CHCH2CH=
CHCH2CH=CH(CH2)3CO2H


O
O OC-R
OH O- O OC-R O
R'-CO O
P R'–CO OC–R"
HO OH O–R"
O
glycerol R– = saturated fatty acid; R–, R'–, R"– = fatty acids:
R'– = unsaturated fatty acid a triacylglycerol
R"– = H: a phosphatidic acid;
R"– = choline, ethanol amine or serine: a glycerophospholipid
O
oleic acid chain
O
O-
O
P choline
palmitic acid chain O chain
O O
N
a phosphatidylcholine O
O
O
stearic
O acid
chains
O O
tristearin
O
FIGURE 5.2
Structures of glycerol; a glycerophospholipid and a triacylglycerol.
risk of coronary artery disease). Most trans fats are actually food industry byprod-
ucts that arise from the catalytic hydrogenation of processed polyunsaturated fats
(“partially hydrogenated vegetable oils”). For example, partial hydrogenation of
soybean or peanut oils produces margarine or shortening with melting points simi-
lar to those of some saturated fats, not only because some chains are in fact satu-
rated in the process, but also because Z to E isomerization of some π-bonds takes
place during the high-temperature catalytic hydrogenation, typically producing fats
with as much as 15% of the more thermodynamically favored transunsaturation
(Fig. 5.3). And unlike Z-configured fats, an E-double bond in a fatty acid chain has
a relatively linear conformation similar to saturated fat chains; this allows closer
aggregation of the hydrocarbon tails and increased melting points. So why hydroge-
nate a vegetable oil and turn it into a (less healthy) synthetic fat with a higher melt-
ing point? As is usually the case, it’s a combination of marketing and demand; some
people prefer spreading a solid (like butter) on their bread rather than pouring an oil
over it (even though doing so would probably be better for their health). Figure 5.3
illustrates the generation of a typical trans fat
Waxes are solid lipids produced by many plants, insects, and other animals and
contain multiple components, many of which are high molecular weight esters de-
rived from even-numbered fatty acids (16–36 carbons) and reduced “fatty alcohols”


O
linoleic acid chain (Z,Z)-9,12

O
H2 Ni catalyst
high T
elaidic acid chain (E)-9

O
FIGURE 5.3
Formation of a trans fat during partial hydrogenation of a polyunsaturated fat.
O
triacontyl hexadecanoate
(melissyl palmitate, component of beeswax)
O
hexadecyl hexadecanoate
(cetyl palmitate, component of spermacetti)
O
triacontyl hexacosanoate
(melissyl cerotate, component of carnauba wax)
FIGURE 5.4
Structures of some common ester components of various natural waxes.
(16–36 carbons). Some common examples are beeswax, the main component of
which is an ester derived from 1-triacontanol (melissyl alcohol, C30) and hexadec-
anoic (palmitic, C16) acid; spermacetti, derived from sperm whale oil and which
consists primarily of the ester derived from 1-hexadecanol (cetyl alcohol) and pal-
mitic acid; and carnauba wax, one of the hardest and most widely used waxes which
among other components, contains the ester derived from melissyl alcohol and
hexacosanoic (cerotic, C26) acid (Fig. 5.4).
Phospholipids, triacylglycerols, and waxes all depend on fatty acid bio-
synthesis to provide the basic saturated hydrocarbon chains which can then
be modified to yield the unsaturated, branched, or reduced components of
these lipids. Much as the mechanism of nonphotosynthetic glucose synthesis
(gluconeogenesis) was essentially the reverse of the catabolic degradation of
glucose (glycolysis), fatty acid biosynthesis turns out to be essentially the reverse

of the important catabolic process for degradation of fats known as β-oxida-

Bioorganic Synthesis 190 tion. In that process, saturated fatty acids released by triacylglycerol hydrolysis
are degraded two carbons at a time into individual acetyl-CoA units which can
then enter the citric acid cycle for further catabolism to CO2. Thus, one can
think of triacylglycerols as energy storage units for on-demand release of fatty
acids that can be degraded into acetyl-CoA for fuel. Unsaturated fatty acids are
slightly more complicated to degrade, requiring additional enzymes at certain
steps to isomerize double bonds into appropriate positions so that the normal
β-oxidation process can continue. But we won’t examine β-oxidation in detail;
we should simply keep in mind when we consider the mechanistic details of
saturated fatty acid biosynthesis, that we need only run that process in reverse
to gain some understanding (at least mechanistically) of how fatty acids are ulti-
mately metabolized in the β-oxidation process.
SATURATED FATTY ACID BIOSYNTHESIS:

IT ALL STARTS WITH ACETYL-COA
Just as β-oxidation ends with acetyl-CoA, biosynthesis of saturated fatty acids begins
with acetyl-CoA. And it is nature’s use of this fundamental two-carbon building
block that is ultimately responsible for the familiar even-numbered constitution
of most fatty acids. We saw previously in Chapter 2 how acetyl-CoA reacts with
N-carboxybiotin to undergo a carboxylation reaction via its enolate ion, leading
to the important derivative, malonyl-CoA (Fig. 5.5). It is the combination of both
acetyl-CoA and malonyl-CoA, acting in tandem that ultimately sets the process of
fatty acid biosynthesis into motion.
As shown in Fig. 5.6, both acetyl-CoA and malonyl-CoA units undergo nu-
cleophilic acyl substitution by thiol groups of an acyl carrier protein (ACP) and a
second enzyme component, both of which are bound together within the fatty acid
synthase (FAS) complex, a multifunctional enzyme system that contains within it
all the catalytic components required to facilitate the entire process. The loading
component of the process, mediated by a malonyl/acetyl transferase component or
CoAS
H
O O O O
O O
C
O N NH N NH CoAS
- biotin
H H CoAS O
H H O
R R C malonyl CoA
S O
S
N-carboxybiotin
FIGURE 5.5
N-carboxybiotin and carboxylation of acetyl-CoA to give malonyl-CoA.


HS–Enz O
acetyl CoA 1
starter unit SCoA fatty acid loading S Enz
synthase
O O complex
2 HSCoA O S ACP
HS–ACP malonyl/acetyl
O SCoA transferase
malonyl CoA O O
FIGURE 5.6
Loading of acetyl-CoA and malonyl-CoA units in step 1 of fatty acid biosynthesis.
O S Enz
O
2
S Enz Claisen S Enz
condensation S ACP
O S ACP S ACP
O O
CO2 O
O β-ketosynthase β-ketothioester
O
tetrahedral
intermediate
FIGURE 5.7
Claisen condensation in step 2 of fatty acid biosynthesis.
domain of the FAS enzyme, constitutes step 1 in the fatty acid biosynthesis process,
producing two enzyme-bound thioesters, acetylSEnz and malonyl-ACP.
Once brought together within the complex, the malonyl-ACP unit undergoes a
decarboxylation reaction to yield an ester enolate ion. This enolate acts as a nucleo-
phile (Fig. 5.7) to attack the carbonyl group of the nearby starter unit, acetylSEnz,
in what is formally a Claisen condensation in step 2. The domain of the FAS enzyme
that mediates this essential C–
C bond- β-ketosynthase—is aptly
forming step—
named, as it facilitates the formation of the usual product from a Claisen condensa-
tion, namely a β-ketoester (actually a β-ketothioester in this case).
Step 3 in the process is an NADPH reduction of the ketone function of the
β-ketothioester (mediated by a β-ketoreductase domain) to yield a β-hydroxythioester
intermediate. Note that delivery of hydride is stereospecific in this reaction, occur-
ring at the Si face of the ketone function (Fig 5.8).
As might be expected, such β-hydroxycarbonyl compounds are susceptible to
dehydration (Fig. 5.9), and this is the process seen in step 4, facilitated by a dehy-
dratase domain, yielding the corresponding α,β-unsaturated thioester known as
trans-crotonyl-ACP, a thioester of trans-crotonic acid (the common name for 2-
butenoic acid).
In step 5, the trans-crotonyl-ACP from the previous step undergoes a reaction
typical of α,β-unsaturated carbonyl systems; namely, an enoyl reductase-mediated
conjugate reduction, as seen in Fig. 5.10. This involves conjugate nucleophilic addi-
tion of hydride from NADPH at the β-carbon of the thioester, affording a thioester

S S
Enz Enz
3
NADPH/H+
S ACP S ACP
β-keto-
reductase
O O OH O
β-ketothioester β-hydroxythioester
FIGURE 5.8
Ketone reduction by NADPH in step 3 of fatty acid biosynthesis.
S Enz S S Enz
B: Enz
H
4
S ACP S ACP S ACP
dehydratase
H2O
OH O B–H OH O O
β-hydroxythioester trans-crotonyl-ACP
(α,β-unsaturated thioester)
FIGURE 5.9
Dehydration to trans-crotonyl-ACP in step 4 of fatty acid biosynthesis.
S Enz
S Enz 5 S Enz
NADPH/H+ B–H
enoyl S ACP
S ACP S ACP
reductase
(conjugate addition O
H: O O
of hydride) butyryl ACP
trans-crotonyl-ACP thioester enolate (saturated thioester)
FIGURE 5.10
Conjugate reduction of trans-crotonyl-ACP to butyryl ACP in Step 5 of fatty acid biosynthesis.
S Enz 6
acyl O O
HS Enz
transfer H2O
S ACP S Enz OH +
butyric acid HS ACP
H-B
O HS ACP
saturated thioester
FIGURE 5.11
Acyl transfer in step 6 of fatty acid biosynthesis, followed by hydrolysis.
enolate intermediate which upon protonation yields the corresponding saturated

thioester product, butyrylACP.
The sixth step of the sequence involves an intramolecular acyl transfer from the
acyl ACP thiol function to the adjacent enzyme thiol function as shown in Fig. 5.11.
What happens next depends on what outcome is required. For example, if the satu-
rated acyl–SEnz bond were to undergo simple hydrolysis, the corresponding short-
chain carboxylic acid, butyric acid, would be liberated.


four carbons O 1 Repeat
six carbons
O
loading S Enz Steps 2–6
S Enz S Enz
malonyl CoA O S ACP
HS ACP HS ACP
O O Repeat
Steps 1–6
O
eight carbons
S Enz
Repeat
Steps 1–6 HS ACP
x4
then H2O
O HS Enz
sixteen carbons
+
OH HS ACP
palmitic acid
FIGURE 5.12
Sequence repetition for chain extension in biosynthesis of saturated fatty acids.
However, if loading of a new malonyl-CoA unit onto the fatty acid synthase com-
plex should occur (i.e., a repeat of step 1) instead of hydrolysis, this would set the stage
for subsequent repetition of steps 2 through 6, resulting in extension of the chain
from four carbons to six carbons (Fig. 5.12). Another loading step followed by repeti-
tion of steps 2 through 6 would extend the chain further, to eight carbons. Finally, if
an additional four cycles of malonyl-CoA loading followed by steps 2 through 6 cul-
minated in a final hydrolysis step, the resulting carboxylic acid would contain a total
of 16 carbons (palmitic acid). Thus, we see that by sequential loading of malonyl-CoA
units and subsequent Claisen condensations, reductions, dehydrations, conjugate re-
ductions, and acyl transfers, the chain grows by two carbons per cycle, always yield-
ing a fatty acyl product with an even number of carbon atoms. While this may be a
somewhat simplified representation of a complex biochemical system, the emphasis
here on the involvement of a number of familiar organic reactions adequately repre-
sents the fundamental chemistry at the core of this important process.
Reversal of the steps outlined above constitutes the chemistry associated with the
degradative β-oxidation of saturated fatty acids to acetyl-CoA. Figure 5.13 uses the
degradation of palmitic acid (as palmitoylCoA) to illustrate the process, but note
some important caveats: i) FAD → FADH2 is used for the introduction of the enoyl
thioester double bond and ii) NAD+ is used for the alcohol oxidation step (FA syn-
thesis carries out the corresponding reduction using NADPH/H+). Otherwise, the
organic chemistry of these two processes is nearly identical.
In some fatty acid synthase (FAS) systems, palmitic acid is the end prod-
uct, though shorter chains may be obtained along the way by simple hydrolysis.
Subsequent chain extension of palmitic acid can occur via its coenzyme A deriva-
tive (palmitoylCoA) as a starter unit along with malonyl-CoA units as extenders.
In other systems, odd-numbered fatty acids, though rare, can be produced; one ex-
ample is margaric acid (heptadecanoic acid), a trace component found in ruminant

O OH O

sixteen carbons sixteen carbons
sixteen carbons H2O
palmitoyl CoA SCoA SCoA
5 conjugate 5
FAD FADH2 conjugated enoyl thioester addition β−hydroxythioester
NAD +
B: H O SCoA O O O
sixteen carbons
HSCoA
SCoA SCoA
5 5
H B β−ketothioester
Retro-Claisen condensation
O O O
fourteen carbons
8
SCoA + SCoA SCoA
4 sequence
repetition
FIGURE 5.13
Reversal of steps in the β-oxidation pathway for degradation of palmitoylCoA to acetyl-CoA.
milk. The same sequence of reactions is involved as for palmitic acid biosynthesis,
except that the starter unit is propionyl-CoA instead of acetyl-CoA. Subsequent
cycles of malonyl-CoA chain extension via condensation, reduction, dehydration,
and conjugate reduction will thus produce an odd-numbered fatty acid chain, re-
gardless of the number of cycles. This is illustrated in Fig. 5.14 using bolded bonds
for the three carbons derived from the propionyl-CoA starter unit.
BRANCHED FATTY ACIDS: DIFFERENT ROUTES

AND DIFFERENT RESULTS
While most fatty acids are linear in constitution, some consist of chains containing
one or more branches, usually methyl groups. A number of different routes to such
structures are illustrated in Fig. 5.15. In route 1, a branched starter unit such as iso-
butrylCoA, 2-methylbutyrylCoA or isovalerylCoA is loaded and extended in the
usual fashion by sequential additions of malonyl-CoA units, leading to fatty acids with
methyl branching either one or two carbons distant from the C-terminus of the chain.
In route 2, acetyl-CoA is the starter unit, while methylmalonyl-CoA serves as the
chain extender, leading to a hydrocarbon backbone with repeating methyl branches
on alternate carbons. Route 3 involves the use of an existing unsaturated fatty acid and
introduces a methyl branch via SAM alkylation of the unsaturated fatty acid double
bond, followed by a 1,2-H shift and NAPDH reduction of the resulting carbocation.
Ring-containing fatty acids are rare but are also known, and Fig. 5.16 illustrates
two examples. Hydnocarpic acid, an antimicrobial component of chaulmoogra oil
which was used as an early and effective treatment for Hansen’s diesease (leprosy),
utilizes a cyclic starter unit, cyclopent-2-enyl carboxylCoA derived from the non-
peptidic amino acid precursor cyclopentenyl glycine, followed by chain extension
with five malonyl-CoA units and hydrolysis. Other terminal cycloalkyl fatty acids

O O

HS-Enz
propionyl lCoA 1
SCoA S Enz
starter unit fatty acid loading
synthase
O O complex
2 HSCoA O S ACP
HS-ACP malonyl/acetyl
O SCoA transferase O
O
malonyl-CoA
Steps 2–6
O
five carbons
S Enz
Repeat HS ACP
Steps 1–6
O
seven carbons
S Enz
etc.
HS ACP
O
seventeen carbons
OH
margaric acid
FIGURE 5.14
Propionyl-CoA starter unit and sequence repetition for biosynthesis of margaric acid.
are also known (cyclohexyl and cycloheptyl, for example) and arise in a similar fash-
ion from chain extension of the corresponding cycloalkyl carboxylCoA starter units.
Deuterium labeling studies show that alkylation of the double bond of oleic acid by
SAM leads to the indicated carbocation which is quenched by methyl group depro-
tonation and subsequent cyclopropane ring closure rather than a 1,2-H shift and re-
duction as was previously outlined (Fig. 5.15), yielding dehydrosterculic acid. Also
worth noting are the rare and highly strained ladderane lipids which include struc-
tures such as pentacycloanammoxic acid; all possess three or five fused cyclobu-
tane rings at the C-terminus of a linear carbon chain. Produced by certain deep-sea
ammonium-oxidizing (anammox) bacteria important to the oceanic nitrogen cycle,
ladderane lipids are of unknown biosynthetic origin and may prove to be unique
biomarkers for the anaerobic ammonium oxidation process.
MONO- AND POLYUNSATURATED FATTY ACIDS:

PUTTING IN THE “ESSENTIAL” DOUBLE BONDS
Most unsaturated fatty acids are derived from saturated fatty acids by the introduc-
tion of one or more double bonds in a dehydrogenation process usually mediated by

O O
Bioorganic Synthesis 196 SCoA OH

n-1
isobutryl CoA
O O
O O
O SCoA
Route 1 SCoA
starter units n malonyl CoA OH
extender units n-1
2-methylbutyryl CoA
O O
SCoA OH
isovaleryl CoA n-1
O O
O O
Route 2 O SCoA
starter unit SCoA OH
n methylmalonyl CoA n-1
acetyl CoA
extender units
H3C SAM O O
n n OH OH
Route 3 via n n
SAH
unsaturated fat 1,2-H shift then
NADPH/H+
O
n n OH
FIGURE 5.15
Different starter or extender units or SAM methylation for branched fatty acid biosynthesis.
so-called desaturase enzymes which also control the position at which the double
bond will be introduced. As we will see, there are different mechanisms for the in-
troduction of double bonds in fatty acid chains and these can vary depending on
whether the producing organism is animal, plant, or bacteria. We will also see that
animals and plants differ in their ability to introduce double bonds at specific posi-
tions in a fatty acid chain. But before we go there, let’s take a brief look at the two
most important methods for desaturation of fatty acid chains.
AEROBIC VERSUS ANAEROBIC ROUTES

TO DESATURATION
The basic route to the introduction of unsaturation in a fatty acid via aerobic oxida-
tion is illustrated for the conversion of stearic acid to oleic acid in Fig. 5.17. As the
name suggests, the aerobic mechanism requires O2 and is believed to involve diiron-
oxygen complexes, though the proposed structures are speculative. Note the syn ste-
reochemistry of this oxidative dehydrogenation process; the enzyme catalyzing the
process is referred to as a Δ9 desaturase, with Δ9 referring to desaturation occurring
at the 9-position relative to the carboxyl carbon. In plants, the fatty acid is ACP-
bound for the desaturation process, while in animals and fungi it is CoA-bound.

O O

5 malonyl CoA
SCoA OH
extender units
hydnocarpic acid
cyclopent-2-enyl-
carboxyl CoA
H :B
H3C SAM O O
OH OH
oleic acid SAH
OH
dehydrosterculic acid
O
?
precursors
OH
pentacycloanammoxic acid
FIGURE 5.16
Ring-containing fatty acids: hydnocarpic acid, dehydrosterculic acid, and
pentacycloanammoxic acid.
stearic acid (ACP-bound)

ACP ACP
H H ∆9 H H
H H O desaturase H O
O OH
FeIV FeIV FeIV FeIII
O O
oleic acid (ACP-bound)

ACP
O2
H H
O
H H
2e-, 2H+
FeIII FeIII O
FeIII FeIII
O FeIII FeIII
H2O H2O O
FIGURE 5.17
Possible Fe-mediated mechanism for aerobic fatty acid desaturation in plants.
The production of unsaturated fatty acids by anaerobic bacteria requires a non-

oxidative mechanism; quite sensibly, such organisms utilize an intermediate already
available during normal chain extension in the usual fatty acid biosynthetic process.
As shown in Fig. 5.18, the process for the production of oleic acid involves inter-
ruption of the usual fatty acid chain extension process for 12 carbons at the α,β-un-
saturated thioester stage. Instead of the extension process continuing with the usual

conjugate

reduction X B:
acetyl CoA/malonyl CoA, H H H-B
usual chain
H E SEnz H SEnz
extension process isomerization
H H
conjugated H O
H O enoyl thioester
3 malonyl CoA
extender units H H
oleic acid
OH + reductions, SEnz
H dehydrations, H Z
conjugate non conjugated
H O reductions, etc. H O enoyl thioester
FIGURE 5.18
Nonoxidative desaturation mechanism for production of oleic acid by anaerobic bacteria.
R = OH (free acid)
COR
R = SCoA (for modification in animals)
R = SACP (for modification in plants)
9 oleic acid (C18)
desaturation desaturation 6
towards towards
COR methyl end carboxyl end COR
in plants in animals
9 12 ∆12 desaturase ∆6 desaturase 9
linoleic acid cis,cis-6,9-octadecadienoic acid
FIGURE 5.19
Divergence in plant vs. animal FA desaturation pathways and biosynthesis of linoleic acid.
conjugate reduction of the enoyl thioester intermediate, a double-bond isomeriza-

tion takes place, converting the conjugated enoyl thioester into a nonconjugated
enoylthioester with Z geometry at the double bond. At this point, normal fatty acid
chain extension then resumes for three additional cycles of malonyl-CoA addition,
with the attendant reductions, dehydrations, and conjugate reductions terminating
at 18 carbons to ultimately produce oleic acid.
The anaerobic process can produce only monounsaturated chains, but the aero-
bic unsaturation process can proceed further from mono-to di-, tri-or polyunsatu-
rated chains, usually with a nonconjugated relationship between the double bonds
(“methylene-separated”). While both plants and animals can produce multiply un-
saturated fatty acid (FA) chains, there is a distinct difference in positional selectiv-
ity for further double bond introduction. In plants, further oxidative desaturation
of FAs (as their –S-ACP thioesters) usually occurs from an existing double bond
toward the methyl end of the fatty acid chain, while in animals, further desaturation
of FAs (as their –S-CoA thioesters) occurs from an existing double bond toward
the carboxyl end of the fatty acid chain (Fig. 5.19). As it turns out, two fatty acids
important to human health, linoleic acid and α-linolenic acid, are both derived
from oleic acid; linoleic acid (shown in Fig. 5.19) is obtained by introduction of one
additional double bond into oleic acid at the 12-position (i.e., toward the methyl end
of the chain).
Further oxidative desaturation of linoleic acid at the 15-position (again, toward
the methyl end of the chain) produces α-linolenic acid, the world’s most prevalent

linoleic acid

∆6 desaturase
(essential ω−6 FA)
in animals
6
COR
∆15desaturase malonyl-CoA
in plants chain
9 12 extension
γ-linolenic acid
8
COR COR
9 12 15 11 14
α -linolenic acid ∆5 desaturase dihomo-γ-linolenic acid (C20)
(essential ω-3 FA) in animals
8 5
COR important C20
FAs required
∆6 desaturase for biosynthesis
in animals of prostaglandins
11 14
and other
arachidonic acid (C20)
eicosanoids
6 malonyl-CoA 8 5
chain COR
COR extension
then ∆5
9 21 15 desaturase 11 14 17
stearidonic acid in animals eicosapentaenoic acid (C20)
FIGURE 5.20
Biosynthetic modifications of linoleic, α-linolenic, and γ-linolenic acids.
fatty acid (Fig. 5.20). Since animals can only introduce additional double bonds
toward the carboxyl end of the chain relative to an existing double bond, this means
that humans cannot biosynthesize either of these two fatty acids (animals lack the
required Δ12 and Δ15 desaturase enzymes). Thus, linoleic and α-linolenic acids are
referred to as essential fatty acids, since they are essential to human health but can
only be obtained in the diet from plant sources. In nutritional literature, linoleic
acid is often referred to as an omega-6 fatty acid (or ω-6), using a Greek alphabet
nomenclature system. Here, the omega-carbon of a fatty acid is identified as the
one furthest from the carboxyl end of the chain; so in the case of linoleic acid, a
double bond is located starting at the sixth carbon relative to the omega-carbon
(methyl terminus) of the chain. Similarly, α-linolenic acid would be classified as an
omega-3 fatty acid using this nomenclature system. Figure 5.20 also illustrates the
divergence of further desaturation of fatty acids in plants versus animals and the
sequences involved in producing some other important polyunsaturated fats. Once
linoleic and α-linolenic are obtained in the diet, animals can further desaturate their
chains toward the carboxyl end. Thus, linoleic acid (all cis-9,12-octadecadienoic
acid) is converted by desaturation at the 6-position to give γ-linolenic acid, which
can be used as a starter unit (as its SCoA derivative) for one additional cycle of fatty
acid chain extension. The resulting C20 product, dihomo-γ-linolenic acid, can also
undergo a subsequent desaturation at what is now the 5-position of the extended
C20 chain (again toward the carboxyl end), leading to arachidonic acid. A similar
sequence of chain extension and Δ5 desaturation of α-linolenic acid yields eicosa-
pentaenoic acid. All three of these C20 FAs are important as precursors for the pro-
duction of prostaglandins and other structurally related C20 signaling molecules
known as eicosanoids which are important compounds in several highly complex

systems of the body, exerting control over inflammatory and immune responses
Bioorganic Synthesis 200 among others. We will shortly take a look at some of the biosynthetic chemistry
involved in their production, but first we conclude our examination of unsaturated
fatty acids by briefly considering examples of some unusual acetylenic and hetero-
cyclic structures that represent further modified fatty acids.
FURTHER DESATURATION OF FATTY ACIDS:

TRIPLE BONDS AND RINGS
Further desaturation of fatty acids at existing double bond positions can lead to the
introduction of one or more triple bonds. Such acetylenic fatty acids, though less
common and far less stable than their olefinic precursors, are formed in an oxida-
tive desaturation process using O2 and a nonheme diiron complex analogous to that
used in the introduction of double bonds and mediated by acetylenase enzymes,
some of which have both desaturase and acetylenase functions. A typical system is
shown in Fig. 5.21 in which the cis-12 double bond of linoleic acid is further desatu-
rated to give the corresponding acetylenic derivative, crepenynic acid, which makes
up about 70% of the content of crepis alpina seed oil. Matricaria acid, an antifeedant
for both the pink bollworm (a serious cotton pest) and the tobacco hornworm, is de-
rived from typical modifications of crepenynic acid as shown. Further desaturations
and an allylic isomerization of the cis double bond from the 9-to the 10-position are
followed by an allylic oxidation that introduces a ketone function at the 9-position.
COR O 2, NADPH/H+ COR

∆12 -acetylenase
R = OH (free acid) crepenynic acid
linoleic acid R = OPL (phospholipid)
O 2, NADPH/H+
further sequential 14
∆
oxidative desaturations 14 -desaturase
∆ -acetylenase
COR
allylic COR
isomerization
O 2, allylic oxidation then NADP+

(C-H to CH-OH) (CH-OH to C=O)
O O
O 2, NADPH/H+
COR COR
∆16 -desaturase
O 2, NADPH/H+ Baeyer–Villiger
O oxidation
OH O
O COR
H2O
ester hydrolysis
matricaria acid
FIGURE 5.21
Biosyntheses of acetylenic FAs: crepenynic acid and its conversion to matricaria acid.

Subsequent desaturation to afford a second cis-double bond at the 16-position and a

Baeyer–Villiger oxidative ketone-to-ester conversion terminates in ester hydrolysis,
affording the polyunsaturated acetylenic FA natural product. The methyl ester of
matricaria acid has also been found to be a powerful skin-bleaching agent, making
it a potentially useful compound for the treatment of skin hyperpigmentation.
Other examples of polyacetylenic fatty acids include 13,14-dihydrooropheic acid,
isolated from the stem bark of the Indonesian plant Mitrephora celebica and which
has shown remarkable activity against methicillin-resistant strains of Staphylococcus
aureus (MRSA). Similar polyacetylenic FA derivatives (Fig. 5.22) are also thought
to be involved in the biosynthesis of certain natural products containing O-and
S-heterocyclic rings. Wyerone, a potent antifungal acetylenic keto ester isolated
from faba bean shoots contains a furan ring which is believed to arise from H2O
addition across a conjugated diyne precursor as shown in Fig. 5.22. Finally, a pho-
totoxic acetylenic dithiophene ester was isolated from Dyssodia tageoides along with
what is believed to be its precursor, 3,5,7,9,11-dodecapentayn-1-yl-stearoate; the
dithiophene ring system presumably forms via sequential additions of H2S across
the conjugated polyacetylenic ester chain. While other furan and thiophene-con-
taining FA derivatives are presumed to be derived in a similar fashion, a number are
also known to be formed by alternate mechanisms.
There are many additional types of fatty acids, including those containing halo-
gens, aldehyde, and ketone groups, hydroxyl and alkoxyl groups, amides, dicarbox-
ylic acids, and others too numerous to treat here. But before we move on to the
core of the polyketide pathway to natural products, we conclude our examination
of modified fatty acids by returning to arachidonic acid and related C20 FAs and the
13,14-dihydrooropheic acid OH
H+
OCH3
H2O H
O
O
O O
wyerone
H :B
O 2H2S
14
3,5,7,9,11-dodecapentayn-1-yl stearoate
O
H+
i) oxidation H
O
S S
ii) acetylation
O S
from Dyssodia tagetoides 14
OAc H :B
FIGURE 5.22
Dihydroorpheic acid and polyacetylenic precursors to furan and thiophene natural products.

role they play in the formation of the important group of signaling molecules known
Bioorganic Synthesis 202 as the eicosanoids.
PROSTAGLANDINS, THROMBOXANES, AND

LEUKOTRIENES: THE POWER OF OXYGENATED FAS
As seen in the previous section, oxidative transformations of various types can trans-
form fatty acids into some unusually bioactive substances that bear only a passing
resemblance to their relatively simple precursors. A most striking example of this is
seen in the so-called eicosanoids, a group of powerful bioactive molecules derived
from the essential C20 fatty acids arachidonic acid, dihomo-γ-linolenic acid, and
eicosapentaenoic acid that we saw earlier in Fig. 5.20. Of these three, arachidonic is
the most important player, as it serves as the essential precursor to most of the pros-
tanoid family of eicosanoids which includes the prostaglandins, prostacyclins, and
thromboxanes, examples of which are illustrated in Fig. 5.23 along with the stan-
dard abbreviations used for the constitutions of the various C-5 ring systems found
in different prostaglandins (PGs) and thromboxanes (TXAs).
CO2H
O O
CO2H
HO HO
OH OH
PGE2, a prostaglandin PGI2, a prostacyclin
configuration of ring
CO2H
O
O PG E 2 number of side-chain
double bonds
OH
TXA2, a thromboxane a prostaglandin
O O O OH O OH
O HO HO
PGA PGB PGC PGD PGE PGF
O O OH
O
O O
O O
HO HO
PGG, PGH PGI PGJ TXA TXB
FIGURE 5.23
Examples of a prostaglandin, a prostacyclin, a thromboxane, and prostanoid ring abbreviations.


CO2H
dihomo-γ-linolenic acid Type-1
OH
CO2H
arachidonic acid Type-2
OH
CO2H
eicosapentaenoic acid Type-3
OH
FIGURE 5.24
Origins of Type-1, Type-2 and Type-3 prostanoids from the three different C20 EFAs.
The examples shown in Fig. 5.23 are all members of the so-called “Type-2” pros-
tanoids which are derived from arachidonic acid and are the most important of the
three types. Type-1 and Type-3 prostanoids are derived from the two remaining C20
EFAs, as shown in Fig. 5.24. Type-1 prostanoids have only one side chain double
bond, while the others have two and three side chain double bonds, respectively.
Thus, the numeral 1, 2, or 3 following the abbreviation for a given PG or TX com-
pound not only indicates the number of double bonds in the side chains but is also
indicative of a given prostanoids EFA precursor.
The prostaglandins were originally isolated from the fluid of sheep prostate glands,
hence the name “prostaglandin.” These and other prostanoids have subsequently
been found in most human tissues, though only in trace amounts. Prostaglandins
exert their effects on many different systems in the body, including the constriction
or dilation of smooth muscle, the induction of uterine contraction during labor,
and the regulation of inflammatory response, among many others. While extremely
potent compounds which exert their effects at very low concentrations, they are also
relatively short-lived and so mainly act locally at the site of production.
The biosynthesis of the Type-2 prostaglandin PGH2 is outlined in Fig. 5.25.
Following hydrolytic release of arachidonic acid from a phospholipid via phospho-
lipase A2, the process begins with an oxidative H-atom abstraction mediated by a
PGH2 synthase complex consisting of both a cyclooxygenase (COX) and a peroxidase
component. The former may be one of two types, either COX-1 or COX-2, and it
is their inhibition by aspirin or other COX-1/COX-2 inhibitors such as nonsteroi-
dal anti-inflammatory agents (NSAIDs like ibuprofen, naproxen, or specific COX-2
inhibitors like Celebrex) that act to reduce inflammation through the inhibition of
this biosynthetic pathway. Addition of molecular oxygen to the allylic radical and
subsequent cyclization (hence the term cyclooxygenase) initiated by addition of a

phospholipid

phospho-
CO2H O2
lipase A2 H H
H H
PGH synthase
COX-1/COX-2
arachidonic acid (C20) O
O
O O2 O2
O H
O O
O
O O
H atom abstraction O O O
O O
CO2H CO2H
O reduction O
(R-OOH to
R-OH)
O-OH PGG2 OH PGH2
FIGURE 5.25
Oxidative transformations of arachidonic acid leading to a key prostanoid, PGH2.
second O2 molecule is followed by H-atom abstraction to produce the cyclic per-

oxide derivative PGG2 which undergoes a subsequent hydroperoxide-to-alcohol
reduction to give PGH2. Though unstable, PGH2 is of great significance as it is a key
intermediate which goes on to form several other important PG or TX derivatives
by subsequent transformations.
Mechanisms for the conversion of PGH2 into PGF2 or PGE2 by nucleophilic ring
opening of the cyclic peroxide component are shown in Fig. 5.26. Nucleophilic attack
by hydride from NADPH and resultant cleavage of the O–O bond is accompanied
by protonation to give the cis-1,3-cyclopentanediol component of PGF2 (PGF2α in
prostanoid nomenclature, where α indicates the natural S configuration at the chiral-
ity center bearing the side chain hydroxyl group). Naturally occurring PGF2 is also
known in medicine as dinoprost; its function in causing uterine contractions makes
it useful for the induction of labor at term. It can also act as a bronchodilator and
an abortifacient.
For the formation of PGE2, one proposed mechanism involves nucleophilic
attack on the PGH2 cyclic peroxide linkage by the sulfhydryl group of the important
tripeptide glutathione (abbreviated GSH, as its anion GS–), leading to an interme-
diate that undergoes oxidative elimination and regeneration of GS– to afford the
corresponding β-hydroxycyclopentanone ring system. PGE2, known in medicine as
the drug dinoprostone, also exhibits important effects in the induction of fever and,
like PGF2, can be used to induce labor at term or as an abortifacient. PGE2 is also a
vasodilator, causing the relaxation of smooth muscle. A number of synthetic ana-
logues of both PGF2 and PGE2 have emerged over the years such as carboprost and
gemeprost which have proven to be useful primarily in obstetric medicine, either
for labor induction or as abortifacients.

-SG

O O
CO2H GS-H CO2H
O O
(glutathione)
H+
PGE2 synthase
OH PGH2
OH
NADPH/H+ PGF synthase

:H- :B
GS-O H
O
CO2H CO2H
O
H+
HO
OH GS- OH
OH O
CO2H CO2H
HO PGF2 (dinoprost) HO PGE2 (dinoprostone)

OH OH
OH
synthetic analogue O
synthetic analogue
CO2H CO2CH3
HO
HO
HO
OH
carboprost gemeprost
FIGURE 5.26
PGF2 or PGE2 via ring opening of PGH2 plus synthetic analogues carboprost and gemeprost.
For the conversion of PGH2 into prostacyclin (PGI2), a diradical mechanism

via homolytic cleavage of the cyclic peroxide O–O bond, followed by cyclization
and H-atom abstraction is usually invoked (Fig. 5.27). Prostacyclin is also known as
the drug epoprostenol in medicine, being used as a very potent inhibitor of platelet
aggregation; it was also the first prostaglandin the formation of which aspirin and
other NSAIDs were shown to be able to limit through COX-1/COX-2 inhibition.
Epoprostenol has also been found useful for the treatment of pulmonary hyperten-
sion, being administered either subcutaneously or by inhalation. As with PGE2 and
PGF2, synthetic analogues of PGI2 such as iloprost and cicaprost are finding in-
creasing use for treatment of pulmonary hypertension and also certain autoimmune
diseases such as scleroderma. Such analogues help to address the short half-life of
the natural drug by removal of its labile enol ether function which, like an acetal, is
subject to hydrolysis which deactivates PGI2.
Finally, we consider the conversion of PGH2 into the potent vasoconstrictor
and platelet aggregation promoter, thromboxane A2 (TXA2). Here, the mechanism
is again formulated as a cleavage of the O–O bond of the cyclic peroxide ring ac-
companied by adjacent C–C bond cleavage to yield an alkoxyradical and an allylic


O
HO
O
CO2H O2 O
O
NADPH
OH PGH2
O OH
synthetic analogues O
O
address PGI2 deactivation via
enol ether hydrolysis: OH
HO
H
H+
OH O -H O
O O H2O +
+H
O OH
HO OH prostacyclin (PGI2)
O O O
HO HO
HO HO
HO iloprost cicaprost OH
FIGURE 5.27
PGH2 to PGI2 conversion; two synthetic PGI2 analogues, iloprost and cicaprost.
radical (Fig. 5.28). Single electron transfer from the allylic radical leads to a dipolar
species composed of an allylic cation and an alkoxy anion, the latter of which acts as
a nucleophile to attack the proximate aldehyde carbonyl. The resulting alkoxy anion
then traps the allylic carbocation, leading to formation of the bicyclic acetal struc-
ture of TXA2. This strained acetal has a very short half-life and is rapidly hydrolyzed
to a bioinactive hemiacetal derivative, TXB2. Because of its rapid hydrolysis, TXA2
functions primarily in tissues surrounding its site of production. It is thought to
be particularly important during tissue injury and inflammation, though its potent
modes of action also implicate it in various blood-clotting (thrombosis) and angina-
related events. Like other prostanoids derived from PGH2, production of TXA2 is
also suppressed by aspirin or other COX inhibitors.
We conclude this section with a brief look at the formation of another group of
arachidonic acid metabolites known as the leukotrienes, a group of compounds
intimately linked to the effects of inflammatory response. The name is related to
leukocytes (white blood cells) from which these compounds are produced; the
name is also indicative of the characteristic presence of three conjugated double


O
CO2H CO2H
O TXA2
O
synthase
OH PGH2
OH
cleavage
:O O
electron CO2H
CO2H
transfer
O O
OH OH
cyclization HO
CO2H H O/H+ CO2H

O 2
O acetal HO O
acetal hydrolysis
OH TXA2 hemi-acetal OH TXB2
linkage
linkage
FIGURE 5.28
Conversion of PGH2 to TXA2 and its subsequent hydrolysis to inactive TXB2.
H
O O
CO2H O2 CO2H
H H 5-lipoxygenase H H
arachidonic acid (C20) O O

H+
O OH
O CO2H
CO2H H
H
LTA4 H2O B:
FIGURE 5.29
5-Lipoxygenase-mediated biosynthesis of leukotriene A4 (LTA4) from arachidonic acid.
bonds within the structures. Unlike the biosynthesis of prostanoids, which utilizes
cyclooxygenase enzymes, initial leukotriene biosynthesis employs a 5-lipoxygenase
enzyme, the action of which initially leads to formation of leukotriene A4 (LTA4) as
shown in Fig. 5.29 (as in the prostanoids, the numeric subscript indicates the total
number of double bonds present within a given LT structure).
LTA4 is subsequently converted to two other important leukotrienes, as illus-
trated in Fig. 5.30. The action of the enzyme LTA4 hydrolase leads to remote nu-
cleophilic attack by water at the furthest end of the conjugated triene system of
LTA4, resulting in conjugate addition and epoxide ring opening to produce LTB4,
a potent leukotriene associated with the immune response. LTB4 acts in part by
helping to direct white blood cells to affected tissues; however, elevated levels of
LTB4 have been linked to a variety of inflammatory and allergic conditions, espe-
cially asthma, ulcerative colitis, psoriasis, and rheumatoid arthritis among others,

:B
H
O H H B
O O
CO2H CO2H
H 2O
LTA4
LTA4
hydrolase
glutathione LTC4 synthase
(GSH)
H B
O HO
CO2H
CO2H
S-H :B
OH LTB4
Glu Cys Gly
OH OH OH
CO2H
H2O H2O
S LTC4 - glu S LTD4 S LTE4
- gly
Cys Gly Cys Cys
Glu Gly
FIGURE 5.30
Conversion of LTA4 to LTB4 or LTC4 and sequential LTC4 hydrolysis to produce LTD4 and LTE4.
making the LTB4 receptor an ongoing target for intensive drug research. Direct
nucleophilic attack on the epoxide ring of LTA4 by the cysteine thiol function of
the tripeptide glutathione (Glu-Cys-Gly) leads to formation of LTC4 which, along
with its metabolites LTD4 and LTE4, make up the slow-reacting substance of ana-
phylaxis (SRS-A) that initiates this potentially life-threatening condition of ex-
tensive allergic inflammatory reaction. The SRS-A combination is estimated to be
5000 times more potent than histamine for triggering inflammatory response and
though slower in onset, has a longer duration of action. LTD4 and LTE4 are pro-
duced from LTC4 by sequential degradation of the glutathione component, first
through hydrolytic loss of Glu to give LTD4, then loss of Gly from LTD4 to yield
LTE4, as shown.
We’ve presented here a bare minimum of examples of biosynthesis and subse-
quent modification of fatty acids in this section to at least have enough background
to move to the main event in this chapter, namely the closely related chemistry as-
sociated with the biosynthesis of the diverse family of bioactive natural products
derived from the so-called polyketide pathway.
POLYKETIDE BIOSYNTHESIS: MORE STARTER UNITS

AND EXTENDER UNITS, BUT WITH A TWIST
As mentioned at the beginning of the chapter, polyketide research dates all the way
back to the late 1800s with the work of J. N. Collie and others. In the following
years, many new compounds that fit the structural peculiarities associated with this

class of natural products were isolated and characterized from fungi and bacteria,

mainly by organic chemists using standard organic laboratory techniques. But it
wasn’t until the early 1950s that A. J. Birch (also an organic chemist) began a serious
examination of the possible biosynthetic mechanisms associated with production of
these compounds, inspired in part by the known chemistry of fatty acid biosynthe-
sis. Birch’s initial proposal became known as the acetate hypothesis: “Polyketides
are formed by the head-to-tail linkage of acetate units, followed by cyclization by
an aldol reaction or by acylation to phenols” (Birch & Donovan, 1953). By using
radiolabeling techniques in their early work, Birch and others were able to show
that polyketide compounds derived from the same linkage of acetate units that was
already known to be employed in primary metabolism pathways in the organisms
producing the compounds. As we will see shortly, Birch’s insight derived in part
from his knowledge of the kinds of organic reactions a hypothetical polyketide
compound might be expected to undergo and the resulting structural features that
would result from those reactions. In other words, working backward from a natural
product structure, he could visualize how standard organic transformations operat-
ing on a “polyketide” precursor could give rise to that product and that the origin
of the “ketide” units in the polyketide would almost certainly be individual acetate
units which had been linked together in some fashion.
The gist of this concept is illustrated in Birch’s proposed biosynthesis of orsell-
inic acid, an aromatic compound isolated from the fungus Penicillium griseofulvum
(Fig. 5.31). As can be seen, if the phenolic “enol” hydroxyl groups of orsellinic acid
are envisioned in their keto forms, the remaining ring double bond could arise by
a dehydration of a tertiary alcohol precursor which itself could be produced by an
intramolecular aldol addition between carbonyl units of a folded “polyketide” pre-
cursor, which in turn could then be envisioned as arising from the linkage of four
individual acetic acid units by some undetermined biosynthetic mechanism.
How could one support such a hypothesis experimentally? In Birch’s early work,
he made use of acetic acid (as acetate) which had been radiolabeled at the carboxyl
FIGURE 5.31
Retrosynthetic analysis of orsellinic acid leading to a “polyketide” derived from four acetic acids.


CH3 O CH3 O
O
Penicillium O
OH OH
H3C OH
griseofulvum
14C-labeled acetic acid O O HO OH
14C-labeled orsellinic acid
FIGURE 5.32
Location of 14C atoms in orsellinic acid derived from 14C-labeled acetic acid.
carbon (i.e., 14C-labeled) and which was then provided to the organism producing
the orsellinic acid. Such isotope feeding studies were then followed by isolation of
the natural product which was then subjected to a series of systematic chemical
degradation reactions. Careful measurement of the radioactivity content of the deg-
radation products from different reactions allowed the unambiguous determination
of the specific positions in orsellinic acid where 14C atoms had been incorporated by
the organism into the structure. As Fig. 5.32 illustrates, the pattern of 14C substitu-
tion anticipated by the acetate hypothesis was confirmed in these experiments. In
later work, patterns of incorporation of 13C-labeled acetate into many other struc-
tures has been more conveniently analyzed through the use of 13C NMR techniques;
such isotope incorporation studies continue to be an essential part of ongoing stud-
ies of the chemistry of biosynthetic and metabolic pathways, as we will see shortly.
Of course, the above analysis does not address the more important question
of how individual acetate units might become linked together to form the sorts of
polyketide intermediates that could undergo subsequent condensations, aromatiza-
tions, and other reactions leading to various natural product structures. As it turns
out, a likely answer to this question could be found by envisioning some simple
modifications of the chemistry associated with the biosynthesis of fatty acids, a pro-
cess which was already well understood by the time Birch began his initial investi-
gations. As Fig. 5.33 illustrates, individual acetate units could be readily assembled
into even-numbered polyketide chains of varying length via a modified fatty acid
pathway in which the usual β-ketothioester intermediate arising from Claisen con-
densation of acetyl-CoA and malonyl-CoA units (enzyme-bound) is prevented from
undergoing the usual sequence of ketone reduction, dehydration, and conjugate re-
duction that normally precedes acyl transfer and chain extension. In this way, malo-
nyl-CoA extender units could be added sequentially to a growing polyketide chain
rather than to a growing saturated fatty acid chain. While the scheme presented in Fig.
5.33 is general and rather simplified, it illustrates the basic chemistry associated with
certain kinds of polyketide synthase (PKS) systems which, as it turns out, closely
resemble fatty acid synthase (FAS) systems in both composition and chemistry.
We will find this modified fatty acid pathway operating mainly in bacteria, fungi,
and plants rather than in animals (unlike the terpenoid pathway which is more widely
distributed). Later on we’ll sort out the different types of PKS systems that are found in
various organisms and the different kinds of natural products that they can produce.


HS-Enz O
acetyl CoA 1
SCoA S Enz
starter unit polyketide loading
synthase
O O complex
2 HSCoA O S ACP
HS-ACP
O SCoA
malonyl CoA O O
S Enz O O
2
S Enz Claisen S Enz
S ACP condensation
S ACP O S ACP
O O
O CO2
β-ketothioester tetrahedral O
β-ketosynthase O
intermediate
NOTE:
at this stage, S Enz a diketide
NO reduction, 3 O
acyl O
dehydration,
or conjugate reduction transfer
S ACP S Enz
H-B
O O HS ACP
a triketide O O
O O O Repeat 1
Steps 2,3 S Enz loading
S Enz
malonyl-CoA
Repeat O S ACP
Steps 1-3 HS ACP
x5 O O
then H2O a polyketide
O O O O O O O O O
OH
FIGURE 5.33
Simplified, generalized diagram of chain extension sequence in polyketide biosynthesis.
But for now, we focus initially on some simple polyketide structures assembled ac-
cording to the simplified scheme above to learn how different kinds of reactions or
even different chain folding patterns can lead to natural products of very different
structure, even though they may arise from the same initial polyketide precursor.
AROMATIC POLYKETIDE NATURAL PRODUCTS:

PHENOLS AND RELATED STRUCTURES
The polyketide pathway is one of nature’s most important sources of aromatic com-
pounds, especially phenols of widely varying structure; the versatility of polyketide
cyclizations and aromatizations stems in part from the multifunctional nature of
a given polyketide chain operating in conjunction with enzymes which may en-
force different patterns of chain folding as well as positional selectivity for deproton-
ations and cyclizations. Thus, a single precursor polyketide chain may yield different

acetyl-SEnz + 3 malonyl-SEnz
Bioorganic Synthesis 212 PKS
O O O O
folding pattern 1 folding pattern 3
H3C SEnz
folding pattern 2
O
CH3 O EnzS O EnzS O
O O
O
SEnz H3C
deprotonation H3C Odeprotonation
O O here here O O
deprotonation
β-diketone formation here
thioester formation β-diketone formation
enolate enolate enolate
O
CH3 O EnzS
O EnzS O
O
O O
SEnz H3C
O
H3C
O O O O
intra- intra-
molecular - HSEnz intra-
molecular - H2O molecular - HSEnz
aldol O-acylation
Claisen
CH3 O O O O
SEnz O O H3C
O O H3C O O O
keto to H2O, keto to

enol - HSEnz enol
OH O OH
CH3 O
O O H3C
OH
H3C O HO OH
HO OH
orsellinic acid tetraacetic acid lactone phloroacetophenone
FIGURE 5.34
Different folding patterns and reaction modes for a tetraketide leading to different products.
products from different organisms, or a mixture of different products from the same
organism via a thioester enolate or any one of several different β-diketone enolates
that may be available via enzyme-specific deprotonation reactions. Different prod-
ucts will require different chain folding patterns which may then lead to different
intramolecular condensations or other reactions.
As shown in Fig. 5.34, for orsellinic acid, folding pattern 1 can lead to for-
mation of a thioester enolate ion which can cyclize via intramolecular aldol con-
densation followed by thioester hydrolysis and keto-to-enol tautomerization to
produce the phenolic product. The same tetraketide precursor can follow folding
pattern 2, undergoing intramolecular cyclization via O-acylation of the indicated
β-diketone enolate ion, which leads directly to tetraacetic acid lactone (an α-pyrone)
which has been isolated from a number of different sources such as Penicillium

stipitatum along with small amounts of orsellinic acid and its decarboxylation product,

orcinol (5-methylbenzene-1,3-diol). Folding pattern 3 and the indicated β-diketone
enolate ion allows an intramolecular Claisen condensation, affording the cytotoxic
fungicide phloroacetophenone after keto-to-enol tautomerization and aromatiza-
tion. While the actual intermediates and precise sequence of events involved are
undoubtedly more complex, a glance at this somewhat simplified analysis allows us
to quickly see the predictive power of the acetate hypothesis and the synthetic power
inherent in the polyketide pathway to natural products.
A somewhat more complex example of alternative polyketide chain folding pat-
terns leading to different aromatic products is illustrated in Fig. 5.35. Both alternar-
iol, a toxic metabolite of Alternaria fungi that is an important contaminant of fruits
and grains, and rubrofusarin, an antimycobacterial compound with antiestrogenic
properties from Fusarium graminearum, are derived from a common heptaketide
intermediate that undergoes different enzyme-specific modes of cyclization and aro-
matization (with an additional postcyclization SAM methylation for rubrofusarin).
It is worth noting that the acetate hypothesis has often been quite useful in resolv-
ing conflicts over structural assignments when two alternative structures for a natural
product are possible. Prior to the advent of highly sophisticated instrumental analy-
sis techniques, choosing between or among similar proposed structures for a newly
isolated natural product could be quite problematic (and occasionally still is). Using
alternariol as an example, consider the two proposed structures, I and II, as shown
in Fig. 5.36. These compounds are constitutional isomers of one another and either
might be reasonable to assign to alternariol; both would have very similar IR, 1H
NMR and 13C NMR spectra and would be difficult to distinguish from one another
on that basis. Of course, one way to decide the matter would be to independently
synthesize the proposed structures and then compare a sample of each with a sample
of the naturally occurring compound for melting point, NMR spectra, and so on,
(this was a common technique in earlier natural products chemistry and is still used
for structure confirmation in some cases). However, this would involve a great deal of
time and effort in the laboratory; fortunately, the decision is made much easier by ap-
plication of the acetate hypothesis. One can readily derive a single folded polyketide
chain precursor for structure I, while structure II cannot be derived without resorting
to the use of two separate chains which would have to be linked together in a very
specific and far more complicated sequence. While not an absolute proof, the acetate
hypothesis, along with a bit of work with pencil and paper, quickly leads to a predic-
tion of structure I as being far more likely than structure II for this compound.
ISOTOPIC LABELING STUDIES: BIOSYNTHETIC

INSIGHTS VIA 13 C NMR
As mentioned earlier, one way to demonstrate how linear polyketide precursors are
cyclized to yield aromatic products is through the use of 13C NMR. One might expect

acetyl-SEnz + 6 malonyl-SEnz
Bioorganic Synthesis 214 PKS
O O O O O O O
H3C SEnz
folding folding
pattern 1 pattern 2
O
CH3 O
O a O CH3
O
O b
O O
O O
SEnz O SEnz O
O O
aldol-type a: aldol-type b: Claisen
(Knovenagel) condensation
condensations condensation
(Knovenagel)
O
CH3 O O O CH3
O O SEnz O O O
O
keto-to-enol keto-to-enol
aromatization aromatization
OH SAM
OH
HO O
CH3 CH3
OH
OH SEnz OH OH O
HO O
cyclization i. cyclization ii. SAM
-HSEnz -H2O methylation
OH
H3CO O CH3
CH3
OH
OH OH O
HO O O
alternariol rubrofusarin
FIGURE 5.35
Different folding patterns and reaction modes leading to alternariol vs. rubrofusarin.
that adjacent 13C atoms would be able “talk to each other” via spin-spin coupling,
just as adjacent H atoms are able to do in 1H NMR, but recall that the natural abun-
dance of 13C is only about 1.1%, so for 13C NMR, 13C–13C coupling is insignificant
since the probability of two 13C atoms being adjacent to one another is vanishingly

OH

OH
OH OH
HO O O HO O O
I II
O
O O
O O O
SEnz
O O
O EnzS O
O O O
SEnz
more likely less likely
O
FIGURE 5.36
Two proposed structures for alternariol (I vs. II) clarified by the acetate hypothesis.
small. Also recall that 13C–H coupling (which is significant) is usually removed by
irradiation with a separate rf source, thereby eliminating any signal splitting due to
such coupling, leaving a spectrum with single peaks for each nonequivalent carbon
atom within a given structure. It is also useful to recall that signals from 13C atoms
which are not bonded to a H atom usually have a smaller intensity when compared
to signals from 13C atoms which are bonded to H atoms, often making these signals
relatively easy to distinguish from one another (this is also one reason why the area
under peaks in 13C NMR spectra are not “integrated” as they are in 1H NMR). With
all the above in mind, consider Fig. 5.37, which provides a simple example of the use
C NMR in natural product studies.
13
In the simulated spectrum of natural alternariol (spectrum A) with C atoms

numbered as shown, we can see a total of 14 signals, one for each C atom in the
structure (since no two C atoms are equivalent in this compound). We can now
compare this spectrum with simulated spectrum B, which is of alternariol isolated
from a producing organism which had been fed 1-13C-labeled acetate. As expected,
the signal for each of the odd-numbered carbon atoms in the structure has been
enhanced in spectrum B. While not a dramatic result, it clearly illustrates how 13C
incorporation serves to confirm a polyketide pathway origin for the product.
Further use of 13C NMR is illustrated in Fig. 5.38, again with two simulated spec-
tra (A and B) of alternariol. Spectrum A is that of alternariol derived from a produc-
ing organism which has been fed 2-13C-labeled acetate. As expected, we now see
enhanced signals for each of the even-number carbons in the structure. The more
significant spectrum, however, is spectrum B which is the result of a feeding study
employing acetate which has been doubly labeled (1,2-13C-labeled acetate). This
spectrum requires some additional explanation. First of all, when 1,2-13C-labeled


14CH OH
3 6
5 4
13 14CH 6 4
O 3 5
12 O 7
3
8 7 3
O O 12
OH
O-
9 2 13 8
H3C 2
O 11 O SEnz 11
acetate 10
1 HO 9 O 1 O
O 10
6
12 10
14
9 7 13
1 3 11 2
5 8
180 160 140 120 100 80 60 40 20 0

PPM
O
14CH OH
3 6
13 5 4
14CH 6 4
3 5
O 12 O 7
3
8 7 3
9 O O 12 13 OH
O-
2 8 2
H3C O 11 O 11
10 SEnz
1-13C-labeled acetate 1 HO 10
9 O 1 O
O
6
12 10
4
13
3 11 9 7 14
5
B 1
2
8
180 160 140 120 100 80 60 40 20 0

PPM
FIGURE 5.37
Simulated C NMR of natural alternariol (A) and alternariol via 1-13C-enriched acetate (B).
13
acetate is incorporated into a structure (depicted with bold bonds), we would not
expect to see selected signal enhancements. But unlike the 13C NMR spectrum of
natural alternariol, in which no 13C–13C coupling would be expected, we should
now see such coupling, since some of the compound will contain the doubly la-
beled acetate with adjacent 13C atoms. Keeping in mind that the degree of incorpora-
tion of isotopically labeled precursors by organisms in such studies is usually very
low compared to incorporation of normal starting materials, we will not expect to
see 13C–13C coupling between adjacent doubly-labeled acetate units, since the prob-
ability of two doubly labeled units being incorporated next to one another within
the same structure is vanishingly small. Thus, we should only see13C–13C splitting
between carbon atoms within a given doubly labeled acetate unit. It turns out that


14 OH
6
5 4
13 14 6 4
O 5
12 O 7
3
8 7 3
O O 12
OH
O-
9 2 13 8 2
O 11 O SEnz 11
2-13C-labeled acetate 10
1 HO 9 O 1 O
O 10
6
12 10
14
4
A 2
8
9 713
1 3 11
5
180 160 140 120 100 80 60 40 20 0

PPM
O
14 OH
6
13 5 4
14 6 4
5
O 12 O 7
3
8 7 3
9 O 2 O 12
13 8 OH
O-
2
O 11
10
O SEnz 11
1,2-13C-labeled acetate 1 HO 9 O 1 O
O 10
J 7,8 J 13,14
J 13,14 6
12 10
J 7,8
4
14
B
9 7
1 3 11 2
5 13 8
180 160 140 120 100 80 60 40 20 0

PPM
FIGURE 5.38
Simulated C NMR of alternariol from 2-13C-enriched acetate (A) and selected coupling constants
13
from 13C NMR of alternariol derived from 1,2-13C doubly labeled acetate (B).
coupling constants for 13C–13C splitting are on the order of 50–80 Hz, so they are
more than large enough to distinguish in such spectra and are sufficiently variable
and environment-dependent that we should be able to match up a signal in one
region of the spectrum to another to which it is coupled elsewhere in the spectrum,
based on the unique line spacings involved. Spectrum B illustrates this for two se-
lected cases: i) the coupling between the aromatic ring carbon at position 13 and the
methyl group carbon at position 14 and ii) the coupling between the aromatic ring
carbons at positions 7 and 8 which link the two aromatic rings together. For i), most of
resonance at ~21 ppm from the methyl group comes from the natural abundance13C,

but some must also come from doubly labeled acetate incorporated into the struc-
Bioorganic Synthesis 218 ture. This portion of the signal must in turn be split by the adjacent doubly labeled
acetate carbon at position 13, and therefore should appear as a doublet with line
spacing characteristic of the coupling constant J13,14. If we look further down field
for the resonance from the aromatic ring carbon at position 14, we again see the
satellite peaks on either side of the main peak with the characteristic line spacing
again corresponding to J13,14 due to the expected splitting of this signal by the methyl
carbon. A similar analysis applies to case ii) for the observed splitting for the signals
associated with the aromatic ring carbons at positions 7 and 8.
We can see how this kind of doubly labeled acetate experiment could help sort
out different possible folding patterns for certain polyketide cyclizations, as illus-
trated in Fig. 5.39. The polyketide precursor we saw earlier for biosynthesis of ru-
brofusarin should actually be able to adopt either of two possible folding patterns
that would lead to the same product. Does the mediating enzyme for rubrofusarin
production force the heptaketide precursor into folding pattern (1) or folding pat-
tern (2), or is the product produced by a random mixture of the two possible folding
patterns? This might seem like a very difficult question to answer, but it turns out
that the 13C NMR of the experimentally observed product from a 1,2-13C-labeled
O-
1,2-13C-labeled acetate
O O O O O O O
SEnz
folding folding
pattern (1) pattern (2)
SEnz O
O O O
O
O vs.
O O
O O SEnz O
O O O
H3CO O CH3 H3CO O CH3 This structure,

from folding
pattern (2), is
vs. consistent with
observed
OH OH O OH OH O 13C NMR
splitting patterns
rubrofusarin (1) rubrofusarin (2)
FIGURE 5.39
Different possible folding patterns for rubrofusarin; folding pattern (2) implied by 13C NMR.

acetate feeding study shows isotope incorporation and coupling patterns consistent

only with folding pattern (2) in this case. As might be expected, this has been just a
small taste of how isotope incorporation experiments for NMR studies involving not
only 13C but also isotopes of H, O, N, and other atoms can be used to probe some of
the most intimate details associated with biosynthetic mechanisms and pathways.
We’ll see more later on.
FURTHER MODIFICATION OF POLYKETIDES:

ALKYLATIONS, OXIDATIONS, REDUCTIONS,
AND DECARBOXYLATIONS
Relatively few aromatic polyketide-derived natural products arise from a process

that terminates after simple polyketide cyclization and aromatization. In most cases,
a variety of additional structural modifications take place either before or after cy-
clization/aromatization. For instance, we saw previously that following polyketide
folding, cyclization, and aromatization, rubrofusarin biosynthesis required a final
step involving S-adenosylmethione (SAM) methylation of a phenolic hydroxyl
group (Fig. 5.35). Methylations of both O-and C-atoms are quite common compo-
nents of various biosynthetic schemes, as in the final O-methylation of phloroace-
tophenone to give the antifungal xanthoxylin or the regiospecific C-methylation
of the same tetraketide precursor followed by cyclization and aromatization to give
methylphloroacetophenone (Fig. 5.40), a compound which we will see later in the
biosynthesis of the antibiotic, usnic acid.
Another important and commonly observed alkylating agent is DMAPP which
figured prominently in the terpenoid pathway and, like SAM, may serve as an al-
kylating agent for carbon or oxygen, though C-alkylations tend to predominate.
An example of sequential polyketide alkylations is shown in Fig. 5.41 in which the
triphenolic ketone derived from a pentaketide precursor may be directly alkylated
by SAM to give eugenone, a fragrant component of oil of cloves, or may undergo
further cyclization followed by DMAPP ring alkylation to give peucenin, a major
chromone component of sneezewood and a potent acetylcholinesterase inhibitor.
Additional modifications of peucenin serve to illustrate some examples of some
other typical postcyclization transformations, especially formation of benzofuran-
type heterocycles. The dimethylallyl moiety is frequently a target of oxidative epoxi-
dation in biosynthesis, leading to side chain cyclizations, as illustrated in Fig. 5.42.
Both khellin, a diuretic herbal extract used in ancient Egypt as a remedy for renal
colic, and visnagin, a potent vasodilator used in the treatment of angina and asthma,
are obtained from a widely distributed member of the carrot family, Ammi visnaga.
Epoxidation of peucenin followed by intramolecular nucleophilic cyclization leads
to the indicated tertiary alcohol which may then undergo oxidation of the C5 ring
at the benzyllic position to give the corresponding diol. A 1,2-elimination driven
by aromatization and loss of acetone gives the intermediate benzofuran derivative

O EnzS
O
H3C
deprotonation deprotonation
here O O here
cyclization then aromatization SAM C-methylatio n
O EnzS O
O OH
H3C
H3C
O O
HO OH
CH3
phloroacetophenone
2 X SAM O-methylation cyclization then aromatization
O OH O OH
H3C H3C
H3CO OCH3 HO OH
CH3
xanthoxylin methylphloroacetophenone
FIGURE 5.40
Examples of C-and O-methylations via SAM either before or after polyketide cyclization.
which may then undergo SAM methylation to yield visnagin or subsequent aromatic
ring oxidation to a hydroquinone derivative which is then converted to khellin by
SAM methylation of both phenolic hydroxyl groups. While use of khellin is quite
limited due to a number of adverse side effects, the synthetic analogue amikhelline
has seen use as a vasodilator and in the treatment of colic.
Another interesting and far more complex example of structural modification
is seen in the biosynthesis of tajixanthone, a polyketide-derived antimicrobial
compound of considerable interest (Fig. 5.43). Note the use of a ketone-to-alco-
hol reduction and dehydration sequence prior to polyketide cyclization—the usual
pattern followed when aromatic structures appear in which oxygens have been re-
moved (sequence A). After cyclization and aromatization (sequence B), introduc-
tion of oxygens via the usual C–H to C–OH oxidation (typically a postcyclization
process) is followed by a hydroquinone-to-quinone dehydrogenation (sequence C)
to yield the modified polyketide which then undergoes hydrolysis to the free car-
boxylic acid. A typical ortho-hydroxybenzoic acid-type decarboxylation followed
by sequential DMAPP alkylations on both carbon and oxygen centers introduces
additional side chains. A Baeyer–Villiger-type oxidation and a lactone carbonyl
reduction are suggested for producing the intermediate hemiacetal (sequence E).

O O O

O O O
O Claisen
O O O enolizations
SEnz
OCH3 O O OH O O
3 X SAM
H3CO OCH3 HO OH
eugenone
OH O OH O OH O
PPO H
- H2O
O O HO O HO OH
OH O
H :B
OH O OH O
keto-to-enol
H tautomerization
O O HO O
peucenin
FIGURE 5.41
SAM O-methylation or DMAPP C-alkylation leading to eugenone or peucenin.
The open hydroxy aldehyde form must then undergo a conformational rotation to
bring the aldehyde and the dimethylallyl functions into the proximity required for
an “ene reaction” cyclization (somewhat like a Diels–Alder reaction) to produce the
substituted benzopyran ring system; condensation between the now adjacent phe-
nolic hydroxyls may be thought of as involving a conjugate-type addition of one
hydroxyl group to the aryl ketone function of the adjacent ring followed by loss of
water to produce the indicated xanthone core (sequence F). A final oxidative epoxi-
dation completes the sequence of structural modifications to produce tajixanthone.
This is a good example of a complex structure which at first glance might not appear
to be of polyketide origin, but which contains several key structural features that
arise via a series of structural modifications commonly seen in a variety of other
natural product biosynthetic sequences.
OTHER OXIDATIVE MODIFICATIONS OF AROMATIC

RINGS: EXPANSION OR CLEAVAGE PROCESSES
In the biosynthesis of tajixanthone, we saw an example of a Bayer–Villiger-type oxi-

dation process leading to formation of the xanthone core of the structure. Oxidative


B H
OH O OH O
O
O2
mono-
HO O oxygenase O O
peucenin
H :B
Enz Fe Enz Fe
OH OH O O OH O
H
HO HO
O2
O O O O
O
H 2O
OH O OCH3 O
OH O
O2 2 X SAM
mono-
oxygenase O O O O
O O
OH OCH3
SAM Cl khellin
N K2CO3
OCH3 O OH O
amikhellin
(synthetic
O O O O khellin
visnagin N analogue)
O
FIGURE 5.42
Modifications of peucenin to produce visnagin, khellin, and synthetic amikhelline.
ring expansions or cleavage reactions mediated by mono-or dioxygenase enzymes

represent pathways for rather dramatic structural modification of other aromatic
polyketide structures. For example, the tropolone core of the fungal metabolite
stipitatic acid is derived from the simple aromatic polyketide, 3-methylorsellinic
acid, which undergoes an oxidative ring expansion via a pinacol–pinacolone-type
rearrangement as shown in Fig. 5.44. Feeding studies show that orsellinic acid itself
is not converted to stipitatic acid, indicating that SAM methylation of the polyketide
precursor must precede cyclization and aromatization. The anhydride, stipitatonic
acid, has been shown to be an intermediate.
A simple example of oxidative aromatic ring cleavage is found in the biosynthesis
of the antifungal metabolite xanthofusin, as shown in Fig. 5.45. The addition of
two methyl groups derived from SAM to a triketide precursor is followed by the
usual cyclization and aromatization to give 2,4-dimethyl-1,3-benzenediol as an in-
termediate. Further ring hydroxylation is followed by a dioxygenase-mediated ring
cleavage to a ketoacid which can undergo conformational rotation to bring an enol
hydroxyl group into proximity with the carboxyl function. Cyclization with loss of

O O O O O O O O

O A
EnzS 1) reduce EnzS
8 O- O O O O O O
2) dehydrate
O
cyclize aromatize B
O OH O OH C O OH OH OH
D
oxidations
decarbox- HO (C-H to C-OH) EnzS
ylation
dehydrogenation
2 DMAPP (hydroquinone
OH O to quinone)
CO2 and thioester hydrolysis
E OH O OH OH O OH
OH O OH
Bayer–Villager-type
oxidation
then C=O to
CH-OH reduction O HO
O O HO O H
O O
OH F H
O OH OH
G O OH O
O ene-type H O OH
O2 O cyclization
mono- and ether O
O oxygenase condensation
O
xanthone OH
O core HO
tajixanthone
FIGURE 5.43
Octaketide cyclization and subsequent modifications to produce tajixanthone.
water leads to the keto-lactone product as shown. Once again, a quick glance at the
structure of xanthofusin does not immediately suggest a polyketide origin, remind-
ing us that relatively simple modifications following polyketide cyclization and aro-
matization can sometimes lead to dramatic structural changes.
OXIDATIVE COUPLING OF PHENOLS: FORMATION

OF ARYL–A RYL BONDS
Formation of C–C bonds between aromatic rings has been a long-standing chal-
lenge in organic chemistry due to both the lack of available nucleophilic aryl systems
and to resistance to many types of nucleophilic substitution reactions (SN1, SN2, etc.)
for aryl rings bearing leaving groups typical for such reactions. Nature overcomes
these limitations in many situations by linking aryl rings to one another through the
coupling of aryl radicals which are readily formed via the oxidation of phenols. Such
phenolic coupling reactions are quite common in biosynthesis and may occur in
either an intermolecular or an intramolecular fashion. An example of the former type
is shown in Fig. 5.46 for the oxidative dimerization of methylphloroacetophenone

O CH3

O CH3
acetate SAM O
+ 3 malonate O
SEnz SEnz cyclization,
H3C aromatization,
hydrolysis
O O O O
B: HO CH3
O2 HO CH3 HO CH3
O2
H-O
HO mono- mono-
HO
CO2H oxygenase CO2H oxygenase H3C CO2H
B-H
O OH OH
pinacol– 3-methylorsellinic acid
pinacolone type
rearrangement H2O
CH3 CH3
HO HO OH
HO
O2
CO2H O CO2H O CO2H

O
O OH OH
HO lactone then
formation oxidation
O
tropolone O O O
HO OH HO HO
OH O
OH H2O
O O O O
CO2 O
OH (keto form is OH OH
stipitatic acid a β-ketoacid) stipitatonic acid
FIGURE 5.44
Oxidative ring expansion via pinacol–pinacolone rearrangement leading to stipitatic acid.
to produce usnic acid, a yellow, bitter-tasting compound produced by lichen which

has potent antibiotic activity against gram-positive bacteria and also some antiviral,
anti-inflammatory, and analgesic activity.
A typical O2/CYP H atom abstraction is shown for generation of the required
phenoxy radicals with their expected resonance contributors. The key C–C bond-
forming step in the scheme is coupling of an ortho C-radical resonance form with
a para C-radical resonance form, followed by keto–enol tautomerization (note
that only one of the two rings can aromatize). The subsequent generation of the
benzofuran ring system is represented as occurring via a cyclic hemiacetal forma-
tion followed by simple dehydration, though other mechanisms are plausible. This
sequence produces the α,β-unsaturated cyclohexadienone ring system of usnic
acid, which can exist as a mixture of keto–enol tautomers of what is formally the
6,6-disubstituted-4-cyclohexene-1,3-dione ring component of the structure.
An important example of intramolecular oxidative phenolic coupling is seen in
the biosynthesis of griseofulvin, a potent antifungal agent that is widely used for
treatment of ringworm, jock itch, and athlete’s foot as well as fungal infections of the
scalp, fingernails, and toenails (Fig. 5.47).
Note the unusual chlorination step (mechanism undetermined) that immediately
follows the initial polyketide cyclization-aromatization steps and the sequential SAM

reduce

dehydrate
FIGURE 5.45
Oxidative aromatic ring cleavage and lactone cyclization in biosynthesis of xanthofusin.
methylations. The key C–O bond-forming step arises from phenol oxidations followed
by an intramolecular trapping of a para C-radical resonance form by a phenoxy
O-radical. NADPH conjugate addition of hydride to the resulting 2,5-cyclohexadienone
ring system affords the natural product as a single diastereomer (Fig. 5.47).
Anthraquinone natural products are widely distributed examples of acetate-
derived polyketides which can undergo a variety of postcyclization modifications,
including various oxidative couplings. Biosynthesis of emodin anthrone, which illus-
trates a common route to precursors of natural anthraquinones, involves a poyketide
cyclization and aromatization sequence with specifically timed steps for decarboxyl-
ation and dehydration which lead to a precursor anthrone structure (Fig. 5.48).
The various fates of emodin anthrone are illustrated in Fig. 5.49. As shown, direct
oxidation of the anthrone methylene group (path A) leads directly to emodin, a
natural anthraquinone derived from rhubarb root extracts and other plant sources
which is sometimes used as an alternative medicine treatment for inflammation,
constipation, or other conditions. An alternative oxidation of emodin anthrone
(path B) leads to H atom abstraction and coupling of two resulting radicals to pro-
duce the dimeric form, emodin dianthrone. Further oxidative dehydrogenation of
the dianthrone can then lead to formation of emodin dehydrodianthrone. With
this nearly planar structure, adjacent phenols found in the upper and lower tricyclic
ring systems are held in close proximity, favoring sequential intramolecular oxida-
tive phenolic coupling reactions. The first coupling leads to C–C bond formation and
production of protohypericin; this is then followed by a second oxidative phenolic
coupling to produce hypericin, the main component and one of the active princi-
pals in extracts of St. John’s wort (Hypericum perforatum), a formulation which has
successfully competed in recent years with prescription medications as an effective

O O O O
Bioorganic Synthesis 226 acetate CH3

H3C
CH3
SAM O cyclize
O
+ 3 malonate aromatize
O SEnz O SEnz
OH O OH O
OH O
H3C H3C
H3C CH3 O2 CH3
CH3 2 2
2 CYP
HO O-H O HO OH
HO O OH
phenoxy methylphloroacetophenone
radical Enz Fe Enz Fe
H3C OH
OH O H3C OH
OH O
H3C H H3C
HO CH3 ortho–para HO
+ CH3
coupling
O O HO O
O O HO O
CH3 para radical CH3
ortho radical
H3C OH H3C OH
O O OH O
H3C H3C
HO HO
CH3 CH3
O O OH
OH O OH O
CH3 CH3
(+)-usnic acid
H3C OH H3C OH
OH O OH O
H3C H3C
HO
CH3 HO CH3
O O O H2O O O O
CH3 OH
CH3 H+ H
FIGURE 5.46
Intermolecular oxidative phenolic coupling of methylphloroacetophenone to produce usnic acid.
antidepressant. Various studies have reported other effects for hypericin including
antineoplastic, antitumor, and antiviral activities (including against HIV and hepati-
tis C viruses), making hypericin a target of ongoing pharmacological research.
The forgoing introduction to phenolic oxidative coupling processes, while of ob-
vious importance to the biosynthetic chemistry of the acetate pathway, will have
even greater significance later on. As we move ahead in the next two chapters to ex-
amine the details of the shikimate pathway and the biosynthesis of alkaloid natural
products, we’ll see that oxidative phenolic coupling and some closely related oxida-
tive processes play a central role in the formation of key C–C bonds in both of these
important systems. Until then, we have some final crucial elements of the polyketide
pathway to examine. In doing so, we will move beyond the forgoing families of
natural products derived mainly from acetate and malonate precursors, eventually

O O O OH OH (2)

SAM(1)
acetate cyclize
+ 6 malonate O O
aromatize H3C O-H
H3C O HO (1)
SEnz O-H
O
OH OCH3 OH OCH3
ortho
chlorination SAM(2)
(electrophilic
H3C
H3CO
H3C O-H or radical?) H3CO O-H
O-H O-H
Cl
O2 CYP
OH OCH3
OH OCH3
H3C O-H
H3CO O O
O-H O H3CO O
Cl H3C
Enz Fe Cl
Enz Fe
OH H3CO OH OCH 3
O
H3CO O
H3CO O O
Cl H3C H3C
Cl
SAM
OCH3 H CO
OCH3 H CO 3
3
NADPH O
O
(conjugate O
O reduction) H3CO
H3CO
H C H+ Cl H3C
Cl H:- 3
(+)-griseofulvin
FIGURE 5.47
Intramolecular oxidative phenolic coupling in biosynthesis of the potent fungicide griseofulvin.
reaching out beyond aromatic structures to embrace branched polyols, large ring
lactones, and polyether structures of special significance. To produce such a vast
array of additional compounds requires further structural modifications of emerg-
ing polyketide chains as well as the use of different starter and/or extender units. To
begin, we’ll take a look at some simple examples of the latter modification and then
move on to some more complex systems.
THE USE OF OTHER STARTER GROUPS: FROM CANCER

DRUGS AND ANTIBIOTICS TO POISON IVY
One of the most important of all cancer chemotherapy drugs currently in worldwide
clinical use is doxorubicin, a highly modified aromatic polyketide glycoside pro-
duced by fermentation of a mutant strain of Streptomyces peucetius, a soil microbe

O O O O

OH OH O O
SEnz
acetate OH
O O O
+ 7 malonate
O HO CO2
OH
OH O OH OH O O OH OH O
H2O
HO HO HO
emodin anthrone
FIGURE 5.48
Polyketide cyclization and modifications in biosynthesis of emodin anthrone.
OH O OH
OH O OH
O2
A
(C–H to
C–OH to HO
HO C=O) O
emodin anthrone B emodin
O2
OH O OH OH O OH
O2 HO HO
H H
HO HO
H2O
OH O OH OH O OH
emodin dianthrone
OH O OH OH O OH OH O OH
HO O2 O2 HO
HO
HO oxidative HO oxidative HO
phenolic phenolic
coupling coupling
OH O OH OH O OH OH O OH
emodin dehyrodianthone protohypericin hypericin
FIGURE 5.49
Oxidative transformations of emodin anthrone in biosynthesis of emodin and hypericin.
originally found in Italy. An abbreviated biosynthetic scheme for doxorubicin, which

begins with a propionate starter unit instead of an acetate starter unit, is shown
in Fig. 5.50. Note the initial formation of an anthrone ring which is then oxidized
to an anthraquinone structure prior to aldol-initiated assembly of the cyclohexane
(D) ring, which is the focus of most of the remaining modifications.
Oxidation of the C-ring and reduction of D-ring ketone is followed by glyco-
sylation of the D-ring benzylic alcohol with the aminosugar L-daunosamine (via
TDP-L-daunosamine) which is added prior to subsequent decarboxylation, further


SCoA
O 9 X malonyl CoA SAM

propionyl CoA
O2
O SEnz
reduction O OH
O i) NADPH
O O
ii) cyclize
O O O
iii) aromatize
O O O O OH O OH O
i) oxidation iii) aldol
- CO2 ii) SAM
O2
O OH O OCH3
O OH O2 O
i) NADPH
ii) oxidation
OH A B C D OH
iii) TDP-L-
daunosamine
iv) ester
OH O OH O hydrolysis OH O OH O reduction
O
SAM i) decarboxylation
NH2 ii) oxidation
OH iii) SAM
O OH O O OH O
O2
OH
oxidation
OH OH
OCH3 O OH O OCH3 O OH O
O O
NH2 NH2
OH daunorubicin OH doxorubicin
FIGURE 5.50
Abbreviated biosynthetic scheme for daunorubicin and its conversion to doxorubicin.
oxidation of the D-ring ethyl group (from the propionate starter unit) and SAM
methylation of the A-ring hydroxyl to give daunorubicin, the original anthracycline
drug produced by a nonmutant S. peucetius strain. A final oxidation of the D-ring
methyl ketone function via the mutant strain produces the more powerful doxoru-
bicin. Both drugs are members of a family of related polyketides collectively known
as anthracycline antibiotics and represent some of the most effective anticancer
agents known, in part because of their activity against so many different types of
cancer. All work by binding directly to DNA base pairs via intercalation and also by
inhibiting DNA repair. Doxorubicin is commonly used to treat many different types
of cancers, including those of the bladder, stomach, lung, breast, and ovaries; it is
also effective for treatment of both Hodgkin’s and non-Hodgkin’s lymphomas and
certain forms of leukemia. Aside from the usual toxic side effects of cancer chemo-
therapy agents, most anthracyclines, including doxorubicin, exhibit a severe form
of cardiotoxicity (causing heart damage) which limits the total amount of the drug

O OH O

O OH O
O OH O OH
OH
OH OH
OH
OH
OCH3 O OH O
OCH3 O OH O
O OH O O
O
O
NH2
NH2 F3COCO
NH2 idarubicin O O pirarubicin valrubicin
OH
N OH
H
O OH O O OH O NH O OH
OH
OH NH2
OCH3 O OH O O OH O
O O NH O OH
NH2 H
esorubicin HO HO amrubicin N OH mitoxantrone
FIGURE 5.51
A group of synthetic and semisynthetic anthracycline analogues currently in clinical use.
which can be administered over a lifetime. Reduction of this cardiotoxicity has been
the main focus of many years of research effort in the development of synthetic or
semisynthetic analogues, several of which are shown in Fig. 5.51. All of these ana-
logues are currently in clinical use.
Another group of biologically important polyketides are the tetracycline anti-
biotics. While somewhat similar in structure to the anthracyclines, both the starter
unit and subsequent cyclization/aromatization patterns are different, with many ad-
ditional transformations involved in producing the core structures (Fig. 5.52). The
malonamyl-CoA starter unit, derived from malonyl-CoA, glutamine, and ATP,
condenses with nine units of malonyl-CoA in the usual fashion to produce the
polyketide precursor which, as in the anthracyclines, undergoes a single NAPDH
reduction prior to cyclization and aromatization, though in this case of the tetra-
cyclines, the folding pattern is different, leading to D-ring aromatization as shown.
The resulting aromatic tetracycle, pretetramide, then undergoes B-ring SAM
methylation and a sequence of C-ring oxidations, the first to introduce a hydroxyl
group and the second to dehydrogenate the resulting hydroquinone to the qui-
none oxidation state. The resulting 4-keto-6-methylpretetramide then undergoes
conjugate addition of water at the indicated position, followed by a PLP-mediated
transamination of one of the C-ring ketone functions to introduce the amino group
of anhydrotetracycline. This is followed by sequential SAM methylations of the
amino group and oxidation at the methyl-bearing B-ring carbon to produce the cor-
responding tertiary alcohol of 5a,11a-dehydrotetracycline. This key intermediate
then either directly undergoes NADPH conjugate reduction at the indicated posi-
tion to produce tetracycline (via path A), or undergoes C-ring oxidation to an al-
cohol prior to the aforementioned NADPH conjugate reduction (via path B) which
then produces oxytetratcycline. A third member of the group, chlorotetracycline,


O O
H2N SCoA 9 X malonyl-CoA

malonamyl CoA SAM O2
O SEnz i) NADPH OH
ii) cyclize
O O O O A B C D
iii) aromatize NH2
NH2
reduction
OH OH OH OH O
O O O O O
pretetramide
i) SAM iii) oxidation
ii) oxidation (hydroquinone to
(C-H to C-OH) quinone)
SAM
O2 'NH3'
H3C NH2 CH3 O
i) H2O
H (conjugate
OH OH
addition)
NH2 ii) trans- NH2
amination
OH (PLP)
OH OH O O O OH OH O O O
H2O
anhydrotetracycline
4-keto-6-methylpretetramide
i) SAM X2 ii) oxidation
conjugate
reduction O2(b only)
(a and b) H3C OH N(CH3)2 H3C OH N(CH3)2
H H H
OH OH
A
NH2 NADPH NH2
only OH
OH
OH O O O O OH O OH O O
5a,11a-dehydrotetracycline tetracycline
oxidation
B para
then NADPH chlorination,
then same
H3C OH OH N(CH3)2 Cl H C OH N(CH3)2 sequence
3 H
H H H as for
OH OH
tetracycline
NH2 NH2
OH OH
OH O OH O O OH O OH O O
oxytetracycline chlortetracycline
FIGURE 5.52
Abbreviated biosynthetic scheme for tetracycline and other tetracycline antibiotics.
is derived from 4-keto-6-methylpretetramide via A-ring chlorination para to the

phenolic hydroxyl group followed by the same sequence of transformations involved
in production of tetracycline; tetracycline itself is actually produced via a mutant
bacterial strain that blocks the chlorination step leading to chlortetracycline, the
first of these compounds to be isolated in the mid 1940s.
The tetracyclines are important broad-spectrum, orally active antibiotics which
have been widely used in both human and animal medicine for many years. As with
all such drugs, their widespread use has led to the development of tetracycline-
resistant bacterial strains, creating a need for derivatives and analogues capable
of overcoming resistance mechanisms. Doxycycline and minocycline (Fig. 5.53)
are semisynthetic examples of so-called second-generation tetracyclines that, like

H3C OH N(CH3)2 N(CH3)2 N(CH3)2

H H H H
OH OH
NH2 NH2
OH OH
OH O OH O O OH O OH O O
doxycycline minocycline
(CH3)3C N(CH3)2 N(CH3)2

H H
NH OH
NH2
O N
H OH
OH O OH O O
tigecycline
FIGURE 5.53
Second-and third-generation tetracyclines for antibiotic-resistant bacteria strains.
the natural tetracyclines, are orally active and have proven to be clinically valuable
for treating a wide variety of antibiotic-resistant infections. More powerful third-
generation derivatives like tigecycline have come into use only since the mid-
2000s; tigecycline is especially valuable in its activity against methicillin-resistant
Staphylococcus aureus (MRSA) strains, but must be given intravenously.
Long chain carboxylic acids are also sometimes employed as starter units for
the production of aromatic polyketide products. One example of this is seen in the
biosynthesis of the urushiols, a group of very potent allergen oils produced by a
number of different plants including poison oak, poison ivy, and poison sumac
(Fig. 5.54). Each member of this group of polyphenols consists of a basic catechol
ring with a C15 or C17 side chain which may be either saturated or mono-, di-or
triunsaturated, depending on the carboxylic acid starter unit. Mixture compositions
vary with different plant sources, but structures with longer unsaturated chains tend
to produce more significant allergic reactions in most individuals. The biosynthesis
of the urushiol shown below begins with a monounsaturated C16 starter unit, palmi-
toleic acid (enzyme-bound) which is condensed with three units of malonyl-CoA
in the usual fashion to produce the indicated triketide derivative. Carbonyl reduc-
tion followed by cyclization, dehydration, and aromatization produces the aromatic
polyketide, anacardic acid. Decarboxylation of the ortho-hydroxybenzoic acid ring
followed by ortho-oxidation to the corresponding catechol gives the unsaturated
urushiol shown.
Use of a less familiar carboxylic acid starter unit, para-hydroxycinnamic acid,
provides a preview of one of the many basic components available from the shi-
kimate pathway (the subject of our next chapter) and how compounds derived
from different pathways may intersect in some organisms to produce hybrid natu-
ral products of mixed biosynthetic origin. As shown in Fig. 5.55, enzyme-bound
para-
hydroxycinnamic acid may condense with three malonyl-CoA units as


H H O H H
H H
SEnz ∆9 SEnz
desaturase
palmitic acid (as thioester) palmitoleic acid
3 X malonyl-CoA
O O O
OH
HO i) NADPH EnzS
H H H H O
ii) cyclize
O
aromatize
anacardic acid (- H2O) reduction
hydrolyze
decarbox- OH OH
ylation CO2 HO
H H O2 H H
NADPH
urushiol
FIGURE 5.54
Biosynthesis of urushiol, the active principal in poison ivy.
HO
HO
3 X malonyl CoA O
SEnz
O
EnzS
O
p-hydroxycinnamic acid
(enzyme bound) O O
cyclize
Knoevenagel aromatize
hydrolyze
HO HO
OH OH
decarboxylate
HO
CO2
resveratrol OH O OH
FIGURE 5.55
Biosynthesis of resveratrol, a mixed pathway polyphenolic natural product.
usual to produce the unique triketide structure shown. The usual Knoevenagel-
type condensation and aromatization is again followed by decarboxylation of the
ortho-hydroxybenzoic acid intermediate to produce the controversial polyphenolic
metabolite, resveratrol.
Resveratrol is found in the skins of red grapes and in other fruits and has often
been singled out as a component of red wine that may be partially responsible for
the so-called “French paradox,” the well-known observation of relatively low mor-
tality from coronary heart disease among the French in spite of the fairly high levels

of saturated fats in their diet. Resveratrol is just one of many red wine polypheno-
Bioorganic Synthesis 234 lic compounds which studies have shown to have antioxidant, anti-inflammatory,
and other potentially beneficial effects, though the amounts required for such ef-
fects usually far exceed what would be obtainable from wine consumption. Even
more controversial are claims made by dietary supplement suppliers that resveratrol
consumption may lead to increased life span due to its antioxidant properties. In
preliminary studies, resveratrol feeding has been found to extend the lifespans of
certain worms, fruit flies, and fish, but there is only limited evidence at present to
suggest that such effects will be seen in higher animals.
MORE ON POLYKETIDE SYNTHASE (PKS)

SYSTEMS: INCREASING PRODUCT DIVERSITY
We noted earlier in the chapter the close relationship between fatty acid synthase
(FAS) enzyme systems and polyketide synthase (PKS) systems. Certainly, both
enzyme families catalyze Claisen condensations between acetyl-CoA and malonyl-
CoA units to yield enzyme-bound β-ketoacyl thioesters. The simplified diagram
shown earlier (Fig. 5.33) presented a PKS as essentially an FAS system lacking the
usual components required for ketone reduction (β-ketoreductase), alcohol dehy-
dration (dehydratase), and α,β-unsaturated thioester conjugate reduction (enoyl re-
ductase), and this has been the basic pattern of polyketide biosynthesis up to this
point. But there is much more to the story than starter units, extender units, and
polyketide folding patterns leading to phenolic natural products. To better under-
stand some of the more complex aspects of the biosynthesis of many nonaromatic
polyketide natural products, we have to look a bit more closely at certain features
of different types of PKS systems and how they work. The discussion that follows is
limited and simplified, as it is intended mainly to provide a modest familiarity with
some of the concepts, terminology, and abbreviations associated with the enzymol-
ogy of PKS systems. While a great deal of important detail is omitted, it is hoped
that what is presented will help to deepen the reader’s understanding of the organic
chemistry involved in the transformations catalyzed by these systems.
Most of the aromatic polyketide chemistry we have seen so far has been char-
acteristic of bacteria employing a type II PKS system (as illustrated previously in
Fig. 5.33) or by plants employing a type III PKS system. In these systems, one or
more modules (essentially organized collections of enzyme active sites required for
catalyzing the various reactions involved in the polyketide assembly or modification
process) are employed in an iterative assembly mechanism; that is to say, the active
sites of a given module are reused repeatedly to produce a growing polyketide chain.
Recall that for the aromatic natural products we have seen, the PKS system loads a
starter unit (usually acetyl-CoA), then loads an extender unit (always malonyl-CoA)
adjacent to the starter unit, catalyzes a Claisen condensation between the two to
produce a β-ketothioester which is then transferred back to the adjacent starter unit

position to make way for the loading of a second malonyl-CoA extender unit, after

which the entire process is repeated over and over to produce a growing polyketide
chain. This kind of “back-and-forth” assembly mechanism is also employed by fungi
using so-called iterative type I PKS systems. Table 5.2 summarizes some of the fea-
tures of the different PKS systems currently known.
Another group of PKS enzymes used by bacteria, called modular type I PKS
systems, employ a different kind of mechanism that is more like an “assembly line”
process for generating a growing polyketide chain rather than the “back-and-forth”
iterative mechanism. Here, the enzyme has multiple modules, each of which car-
ries out one or more specific functions or modifications, after which the substrate
molecule is passed on to the next module and so on (more on this later). The prod-
ucts produced by both iterative and modular type I PKS systems are often (but not
always) nonaromatic compounds that may be linear, cyclic, polycyclic, or some
combination of these. But it is important to keep in mind that all types of PKS sys-
tems can produce a variety of different product structures, so it is not really possible
to classify polyketide natural product structures strictly according to the type of PKS
systems involved in their production. Fortunately, much of the chemistry involved
in all types is very similar, relying primarily on Claisen condensations of repeated
acetate or propionate units for the basic assembly process, often accompanied by
familiar FAS-type modifications.
Recall that in the formation of most aromatic polyketides, we only occasionally
saw additional modifications to the growing polyketide chain (such as a reduction
or a methylation, etc.) prior to cyclization and aromatization; most of the modifica-
tions were postcyclization/aromatization processes. With type I PKS systems, it is
more common to see growing polyketide chains undergoing modifications more
Table 5.2 Types and Features of Different Polyketide Synthase (PKS) Systems
PKS Group Structure Assembly Mechanism Used by Products
Iterative Single protein, Iterative; repeated re-use Fungi Linear, cyclic,

type I single module of module active sites others
by growing chain
Modular Single protein, Linear; single use of Bacteria Linear, cyclic
type I multiple modules module active sites, macrolides
growing chain passed
on to next module
Type II Multiprotein, single Iterative; single or Bacteria Aromatics
module per protein repeated use of module
active sites by growing
chain
Type III Single protein, Iterative; repeated re-use Bacteria Aromatics,
multiple modules of module active sites by plants, others
growing chain fungi

akin to those we saw earlier in FAS systems, such as reductions, dehydrations, or

Bioorganic Synthesis 236 conjugate reductions as the chain grows. We say that these PKS systems typically
employ one or more modules with multiple domains of catalytic activity. If modules
are described as organized groups of different domains (enzyme active sites), it is
helpful to think of a domain as simply a region or part of a given enzyme structure
that functions more-or-less independently from the rest of the enzyme, performing
a specific catalytic function (often requiring various coenzymes) to facilitate a spe-
cific process such as loading, condensation, reduction, dehydration, and so on. A list
of some familiar catalytic domains of significance in various polyketide synthase
systems, along with commonly used abbreviations, is given in Table 5.3.
A convenient way to think about iterative PKS systems is to divide them into fully
reducing, partially reducing, and nonreducing groups. A fully reducing PKS system,
one in which KR, DH, and ER domains operate will closely resemble FAS systems,
though different starter or extender units may be employed. A partially reducing
PKS would produce products with only partially reduced structural segments (al-
cohols or alkenes) while a nonreducing PKS would lead to “true” polyketides, that
is, multiple ketone functions as seen in our earlier examination of precursors to
aromatic polyketides. Modular PKS systems, as we will see, can produce products
with a mixture of structural segments that may be fully reduced, partially reduced,
or unreduced.
To illustrate an excellent example of a fungal iterative type I PKS system in opera-
tion (another “back-and-forth” construction system), we will look at biosynthesis
of the cholesterol-lowering drug, lovastatin (trade name Mevacor), a nonaromatic
polyketide natural product obtained from oyster mushrooms. Lovastatin and similar
statin drugs have a powerful inhibitory effect on HMG-CoA reductase, the enzyme
Table 5.3 Some Catalytic Domains and their Functions in PKS Systems
PKS Domain Abbrev. Synthetic Function of Active Site
Acyltransferase AT Loads starter or extender units

Acyl carrier ACP Anchors growing polyketide chain as thiol ester
protein
β-Ketoacyl KS Promotes Claisen condensation between starter/
synthase extender units
β-Ketoacyl KR Reduces β-ketothioester to β-hydroxythioester
reductase (via NADPH)
Dehydratase DH Dehydrates β-hydroxythioester to α,β-unsaturated
enoyl thioester
Enoyl reductase ER Promotes conjugate reduction of enoyl thioester
to saturated thioester (via NADPH)
Methyltransferase MT Transfers methyl groups to a growing polyketide
chain (via SAM)
Thioesterase TE Releases final polyketide product from enzyme

which mediates NADPH reduction of hydroxymethylglutarylCoA, a key intermedi-

ate in the production of mevalonic acid (MVA) that, you will recall, is required for
terpenoid (and therefore cholesterol) biosynthesis (see Fig. 4.5). All of the statin
drugs shown in Fig. 5.56 are obtained by fermentation processes with the exception
of atorvastatin (trade name Lipitor) which is a synthetic drug retaining some of the
key structural features of the natural statin products.
Lovastatin biosynthesis is mediated by lovastatin nonaketide synthase (LovB),
a multifunctional type I PKS enzyme that contains active KS, AT, DH, MT, KR, and
ACP domains but an inactive ER domain. Because of this, a separate ER enzyme
known as LovC is required whenever an α,β-unsaturated thioester reduction step
(via NADPH) is required during the biosynthesis. The process begins with forma-
tion of a simple diketide (Fig. 5.57) via loading of acetyl-CoA and malonyl-CoA
units onto LovB as usual, but in this case, the KS (Claisen condensation) operation is
followed by ketone reduction (KR) and dehydration (DH) to give an α,β-unsaturated
diketide intermediate. This same sequence of steps (which bears obvious similarity
to steps in fatty acid biosynthesis) is repeated with a second malonate unit to give
the doubly unsaturated triketide intermediate as shown. After addition of a third
malonate unit, the KR and DH domains operate as usual, but in this step the LovC
enzyme is employed for NADPH reduction of the α,β−double bond of the triene
tetraketide, leaving a conjugated diene component intact (a segment of the molecule
that will be of paramount importance later in the sequence). This is followed by
action of LovB’s MT (methyl transferase) domain which installs a methyl group (via
SAM alkylation of the β-dicarbonyl enolate ion) at the α-carbon, producing the final
form of the tetraketide intermediate. Repetition of the previous malonyl-CoA, KS,
HO O HO O HO O
O O O
O O O
O O O
H H H
lovastatin (Mevacor) mevastatin (Compactin) simvastatin (Zocor)

HO OH
HO OH
O OH O
OH O F
O N
H
H
N
HO O
pravastatin (Pravachol) atorvastatin (Lipitor)
FIGURE 5.56
Structures of some important statin drugs (HMG-CoA reductase inhibitors).

malonyl CoA O O

LovB malonyl CoA
acetyl CoA
KS, KR, DH EnzS KS, KR, DH EnzS
diketide triketide
O SEnz
O SEnz
malonyl CoA malonyl CoA
KS, KR, DH, KS, KR, DH,

LovC LovC, MT
tetraketide
pentaketide
FIGURE 5.57
Early steps leading to production of the pentaketide intermediate in lovastatin biosynthesis.
O SEnz
SEnz
malonyl CoA
O
KS, KR, DH
(s-trans diene)
pentaketide
O SEnz SEnz
H
O
[4+2]
Diels-
Alder
(s-cis diene)
H
cyclic hexaketide
FIGURE 5.58
Intramolecular Diels–Alder leading to a trans-decalin-based lovatstatin hexaketide intermediate.
KR, DH, and LovC conjugate reduction steps then produces a pentaketide interme-
diate with the indicated structure.
Continuing on, we see one of the key steps (Fig. 5.58) in the overall biosynthetic
sequence. Extension of the pentaketide chain introduces a sixth malonate unit, ac-
companied by ketone reduction and dehydration to the α,β-unsaturated thioester
intermediate. At this point, a folding mode which brings the s-cis conformer of the
conjugated diene portion of the hexaketide into close proximity with conjugated
enoylthioester double bond leads to an intramolecular Diels–Alder reaction, pro-
ducing the decalin ring system, a structural feature that will be carried all the way
through to the end of the biosynthetic scheme. While somewhat surprising, this
is just one of a number of examples of the use of biological Diels–Alder processes
in the construction of various natural products. Note that the sequence pairs an
electron-rich diene component (alkyl substitution at each end) with an electron-
poor dienophile component (thioester electron-withdrawing group) as typically
seen in more familiar laboratory Diels–Alder reactions.


O SEnz HO
O SEnz SEnz
cyclic OH OH
hexaketide
malonyl CoA malonyl CoA malonyl CoA lactone
H formation
KS, KR, DH KS, KR H KS, KR H
LovC
H HSEnz
heptaketide H H
octaketide nonaketide
HO O HO O HO O
O O O
dehydration O2
NADPH
H H2O H (allylic H
oxidation
with
rearrangement)
OH H
monacolin L
H :B H+ dihydromonacolin L
HO O HO O
O O O
acetyl CoA KS, MT O
+ malonyl CoA KR, DH, SEnz
O2 OH
ER
O
H H
NADPH OH
(C–H to H
C–OH)
monacolin J lovastatin
FIGURE 5.59
From hepta-and octa-to nonaketide and postcyclization modifications to give lovastatin.
As the process continues, we see a third LovC-mediated enoyl reduction (ER)

step occurring at the heptaketide stage, resulting in a total of three fully reduced
segments within the growing polyketide chain (Fig. 5.59). In moving through
the remaining two stages of malonyl-CoA addition, we see that only KS and KR
domains are employed, leading to sequential ketone-to-alcohol reductions and
thus a final nonaketide that retains two secondary alcohol functions in the usual
1,3-relationship. It is at this stage that the nonaketide is released from the LovB
enzyme via intramolecular nucleophilic acyl substitution with expulsion of LovB-
SH to form the lactone dihydromonacolin L.
At this juncture, a C–H to C–OH oxidation takes place with allylic rearrange-
ment to produce an intermediate allylic alcohol which then undergoes a 1,4-type
dehydration process to introduce the conjugated diene component of the advanced
intermediate monacolin L. A final C–H to C–OH oxidation produces monacolin J
by introduction of a hydroxyl group adjacent to the decalin ring junction. The bio-
synthesis is completed by esterification of this final hydroxyl group by a methyl-
substituted diketide intermediate which is produced in a separate biosynthetic op-
eration, leading directly to lovastatin as shown.

MODULAR TYPE I PKS COMPLEXES AND MACROLIDE

Bioorganic Synthesis 240 ANTIBIOTICS: ERYTHROMYCIN BIOSYNTHESIS
Perhaps the most interesting type of polyketide assembly mechanism is found in

the modular type I PKS systems. These “assembly line” multienzyme complexes
are involved in the production of a class of antibiotics known as the macrolides
which are essentially large-ring (macrocyclic) polyfunctional lactone structures,
the most important of which is erythromycin. Among the antibiotics most useful
for those who have allergic reactions to penicillin, erythromycin is the most widely
employed antibiotic of its type, with a complex mechanism of action that ultimately
interferes with the ability of bacteria to produce certain proteins essential to their
growth and reproduction. The PKS enzyme system responsible for the biosynthe-
sis of 6-deoxyerythronolide B, the key polyketide precurusor to erythromycin,
is 6-deoxyerythronolide B synthase or DEBS, a diagram of which is shown in
Fig. 5.60. The enzyme consists of three linked polypeptide subunits, DEBS-1, DEBS-2,
and DEBS-3, each of which is, in turn, composed of two segments or modules with
specific catalytic domains. DEBS-1 also contains a loading module with AT and
ACP domains for introduction of a propionyl-CoA starter unit, while DEBS-3 con-
tains an “end” module with a TE domain that terminates the biosynthesis by facili-
tating the liberation of the final polyketide from the enzyme via lactone formation.
Each module in a DEBS subunit is capable of adding only a single
S-methylmalonyl-CoA extender unit to the growing polyketide chain and therefore
must pass the chain along to the next module, then to the next subunit, and so on
until reaching the end module. Because of this modular “assembly line” mechanism,
it is possible to “read” the enzyme’s modular organization and predict the length of
the final polyketide chain that will be produced prior to lactone cyclization. Thus in
the case of DEBS, since there are six assembly modules present and each can cata-
lyze only one Claisen condensation (via its KS domain) for chain growth, the final
product prior to cyclization must be a heptaketide (module 1 produces a diketide,
module 2 a triketide and so on in going from left to right). Furthermore, by “read-
ing” the domain organization of each module, one can readily identify the chain
modifications that will occur going forward as the assembly progresses. A diagram
of the DEBS-1 subunit with its loading and extension modules and the reactions
they carry out is shown in Fig. 5.61, along with a depiction of the actual nature of
the long, flexible acyl-S-ACP linkage (denoted by a wavy bond in the ACP domain)
used by these PKS systems (the same linkage is used in FAS systems).
As can be seen in Fig. 5.62, the long, flexible acyl-S-ACP linkages are required not
only to allow the loaded methylmalonyl-CoA to reach across the system to engage
the adjacent thioester units for Claisen condensations, but also to allow the grow-
ing chain to reach the KR domains in modules 1 and 2 for catalysis of the ketone
reductions (via NADPH). Note the inversion of configuration at the methyl-bearing
carbon of the diketide (which is subsequently epimerized back to the S configuration

DEBS (6-deoxyerythronolide B synthase), a modular type 1 PKS enzyme
DEBS-1 Subunit DEBS-2 Subunit DEBS-3 Subunit
Extension Extension Extension Extension Extension Extension

Loading Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 End
AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP KS AT DH ER KR ACP KS AT KR ACP KS AT KR ACP TE
SH SH SH SH SH SH SH SH SH SH SH SH SH SH SH SH SH SH SH SH OH
direction of "assembly line" synthesis
O O
HO OH
propionyl CoA post assembly
DEBS OH OH
oxidations
+ N
various modifications HO
6 methylmalonyl CoA glycosylations
during assembly, then O OH O O O
and methylation
release of polyketide O
from DEBS via lactonization O OH O O
OH
erythromycin A O
6-deoxyerythronolide B
FIGURE 5.60
Overview of organizational structure of DEBS enzyme for biosynthesis of 6-deoxyerythronolide B.

DEBS-1 Subunit
Bioorganic Synthesis 242 Loading

Extension
Module 1
Extension
Module 2 Loading
Extension
Module 1
Extension
Module 2
propionyl CoA
AT ACP KS AT KR ACP KS AT KR ACP AT ACP KS AT KR ACP KS AT KR ACP
AT
loding
SH SH SH SH SH SH SH SH module S SH SH SH SH SH SH SH
O loading
module AT ACP
Extension Extension Extension Extension

Loading Module 1 Module 2 Loading Module 1 Module 2
ACP KS
extension
module 1
SH SH S SH SH SH SH SH SH SH SH
S SH SH SH SH
O O O
AT O
-O S-CoA extension
module 1
S-methylmalonyl-CoA
AT ACP
extension
module 1
SH SH S S SH SH SH SH SH SH S SH S SH SH SH
O O O O
O O
O- O-
O O
ACP
S O O Ser-ACP
O N N P
S H H O OH
O OH
O
O O- long, flexible pantothenic acid linker
O- in acyl-ACP bond; similar to acyl-CoA
FIGURE 5.61
Initial loading in extension module 1 of DEBS-1 subunit and nature of acylACP thioester linkage.
prior to module 2 chain extension) and the stereospecific ketone reductions leading
to the triketide intermediate. Note also that inversion at the methyl-bearing carbon
does not occur during the second Claisen condensation.
At this point in the synthesis, the reduced triketide must now be passed forward
from module 2 of DEBS-1 to module 3 of the DEBS-2 subunit, as shown in Fig. 5.63.
Note that module 3 is relatively short as it lacks a KR domain. This means that only
chain extension via the Claisen condensation can occur in this module, leaving the
resulting tetraketide intermediate with its β-ketone function intact. Now the chain is
ready to be passed further along the “assembly line” to extension module 4.
Moving forward, we now notice (Fig. 5.64) that extension module 4 not only
has a KR domain as previously seen in modules 1 and 2, but also has active DH
(dehydratase) and ER (enoyl reductase) domains. This is the same familiar set of
catalytic domains found in the fatty acid synthase (FAS) systems we examined ear-
lier. And once again, the importance of the long, flexible ACP thioester linkage is
evident, as the pentaketide from the Claisen condensation must also be able to reach
the KR, DH, and ER catalytic domains for the required ketone reduction, alcohol

DEBS-1 Subunit

Claisen
extension
module 1
SH SH S SH S SH SH SH SH SH SH SH S SH SH SH
O O CO2
O
O O epimerization
O- (R to S)

KR
extension
module 1
SH SH SH SH S SH SH HS SH SH SH SH SH SH
S SH
O O
diketide
HO ACP KS
O
extension
module 2

methyl
malonyl CoA
AT
S
SH SH SH SH SH S SH SH SH SH SH SH SH S SH
O O O
AT ACP
HO HO O
O-

Claisen
CO2
SH SH SH SH SH SH SH S then KR SH SH SH SH SH S SH S
O O O
HO HO O
triketide O-
HO
FIGURE 5.62
Diketide and triketide transformations for extension modules 1 and 2 of the DEBS-1 subunit.
dehydration, and conjugate reduction steps. This sequence of events leads to a seg-
ment of the growing chain that is now fully reduced (no oxygen at the β-carbon).
The growing chain is ready to be passed along to extension module 5 of the DEBS-
3 subunit. If we read the domain organizations correctly, we should be able to de-
termine that the transformations occurring in modules 5 and 6 will be more or less
identical to those we saw previously in modules 1 and 2 (except for methyl group ste-
reochemistry) as the process proceeds from a penta-to a hexa-to a final heptaketide
of readily predictable structure as shown in Fig. 5.65. After the final ketone reduction
step, this heptaketide is finally transferred by nucleophilic acyl substitution to the
final TE (thioesterase) domain of the terminal module. Note that this final linkage to
the heptaketide chain is not a thioester but rather a simple ester linkage formed via
the side chain primary alcohol function of a serine residue contained within the final
TE domain.

FIGURE 5.63
Transfer from DEBS-1 to DEBS-2 and module 3 triketide-to-tetraketide chain extension.


Module 3 Module 4 ACP to KS Module 3 Module 4
transfer
KS AT ACP KS AT DH ER KR ACP KS AT ACP KS AT DH ER KR ACP
and methyl
malonyl-CoA
SH SH S SH SH SH loading SH SH SH S SH S
O O O
O O O
O-
HO HO
tetraketide
Claisen
HO HO

Module 3 Module 4 Module 3 Module 4
1. KR (NADPH)
KS AT ACP KS AT DH ER KR ACP KS AT ACP KS AT DH ER KR ACP
2. DH
3. ER (NADPH)
SH SH SH SH SH S SH SH SH SH SH S
O O
ER DH KR
HO O
O O
HO HO
pentaketide
HO HO
FIGURE 5.64
Module 4 chain extension with additional modifications via active KR, DH, and ER domains.
While this sequence has finally brought us to the end of the modular system, the
enzyme still has one final and very critical operation to perform, and so the stage has
been set for the final operation of the PKS assembly line, namely, the release of the
polyketide chain from the enzyme structure itself.
As shown in Fig. 5.66, deprotonation of the indicated alcohol function leads to
an alkoxide which can act as a nucleophile for intramolecular attack on the terminal
ester carbonyl, resulting in lactone formation and production of the free macrolide,
6-deoxyerythronolide B. The remaining oxidation, glycosylation, and methylation
steps leading to erythromycins A, C, and D are all postcyclization processes cata-
lyzed by individual enzymes. Thus, 6-deoxyerythronolide B is the aglycone precur-
sor to all other members of the family. The unusual sugars employed for the gly-
cosylation steps (both as TDP-sugars derived from glucose) are L-mycarose and
D-desoamine; the final SAM step leading from erythromycin C to erythromycin A
converts L-mycarose to its 3-O-methyl derivative L-cladinose.
GENETIC MANIPULATION OF MODULAR PKS

SYSTEMS: RATIONAL DRUG MODIFICATION
It has long been a goal of drug discovery research to find ways to control or tailor
the machinery of organisms that produce bioactive molecules in order to generate

DEBS-3 Subunit

Extension Extension methyl Extension Extension
pentaketide Module 5 Module 6 End malonyl-CoA Module 5 Module 6 End
transfer loading
from DEBS-2 KS AT KR ACP KS AT KR ACP TE KS AT KR ACP KS AT KR ACP TE
(module 4 ACP)
to DEBS-3
(module 5 KS) S SH SH SH SH SH OH S SH S SH SH SH OH
O O O
O
O-
O O
HO HO Claisen,
pentaketide then KR
HO HO
Module 5 Module 6 End module 5 ACP Module 5 Module 6 End
to module 6 KS
KS AT KR ACP KS AT KR ACP TE KS AT KR ACP KS AT KR ACP TE
methyl
malonyl-CoA
SH SH SH S H loading SH SH S SH SH SH OH
O O O
HO O HO
O-
O O
HO HO
Claisen,
then KR hexaketide
HO HO

Module 5 Module 6 End Module 5 Module 6 End
final transfer
KS AT KR ACP KS AT KR ACP TE KS AT KR ACP KS AT KR ACP TE
to TE domain
SH SH SH SH SH S OH SH SH SH SH SH SH O
O O
HO HO
HO HO
O O
HO HO
heptaketide
HO HO
FIGURE 5.65
Final sequence of penta-to-heptaketide DEBS-3 chain extensions and transfer to TE domain.
analogues of known natural products or to produce, through biosynthesis, entirely

new and novel compounds that may then be screened for potential therapeutic bio-
activity. We briefly saw a relatively simple but successful example of this concept
earlier when we noted that fermentation-based production of the highly active an-
thracycline anticancer compound doxorubicin was accomplished through use of
an intentionally mutated strain of the organism that produced the original but less
active anthracycline daunorubicin. In this context, the discovery of the modularity
of certain PKS enzyme systems together with modern genetic engineering tools for


TE DEBS
H+
O
O
O O
HO
OH
HO lactone OH O2 OH
formation
O OH O OH
O O OH O OH
HO 6-deoxyerythronolide B erythronolide B
H-O heptaketide TDP-mycarose TDP

B:
O
O
OH
OH TDP-desoamine
OH
OH
N
HO TDP O OH
O O O
OH
OH O O
O O
OH
OH
3-O-mycarosyl O
erythromycin D O erythronolide B
O2
O O
HO OH HO OH
OH SAM OH
N N
HO HO
O O O O O O
OH OCH3
O O O O
OH OH
O O
erythromycin C erythromycin A
FIGURE 5.66
Erythromycins via release from DEBS, then oxidations, glycosylations, and a methylation.
manipulation of gene sequences involved in production of PKS enzymes has led to

something of a revolution in the field of rational drug modification. This field is both
large and quite complex, so a detailed examination is clearly beyond the scope of this
chapter; nevertheless, a limited discussion of this crucial topic with some represen-
tative examples is both important and instructive.
Modular PKS systems are particularly amenable to genetic manipulation through
a number of different approaches. Gene sequences responsible for generating specific
catalytic domains can be modified or eliminated, thus “knocking out” certain domain
activities within a given PKS module. For example, if the ER domain in module 4 of
DEBS-2 is deactivated in this way, all remaining steps in the biosynthesis still occur as
usual, leading to a 6-deoxyerythronolide analogue with unsaturation at the position
indicated in Fig. 5.67. Similar macrolide structural modifications have been achieved

Extension Extension

O
Module 4 Module 4
1. KR
(NADPH)
KS AT DH ER KR ACP KS AT DH ER KR ACP
2. DH
OH
SH SH S SH SH S
O O O OH
O O OH
avermectin PKS-DEBS PKS hybrid O

Extension Extension
Loading Module 1 Module 2
AT ACP KS AT KR ACP KS AT KR ACP

OH
SH SH S SH S SH SH SH O OH
O O
isobutyryl O OH
starter O
unit
O-
rapamycin PKS-DEBS PKS hybrid O
Extension Extension
Module 5 Module 6 End
ACP to TE
transfer
KS AT KR ACP KS AT KR ACP TE
OH
then normal
SH SH SH OH cyclization
SH S S O OH
O O
malonyl
extender O OH
HO O unit
O-
FIGURE 5.67
6-Deoxyerythronolide analogues via engineered DEBS modifications.
through use of so-called hybrid modular PKS systems which can be engineered by
splicing portions of different natural PKS gene clusters together, leading to produc-
tion of hybrid PKS enzymes with novel domain organizations. For instance, replac-
ing the loading module (AT, ACP) of DEBS with the loading module from another
natural PKS enzyme (one which produces avermectin) leads to a hybrid PKS enzyme
system that, like the avermectin PKS, is selective for an isobutyrylCoA starter unit
rather than the usual propionyl-CoA starter unit. This loading domain substitution
ultimately leads to production of a 6-deoxyerythronolide analogue that incorporates
the isobutyryl group at the expected position, as shown. A similar modification in-
volving substitution of the DEBS-3 module 6 AT domain with the AT domain from
module 2 of a PKS enzyme responsible for rapamycin synthesis leads to a hybrid
enzyme with an AT6 domain which, like the rapamycin AT2 domain, is specific
for incorporation of a malonyl-CoA unit rather than the usual S-methylmalonyl-
CoA unit. This domain substitution leads to production of a 6-deoxyerythronolide
analogue lacking a methyl group at the indicated position. It is worth noting that
when this compound was isolated and fed to an organism capable of completing the

DEBS-1-TE

OH
Extension Extension
Loading Module 1 Module 2 End
2 x methyl-
malonyl CoA
AT ACP KS AT KR ACP KS AT KR ACP TE
then
cyclization O O
SH SH S SH SH SH SH SH OH
O
DEBS-1-TE with modified AT1 domain

favoring malonyl CoA incorporation
OH
Extension Extension
Loading Module 1 Module 2 End
1 x malonyl CoA
1 x methyl malonyl CoA
AT ACP KS AT KR ACP KS AT KR ACP TE
then cyclization
O O
SH SH S SH SH SH SH SH OH
O
FIGURE 5.68
A truncated DEBS-1-TE and an AT1-modifed version leading to small-ring lactones.
required oxidation and glycosylation steps, the resulting erythromycin analogue re-
tained significant antibacterial activity. Further advances in cloning and characteriza-
tion of PKS-containing gene clusters for most of the major polyketide product groups
has led to many additional examples of such hybrid construction.
Further interesting work has been carried out using truncated versions of the
DEBS system in which a TE domain is incorporated onto the end of the DEBS-1
component which therefore terminates polyketide formation after only two cycles
of chain extension. The resulting cyclized triketide lactone (Fig. 5.68) represents
a potentially useful chiral building block for laboratory organic synthesis, as it
is produced as a single diastereomer carrying four contiguous chirality centers.
Similarly, genetic modifications have been employed to replace the AT1 domain
with another which favors loading malonyl-CoA over methylmalonyl-CoA as
an extender unit, ultimately leading to formation of the indicated chiral lactone
lacking a methyl group at C-4. These examples serve to illustrate that while still
in its infancy, the field of genetic engineering for the production of enzyme sys-
tems designed to produce specifically tailored polyfunctional organic compounds
will surely play a key role in the future of industrial and pharmaceutical organic
synthesis.
SOME FINAL PKS PRODUCTS OF MEDICINAL IMPORTANCE
With our additional insight into how different PKS stems operate, we can now take
a brief look at some final polyketide natural products of relatively complex struc-
ture and be able to infer many of the details associated with their assembly without

the need for extensive explanation or analysis. We begin with the assembly of two
Bioorganic Synthesis 250 polyene antifungal agents of special significance, amphotericin B and nystatin A.
As shown in Fig. 5.69, the two are structurally very similar, differing only in their
oxygenation patterns as referred to in the positions indicated at R1–R4. Both uti-
lize a combination of acetyl-CoA, malonyl-CoA and methylmalonyl-CoA units
assembled in the indicated sequence with each of KR, DH, and ER domains op-
erating alone or in combination in identical ways at various locations throughout
the structures. The only exception to this is variation in oxygenation patterns at
positions R1–R4 which are determined by operation of different domain combi-
nations associated with each of the respective synthase enzymes, amphotericin
PKS or nystatin PKS. Once the polyketides are assembled, release from the PKS
enzyme is again achieved through lactone formation which is accompanied by in-
tramolecular hemiacetal formation at the position indicated. The postcyclization
transformations involve sequential oxidations at the indicated methyl group for
both, with additional C–H to C–OH oxidation occurring on the ring at position
R2 for amphotericin B or at position R4 for nystatin A. Both undergo a final gly-
cosylation at the allylic alcohol function via a GDP-derivative of the aminosugar
mycosamine.
O O O O O
-O C -O C -O C -O C
SCoA 2 SCoA 2 SCoA 2 SCoA 2 SCoA
starter 2x extender 8 x extender extender 8 x extender
EnzS R2 R4
OH OH
lactone amphotericin PKS
O O
OH OH R1 R3 OH or
formation HO HO
nystatin PKS
via amphotericin PKS: R1 = H, R2 = H, R3 = OH, R4 = H OH

via nystatin PKS: R1 = OH, R2 = R3 = R4 = H
O2 (amphotericin) R
2 R4 O2 (nystatin)
OH
O OH
O2, NADPH
hemiacetal HO O OH OH R1 R3 OH O
formation
O2 (both)
OH glycosylation (both)
R2 R4
OH GDP-D-
O OH
mycosamine
HO O OH OH R1 R3 OH O
CO2H
amphotericin B: R1 = H, R2 = OH, R3 = OH, R4 = H O O
nystatin A: R1 = OH, R2 = H, R3 = H, R4 = OH
HO OH
NH2
FIGURE 5.69
Biosynthesis of the antifungal polyene macrolides amphotericin B and nystatin A.

Originally extracted from Streptomyces nodosus, amphotericin B is so named be-

cause of amphoteric properties associated with the presence of both an acidic car-
boxyl function and a basic amino group (on the sugar moiety). The drug targets
sterol-containing fungal cell membranes and is administered intravenously. While
an important treatment for certain systemic and life-threatening fungal infections
such as valley fever in the southwest United States, the potentially serious side effects
of amphotericin B therapy make careful control of dosage essential. By comparison,
the toxicity of nystatin A is much lower since it is mainly administered orally and
its absorption by this route is minimal, making it especially useful for treatment of
intestinal fungal infections. It is also a common component of topical creams for the
treatment of fungal infections of the skin, usually in combination with a steroidal
component used to reduce associated inflammation.
Monensin A, a polyether antibiotic isolated from Streptomyces cinnamonensis,
is an ionophore that can form stable complexes with a number of different mon-
ovalent metal cations such as Li+, Na+, K+, Rb+, and Ag+. The compound exhibits
antibiotic, antimalarial, and other biological activities, and its valuable antibacterial
properties have led to its wide use, mainly in the beef and cattle industries. Its bio-
synthesis is outlined in Fig. 5.70.
O O O O O
-O C -O C -O C -O C
2 SCoA 2 2 2 SCoA
SCoA SCoA SCoA
starter 2x extender extender extender extender
O O O O
- -O C
monensin O2C SCoA 2 SCoA
-O C
2
-O C
2
SCoA SCoA
PKS 3x extender 2 x extender extender extender
HO
O
OH
HO
thioester hydrolysis
EnzS
O hemiacetal formation
O
HO
HO
O OH O O OH
epoxidations
HO
HO HO
O O
O O
O HO
premonensin
O
HO HO
O O O O
O SAM O
O O
HO O2, NADPH
H3CO
O O
HO HO
HO HO
O O monensin A
HO
FIGURE 5.70
Biosynthesis of the polyether antibiotic monensin A.


HO O O
O O O O O O
O O K+
K+
O
O O O O O
HO O O
HO
O monensin A HO 18-crown-6 18-crown-6 K+ complex
FIGURE 5.71
Comparison of monensin A with a crown ether potassium ion complex.
Ionophores are able to bind and transport metal ions across hydrophobic lipid
membranes. In that context, note the structural similarity between monensin A and
18-crown-6, a member of the “crown ether” group of synthetically prepared iono-
phore macrocyclic polyethers developed by Charles Pedersen of DuPont, for which
he received a share of the Nobel Prize in chemistry late in his life. Binding occurs via
interaction between the metal cation and ether oxygen lone pairs (Fig. 5.71).
LOOKING AHEAD
The foregoing examination of various classes of polyketide natural products repre-

sents a mere tip of the iceberg with regard to product diversity from this remark-
ably complex and ever-expanding area of biosynthetic chemistry. There is so much
more to explore, particularly with respect to the many additional important com-
pounds that may be produced when different biosynthetic pathways intersect. We
saw a simple example of this earlier when we looked at the biosynthesis of poison
ivy’s active principal, urushiol (Fig. 5.54) which utilized an aromatic component, cin-
namoylCoA, as a starter unit in the polyketide assembly process. At that point, we
mentioned that this aromatic component was derived from a different pathway—the
shikimic acid pathway. And it is no coincidence that this hybrid polyketide product
was produced by a plant rather than by the bacteria or fungi which produce most of
the polyketides we have seen. To better understand this connection, we move on in
our next chapter to explore this unique biosynthetic pathway that operates only in
plants and that represents the second major source of aromatic compounds produced
by nature.
STUDY PROBLEMS
1. Propose a series of experiments to rule out biosynthesis of methylphloroaceto-

phenone via a direct C-methylation of phloroacetophenone (see Fig. 5.40).
2. Suggest a biosynthetic sequence to produce 6-methylsalicylic acid.

CO2H

H3C OH
6-methylsalicylic acid
3. What are the R/S stereochemical assignments of the chirality centers in griseoful-
vin? The NADPH conjugate addition of hydride in the final step occurs at which
face of the prochiral ring pi-bond?
OCH3 H3CO
H3CO O
H3C
Cl
(+)-griseofulvin
4. Propose a triketide-based biosynthesis of 6-methoxymellein, an antifungal me-

tabolite isolated from carrots.
OH O
H3CO
6-methoxymellein
5. Propose a mechanism for the laboratory oxidative conversion of 4-methylphenol

to yield Pummerer’s ketone.
CH3
H3C O
K3[Fe(CN)6]
2 H3C
(oxidation)
O
OH
Pummerer's Ketone
6. Propose a reasonable mechanism for the oxidation step involved in conversion of

anhydrotetracycline to 5a,11a-dehydrotetracycline.

H3C
H3C OH
O2, NADPH
monooxygenase
enzyme
OH OH O OH O O
7. Emodin is a precursor to skyrin, a chiral natural product. Show how skyrin is

formed from emodin and explain how this compound can be chiral even though
it has no chiral carbons.
OH O OH
OH O OH
HO CH3
O
O
HO CH3 HO CH3
O
emodin
OH O OH
skyrin
8. The acetate hypothesis has proven useful in resolving questions related to cor-
rect structural assignments for many natural products when alternate structural
assignments were consistent with available spectroscopic data. Which structure
(A or B) would you predict to be the correct structure of natural product X,
assuming it is biosynthesized in a normal way via the polyketide pathway?
(Note that a methylation and an oxidation would be required to produce X after
polyketide cyclization.)
O OH O
H3CO O OH H3CO O
vs.
OH
CH3 O OH O CH3 O O
A B
9. Compound B is formed from compound A which is initially produced via the

polyketide pathway.

OH

OH
O OCh3
∗ ∗
H3C OH H3C O
A CO2H B
a) Outline the likely steps involved in the biosynthesis of A.

b) When the indicated carbon of A is labeled with 13C(*) and fed to the organ-
ism producing the metabolite, only 13C-labeled B is produced. Keeping in
mind the potential symmetry of certain possible intermediates, what does
this result say about the reactions involved and especially about the order of
the steps in which A is probably transformed into B?
10. Provide the missing structures to complete the following reaction scheme
(hint: the second step involves conjugate addition of water as a nucleophile).
O
H2O
RCH2CH2CH2CH2CSCoA
FAD FADH2
NAD+
NADH/H+
O O
CoASH
RCH2CH2CSCoA + CH3CSCoA
a) In the first step of this process, a dehydrogenation takes place which involves ini-
tial formation of a thioester enolate ion, followed by delivery to FAD of hydride
as the pro-R β-hydrogen atom of the thioester enolate. The resulting FADH–
anion then accepts a proton from a suitable donor to complete the process.
Outline the correct structures and mechanisms involved in this overall trans-
formation (use an abbreviated structural representation for FAD that shows the
key structural elements involved in the dehydrogenation mechanism).
b) The last step is a retro-Claisen condensation. Show a mechanism including
key intermediates.
11. The compound on the left reacts with geranyl diphosphate (GPP) to give the
compound shown. Propose a mechanism for this biosynthesis of the phenolic
component, then show its reaction with GPP and subsequent steps involved in
converting it to the compound shown.

Bioorganic Synthesis 256 OH

O OH
HO GPP
steps
O
OH
12. The polyketide metabolite A has recently been isolated from a lichen. Indicate
a biosynthesis of A starting from acetyl-CoA. In your answer indicate the se-
quence of events and a mechanism for aromatic and lactone ring formation.
A O OH
13. Suggest a reasonable polyketide-based biosynthesis of the macrocyclic lactone

shown, produced by Steptomyces venezuelae (indicate starter unit, extension
units and any transformations of the intermediate polyketide required prior to
lactone cyclization).
CH3
H3C CH3
O
O OH
CH3 CH3
14. Prostacyclin (PGI2) is a potent vasodilator and inhibitor of platelet aggregation

produced by vascular endothelial cells. Decreased production of prostacyclin is
associated with platelet aggregation and the formation of blood clots (throm-
bosis), a primary cause of heart attacks and stroke. Outline the biosynthesis of
prostacyclin from PGH2, and write a mechanism to show how it is transformed,
in vivo, into the compound shown.
HO2C HO
CO2H
O H+
O
H2O
HO
OH
OH OH
prostacyclin

15. Mechanistically similar to the hydroperoxidation of arachidonic acid, linolenic

acid forms a hydroperoxide in the presence of the enzyme 13-lipoxygenase. This
hydroperoxide can undergo several transformations, one of which involves a 1,2
C-to-O cationic migration somewhat similar to those seen in Baeyer–Villiger
oxidations. Such rearrangements are the source of numerous C6 aldehydes and
alcohols found in nature. Outline a reasonable mechanism for how such a rear-
rangement of hydroperoxide A in aqueous solution would lead to the two alde-
hyde products B and C shown (the migrating carbon is starred in both A and B).
Start it off by protonating the OH group of the hydroperoxide.
CO2H H+ CO2H
O
H2O O +
* * H C
O OH B H
A
16. Use equations to show how a sequence of 1) oxidation, 2) dehydrogenation and

3) Baeyer–Villiger oxidation could transform A into B, a precursor to penicil-
linic acid. Show mechanistic details of the Baeyer–Villiger oxidation step.
O
H3CO OH H3CO
1) 2) 3)
O
O
A CH3 B H3C
17. Assuming the producing organism was fed doubly labeled 13C acetate, show the
expected polyketide folding pattern and as well as the expected labeling pat-
tern in the product shown below (for labeling, use bolded bonds as shown in
Fig. 5.39).
HO OH
OH CH3 O
OH

6 The Shikimate Pathway

Biosynthesis of Phenolic Products
from Shikimic Acid
I think my strongest suit in science has been critical, logical analysis, leading to a single but
decisive experiment, although a systematic program, pursuing the shikimate pathway, has
probably contributed most to my scientific reputation
—Bernhard Davis (Microbiologist, 1916–1994)
Like other amino acids, the aromatic amino acids phenylalanine, tyrosine, and
tryptophan are vitally important for protein synthesis in all organisms. However,
while animals can synthesize tyrosine via oxidation of phenylalanine, they can syn-
thesize neither phenylalanine itself nor tryptophan and so these essential amino
acids must be obtained in the diet, usually from plant material. Though many
other investigators made significant contributions in this area over the years, it
was Bernhard Davis in the early 1950s whose use of mutant stains of Escherichia
coli led to a full understanding of the so-called shikimic acid pathway that is used
by plants and also by some microorganisms for the biosynthesis of these essential
amino acids. The pathway is almost completely devoted to their synthesis for pro-
tein production in bacteria, while in plants the pathway extends their use to the
construction of a wide array of secondary metabolites, many of which are valu-
able medicinal agents. These secondary metabolites range from simple and famil-
iar compounds such as vanillin (vanilla flavor and fragrance) and eugenol (oil of
clove, a useful dental anesthetic) to more complex structures such as pinoresinol,
a common plant biochemical, and podophyllotoxin, a powerful cancer chemo-
therapy agent (Fig. 6.1).
258

CO2H

CO2H CO2H NH2
NH2 NH2
HO N
H
phenylalanine tyrosine tryptophan
O
H3CO H3CO
H
HO HO
vanillin eugenol
OH
OH O
O
O
OCH3 O
O
O
H3CO OCH3
HO OCH3
OCH3 pinoresinol podophyllotoxin
FIGURE 6.1
Aromatic amino acids and some secondary metabolites from the shikimic acid pathway.
WHAT IS SHIKIMIC ACID?
Earlier in Chapter 3, we encountered two important intermediates, erythrose-4-

phosphate and phosphoenolpyruvate (PEP), each of which was derived from a
different pathway utilized in carbohydrate metabolism. Erythrose-4-P was an in-
termediate in one of the steps of the pentose phosphate pathway while hydrolysis of
PEP to pyruvic acid was the final step in glycolysis (Fig. 6.2).
These two simple intermediates provide the seven carbon atoms required for
construction of shikimic acid itself. The two are linked to one another via a sequence
of enzyme-mediated aldol-type reactions, the first being a bimolecular reaction and
the second an intramolecular variant that ultimately leads to a cyclic precursor of
shikimic acid known as 3-dehydroquinic acid as shown in Fig. 6.3.
Subsequent dehydration of 3-dehydroquinic acid leads to 3-dehydroshikimic
acid which then leads directly to shikimic acid via NADPH reduction. It should
also be noted that this intermediate may undergo several additional transforma-
tions (Fig. 6.4) such as dehydration, one of several routes to protocatechuic acid,
a naturally occurring antioxidant and anti-inflammatory polyphenol, or dehydro-
genation by the action of NADP+ to give 3-didehydroshikimic acid, an unstable
tautomer rapidly converted to gallic acid, a major component (along with glucose)
of tannic acid and other tannins derived from wood barks and other sources and
used in the textile, wood, and food industries. Decarboxylation of gallic acid either

CH2OH

CH2OH
O H N H
2 N CHO
HO H
HO H H OH
H OH - H2 O :B retroaldol
i) H O H H OH
H OH imine
formation H OH H CH2OP
HOH2C N
H OH erythrose-4-P
H OH
CH2OP HO H
CH2OPH2
sedoheptulose-7-P
O OH O OH
O OH C C
C
ii) C OP - H2O C OP
H C OP
CH2-OH CH2
B: CH2OH
B phosphoenol-
H
2-phosphoglycerate pyruvate (PEP)
FIGURE 6.2
Production of: i) erythrose-4-P from the pentose phosphate pathway and ii) phosphoenolpyruvate
(PEP) from the glycolytic pathway.
O O O
OH CO2H
C H 2O CO2H
B:
PEP C O-P 1,2-elimination
H OH OH HO OH
aldol
CH 2
(bimolecular)
B OH POH OH
CH=O H PO
H OH
H OH CO2H
HO CO2H
CH2OP
B
erythrose-4-P H O
aldol OH
(intramolecular)
O OH
B: H-O OH
OH
3-dehydroquinic acid
FIGURE 6.3
Sequential aldol additions in biosynthesis of 3-dehydroquinic acid.
enzymatically or by heating leads to pyrogallol, a phenolic reducing agent used

in early photographic processes and still widely employed as a stoichiometric O2
scavenger.
SHIKIMIC, CHORISMIC, AND PREPHENIC ACIDS

AT THE HEART OF THE PATHWAY
Getting back to shikimic acid itself, we find that this key intermediate must un-
dergo a number of additional transformations on its way to finally being converted
into phenylalanine or tyrosine. Some have even suggested that the pathway might


CO2H CO2H
HO CO2H
B: H
NADPH
O OH O OH HO OH
H2O
OH OH OH
3-dehydroquinic acid 3-dehydroshikimic acid shikimic acid
H2 O
NADP +
CO2H CO2H CO2H
O O HO OH
HO HO OH CO2
OH OH
OH OH
protocatechuic acid 3-didehydroshikimic acid gallic acid pyrogallol
FIGURE 6.4
Formation of shikimic acid and other transformations of 3-dehydroshikimic acid.
be more suitably named after one of the subsequent intermediates, but names
tend to stick over the years, and no matter how we look at it, the end products
of the pathway ultimately lead back to shikimic acid as the pivotal source. The
first of the subsequent intermediates, chorismic acid, is formed by initial ATP
phosphorylation to give shikimic acid-3-phosphate. Subsequent addition of
the 5-hydroxyl group across the π-bond of phosphoenolpyruvate (PEP) gives 5-
enolpyruvylshikimic acid-3-phosphate; sequential 1,2-and 1,4-eliminations of
phosphate (POH) complete the conversion of shikimic acid to chorismic acid as
shown in Fig. 6.5.
At this point, we stop to take a brief overview of the key reaction involved in the
subsequent transformation of chorismic acid into prephenic acid, at which point we
will be well on our way to the final targets, phenylalanine and tyrosine.
THE CLAISEN REARRANGEMENT: ALLYL VINYL

ETHERS IN A CHAIR
One of the oldest and most important C–C bond forming reactions in organic
chemistry is a process known as the Claisen rearrangement, originally reported by
L. Claisen in 1912. As the first known example of the so-called [3,3]-sigmatropic re-
arrangements, the Claisen rearrangement is formally a pericyclic reaction, mean-
ing that the transformation involves a cyclic transition state which facilitates the

CO2H CO2H CO2H :B

H+ H
HO OH ATP PO O-H PO CO2H PO O CO2H

ADP OP
OH PEP OH
OH :B H+
5-enolpyruvyl
shikimic acid shikimic acid-3-phosphate shikimic acid-3-phosphate
1,2-
elimination
CO2H CO2H :B POH
1,4- H
elimination
O CO2H H+
POH PO O CO2H
OH OH
chorismic acid
FIGURE 6.5
Conversion of shikimic acid to chorismic acid.
3 3 heat
O O
3 heat 2 3
2
O O
1 2 1 2
1 1 O heat O
allyl vinyl ether γ,δ− unsaturated
carbonyl
chair transition state and orbital overlap
FIGURE 6.6
Some key features of the Claisen rearrangement of allyl vinyl ethers.
intramolecular rearrangement of bonding electrons among the atoms of the reac-

tant. In the case of the Claisen rearrangement, the reactant is an allyl vinyl ether
for which rearrangement of the electrons via the cyclic transition state leads to a
γ,δ-unsaturated carbonyl derivative as shown in Fig. 6.6. Furthermore, it has been
shown that the so-called chair transition state for the Claisen rearrangement is
one which leads to the most rapid reaction via advantageous overlapping of orbitals
in the reactant and product. Because of the thermodynamic stability of the C=O
bond formed, the reaction is irreversible, unlike the related Cope rearrangement of
1,5-dienes. The [3,3]-sigmatropic rearrangement designation is related to the ob-
served movement of a sigma bond between atoms designated 1 and 1 to the atoms
numbered 3 and 3 in the diagram.
Like other sigmatropic rearrangements, there are no typical reactive interme-
diates involved in the Claisen rearrangement (i.e., no carbocations, no radicals,
etc.). The reaction is a concerted one, meaning that all the bond-making and bond-
breaking events occur simultaneously. This makes the Claisen rearrangement a ste-
reospecific reaction: optically pure reactants will yield optically pure products when
bonds to existing chirality centers are broken and new chirality centers are formed
in the process.

CO2H

HO2C
chorismate
mutase O O
~108 rate
O CO2H acceleration OH chair-like
CO2H
conformation
OH
chorismic acid
HO2C
O O
CO2H OH chair-like
conformation
CO2H
HO2C
HO2C
O
O
CO2H OH
OH synthetic transition state mimic
prephenic acid and enzyme inhibitor
FIGURE 6.7
Chair-like transition state in the Claisen rearrangement of chorismic acid to prephenic acid and a
transition state mimic and enzyme inhibitor.
CONVERSION OF CHORISMIC ACID TO PREPHENIC ACID
The chorismate mutase-catalyzed Claisen rearrangement of chorismic acid to pre-

phenic acid (Fig. 6.7) is one of the few examples of an enzyme-catalyzed pericyclic
reaction occurring in nature. The reaction shows an enormous rate acceleration—
on the order of 108—relative to the uncatalyzed reaction, due in part to the ability
of the enzyme to bind chorismic acid in the ideal chair-like conformation required
by the Claisen rearrangement. Evidence to support this includes the significant
rate retardation observed with the introduction of a synthetic enzyme inhibitor
whose shape mimics the chair-like transition state believed to be involved. Again,
note the stereospecificity of the reaction, with optically pure chorismic acid yield-
ing optically pure prephenic acid, the key pathway precursor to both phenylala-
nine and tyrosine.
CONVERSION OF PREPHENIC ACID

TO PHENYLALANINE OR TYROSINE
As shown in Fig. 6.8, prephenic acid serves as a branch point in the pathway,
leading to either phenylpyruvic acid via a decarboxylative 1,4-dehydration or
to 4-hydroxyphenylpyruvic acid via a decarboxylative 1,4-
dehydrogenation,


CO2H
H
HO O CO
prephenic acid
H :B H :B
O O
O C O
C
OR
H H
HO CO2H HO O CO2H
O
NADP +
H B
CO2 CO2
H2O NADPH
CO2H CO2H
O O
HO
phenylpyruvic acid 4-hydroxyphenylpyruvic acid
PLP transamination PLP transamination
CO2H CO2H
NH2 NH2
HO
phenylalanine tyrosine
FIGURE 6.8
Steps in the conversion of prephenic acid to phenylalanine or tyrosine.
depending on which enzyme system is employed. Each of these products in turn may
undergo PLP-mediated transamination in the usual fashion to convert the respec-
tive α-ketoacids to their corresponding L-α-amino acids, that is, phenylalanine or
tyrosine. In some instances, transamination of prephenic acid (to give arogenic
acid) has been found to precede the decarboxylative elimination steps, constitut-
ing yet another pathway to phenylalanine and tyrosine. Recall that animals lack the
shikimic acid pathway but can synthesize tyrosine by oxidation of phenylalanine
obtained in the diet.

MORE USES FOR CHORISMIC ACID

Before moving on to the many compounds derived from phenylalanine or tyrosine,
we should briefly consider some additional transformations of chorismic acid, in-
cluding its role in the biosynthesis of tryptophan, the third amino acid from this
pathway. As shown in Fig. 6.9, chorismic acid may be diverted from the chorismic-
to-prephenic acid Claisen rearrangement via several different routes (A–C among
others), each of which ultimately leads to the formation of a biologically important
benzoic acid derivative.
Path A (Fig. 6.9) involves a 1,4-addition–elimination sequence with water acting
as both nucleophile and leaving group, leading to an intermediate that undergoes
subsequent aromatization via a 1,2-elimination of pyruvic acid to afford salicylic
acid, the well-known aspirin precursor and active agent in medicinal extracts of
chorismic acid
A B C
CO2H
CO2H CO2H
H2N NH3
H2O
O CO2H
O CO2H O CO2H
OH
OH OH
H B
H B H B
H2O
H2O, H+
H2O, H+
CO2H
H :B
CO2 H :B NH2 CO2H
H H
H B H B
OH NH2
O CO2H
O CO2H O CO2H
NH3
O H2N, H+ O
CO2H CO2H CO2H
H B
CO2H CO2H
OH NH2
O CO2H
H NH2
B:
O
salicylic acid anthranilic acid
CO2H
CO2H
NH2
p-aminobenzoic acid (PABA)
FIGURE 6.9
Other transformations of chorismic acid to salicylic acid, PABA, and anthranilic acid.


willow bark whose recorded use dates as far back as the 5th century B.C. Salicylic
acid is also an important plant hormone with roles in photosynthesis, growth, and
ion transport, among many others. Paths B and C each lead to simple aromatic
amino acids: path B involves a 1,4-addition–elimination sequence with water as
leaving group and an enzyme side chain amino group as nucleophile. The inter-
mediate obtained then undergoes subsequent 1,4-addition–elimination with NH3
(from glutamine) as nucleophile and the enzyme side chain amino function acting
as leaving group. Subsequent 1,2-elimination and aromatization (again with loss of
pyruvic acid) affords para-aminobenzoic acid (PABA), an important precursor to
folic acid (vitamin B9). Path C differs from path A only in the use of glutamine-
derived NH3 as nucleophile instead of water in the initial 1,4-addition–elimination
sequence. 1,2-Elimination of pyruvic acid again leads to aromatization and forma-
tion of anthranilic acid which, as we will see, plays a key role in tryptophan bio-
synthesis in a series of reactions that initially converts anthranilic acid to the simple
N-heterocycle indole, as shown in Fig. 6.10.
The sequence begins with a nucleophilic displacement of diphosphate from the
anomeric carbon of 5-phosphoribose diphosphate by the anthranilic acid amino
group nitrogen to give an N-glycoside (i). Ring-opening protonation of the furan
ring oxygen atom of (i) leads to an imine intermediate (ii) which may be envisioned
HO H O OH
H OH HO OH
CO2H PPO HO OH
H
H O OP OH O
H
5-phosphoribose O OP
NH2 diphosphate N N OP
H H imine HO
anthranilic acid (i) H B (ii)
OH H B OH
O OH OH
B: H O OH HO
HO OP O O
O
OH N OP
N OP H HO (iii)
N (v) H HO
(iv) enamine
H α−aminoketone
enol
CO2
H B OH OH
HO B H OP
OP
OH OH
H
N H2O N
H H
:B (vii)
(vi)
:B
O H
H OP
retroaldol
N OH
H CHO
indole N
H OH H (viii)
CH2OP
FIGURE 6.10
Conversion of anthranilic acid to indole.

as undergoing a series of tautomerizations, imine-to-enamine (iii), with the re-

sulting enamine also serving as an enol due to the hydroxyl group present on the
enamine double bond. This enol (iii) in turn can give rise to an enol-to-ketone tau-
tomerization ultimately leading to an α-aminoketone intermediate (iv). To form the
five-membered ring required for the indole nucleus, a subsequent decarboxylative
ring-forming reaction may be viewed as involving an intramolecular enamine-type
aldol reaction with nucleophilic attack by the C-6 ring “enamine” of (iv) at the ad-
jacent ketone function to give cyclic intermediate (v). This is followed by decar-
boxylation of (v) to restore the aromaticity of the C-6 ring in intermediate (vi) and
then by a simple dehydration to give the 3-substituted indole aromatic heterocycle
(vii) as shown. Finally, protonation at the 3-position of (vii) gives an iminium ion
(viii) which may be viewed as undergoing what is formally a retroaldol reaction to
produce the parent indole structure with concurrent loss of the three carbon side
chain as 3-phosphoglyceraldehyde, a familiar intermediate previously seen in glu-
cose metabolism (Fig. 3.25).
Next, we consider the final conversion of indole to tryptophan as shown in
Fig. 6.11. This sequence begins with condensation of the amino acid serine with
PLP (protonated form) to give the usual intermediate as shown. The attached PLP
system acts essentially as an electron withdrawing function which facilitates a de-
hydration reaction (i.e., a 1,2-elimination) to give the unsaturated derivative which
then undergoes an enamine-type nucleophilic attack by the indole C-5 ring to give
the required C–C bond formation at position 3. Proton loss for restoration of the
indole ring aromaticity is accompanied by protonation at the α-position of the car-
boxylic acid to give the familiar α-amino acid-PLP derivative. This imine linkage
is essentially the same PLP derivative that would be observed in transamination of
:B
B H H CO2H
CO2H
HO
N N
CO2H PLP N H
HO
OH
NH2 OH H2O PO
PO
serine
N
N H
H
B:
H B
H
CO2H CO2H CO2H
N N
NH2 H2O N N
H OH H OH
PO PO
N PLP
H
tryptophan N N
H H
FIGURE 6.11
Final conversion of indole to tryptophan via serine and PLP.

tryptophan itself. Thus, subsequent hydrolysis of the imine linkage regenerates PLP
Bioorganic Synthesis 268 and releases L-tryptophan as shown.
We will see a number of uses of this essential amino acid as a building block
for alkaloid biosynthesis and for the production of neurotransmitters, neurohor-
mones, and other important biochemicals when we consider alkaloid biosynthesis
in the following chapter. But for now, we must return to consideration of the many
fates that await the amino acids phenylalanine and tyrosine, as these are the pre-
dominant building blocks used for the construction of a remarkably diverse array of
biologically and medicinally important secondary metabolite compounds that are
produced by the shikimic acid pathway.
SHIKIMIC ACID PATHWAY PRODUCTS FROM

PHENYLALANINE AND TYROSINE: AN OVERVIEW
A simplified overview of the pathway and how its intermediates are partitioned into
various products is shown in Fig. 6.12. Aside from the alkaloid products that will
be treated separately in the next chapter, we will see that both phenylalanine and
tyrosine will undergo modifications to afford various phenyl C3 compounds via
reductions, deaminations, oxidations, and other transformations. Some phenyl C3
Chorismic Acid Shikimic Acid

isoflavonoids
modify,
structural
Prephenic Acid rearrangement
coumarins, flavonoids
lignans,
lignin, others
phenylalanine,
tryptophan phenyl C1
tyrosine
compounds
intersect
with
modify, cyclize, polyketide
modify modify modify modify, dimerize, pathway
cleave or polymerize
aromatic phenyl C3
alkaloids compounds
OH OCH3
H3CO O
OH
H3CO HO O O HO OH
anethole, umbelliferone, guaiacylglycerol-β-
a phenyl C3 compound a coumarin coniferyl ether, a lignan
OH
HO O HO O
HO CO2H OH
OH
OH O O
OH
gentisic acid, luteolin, daidzein,
a phenyl C1compound a flavonoid an isoflavonoid
FIGURE 6.12
A brief overview of some product classes derived via the shikimic acid pathway.


compounds may then undergo further modifications to give cyclic products such as
coumarins. Phenolic oxidative dimerization of certain phenyl C3 compounds can
lead to a wide variety of so-called lignan compounds, while their oxidative polym-
erization yields an abundant plant structural material known as lignin. Phenyl C3
compounds may also undergo various modifications followed by oxidative cleav-
age to yield a variety of simpler phenyl C1 compounds, while others may be em-
ployed as unique starter units for polyketide chain extensions, modifications, and
cyclizations to yield compounds known as flavonoids, some of which may then be
structurally rearranged to yield the so-called isoflavonoids. While other classes of
compounds are known, we will focus primarily on those shown here since these
include the majority of the most important products produced by this pathway.
PHENYLPROPANOIDS: A LARGE FAMILY
OF PHENYL C 3 COMPOUNDS
As we will see, many of the products from the shikimic acid pathway are so-called
phenyl C3 compounds or phenylpropanoids which are derived from phenylala-
nine or tyrosine. The initial phenylpropanoid which serves as the building block
for many others is trans-cinnamic acid, a product obtained via a 1,2-elimination
(deamination) of the elements of NH3 from L-phenylalanine as shown in Fig. 6.13.
This anti elimination process is catalyzed by the enzyme phenylalanine ammonia-
lyase (PAL).
Figure 6.14 outlines the steps involved in biosynthesis of four other important
shikimic acid pathway products, namely 4-coumaric acid, caffeic acid, ferrulic
acid, and sinapic acid. These compounds are sometimes referred to as cinnamic
acid derivatives since they all share the same trans-1-phenylpropenoic acid struc-
tural core and are all derived from trans-cinnamic acid via a series of simple oxida-
tions and SAM methylations (4-coumaric acid is also known as 4-hydroxycinnamic
acid). Note that while the main substrate for the PAL enzyme system is phenylala-
nine, it may also be employed for direct production of 4-coumaric acid via deamina-
tion of L-tyrosine, though the primary route to this product is via direct oxidation
of trans-cinnamic acid.
B:
H H
CO2H phenylalanine CO2H
ammonia lyase CO2H
NH2 (PAL) H NH2
NH3
L-phenylalanine B H trans-cinnamic acid
FIGURE 6.13
Biosynthesis of trans-cinnamic acid from phenylalanine.

FIGURE 6.14
Biosynthesis of coumaric, caffeic, ferulic, and sinapic acids.
PHENYLPROPANOIDS: REDUCTION OF ACIDS
TO PHENYL C 3 ALDEHYDES AND ALCOHOLS
In addition to simple esters such as ethyl cinnamate, plants produce a wide variety
of other phenyl C3 acid derivatives, many of which involve simple reduction of the
carboxylic acid moiety. The transformations involved are illustrated in Fig. 6.15 for
the conversion of trans-cinnamic acid into cinnamaldehyde and cinnamyl alcohol.
The same sequence of reactions may be applied to the other phenyl C3 acids to pro-
duce the corresponding aldehydes and alcohols as shown. Cinnamaldehyde is the
organic compound responsible for the familiar flavor and fragrance of cinnamon
which may be obtained from the bark of the cinnamon tree. It is also widely used as
a fungicide in agriculture due to its relatively low toxicity.
Conversion of these acids to their aldehydes is achieved via NAPH reduction of
the corresponding CoA thioesters to a thiohemiacetal which dissociates to give the
aldehyde and HSCoA. Subsequent conversion of the aldehydes to the alcohols is
likewise via the usual NADPH reductions. These alcohols are sometimes referred to
as monolignols, since they serve as the monomers used in production of the plant
biopolymer lignin which we will discuss shortly.
REDUCTION OF PHENYL C 3 ALCOHOLS

TO PHENYLPROPENES
Phenylpropenes are widely distributed plant aromatics derived from phenyl C3 alco-
hols (lignols) and are components of many different essential oils. All share the same
basic phenylpropene (allyl benzene) core and differ mainly in the degree, position,
and/or alkylation pattern of ring oxygens. Examples include myristicin from nutmeg
oil, estragole found in basil and pepper, and its isomer anethole, isolated from anise
and fennel and responsible for the familiar flavor and fragrance of licorice (Fig. 6.16).
Evidence suggests that in some instances, the NADPH reduction of lignols to
phenylpropenes proceeds via initial conversion of the lignol to its acetate ester,


O B: H B
CO2H HO SCoA
CoASH SCoA NADPH
H
trans-cinnamic acid H2O

CoASH
OH O
coumaric acid
caffeic acid NADPH H
ferulic acid
sinapic acid
same cinnamyl alcohol cinnamaldehyde
sequence
OH OH OH
OH
H3CO
HO HO HO
HO
OH OCH3 OCH3
4-coumaryl alcohol caffeyl alcohol coniferyl alcohol sinapyl alcohol
FIGURE 6.15
Reductions of cinnamic acids to their corresponding aldehydes and alcohols.
O O
O O
core structure of
phenylpropenes HO HO
(allyl benzenes)
OCH3 OCH3
coniferyl acetate
NADPH
CH3CO2-
O OCH3
O H:
A B
myristicin HO A
OCH3
eugenol B H
O
H3CO
estragole OCH3
quinone methide
HO (protonated form)
H3CO OCH3
anethole isoeugenol
FIGURE 6.16
Some common phenylpropenes and conversion of coniferyl acetate to eugenol or isoeugenol.
followed by loss of acetate to give a quinone methide intermediate. Conjugate de-

livery of hydride from NADPH may occur via either path A or path B as shown
in Fig. 6.16 for reduction of coniferyl acetate. Path A leads to the usual allyl ben-
zene derivative, in this case eugenol, while path B affords the corresponding con-
jugated isomer isoeugenol which accompanies eugenol isolated from sources such
as oil of clove or oil of nutmeg. Other phenylpropenes may arise by direct hydride

displacement of lignol acetate or alcohol functions (in protonated form) rather than
Bioorganic Synthesis 272 proceeding via the quinone methide mechanism.
LIGNANS AND LIGNIN: OXIDATIVE PHENOLIC

COUPLING WITH A TWIST
We saw in the previous chapter an example of bimolecular oxidative phenolic cou-

pling in the biosynthesis of usnic acid (Fig. 5.46). We now revisit this type of oxida-
tive C–C bond-forming reaction within the context of the chemistry of the phenolic
lignols we have just examined. Figure 6.17 shows how the initially formed phenoxy
O-radical from any of the three indicated lignols leads to the usual ortho and para
C-radical resonance forms, but in the case of the lignols, the propenyl side chain of
the oxidized phenol permits further electron delocalization to yield an additional
contributor referred to here as the allyl C-radical resonance form as shown.
These radicals may be coupled together in several different combinations to
form C–C bonds. The resulting products are examples of lignans—essentially lignol
dimers—that are widely distributed in nature and have many interesting proper-
ties. Several lignans have been found to act as phytoestrogens (compounds that can
mimic the hormonal activity of estrogen) and are currently being studied as poten-
tial agents for cancer prevention, especially breast cancer.
CONIFERYL ALCOHOL OXIDATIVE COUPLING:

ALLYL C-R ADICAL + ALLYL C-R ADICAL
The term “lignan” is sometimes specifically applied to products arising from lignol
oxidative coupling at the central carbon of the phenyl C3 side chain. When coupling
OH OH OH
X X X
HO O O
e-, H+
Y Y Y
X, Y = H: 4-coumaryl alcohol O-radical ortho C-radical
X = H, Y = OCH3: coniferyl alcohol
X, Y = OCH3: sinapyl alcohol
OH OH OH
X X X
O O O
Y Y Y
allyl C-radical para C-radical ortho C-radical
FIGURE 6.17
Resonance forms of lignol phenoxy radicals.

OH

H3CO
OH O
HO O HO
2e-, 2H+ allyl OCH3
OCH3 allyl
2 x coniferyl alcohol C-radical
C-radical
O H+
OH H3CO O
H3CO OH
H
H HO
OCH3 2H + O
HO H+ OCH3
O
(+)-pinoresinol
FIGURE 6.18
Biosynthesis of (+)-pinoresinol via allyl C-radical/allyl C-radical coupling.
follows some other combination of lignol radicals, the products may be referred to
as neolignans. The distinction is not a terribly significant one, so we will stick with
referring to all such products as lignans while pointing out examples of products for
which the neolignan designation might be used.
One example of an important lignan arising via what we term allyl C-radical/
allyl C-radical coupling is the formation of (+)-pinoresinol from coniferyl alco-
hol as outlined in Fig. 6.18. In this scheme, C–C coupling of the radicals leads
to two highly reactive intermediate quinone methide structures which then un-
dergo intramolecular nucleophilic conjugate addition of the adjacent hydroxyl
oxygens. This results in rearomatization of the phenolic rings accompanied by
formation of the fused bicyclic tetrahydrofuran ring system shown. As we will
see shortly, this lignan serves as the starting material for subsequent biosynthetic
modifications leading to formation of the important cancer chemotherapy agent,
podophyllotoxin.
CONIFERYL ALCOHOL OXIDATIVE COUPLING: ORTHO

C-R ADICAL + ALLYL C-R ADICAL
Oxidative C–C dimerization of coniferyl alcohol via ortho C-radicals and allyl
C-radicals leads to another quinone methide-type intermediate which under-
goes subsequent aromatization via the intramolecular conjugate addition of an
adjacent phenolic alcohol function to yield (−)-dehydrodiconiferyl alcohol
as shown in Fig. 6.19. This 2-aryl dihydrobenzofuran compound and its many
structurally related derivatives are promising lead structures for pharmaceuti-
cal research, having shown a broad spectrum of biological and pharmacological
activities such as antioxidant, antitumor, antimicrobial, and antiHIV activities
among others.

H3CO

OH
OH
O O
allyl
C-radical
HO H3CO
2e-, 2H+ ortho C-radical
OCH3 OH
2 x coniferyl alcohol
keto-to-
enol
OH
OH
OH H3CO
H3CO
HO O HO OH
O BH
B:
(-)-dehydrodiconiferyl OCH3 H3CO
alcohol
FIGURE 6.19
Biosynthesis of (−)-dehydrodiconiferyl alcohol via ortho C-radical/allyl C-radical coupling.
CONIFERYL ALCOHOL OXIDATIVE COUPLING:

O-R ADICAL + ALLYL C-R ADICAL
As shown in Fig. 6.20, a different combination of coniferyl alcohol radicals can lead
to formation of a C–O ether linkage rather than a C–C linkage, yielding yet another
quinone methide intermediate that in this case is quenched and aromatized via con-
jugate addition of a water molecule to yield the lignan guaiacylglycerol-β-coniferyl
ether. Unlike the other lignans, the stereochemistry at the chirality centers of this
compound is usually revealed as a mixture of erythro and threo diastereomers and like
dehydrodiconiferyl alcohol, this lignan and its derivatives continue to be evaluated
as potential pharmaceutical lead compounds due a spectrum of biological activities.
LIGNIN: A PLANT POLYMER AND MAJOR

SOURCE OF CARBON
As we have seen, radical combinations from oxidized lignols follow different paths
of dimerization to yield some interesting organic compounds. Of course, other radi-
cal combinations from lignols are also possible and a variety of such linkages are
involved in the formation of lignin, a plant biopolymer with structural segments
closely related to the lignan structures we have already seen. A hypothetical struc-
tural segment of lignan is shown in Fig. 6.21 in which some of the individual link-
ages between lignol monomer units are readily identifiable. The detailed structure
of lignin itself is difficult to specify, since it may vary from source to source and may
also vary depending on techniques employed in its isolation or degradation.
Lignin is the second most abundant biopolymer after cellulose, making up
about 30% of the dry mass of wood, the remainder being mostly cellulose along

OH

OH OCH3
O O
OCH3
HO allyl C-radical
2e-, 2H+ O-radical
OCH3 OH
2 x coniferyl alcohol
OH OH H2O OH
OCH3 OCH3
O O
HO O
OCH3 OCH3
H+ H B
guaiacylglycerol-β- OH
OH
coniferyl ether
FIGURE 6.20
Biosynthesis of guaiacylglycerol-β-coniferyl ether via O-radical/allyl C-radical coupling.
OH
H3CO O-lignin
OH
OCH3
O OCH3
O
HO
OH OCH3 O
OH
lignin-O OH
lignin-O O OCH3
OH O
HO
OCH3 H3CO O O-lignin
HO
OH OH OCH3
O HO
O
H3CO OH OH
O
O O O O-lignin
O
OCH3 OH O
OCH3
O O
H3CO
H3CO O OH
OCH3 O O-lignin
HO OH OCH3
lignin-O OH
FIGURE 6.21
Hypothetical structural segment of the biopolymer lignin.
with smaller amounts of resins containing lower molecular weight organics such
as terpenes. Unlike cellulose and other biopolymers, lignin has an irregular,
nearly random structure. The three principal lignol monomers (monolignols),
p-coumaryl, sinapyl, and coniferyl alcohols, are connected to one another via link-
ages that arise via oxidative coupling processes similar to those utilized in the forma-
tion of lignans. Unlike lignans however, whose structures are enzyme-specific and

therefore enantiomerically pure, lignin stereocenters are racemic. So while the basic
Bioorganic Synthesis 276 building blocks for lignans and lignin are the same, lignin itself is not built up from
preformed lignans but rather from a stereorandom polymerization of monolignols.
Given its structural elements, one would expect lignin to be a tough, durable poly-
mer that would be difficult to break down. Certainly, wood owes much of its useful-
ness as a renewable source of building materials and fuels to its high lignin content.
PODOPHYLLOTOXIN BIOSYNTHESIS: ARYLTETRALIN
LIGNANS FROM THE AMERICAN MAYAPPLE
We now turn to a brief examination of an important class of bioactive lignans

known as the aryltetralins. The term is derived from tetralin, the common name
for the familiar fused bicyclic arylcyclohexyl ring system which forms the core of
these lignan structures. They are produced in significant quantities by plants of
the genus Podophyllum, among which is the American mayapple, Podophyllum
peltatum (also known as mandrake), which grows abundantly in eastern North
America and produces an extremely important aryltetralin known as podophyl-
lotoxin. Its principal biological activity involves the blocking of cell mitosis (cell
division); it is widely used in topically applied creams for the treatment of certain
viral warts.
The biosynthetic process for podophyllotoxin (Fig. 6.22) begins with sequential
NADPH reductions of the fused tetrahydrofuran rings of the lignan (+)-pinoresinol
whose structure and synthesis we described earlier. The resulting tandem reduc-
tions lead to the diol (−)-secoisolariciresinol. Selective NADP+ oxidation of one
of the alcohol functions leads to the corresponding aldehyde which forms a cyclic
hemiacetal or lactol. Further NADP+ oxidation of the lactol yields the correspond-
ing lactone (−)-matairesinol, a key intermediate product which undergoes a
series of aryl ring oxidations and SAM methylations proceeding through the in-
termediate compounds thujaplicatin (see structure for position numbering) and
4’,5’-dimethylthujaplicatin whose ortho-methoxyphenol moiety then undergoes
standard oxidative conversion to a methylenedioxy unit, yielding (−)-yatein. The
precise details associated with the subsequent oxidative cyclization of yatein are not
fully known, but an intramolecular electrophilic aromatic substitution is most likely
involved, leading to (−)-deoxypodophyllotoxin which is then finally hydroxylated
at the 4-position of the cyclohexane ring to yield (−)-podophyllotoxin as shown.
Most significantly, podophyllotoxin is used as the starting material for produc-
tion of the semisynthetic cancer chemotherapy drugs etoposide and teniposide
(Fig. 6.23). While podophyllotoxin acts as a mitosis inhibitor, etoposide and teni-
poside have an entirely different mode of action, interfering with the action of the
enzyme topoisomerase II which assists in the unwinding and reconnection of DNA
strands.

H3CO

OCH3 OH
HO H+ OH
HO
H NADPH H O H: NADP+
O
H+
O H H: O H OCH3
H+
(+)-pinoresinol OH OH
H3CO (–)-secoisolariciresinol
H3CO H3CO H3CO
OH
O O
O
HO HO HO
O2 O NADP+ OH
NADPH
OCH3 OCH3 OCH3

OH OH OH
(–)-matairesinol
4 O
H3CO 3 H3CO
O O O
1 2 O
HO HO
1' O O O
2' 2 SAM O2 O2
5' NADPH -H , - e-
HO 3' OCH H3CO OCH3 H3CO OCH3
4' 3
OH OCH3 OCH3
thujaplicatin 4',5'-dimethylthujaplicatin (-)-yatein
OH
O O O
O O electrophilic O
O aromatic
O substitution O
O2 O (cyclize)
O O
NADPH
H+
OCH3 H3CO OCH3 H3CO OCH3
H3CO
OCH3 OCH3 OCH3
(–)-podophyllotoxin (–)-deoxypodophyllotoxin
FIGURE 6.22
Biosynthesis of (−)-podophyllotoxin from (+)-pinoresinol.
These important chemotherapy agents have seen wide clinical use for the treat-
ment of small and large cell lung, testicular, pancreatic, and stomach cancers, as
well as in the treatment of myeloid leukemias, lymphomas, and Kaposi’s sarcoma.
The key step in the production of these drugs is conversion of podophyllotoxin
to 4’-demethylepipodophyllotoxin. This is achieved synthetically by demethyl-
ation of podophyllotoxin’s 4’-methoxyl group and epimerization at the 4-hydroxyl
group position via treatment with gaseous HBr followed by aqueous BaCO3; the
remaining steps involve protection of the phenolic hydroxyl group, formation of
a specific glycoside linkage at the 4-hydroxyl group and final deprotection back
to the free 4’-phenol. Ultimately, epimerization at the 4’-position has been shown
to be responsible for the remarkable shift in mode of action from mitosis inhibi-
tion for podophyllotoxin to topoisomerase II inhibition for both etoposide and
teniposide.

OH OH
Bioorganic Synthesis 278 O O
O O
O i) HBr O
O ii) BaCO3 O
H2O
acetone
H3CO OCH3 H3CO OCH3
OCH3 OH
podophyllotoxin 4'-demethylepipodophyllotoxin
glycoside formation
OH
HO O R
O
O O
O
etoposide: R = CH3
O
O teniposide: R = S
O
H3CO OCH3
OH
FIGURE 6.23
Conversion of podophyllotoxin to 4’-demethylepipodophyllotoxin, etoposide, and teniposide.
CLEAVAGE OF CINNAMIC ACIDS TO PHENYL C 1

COMPOUNDS: DIFFERENT ROUTES, SIMILAR
OUTCOMES
In addition to the formation of phenyl propenes and other phenyl C3 compounds,

the cinnamic acid group also serves as a source of simple phenyl C1 compounds
that may be obtained by oxidative cleavage of propenoic acid double bond. One
commonly cited cleavage sequence is illustrated in Fig. 6.24 for biosynthesis of both
vanillin, the compound from vanilla bean extract responsible for vanilla flavor and
fragrance, and vanillic acid.
This cleavage mode proceeds via the corresponding CoA thioester derivatives
which undergo conjugate addition of water, NADP+ oxidation to the β-ketothioester
then, in a sequence analogous to the β-oxidation process involved in fatty acid me-
tabolism, addition of CoASH to the ketone function to give a hemithioacetal which
then undergoes a retro-Claisen condensation. The resulting phenyl C3 thioesters

O O

H3CO H3CO
OH HSCoA SCoA
HO HO
H2O
ferulic acid H2O (conjugate
addition)
OH O
O O
H3CO
H3CO NADP + SCoA
SCoA
HO
HO O
HSCoA H3CO
H
NADPH
B: H O HO
O O retro- H3CO vanillin NADP+
CoAS SCoA
H3CO Claisen O
SCoA H3CO
O HO OH
HB H2O
HO
SCoA
HO
vanillic acid
FIGURE 6.24
Proposed routes for cleavage of ferulic acid to vanillin and vanillic acid.
may then undergo NADPH reduction to the corresponding aldehydes or simple

hydrolysis to yield the acids as shown in Fig. 6.24, though these acids may also be
formed via oxidation of the aldehydes themselves.
A second, often cited, cleavage mechanism also involves conjugate addition of
water to the cinnamic acids, but does not involve CoA thioester derivatives and re-
quires a retroaldol rather than a retro-Claisen condensation. Two alternative routes
utilizing this sequence are illustrated in Fig. 6.25 for the conversion of cinnamic acid
to salicylic acid from which aspirin (acetyl salicylic acid) is commercially prepared
by treatment with acetic anhydride. Biosynthetically, SAM methylation of salicylic
acid leads to methyl salicylate (wintergreen oil) which serves as both a natural
flavor and a fragrance additive for candies and gums and as well as a “deep heating”
agent and analgesic used in various topically applied creams and liniments.
COUMARINS: SWEET-S MELLING BENZOPYRONES
Another group of compounds derived from cinnamic acids may be formed when
the aryl ring position ortho to the propenoic acid side chain is oxidatively hydrox-
ylated. As shown in Fig. 6.26, such ortho-hydroxylation of cinnamic acid leads to
2-coumaric acid as the usual (E) isomer. However, conjugated π-bonds such as these
are susceptible to reversible photochemical isomerization (by ambient sunlight in
plants), and when such isomerization occurs for 2-coumaric acid, the Z-isomer may
then undergo irreversible intramolecular condensation to yield the correspond-
ing lactone known as coumarin, which is an example of a benzo-2-pyrone (the


B: H O O
H2O
OH OH
(conjugate
cinnamic acid addition) HB
O2 NADPH retro-
aldol
CH3CO2H
O
O
OH
H2O H
(conjugate OH
addition)
2-coumaric acid benzaldehyde
retro-
aldol
CH3CO2H NADP +
O O
NADP +
OH O2 OH
NADPH
OH
salicylic acid benzoic acid
SAM
O O
OH OCH3
acetic
anhydride
OCOCH3 OH
acetyl salicyclic acid (aspirin) methyl salicylate
FIGURE 6.25
Conversions of cinnamic acid and 2-coumaric acid to salicylic acid and derivatives.
carbonyl carbon is at the 2-position relative to the ring oxygen). Derivatives with the
same basic ring system are often referred to generically as substituted coumarins.
Coumarin itself, produced by many different kinds of plants, is responsible for the
familiar sweet smell of newly mown hay and is thought to be produced by plants
as a defense chemical, acting as deterrent to grazing animals due to its bitter taste.
A similar sequence for 4-coumaric acid leads to another coumarin known as um-
belliferone, a powerful UV-absorbing compound found in plants such as coriander
and carrots and which is used in some sunscreen preparations. It also serves as the
biosynthetic precursor to a variety of substituted derivatives with interesting bio-
logical properties (see Problem 6.4).
When moldy grasses are acted upon by certain fungi, the presence of couma-
rin can lead to formation of 4-hydroxycoumarin (Fig. 6.27) via conjugate addition
of water to 2-coumarylCoA followed by NADP+ oxidation and cyclization. A sub-
sequent aldol condensation in the presence of formaldehyde can then produce an
unsaturated cyclic β-ketoester which undergoes conjugate addition by the enolate of
a second molecule of 4-hydroxycoumarin, producing a compound that is formally
two 4-hydroxycoumarin units linked together by a bridging methylene unit. This


O
O2
OH OH
NADPH
OH
cinnamic acid (E)-2-coumaric acid
hν
O
O O H2O OH OH
coumarin (Z)-2-coumaric acid
same as above
OH
HO O O
HO
4-coumaric acid umbelliferone
FIGURE 6.26
Formation of coumarins from cinnamic acid or 4-coumaric acid.
compound is known as dicoumarol, an important anticoagulant agent that acts by

depleting levels of vitamin K (clotting factor) in the blood. The presence of dicou-
marol in contaminated sweet clover or hay was found to be responsible for many
historical cases of fatal internal bleeding in grazing cattle; this discovery led to ex-
tensive research into its mode of action and ultimately to its use as an anticoagu-
lant for the treatment of deep vein thrombosis in humans, though it has since been
largely displaced by the use of heparin in conjunction with various synthetic deriva-
tives of 4-hydroxycoumarin such as warfarin (trade name Coumadin). It should be
noted that coumarin itself has no anticoagulant activity.
MIXED PRODUCTS: COMBINING THE SHIKIMATE,

POLYKETIDE, AND TERPENOID PATHWAYS
We saw in the previous chapter an example of the use of para-hydroxycinnamic acid

(4-coumaric acid) as the starter unit for a polyketide chain extension, cyclization,
aromatization, and decarboxylation sequence in the production of the mixed poly-
phenolic stilbene (1,2-diphenylethylene) derivative resveratrol (see Fig. 5.55). As it
turns out, a remarkably diverse array of secondary metabolites make use of shikimic
acid pathway products as starter units for familiar polyketide construction sequences
and further structural elaboration via terpenoid building blocks. So we are now in


O OH O
NADP+
SCoA H2O SCoA
(conjugate OH O
OH addition) OH CoAS
2-coumarylCoA
:B O
H OH
O
H2C O HB
H2C O
aldol O O
O O
O O
4-hydroxycoumarin
O HB BH B: H O
OH O
H
:B H 2O
O O
O O O O
enolize
OH OH
OH O
O OO O
O O
dicoumarol
warfarin (synthetic analogue)
FIGURE 6.27
Formation of dicoumarol from 4-hydroxycoumarin; structure of warfarin.
a position to examine a number of such products in light of what we have already

learned so far about the shikimate, polyketide, and terpenoid biosynthetic pathways.
KAVALACTONES: NATURAL SEDATIVES
FROM THE SOUTH PACIFIC
Kava is the common name for the western Pacific pepper plant, Piper methysticum,
the roots of which are extracted to produce an intoxicating or sedating beverage of
the same name (kava or kava–kava) consumed by much of the native island popu-
lations in the South Pacific. The active ingredients in these extracts are members
of a group of compounds known as styrylpyrones (styrene + pyrone), structurally
similar to stilbenes but with a pyrone ring in place of one of the aryl rings. Several of
these so-called kavalactones (or kavapyrones) are shown in Fig. 6.28. While many
examples are known, the main structural types are represented by yangonin, kavain
(which has a reduced pyrone ring), dihydrokavain (with reduced pyrone and styryl
groups), and methysticin (with a methylenedioxy-substituted aromatic ring).
A typical biosynthetic scheme is shown in Fig. 6.29. 4-Coumaric acid undergoes
two cycles of malonyl-CoA chain extension followed by intramolecular cyclization

H 3 CO

H
O O O O
yangonin kavain
OCH 3 OCH3
O
O
H
O O H
O O
dihydrokavain methysticin
OCH3
OCH3
FIGURE 6.28
Representative kavalactone structures: yangonin, kavain, dihydrokavain, and methysticin.
HO
HO
O
2 x malonyl CoA O SCoA
SCoA
H
4-coumaric acid O
H
(as CoA thioester)
O
cyclize
H3CO HO
O O O O
enolize
2 x SAM
yangonin
OCH3 O
FIGURE 6.29
Biosynthesis of the kavalactone yangonin, a mixed pathway natural product.
to give the pyrone structure; this is followed by enolization of the pyrone ketone
carbonyl and SAM methylation of the enol and phenolic hydroxyl groups to afford
yangonin.
The effects of these secondary metabolites are similar to those associated with
antianxiety benzodiazepines such as Xanax, Valium, or Librium. However, kavalac-
tones are currently regulated or banned in many countries due to reports of signifi-
cant liver damage in some users. Interestingly, recent data has suggested that dietary
supplements, many of which are herbal extracts, now account for nearly 20% of all
drug-related liver injuries seen in U.S. hospitals, raising new concerns about the lack
of regulation of such compounds for use as dietary supplements or in alternative
medicine. In some instances, “natural” products can lead to adverse interactions

with prescription medications and should always be used with caution and under
Bioorganic Synthesis 284 the supervision of a physician.
FLAVONOIDS: STRUCTURALLY DIVERSE
PLANT POLYPHENOLICS
The flavonoids constitute a very large class of phenolic natural products derived
mainly from condensation of 4-coumarylCoA and malonate units. Over 4500 ex-
amples have been identified up to the present time in a variety of forms representing
some of the most abundant of all plant-derived polyphenolics. Many contribute to
plant and flower coloration which can serve a role in the attraction of pollinating
insects, while others may act as bitter-tasting antifeedants, antifungal, or antibiotic
agents, or even as UV absorbers. Given their remarkable abundance, flavonoids
likely have other properties or play other roles in plant biochemistry that are yet
to be discovered which makes them obvious targets for investigation as potential
medicinal agents. The diversity of principal flavonoid structural types and their bio-
synthetic relationships are illustrated in Fig. 6.30.
Many flavonoids have already been intensively studied in terms of their potential
as antiviral, antibiotic, and anticancer agents. For instance, some studies suggest that
the lower incidence of both breast and prostate cancers in vegetarians may be related
OH O O
O O O
chalcone flavanone flavone
O
O
O
OH
O
O isoflavone
O
aurone dihydroflavonol
O O
O
OH
O OH O
flavonol anthocyanin isoflavanone
FIGURE 6.30
Principal structural types and relationships in the flavonoid series.

HO OH OH

CH3 OH
O O
H
O O
H H
HO HO HO
estradiol genistein daidzein
FIGURE 6.31
Structural similarities of estradiol with the phytoestrogens genistein and daidzein.
to consumption of dietary isoflavonoids such as daidzein and genistein. These com-

pounds, abundant in sources such as soybean, tofu, and fava bean, are two examples
of plant products known as phytoestrogens, so-called because of their structural
resemblance to the estrogen hormone estradiol and their ability to bind to an estro-
gen receptor site (Fig. 6.31). Clearly flavonoids and isoflavonoids are of significant
medicinal potential, so the following sections will explore their biosynthetic origins
and some further transformations.
THE CHALCONE-T O-F LAVANONE-T O-F LAVONE

SEQUENCE: FORMATION OF APIGENIN
Given the vast number of known flavonoids and their structural diversity, we can only
briefly consider representative examples from specific classes to help illustrate some
of the general biosynthetic details involved in their production. To begin, apigenin
will serve as a particularly interesting example of a flavone compound under active
investigation due to its potentially valuable biological activity. Its biosynthesis is out-
lined in Fig. 6.32. The sequence again begins with a polyketide chain extension of
4-coumarylCoA with three units of malonyl-CoA. Note that the resulting polyketide
is the same one employed in the biosynthesis of resveratrol (see Fig. 5.55), but in-
stead of a folding pattern leading to cyclization via a Knoevenagel-type conden-
sation, the chalcone synthase-mediated folding pattern leads to cyclization via a
Claisen-type condensation, affording apigenin chalcone.
The chalcone then undergoes an intramolecular conjugate addition to the
α,β-unsaturated ketone function by the adjacent phenolic hydroxyl group, lead-
ing to a flavanone derivative known as naringenin. Conversion of this flava-
none to the flavone apigenin is accomplished via an O2/2-oxoglutarate-type
C–H to C–OH oxidation. The intermediate β-hydroxyketone thus formed then
undergoes a dehydration to afford the α,β-unsaturated derivative, apigenin, a
flavonoid found in many fruits and vegetables including celery and parsley. Like
many other flavanones and flavanols, apigenin has been shown to be a potent
inhibitor of CYP2C9, a P450 enzyme involved in oxidative metabolism of var-
ious pharmaceutical and medicinal agents in the body. It is also one of only
a few compounds known to activate proteins responsible for the transport of

OH OH

O O
3 x malonyl-CoA
CoAS
O 4-coumarylCoA O SCoA O
Claisen
condensation HSCoA
OH OH
HO OH enolize x 3 O O
OH O apigenin chalcone O O
:B
OH OH
H
H
HO O conj
. HO O
addition
then
enol-to-
ketone
OH O OH O naringenin
HB
O2 2-oxoglutarate
OH
OH
BH
8 OH
HO O
HO O
7
6 :B
H2O
H
OH O apigenin
OH O
FIGURE 6.32
Formation of apigenin chalcone, naringenin, and apigenin.
monoamine neurotransmitters such as serotonin, dopamine, and norepineph-

rine. Structurally, apigenin also serves as the aglycone component of a number
of known O-glycosidic derivatives such as apigetrin (apigenin 7-O-glucoside),
and two unusual C-glucoside examples, vitexin (apigenin 8-C-glucoside) and
isovitexin (apigenin 6-C-glucoside; see Fig. 6.32 for apigenin ring position
numbering).
THE FLAVANONE-T O-D IHYDROFLAVONOL-

TO-A NTHOCYANIN SEQUENCE: FORMATION
OF PELARGONIDIN
Many flavonoids serve as color-producing components in various flowers and

fruits. An example is the variety of shades of yellow or orange that can result

OH OH

H H
HO O HO O
O2
2-oxo- OH
glutarate
OH O OH O
naringenin dihydrokaempferol
HB NADPH
OH
OH
OH
HO O H
HO O
O2
H :B
OH 2-oxo-
glutarate OH
OH OH
OH OH
HB
leucopelargonidin
2 x H2 O
5'
4' OH OH
HO O 3' HO O
1 2 UDP-glucose
3 OH OGlu
OH OH
pelargonidin pelargonidin-3-glucoside
FIGURE 6.33
Formation of pelargonidin (an anthocyanidin) and its 3-glucoside (an anthocyanin).
from variations in substitution patterns present in a number of different flavones.

Perhaps the most striking color-producing flavonoids are the anthocyanidins, an
oxonium ion-containing group of flavonoids that come in virtually every color but
green. The biosynthesis of pelargonidin, a salmon-pink anthocyanidin, is outlined
in Fig. 6.33.
The sequence begins with a 2-oxoglutarate-dependent C–H to C–OH oxidation
of the saturated ring of naringenin to give dihydrokaempferol whose ketone func-
tion undergoes NADPH reduction to afford leucopelargonidin. This compound
also undergoes a 2-oxoglutarate-dependent C–H to C–OH oxidation which is
followed by sequential dehydrations to yield the anthocyanidin derivative pelar-
gonidin. Glucosylation at the 3-position hydroxyl group gives the corresponding
glucoside. Such sugar-substituted anthocyanidins are known as anthocyanins. Both
groups of compounds are largely responsible for many of the vibrant shades of red,
violet, and blue found in flowers and fruits, with the anthocyanins being especially
important in the coloration of autumn leaves. Interestingly, flower colors have been
successfully manipulated commercially by altering some of the genes and enzymes
involved in flavonoid biosynthesis.

THE FLAVANONE-T O-I SOFLAVANONE-T O-I SOFLAVONE

Bioorganic Synthesis 288 SEQUENCE: FORMATION OF GENISTEIN
Isoflavonoids are essentially regular flavonoids which have undergone a structural

rearrangement in which the 4-coumarylCoA-derived phenol ring of a flavanone
has undergone an oxidatively driven 1,2-aryl migration to yield an isoflavanone.
Subsequent dehydration of the isoflavanone yields the unsaturated isoflavone.
A typical sequence, illustrated in Fig. 6.34, begins with naringenin (a flavanone)
undergoing a typical C–
H to C–
OH oxidation via O2/
NADPH at the ketone
α−position. This leads to a radical intermediate that, prior to capture of hydroxyl
oxygen, undergoes a 1,2-aryl shift via a bridged radical intermediate. The result-
ing rearranged radical is then quenched as usual to give the hydroxylated product
2,4’,5,7-tetrahydroxyisoflavanone. A final dehydration step gives the isoflavone ge-
nistein. An analogous sequence starting with the flavanone liquiritigenin leads to
formation of the isoflavone daidzein.
OH
OH
HO O O2 HO O
NADPH
OH O naringenin
OH O
HO O
1,2-aryl HO O OH
migration
OH O
OH
OH O
8 1
HO O 2 OH
HO O
7
6'
6
4 3 1' 5'
5
OH O 4'
2' H2O OH O
OH
3' OH
2,4',5,7-tetrahydroxyisoflavanone genistein
OH HO O
HO O
O
OH
O liquiritigenin daidzein
FIGURE 6.34
Isoflavonoid biosynthesis via 1,2-aryl migration; formation of genistein and daidzein.

ISOFLAVANOID STRUCTURAL MODIFICATIONS:

PRODUCTION OF ANTIMICROBIAL PHYTOALEXINS
The isoflavonoids may be further metabolized to yield a remarkable array of in-

teresting structures, many of which have significant biological activity. A series
of some typical transformations is illustrated in Fig. 6.35, beginning with
2,4’,7-trihydroxyisoflavanone. While dehydration leads directly to daidzein,
SAM methylation of the 4’-
hydroxyl group followed by dehydration yields
the isoflavone formononetin. Aryl ring hydroxylation of formononetin yields
2’-hydroxyformononetin which then undergoes both conjugate reduction and
ketone-to-alcohol reduction by NADPH to give a triol intermediate which cyclizes
with loss of water to yield medicarpin, a member of a large class of such tetracyclic
isoflavonoids known as pterocarpans. The pterocarpans are examples of phytoalex-
ins, antimicrobial phenols, or polyphenols that are produced by plants at the site of
an attack by pathogens. Their antifungal and antibiotic properties have made them
obvious targets for extensive pharmaceutical research over the years.
The parallel sequence shown in Fig. 6.35 involves the oxidative hydroxylation
and conjugate reduction of daidzein to yield 2’-hydroxydihydrodaidzein. This
compound forms a cyclic hemiacetal derivative which readily undergoes dehy-
dration to yield 3,9-dihydroxypterocarpene, which is effectively an unsaturated
pterocarpan. Further oxidation of the cyclic ether linkage of the pterocarpene
(mechanism as yet undetermined) yields 3,9-dihydroxycoumestan, more com-
monly known as coumestrol, found mainly in red clover, alfalfa, soybean, and
kudzu leaf. Its structural similarity to estradiol is obvious and like other phytoes-
trogens, it has a binding affinity for the estrogen receptor (higher than that of
genistein). Its mode of action is related to the inhibition of certain enzymes in-
volved in steroidal hormone biosynthesis, leading to modulation of their produc-
tion. In model studies, coumestrol has shown some potential for the reduction of
osteoporosis-related bone loss.
A recurring theme in isoflavonoid biosynthesis is the formation of products
which have undergone alkylation with one or more prenyl groups from the terpe-
noid biosynthetic pathway (Chapter 4). These groups may then undergo further
modifications via oxidations and/or cyclizations to yield a remarkable array of
higher molecular weight derivatives, some of which show promise as therapeutic
agents. A sampling of such compounds is shown in Fig. 6.36.
An interesting geranyl derivative of daidzein is corylifol A, a compound found in
the seeds of Cullen corylifolium. Glabroisoflavanone A, a minor constituent found
in extracts used in the production of licorice, is just one of many examples of prenyl-
substituted isoflavanones in which a prenyl side chain has undergone subsequent
cyclization to yield a pyran-type ring. Eryvarin S, a prenyl-substituted isoflavone
and erypoegin H, a prenylated pterocarpene, have both exhibited activity against
multiple methicillin-resistant Staphylococcus aureus (MRSA) strains; erypoegin H

HO O OH HO O
O H2O O
OH daidzein OH
2,4',7-trihydroxyisoflavanone
O2 , then NADPH
SAM H 2O NADPH conj. reduction
HO O HO O
H
O O
OCH3 HO OH
formononetin
2'-hydroxydihydrodaidzein
O2 NADPH
HO O
O H
HO
O OH
HO OCH3 HO O
2'-hydroxyformononetin
NADPH then NADPH
(conj. reduction) (C=O to CHOH) H 2O
HO O O
H
HO
O OH
H
OH 3,9-dihydroxypterocarpene
HO OCH3
O2
H2O
O
O H O
HO HO
H O OCH3 OH
O
medicarpin coumestrol
FIGURE 6.35
Transformation of formononetin and daidzein into medicarpin and coumestrol.
has also shown promising activity against vancomycin-resistant enterococci (VRE)

strains. Many examples of prenylated pterocarpans have also been isolated; rautan-
diol B shows how such prenylation may be followed by oxidative cyclization lead-
ing to formation of furan-type ring systems. Like coumestrol, hedysarimcoumestan
D is an example of a phenolic coumestan, but with additional prenyl substitution.
Mild anthelmintic activity (activity against parasitic worms) has been demonstrated


HO O
O O
OH
O
OH O
corylifol A OH
glabroisoflavanone A
HO O HO
O OH
erypoegin H
O
eryvarin S OH
O
O
O H
HO
O O OCH3
H O OH
OH
HO rautandiol B hedysarimcoumestan D
H3CO O
OH
O O
O
H 3C
OH
OH O
OH
triquetrumone A 7-O-methylmanuifolin K
OH
FIGURE 6.36
Some examples of structural variety in alkylated isoflavonoids.
by triquetrumone A, a member of the coumaronochromone group of isoflavo-

noids which shows how both cyclized and open prenyl chains may be incorporated
within a single structure. Finally, the less common incorporation of a tertiary prenyl
group is shown in the structure of 7-O-methylmanuifolin K, an isoflavan that has
been shown to be comparable to amphotericin B in its activity against Naegleria
fowleri, an amoeba responsible for a rare but often fatal disease, primary amoebic
meningoencephalitis.
The oxidative cyclizations employed in conjunction with prenyl side chain in-
corporation usually involve epoxidation followed by nucleophilic ring opening and
subsequent 1,2-elimination of water. A reasonable mechanism summary leading
to dihydrobenzofuran, benzopyran, and benzofuran ring systems is shown in
Fig. 6.37. Note that the mechanism leading to the benzofuran nucleus also involves
a benzylic oxidation followed by loss of acetone and water.


O2
NADPH O
HO HO
O vs. O
HO HO
HO
O HO O O
H2O O H2O
dihydrobenzofuran benzopyran
O2 NADPH
HO
O O O O
+ H2O benzofuran
H
FIGURE 6.37
Prenyl side chain cyclization and modification mechanisms.
ROTENOIDS: FISH POISONS FROM ISOFLAVONES
The rotenoids are a class of plant compounds derived from isoflavones that play a
role in plant defense against insect attack. Six plant-derived rotenoids are known
to occur naturally in Derris eliptica (Southeast Asia) and either Lonchocarpus utilis
or L. urucu (South America) and all are similar in chemical structure. The most
common member of the family is rotenone which has been used for centuries by
native populations as a potent fish poison. Its biosynthesis from formononetin is
shown in Fig. 6.38.
After two sequential aryl ring hydroxylations and SAM methylations, an
additional oxidation takes place at a methoxyl methyl group. One proposed
sequence for subsequent cyclization of the resulting radical proceeds via its addi-
tion to the adjacent α,β-unsaturated enone and subsequent radical quenching by
a H-atom donor to yield the ketopyran ring as shown (other cyclization mecha-
nisms have also been proposed). The remaining steps involve ortho-alkylation
of the phenolic ring by DMAPP to yield rotenoic acid. This is followed by a final
oxidative cyclization of the prenyl side chain to give the 5-membered ring of ro-
tenone. Another rotenoid known as duegelin differs from rotenone only in that
the final prenyl side chain cyclization step leads to a 6-membered pyran rather
than a 5-membered furan ring system. The cyclization mechanisms involved
have not been fully determined but appear to be more complex than those in-
volved in formation of similar ring systems we have seen in other isoflavonoid
derivatives.

HO O HO O

O2, NADPH
then SAM
O O
OCH3 OCH3
formononetin O2, NADPH
OCH3
then SAM
HO O H2C HO O
O OCH3
O2
H-atom
O abstraction
O
OCH3 OCH3
OCH3 OCH3
H H
HO O HO O
O O
H-atom
donor
O H
OCH3 O
OCH3
OCH3 DMAPP OCH3
H H
O O oxidative HO O
O cyclization O
(mechanism
unknown)
H H
O O
OCH3 OCH3
rotenone OCH3 rotenoic acid OCH3
FIGURE 6.38
Biosynthesis of the fish poison rotenone from the isoflavone formononetin.
A dusty solid when isolated, rotenone is also useful as a biodegradable insecti-

cide that is effective for control of a variety of beetles and other common garden
pests. It is also used in veterinary medicine for treatment of mites, but because it
is irritating to the skin, eyes, and upper respiratory tract, it has seen only limited
use in human medicine as a topical treatment for head lice and other parasites. In
animal studies, rotenone has been shown to interfere with both the electron trans-
port chain and with tubulin self-assembly and can also promote Parkinson’s-like
symptoms.
LOOKING AHEAD
We have only just scratched the surface of the incredible diversity of organic com-
pounds produced by the shikimic acid pathway. Now that we are armed with some

of its basics and have a better understanding of how certain amino acids can be
Bioorganic Synthesis 294 transformed into remarkably complex organic structures by relatively simple or-
ganic reactions, we can move on to Chapter 7 which deals with another group of
compounds that utilize amino acids as building blocks and which are historically
the most important of all secondary metabolites in terms of their impact on human
health: the alkaloids.
STUDY PROBLEMS
1. Many 2-arylbenzofuran natural products such as the one shown here are potent
anti-inflammatory and cytotoxic agents. Using cinnamoyl-CoA (from the shi-
kimic acid pathway) as a starter unit, three malonyl-CoA units and any other
building blocks required, propose a reasonable biosynthesis of the indicated
compound.
O OH
SCoA CH3
O
OH
2. In the naturally occurring form of podophyllotoxin, the C6–C5 lactone ring junc-
tion is not the most thermodynamically stable form and is readily isomerized to
its more stable form. Suggest a structure and a mechanism for the isomerization.
OH
O
O
O
H3CO OCH3
OCH3
podophyllotoxin
3. The cyclization of yatein (Fig. 6.22) to deoxypodophyllotoxin involves a benzylic

carbocation intermediate. A quinone methide-type carbocation intermediate
has also been invoked for this process. Draw a likely structure for it. Would this
structure be more stable than the simple benzylic carbocation shown in Fig. 6.22?
Why or why not?

4. Bergaptol is an example of one of several furanocoumarins that occur naturally

in grapefruits and are responsible for the so-called “grapefruit juice effect” that
arises from their interference with certain CYP-type enzymes that assist in the
metabolism of various drugs including statins (such as Liptor and Mevacor) and
benzodiazepams (such as Valium and Xanax) among others. Outline a likely bio-
synthesis of bergaptol from umbelliferone, DMAPP and other co-factors.
OH
O O O
bergaptol
5. Warfarin (brand name Coumadin) is a coumarin derivative originally produced

and used as a rat poison in the 1940s. It is a synthetic analogue of dicouma-
rol (Fig. 6.27) and derives its name from the original source of funding for its
discovery (Wisconsin Alumni Research Foundation plus –arin from coumarin).
Warfarin is widely used in human medicine as a highly effective anticoagu-
lant for the prevention of thrombosis (blood clots). It can be synthesized from
2-hydroxyacetophenone, diethyl carbonate, benzaldehyde, and acetone as shown.

O
O O
O
CH3 B
1 OCH2CH3 2
+
H3CH2CO OCH2 CH3 CH3CH2O + CH3CH2OH
OH CH3CH2O
OH A
2-hydroxyacetophenone diethylcarbonate
O OH O
O
4
H + 3 B + C
H3C CH3 C
CH3CH2O
CH3CH2O
acetone
O O
benzaldehyde O
warfarin

Given that all the laboratory reactions can be carried out using a simple base such

as ethoxide ion, answer each of the following:
a) Provide a mechanism for the condensation involved in Reaction 1 to
produce A.
b) Determine the structure of B derived from A via Reaction 2.
c) Provide a mechanism for the condensation involved in Reaction 3 to
produce C.
d) Provide a likely mechanism for reaction of B with C to produce warfarin.
6. Gingerol (C27H26O4) is a spicy, pungent yellow oil isolated from fresh ginger.
O OH
HO
OCH3 gingerol
a) Propose a reasonable sequence for biosynthesis of gingerol from ferulic acid

CoA and 3-ketooctanoic acid CoA (among the required reactions are a Claisen
condensation and NADPH reductions). Think carefully about sequence.
b) The cooking process can transform gingerol into zingerone (C11H14O3).
The reaction involved is a retroaldol addition. What is the likely structure of
zingerone?
c) Shogaol (C27H24O3) is structurally related to gingerol but much spicier. It is
formed when ginger is heated and dried. What is a likely structure for shogaol?
7. Propose a reasonable sequence for biosynthesis of liquiritigenin. What is its
configuration as shown?
OH
HO O
O liquiritigenin
8. Silibin A is a so-called flavonolignan compound that has been shown to help

protect liver cells from certain toxins and has also been shown to possess antican-
cer activity against breast, prostate, colon, and lung cancer cells. Its structure is
a hybrid of the dihydroflavonol taxifolin and the lignol coniferyl alcohol which
have been joined via an oxidative coupling process similar to that used in the
formation of lignans. Propose a mechanism for biosynthesis of silibinin A.

OH

OH
OCH3
O
OH HO O OCH3
coniferyl alcohol O
+ OH OH
OH
OH O silibin A
HO O
OH
OH
OH O taxifolin
9. Isolation of rautandiol B (Fig. 6.36) also yields a constitutional isomer, rautan-

diol A. Both are pterocarpans formed in a single step from the same immediate
precursor and both have a phenolic hydroxyl group. The remaining hydroxyl
group of rautandiol B is a tertiary alkanol, but for rautandiol A, it is a second-
ary cycloalkanol which can exist as two stereoisomers. Propose likely structures
for the rautandiol A stereoisomers and a sequence for their biosynthesis from
vestitone.
HO O
rautandiol A
O
vestitone HO OH
10. Cyanidin can undergo an NADPH reduction to afford epicatechin.
OH OH
HO O HO O
OH
OH OH
OH OH
cyanidin epicatechin
a) Show a reasonable mechanism for this hydride reduction.

b) Show a plausible mechanism for the reaction of cyanidin with epicatechin to
give the dimeric structure shown.

OH

HO O
OH
OH OH
OH
HO O
OH
OH
11. Eugenol and isoeugenol are involved in the biosynthesis of a number of interest-
ing lignan compounds. One unusual example is guaianin, shown here. Show
how the two isoeugenol units could be joined together via oxidative coupling,
then propose a structure for compound A that is a likely transient intermediate
in the biosynthesis and finally show how A can then be further transformed into
guaianin. Note that the alcohol function of guaianin is derived from the H2O
incorporated into A.
H3CO
2
HO
isoeugenol
OCH3 H O OCH3
2 HO H
H3CO A
O
O O
O O guaianin

7 Biosynthesis
of Alkaloids and Related
Compounds
I could imagine his [Sherlock Holmes] giving a friend a little pinch of the latest vegetable al-
kaloid, not out of malevolence, you understand, but simply out of a spirit of inquiry in order
to have an accurate idea of the effects. To do him justice, I think that he would take it himself
with the same readiness. He appears to have a passion for definite and exact knowledge …
of botany—well up in bella-donna, opium, and poisons generally; of chemistry—profound
—Sir Arthur Conan Doyle (A Study in Scarlet, 1887)
Though definitions may vary from source to source, the term alkaloid generally
refers to members of a large set of naturally occurring, slightly basic (i.e., alkaline)
nitrogen-containing organic compounds. Generally excluded from this group are
amino acids, peptides, proteins, N-containing carbohydrates, and nitrogenous bases
used in the construction of nucleotides. Though a small number are produced by
animals or microorganisms, the vast majority of alkaloids are plant-produced com-
pounds possessing a remarkably diverse range of structural features, from simple
cycloaliphatic amines to highly complex polycyclic N-heterocycles. Some represen-
tative alkaloids are shown in Fig. 7.1.
Alkaloid-containing plants and their extracts have been used by humans for
thousands of years, mainly on the basis of their stimulant, therapeutic, or poisonous
properties. References to plants containing compounds such as morphine (from
opium poppies), strychnine (from seeds of the Strychnos nux-vomica tree), ephed-
rine (from the plant Ephedra chinensis), and coniine (from the poison hemlock
plant) may be found in some of our earliest known writings. Today, it has been esti-
mated that the health care of over 5 billion people worldwide benefits from the use of
300

301 Biosynthesis of Alkaloids and Related Compounds

OCH3
N
H3C O N
CH3 N
cocaine O nicotine H coniine
N
(coca leaf (tobacco (hemlock
narcotic) toxin) neurotoxin)
HO
H3CO
NHCOCH3
NH2
HO O H3CO
OH dopamine NCH3 H3CO
(neurotransmitter) morphine colchicine O
HO (poppy-derived (anti gout
analgesic) drug) OCH3
N
OH
N N HO N
H
H
H3CO2C
H3CO
H3CO OCOCH3
vinblastine N quinine
(anti cancer CH3 OH N (anti malarial
drug) H3CO2C drug)
FIGURE 7.1
Some representative structures of alkaloids with significant biological activities.
plant-based medicinal agents, many of which are alkaloids. With that in mind, it is
worth noting concerns that deforestation, environmental damage, large-scale devel-
opment, and unregulated harvesting programs may ultimately lead to the extinction
of hundreds of known medicinal plants and perhaps even more whose medicinal
properties have yet to be discovered, thereby endangering the prospects for future
discoveries of new curative agents for the benefit of all humankind.
As a scientific field, alkaloid chemistry itself dates back to the early 1800s
with the first isolation of pure crystalline morphine from opium. This milestone
achievement allowed the delivery of accurate, therapeutic doses of a drug that
was immensely valuable for the relief of pain but which could also lead to fatal
overdoses when administered from simple extracts of variable composition and
strength. The subsequent rapid development of increasingly sophisticated tech-
niques for the isolation and purification of the active components (often alkaloids)
from many other medicinal plants essentially spawned the field of organic chemis-
try. By 1886, less than 70 years after the isolation of morphine, laboratory synthesis
of the alkaloid coniine had been accomplished; further triumphs in the synthesis
of increasingly complex natural products quickly followed, ultimately launching
the fields of synthetic organic and medicinal chemistry. By the end of the first
decade of our own century, well over 12,000 different alkaloid structures had
been identified. Such a staggering number obviously requires us to limit ourselves

here to an abbreviated introductory approach only; readers are therefore strongly

Bioorganic Synthesis 302 encouraged to consult some of the outstanding general reference works listed at
the end of the book for a more detailed and thorough treatment of this vast and
challenging topic.
ALKALOID STRUCTURE: THE IMPORTANCE

OF N-H ETEROCYCLES
Alkaloids are generally derived from modification of various amino acids, though
not always the more familiar amino acids used in peptide and protein formation.
Among many others, shikimate-derived amino acids are important precursors in
the construction of alkaloids, with terpenoid or polyketide components also in-
corporated in many instances. Thus, the alkaloids themselves are not necessarily
thought of as being derived from a distinct biosynthetic pathway of their own, but
rather from a set of bioorganic transformations operating on amino acid build-
ing blocks and other components provided by each of the three main biosynthetic
pathways we have already encountered. Because alkaloid structures are quite vari-
able and not readily classified, it is often convenient to group them according to the
amino acid precursor from which they were originally derived. As noted earlier,
most transformations of amino acid precursors ultimately lead to alkaloid structures
containing one or more N-heterocyclic rings. With these points in mind, Table 7.1
summarizes the most important amino acids involved in alkaloid biosynthesis
along with the most commonly encountered N-heterocyclic ring systems that each
of them produces.
The above listing is by no means exhaustive, but it does present a reasonable
overview of some of the main sources of alkaloid structure. Amination reactions
of acetate-and terpene-derived precursors as well as purine-derived alkaloids and
selected β-lactam antibiotics will also be treated briefly.
ALKALOIDS NOT DERIVED FROM AMINO ACIDS:

AMINATION REACTIONS, POISONS, AND VENOMS
Before we delve into the chemistry of alkaloids that make extensive use of amino
acids, we begin our examination with some examples of simple alkaloids derived
from the acetate pathway, with nitrogen being provided by transamination. One
such alkaloid is the neurotoxin coniine, which we encountered previously (Fig. 7.1).
The biosynthesis of this structurally simple 2-alkylpiperidine from octanoic acid is
outlined in Fig. 7.2. The process begins with oxidation of octanoic acid to give a
ketone at position 5; this is followed by the usual sequence for thioesterification
and reduction of carboxylic acid functions to give 5-oxo-octanal. Alanine trans-
amination of the aldehyde function then yields 5-oxo-octylamine which undergoes
intramolecular imine formation to give γ-coniceine. Finally, NADPH reduction of

Table 7.1 Heterocyclic Alkaloid Ring Systems from Amino Acid Precursors

Amino Acid Heterocyclic Alkaloid Ring Systems
N
CO2H
H2N
N N
ornithine NH2
pyrrolidines pyrrolizidines tropanes
H2N CO2H
N N N
lysine NH2
piperidines quinolizidines indolizidines
CO2H
NH
NH2 N NH
X
benzyl
X = OH, tyrosine isoquinolines tetrahydro- tetrahydro-
X = H, phenylalanine isoquinolines isoquinolines
CO2H
N
NH2
NH
N N N N
H H H H
tryptophan indoles pyrroloindoles β-carbolines
CO2H
N
NH2 N N N
anthranilic acid quinolines quinazolines acridines
CO2H H CO2H H
N N
or or
NH2
N N N N
nicotinic acid histidine pyridines imidazoles
trans-
oxidation reduction amination
H H2N O
HO O O O CO2H CO2H
octanoic acid 5-oxo-octanal 5-oxo-octylamine
NH2 O
(caproic acid)
imine
H NADPH formation
N N
H
coniine γ-coniceine H2O
FIGURE 7.2
Biosynthesis of the neurotoxin alkaloid coniine.
the imine linkage affords coniine as shown. Note also that SAM methylation of coni-
ine affords the related compound N-methylconiine (not shown) which is also often
present, though in much smaller amounts.
Most notable as the poisonous hemlock alkaloid used in the death of Socrates in
399 B.C., coniine is extremely toxic to animals with a fatal dosage of less than 200 mg
for humans. Notorious for causing death by slow suffocation, coniine consumption

Bioorganic Synthesis 304 N N

H n H n
n = 4, cis-solenopsin A n = 4, trans-solenopsin A
n = 5, cis-solenopsin B n = 5, trans-solenopsin B
n = 6, cis-solenopsin C n = 6, trans-solenopsin C
O
O
8x
O O reductions,
malonyl-CoA
SCoA dehydrations,
O O reductions, O O
4 4
HO2C decarboxylation
trans-
amination
reduction imine
formation
N N
H 4 4 NH2O 4
H2O
cis-solenopsin A
FIGURE 7.3
Proposed biosynthetic scheme for cis-solenopsin A, an alkaloid component of fire ant venom.
induces a gradual sequence of drowsiness, tingling, or burning sensations of the

skin, lack of muscle control, nausea, excessive salivation, and rapid pulse eventually
followed by fatal respiratory paralysis. Salts of coniine have reportedly shown some
local analgesic action in the symptomatic relief of intense itching, but the toxicity of
coniine generally makes its use for such purposes far too dangerous.
Polyketide-derived precursors can also play a role in alkaloid biosynthesis. An
interesting example is a group of 2-methyl-6-alkyl-substituted piperidines known as
the solenopsins. These alkaloids are unusual in that they are among the very few
insect-produced alkaloids known to us. As the toxic defense components of fire ant
venom, these compounds are ultimately responsible for the extremely painful welts
produced by fire ant stings. While the details of their biosynthesis are not fully known,
careful studies have confirmed their polyketide origin and that they are produced by
the insects themselves rather than being present due to ingestion from a plant source.
A reasonable biosynthetic sequence similar to that of coniine is outlined in Fig. 7.3.
In addition to painful welts, allergic reactions to the solenopsins in fire ant stings can
lead to anaphylactic shock if not properly treated. Nevertheless, these compounds have
been investigated extensively for their surprising ability to inhibit formation of new
blood vessels (angiogenesis). The solenopsins have also been found to have insecticidal,
antibacterial, and antifungal properties and also show promise as inhibitors of nitric
oxide production, comparing favorably in potency with other widely used inhibitors.
AMINO ACIDS AND MANNICH REACTIONS: IMPORTANT

KEYS TO ALKALOID BIOSYNTHESIS
A great deal of postassembly modification of alkaloids occurs via oxidative processes

and these can vary a great deal depending on class and plant source. Nevertheless,

there are some fundamental transformations employed in the initial assembly of al-

kaloid structures that are used over and over again in various biosynthetic contexts.
Several of these reactions are outlined in general terms in Fig. 7.4. In order to utilize
amino acids as building blocks for alkaloid biosynthesis, they are usually first con-
verted either into amines via PLP-mediated decarboxylation, or into α-ketoacids
via PLP-mediated transamination. Transamination is usually then followed by TPP
ylide-mediated decarboxylation to produce the corresponding aldehydes. With
primary amines and aldehydes thus readily available, a simple condensation reac-
tion leads to the formation of imines which play a central role in the construction
of many higher-molecular weight amines from simpler building blocks. Beyond
this simple beginning, we then find one of the most important C–C bond forming
reactions in alkaloid biosynthesis—the so-called Mannich reaction. The classic
laboratory version of this reaction makes use of three components: 1) an amine or
ammonia; 2) a nonenolizable aldehyde or ketone; and 3) an enolizable aldehyde or
ketone. The first two components condense to form an imine, while the third com-
ponent serves as a carbon nucleophile, usually as an enol or enolate ion, which at-
tacks the electrophilic imine linkage, thereby forming a carbon–carbon bond and
the resulting amine product as shown in Fig. 7.4.
In alkaloid biosynthesis, the first two components—an amine and an aldehyde
derived via the decarboxylation and transamination chemistry shown in Fig. 7.4—
condense to form the corresponding imine required for a biological Mannich reac-
tion. The nucleophilic component (represented here by a generic nucleophile, Nu:–)
may be a β-dicarbonyl enol, a thioester enolate from acetyl CoA or similar sources,
or the nucleophilic enol of a phenol ring. The combination of such components
leads to a Mannich-derived alkaloid amine. As we will see, this combination of com-
ponents can be used in both inter-and intramolecular versions of the biological
R CO2H R
PLP
NH2 NH2 R
amino acid CO2 primary amine
condensation
N
R' CO2H PLP R' CO2H TPP ylide R' H H2O R' H
imine
NH2 trans-
O O
amination CO2
amino acid α-ketoacid aldehyde H+
R' H R R
Nu:
Nu N R R' N R' N
Mannich H
Nu: H
H reaction
H
Mannich product H
protonated
imine
FIGURE 7.4
Mannich reaction via amino acid transformations, imine formation and nucleophilic addition.

Mannich process, resulting in formation of alkaloid products with a variety of

Bioorganic Synthesis 306 unique structural features.
ALKALOIDS FROM ORNITHINE: TROPANES VIA

THE MANNICH REACTION IN ACTION
In biochemistry, the urea cycle (sometimes also referred to as the ornithine cycle) is
a metabolic pathway that allows for the excretion of excess nitrogen via production
of urea. One of the reactions of this pathway is the arginase-mediated production of
urea from the peptidic amino acid L-arginine. Extrusion of urea from its structure
converts arginine into L-ornithine, a nonpeptidic amino acid which, in addition
to other important roles, serves as a precursor to a number of important alkaloids,
including those of the tropane family. Some of the reactions involved in biosynthesis
of tropinone and tropine are outlined in Fig. 7.5.
Initial PLP-mediated decarboxylation of ornithine gives the 1,4-diamine putres-
cine whose foul (putrid) odor is associated with the smell of decaying flesh. SAM
methylation leads to N-methylputrescine which is converted to the corresponding
aminoaldehyde by a diamine oxidase-mediated oxidative process. Intramolecular
imine cyclization leads to formation of an important 5-membered ring intermediate,
N-methyl-Δ1-pyrrolinium cation, which is the substrate for a subsequent Mannich
reaction using malonyl-CoA as the carbon nucleophile. Addition of a second mal-
onyl-CoA via Claisen condensation extends the side chain by two carbons to give a
diketide-substituted N-methylpyrrolidine derivative. Oxidation at the 2-position of
the pyrrolidine ring gives a hydroxyl-substituted derivative (shown here in a more
useful conformation) which then undergoes dehydration to afford a substituted
N-methylpyrrolinium ion. Hydrolysis of the thioester yields a β-ketoacid which
serves as the nucleophile for a second intramolecular Mannich reaction, forming
a bicyclic β-ketoacid which decarboxylates as expected to afford the key alkaloid
ketone known as tropinone. A final NADPH reduction of the ketone function af-
fords tropine as shown.
A number of synthetic alkaloid derivatives of tropine have been prepared and
marketed, mainly as anticholinergic drugs for the treatment of Parkinson-like
symptoms. Tropine also serves as the biosynthetic precursor to the natural tro-
pane alkaloids hyoscyamine (atropine) and hyoscine (scopolamine) as outlined in
Fig. 7.6. The hydroxyl group of tropine initially undergoes condensation with the
CoA thioester of phenyllactic acid. The resulting ester, littorine, then undergoes
an unusual oxidative rearrangement of the hydroxyl ester core to yield hyoscya-
mine. Subsequent sequential oxidations of the tropine ring of hyoscyamine yield
the epoxide function in the structure of hyoscine. Note the change from an axial to
an equatorial position for the N-methyl function in the hyoscyamine-to-hyoscine
transformation.

CO2H diamine
H2N PLP SAM H3C
H2N N oxidase
H
NH2 NH2 FAD, O2
NH2
ornithine putrescine N-methylputrescine H2O
CO2
O
imine
malonyl-CoA formation H3C O
N SCoA N
Mannich N
H
CH3 CH3 H2O H
NH3
N-methyl-∆1-
pyrrolinium cation
O O O
malonyl- O2 N
Co A H3C
N SCoA Claisen SCoA NADPH
N HO O
CH3 COSCoA
CH3
N N
N H2O H3C
H3C H3C
O O
OH
CO2H COSCoA H2O
CO2H HSCoA
N N N
H3C NADPH
Mannich H3C H3C
O O
CO2H CO2
tropinone tropine OH
FIGURE 7.5
Conversion of ornithine to tropine via N-methyl-Δ1-pyrrolinium cation and Mannich reactions.

Bioorganic Synthesis 308 N N

H3C + SCoA H3C
OH
OH
OH O
tropine (R)-phenyllactyl-CoA littorine
O
rearrangement
CH3
N O2 , N
OH oxoglutarate H3C OH
O
X2 O
O
O O
hyoscine hyoscyamine
FIGURE 7.6
Conversion of tropine to hyoscyamine (atropine) and hyoscine (scopolamine).
OH
tropine OH tropine
O2, P450
O Ph O
littorine 2-electron Ph
oxidation
O H
O2, 1-electron O
O OH
P450 oxidation tropine
NADPH tropine
O O
OH Ph Ph
tropine OH tropine OH tropine O O
O Ph O Ph O hyoscyamine hyoscyamine
Ph aldehyde
O O O H
FIGURE 7.7
Proposed mechanisms for the oxidative rearrangement of littorine to hyoscyamine.
The conversion of littorine to hyoscyamine, as shown in Fig. 7.7, is an interest-

ing rearrangement which has been studied extensively. The process has been shown
to occur via a P450-type benzylic oxidation that initially leads to formation of hyo-
scyamine aldehyde as an intermediate. The aldehyde is subsequently reduced to the
corresponding alcohol via the usual NADPH reduction process. Some evidence sug-
gests that hydroxylation at the benzylic position of littorine may occur prior to the
rearrangement, but the precise mechanistic details of the oxidative process remain
to be determined. Given that examples of P450 oxidations involving two-electron as
well as one-electron oxidative processes are now known, reasonable mechanisms for
this rearrangement have been proposed that involve either a carbocation or a radi-
cal as a likely intermediate. The carbocation process would involve a 1,2-shift and
proton loss similar to that seen in pinacol–pinacolone-type rearrangements, while
the radical process would require formation of a transient cyclopropane intermedi-
ate which would fragment to a rearranged radical and loss of a H atom. Both pro-
cesses would lead to the same aldehyde intermediate as shown in Fig. 7.7.
Hyoscyamine and hyoscine are sometimes referred to as the “nightshade alka-
loids,” owing to deadly nightshade (Atropa belladonna) as a primary plant source,
though these compounds may also be found in mandrake, jimson weed, and

henbane among others. They have a long history in folk medicine, beginning with

the use of mandrake extracts for anesthesia by the early Greeks. More notorious
were the potent hallucinatory effects which these alkaloids provided when used in
the so-called “flying ointments” allegedly employed in witchcraft practices of the
Middle Ages. Interestingly, the term belladonna (beautiful woman) was originally
associated with the pupil dilating effects of atropine (the racemic form of hyoscya-
mine) and its use by women during the Renaissance to achieve a striking cosmetic
look for their eyes. In modern ophthalmology, use of atropine for pupil dilation has
diminished in favor of compounds whose effects are of shorter duration. Atropine is
still widely used as a preanesthetic medication to reduce salivary secretions during
surgeries, as an antidote for certain types of poisonings, and for the treatment of
colon spasms. Hyoscine, also known as scopolamine, is sometimes used for the
treatment of severe nausea and motion sickness, usually via a transdermal patch.
From 1915–1960, a mixture of morphine and hyoscine was commonly used in hos-
pital anesthesia to block the pains associated with childbirth. Popularly known as
“twilight sleep,” administration of this mixture gradually fell into disfavor as “natural
childbirth” methods gained wider acceptance as a means of reducing the exposure of
both mother and infant to the systemic effects of such anesthetic agents.
Certainly the most infamous of the tropane alkaloids is cocaine, an addictive
CNS-stimulant narcotic derived from leaves of the South American coca plant. Its
biosynthesis, outlined in Fig. 7.8, again involves the ornithine-derived N-methyl-
Δ1-pyrrolinium cation and its elaboration via sequential malonyl-CoA additions,
O O
malonyl-CoA
N N SCoA
Mannich N SCoA
CH3 CH3
CH3
N-methyl-∆1-
pyrrolinium cation
COSCoA O O malonyl-
OH O2 CoA
N
H3C NADPH N SCoA Claisen
H2O O CH3
COSCoA
COSCoA CO2H
N Mannich H2O
H3C N N
H3C H3C
OH HSCoA
O O
CO2CH3 COSCoA
CO2CH3 CO2CH3
N NADPH SAM
H 3C O O N
N H3C
H3C OH
HSCoA O
cocaine methyl ecgonine
FIGURE 7.8
Conversion of N-methyl-Δ1-pyrrolinium cation to the coca leaf alkaloid cocaine.

but yielding the diketide of opposite configuration at the pyrrolidine ring side chain
Bioorganic Synthesis 310 relative to the diketide formed in tropine biosynthesis (Fig. 7.5). Though oxidation
and dehydration to a substituted pyrrolinium ion proceeds as before, the subsequent
Mannich cyclization proceeds via the CoA ester rather than via the free acid, and
subsequent hydrolysis to the β-ketoacid is immediately followed by SAM methyla-
tion rather than decarboxylation to give a methyl ester. NADPH reduction of the
ketone function yields the equatorial alcohol derivative known as methyl ecgonine.
A final esterification of the alcohol with benzoylCoA gives the corresponding ben-
zoate ester, cocaine.
Coca leaves have been chewed by South American natives for hundreds of years
primarily for stimulation, relief of hunger, and for use in various religious ceremo-
nies. Cocaine itself was not isolated until 1860 but in little more than twenty year’s
time it found clinical use in ophthalmic surgery as the first truly modern local an-
esthetic. The subsequently recognized toxic and addictive effects of cocaine and its
derivatives ultimately led to strict limitation of its use, though it still finds applica-
tion in ophthalmic procedures and in surgeries involving the ear, nose, and throat.
As an alkaloid narcotic of abuse, cocaine is rivalled only by heroin, with as many as
14% of U.S. adults having reportedly used the drug at least once. As an amine salt,
such as the hydrochloride, cocaine is fairly water soluble and it is in this form that it
is “snorted” or dissolved in water and injected. So-called “crack” cocaine is actually
the corresponding “free base” of the amine rather than a salt and it is in this form
that cocaine is “smoked” by heating and inhaling its vapors. The effects of cocaine
are many and varied, ranging from an increased sense of energy, general euphoria,
and feelings ranging from superiority to hyperactivity, pupil dilation, rapid speech,
and elevated pulse among others. Tolerance, intense cravings, and addiction usually
follow extended use and withdrawal is usually multisymptomatic and exceedingly
disagreeable.
PYRROLIZIDINE ALKALOIDS: POISON
PLANTS AND INSECT DEFENSE
Alkaloids of the pyrrolizidine group are also derived from putrescine, but unlike
the tropanes, are surprisingly common antiherbivore defensive compounds found
in over 6000 plants worldwide, which are regarded as the most common poison-
ous plants known to affect wildlife, livestock, and humans. Of the approximately
660 known compounds in this group, some show carcinogenic or mutagenic prop-
erties, but roughly half of them are readily metabolized to compounds which are
significantly toxic to the liver, representing a potentially important health problem
for humans using certain medicinal herbs such as borage leaf, comfrey, or coltsfoot
which are known to contain small quantities of pyrrolizidines. The biosynthesis of
retronecine, the core compound from which many other pyrrolizidines are formed,
is shown in Fig. 7.9.

H2N
HN 3 NH2
H2N H2N
NAD+ N NH2 NADH HN 4 NH2 FAD,O2
spermidine 4
H2O
H2N + H2N diamine
NH2
NH2 homospermidine oxidase
putrescine
HO O
H
imine
FAD,O2 formation O HN
Mannich N 4 NH2 4 NH2
N H2O H2O
N
H+
O
H OH HO OH
H H H
NADPH oxidation
N N dehydrogenation N
trachelanthamidine retronecine
HO OH HO
HO HO O
O O O O
CH3
O O O O
O O O
O O O H
H H N
N danaidone
N N N
CH3
monocrotaline senecionine doronenine senkirkine
FIGURE 7.9
Biosynthesis of retronecine and some of its pyrrolizidine alkaloid derivatives.

Again, intramolecular imine formation and the Mannich reaction figure promi-
Bioorganic Synthesis 312 nently here as ring-forming processes. Though other pyrrolizidine structures are
known, many are diester derivatives of retronecine and related structures generally
referred to as necines. The various carboxylic acids with which necines condense
to form these esters are known as necic acids. Compounds such as senecionine
and doronenine provide representative examples of these toxic diesters, while
the carcinogenic senkirkine serves an example of a pyrrolizidine with cleavage of
the usual bicyclic ring junction. Aside from their general toxicity, some pyrroli-
zidines are also known to play an ecological role as antifeedants in insects such as
the larvae of the ornate moth which feeds on plants containing monocrotaline,
secreting the stored compound as a toxic defense against predators. Other pyrroli-
zidines are consumed and converted biochemically by certain male butterflies into
derivatives such as danaidone which acts as a female “flight arrestant” for mating
purposes.
PIPERIDINE-T YPE ALKALOIDS DERIVED FROM LYSINE
We saw earlier how the piperidine alkaloid coniine was derived via an amination
reaction of 5-oxo-octanoic acid. Most other piperidine alkaloids are actually de-
rived from the amino acid lysine via a PLP-mediated decarboxylation, oxidation,
and imine formation sequence involving a 6-membered rather than a 5-membered
cyclic iminium ion intermediate, as illustrated in Fig. 7.10 for the biosynthesis of the
piperidine alkaloids pelletierine and pseudopelletierine.
Decarboxylation of lysine affords the 1,5-diamine known as cadaverine whose
foul smell, like that of putrescine, is associated with decaying flesh. A diamine
oxidase-mediated oxidation of cadaverine affords the corresponding aminoaldehyde
which then undergoes intramolecular imine formation and protonation to yield Δ1-
piperidinium cation, the key 6-membered ring intermediate. The remaining steps
are analogous to those shown earlier (Fig. 7.5) for the formation of tropinone and its
precursors. Pelletierine is a mild poison that is active against intestinal tapeworms;
pseudopelletierine, a homologue of tropinone, is usually isolated along with pel-
letierine and although a mixture is often administered for tapeworm treatment, the
antiparasitic activity resides only in pelletierine.
Some significant medicinal compounds contain piperidine-type alkaloids which
have been used as building block components of larger structures. A good example
of this is the incorporation of L-pipecolic acid in the structure of the polyketide
macrolide rapamycin (sirolimus), a powerful immunosuppressant drug used in
organ transplants and as a coating in coronary stent formulations. The biosynthesis
of L-pipecolic acid from lysine, and its incorporation in the structure of rapamycin
are shown in Fig. 7.11. Note the retention of the lysine carboxyl group throughout
which distinguishes this route to piperidines from those in which decarboxylation
of lysine precedes alkaloid formation.

diamine
H2N CO2H PLP H2N NH2 oxidase
FAD, O2
H2O
lysine NH2 cadaverine
CO2
imine
H+ formation O NH2
N N H2O H NH3
H
∆1-piperidinium cation
OH
O O
Mannich H2O
N N N
SCoA H+ CO2,
H H HSCoA H
O SCoA
O (+)-pelletierine
O
N
H3C N SAM
H3C
OH HO N
O then O2,
H2O CH 3 NADPH
pseudopelletierine
FIGURE 7.10
Conversion of lysine to cadaverine and biosynthesis of pelletierine and pseudopelletierine.

H2 N CO2H H2N CO2H

PLP
transamination
lysine NH2 O
imine
NADPH formation H 2N O
N CO2H N CO2H H2O CO2H

H
L-pipecolic acid HO
H3CO
O O OH
N
O O O
O H3CO
HO
O OCH3
rapamycin
FIGURE 7.11
Origin of L-pipecolic acid in the macrolide immunosuppressant rapamycin.
As might be expected from its structural simplicity, L-pipecolic acid is not ex-
clusively a plant secondary metabolite as it is produced in a variety of organisms
via metabolism of lysine. However, it serves as the basic building block for certain
indolizidines, a relatively small alkaloid group of pharmaceutical interest primarily
because of the tri-and tetrahydroxy alkaloids swainsonine and castanospermine.
Abbreviated biosynthetic routes are shown in Fig. 7.12.
Beginning with L-pipecolylCoA, one malonyl-CoA extension gives the diketide
derivative which is reduced to a ketoaldehyde. Iminium ion formation and reduc-
tion give 1-indolizidinone which is further reduced to diastereoisomeric alcohols.
One diastereomer undergoes three sequential C–H oxidations to give the tetra-
hydroxy alkaloid castanospermine, while the other alcohol disastereomer is first
oxidized to a diol and then to an iminium ion which upon reduction inverts the
stereochemistry at the ring junction. A final C–H oxidation gives the trihydroxy
alkaloid swainsonine.
The intoxicating and toxic gastrointestinal effects of “locoweed” on grazing ani-
mals are due in part to the swainsonine and castanospermine content of certain
prairie plants. Both are known inhibitors of enzymes responsible for hydrolysis of
glycoside linkages, especially N-glycoside linkages associated with glycoprotein for-
mation, and their ability to interfere with formation of the viral glycoprotein coating
of HIV has generated considerable interest. Swainsonine can also act as an appetite
suppressant and has shown some promise both as an anticancer agent and in the


thioester
H malonyl-CoA H reduction
SCoA O O
N N ? N
H H H H
O HSCoA H
CoAS
L-pipecolylCoA
O O
H OH O O
H H
C=O iminium ion iminium ion
reduction reduction formation
N N N ?
C=O H2O
1-indolizidinone
reduction
OH OH
H OH H
sequential C–H HO
oxidations
N N
HO
castanospermine
OH OH OH
H amine-to- OH H
C–H iminium ion C–H
oxidation oxidation oxidation
OH OH OH
N N iminium ion N
reduction
swainsonine
FIGURE 7.12
Proposed routes to the indolizidine alkaloids castanospermine and swainsonine.
promotion of postchemotherapy bone marrow replenishment. Both compounds

have generated substantial activity among synthetic and medicinal chemists over
the years as lead structures in a continuing quest for analogues with increased activ-
ity and diminished toxicity.
QUINOLIZIDINE ALKALOIDS: LIVESTOCK
POISONS FROM CADAVERINE
The quinolizidine alkaloids, also known as the lupin alkaloids, have one or more ni-
trogen atoms shared by two rings and so are similar in structure to the indolizidines.
Plant producing species are abundant and often act as poisons for grazing livestock,
especially sheep. The presence of these alkaloids in lupin bean makes what might
otherwise be a useful protein source problematical for human consumption. Unlike
the indolizidines, studies have shown that the quinolizidines incorporate two or
more cadaverine molecules as symmetrical precursors via use of Δ1-piperidinium
cation or its corresponding enamine. While many biosynthetic details remain to be
worked out, some of the proposed transformations involved in production of lupin-
ine, lupanine, and sparteine from various studies are shown in Fig. 7.13.
Cadaverine, which is first converted to its aminoaldehyde via oxidative deami-
nation, condenses to form the cyclic imine. The protonated form, Δ1-piperidinium
cation, then participates in an initial Mannich reaction with its enamine tautomer; hy-
drolysis of the resulting iminium ion gives an amino aldehyde side chain on the newly
formed piperidine ring. Oxidative deamination of the side chain to a dialdehyde is then

oxidative imine NH
deamination formation H+
cadaverine
H2O N N N
imine H H enamine
∆1-piperidinium
cation H
H O H O N
H H H
oxidative
deamination H2O Mannich
NH NH NH
H NH3
O NH2
H
OH N
H O
imine H
H N
formation 2 NADPH
H
H2O N O (+)-lupanine
N
(–)-lupinine
imine to enamine oxidation
H O
H H O
H
H Mannich H imine N
HN formation
N N then N
N 2 NADPH H
(+)-sparteine
FIGURE 7.13
Proposed conversion of cadaverine to the quinolizidine alkaloids lupinine, lupanine, and sparteine.

followed by cyclization and imine formation. The resulting aldehyde-iminium ion is

then either completely reduced by NADPH to produce lupinine or is isomerized to its
corresponding enamine tautomer which may then serve as the nucleophile in a second
Mannich reaction with Δ1-piperidinium cation to form an amino-aldehyde-iminium
ion that is further cyclized and reduced to give sparteine. The structurally related
quinolizidine lupanine would initially appear to be derived from oxidation of sparte-
ine, but apparently arises via oxidation of iminium ion precursors. Sparteine itself was
formerly used as a diuretic and cathartic but sees little use for these purposes in recent
times. All members of this class are bitter tasting alkaloids that play a role in the defense
mechanisms of plants by acting as effective feeding deterrents for herbivores.
ALKALOIDS FROM PHENYLALANINE: FROM
NEUROTRANSMITTERS TO DECONGESTANTS
AND NARCOTICS
Phenylalanine and tyrosine are closely related to one another structurally, and in
animals, tyrosine can be produced by oxidation of phenylalanine. On the other
hand, plants produce these amino acids separately via a common intermediate in
the shikimic acid pathway. In terms of plant alkaloid biosynthesis, those derived
from tyrosine constitute a much larger group and are of special significance. Though
fewer in number, plant alkaloids derived from phenylalanine are nevertheless quite
abundant and are of particular interest from a pharmacological perspective.
The Chinese described ma huang and its medicinal uses as far back as 2700 B.C.
First isolated in 1887, derived from Ephedra plant species with the active component
ephedrine, this simple alkaloid came into widespread use only during the last cen-
tury. Most producing plants yield a mixture of alkaloid diastereomers, with ephed-
rine itself usually the predominant component. Studies have demonstrated that
phenylalanine provides the phenylC1 component via benzoylCoA, with the remain-
ing two carbons incorporated via TPP-mediated decarboxylation of pyruvic acid and
nucleophilic acyl substitution to produce 1-phenyl-1,2-propanedione as shown in
Fig. 7.14. A transamination process then produces cathinone which undergoes
carbonyl reduction to produce norephedrine and norpseudoephedrine, with
the traditional “nor” prefix in alkaloid nomenclature indicating the absence of an
N-methyl group relative to the parent alkaloid structures which, in this case, are those
of ephedrine and pseudoephedrine formed as usual via subsequent SAM meth-
ylation. An additional SAM methylation produces the remaining pair of diastereo-
meric derivatives known as N-methylephedrine and N-methylpseudoephedrine.
Note the similarity in structure to the synthetic alkaloids amphetamine and meth-
amphetamine, the latter of which (generically known as “crystal meth”) is a devas-
tatingly addictive and destructive drug of abuse.
Ephedrine is quite useful for the relief of symptoms associated with asthma due to
its long lasting effect as a bronchodilator. Its effect on mucous membranes also makes


CO2H
SCoA
NH2
L-phenylalanine benzoylCoA
R
O CH3 N
R
O
TPP ylide H3C N
OH benzoylCoA S R'
O
H 3C
S H :B
O HO R'
CO2 HSCoA
pyruvic acid
TPP ylide
OH OH O O
trans-
NADPH amination
+ (SAM)
NRR' NRR' (SAM) NH2 O
(–)-norephedrine (+)-norpseudoephedrine (–)-cathinone 1-phenylpropane-

(R = R' = H) (R = R' = H) 1,2-dione
(–)-ephedrine (+)-pseudoephedrine
(R = H, R' = CH3) (R = H, R' = CH3)
(–)-N-methyl (+)-N-methyl NHR
ephedrine pseudoephedrine
(R = R' = CH3) (R = R' = CH3) amphetamine (R = H) and methamphetamine (R = CH3)
FIGURE 7.14
Biosynthesis of the ephedrine alkaloid group and structures of the synthetic amphetamines.
it useful as a nasal decongestant for the treatment of symptoms associated with hay
fever and other allergies. Pseudoephedrine (trade name Sudafed) is likewise useful in
this regard and is incorporated into many formulations for cough syrups and other
cold remedies, though its availability is limited due to its ready conversion into meth-
amphetamine via various reductive methods. A mixture of norephedrine and norp-
seudoephedrine, commonly known as phenylpropanolamine (PPA), has similar
properties and was once widely used in over-the-counter (OTC) cold medicine for-
mulations in the United States but was banned in the mid-2000s due to an increased
risk of stroke in younger women. PPA is still in use in some European countries and
is also used in veterinary medicine for treatment of urinary incontinence in dogs.
ALKALOIDS FROM TYROSINE: THE PICTET–S PENGLER

REACTION IN ALKALOID BIOSYNTHESIS
Tyrosine is the basic building block for an enormous number of different alkaloid
structures. The simplest of such alkaloids are typified by compounds such as tyra-
mine, hordenine, dopamine, and adrenaline, as shown in Fig. 7.15.
The biosynthesis of these β-arylethylamines is straightforward, beginning with
PLP-mediated decarboxylation to the corresponding amines followed by alkylations
or oxidations. Decarboxylation of tyrosine leads directly to tyramine which occurs
widely in plants. Interestingly, while commonly found in the diet, tyramine-rich
foods may cause dangerous hypertension crises in individuals taking antidepressant
monoamine oxidase inhibitors (MAOIs), since MAOs are required to properly


FIGURE 7.15
Conversion of tyrosine to tyramine, hordenine, L-DOPA, dopamine, noradrenaline, and
adrenaline.
metabolize tyramine. In barley, double N-alkylation of tyramine via SAM affords

hordenine, a common alkaloid that is widely sold as a nutritional supplement, usu-
ally marketed as a weight loss aid though there are no valid studies in humans to
support such claims. In addition to being an essential drug for the treatment of
Parkinson’s disease, L-DOPA, derived from ring hydroxylation of tyrosine, is an im-
portant catecholamine neurotransmitter and precursor to the other important CNS
catecholamines dopamine, noradrenaline (norepinephrine), and adrenaline (epi-
nephrine) via subsequent decarboxylation (to give dopamine) followed by oxidation
(to give noradrenaline) and then SAM N-alkylation (to give adrenaline).
The condensation of β-arylethylamines like tyramine or dopamine with vari-
ous aldehydes or ketones leads to imine derivatives which may undergo a biological
version of an old but very useful intramolecular cyclization known as the Pictet–
Spengler reaction. The general process is outlined in Fig. 7.16 for the biosynthesis
of two different tetrahydroisoquinoline alkaloids. Lophocerine (one of a group of
dopamine-derived cactus alkaloids that includes the psychedelic phenethylamine
mescaline as shown) is formed via initial SAM O-alkylation of dopamine followed by
condensation with 3-methylbutanal. The resulting imine then undergoes the Pictet–
Spengler cyclization which is essentially an intramolecular electrophilic aromatic
substitution onto an electron-rich aryl ring by a protonated imine function serving as
the electrophilic component. Similarly, reaction of dopamine with acetaldehyde (an
ethanol metabolite) leads to a similar sequence of condensation and cyclization, af-
fording salsolinol, traces of which may be detected in humans as the result of alcohol
consumption.
(S)-R ETICULINE: A VERSATILE PICTET–S PENGLER-

DERIVED BENZYLTETRAHYDROISOQUINOLINE
Probably the most important alkaloid building blocks derived from the Pictet–
Spengler process are the so-called benzyltetrahydroisoquinolines. In particular,

H3CO H3CO H3CO

O2 2 x SAM
NH2 NADPH NH2 NH2
HO HO H3CO
OH OCH3
mescaline
H3CO
SAM H CO
Pictet– 3
SAM N Spengler
HO H NH
then
H H+ HO
HO
O lophocerine
NH2
HO
dopamine H HO
Pictet– HO
O Spengler
N NH
HO H H+ HO
salsolinol
FIGURE 7.16
Dopamine-derived mescaline biosynthesis and Pictet–Spengler formation of lophocerine and
salsolinol.
(S)-reticuline is a remarkably versatile member of this family that serves as a pre-

cursor to a remarkably diverse array of polycyclic alkaloid structures, as shown in
Fig. 7.17.
Before briefly examining just a few of the important products derived from this
essential compound, we take a look at how it is assembled from tyrosine as outlined
in Fig. 7.18.
The process begins with conversion of tyrosine to dopamine as before. For the Pictet–
Spengler reaction, a carbonyl compound is required for condensation with dopamine
and in this case, tyrosine is again the source providing p-hydroxyphenylethanal via
transamination to the corresponding α-ketoacid followed by TPP-mediated decarbox-
ylation. After condensation, the resulting imine undergoes the usual Pictet–Spengler
cyclization to yield the initial benzyltetrahydroisoquinoline product (S)-norcoclau-
rine. Sequential SAM N- and O-methylations followed by ortho-hydroxylation of the
phenolic ring component affords (S)-3’-hydroxy-N-methylcoclaurine. A final SAM
O-methylation leads directly to (S)-reticuline as shown.
OXIDATIVE COUPLING IN ALKALOID BIOSYNTHESIS:

BIOSYNTHESIS OF CORYTUBERINE AND MORPHINE
An important route to a number of different alkaloids derived from benzyltetrahy-

droisoquinolines involves a process we have encountered before, namely phenolic
oxidative coupling. A simple example of this route to polycyclic alkaloids is the direct
intramolecular oxidative coupling of (S)-reticuline to produce (S)-corytuberine, a
member of the so-called aporphine family of alkaloids, as shown in Fig. 7.19.

H3CO O

H3CO NCH3
N O
H3CO
NCH3 H H
H3CO HO OCH3
H O
papaverine HO noscapine
H3CO O
H3CO H3CO
OCH3
O (S)-reticuline H3CO
O
H3CO
O NCH3 H3CO HO H N
H3C
NCH3
HO
O H O
HO OCH3
O NCH3
protopine H corydaline OCH
H3CO 3
HO
corytuberine morphine
FIGURE 7.17
Structural diversity and complexity of polycyclic alkaloids derived from (S)-reticuline.
HO HO
O2 PLP
NADPH NH2 NH
CO2 HO HO
L-tyrosine dopamine
trans- H
TPP H2O
amination
O HO
α-keto- gluta- CO2 HO
glutarate mate p-hydroxyphenylethanal
H3CO H3CO HO
NCH3 NCH3 NH
HO HO HO
H H H Pictet–
HO SAM HO O2 SAM Spengler
NADPH SAM
H3CO HO HO
(S)-reticuline (S)-3'-hydroxy-N- (S)-norcoclaurine
methylcoclaurine
FIGURE 7.18
Conversion of tyrosine to the benzyltetrahydroisoquinoline alkaloid (S)-reticuline.
Assuming the indicated conformation for (S)-reticuline, oxidative formation of

the phenoxy radical pair leads to an obvious opportunity for ortho–ortho resonance
contributors to participate in intramolecular C–C bond formation. The resulting
diketone rapidly tautomerizes to the corresponding diphenolic (S)-corytuberine
product.
Other variations on this theme are well known in alkaloid biosynthesis, but cer-
tainly one of the most important is the intramolecular oxidative phenolic coupling
process involved in the biosynthesis of opium-poppy-derived morphine, an extremely


H3CO H3CO H3CO
ortho
NCH3 NCH3 NCH3
HO O2 O O
H H H
HO O O
2e–, 2H+
ortho
H3CO H3CO H3CO
(S)-reticuline
H3CO H3CO
NCH3 keto– NCH3

HO enol O
H H H H
HO x2 O
H3CO H3CO
(S)-corytuberine
FIGURE 7.19
Intramolecular ortho–ortho oxidative coupling of (S)-reticuline to give (S)-corytuberine.
H3CO H3CO
NCH3 NCH3 NADP+ NCH3 NADPH NCH3

HO HO
HO
H H :H– H
HO
H3CO H3CO
(S)-reticuline (R)-reticuline
FIGURE 7.20
NADP+ oxidation of (S)-reticuline to an iminium ion and its NADPH reduction to (R)-reticuline.
potent analgesic alkaloid which, in spite of its highly psychoactive and dangerously
addictive properties, will be found on any list of pharmacological compounds con-
sidered essential for basic health care delivery. As shown in Fig. 7.20, the biosynthetic
route to morphine begins with a two-step inversion of configuration of (S)-to-(R)-
reticuline via NADP+ oxidation to an iminium ion which is then reduced by NADPH
addition of hydride to the iminium ion Si face. While involved in biosynthesis of other
alkaloids, (R)-reticuline finds its most important role within the context of morphine
production.
The phenolic oxidative coupling step in morphine biosynthesis is illustrated in
Fig. 7.21. It begins with (R)-reticuline in a conformation that brings the two phe-
nolic rings together in an ortho–para orientation with respect to one another. For
convenience, one methoxyl group is bolded to allow the reader to track how the
mediating enzyme folds the usual representation of (R)-reticuline into the required
conformation prior to oxidative coupling. As in the corytuberine biosynthesis, the
product resulting from the intramolecular coupling is initially a diketone, but in
this case, the 2,5-cyclohexadienone form of the lower ring cannot tautomerize from
ketone to enol form since the 4-position is quaternary. Enolization of the upper ring


H3CO H3CO H3CO
NCH3 HO O2
HO O
H
HO NCH3 NCH3
H 2e–,2H+
H
H3CO H3CO H3CO

(R)-reticuline OH O
H3CO H3CO H3CO
ortho
HO keto– ortho–
enol O para O
H
NCH3 NCH3 coupling NCH3
H H H
H3CO H3CO para
H3CO
O O O
salutaridine
FIGURE 7.21
Intramolecular ortho–para oxidative phenolic coupling in formation of salutaridine from
(R)-reticuline.
leads to the usual phenolic structure, affording the intermediate product salutari-
dine as shown.
To complete the transformation to morphine, the 2,5-cyclohexadienone ketone
function of salutaridine is first reduced to the corresponding alcohol via NADPH,
as shown in Fig. 7.22, affording salutaridinol. The alcohol function is then trans-
esterified to an acetate derivative via reaction with acetyl-CoA. This intermedi-
ate acetate ester, 7-O-acetylsalutaridinol, undergoes a spontaneous nucleophilic
attack by the upper ring phenolic hydroxyl group, with the allylic acetate func-
tion serving as the leaving group in what is essentially an SN2’-type nucleophilic
substitution. The resulting product, thebaine, is a minor constituent of opium
extract that is nevertheless significant since it serves as a useful precursor to a
large number of important semisynthetic opiate derivatives such as oxycodone
and naloxone (about which more will be said shortly). An O2/2-oxoglutarate oxi-
dation of the enol ether methyl group leads to demethylation and formation of an
enol which tautomerizes to the corresponding ketone function of neopinone, a
nonconjugated β,γ-unsaturated cyclohexenone which then undergoes spontane-
ous isomerization to give the conjugated α,β-unsaturated ketone function of co-
deinone. NADPH reduction of the codeinone ketone function affords the useful
and familiar antitussive agent codeine, which upon further O2/2-oxoglutarate oxi-
dative demethylation of the upper ring methoxyl group finally affords morphine
as shown.
Among the oldest of plant-derived alkaloids harvested for use by humans and
the first plant alkaloid isolated in pure form (in 1804), morphine is found only
in opium poppy (Papaver somniferum) and a few related species in which it is
known to act (along with other opioid compounds) as a chemical defense against


H3CO
H3CO H3CO
HO
HO HO acetyl-CoA NCH3
NADPH H
NCH3 NCH3
H H H3CO
H3CO H3CO O
O OH
salutaridine salutaridinol O
7-O-acetylsalutaridinol
H3CO H3CO H3CO
enol- O2
O to-ketone O O
NCH3 2-oxo- NCH3
NCH3 glutarate
H H H CH3CO2H
O HO H3CO
neopinone thebaine
H3CO H3CO HO
isomerize O NADPH O2
O O
(non- NCH3 NCH3 2-oxo- NCH3
enzymatic) H H glutarate H
H
O HO HO
codeinone codeine morphine
FIGURE 7.22
Conversion of salutaridine to thebaine and the principal route from thebaine to the opium poppy
alkaloids codeinone, codeine, and morphine.
herbivores. Among its many physiological properties, morphine readily induces sleep
in humans and its name was originally derived from Morpheus, the god of dreams
in ancient Greek mythology. While both morphine and codeine are widely used in
medicine, morphine’s unrivaled ability to relieve severe and otherwise unbearable
pain makes it one of the most indispensable drugs for post-surgical applications
and for alleviation of the intense pain and respiratory anxiety often associated with
certain late-stage cancers. This action is achieved by its ability to strongly bind to the
so-called opioid receptors present in humans and other animals. While codeine is
considered moderately addictive, morphine is a highly addictive substance associ-
ated with strong physical and psychological dependence and rapid development of
increasing levels of tolerance with extended use. Interestingly, both morphine and
codeine continue to be obtained primarily from plant extracts since commercially
viable synthetic routes have yet to be developed in spite of many years of effort.
THE MORPHINE RULE
In addition to morphine, there are many structurally related opioid analgesics, both nat-
urally occurring and synthetic. As it turns out, there are some basic structural features
present in morphine that allow it to bind strongly to the opioid receptors and which par-
tially account for its unusually potent analgesic properties. These fundamental structural

HO

3 3
4 4
O 1 1
NCH3 2 NCH3
2 H
H
HO The Morphine Rule:
1) a tertiary nitrogen with a small
morphine
alkyl group directly attached;
2) a quaternary carbon;
3) an aryl ring (or equivalent) attached
to the quaternary carbon;
4) a 2-carbon spacer between the nitrogen
and the quaternary carbon.
FIGURE 7.23
The morphine rule: structural requirements for active opioid analgesics.
NH2
O O
O N
H3CH2CO H3CH2CO
N
NCH3 N
pethidine (Demerol) anileridine fentanyl
OCH3
OCH3
O N O N(CH3)2 N(CH3)2
HO
N S
sufentanyl methadone tramadol
FIGURE 7.24
Representative synthetic opioid analogues illustrating features of the morphine rule.
requirements, summarized in the so-called morphine rule, are illustrated in Fig. 7.23 as
follows: 1) a tertiary nitrogen with a small alkyl group directly attached; 2) a quaternary
carbon; 3) an aryl ring (or equivalent) directly attached to the quaternary carbon; and 4)
a 2-carbon spacer located between the nitrogen and the quaternary carbon.
The morphine rule has proven to be a useful if limited guide in the structural for-
mulation of many different synthetic or semisynthetic analogues that not only retain
the opioid analgesic properties of morphine, but in some cases even vastly exceed
the parent compound in terms of pharmacological potency. Though far too great
in number to be fully included and discussed here, Fig. 7.24 shows a representative
sampling of such structural analogues.
Among the earliest synthetic morphine analogues were pethidine and anileri-
dine, both of which contain a piperidine ring as a means of providing a two-carbon

spacer between nitrogen and quaternary carbon for the morphine rule. Pethidine
Bioorganic Synthesis 326 was developed in the late 1930s and was originally believed to be more effective as
an analgesic and less harmful than morphine, though it was subsequently found
to be similarly addictive and to also produce a relatively toxic metabolite, which
now limits its use. Developed later in the 1950s, anileridine is structurally similar
to pethidine and is still used for treatment of moderate to severe pain in certain
instances. Fentanyl, developed still later and the most widely used of all synthetic
opioids, is 50–100 times more potent than morphine and remains an essential tool
for the treatment of severe, chronic, or “breakthrough” pain and as a preoperative
anesthetic. A powerful derivative of fentanyl, sufentanyl is 500 times more potent
than morphine and, like fentanyl, is useful as both an analgesic and as a presur-
gical anesthetic. Low doses of both of these piperidine-based analogues are often
administered via transdermal patches for the treatment of chronic, severe pain.
Methadone, which incorporates all four components of the morphine rule, can be
used medically as an analgesic, but is used mainly for the treatment of opioid depen-
dence. By blocking or reducing the euphoric effects of morphine, heroin, and simi-
lar drugs, methadone administration can help to reduce the withdrawal symptoms
experienced by patients addicted to such alkaloids, thereby increasing the chances
for overcoming the addiction. A final example of a fully synthetic opioid analogue is
tramadol which, interestingly, was also recently discovered to be produced naturally
in the roots of the African pin cushion tree. A relatively simple but widely prescribed
synthetic, tramadol is about as effective as morphine for the treatment of moderate
pain, though less so for severe pain.
Equally important among opioid analogues are the semisynthetic derivatives.
Many of these are derived through synthetic manipulation of the morphine precur-
sor thebaine as shown in Fig. 7.25.
Codeine, the most widely used of all opioids, is naturally occurring but prepared
mainly by synthetic methylation of morphine. However, it may also be produced
from thebaine which is available from certain plant strains that produce little or no
morphine. As a semisynthetic opioid dating from 1917, oxycodone has developed a
reputation in recent years as a drug of abuse in spite of its widely recognized benefits
as a powerful and useful pain-reliever. Although oxycodone-related dependence and
withdrawal are significant problems, the drug is second only to morphine in its use
for treatment of severe pain associated with various cancers. By contrast, naloxone is
one of the most important of all opioids, not for its analgesic properties, but rather for
its ability to bind and block the opioid receptors without eliciting its own biological
response, making it the front-line antagonist for the treatment of opioid overdose.
As a final example of the power of semisynthetic analogues, we note the veterinary
opioid etorphine, which has been shown to be 5000–10,000 times more potent than
morphine. Useful mainly for large animal sedation, darts with as little as 1 mg may
be used for sedation of a typical rhinoceros and as little as 4 mg for a typical elephant.
This extraordinary potency is related to etorphine’s unusually strong binding affinity

H3CO H3CO

O O
NCH3 NCH3
H H
H3CO OH
HO O
codeine oxycodone
O
NCH3 HO
H
HO
H3CO
O
thebaine NCH3
O H
N
H H3CO
OH
O
HO
naloxone etorphine
FIGURE 7.25
Important representative semisynthetic opioids derived from thebaine.
for the opioid receptors, a property which makes etorphine far too dangerous for
use in human medicine. Approved only for veterinary use, kits for its administration
always include separate dosages of diprenorphine, an antagonist similar to but far
more powerful than naloxone, for immediate use in case of accidental administration.
ALKALOIDS FROM TRYPTOPHAN: ADVENTURES
IN INDOLE ALKALOID STRUCTURAL COMPLEXITY
The amino acid tryptophan gives rise to a remarkably large group of structures often
simply referred to as the indole alkaloids since the indole nucleus is the basic het-
erocyclic ring system common to most of these compounds. Within this large family
are many subgroups ranging from relatively simple modified tryptamine alkaloids
to much more complex derivatives such as the DMAPP-derived ergot alkaloids and
the so-called terpenoid indole alkaloids which are derived in part from secologain
(a terpenoid product we encountered briefly in Chapter 4) and which include the
vinca, strychnos, and quinine-type alkaloids. We begin this section with the simple
tryptamine derivatives serotonin and melatonin, as shown in Fig. 7.26.
Tryptamine is a simple monoamine alkaloid derived from PLP-mediated decar-
boxylation of tryptophan. It is found in animals, plants, and fungi and is believed
to play a role as both a neuromodulator and a neurotransmitter, in part by affecting
levels of serotonin, which is biosynthesized via simple ring oxidation of tryptophan
to 5-hydroxytryptophan followed by PLP-mediated decarboxylation. Serotonin
plays a variety of significant roles as a monoamine neurotransmitter including
regulation of mood, sleep, appetite, and other behavioral states. SSRIs (selective se-
rotonin reuptake inhibitors) are widely used antidepressant drugs designed to help

CO2H CO2H
Bioorganic Synthesis 328 HO O2,

NH2 NH2 PLP NH2
NADPH
N N CO2 N
H H H
5-hydroxytryptophan tryptophan tryptamine
HO NH2 H3CO NHCOCH3

PLP acetyl-CoA
then
N SAM N
CO2 H H
serotonin melatonin
FIGURE 7.26
Simple derivatives of tryptophan: the neurochemicals tryptamine, serotonin, and melatonin.
CH3-SAM
H3C H3C
NH2
NH2
NH2 NH
SAM
N
H N N H
N H H
tryptamine H SAH
O O
H3C H3C
H3CHNCO HO
H2N OPP
NCH3 NCH3 2 x SAM
then SAM then O2,
N H (N-methylation) N H NADPH
CH3 CH3
physostigmine
FIGURE 7.27
Conversion of tryptamine to the calabar bean alkaloid physostigmine.
moderate mood by increasing serotonin levels in the brain. Serotonin is also avail-
able from a variety of food sources such as mushrooms, fruits, and vegetables. N-
acetylation and SAM methylation of serotonin affords the closely related hormone
melatonin. In humans, melatonin plays an important role in the sleep–wake cycle.
Light availability at the retina of the eye inhibits melatonin production in the pineal
gland while darkness favors its production. Melatonin also serves as a powerful an-
tioxidant, helping to protect both mitochondrial and nuclear DNA from the damag-
ing effects of oxygen and nitrogen radical species.
Another relatively simple tryptamine derivative is physostigmine, an indole alka-
loid isolated from the calabar bean. The presumed pathway to this alkaloid involves
SAM methylation of the tryptamine indole ring at the 3-position to afford an im-
inium ion which then undergoes intramolecular nucleophilic attack by the adjacent
primary amino group, forming a tricyclic 6-5-5 ring system as shown in Fig. 7.27.
Subsequent SAM N-methylations are followed by an aryl ring hydroxylation with
the resulting phenolic oxygen being converted to a carbamate-type linkage by reac-
tion with carbamoyl phosphate. A final N-methylation of the carbamate function
affords physostigmine. An older drug now used primarily as an antidote for severe

poisoning by anticholinergic agents, it has also long been used topically for the treat-

ment of glaucoma and has more recently been studied for potential use in the treat-
ment of Alzheimer’s disease. Physostigmine also has an interesting history from a
chemical synthesis standpoint, one that we will touch on briefly in Chapter 8.
Derived from tryptophan and DMAPP and produced primarily by certain
fungi, the ergot alkaloids, a family of structurally complex indole alkaloids, have
a remarkable array of important biological activities The ergots are usually divided
into two groups: the structurally simple clavine-type alkaloids, of which agro-
clavine is an example; and the core compound lysergic acid from which is derived
a variety of simple amide and cyclic peptide amide derivatives, both naturally
occurring and synthetic. The proposed pathway for formation of both agroclavine
and lysergic acid (as shown in Fig. 7.28) is complex and has been much studied,
H
DMAPP
CO2H H+
HN HN CO2H
HN HN
H2N
H2N
SAM
(N-alkylation)
B:
H
O2,
OH NADPH
HB then 1,4-
H2O
elimination/
CO2H CO2H dehydration CO2H
HN HN HN
H3CHN H3CHN H3CHN
dimethylallyl-L-abrine
O2 NADPH
OH OH
O H+
NHCH3 NHCH3 B: NHCH3

CO2H H+ OH
epoxide
ring- then
opening O CO2 NAD+ E-to-Z
HN HN HN
chanoclavine-I
CO2H
O
CH3-to- H imine
NCH3 CO2H oxidation NCH3 formation NHCH3
H then π-bond H then
isomerizaton NADPH
(imine-to-
amine)
HN HN HN
lysergic acid agroclavine chanoclavine-I
aldehyde
FIGURE 7.28
Biosynthetic origin of the ergot alkaloids agroclavine and lysergic acid.

though many details are lacking. Beginning with electrophilic aromatic substitu-
Bioorganic Synthesis 330 tion by DMAPP-derived dimethylallyl cation on the aryl ring of tryptophan, a sub-
sequent SAM N-methylation affords dimethyllallyl-L-abrine which can undergo
a benzylic oxidative hydroxylation at the dimethylallyl side-chain with subse-
quent 1,4-dehydrative elimination to produce a conjugated diene. Epoxidation
and acid-catalyzed epoxide ring-opening to a benzylic cation is followed by de-
carboxylation and nucleophilic ring closure. The resulting chanoclavine-I then
undergoes NAD+ oxidation and E-to-Z isomerization to give chanoclavine-I al-
dehyde. Intramolecular cyclization to an imine and subsequent NADPH reduc-
tion gives agroclavine whose methyl group then undergoes sequential oxidations
to give the carboxylic acid derivative. A final allylic rearrangement of the ring
π-bond placing it in conjugation with the aryl ring completes the final conversion
to lysergic acid as shown.
There are over a dozen known, naturally occurring cyclic tripeptide amide
derivatives of lysergic acid, many of which are important medicinal agents. Both
ergotamine and ergostine (Fig. 7.29) have long been used for the treatment of
migraine headaches, while ergocryptine is used as starting material for preparation
of the valuable brominated derivative known as bromocryptine which is used in
the treatment of Parkinson’s disease, pituitary tumors, and several other conditions.
Semisynthetic derivatives of lysergic acid include the notorious psychedelic hallu-
cinogen lysergic acid diethylamide (LSD), which is a controlled substance, while
other simple amide derivatives include ergometrine, an invaluable medicinal agent
widely used in obstetrics for the prevention of postchildbirth bleeding and to assist
in placenta expulsion. Cabergoline is frequently used in place of bromocryptine for
the management of pituitary tumors as well as symptoms of Parkinson’s disease, a
H H H
HO HO HO
O O O
N N N
CONH CONH CONH
N N N
O O O
O O O
NCH3 Ph NCH3 NCH3
H ergotamine H ergonine H ergocryptine
(+)-lysergic acid
CON(CH2CH3)2
CONH CON SCH3
OH
NCH3 N(CH3)2
H H
NCH3 N N
H
H H H
lysergic acid
diethylamide (LSD) ergometrine cabergoline pergolide
FIGURE 7.29
Naturally occurring (upper) and semisynthetic (lower) medicinal derivatives of lysergic acid.

condition for which pergolide was also once widely prescribed—it is no longer ap-

proved for use in the United States due to potential heart valve complications.
In addition to their importance to the pharmaceutical industry and human
health, certain ergot alkaloids are the principal toxins involved in many historic epi-
demics of livestock and human poisonings due to consumption of infected grasses
or grains. Known in the middle ages as St. Anthony’s Fire, the symptoms of ergot
poisoning or ergotism, as it is referred to in modern times, are divided into two
groups, the first being characterized by severe convulsions and mental psychosis and
the second by development of gangrenous tissue resulting from vasoconstriction,
especially at the fingers or toes. Interestingly, some reports have even speculated
that the convulsive and psychotic symptoms displayed by supposedly “bewitched”
individuals described during the Salem witch trials were actually victims of ergotism
arising from the consumption of moldy grains.
PICTET–S PENGLER-T YPE REACTIONS

OF TRYPTAMINE: β-C ARBOLINES AND INDOLE
TERPENE ALKALOIDS
We have previously seen the importance of the Pictet–Spengler reaction in alka-

loid biosynthesis, particularly for those derived from phenylalanine and tyrosine.
An interesting variant of the reaction is also observed in a number of processes
involving tryptamine as the amine component. A useful example that illustrates the
fundamentals of tryptamine-based Pictet–Spengler processes is shown in Fig. 7.30
for the biosynthesis of harmalan and harmine, two representative members of the
so-called harmala group of β-carboline alkaloids of which about ten are known.
In this instance, tryptamine condenses with the ketone function of pyruvic acid to
form the usual imine intermediate. Here the indole C-5 ring rather than the C-6
aryl ring attacks the electrophilic imine linkage leading to cyclization, proton loss,
and restoration of indole aromaticity to afford the basic 6-5-6 ring system char-
acteristic of the β-carbolines. Oxidative decarboxylation of the initial aromatized
O
H+
NH2 CO2H N NH
imine CO2H CO2H

N formation N N H
H H H
tryptamine
NADP + NADPH, CO2
NADP +
N oxidation, N N H
SAM, then O
oxidation
H3CO N N NADPH, N
H H CO2 H OH
harmine harmalan
FIGURE 7.30
Conversion of tryptamine to the β-carboline alkaloids harmalan and harmine.

cyclization product leads directly to harmalan. Further modifications via aryl ring
Bioorganic Synthesis 332 hydroxylation, SAM methylation, and dehydrogenation give the fully aromatic tri-
cyclic derivative harmine. Produced by a number of different plants and also by ani-
mals, the harmala alkaloids, especially harmine, are reversible monoamine oxidase
inhibitors (MAOIs) that affect the central nervous system; all are closely related
structurally and several are known to be the psychoactive components of certain
ritual “magic” potions prepared from native plants and used historically by indig-
enous Amazonian groups.
Also assembled utilizing the tryptamine variant of the Picet–Spengler reaction
are the indole terpene alkaloids. As the name implies, these compounds incorpo-
rate a terpene component which serves as the carbonyl partner for condensation
with tryptamine to form the usual imine component required for the cyclization
process. The iridoid terpene component, commonly known as secologanin, is one
which we have briefly encountered previously in Chapter 4 and which is used in the
construction of all members of this alkaloid group. A proposed pathway to secolo-
ganin from geraniol is outlined in Fig. 7.31.
The process begins with conversion of geraniol to 8-hydroxygeraniol. NAD+
oxidation of both alcohol functions then leads to the corresponding dialdehyde.
NADPH conjugate reduction of the upper α,β-unsaturated aldehyde gives an eno-
late ion which then serves as a nucleophile for intramolecular conjugate addition
to the lower α,β-unsaturated aldehyde function. The resulting cyclized dialde-
hyde, irododial, cyclizes further via a cyclic hemiacetal form to give nepetalactol.
H H
H:
OH O O
OH
P450 2 NAD+ NADPH
allylic (CHOH conjugate
oxidation to C=O) O addition O
OH
geraniol 8-hydroxygeraniol H H
8-oxogeranial H+
enolate
conjugate
addition
H
H H OH H H
OGlu OH
UDP-Glu O2 O
O H O NADPH O OH
H H H
UDP X2
HO2C HO2C
7-deoxyloganic acid 7-deoxyloganetic acid nepetalactol irododial (enol)
HO HO
O
H HO HO H OGlu
OGlu H
O2 O2 H
NADPH, NADPH
O H O
then SAM H
H H H2O
H3CO2C H3CO2C
oxidative cleavage of C-5 ring
loganin secologanin
FIGURE 7.31
Simplified scheme for biosynthesis of the iridoid secologanin from geraniol.

Oxidation of the allylic methyl group of nepetalactol to the carboxylic acid gives

7-deoxyloganetic acid whose lactol hydroxyl group subsequently undergoes gluco-
sylation via UDP-Glu to give 7-deoxyloganic acid. A C-5 ring hydroxylation affords
loganin which finally undergoes an oxidative cleavage of the C-5 ring to give the
iridoid terpene, secologanin.
Condensation of secologanin with tryptamine followed by a Pictet–Spengler-
type cyclization affords the key intermediate strictosidine, which in turn serves as
the starting material for assembly of a vast number of structurally diverse bioac-
tive alkaloids, many of which are important medicinal agents. Figure 7.32 provides
an overview of some of the more important members of the strictosidine-derived
indole terpene alkaloid family, of which over 2000 members have been isolated to
date. Given the sheer numbers involved, we must limit ourselves to examining a few
representative members that illustrate some of the types of transformations that are
likely to be common to others. As before, not all of the pathways shown have been
fully elucidated and the mechanisms proposed are ones which make reasonable
chemical sense given the limited amount of information available.
We begin with ajmalicine (also known as raubasine), an alkaloid found in a va-
riety of plants such as Rauwolfia spp. and which is used in the treatment of hyper-
tension (high blood pressure). The proposed pathway (Fig. 7.33) commences with
deglucosylation of strictosidine to give the corresponding free hemiacetal of which
H O
NH2 OGlu
+
N O
H3CO2C
H
tryptamine secologanin
H3CO O
HO N
N Pictet–Spengler H2O,
H Reaction N
H3CO H+ camptothecin O
(anticancer agent)
N
quinine OH O
(antimalarial) NH
N
H OGlu N
N
H H
H O
H
N H H3CO2C N
O H
H strictosidine ellipticine
O strychnine (anticancer agent)
(fatal poison)
H N N
N
HO N N N
H H CO2CH3
CO2CH3 N H
vincamine H H catharanthine
H O (anticancer agent
(vasodilator)
N precursor)
H3CO2C H H
ajmalicine aspidospermine
(antihypertensive) (respiratory stimulant)
FIGURE 7.32
Diversity of bioactive or medicinal alkaloid structures derived from strictosidine.


NH
H OH NH
N H H O
H2O H
strictosidine H
strictosidine O hemi-
Glu aglycone acetal OH
H3CO2C
H3CO2C hydroxy-
aldehyde
N H+ iminium O
N H NH
ion H
N isomerize formation H
H H
H
dehydro- OH
H+ OH H2O OH
geissoschizine
H3CO2C
H3CO2C H3CO2C
N N N
H
N NADPH N
H H H H
H
OH O O
H+ H H
H3CO2C H3CO2C H3CO2C

cathenamine ajmalicine
FIGURE 7.33
Conversion of strictosidine to the alkaloids cathenamine and ajmalicine.
one possible open form is an enol-aldehyde as shown. Condensation of the aldehyde

with the ring amino group leads to an iminium ion which undergoes allylic isomeri-
zation to afford the important α,β-unsaturated iminium ion intermediate, dehydro-
geissoschizine. Subsequent conjugate addition to the unsaturated iminium ion by
the adjacent enol hydroxyl group gives the precursor alkaloid cathenamine which
undergoes enamine linkage reduction by NADPH to afford ajmalicine.
Dehydrogeissoschizine also plays an important role in the construction of
catharanthine and vindoline, two separate alkaloid components which are subse-
quently modified and coupled to form the remarkably complex structures of the
potent cancer chemotherapy agents vinblastine and vincristine, two members of
the so-called vinca alkaloids isolated from the Madagascar periwinkle plant. Like
taxol (Chapter 4) and podophyllotoxin (Chapter 6), vinblastine and vincristine are
compounds which inhibit cell division (mitosis) through disruption of the forma-
tion of the microtubules which help cells to pull apart during division. These unusual
alkaloids are highly effective in the treatment of a variety of carcinomas including
lung, breast, and testicular cancers as well certain lymphomas and leukemia; both
rank among the most important antitumor agents available to modern medicine.
A highly abbreviated scheme for conversion of dehydrogeissoschizine to catharan-
thine and vindoline and their subsequent modification and coupling to form vin-
blastine and vincristine is shown in Fig. 7.34


N
many steps
N
H H
N OH
OH H
dehydrogeissoschizine CO2CH3
H3CO2C stemmadenine
N
H
N N
N OH +
N H+ N H2O H H
H CO2CH3 H CO2CH3
CO2CH3
dehydrosecodine
N N
Diels–Alder many steps

H
[4+2]
N N
H CO2CH3
H CO2CH3 tabersonine
N
N N
Diels–Alder
H
N [4+2] N H3CO CO2CH3
H CO2CH3 H CO2CH3 + N H OH
CH3 CO2CH3
catharanthine vindoline
N
modify catharanthine,
couple to vindoline
N N
H OH
H3CO2C
vinblastine (R = CH3)
H
H3CO CO2CH3 oxidation
N OH
R H vincristine (R = CHO)
CO2CH3
FIGURE 7.34
Formation and linkage of catharanthine and vindoline to give vinblastine and vincristine.
A complex multistep sequence (not shown) initially converts dehydrogeissoschi-

zine to stemmadenine which then apparently undergoes a dehydration-driven ring
cleavage facilitated by formation of an iminium ion and an α,β-unsaturated ester
function. The resulting conjugated iminium ion can then undergo loss of a proton
to form the conjugated enamine product dehydrosecodine as a key intermediate.
Subsequent conversion to either tabersonine or catharanthine may be conveniently
envisioned as occurring via intramolecular Diels–Alder reactions arising from alter-
nate folded conformations of dehydrosecodine as shown. A multistep sequence of
oxidations and alkylations presumably then converts tabersonine to vindoline. At
this point, additional modifications of catharanthine (not shown) are followed by a
coupling reaction with vindoline that may involve electrophilic aromatic substitu-
tion as a means to form the key C–C bond between the two components, thereby
producing vinblastine. Formation of vincristine occurs via postcoupling oxidation
of the N-methyl group of vinblastine to the corresponding N-formyl derivative as

shown. Though not always obvious from their structures, the formation of strych-
Bioorganic Synthesis 336 nine, vincamine, aspidospermine, and ellipticine (Fig. 7.32) is also known to
involve dehydrogeissoschizine-derived intermediates, though many of the steps
involved are not well understood.
In our final example for this section, we examine the construction of the impor-
tant antimalarial antiparasitic alkaloid known as quinine. The significance of qui-
nine as one of the earliest and most effective antimalarial drugs is enormous from a
variety of perspectives. Historically, quinine is among the oldest of alkaloids known
to have important curative properties and it remains in widespread use throughout
the world in modern times. In addition to its medicinal significance, the intricate
molecular architecture of quinine has made it one of the most important and chal-
lenging targets of the modern era of laboratory total synthesis of structurally com-
plex natural products, from the 1940s up to the present. In terms of the ongoing
impact of malaria itself on human health, it is worth noting that annual cases of
this mosquito-borne parasitic infection number anywhere from 300 to 500 million
worldwide, mostly in sub-Saharan Africa where it continues to claim over half a mil-
lion lives each year, mostly among children.
As outlined in Fig. 7.35, a proposed biosynthetic sequence again begins with
strictosidine aglycone, a hemiacetal which in its hydroxyaldehyde form can un-
dergo an intramolecular iminium ion cyclization, followed by NADPH reduction
to afford the corresponding bicyclic amine derivative. Hydrolysis of the methyl ester
of the β-dicarbonyl side chain of the system affords a β-oxocarboxylic acid which,
like β-ketoacids, can undergo spontaneous decarboxylation to give the intermedi-
ate aldehyde corynantheal. Oxidation of the bicyclic tertiary amine ring system of
corynantheal gives an iminium ion which can then undergo hydrolysis and cleavage
to an amino dialdehyde. Conformational rotation and subsequent condensation is
then followed by NADPH reduction of the resulting bridged bicyclic iminium ion,
affording the corresponding indole aldehyde cinchonaminal as shown. Cleavage of
the indole C-5 ring is shown as occurring via protonation and subsequent hydro-
lysis of the resulting iminium ion, leading to an amino ketoaldehyde intermediate.
Once again, conformational rotation and condensation of the aryl amino group with
the aldehyde function leads to formation of the corresponding cyclic imine. This
dihydroquinoline ring system can then undergo oxidative aromatization to the cor-
responding quinoline derivative, affording the important intermediate alkaloid cin-
chonidinone whose ketone function may then undergo subsequent reduction via
NADPH, leading directly to cinchonidine. The remaining steps involve oxidative
hydroxylation of the cinchonidine aryl ring followed by the usual SAM methylation
to finally afford the methoxylated derivative of cinchonidine known as quinine.
The most important of the so-called cinchona alkaloids found in the bark of the
cinchona tree, quinine is the active principal in cinchona bark which was used for
centuries to treat fever-related shivering and other maladies by indigenous peoples
of Bolivia and Peru where the cinchona tree is found. In the early 1600s, Jesuit


NH NH
OH O
N H N H
H H
strictosidine O hemi- OH
aglycone acetal hydroxy-
H3CO2C H3CO2C aldehyde
N NADPH iminium O
iminium ion N ion NH
reduction formation
N O H
H then H
H
enol-to- OH OH
aldehyde H2O
CO2CH3
H 2O H3CO2C H3CO2C
CH3OH,
CO2
O
N H
N N
oxidation iminium ion
N H O amine-to- hydroylsis
H H H
iminium ion
corynantheal O
O
iminium ion formation

O H+ O O
H
indole NADPH
protonation
iminium ion N
reduction
N N N
H N H H N H H
H
cinchonaminal
H2O iminium ion
hydrolysis
H
O O O
H H N
H N H
imine
formation
H2N O H2N
H N H2O N
O
oxidation
HO HO O
N N N
H oxidative H H
H3CO hydroxylation NADPH
then SAM reduction
N N N
quinine cinchonidine cinchonidinone
FIGURE 7.35
Possible steps for the conversion of strictosidine aglycone to cinchonidine and quinine.
missionaries reportedly sent samples of cinchona bark back to Rome to be tested

for possible use in the treatment of shivers associated with malarial fevers, leading
to the subsequent discovery of its unrelated antimalarial curative properties, for
which it was widely known as Jesuit bark throughout the 17th and 18th centu-
ries. The use of this bitter-tasting alkaloid in more recent times is most often re-
served for treatment of malarial infections arising from strains of the Plasmodium
parasite which have become resistant to more modern antimalarial drugs, though
the low cost of quinine still makes it the treatment of choice in economically

underdeveloped regions of the world and the gold standard for evaluation of the
Bioorganic Synthesis 338 antiplasmodic activity of other potential antimalarial drugs. Quinine has also been
used in the treatment of both lupus and rheumatoid arthritis, though structurally
related synthetic antimalarials such as hydroxychloroquine have largely displaced
it for such purposes. Quinine is also present in small amounts as the bittering agent
used in tonic waters.
ALKALOIDS FROM NICOTINIC ACID: TOXIC ADDICTIVE

DERIVATIVES OF A COMMON NUTRIENT
Nicotinic acid, also known as niacin and vitamin B3, is formally pyridine-3-
carboxylic acid, the amide derivative of which is incorporated into the familiar
redox cofactors NAD+ and NADP+ that we have seen so often. As an essential
nutrient in the human diet, nicotinic acid is produced by both plants and animals,
though by very different biosynthetic routes, as outlined in Fig. 7.36. Animals pro-
duce the compound starting from L-tryptophan via oxidative cleavage of the indole
C-5 ring to yield the ketoamide N’-formylkynuerenine as shown. Hydrolysis of
the formamide linkage affords kynurenine, a key intermediate in the metabolism
of tryptophan. In this sequence, kynurenine is oxidized to the phenolic deriva-
tive 3-hydroxykynurenine which then undergoes a PLP-mediated cleavage of the
amino acid side chain, affording 3-hydroxyanthranilic acid along with L-alanine
as a byproduct. Intramolecular dioxygenase-mediated oxidative cleavage of the
phenolic ring affords the corresponding oxo-dicarboxylic acid whose enamine
function may tautomerize to the imine, thereby allowing a C–C bond rotation
and subsequent imine cyclization and aromatization, affording quinolinic acid
which is the key intermediate in both animal and plant pathways; decarboxyl-
ation directly affords nicotinic acid. The chemistry employed by plants is much
simpler, employing an enamine tautomer of iminoaspartic acid along with
3-phosphoglyceraldehyde which are combined via what is essentially an enamine
alkylation process. The resulting intermediate undergoes an imine cyclization
similar to the previous example, affording 4,5-dihydro-5-hydroxyquinolinic acid
which undergoes dehydration to quinolinic acid and decarboxylation to nicotinic
acid as before.
Nicotinic acid (usually identified as niacin for these purposes) is still used as one
of the oldest cholesterol-reducing compounds, helping to control both LDL (low
density lipoprotein) and triglyceride levels while raising HDL (high density lipopro-
tein) levels. While rapidly metabolized and excreted, it is a strong vasodilator that
often causes a disagreeable flushing and prickling of skin, particularly of the face,
which can be partially diminished through the use of controlled-release formula-
tions of the drug. However, two recent studies have now led to warnings about the
use of niacin for control of cholesterol, citing increased rates of death, infections,
gastrointestinal disorders, and other significant problems. These findings may well

CO2H

HO2C
CO2H
NH2 NH2
O2 O O
NH2
O N CHO
dioxygenase H
N enzyme
H (in animals) N N'-formylkynurenine
H
tryptophan H2O amide
hydrolysis
CO2H CO2H
CO2H NH2 NH2

PLP O2
O O
NADPH
NH2 L-ala NH2 NH2
OH OH kynurenine
3-hydroxyanthranilic acid 3-hydroxykynurenine
H
CO2H CO2H CO2H CO2H
O2
dioxygenase NH2 O O NH
O O NH2 O HN O
enzyme O
O OH OH OH HO
imine
cyclization H2O
CO2H CO2H CO2H

aspartic FAD
acid
HN CO2H N CO2 N CO2H
iminoaspartic acid nicotinic acid quinolinic acid
(in plants) dehydration H2O

OP
H
HO CO2H HO CO2H imine HO CO2H
enamine cyclization
alkylation
O H2N CO2H O HN O N CO2H
H2O
3-phospho- HO 4,5-dihydro-5-hydroxy-
glyceraldehyde quinolinic acid
FIGURE 7.36
Different biosynthetic routes to the formation of nicotinic acid, both via quinolinic acid.
eventually lead to niacin being recommended for use only for high-risk heart pa-
tients who cannot tolerate statins or for those who have otherwise uncontrollable,
high triglyceride levels.
The principal alkaloids derived from nicotinic acid are the so-called tobacco al-
kaloids nicotine and anabasine. A reasonable biosynthetic pathway for these com-
pounds is outlined in Fig. 7.37.
The sequence commences with a 1,4-reduction of nicotinic acid by NADPH in
a process somewhat analogous to the 1,4-reduction of benzoic acid by the action of
Na/NH3 in the familiar Birch reduction process. The resulting 3,6-dihydronicotinic
acid undergoes decarboxylation to afford 1,2-dihydropyridine, which is effec-
tively a cyclic enamine. For simplicity, the enamine is shown in a dipolar resonance
form as it acts as a nucleophile to attack either N-methyl-Δ1-pyrrolinium cation
or Δ1-piperidinium cation, both of which we have encountered previously in the
biosynthesis of ornithine-and lysine-derived alkaloids. In both cases, the resulting

H+

CO2H CO2H CO2H CO2H
NADPH
N H:
N N N
nicotinic acid 3,6-dihydro-
nicotinic acid
similar to Birch reduction H+
of benzoic acid: CO2H
CO2H CO2H
Na, NH3
ethanol
(2e-, 2H+)
O
CO2H
H+
O
N N H
CO2 N N
H H
H
dipolar form 1,2-dihydro-
pyridine
(an enamine)
H
N N
CH3 CH3
N
N CH3 H+ H N NADPH N
H (from nicotine
putrescine)
NADP+
H
N N
H H
N N N NADPH N
H H H+ H anabasine
(from
cadaverine) NADP+
FIGURE 7.37
Conversion of nicotinic acid to the tobacco alkaloids nicotine and anabasine.
substituted dihydropyridine intermediates undergo NADP+ oxidation to yield the

corresponding C10H14N2 alkaloid products.
While found in a number of plants, nicotine constitutes up to 3% of the dry
weight of the tobacco plant, usually accompanied by smaller amounts of anabasine
and related structures. The alkaloid itself is surprisingly toxic with a reported lethal
dose for humans of only about 30–60 mg. Nicotine is also highly addictive, as any
tobacco user well knows. Its physical and psychological effects are highly variable,
ranging from stimulant-like effects at lower doses to more sedative-like effects at
somewhat higher doses. Nicotine is also a powerful vasoconstrictor that increases
blood pressure and can lead to severe vascular damage with long exposure. Post-
operative use of nicotine is particularly problematic, as its vasoconstrictive action
can seriously compromise wound healing. In recent years, nicotine-infused trans-
dermal patches and chewing gums have found wide use for assisting in gradual with-
drawal from chronic tobacco use, although long-term success rates for such therapy
remain somewhat controversial. Measurement of urine levels of nicotine, anabasine,
and various metabolites have been used as a measure of the extent of tobacco use (or

lack thereof) in patients during nicotine replacement therapy. Historically, nicotine

has also served as an important insecticide, though its use has diminished in recent
years with the advent of cheaper and more effective alternatives.
ALKALOIDS FROM ANTHRANILIC ACID: FROM

TRYPTOPHAN TO QUINOLINES AND ACRIDINES
We have seen previously in Chapter 6 how the shikimate product anthranilic acid
serves as a key component for the biosynthesis of both indole and tryptophan (see
Figs. 6.10 and 6.11). Anthranilic acid also serves as a template for structural elabora-
tion into other complex alkaloid structures, particularly examples of quinoline-and
acridine-type alkaloids. This is achieved by employing the CoA derivative of anthra-
nilic acid, anthranoylCoA, as a starter unit for polyketide chain extension reactions
as shown in Fig. 7.38. The first example involves addition of a single malonyl-CoA
unit followed by cyclization to the corresponding lactam derivative quinoline-2,4-
(1H,3H)-dione. Subsequent sequences of oxidations, SAM or DMAPP alkylations
and other transformations (not shown) lead to the quinoline-type alkaloids cas-
miroin and dictamnine, the latter of which has shown both antifungal and smooth
muscle contractant activity. The second sequence leading to the acridine-type alka-
loid acronycine begins with condensation of anthranoylCoA with three units of mal-
onyl-CoA to give a triketothioester which undergoes a Claisen condensation to form
a familiar cyclic triketone system which is shown as undergoing an intramolecular
iminium ion cyclization and subsequent aromatization to give 1,3-dihydroxy-10-
methylacridin-9(10H)-one. Final conversion to acronycine is via SAM and DMAPP
alkylations followed by an acid-catalyzed side-chain cyclization. The anticancer activ-
ity of acronycine has led to exploration of more active derivatives based on this core
structure, leading to the highly active synthetic analogue S 23906-1 which reportedly
exhibits a novel spectrum of antitumor activities compared with many other antican-
cer drugs and which is currently (in 2016) in phase 1 clinical trials.
ALKALOIDS FROM HISTIDINE: FROM SIMPLE

AMIDES TO GLAUCOMA DRUGS
The amino acid histidine apparently serves as the precursor to a number of alka-
loids containing the imidazole ring function, although the biosynthetic details are
not always well known. Some of these products derive from histamine which arises
via PLP-mediated decarboxylation of histidine. Histamine itself is an important
neurotransmitter involved in the sleep–wake cycle and is also involved in immune
system function and inflammatory response. It is the familiar target of so-called
“antihistamines” or histamine antagonists which either block histamine receptors
or inhibit its production from histidine, usually to relieve symptoms associated with
allergic responses such as watery nose and itchy eyes. Figure 7.39 outlines possible


O
COSCoA
SAM
O
3 x malonyl-CoA NHCH3
NH2 O
CoAS
1 x malonyl-CoA Claisen HSCoA
O O
O
O
O O
NH2SCoA NHCH3
HSCoA iminium ion

formation H2O
O
O O
H
N O
H
quinoline-2,4(1H,3H)-dione N O
CH3
O2, SAM, etc. DMAPP, O2,
steps SAM, etc. 2 x keto-enol,
steps aromatization H+
OCH3 OCH3 O OH
N O N O N OH
O CH3 CH3
O casmiroin dictamnine 1,3-dihydroxy-10-
(quinoline-type alkaloids) methylacridine-9(10H)-one
O OCH3 SAM DMAPP,

cyclization
O OCH3
N O
CH3
S 23906-1 H3COCO N O
CH3
(synthetic acronycine OCOCH3
analogue antitumor agent) acronycine
(acridine-type
alkaloid)
FIGURE 7.38
Conversion of anthranilic acid to some quinoline and acridine-type alkaloids.
routes to some simple histamine amide derivatives such as dolichotheline, one

of the first imidazole-containing alkaloids isolated. Certainly the most important
examples of the histidine group from a medicinal perspective are the pilocarpus
alkaloids pilosine and pilocarpine, derived from (5-imidazolyl)pyruvic acid via
histidine transamination. After acid reduction to the corresponding keto alcohol
and condensation with either butyric acid (for pilocarpine) or 3-phenylpropanoic

H H

CO2H H H N O
N N NH2 N
PLP
acetyl-CoA
NH2 CO2
N N N
histidine histamine Nα-acetylhistamine
4-oxodecanoyl- cinnamoyl-
CoA CoA
H H isovaleryl- H H
N N O CoA N N O
N H N
H N O
Nα-(4-oxodecanoyl)- N Nα-cinnamoylhistamine
histamine n-C H O
6 13
trans- N
histidine amination dolichotheline
HO R
H H H
N CO2H N N
reductions OH O O
O O O
N N N O
(5-imidazolyl)pyruvic acid H2O
aldol R
H2O
CH3 H CH3 H CH3
N N N
O O2 O NADPH O
N NADPH N (conjugate N
HO (R = Ph) reduction)
H O H O O
Ph R R
pilosine pilocarpine (R = CH3)
FIGURE 7.39
Possible origins of some simple histamine amides and the alkaloids pilocarpine and pilosine.
acid (for pilosine), the resulting esters may undergo intramolecular aldol condensa-
tions and conjugate reductions to form the 5-membered lactone rings, with pilosine
requiring a final C–H to C–OH oxidation at the benzylic position to complete the
sequence.
Pilocarpine is a very important medication which is obtained commercially
from the leaves of Maranham Jaborandi (Pilocarpus microphyllus), a flowering plant
found only in Brazil. Pilocarpine and its salts are especially useful for the treatment
of severe dry mouth associated with radiation treatment of tumors of the head and
neck. It is also widely used in ophthalmology for pupil contraction and in the treat-
ment of glaucoma.
PURINE ALKALOIDS: ADDICTIVE STIMULANTS IN OUR

COFFEE, TEA, AND CHOCOLATE
Purines, simple N-heterocyclic aromatics made up of fused imidazole and pyrimi-

dine rings, are most commonly encountered in some of the deoxyribonucleosides
and ribonucleosides found in DNA and RNA systems. Substituted and tautomeric
forms are also widely found in nature, particularly derivatives of xanthine of which
theophylline (1,3-dimethylaxanthine), caffeine (1,3,7-
trimethylxanthine), and
theobromine (3,7-dimethylxanthine) are the most common. Figure 7.40 outlines
the sequences involved in their biosynthesis from xanthosine.

O O
CH3 O
CH3
N N
HN HN H2O N
SAM HN
O N N N
O N O N N
H H ribose H
O O
HO HO 7-methylxanthine
H2O OH OH
ribose SAM
HO xanthosine HO 7-methyl-
2 x SAM
xanthosine
O O O
CH3 CH3
H3C H
N H3C N N
N N SAM HN
O N N O N N O N N
CH3 CH3 CH3

theophylline caffeine theobromine
FIGURE 7.40
Biosynthetic origins of the purine alkaloids theophylline, theobromine, and caffeine.
Both tea and coffee contain some amount of these substances, though caffeine
is by far the principal component responsible for the CNS stimulant action of these
beverages, with the others appearing in only trace amounts. Caffeine also acts as a di-
uretic (increases elimination of water from the body) though its action in this regard
is weaker than that of either theobromine or theophylline, both of which are found in
significant amounts in cocoa (cacoa) bean. Theophylline is often used in treatments
for asthma and chronic obstructive pulmonary disease (COPD) due to its ability to
induce relaxation of bronchial smooth muscle. Theobromine (which does not contain
bromine) is sometimes used therapeutically as a heart stimulant or as a vasodilator
and is the ingredient responsible for the well-known toxicity of chocolate in dogs, due
mainly to their comparatively slow metabolism of theobromine. Like caffeine, theo-
bromine is mildly addictive, and the irresistible lure of dark chocolate for many devo-
tees is due in part to its high levels of theobromine relative to simple milk chocolate.
CYCLIC AND MACROCYCLIC PEPTIDES: FROM

SWEETENERS TO ANTIBIOTICS AND BEYOND
In addition to the many transformations of amino acids we have seen thus far for the
formation of alkaloids, amino acids can also play an important role in the construc-
tion of complex alkaloid structures in which the amino acid components remain
more or less intact by being linked to one another primarily through the formation
of peptide bonds. This is particularly important, as we will see, in the formation of
some medicinally important cyclic and macrocyclic polypeptides.
A unique group of such alkaloids, known as the 2,5-diketopiperazines, illus-
trates how simple cyclic peptides can arise when two amino acids are linked to

one another via sequential formation of bimolecular and intramolecular peptide

bonds, as shown in Fig. 7.41. A common example of such a structure is found when
the artificial sweetener aspartame, a simple dipeptide methyl ester derived from
phenylalanine and aspartic acid, undergoes cyclization during heating to form the
corresponding product aspartame diketopiperazine, sometimes referred to as
aspartame DKP. Over the years, amateur consumer health advocates have often
implicated aspartame DKP and its metabolic byproducts (along with traces of meth-
anol released during metabolism of aspartame) as being responsible for a host of
alleged side effects ranging from psychological disorders and convulsions to birth
defects and cancers, all supposedly related to ingestion of aspartame, in spite of a
host of exhaustive scientific studies completely supporting its safety at normal levels
of consumption.
Also shown in Fig. 7.41 is brevianamide F, a diketopiperazine formed via cy-
clization of tryptophan and proline which serves as the precursor to a large family
of modified derivatives such as tryprostatin A and tryprostatin B, both of which
are isolated from the marine fungus Aspergillus fumigates. Tryprostatin A has been
found to act as an inhibitor of a key protein that regulates resistance to breast cancer
chemotherapy agents, while tryprostatin B has chemotherapeutic potential due to its
action as a mammalian cell-cycle inhibitor. Both compounds are viewed as impor-
tant targets for further studies in cancer chemotherapy.
The number of larger macrocyclic polypeptides of medicinal importance is sub-
stantial, so only a few representative examples of the principal types can be treated
O
R' O
HO
R'
NH2 HN
NH2
NH
OH R
R 2 H2O
O
O
a 2,5-diketopiperazine
O O
H3CO
O NH2 heat O HN
NH NH
HO HO
CH3OH
aspartame
O aspartame O
diketopiperazine
O
OH
O O
NH2 H R H
HN
tryptophan N N
+ HN HN HN HN
HN
H2N brevianamide F H H
O O
O proline tryprostatin A (R = OCH3)
tryprostatin B (R = H)
FIGURE 7.41
Formation of 2,5-diketopiperazines from aspartame and from tryptophan and proline.

here. Figure 7.42 shows the structures of two such compounds. Valinomycin is a
Bioorganic Synthesis 346 Streptomyces-derived example of a naturally occurring macrocyclic depsipeptide, a
type of peptide compound in which amide (often peptide) bonds are accompanied
by ester linkages. In the case of valinomycin, all the nitrogens and some of the car-
bonyls in the amide linkages are provided by valine (both D-and L-forms) while
L-lactic acid provides the other amide carbonyls. The hydroxyl groups required for
the ester linkages are all provided by incorporation of D-α-hydroxyisovaleric acid.
Valinomycin, like the polyketide monensin A (Fig. 5.71), is a crown ether-like iono-
phore, with antitumor, antifungal, and antibacterial activity, that is able to bind and
transport potassium ions across hydrophobic lipid membranes via interaction of the
metal cation with carbonyl oxygen lone pairs. It has also been shown to be highly
active against the corona virus responsible for severe acute respiratory syndrome
(SARS) in an infected test cell line.
Bacitracin A is one member of a group of nine structurally related broad spec-
trum antibiotics from Bacillus subtilis that acts by disrupting cell wall synthesis
in bacteria. Structurally, the compound is made up of a mixture of both D-and
L-amino acids (see Fig. 7.42 for amino acid labelling) with a linear pentapetide seg-
ment joined to a cyclic heptapeptide component. The assembly of these (and other)
cyclic polypeptides is usually accomplished via a modular multifunctional enzyme
similar to the type I polyketide synthase (type I PKS) systems we encountered pre-
viously (see Table 5.2). The growing peptide chain is passed along from module to
module, terminating in a cyclization step somewhat analogous to the TE domain-
mediated lactone formation step in the biosynthesis of 6-deoxyerythronolide B (see
H HN N
N
O O
O O NH HO O His
HN O O
O
O O NH HN Phe
O O D-Asp
O
O valinomycin
O O NH HN
Asn O
O O NH
O O Ile
HN O O
HN O
O NH2 HN
O D-Orn
NH
bacitracin A Lys HN
O O NH2
HO D-glu NH O
H2N
HN Ile
S
O
Ile N NH O
Cys H
N Leu
O
FIGURE 7.42
Structures of the macrocyclic polypeptide antibiotics valinomycin and bacitracin A.

Fig. 5.66). Bacitracin is frequently blended with the polysaccharide antibiotic neo-

mycin (see Fig. 3.16) and polymyxin B (structurally similar to bacitracin) in topically
applied “triple antibiotic” lotions or ointments for treatment of superficial wounds
or infections. The effectiveness of such applications is suspect, however, and some
research suggests that widespread use of bacitracin, particularly from incorporation
into animal feeds, has contributed to the development of MRSA strains. Ophthalmic
solutions of bacitracin are also widely used for treatment of eye infections.
Another interesting cyclic polypeptide antibiotic isolated from Streptomyces
strains is dactinomycin, a potent but toxic antibiotic that is used primarily in cancer
chemotherapy. Structurally, dactinomycin is essentially a dimer of a cyclic hexa-
peptide initially constructed from 3-hydroxy-4-methylanthranilic acid to which
threonine, D-valine, proline, N-methylglycine, and N-methylvaline are added in
sequence prior to a final lactone cyclization (Fig. 7.43). The resulting cyclic hexapep-
tide then undergoes an unusual type of oxidative phenolic coupling reaction involv-
ing the amino-substituted aromatic ring of the 3-hydroxy-4-methylanthranilic acid
residue, as shown in Fig. 7.43. The mechanism of this process is not fully understood,
but could reasonably involve the sequence shown, beginning with oxidation of the
ortho-aminophenol to the corresponding ortho-quinone imine. The amino group
of a second molecule of the monomer then may act as a nucleophile in a conjugate
addition to the α,β-unsaturated imine to give an enamine which, following a second
oxidation, affords a para-quinone imine. Intramolecular conjugate addition to the
α,β-unsaturated ketone function by the proximal phenolic hydroxyl group results in
formation of the central N,O-containing six-membered ring. Tautomerization and
a final oxidation complete the conversion to the dimeric structure of dactinomycin.
As the first antibiotic found to have anticancer activity, dactinomycin is one of
the oldest of the cancer chemotherapy agents and it is still in wide clinical use today.
Several types of rare childhood cancers such as Wilms’ tumor (a kind of kidney
cancer), rhabdomyosarcoma (cancer of the muscles), and retinoblastoma (a type
of childhood eye cancer) respond to dactinomycin chemotherapy as do certain tes-
ticular, bone, and muscle cancers in adults, often in combination treatment with
other anticancer agents. It is also used for the treatment of gestational trophoblastic
neoplasia, a rare type of pregnancy-related cancer.
An example of more extensive use of phenolic oxidative coupling for the for-
mation of a macrocyclic polypeptide structure is found in the biosynthesis of
vancomycin, isolated from the soil bacterium Amycolatopsis orientalis and one
of the most important clinical antibiotics in use worldwide for the treatment of
serious bacterial infections. Formally classified as a glycopeptide antibiotic, van-
comycin is derived via the assembly of a basic linear heptapeptide composed of
D-leucine, asparagine, two β-hydroxytyrosines, two (p-hydroxyphenyl)glycines, and a
(3,5-dihydroxyphenyl)glycine as shown in Fig. 7.44. Two aryl ring chlorinations
are then followed by oxidative phenolic coupling reactions which form key aryl
C–O–C and aryl C–C bonds found in the macrocyclic core. SAM methylation of


O
CO2H N
NH2 1. threonine NCH3
2. D-valine
3. proline O O2
HN O oxidative
OH 4. N-methylglycine O NCH3 coupling
5. N-methylvaline of two
CH3 6. cyclize to lactone O units
HN O
3-hydroxy-4-methyl
anthranilic acid O CH3
NH2
OH
CH3
O O O O O O
H H
N NH2 NH2 NH NH NH2
O2
OH O OH O O OH
CH3 CH3 CH3 CH3 CH3 CH3
O2 conjugate addition to ortho-quinone
ortho-quinone imine imine
O O O O O O
N NH2 N NH2 N NH2
tautomerization
OH O O OH O O
CH3 CH3 CH3 CH3 CH3 CH3
conjugate addition to
para-quinone imine O O
NCH3 N N H3CN
O O O O
NCH3 H3CN
HN NH
O O O O
O O O2
H3C HN O O NH CH3
N NH2
O O
CH3 CH3
dactinomycin
FIGURE 7.43
Proposed assembly sequence for construction of the macrocyclic antibiotic dactinomycin.
the N-terminal D-leucine residue is then followed by a phenolic hydroxyl glucosyl-

ation. Final formation of a 2-O-glycoside linkage between the glucose residue and
the aminosugar vancosamine completes the sequence.
Often referred to as “the antibiotic of last resort” for gram-positive bacterial in-
fections involving multiple-antibiotic-resistant strains (especially MRSA strains) or
for patients allergic to β-lactam antibiotics such as penicillins and cephalosporins,
vancomycin is a vital weapon in the antibiotic arsenal, particularly in hospital set-
tings. Unfortunately, the widespread use of vancomycin over the years has led to
increasing concerns about the evolution of vancomycin-resistant (VR) bacterial

OH OH

H2N CO2H
H2N CO2H HO OH
D-leucine HO asparagine
HO
OH
H2N H2N CO2H OH H N CO2H
CO2H 2
HO OH
OH
O
CO2H peptide
NH2 O NH2 O HN assembly
H H H
N N N O 2x
N N chlorination
H H
O
O O
HO OH
OH HO
Cl OH Cl
3 x phenolic
addition of OH
oxidative glucose, then HO
coupling, vancosamine OH
then SAM
O
CO2H
NHCH3 O NH2 O HN
H H H
N N N O
N N
H H
O
O O
HO OH
O O
Cl Cl
O O
HO
O
HO O OH
OH
NH2
vancomycin
FIGURE 7.44
Polypeptide assembly, oxidative coupling, and glycosylation in the biosynthesis of vancomycin.
strains, particularly Staphylococcus aureus (VRSA strains). Such concerns have led
to limitations on the use of vancomycin to specific instances such as life-threatening
infections for which less toxic agents are ineffective, certain cases of Clostridium dif-
ficile (C. diff)-related colitis and for precautionary treatment of particularly serious
infections during bacterial identity culturing, among others.
PENICILLINS, CEPHALOSPORINS, AND

CARBAPENUMS: THE ESSENTIAL Β-L ACTAM
ANTIBIOTICS
In 1928, the Scottish scientist Alexander Fleming accidentally discovered a curi-

ous substance with apparent antibacterial activity from cultures of Penicillium fungi.
Dubbed penicillin, this fungal metabolite eventually became the target of intensive
research efforts which would ultimately lead to the development of the world’s first
medically practical antibiotic, though it would take nearly 17 years from the time of

its discovery to determine its unique β-lactam structural feature. The discovery of
Bioorganic Synthesis 350 penicillin may be said to have independently introduced the age of antibiotics, an
era in which countless human lives have been saved over the years. And it is not a
stretch to say that the many wondrous achievements of antibiotic research, develop-
ment, and clinical application in modern medicine have ultimately been the fruit of
Fleming’s early blend of expertise and curiosity.
The β-lactams are the most important group of antibiotics in terms of over-
all usage, accounting for over half of all sales of antibiotics worldwide. As we will
see, this group includes a variety of closely related structures: the penicillins, the
cephalosporins, and the carbapenums, all of which share a β-lactam linkage whose
strained four-membered ring is susceptible to nucleophilic attack by –CH2OH
residues found in bacterial enzymes that catalyze the formation of cell wall cross-
linkages. The resulting acylated enzymes are thus deactivated, leading to relatively
rapid cell death due to cell wall degradation. Penicillin-resistant bacteria are those
which have evolved the ability to produce a substance known as a penicillinase, an
enzyme that catalyzes the hydrolysis of the β-lactam linkage prior to its ability to
interact with these cell wall enzymes. The ever-increasing levels of bacterial resis-
tance to all types of antibiotics are of grave concern to the medical establishment
worldwide and are among the main drivers for the ongoing development of ever
more sophisticated and potent antibiotics.
Figure 7.45 shows the basic assembly steps involved in the biosynthesis of ben-
zylpenicillin (penicillin G) and the versatile and equally important precursor,
6-aminopenicillanic acid, along with a simplified scheme representing the basic
interaction of the β-lactam ring with a bacterial cell wall enzyme.
The biosynthesis begins with the amino acid L-lysine which undergoes trans-
amination to the corresponding aldehyde which is further oxidized to give L-2-
aminoadipic acid. A peptide linkage is then formed with L-cysteine followed
by peptide bond formation with D-valine. The resulting simple tripeptide, L-δ-
(α-aminoadipoyl)-L-cysteinyl-D-valine, then undergoes an intriguing oxida-
tive cyclization involving an enzyme-bound Fe-oxygen species coordinated to the
cysteine thiol function. This complex radical process is catalyzed by the enzyme
isopenicillin N synthase and is believed to involve the steps shown and results in
formation of the β-lactam ring-containing precursor isopenicillin N. At this stage,
a key peptide bond is hydrolyzed to release 2-aminoadipic acid and the essential
core compound 6-aminopenicillanic acid (6-APA) from which other penicillin
derivatives may be produced semisynthetically. Acylation of the amino group by
phenylacetylCoA leads directly to benzylpenicillin, also known as penicillin G,
the initial product of commercial fermentation processes used in the production
of other penicillins. Because of its tendency to undergo acid hydrolysis and struc-
tural rearrangement in the stomach, penicillin G is administered intravenously or
by injection, allowing higher concentrations of antibiotic than are usually available
with more stable penicillin derivatives that are administered orally. Penicillin G is

NH2 H

PLP N
lysine CO2H cysteine HO2C SH
transamination HO2C
then NAD+ NH2 O
oxidation 2-aminoadipic acid O OH
(epimerized
valine from L- to
(II) (III) D-valine)
S Fe Fe
O S O H
HO2C N
OH SH
H O Fe(II), O2
H isopenicillin N NH2 O H
H O N
O N O N synthase
H H
H
HO2C HO2C
HO2C
L-δ-(α-aminoadipoyl)-
L-cysteinyl-D-valine
H2O
(IV) H
H H H2N
Fe HO2C N S
S
S O H2 O
N
NH2 O N
H (II) O
N O 2-amino- CO2H
Fe-OH CO2H
O adipic acid 6-amino-
isopenicillin N
CO2H penicillanic acid
COSCoA
H H H H
N N S
S
O HN O N
O O
O penicillin G CO2H
CO2H CoASH
(benzylpenicillin)
HO
bacterial cell
wall enzyme
(inactive) bacterial cell
wall enzyme
(active)
FIGURE 7.45
Assembly steps for β-lactam formation in the biosynthesis of penicillin G and a simplified
representation of the mode of action of β-lactam antibiotics.
also important because not only can it be commercially produced in large quanti-
ties by fermentation, it can also undergo either enzymatic or chemical hydrolysis to
produce pure 6-APA which can then be converted by simple synthetic acylation of
the amino group to give a variety of penicillin derivatives of diverse potency, stabil-
ity, and specificity. This is important because the early penicillins were active only
against gram-positive bacterial strains while later derivatives were developed which
are also active against gram-negative strains. Figure 7.46 shows a small sampling of
various penicillins derived from 6-APA. Though allergic reaction to penicillins is the
most commonly reported medication allergy, studies suggest only a small percent-
age of reported reactions are due to actual penicillin allergy.
First isolated from cultures of Cephalosporium acremonium in 1948, the cepha-
losporins may be thought of as close cousins to the penicillins. Both contain the
characteristic β-lactam ring, but in the cephalosporins, the sulphur-containing
ring is expanded from five to six atoms in size. The cephalosporin structure is
actually derived from the penicillin ring system itself by a sequence of oxidative


H H
N S
O
O N
O
NH2 CO2H NH2
H H penicillin V H H
N N S
S
O N O N
O H3CO O
CO2H amoxycillin CO2H
ampicillin
H
H2N S
N
O
CO2H
CO2H 6-aminopenicillanic acid
H H (6-APA) H
N S N
N S
O N N
O O
CO2H CO2H
carbenicillin OCH3 mecillinam
H H
N S
OCH3 O N
O
methicillin CO2H
FIGURE 7.46
Some structural variations in penicillin antibiotics derived from 6-aminopenicillanic acid.
transformations sometimes referred to as the penicillin-cephalosporin rearrange-

ment which is outlined in Fig. 7.47 along with the structures of cephalosporin C
and two members of the so-called “first generation” cephalosporins, cephalexin and
cephalothin. Most of the so-called five generations of cephalosporins are derived
from 7-aminocephalosporanic acid (7-ACA) or its less significant deacetoxy de-
rivative (7-ADCA). Both are the cephalosporin equivalents of the similarly versatile
6-APA for semisynthesis in the penicillin family.
Second generation cephalosporins such as cefuroxime and third generation ex-
amples such as cefdinir and cefixime became important partly due their greater
activity against gram-negative bacteria, though at the expense of gram-positive anti-
microbial activity. The later fourth and most recent fifth generation cephalosporins
are true “broad spectrum” antibiotics with activity against both types. The fifth-
generation compound ceftaroline is extremely important due to its activity against
multidrug-resistant VRSA/VISA (vancomycin intermediate Staphylococous aureus)
strains and its unique role as the only β-lactam with activity against MRSA strains.
The structure of ceftaroline shown in Fig. 7.48 is the active form of the drug which is
administered intravenously as ceftaroline fosamil, a prodrug in which the primary
amino group is phosphorylated to improve solubility. Note the unusual zwitterionic
form of fourth and fifth generation compounds.
We conclude this final section of our chapter with the so-called carbapenum class
of wide spectrum β-lactam antibiotics whose importance is related to their resistance

H H H H

HO2C N S PLP HO2C N S
epimerize
NH2 O N NH2 O N
O O
isopenicillin N CO2H penicillin N CO2H
O2 2-oxoglutarate
H H H H H H
N S N S N S
O N O N O N
O e- O O
H CO2H H CO2H CO2H
H+
H H H H
HO2C N S HO2C N S
O2
NH2 ON 2-oxoglutarate NH2 O N O
then O
O CH3COSCoA
deacetoxycephalosporin C CO H cephalosporin C CO2H O
2
hydrolysis hydrolysis
H H
H2N S H2 N S
N N O
O O
CO2H CO2H O
deacetoxy-7-aminocephalosporanic acid (7-ADCA) 7-aminocephalosporanic acid (7-ACA)
NH2
H H H H
N S N S
O N S O N O
O O
cephalexin CO2H cephalothin CO2H O
(Keflex) (Keflin)
FIGURE 7.47
The penicillin-cephalosporin rearrangement: formation of cephalosporin C along with two first
generation semisynthetic derivatives, cephalexin (from 7-ADCA) and cephalothin (from 7-ACA).
to β-lactamase-induced deactivation. They are especially significant as dependable

agents for treatment of infections due to β-lactam-resistant strains of E. coli and
Klebsiella spp., both of which can lead to a variety of diseases including pneumonia,
urinary tract infections, septicemia, and meningitis. Structurally, the carbapenums
derive their name from penum, the common name for the fused four-membered
β-lactam/five-membered sulphur-containing rings of the penicillins; substitution of
carbon for sulphur in the penum ring thus yields a “carbapenum.” A possible scheme
for biosynthesis of thienamycin, produced by Streptomyces cattleya and the first nat-
urally occurring carbapenum to be isolated, is shown in Fig. 7.49.
After intramolecular imine condensation of L-glutamic acid semialdehyde, nu-
cleophilic acylation of the imine linkage by an enolate ion derived from malonyl-
CoA leads to the indicated thioester. SAM methylation (presumably via the thioester
enolate) is followed by intramolecular amide formation to give the β-lactam linkage.
Oxidation of the five-membered ring by O2/2-oxoglutarate gives an α,β-unsaturated
carboxyl which adds an L-cysteine thiol group via conjugate addition. PLP-mediated
decarboxylation of the amino acid side chain and further oxidations introduce the

N OCH3

H H
N S O NH2
O
O N O
OH OCH2CO2H
N O N
N H H CO2H N H H
N S cefuroxime N S
S (second generation) S
O N H2N O N
H2N
O O
cefdinir CO2H cefixime CO2H
H
(third generation) H2N S (third generation)
N O
O
CO2H O
7-aminocephalosporanic acid CH3
(7-ACA)
OCH3 N
N OCH2CH3
H H N
N N S N
S H H
S N S
O N N N N N N
H2N
O H2N O N
S S
CO2- O
CO2-
cefozopran ceftaroline
(fourth generation) (fifth generation)
FIGURE 7.48
Some structural variations in second through fifth generation cephalosporins derived
from 7-ACA.
indicated unsaturation, while a final SAM methylation extends the vinylidene link-
age on the β-lactam ring. A final conjugate addition of H2O completes the sequence
to yield thienamycin.
Among the semisynthetic carbapenums (Fig. 7.50), imipenem was developed as
a stable derivative of thienamycin (which rapidly decomposes in aqueous solution).
Imipenum has a broad spectrum of activity against both gram-positive and gram-
negative bacteria, though it is not active against MRSA strains. Similar to imipenem
in terms of broad-spectrum activity and resistance to β-lactamases, meropenem,
doripenem, and ertapenum are somewhat better tolerated than imipenem when
high dosages are required. All are administered intravenously and, like vancomy-
cin, are considered among the antibiotics of last resort for many serious bacterial
infections, especially those related to the E. coli and Klebsiella spp.-related infec-
tions mentioned above. Unfortunately, the gradual spread of evolving resistance to
carbapenem antibiotics has raised alarms in recent years, making the development
of new antibiotics to combat such resistant strains an imperative for the safety and
health of future generations.
A FINAL LOOK AHEAD
There is a vast amount of information related to alkaloids to which a short chapter

such as this one could never hope to do even partial justice, so we are compelled

O O
imine
formation malonyl-CoA
N CoAS HN
H2N CO2H
H2O
CO2H CO2H
L-glutamic acid SAM
semialdehyde
CH3
H H3C H
H3C O
O2
N 2-oxo- N CoAS HN
glutarate
O O CoASH
CO2H CO2H CO2H
L-cysteine
NH2 H NH2
H3C H H3C
PLP
S CO2H S
N N
O CO2 O
CO2H CO2H
oxidations
OH
H H H
H2C H
H2O SAM
S S
N N S
N
O O
CO2H CO2H O
CO2H
NH2 NH2
thienamycin NH2
FIGURE 7.49
Possible biosynthetic pathway to the carbapenum antibiotic thienamycin.
O
H
OH OH N
N
H H H H
S S
N N
O O
CO2H NH CO2H
imipenem HN meropenem
O
H H
OH N SO2NH2 OH N
N NH
H H H H H
S S
N N
O O HO2C
CO2H CO2H
doripenem ertapenem
FIGURE 7.50
Semisynthetic carbapenem antibiotics: imipenem, meropenem, doripenem, and ertapenem.

to move on at this point to a final chapter that briefly considers how laboratory
Bioorganic Synthesis 356 chemists have harnessed the power of synthetic organic chemistry over the years to
carry out the painstaking, step-wise assembly of some of the most challenging or-
ganic structures produced by the pathways we have studied so far. Looking at these
examples should give us a deeper appreciation for the complexities, demands, and
limitations inherent in such organic synthesis projects, especially when compared
with the elegance, power, and simplicity of nature’s “cellular laboratory” approach to
construction of architecturally complex organic molecules.
STUDY PROBLEMS
1. Propose reasonable mechanisms for conversion of N-(4-oxodecanoyl)-

histamine to the alkaloids glochidine and glochidicine from a common
intermediate.
H H N
N N O
N
? +
N N HN N
N
n-C6H13 O n-C6H13 O
N-(4-oxodecanoyl)-
histamine n-C H O glochidine glochidicine
6 13
2. Suggest an alternative to the route shown in Fig. 7.11 for formation of L-pipecolic
acid from lysine.
H2N CO2H
N CO2H
NH2 H
lysine
L-pipecolic acid
3. The psychedelic alkaloid mescaline is structurally related to the neurotrans-

mitter dopamine and is produced by the peyote cactus. Similarly, psilocybin is
structurally related to the neurotransmitter 5-hydroxytryptamine and is pro-
duced by many species of mushrooms. Both illegal compounds were ritually
used by Native Americans for thousands of years owing to their strong halluci-
nogenic effects which are similar to those produced by LSD. Briefly outline the
likely steps involved in biosynthesis of mescaline and psilocybin from simple
amino acids.


H3C
HO
P N CH3
H3CO NH2 HO O
H3CO
N
OCH3 H
mescaline psilocybin
4. Tubocurarine, a toxic alklaoid component of curare, was once widely used

during surgeries for relaxation of skeletal muscles. Outline a sequence of likely
steps for biosynthesis of tubocurarine from (S) and (R)-N-methylcoclaurine.
H3CO
H3CO H3CO NCH 3

O OH H
NCH 3 NCH 3
HO HO ?
H + H
O
HO HO H
OH
(S)-N-methylcoclaurine (R)-N-methylcoclaurine (H 3C) 2N
OCH 3
tubocurarine
5. Propose a mechanism for conversion of (S)-autumnaline to isoandrocymbine,

an intermediate involved in biosynthesis of the medicinal antigout alkaloid
colchicine.
H3CO
NCH3 HO H3CO
HO
H NCH3 NHCOCH3
?
H3CO H3CO
OCH3 CH3O
O O
H3CO colchicine
H3CO OH OCH3
isoandrocymbine
OCH3
(S)-autumnaline
6. Provide a mechanism for the O2/2-oxoglutarate demethylation of thebaine to

neopinone in the morphine pathway. Why does neopinone spontaneously isom-
erize to codeinone?

H3CO H3CO

H3CO
O O2 O O
NCH3 isomerize
2-oxo- NCH3 NCH3
H H (non- H
glutarate enzymatic)
H3CO ? O O
thebaine neopinone codeinone
7. Metabolism of pethidine, a synthetic analgesic, gives norpethidine, a neuro-

toxin whose build up during high dosage pethidine treatment can lead to seri-
ous complications and even death. Conversion to norpethidine is carried out
mainly by the CYP enzymes. Propose a reasonable mechanism for formation of
norpethidine.
O
O
CYP enzymes
H3CH2CO
H3CH2CO
NH
NCH3
pethidine (Demerol) norpethidine
8. Contact with capsaicin, a component of cayenne and other hot peppers, produces
a strong burning sensation in most tissues. In addition to its use in foods, capsa-
icin is used in a variety of topical ointments and dermal patches for the relief of
pain. Considering the products produced from its hydrolysis, propose a reason-
able biosynthesis of capsaicin from vanillin and suggest a sequence for biosyn-
thesis of the 10-carbon carboxylic acid component, starting with isobutrylCoA.
H3CO
N
H
capsaicin
HO
9. Propose a mechanism for this laboratory transformation:
HO N N
H+
H2O
N OH N O
OH
H3CO H H3CO
O O

10. Compound A is a substituted pyrrole intermediate used in porphyrin biosyn-

thesis. Propose a detailed mechanism to account for the formation of A from
5-aminolevulinic acid as shown (hint: the mechanism requires no co-factors
or reagents other than water and a proton. You need only use imine formation,
imine-enamine tautomerism, and other intramolecular steps including a final
dehydration).
CO2H
CO2H CO2H CO2H
O O
N
H
NH2 NH2 NH2
A
5-aminolevulinic acid
11. It was demonstrated as early as 1917 that the bicyclic alkaloid A could be syn-
thesized in the laboratory from the three-component system shown below.
Propose a mechanism for the formation of A.
N
OHC CHO + CH3NH2 + HO2C CO2H H3C
O A O
12. A proposed biosynthetic route to alkaloid X involves L-

DOPA and 3-
aminopropanal as starting materials. Provide a reasonable mechanistic pathway
leading to X (hint: prior to the final cyclization, an oxidation to an ortho-
quinone may be involved).
CO2H
H H3CO
NH2
HO NH
+ H3CO
O
NH
HO
H X
NH2
13. If 13C-labeled tyrosine was used for biosynthetic labeling experiments, where
would the 13C labeled carbon atoms be located in the resulting morphine?

HO
CO2H
*
O
NH2 NCH3
HO
13 C
*= label
HO
morphine
14. The alkaloid pluviine is transformed into homolycorine by a sequence of

four simple steps. Outline those steps using intermediate structures, and so on
(hint: begin with oxidation at a benzylic carbon).
H3C N
HO
H3CO H3CO
N O
H3CO H3CO
pluviine homolycorine O
15. Show a mechanism for the formation of B from A, then propose a mechanism
for the acid-catalyzed rearrangement of B to give C.
H3CO
H3CO H3CO
NCH3 HO NCH3 NCH3

HO H+ OH
H H H
OCH3 H3CO
H3CO
A OH B C HO
O
16. Benzodiazepines are psychoactive drugs whose core chemical structure is the
fusion of a benzene ring and a 1,4-diazepine ring. Though discovered and
marketed as synthetic drugs (Librium, Valium, etc.), one particular species of
Penicillium mold produces the natural benzodiazepine shown here which shows
selective inhibition against acetylcholinesterase (AChE) and moderate antibi-
otic activity. Identify the likely amino acid precursors to this alkaloid, then sug-
gest a reasonable biosynthetic scheme.
O
CH3
N H
O
N
H O

8 Organic Synthesis in the

Laboratory
To the field of synthetic chemistry belongs an array of responsibilities which are crucial for
the future of mankind, not only with regard to the health, material and economic needs of
our society, but also for the attainment of an understanding of matter, chemical change and
life at the highest level of which the human mind is capable.
—E. J. Corey (Nobel Prize in Chemistry, 1990)
The German chemist Friedrich Wöhler is generally credited with the first labora-
tory synthesis of a known organic compound (urea) from inorganic materials. He
accomplished this by the simple heating of an inorganic salt, ammonium cyanate
(NH4OCN). “I must tell you,” he wrote to his mentor Jöns Jakob Berzelius in 1828,
“that I can prepare urea without requiring a kidney of an animal, either man or dog.”
While this report may seem relatively minor given the structural simplicity of urea,
its impact was revolutionary. For the first time, the preparation and isolation of an
organic compound had been achieved in the absence of the elemental “vital force”
of living systems previously believed to be required for the construction of all such
compounds. This milestone of 19th century organic chemistry was later followed by
many others, including Kolbe’s synthesis of acetic acid in 1847 and Fischer’s synthe-
sis of glucose in 1890. With the support of evolving methods for compound separa-
tion, purification, and spectroscopic analysis, rapid advances in the sophistication
of organic synthesis followed throughout the 20th century, developing in tandem
with an ever-deepening understanding of the underlying organic processes associ-
ated with living systems. While it is certainly true that syntheses of many structur-
ally complex unnatural compounds of theoretical interest are also among the most
361

remarkable achievements in synthetic strategy, tactical execution, and perseverance,

Bioorganic Synthesis 362 the realm of natural products remains the dominant source for the most challeng-
ing and potentially beneficial targets available for such synthetic efforts. Figure 8.1
shows a small selection of some natural (and unnatural) products which have been
produced via synthesis over the years, from Wöhler’s time to the present. Note the
increasing levels of structural sophistication and stereochemical complexity that
have eventually been mastered by practitioners of organic synthesis.
In our own time, the traditional boundaries between organic and biological chem-
istry are disappearing in ways that are likely to transform the design and synthesis of
organic molecules, from the construction of synthetic biologicals designed to act as
biomarkers, biosensors, or drug delivery agents, to the development of molecular
motors, self-replicating macromolecular systems, and even synthetic life forms. But
regardless of their complexity, such efforts will always draw to some extent upon a
vast well of knowledge of reactions, mechanisms, techniques, and accomplishments
accumulated over nearly two centuries of careful and exhaustive research in synthetic
organic chemistry—work often inspired by a drive to emulate the sophistication and
elegance of biosynthetic processes and the remarkable products they produce.
In this brief final chapter, we can touch on only a few of the many essential aspects
of organic synthesis, bearing in mind the introductory nature of our text and the need
to avoid excessive complexity or terminology unfamiliar to the audience. But even
within such limits, we can still obtain a deeper level of appreciation for the signifi-
cance, sophistication and allure of the art and science of synthetic organic chemistry.
WHY WE SYNTHESIZE ORGANIC COMPOUNDS
Organic chemistry is not only a science—it is big business. And the list of industrial
organic compounds and materials manufactured for profit is a long and ever-growing
one: organic conductors and semiconductors, liquid crystals, plastics and polymers,
dyes, lubricants, fuels, propellants, coatings, solvents, explosives, pesticides, fertiliz-
ers, and weed killers are just some of the examples. Many of these commercial prod-
ucts are produced from raw organic materials derived from petroleum while others,
such as flavors, fragrances, food additives, and preservatives or certain components
used in cosmetics and personal care, are partially or wholly derived from natural
sources. Some products may be of limited availability from natural sources but also
relatively simple in terms of their structural features, making commercial synthesis
potentially cost-effective. By contrast, many other naturally occurring compounds,
particularly those used as medicinal agents and pharmaceuticals, can possess re-
markably complicated structures, and so isolation from natural sources via extrac-
tion or fermentation may be almost imperative since commercial synthesis may not
be economically feasible. Alternatively, certain complex products of limited natural
availability may nonetheless be obtainable by chemical modification of a more abun-
dant, structurally similar natural product. Regardless of the context, the synthesis of

363 Organic Synthesis in the Laboratory

HOH
O HO O
HO
HO H H H
H 2N NH2
urea H OH
OH HO
1826 glucose camphor
1903 equilenin
1890 1939
O
N
H
N H OH
OH
H OH
O H
N O OH
H
O cubane
O 1964 O OH
N
quinine strychnine
1954 erythronolide B
1944 1978
OH
OH
O OH
HO O OH OH OH OH O
CO2H
dodecahedrane
1982 O O
amphotericin B
1987
HO OH
HO O
NH2
O O
O OH
HN
O OH
H
O O
HO O H
O H
O
OH CO2-
O
pinnatoxin A amphidinolide F
1998 2013
FIGURE 8.1
A group of selected organic compounds produced by synthesis from 1828 to the present.
organic compounds, from the simplest to the most intricately structured, is a large
and often profitable enterprise that continues to command the attention of organic
chemists in both industry and academia on a worldwide basis.
Our focus here, of course, is mainly on the synthesis of interesting or useful com-
pounds that are known to be produced by biosynthetic processes. With that in mind,
a partial list of some of the many reasons for undertaking such synthetic efforts
includes:
• advancement of new synthetic reactions or methods;

• investigation of approaches to new or intellectually challenging structural
features;

• development or improvement of a commercial synthesis process;

Bioorganic Synthesis 364 • provision of sufficient quantities for biological studies/testing;
• development of processes that mimic biosynthesis (biomimetic);
• determination or confirmation of molecular structure;
• identification of structural features required for biological activity;
• determination of molecular mechanism of biological action;
• synthesis of structural analogues.
At this point, let’s look more closely at several of these points and at some of
the different approaches to synthesis that organic chemists have developed over
the years to overcome some of the considerable obstacles one may encounter when
trying to match the synthetic prowess of nature.
SYNTHETIC CHALLENGES: TOTAL SYNTHESIS
The term total synthesis refers to the complete construction of a natural product
from relatively simple and available organic starting materials. Total synthesis of
complex organics is frequently employed by academic chemists to test or to demon-
strate the utility of new reactions or new approaches to various structural features.
These were the key drivers for total synthesis of many of the more complex examples
shown in Fig. 8.1. A relatively simple example of a total synthesis is Stork’s approach
to grandisol (shown in Fig. 8.2), one of four monoterpenoid components of the
sex attractant for the male boll weevil (Anthonomus grandis), a major pest of cotton
crops in the western hemisphere. This synthesis is especially notable for its unique
approach to formation of the cyclobutane ring, a notoriously difficult ring system to
prepare synthetically.
The synthesis scheme begins with a seven carbon cyanoalkene 8.2a which is
deprotonated at the position α to the cyano group (recall the enhanced acidity at
that position). The resulting anion is then reacted with 2-bromo-1-heptanol THP
(tetrahydropyranyl ether-protected alcohol) in a simple SN2 reaction to give the al-
kylated product 8.2b. Alkene epoxidation then affords 8.2c which again undergoes
deprotonation α to the cyano group, with the resulting anion acting as a nucleo-
phile to attack the epoxide ring in an intramolecular fashion, leading to formation
of the cyclobutane ring of 8.2d as shown. Partial hydride reduction of the cyano
group triple bond with diisobutylaluminum hydride (DIBAL-H, similar in action
to LiAlH4 or NaBH4)) gives an intermediate imine which is then hydrolyzed to the
corresponding aldehyde 8.2e. Wolff–Kishner reduction of the aldehyde carbonyl
to a methyl group gives 8.2f which then undergoes chromic acid oxidation of its
secondary alcohol function to yield the ketone 8.2g. After Wittig reaction to con-
vert the ketone function to the indicated alkene 8.2h, the synthesis is completed by
acidic removal of the THP protecting group to release the primary alcohol func-
tion of the final product, grandisol. It is worth noting that while the stereochemical

(–OH protecting group)
CN NC CN CN
Br OTHP
LiN(iPr)2 OTHP OTHP

mCPBA
(acidic SN2
(epoxidation)
proton c
removal) O
8.2a 8.2b
(acidic
LiN(Si(CH3)3)2 proton
removal)
OTHP CN
CH=O CH=NH CN
OTHP OTHP
H3O + (iBu)2AlH H+ OTHP
OH (imine OH OH
(nitrile-to-
hydrolysis) imine
8.2e H reduction) H d O
H
(epoxide
H2NNH2 (Wolff–Kishner ring opening)
KOH reduction)
OTHP OH
OTHP OTHP
H3C H3C H3C
H3C
H2CrO4 Ph3P=CH2 H3O +
OH (C-OH to O
(Wittig (THP ether
C=O) hydrolysis)
8.2g Reaction)
H 8.2f H 8.2h H
H
(±)-grandisol
FIGURE 8.2
Stork’s total synthesis of grandisol, a terpenoid component of boll weevil sex attractant.

H+
OH
H3C
OH OH
H H
(1R,2S)-grandisol
geraniol
FIGURE 8.3
Presumed enzyme-mediated biosynthesis of (1R,2S)-grandisol.
structure of the final product as shown in Fig. 8.2 is the naturally occurring (1R,2S)
diastereomer, the product actually produced by this synthetic approach is a (±) ra-
cemic mixture of the (1R,2S) and (1S,2R) diastereomers. While several other total
syntheses of grandisol have successfully produced the final product in its optically
pure (R,S) form, we note here that any total synthesis which produces an optically
pure product is usually much more complex and difficult to achieve than one which
produces only a racemic product. Nevertheless, even racemic syntheses of grandisol
must properly address the stereochemical relationship of the three substituents on
the cyclobutane ring.
Compare the multistep nature of the synthetic approach to grandisol above with
the relative simplicity of its biosynthesis from geraniol as outlined in Fig. 8.3. Note
the cyclization of a carbocation intermediate to produce the required 4-membered
ring. Such site-specific protonation and 4-membered ring cyclization cannot be
achieved by any known synthetic processes; they are only feasible when the substrate
is regiospecifically protonated and held in the necessary strained folding pattern by
the enzyme that mediates the process. Furthermore, note that the enzyme-mediated
cyclization leads to formation of only the optically pure (1R,2S) diastereomer.
Nature makes it look so easy.
SYNTHETIC CHALLENGES: SEMISYNTHESIS
Unlike total synthesis, semisynthesis (or partial synthesis) is a process in which a

target compound is produced by synthetic modification or elaboration of a struc-
turally similar compound which is readily available in reasonable quantities from
natural sources. This approach can be especially useful commercially if the target
itself is produced naturally in only limited amounts and possesses multiple chirality
centers or other complex stereochemical features that would make total synthesis
cost-prohibitive. To begin our analysis, let us first consider the complexity involved
in achieving another total synthesis of an important compound, in this case the
steroid progesterone (as outlined in Fig. 8.4), before we illustrate the benefits of a
semisynthetic approach to the same compound.
This lengthy synthesis begins with reaction of 2-methylpropenal with an acety-
lenic Grignard reagent, affording secondary alcohol 8.4a. The alcohol function of

FIGURE 8.4
Johnson’s total synthesis of the sex hormone progesterone.
8.4a is then reacted with 1,1,1-triethoxyethane (triethylorthoformate) in an acid-

catalyzed sequence of alcohol exchange and alcohol elimination to give derivative
8.4b. Recall (from Fig. 6.6) that allyl vinyl ethers of this type may be induced to
undergo the Claisen rearrangement—in this case, by heating. The result here is
formation of ethyl ester 8.4c which is then converted to alcohol 8.4d via standard
LiAlH4 reduction. Oxidation of 8.4d with CrO3-pyridine complex (similar to pyri-
dinium chlorochromate or PCC) then gives aldehyde 8.4e. A Wittig reaction of this
aldehyde with phosphorus ylide 8.4f (prepared via a separate synthetic sequence
requiring many steps) gives the indicated alkene 8.4g. Hydrolysis of the ethylene
glycol acetal functions of 8.4g then leads to diketone 8.4h which is reacted with

base under standard aldol condensation conditions to produce the cyclopentenone

Bioorganic Synthesis 368 derivative 8.4i, with subsequent addition of methyllithium to the ketone function
giving the expected alcohol 8.4j. Acid-catalyzed dehydration of this tertiary allylic
alcohol with trifluoroacetic acid leads to formation of carbocation 8.4k which un-
dergoes a cationic cyclization cascade similar to the one we have seen previously in
the biosynthesis of cholesterol (see Fig. 4.38). The resulting tetracyclic ring system
terminates in the vinylic cation 8.4l which is subsequently trapped by a cyclic car-
bonate ester acting as a nucleophile, giving rise to the more stabilized carbocation
8.4m. Ring opening of this cation via nucleophilic attack by trifluoroacetate ion
quenches the carbocation, giving rise to the mixed carbonate-trifluoroacetate ester
derivative 8.4n. Ester hydrolysis with aqueous base cleaves this complex side chain,
releasing enol 8.4o which rapidly tautomerizes to methyl ketone 8.4p. The synthesis
is completed via ozonolysis of the cyclopentene ring double bond which yields the
corresponding diketone 8.4q. A final intramolecular aldol condensation forms the
required cyclohexenone ring system of progesterone.
It is worth reflecting on the careful synthetic planning and execution required
for this remarkable outcome. Especially noteworthy is the establishment of correct
stereochemistry at the molecule’s six chirality centers, though the product produced
here is racemic progesterone rather than the naturally occurring (+)-enantiomer.
While later (and more sophisticated) variants of this approach have successfully pro-
duced the optically pure product, the original process gives us a reasonably good
idea of the incredible intricacy involved in trying to produce a complex polycyclic
natural product with multiple stereocenters from relatively simple starting materials.
Recall from Chapter 4 that progesterone is produced biosynthetically via a se-
mediated transformations carried out on cholesterol (see
quence of enzyme-
Fig. 4.44). With that in mind, we may compare the above multistep total synthesis
approach to racemic progesterone with one of several known semisyntheses of pro-
gesterone, obtained in this instance from stigmasterol, an abundant plant sterol
similar in structure to cholesterol and available from soybean oil. An outline of this
semisynthetic process is shown in Fig. 8.5.
The stigmasterol hydroxyl group is initially converted to the corresponding
α,β-unsaturated ketone 8.5a by a process known as Oppenauer oxidation, a mild
oxidative method which uses aluminum isopropoxide dissolved in acetone solvent
(which also acts as a hydride acceptor from the secondary alcohol being oxidized).
Under the usual conditions for this oxidation, the nonconjugated β,γ-double bond
also isomerizes into conjugation with the newly formed steroidal ketone. Next, a
simple ozonolysis of the side chain double bond of 8.5a gives the corresponding
aldehyde 8.5b which is then converted to enol acetate derivative 8.5c via treatment
with acetic anhydride. A final ozonolysis to selectively cleave the enol acetate double
bond of 8.5c yields (+)-progesterone. Note that the semisynthetic approach is not
only much simpler than a total synthesis, it also produces optically pure product
rather than a racemic mixture.


H O H
Al 3
H H H H 8.5a
(–)-stigmasterol O3
HO O O
O
O
acetic
O3 O anhydride O
H
NaOAc
H H
O H 8.5c H 8.5b
(+)-progesterone
FIGURE 8.5
Heyl and Herr’s semisynthesis of progesterone from stigmasterol.
Aside from its significant role as the principal female sex hormone involved in
pregnancy, menstruation, and embryogenesis, progesterone is also an important
precursor both biosynthetically and semisynthetically to other important steroids
such as cortisol. This is but one of many known examples of the economical produc-
tion of an important yet relatively scarce natural product via chemical transforma-
tion of a structurally related but more abundant phytochemical (plant chemical).
SYNTHETIC CHALLENGES: BIOMIMETIC SYNTHESIS
In spite of the ingenuity and intricate planning obviously involved, only one of the
two foregoing examples of terpenoid natural product total syntheses actually bears
much resemblance to the process employed by nature for construction of those
target compounds (see the cationic cyclization step in Johnson’s synthesis of pro-
gesterone, Fig. 8.4). An equally sophisticated challenge in total synthesis involves ef-
forts to devise synthetic approaches which closely mimic the biosynthetic chemistry
actually used (or believed to be used) by the producing organism. Such a scheme is
usually referred to as a biomimetic synthesis. In some cases, the work may even use
biochemical reagents or cofactors in an effort to mimic nature as closely as possible.
The justification usually cited for undertaking a biomimetic synthesis is to support
or to challenge a specific biosynthetic proposal, although in some cases compet-
ing proposals have been supported by correspondingly different biomimetic ap-
proaches. Regardless, one benefit of successfully imitating nature is that biomimetic
syntheses are often relatively efficient and elegant compared to other approaches.
It is generally agreed that the first biomimetic synthesis was achieved in 1917 by
Sir Robert Robinson in his unique approach to the construction of the tropane alka-
loid tropinone, as shown in Fig. 8.6 (note the close resemblance to the biosynthetic
process previously outlined in Fig. 7.5). As in the biosynthetic scheme, Robinson’s
synthesis relies on sequential Mannich reactions for formation of the key bonds in


N
CH3NH2 + HO2C CO2H H 3C
OHC CHO +
O
O
CH3NH2 tropinone
2 CO2
O CH3
N CO2H
N
H H3C
CO2H O
HO N CO2H CO2H
N N
CH3 H3C H3C
HO O OH
HO2C CO2H CO2H CO2H
OH
FIGURE 8.6
Robinson’s 1917 biomimetic synthesis of tropinone via sequential Mannich reactions.
H3CO H3CO
H3CO
HO
K3[Fe(CN)6] HO O
NCH3 NCH3 NCH3
HO
O O
desmethyl belladine narwedine
FIGURE 8.7
Barton’s biomimetic oxidative coupling: preparation of narwedine from desmethyl belladine.
the bicyclic ring system and subsequent decarboxylations of the β-ketoacid core to
reach the final product. Also note the simplicity of this process which is achieved in
a single three-component reaction using relatively simple starting materials. This
result strongly supported Robinson’s proposal that chemistry very similar to this
was likely used by nature to produce the tropane alkaloids, a proposal that has sub-
sequently been sustained by numerous meticulous studies over the ensuing years.
This example shows Robinson’s early insight into potential biosynthetic mecha-
nisms involved in the construction of key alkaloids. In 1932 he further proposed
that one of the key steps in the biosynthesis of morphine—formation of salutaridine
from (R)-reticuline—most likely involved an intramolecular oxidative coupling (see
Fig. 7.21). This proposal was eventually confirmed by Barton who also showed that
laboratory oxidation of desmethyl belladine with aqueous potassium ferricyanide
produced narwedine (Fig. 8.7); further reduction of the ketone function to the cor-
responding alcohol afforded galanthamine, an alkaloid which is now commercially
prepared due to its promise as a treatment for mild to moderate cases of Alzheimer’s
disease. Though the chemical yield of narwedine was small (0.97%) from this early

O O

Br DMF O
K2CO3 o
190 C
O OH O O O
(SN2) (Claisen) OH
sesamol sesamol allyl ether 2-allylsesamol
KOtBu DMSO
(isomerization)
O O O
PdCl2
+
NaOAc
O O O OH
CH3OH/H2O
O
(phenolic (E)-2-propenylsesamol
O oxidation)
O O radical O O
coupling
+
O O O O
O O
O 8.7a O
H Diels–
O O Alder
H
O O
O
carpanone O
FIGURE 8.8
Chapman’s biomimetic total synthesis of the lignan product carpanone.
example of biomimetic oxidative coupling, higher yielding oxidations have subse-

quently become a common theme over the years in many total syntheses using a
variety of more sophisticated oxidizing agents.
One of the best examples of such a biomimetic oxidative coupling process in-
volves the total synthesis of a shikimate natural product known as carpanone, a
compound lacking any intrinsic medicinal value but which is endowed with unusual
stereochemical complexity with its five contiguous chirality centers. Chapman’s el-
egant biomimetic approach to this lignan derivative is shown in Fig. 8.8 which begins
with O-allylation of sesamol followed by an aromatic Claisen rearrangement to give
2-allylsesamol. Base-catalyzed isomerization of the side-chain double bond into
conjugation with the aromatic ring then gives the essential starting material (E)-2-
propenylsesamol. Using a Pd-based reagent to achieve oxidative formation of the
corresponding phenoxy radicals, bimolecular C–C coupling of two allyl radical reso-
nance contributors leads initially to the dimer 8.7a which then undergoes an intra-
molecular hetero-Diels–Alder cyclization to afford carpanone directly. The product


H3C O H3C OH H3C OH
O O
HO in vivo H3CO HO
O OH O
SEnz HEnz,
SEnz H2O
OH O OH O OH O
8.9a alternariol
H3C O H3C OH
O HO
HO
H2O/CH3OH
O pH 6-9 O
SEnz Na+ or Mg+2
OCH3 O 25oC OCH3 O
8.9b (>75%) alternariol methyl ether
FIGURE 8.9
Laboratory cyclization and aromatization of a tetraketide to give alternariol methyl ether.
produced was racemic but nevertheless possessed the correct stereochemical rela-
tionships among all substituents at the molecule’s chirality centers. This is an excellent
example of a synthesis designed to confirm a proposed biosynthetic scheme, namely
the presumed formation of carpanone from (E)-2-propenylsesamol via an enzyme-
mediated oxidative coupling and subsequent Diels–Alder cyclization. To assemble
such a structurally complex target compound in such a simple, elegant fashion in the
laboratory remains an impressive achievement in biomimetic synthesis.
Products from the polyketide pathway, especially aromatic polyketides, would
seem to be obvious candidates for simple testing of the acetate hypothesis via a
biomimetic approach to their synthesis. As illustrated in Fig. 8.9, formation of an
advanced enzyme-bound thioester tetraketide precursor 8.9a (proposed earlier as
a possible intermediate arising from a simple heptaketide via condensation, cycliza-
tion, and aromatization) could adopt the indicated folding pattern leading to further
condensation and cyclization to give alternariol as shown. To test this proposal,
preparation of the structurally related ethylthioester tetraketide 8.9b was followed
by treatment under a variety of mildly basic conditions which invariably led to for-
mation of alternariol methyl ether as the sole product in >75% yield as shown.
While not conclusive evidence for a similar mechanism operating in vivo, this ex-
ample demonstrates the usefulness of laboratory synthesis for probing potential
mechanistic pathways in biosynthetic processes.
SYNTHETIC CHALLENGES: STRUCTURAL
REVISION OR CONFIRMATION
Many remarkable advances have been made over the years in spectroscopic deter-
mination of chemical structures, especially in the areas of x-ray crystallography, nu-
clear magnetic resonance (1H and 13C NMR) and mass spectrometry. Nevertheless,
organic synthesis continues to play a significant role in the determination or

confirmation of natural product structures. Interestingly, a typical year finds numer-

ous examples of the use of total synthesis in published revisions of previously misas-
signed natural product structures. These corrections range from minor amendment
of the stereochemical relationships among substituents to significant changes in
atom connectivity, ring size, or other major structural features.
A useful example that confirms one assigned structure for a natural product
while correcting the structure previously assigned to another is provided by a total
synthesis of the natural product pseudodeflectusin which had originally been
isolated from cultures of Aspergillus pseudodeflectus. This structurally interesting
isochroman had been shown to exhibit activity against several human cancer cell
lines, so confirmation of the originally assigned structure, as shown in Fig. 8.10,
was achieved via stereoselective synthesis and spectroscopic comparisons of the syn-
thetic product with the originally isolated fungal metabolite. Interestingly, it was
simultaneously confirmed that the previously assigned structure of another natural
product, aspergione B, was actually incorrect and that pseudodeflectusin and asper-
gione B were in fact the same compound since the full spectroscopic data for both
were identical in all respects. The originally proposed structure for aspergione B was
then independently synthesized as shown from the same precursor used in the total
synthesis of pseudodeflectusin. As expected, the 1H and 13C NMR data were similar
but not identical to those originally reported for aspergione B.
A somewhat simpler example of structural revision of a previously reported com-
pound is shown in Fig. 8.11. Intricatin, an antimutagenic homoisoflavonoid (“homo”
indicates an extra carbon, in this case between the pyranone ring and its attached
phenyl group) was isolated in 1989 from a Mexican desert plant and was originally
assigned the structure shown. However, independent total synthesis of this struc-
ture later showed that the 1H NMR data for the naturally occurring compound and
those of the synthetic material were not the same, leading to a search of the chemical
literature which ultimately revealed that 1H NMR data for intricatin and those of
another natural homoisoflavonoid isolated in 1987, 8-methoxybonducellin, were
identical, indicating that the original structure for intricatin had been misassigned
and that intricatin and 8-methoxybonducellin were in fact the same compound. The
location of a methoxyl group relative to an adjacent phenolic hydroxyl group in
compounds such as these would clearly be difficult to correctly assign via spectro-
scopic data alone and further emphasizes the usefulness of synthesis as an aid in
structure determination.
SYNTHETIC CHALLENGES: FORMAL SYNTHESIS
Formal synthesis of a compound involves development of a new synthetic route

to an advanced precursor which is known to be readily convertible to the target
compound by a previously published reaction or sequence of reactions. An early
example from 1935 is Percy Julian’s formal synthesis of the medicinal alkaloid

O OH O
O O
many synthetic steps all spectroscopic data
H O O also consistent with those
O previously reported for
i)
aspergione B
8.10a
pseudodeflectusin
ii) (AND revised structure for
CH3CH2MgBr then H2O aspergione B)
O
OH OH O O OH O O O
MnO2 O
O O
O
pyridine HO
8.10b 8.10c
OH
O OH O O
DIBAL-H H O
spectroscopic data
NOTconsistent O O O O
with those reported for H2 O O
isolated natural product
incorrect structure 8.10d
previously asssigned
to aspergione B
FIGURE 8.10
Total syntheses to confirm that i) structure of pseudodeflectusin and aspergione B are identical and ii) originally assigned structure for aspergione B is incorrect.


OH
OH OCH3
H3CO O OCH3
H
HO O dimethyl
O sulfate
base, NaHCO3,
aldol acetone O
O spectroscopic data NOTconsistent with
those reported for isolated natural
OCH3 product, so incorrect structure previously
asssigned to intricatin.
HO O OCH3
O
8-methoxybonducellin: spectroscopic data
identical with those reported for isolated intricatin
FIGURE 8.11
Total synthesis of structure originally assigned to intricatin and revision to the structure of
8-methoxybonducellin by comparison of spectroscopic data.
H3C H3C
NHCH3
CH3CH2O CH3CH2O
O Na NCH3
N ethanol N H
CH3 CH3
8.12a (±)-eserethole
H3C H3C
H3CHN O HO
NCH3 CH3NCO AlCl3
NCH3
O H
N N H
CH3 CH3
(±)-physostigmine (±)-eseroline
H 3C
CH3CH2O
N(CH3)2
N
probable structure for Robinson's
incorrect (±)-eserethole
FIGURE 8.12
Julian’s formal synthesis of physostigmine and correction of Robinson’s reported eserethole
synthesis.
physostigmine (see Fig. 7.27 and related text). This remarkable achievement was
the first synthesis of physostigmine and also involved correction of some previously
published work on the same compound by Sir Robert Robinson, the future British
Nobel laureate. Julian, an African-American organic chemist working in relative
obscurity at a small midwestern university, managed to complete the synthesis in 11
total steps over a period of three years. The last few steps of his approach are shown in
Fig. 8.12. A key intermediate compound in the synthesis, eserethole, had reportedly

been synthesized previously by Robinson, but based on his own careful work on re-
Bioorganic Synthesis 376 ductive cyclization of 8.12a using sodium metal in ethanol, Julian recognized that
the compound Robinson had synthesized and identified as eserethole must have
had a different constitution. Julian found that his racemic eserethole had chemi-
cal and physical properties different from Robinson’s racemic material, but after
chemical resolution to separate its enantiomers, his own synthetic l-eserethole
was identical in all respects to the naturally occurring product. Julian’s eserethole
was then converted to the phenolic derivative eseroline by treatment with AlCl3
to dealkylate the ethoxyl group after which he states in his original publication
that “since Polonovski and Nitzberg have described conversion of l-eseroline into
l-physostigmine by treatment of the former with methyl isocyanate, our synthesis
represents a complete synthesis of the alkaloid physostigmine.” This made Julian’s
a formal synthesis of physostigmine (although he also states in a footnote of the
paper that subsequent to submission of the original manuscript, he did in fact
complete the synthesis by repeating the above mentioned reaction of methyl iso-
cyanate with his synthetic l-eseroline in order to provide a requested sample of
pure l-physostigmine). Julian, the first African-American chemist elected to the
National Academy of Sciences, went on to further fame as a highly innovative en-
trepreneurial organic chemist whose many contributions to the field of natural
products chemistry included work that ultimately led to the economical availability
of previously scarce medicinal steroids such as cortisone, progesterone, and others
from stigmasterol and other plant-based sterols.
Another mechanistically interesting example of formal synthesis is shown in
Fig. 8.13 which outlines Evans’s formal synthesis of colchicine, an important medic-
inal alkaloid with anti-inflammatory properties long used as the standard treatment
for flaring symptoms of gout. Evans’s synthesis begins with quinone monoacetal
8.13a which is reacted with a sulphur ylide derived via deprotonation of trimethyl-
sulfoxonium iodide. This nucleophilic ylide adds to cyclic α,β-unsaturated ketones
in 1,4-fashion to give an enolate which then reacts intramolecularly with expulsion
of neutral dimethylsulfoxide (DMSO) to give a ring-fused cyclopropane deriva-
tive, in this case compound 8.13b. Reaction of the ketone function of 8.13b with
Grignard reagent 8.13c yields the corresponding tertiary alcohol 8.13d. Further
treatment of this alcohol with trifluoroacetic acid leads to dehydration and forma-
tion of a carbocation which is then trapped intramolecularly in an electrophilic
aromatic-substitution-type process. Subsequent protonation of the ketone function
then leads to a carbocationic rearrangement with 1,2-migration of the trimethoxyl-
ated aryl ring accompanied by cleavage of the cyclopropane linkage and concurrent
ring expansion of the spiro-fused 6,6 ring system to the fused seven-membered rings
of the tricyclic product 8.13e. This was finally followed by oxidative dehydrogena-
tion of 8.13e with DDQ (dichlorodicyanoquinone) to give the tropolone-containing
derivative desacetamidoisocolchicine which had previously been transformed via
several additional steps to colchicine itself by a number of other investigators.


O O O
H3C S CH2
CH3 H3C
H3C S
OCH3
OCH3 OCH3
O H3CO OCH3
H3CO OCH3 H3CO OCH3
8.13a DMSO
H3CO
H3CO
O
H3CO HO H3CO MgBr

OCH3 8.13c
CH3O
OCH3
H3CO OCH H3CO OCH3
H3CO 8.13d3 8.13b
CF3CO2H
2 CH3OH,
H3CO H3CO H3CO
H2O
H3CO H3CO H3CO

H
CH3O CH3O CH3O
H3CO OCH3
H3CO O O OH
H
H+
H3CO O H3CO H3CO

NH steps DDQ
H3CO H3CO H3CO
CH3O CH3O CH3O
O OCH3 8.13e OCH3
OCH3 O O
colchicine desacetamidoisocolchicine
FIGURE 8.13
Evans’s formal synthesis of colchicine via total synthesis of desacetamidoisocolchicine.
SYNTHETIC CHALLENGES: STEREOSELECTIVE
SYNTHESIS OF OPTICALLY PURE COMPOUNDS
Some of the syntheses we have encountered thus far have successfully assembled
some architecturally challenging target structures, and in most instances these el-
egant synthetic approaches have been used to produce chiral compounds. However,
the result has usually been formation of racemic chiral compounds rather than opti-
cally pure ones. And the production of optically active, single enantiomer products
is extremely important, especially when those products are used medicinally since
the biological activity of compounds is often directly related to their stereochemi-
cal configuration. Addressing this additional obstacle requires us to confront one
of the iron rules of synthetic chemistry: optically inactive reactants cannot produce
optically active products. Put another way, if all reactants, solvents, and catalysts used
in a given synthetic process are either achiral or chiral but optically inactive, they
may combine to produce a compound with one or more chirality centers, but the
compound produced will always be racemic (i.e., optically inactive). You cannot get
true molecular asymmetry out of thin air; it always requires the intervention of a

preexisting asymmetric entity in some fashion, which begs the question: How were
Bioorganic Synthesis 378 optically pure compounds in nature formed in the first place? What was the original
intervening chiral entity that led to formation of single enantiomer L-amino acids
and D-sugars instead of racemic mixtures? No one knows for certain, though there
are many hypotheses on the primordial origins of molecular asymmetry. But even
though we have yet to discover any method in the laboratory that can produce opti-
cally pure compounds from optically inactive reactants, catalysts, and solvents, there
are a number of different methods we can employ that use preexisting asymmetric
agents in some fashion to help us to induce asymmetry at newly formed chirality
centers.
Of course, we know that nature readily produces optically active single enantio-
mer compounds from optically inactive reactants by using enzymes (which again
are already chiral and optically active, being derived from optically pure single enan-
tiomer amino acids) to catalyze bioorganic reactions. So, one method for solving the
dilemma of racemic mixture production would be to employ enzymes for chemical
reactions. But while it is sometimes possible to use enzymes for organic synthesis,
they are notoriously complex, difficult to isolate in useful quantities and do not read-
ily lend themselves to large-scale use. With that in mind, let’s briefly consider some
of the ways that chemical ingenuity has been used to achieve isolation or synthesis
of nonracemic chiral compounds in the chemical laboratory.
RESOLUTION OF ENANTIOMERS TO OBTAIN

OPTICALLY PURE COMPOUNDS
Recall that enantiomers have identical chemical and physical properties and so
cannot be separated by ordinary chemical means (distillation, crystallization, or
ordinary chromatography). The process of separation of enantiomers from one an-
other is called resolution and one approach to resolution involves the use of so-
called resolving agents; these are optically pure chiral compounds (usually natural
products) which can be reversibly attached to the enantiomeric pair to yield a pair of
diastereomers rather than enantiomers. Diastereomers do not have identical chemi-
cal and physical properties and so may be separated from one another by conven-
tional methods. A schematic overview of resolution of enantiomers using a resolving
agent is shown in Fig. 8.14. This scheme shows a racemic mixture of compound A
(i.e., a 50:50 mixture of (R)-and (S)-A) being derivatized by reaction with a single
enantiomer resolving agent, (R)-B. The derivative produced is actually a pair of dia-
stereomers, in this case, a 50:50 mixture of (R,R)-A-B and (S,R)-A-B. Such a mix-
ture of diastereomers is separable in principle by traditional means for separation
of chemical mixtures, that is, distillation, crystallization, or chromatography. Once
separated, each diastereomer undergoes removal of the appended resolving agent,
thereby releasing a pure single enantiomer of A. Again, in principle, the resolving
agent can be recycled for further use. This process can, at best, produce a 50% yield


(R)-A
(R,S)-A optically pure

(R)-enantiomer
racemic mixture 4. Remove
1. Optically pure and recycle (R)-B
resolving agent (R)-B resolving
reacts with (R, S )-A to (R)-B agent
(S)-A
form a bond. Must be
removable later.
(R,R)-A-B optically pure
2. Step to
separate (S)-enantiomer
(R,R)-diastereomer
diastereomers
(R,R)-A-B and (S,R)-A-B 5. Remove (R)-B
+ and recycle
Diastereomeric Mixture ; different resolving
agent
chemical and physical properties. Can (S,R)-A-B
be separated! Distill, crystallize,
chromatograph… (S,R)-diastereomer
FIGURE 8.14
Generic resolution scheme: isolation of pure enantiomers from a racemic mixture via reaction
with a resolving agent and formation of separable diastereomers.
of a desired enantiomer, and in practice, yields are usually lower due to mechanical
losses during derivatization, purification, and resolving agent removal.
Let’s move from a generic representation to a specific example of isolation of a
desired enantiomer by chemical resolution. As shown in Fig. 8.15, the ketone func-
tion of a racemic (R,S) mixture of α-hydroxyketone 8.15a is initially condensed with
pure (S)-1-phenethylamine to yield the corresponding mixture of diastereomeric
imines (R,S)-8.15b and (S,S)-8.15b. When this mixture is dissolved in boiling ethyl
acetate and then allowed to cool to room temperature, crystals of the higher melting
diastereomer (S,S)-8.15b slowly form. Vacuum filtration then affords a 25% yield of
pure (S,S)-8.15b crystals, while the liquors from the process contain a 2:1 mixture
of (R,S)-8.15b and (S,S)-8.15b. Hydrolysis of the imine linkage of the crystalline
(S,S)-8.15b releases pure (S)-8.15a along with the original resolving agent (S)-1-
phenethylamine which can be recovered and recycled if needed. The remaining ma-
terial in the liquors from the crystallization (a mixture of (R,S)-and (S,S)-8.15b
enriched in (R,S)-enantiomer) can be further processed via column chromatogra-
phy or by imine hydrolysis and subsequent reaction if the ketone mixture with pure
(R)-1-phenethylamine followed by separation of diastereomers as above to recover
pure (R)-8.15a as well as some additional (S)-enantiomer which, as it turns out, is
the desired enantiomer from this scheme which is part of a total synthesis of the
aglycone portion of an analogue of the polyketide anticancer antibiotic daunorubi-
cin (see Fig. 5.50 for structural details).
Of course, other methods for separation of enantiomers also exist, including
chiral chromatography (use of a chiral stationary phase in liquid chromatography)
or reactions with enzymes which recognize and catalyze specific reactions with only
one enantiomer of a racemic pair (known as enzymatic resolution). We’ll take a brief
look at this and other uses of enzymes later on.

(S)
(S)
Ph Ph
OCH3 O OCH3 N OCH3 N
(S)
(S/R) Ph (S) (R)
H2N
OH imine OH + OH
formation
S S S S S S
CH3O CH3O CH3O
H2O
racemic 8.15a (S,S)-8.15b (R,S)-8.15b
(S)
Ph
OCH3 O OCH3 N recrystallize by
(S) (S) dissolving in boiling ethyl
H3O+ acetate, cool to 25 oC
OH OH
S S (S) S S (R,S)-8.15b in
CH3O Ph CH3O liquid (liquors)
H2N from crystallization
pure (S)-(-)-8.15a pure (S,S)-8.15b
as crystals, mp 195 oC
FIGURE 8.15
Isolation of pure (S)-enantiomer of 8.15a via resolving agent resolution/crystallization.
USE OF CHIRAL POOL COMPOUNDS FOR SYNTHESIS

OF OPTICALLY PURE NATURAL PRODUCTS
Organic synthesis that uses the chirality inherent in enantiomerically pure start-
ing compounds available from nature has been widely employed for many years.
Such compounds are said to constitute the available chiral pool which is composed
mainly of compounds such as carbohydrates and amino acids as well as certain
abundant chiral terpenes and alkaloids, among many others (Fig. 8.16).
Chiral-pool-based synthesis usually works best if the starting compound is cheap
and closely resembles the target product (some of the previous examples we have
seen of semisynthesis should come to mind). Otherwise, the use of chiral pool start-
ing materials may involve long, multistep syntheses with low overall yields.
A simple example of total synthesis of an optically pure natural product starting
from a chiral pool compound is shown in Fig. 8.17. This sequence outlines prep-
aration of (R)-japonilure, an alkenyl-substituted γ-lactone that is the sex attrac-
tant produced by the female Japanese beetle, a notoriously destructive garden pest
that feeds on the leaves of many important fruit and ornamental plants. Only the
(R)-enantiomer is bioactive as an attractant and it is available commercially in small
traps used for insect control. However, the presence of even small amounts of the (S)-
enantiomer renders the compound completely inactive, making optical purity of the
synthetic product an absolute necessity. The synthesis begins with diazotization of
(R)-glutamic acid as shown. The resulting diazonium ion (R)-8.17a then undergoes
an intramolecular nucleophilic displacement of N2 by the adjacent carboxyl group,
forming the α-lactone (S)-8.17b with inversion of configuration. This strained (S)-
lactone then undergoes a second inversion of configuration via a subsequent in-
tramolecular nucleophilic epoxide ring opening by the remaining carboxyl group


CO2H OHC CH2OH O
NH2 OH
L-alanine D-glyceraldehyde (S,S)-camphor (R)-α-pinene
OH
H
HO2C HO2C CO2H
N
CH3
NHCH3 HO CO2H HO OH
(S)-nicotine (R,S)-ephedrine (R)-malic acid (2R,3R)-tartaric acid
FIGURE 8.16
Examples of chiral pool compounds: amino acids, carbohydrates, terpenes, alkaloids, and others.
H+
O O O O O
HONO inversion O
HO OH HO OH
NH2 N N N2 O
HO
(R)-glutamic acid (R)-8.17a (S)-8.17b
inversion
H H H
H2 SOCl2
O O O O
O O
O Pd-BaSO4 O O
S
H Cl OH
(R )-8.17e (R)-8.17d (R)-8.17c
(H3C)2N N(CH3)2
(Ph)3P=CH(CH2)7CH3 H
THF/HMPA, -60oC O H
O
(Wittig reaction)
H (R)-japonilure
FIGURE 8.17
Chiral pool synthesis of the Japanese beetle pheromone (R)-japonilure from (R)-glutamic acid.
to afford the carboxyl-substituted γ-lactone (R)-8.17c (note that the overall effect
of two sequential inversions of configuration is a net retention of configuration).
Conversion of (R)-8.17c to the corresponding acid chloride (R)-8.17d is followed
by a Rosenmund reduction using H2 and Pd-BaSO4 to afford the corresponding
aldehyde (R)-8.17e. A final Wittig reaction of the aldehyde with a linear nine-
carbon phosphorus ylide provides the alkenyl-substituted final product with mostly
(Z)-configuration at the π-bond as required for naturally occurring (R)-japonilure.
USE OF CHIRAL REAGENTS FOR SYNTHESIS

OF OPTICALLY PURE COMPOUNDS
Another method for introduction of enantioselective chirality into molecules is

via the use of chiral reagents. There are a number of different types which can be


used in organic reactions including chiral reducing agents for reduction of carbonyl
compounds to optically active alcohols, chiral hydroborating agents for hydration
of alkenes to give optically active alcohols, chiral allylborane reagents which can
alkylate carbonyl compounds to give optically active alcohols, and chiral bases for
stereospecific deprotonation reactions.
Reactions which produce chiral but optically inactive products often involve a
step in which a reagent reacts with a planar prochiral reactant (or reactive interme-
diate) such as an unsymmetrical carbonyl or alkene. Racemic mixtures are always
produced as a result of such reactions because two enantiomeric transition states
(nonsuperimposable mirror images) result from approach of the reagent from above
or below the planar reactant. These transition states are of the same energy, so the
two enantiomeric products are formed at identical rates, giving a racemic mixture.
This simplistic but useful analysis is illustrated in the upper portion of Fig. 8.18.
The lower portion of the figure shows use of a generic chiral hydride source for
the same reduction. As can be seen, approach from above or below the plane again
leads to two transition states, but these are diastereomeric transition states (non-
superimposable, nonmirror image) rather than enantiomeric transition states and
H:-
δ- δ- δ- δ-
O H O O H
+
NaBH4
achiral
hydride transition states: same energy
enantiomeric
reagent H:- H+
O H OH HO H
prochiral 2-butanol; chiral but racemic

carbon
H--R*
δ-O δ-
H--R*
O
δ- δ- +
*R--H O
H--R*
chiral
hydride transition states: NOT same energy
reagent H--R* diastereomeric H+
O H OH H R*
HO
+
major minor
prochiral 2-butanol; chiral but non racemic

carbon
FIGURE 8.18
Use of achiral vs. chiral reagents and enantiomeric vs. diastereomeric transition states.

so would be of different energies. The lower energy transition state would form

product faster and would therefore lead to preferential formation of one enantiomer
at the expense of the other (here we assume the chiral entity bearing the hydride is
shed after the addition and protonation steps). Note that this analysis could also be
extended to the use of a chiral alkylating reagent (chiral portion not shed) which
would lead to formation of diastereomeric alcohols with an excess of one diaste-
reomer over the other. Such processes would be termed stereoselective reactions
in that they favor the formation of one stereoisomer at the expense of other pos-
sible stereoisomers. More specifically, the chiral hydride reduction shown would be
termed an enantioselective reaction while the corresponding hypothetical chiral
alkylation reaction would be termed a diastereoselective reaction.
These are components of what is broadly termed asymmetric synthesis which
may be loosely defined as any synthetic process which converts an achiral substrate
into a product with a new chirality center in such a way as to produce stereoisomers
in unequal amounts. This is an enormous subject area and even a modest treatment
of it is far beyond the scope of this chapter. We can nevertheless gain some level of
appreciation for its challenges and accomplishments by considering a few examples
such as those shown in Fig. 8.19 which includes an example of an asymmetric ketone
reduction, an asymmetric aldehyde alkylation and an asymmetric hydroboration
reaction, all of which produce chiral alcohols in stereoisomeric excess.
In the first example, the reduction of the ketone function of an aryl β-ketoester
uses a modified form of lithium borohydride which derives its chirality from in-
corporation of an N-protected form of the amino acid cysteine into its structure.
In the second example, the asymmetric alkylation of propanal involves transfer of
Li NHCH2Ph
O S OH OH
H B
CO2Et O O CO2Et CO2Et
t-BuO (R) (S)
+
(94% yield)
93% 7%
i)
B
OH OH
O
2
(R) + (S)
H (71% yield) 92% 8%
ii)
BH
OH OH
2 +
(S) (R)
iii) then NaOH/H2O2 99% 1%
(98% yield)
FIGURE 8.19
Simple examples of asymmetric alcohol synthesis: i) ketone reduction using a chiral hydride
reagent; ii) aldehyde alkylation using a chiral allyl boron reagent; and iii) alkene hydroboration
using a chiral hydroborating agent.

an allyl group delivered from an allyl borane reagent that derives its chirality from
Bioorganic Synthesis 384 incorporation of two units of the chiral terpene (+)-α-pinene. This terpene is also
the source of chirality for the reagent used in the final example which is asymmet-
ric hydroboration of a simple alkene. Mechanisms and proposed transition state
conformations for these reactions are complex, but the outcomes are impressive in
terms of enantioselectivity.
Suffice it to say that there are a multitude of additional reactions involving chiral
reagents which have been employed as part of many multistep syntheses of complex
natural product structures. We note here that the primary disadvantages of this ap-
proach to asymmetric synthesis are: i) the reagents are often quite expensive; ii) a
stoichiometric quantity of the chiral reagent is required in each case; and iii) reac-
tion workups can often be complicated and time consuming.
USE OF CHIRAL SUBSTRATE CONTROL

FOR STEREOSELECTIVE SYNTHESIS
Another form of stereoselective synthesis involves a factor known as substrate con-

trol. When a chirality center is already present in a substrate molecule and is close
in proximity to a prochiral center undergoing an addition reaction, diastereomeric
transition states are again formed which can lead to stereoselectivity at the newly
formed chirality center. In this case, the resulting products will be an unequal mix-
ture of diastereomers. Nucleophilic additions to carbonyl groups adjacent to existing
chirality centers are the most common examples of chiral substrate control, though
there are many others. The stereochemical outcome of such reactions is usually de-
scribed in terms of the extent of diastereoselectivity observed (excess of one dia-
stereomer over another). Figure 8.20 shows the use of Cram’s rule, one of the more
commonly used models for prediction of the major diastereomeric product to be
expected from nucleophilic addition to a typical carbonyl adjacent to an existing
chirality center. Note that if the existing chirality center is racemic rather than opti-
cally pure, the resulting product will be also racemic.
Many other reactions are useful in the context of this kind of diastereoselectiv-
ity; one of the largest groups to yield useful results in this context are enolate ions.
Enolates of aldehydes, ketones, esters, and related carbonyl compound have been
used extensively to produce chiral diastereomeric products from enolate alkylations,
conjugate additions, aldol-type condensations, and others with often excellent dia-
stereoselectivity. The first example shown in Fig. 8.21 is a simple enolate ion alkyla-
tion process starting from a chiral ester. After treatment of the ester with lithium
diisopropyl amide (LDA) as base, the resulting (Z)-enolate (in this case) is alkylated
with methyl iodide, a reaction in which the methyl group is added to the less ste-
rically hindered face of the enolate ion (away from the phenyl group) to produce
predominantly the (R,R) diastereomer as shown.

L L L L
R R Nu
R Nu Nu R
S Nu Nu S S
M M + M
M S
O OH OH
O major minor
Ph Ph Ph
H Ph
CH3
H CH3MgBr H
CH3
H (S) CH3:- -:CH
3 H
H
(S)
H
H3C (S) (S) + (R)
H3C H3C
H3C H
O OH
O OH
major minor
FIGURE 8.20
Illustration of Cram’s rule for prediction of major vs. minor diastereomeric addition products.

CH3 CH3

(R) OEt (R) OEt (R) OEt (R) OEt
LDA CH3-I (S)
(R)
THF +
Ph O Ph OLi Ph O Ph O
i) (Z)-enolate 89% 11%
O (S) O (R)
(R) m-CPBA (R) (R)
+
OH OH OH
ii) 95% 5%
I- H3C H3C
I2 (S) (S)
H 3C OH H3C OH O + O
(S) (S) I (S) I (R)
I+ O O
O O H 91% H 8%
iIi)
FIGURE 8.21
Examples of substrate control in: i) enolate alkylation; ii) epoxidation; and iii) iodolactonization.
The second example in Fig. 8.21 makes good use of the stereospecificity of alkene
epoxidation (occurs with syn addition) along with the syn directing effect usually
observed with allylic hydroxyl groups, leading to predominant formation of the
diastereomer shown. Other epoxidations of this type, where substituents are alkyl
rather than hydroxyl, tend to be governed mainly by steric effects and so give re-
sults opposite to those shown for the alcohol substrate by adding oxygen to the less-
hindered alkene face. The third example shown is application of a reaction known as
iodolactonization in which a carboxylic acid with an alkene function, usually at the
4-or 5-position, is treated with iodine. Electrophilic addition as usual for halogens
leads to a cyclic iodonium ion which then undergoes intramolecular nucleophilic
attack by the carboxyl group from the less hindered side (opposite side to the chiral
methyl group), leading to iodolactone formation. Many other reactions of chiral
alkenes have been shown to give useful diastereoselectivity in certain contexts, in-
cluding Diels–Alder reactions, hydroborations, and dihydroxylations with osmium
tetroxide.
The principal advantages of substrate control for all these reactions are i) a new
chirality center with often substantial excess of one stereoisomer is produced and
ii) structure of the major isomer can often be predicted using various transition
state models (such as Cram’s rule). The main disadvantage of substrate control is the
often-limited availability or high cost of suitable optically pure starting materials for
the reactions involved.
USE OF CHIRAL AUXILIARIES FOR SYNTHESIS

As mentioned above, the most obvious drawback to substrate control for achiev-
ing stereoselective reactions is the need for optically pure starting compounds to

begin with. One might imagine that many of these substrates would come from

the chiral pool which would tend to limit structural diversity to some extent and
this is often the case. We now consider the use of so-called chiral auxiliaries for
achieving the same results obtainable by more traditional substrate control but with
additional elements of structural diversity. A chiral auxiliary may be thought of as
a chiral compound or subunit which may be reversibly attached to (or incorporated
into) the structure of an achiral compound. This is similar to the use of resolving
agents except, in this case, the attachment of the auxiliary does not produce a mix of
diastereomers since the substrate compound was achiral to begin with. The result-
ing product may be thought of as being temporarily chiral due to the presence of
the auxiliary. This chiral substrate may then undergo the usual reactions that will
show substrate control for diastereoselectivity when forming a new chirality center.
However, after the diastereoselective reaction is completed, the auxiliary is then re-
moved from the substrate (and may be recycled). For the simplest case in which
only one new chirality center has been formed, the result will be formation of a new
compound with often-substantial enantiomeric excess. Figure 8.22 gives a general
scheme for the use of chiral auxiliaries in asymmetric synthesis.
A relatively simple example of the use of a chiral auxiliary for asymmetric syn-
thesis is shown in Fig. 8.23. Recall that Robinson annulation involves the use of
both conjugate addition of an enolate ion to an α,β-unsaturated carbonyl system
and a subsequent intramolecular aldol condensation to form a fused 6-6 bicyclic
ring system (see Fig. 1.52). In the asymmetric variant shown here, the nucleophilic
component for the initial conjugate addition to methyl vinyl ketone is the enamine
8.23b which is formed by isomerization of imine 8.23a which in turn is prepared by
simple condensation of racemic 2-methylcyclohexanone with commercially avail-
able (S)-α-phenethylamine as the chiral auxiliary. After the enamine conjugate ad-
dition (which is sterically directed to the top face of the enamine and away from
the bulky chiral auxiliary), the resulting imine 8.23c is hydrolyzed to remove the
substrate
achiral R
3. Remove
1. Optically pure chiral substrate-(+)-Aux and recycle
auxiliary (+)-Aux reacts 2. Reaction to (+) chiral
with achiral substrate to (+)-Aux auxiliary
produce
form a bond or bonds. R-substitued major diastereomer
Must be removable later. substrate with R
diastereo- +
selectivity (+)-Aux substrate
substrate-(+)-Aux
(+)
temporarily R single enantiomer
chiral substrate
product
substrate-(+)-Aux
(–)
minor diastereomer
FIGURE 8.22
Generic chiral auxiliary scheme: formation of a single enantiomer product from an achiral
substrate via chiral auxiliary attachment, diastereoselective reaction, and chiral auxiliary removal.

Bioorganic Synthesis 388 O (S) N Ph Ph NH

CH3 NH2 CH3 base CH3
H+, -H2O
2-methyl 8.23a 8.23b

cyclohexanone (imine) (enamine)
(racemic) O
conjugate
addition
O
O O Ph N O
NaOCH3 H3O+
CH3 CH3OH CH3 CH3
(48%)
8.23e 8.23d 8.23c

(98% (R), 2% (S))
FIGURE 8.23
Use of an amine chiral auxiliary for an asymmetric version of the Robinson annulation.
chiral auxiliary to give the corresponding diketone 8.23d. A final intramolecular

aldol condensation affords 98% of the (R)-enantiomer of bicyclic Robinson annula-
tion product 8.23e with excellent enantioselectivity.
While chiral auxiliaries have been successfully used in a variety of different re-
action schemes, their use in the formation of chiral enolates for alkylation or aldol
reactions has been most noteworthy. An excellent example of their utility is shown
in Fig. 8.24 which outlines the use of one of the Evans chiral oxazolidinones in a
stereoselective synthesis of Prelog–Djerassi lactonic acid, a product derived from
degradation of certain natural macrolide antibiotics and which is used as a template
for construction of new variants of these and other natural products. Many different
and useful oxazolidinones have been prepared from various chiral amino alcohols,
and in this instance, we see the use of commercially available (1S,2R)-norephedrine
which is converted to the oxazolidinone chiral auxiliary 8.24a by reaction with di-
ethyl carbonate. Deprotonation of 8.24a with n-butyllithium gives the anionic form
which is then acylated with propionyl chloride (serving here as the achiral substrate)
to give the temporarily chiral substrate 8.24b to which the chiral auxiliary has now
been attached. Subsequent deprotonation with LDA (lithium diisopropylamide)
gives the chiral enolate 8.24c which then undergoes asymmetric alkylation with
methallyl iodide which approaches the enolate face away from the substituents on
the auxiliary ring to give 8.24d with excellent diastereoselectivity. Reductive treat-
ment of 8.24d with lithium aluminum hydride to remove the chiral auxiliary affords
the chiral alcohol 8.24e as shown.
The process in Fig. 8.24 continues by oxidation of alcohol 8.24e to aldehyde 8.24f
which is then subjected to a second reaction employing the same acylated chiral


O O O
Li+
OH
(EtO)2C=O HN O n-BuLi Cl *
(R) (S) N O NAux
(R) (S) achiral
H2N Ph 8.24b
substrate temporarily
Ph Ph
(1S ,2R )-norephedrine 8.24a chiral substrate
an oxazolidinone
chiral auxiliary
Li
O O O
I
(S) LDA
(S) LiAlH4 *
NAux N O lithium
OH removal alkylation enolate
of chiral formation
auxiliary 8.24d Ph
8.24e
8.24c
chiral enolate
DMSO
SO3-pyr
Bu2B
O (CH3)3SiO O
OH O
(S) * *
NAux (S) (R) * (CH3)3Si-X NAux
O (S) NAux
8.24g alcohol
chiral protection
8.24f boron enolate 8.24i
8.24h
for aldol addition
hydroboration, then remove
O O oxidation OH protection
oxidize
O O O O CH2OH OH OH O
LiOH to CO2H
removal * then (R) (S) (R) *
OH NAux (S) NAux
of chiral cyclize
auxiliary to lactone
Prelog–Djerassi
8.24k 8.24j
lactonic acid
FIGURE 8.24
Use of Evans oxazolidinones as chiral auxiliaries for enolate alkylation and aldol addition reactions
in an asymmetric synthesis of Prelog–Djerassi lactonic acid.
auxiliary as before but which in this case had been converted to chiral boron eno-
late 8.24g. Here the enolate adds to the less hindered face of the aldehyde func-
tion of 8.24f in an asymmetric aldol addition process to give the corresponding
β-hydroxyamide 8.24h in which two additional chirality centers have now been
added, again with excellent diastereoselectivity. The alcohol function is then tem-
porarily protected as trimethylsilyl ether derivative 8.24i which undergoes a ste-
reospecific hydroboration–oxidation sequence which, after removal of the previous
protecting group, produces diol 8.24j in which yet a fourth chirality center has now
been introduced, again with excellent diastereoselectivity. After recrystallization to
remove minor diastereomeric impurities, the synthetic sequence is completed by
oxidation of the primary alcohol function of 8.24j to give the carboxylic acid which
spontaneously cyclizes to produce the corresponding lactone 8.24k. Finally, removal
of the chiral auxiliary by treatment with LiOH in methanol yields the target, Prelog–
Djerassi lactonic acid, which was found to be >99.9% diastereomerically pure.
Derivatives of the chiral auxiliary (−)-8-phenylmenthol have also been used in
a variety of ways for asymmetric synthesis. One example is shown in Fig. 8.25 in
which 8-phenylmenthol is reacted with pyruvic acid to form 8-phenylmenthol py-
ruvate ester. Reaction of the ketone function with Grignard reagent 8.25a leads
to formation of the tertiary alcohol 8.25b as a single diastereomer. Removal of the


MgBr
O O
HO
Ph 8.25a Ph
O Ph
O O OH
OH H+, -H2O -78 oC, Et2O
(–)-8-phenylmenthol O
8-phenylmenthol
pyruvate ester 8.25b
LiAlH4 THF
O
acetal
o HO OH
formation HO OH O3. -78 C (S)
O (intramolecular) CH2Cl2,
then
(S)(–)-Frontalin 8.25d (CH3)2S 8.25c
O
> 99% (S)
FIGURE 8.25
Whitesell’s synthesis of optically pure frontalin using the chiral auxiliary 8-phenylmenthol.
chiral auxiliary via lithium aluminum hydride reduction affords diol 8.25c which is
optically pure. Ozonolysis of the alkene function leads to the corresponding keto-
diol 8.25d which is not isolated but spontaneously undergoes intramolecular cyclic
acetal formation to yield the pure (S)-enantiomer of frontalin, an aggregation pher-
omone of a number of different species of beetles including the Western pine beetle,
a particularly destructive pest of North American pine forests.
A representative sample of several other important chiral auxiliaries widely used
in asymmetric synthesis is shown in Fig. 8.26, though exploring further details of
their use is beyond the scope of our chapter. To summarize, we may say that chiral
auxiliaries represent a different and very useful approach to substrate control for
diastereoselective reactions. One advantage to this approach is that unlike typical
substrate control, the chiral auxiliary need not resemble the structure of the syn-
thetic target since the auxiliary is subsequently removed after the diastereoselective
reaction(s). It is also worth noting that a chiral auxiliary may also act as a built-in
resolving agent to aid in the separation of minor diasteromeric products prior to its
removal. Disadvantages include the need for stoichiometric amounts of the chiral
auxiliary, the need for a chemically viable point of attachment on the original sub-
strate, and the need for additional steps to remove the auxiliary under conditions
that will not adversely affect the target compound being produced.
USE OF CHIRAL CATALYSIS FOR SYNTHESIS

The gold standard for creation of new chirality centers with a high level of optical purity
in organic reactions is, of course, the enzyme-mediated process. Since the chiral enzyme
is used as a catalyst only, it can provide the chiral environment needed to induce asym-
metry again and again without being consumed or affected in any way. Chiral catalysis
is an area of synthetic organic chemistry that tries to duplicate nature’s own approach
to asymmetric synthesis by employing a chiral component in catalytic rather than

Ph

O
N N
OH H H
OR
trans-2-phenyl proline chiral
cyclohexanol esters 2,5-dimethylpyrrolidines
Ph
O
N NR
N
NH2 OCH3 R
S OCH3
(S)-1-amino-2-methoxy O2
methylpyrrolidine (SAMP) camphorsultams chiral oxazolines
CH3 O
S
N NH2
NHCH3
NH2 OCH3
OH
(R)-1-amino-2-methoxy pseudoephedrines chiral tert-butanesulfinamides
methylpyrrolidine (RAMP) (all diastereomers)
FIGURE 8.26
Additional chiral auxiliaries widely used in asymmetric synthesis.
stoichiometric quantities that will not only accelerate a particular reaction but will also
lead to enantioselectivity at newly developed chirality centers. The approach used most
often (though not exclusively) involves the use of a chiral catalyst that can bind a spe-
cific reagent, then deliver that reagent to a planar, prochiral substrate molecule in such
a way that the reagent reacts preferentially with one of the two available faces (Si or Re)
of the substrate. After release of the reagent and production of the chiral reaction prod-
uct, the catalyst is free to bind more reagent and initiate another cycle of the catalytic
process. A general scheme for this type of catalysis is presented in Fig. 8.27.
No area of asymmetric organic synthesis has received more attention than the
development of effective and efficient chiral catalysis. The benefits are obvious: i) a
single chiral molecule employed in a catalytic fashion can lead to the production of
millions of optically active product molecules; ii) chiral catalysts can, in principle, be
economically recycled for further use; and iii) their use can reduce or eliminate most
of the waste or inefficiency associated with the processing and separation of enantio-
mers via resolution, the addition and removal of chiral auxiliaries, or the use of stoi-
chiometric quantities of chiral reagents. As before, the key to their success lies in their
ability to form diastereomeric reaction transition states of unequal energy, resulting
in significant preference for one enantiomeric product at the expense of the other.
The earliest examples of successful development of chiral catalysis are found in
the area of catalytic asymmetric hydrogenation of double bonds. Such reactions
are of significant versatility since they can be applied to the reduction of prochiral
alkenes, ketones, imines, enamines, aromatic heterocycles, and many other types
of unsaturated substrates. Figure 8.28 outlines two examples of such processes.
The first is the commercial synthesis of the aromatic amino acid L-DOPA via


prochiral
reactant
chiral chiral
catalyst catalyst
reagent
reagent
prochiral
reactant
chiral
reagent
chiral product
catalyst
FIGURE 8.27
Generic catalytic cycle involving a prochiral reactant, a chiral catalyst, and a reagent.
catalytic hydrogenation of the enamide precursor (Z)-2-acetamido-3-(4-acetoxy-3-

methoxyphenyl)acrylic acid to give intermediate reduction product 8.28a in high
optical purity. This is followed by ester and amide hydrolysis and methoxyl group de-
methylation to give L-DOPA, one of the most important pharmaceutical agents for
the treatment of Parkinson’s disease. The reduction employs a rhodium-based cata-
lyst which employs containing a phosphine-based ligand known as (R,R)-DiPAMP.
Chelation of the metal by the phosphorus atoms of this chiral diphosphine creates
the chiral environment in which both substrate and reagent are brought together for
delivery of H2 preferentially to the Re face of the alkenyl system.
In the second instance, catalytic hydrogenation of the unsaturated 2-(6-
methoxynaphthalen-2-yl)acrylic acid in the presence of a ruthenium catalyst coor-
dinated to another chiral diphosphine ligand, (R)-BINAP, affords the (S)-enantiomer
of naproxen, a common over-the-counter NSAID (nonsteroidal anti- i nflammatory)
that acts as a COX-1 and COX-2 inhibitor, similar in action to the structurally re-
lated compound ibuprofen which, while also containing a 2-arylpropanoic acid func-
tion, is marketed as a racemic mixture. For naproxen, only the (S)-enantiomer has
anti-inflammatory activity; the (R)-enantiomer is inactive in that regard but can be
toxic to the liver, making an enantioselective synthesis essential for pharmaceutical
purposes.
Both of the above hydrogenation reactions are examples of the use of homoge-
neous catalysis, a process in which a reaction catalyst and all reactants are all brought
together and dissolved in solution (i.e., are present in the same phase). Recall that
typical hydrogenation processes employ an insoluble, finely divided transition metal

H3CO CO2H [Rh(R,R)-DiPAMP)COD]BF4, (cat.) H3CO (S) CO2H
NHCOCH3 H2, 3 atm NHCOCH3

H3COCO H3COCO
8.28a
(Z)-2-acetamido-3-(4-acetoxy-
98% yield, 98% (S)
3-methoxyphenyl)acrylic acid
H O+
3
OCH3
L
Rh BF4- L, L = HO CO2H
P P
L
chiral catalyst Ph Ph OCH3 NH2
(COD = 1,5-cyclooctadiene) (R,R)-DiPAMP HO
L-DOPA
CO2H Ru(OCOCH3)2(BINAP) (cat.) CO2H

H2, 135 atm, CH3OH
H3CO H3CO
(S)-naproxen
2-(6-methoxynaphthalen-2-yl)acrylic acid
> 98% (S)
O L
O
Ru (Ph)2P
L, L=
O L (Ph)2P
O
chiral catalyst
(R)-BINAP
FIGURE 8.28
Examples of catalytic asymmetric hydrogenation in the synthesis of L-DOPA and (S)-naproxen.

such Pt, Pd, Ni, or others and that the reduction process actually occurs on the sur-
Bioorganic Synthesis 394 face of the metal itself. Such a process is referred to as heterogeneous catalysis since
the metal catalyst is never in solution along with the reactants. Dissolution of a metal
in homogeneous catalysis is achieved when one or more chelating organic ligands
are dissolved in an organic solvent along with the metal catalyst. Chelation with the
metal leads to a catalyst-ligand complex that is predominantly organic in structure,
allowing the metal to come into solution along with reactants and reagents. When
chiral ligands are used, all reactants and reagents may come into intimate contact
with the metal catalyst within a chiral environment created by the coordinating li-
gands, leading to the kind of enantioselectivity observed above.
While catalytic asymmetric hydrogenation of ketones is also feasible, a more gen-
eral catalytic approach to such reductions involves the use of borane-THF complex
as the reducing agent along with a catalytic amount of a chiral oxazaborolidine,
compounds usually derived from transformations of the amino acid proline. These
so-called CBS catalysts (CBS is derived from Corey–Bakshi–Shibata, authors of the
original publications describing their synthesis and use) are especially enzyme-like
in their behavior because they bind both the substrate and the reagent. A general
scheme for ketone reductions via this methodology and several simple examples
are shown in Fig. 8.29. Since their initial introduction, several varieties of CBS-type
catalysts have been developed and have found wide use in the asymmetric synthesis
of many complex natural products and pharmaceutical agents.
Among catalytic asymmetric C–C bond forming reactions, one of the simplest
and most useful processes is the carrying out of certain aldol condensations in the
presence of a catalytic amount of the amino acid proline. While mechanistic de-
tails remain uncertain, involvement of an iminium ion–enamine equilibrium is
likely, leading to good-to-excellent levels of enantioselectivity. A useful example
is shown in Fig. 8.30 for the proline-catalyzed formation of Wieland–Miescher
ketone, a key chiral intermediate which has been used as a starting material for the
total synthesis of a number of steroids and other important pharmaceutical agents.
Following conjugate addition of methyl vinyl ketone to the enolate derived from
2-methyl-1,3-cyclohexanedione, intramolecular aldol condensation of the symmet-
ric triketone 8.30a in the presences of catalytic L-proline in DMSO gives mainly the
(S)-enantiomer of the unsaturated diketone product as shown. This is but one of
many examples of the use of proline-catalyzed aldols for enantioselective formation
of useful synthetic intermediates and products.
Alkene epoxidation is a very useful means of oxygen incorporation into organic
compounds. An important feature of the reaction is its stereospecific delivery of
oxygen to the alkene in a concerted “syn” fashion. That stereospecificity makes it
an ideal candidate for exploitation in a potential asymmetric variant. As it turns
out, such a variant has turned out to be one of the most important of all catalytic
asymmetric processes which is known as the Sharpless epoxidation. This reaction
converts allylic alcohols to chiral epoxy alcohols with excellent enantioselectivity

R H
R
O
O
BH3-THF H OH
RL RS B N H3O +
CBS catalyst O R' BH2
(large) (small) RL RS
H
RL
RS > 90% enantioselectivity
H R R
typical substituents
O R = phenyl, substituted phenyl
N B
R' = H, CH3, n-butyl
CBS R'
catalyst
Examples: (BH3-THF; CBS catalyst: R = phenyl, R' = CH3)
O H OH O H OH
i) ii)
98% (R)
98% (R)
O H OH O H OH
iii) iv)
CO2CH3 CO2CH3
93% (R) 97% (R)
FIGURE 8.29
Catalytic asymmetric ketone-to-alcohol reductions using BH3-THF and chiral CBS catalysts.


O O
O (cat.)
N CO2H
O H
AcOH(aq) DMSO
O
O conj. addition O 25 oC
2-methyl-1,3- 8.30a Wieland–Miescher ketone
cyclohexanedione pure (S), 57% yield
FIGURE 8.30
Asymmetric synthesis of Wieland–Miescher ketone via proline-catalyzed aldol condensation.
HO CO2Et
tBu O
H
O
HO CO2Et
D-(–)-DET
R3 R2 R2 O
L-(+)-DET D-(–)-DET R3 R2
R3
HO 1 R1 Ti(O-iPr)4
O R Ti(O-iPr)4
HO HO R1
tBuOOH, CH2Cl2 tBuOOH, CH2Cl2
70–90% yield 70–90% yield
> 95% -20oC -20 oC
> 95%
enantioselectivity enantioselectivity
HO CO2Et O R2
tBu H R3
O R1
HO CO2Et
tBu O O O simplified catalyst
L-(+)-DET Ti complex
O O
EtO2C CO2Et
FIGURE 8.31
Structural prediction model for enantioselective Sharpless epoxidation of allylic alcohols.
in most cases. The catalyst system is derived from the use of titanium isopropox-
ide which undergoes replacement of its four isopropoxide ligands with the –OH
functions of either enantiomer of relatively inexpensive diethyl tartrate ((+)-DET
or (−)-DET) along with the –OH groups of both t-butyl hydroperoxide (TBHP or
tBuOOH, the oxidizing agent) and the allylic alcohol substrate. Face-selective deliv-
ery of the active oxygen from tBuOOH to the alkene function in this chiral catalyst
environment occurs with excellent enantioselectivity that is dependent on which
DET enantiomer is used. Thus, either stereoisomer of the resulting epoxy alcohol
can be selectively produced, making this a process of enormous synthetic utility.
Figure 8.31 provides a general structural prediction model for the Sharpless epoxi-
dation system along with a greatly simplified representation of the structural com-
ponents of the catalyst complex.
Extensive use has been made of the Sharpless epoxidation in total synthe-
sis schemes over the years and even a partial listing of its applications would be
daunting. A simple example of its use as shown in Fig. 8.32 will serve our purposes.
Epoxidation of the allylic alcohol (Z)-2-tridecen-1-ol using D-(−)-DET in the
Sharpless catalyst complex gives the corresponding epoxide (2R,3S)-2,3-epoxy-
1-tridecanol with excellent enantioselectivity. Oxidation of the primary alcohol
with pyridinium dichromate (PDC, similar to PCC) gives the corresponding

O O
D-(–)-DET PDC
OH O
OH Ti(O-iPr)4 9 CH2Cl2
9
9 tBuOOH, CH2Cl2
(2R,3S)-2,3-epoxy- (2S,3S)-2,3-epoxy
(Z)-2-tridecen-1-ol –20 oC
1-tridecanol tridecanal
(>95% enantioselectivity)
(Ph)3P Wittig
reaction
O O
H2
RhCl 9
(+)-disparlure
(7S,8R)-7,8-epoxy-2-methyl-
5-octadecene
FIGURE 8.32
Enantioselective synthesis of (+)-disparlure via Sharpless asymmetric epoxidation.

epoxyaldehyde as shown. A subsequent Wittig reaction with the indicated phos-

Bioorganic Synthesis 398 phorous ylide leads to an epoxyalkene (as a mixture of (Z) and (E) isomers) which
then undergoes catalytic hydrogenation to afford the saturated natural product,
(+)-disparlure ((7R,8S)-7,8-epoxy-2-methyloctadecane). Produced in minute quan-
tities by the female gypsy moth for attraction of males, this potent sex attractant has
proven to be useful for the baiting of traps to monitor expanding populations of this
enormously destructive forest pest. In tests, it was found that the (+)-enantiomer is
significantly more potent as an attractant than racemic material, making an enanti-
oselective synthesis quite valuable.
The synthetic value of the catalytic asymmetric epoxidation reaction is nicely
complemented by another process known as the Sharpless asymmetric dihy-
droxylation (ADH) reaction. Recall that the familiar conversion of alkenes to syn
vicinal diols via reaction with osmium tetroxide (OsO4) is most useful when the
expensive and highly toxic OsO4 is used in only catalytic amounts in conjunc-
tion with a stoichiometric oxidant that reoxidizes the reduced form of osmium
back to the OsO4 oxidation state to continue the catalytic cycle. After extensive
work, Sharpless found that the use of certain chiral alkaloids could not only ac-
celerate these reactions but could also produce diol products with a high level of
enantioselectivity. The now commercially available “AD-mixtures” for this chem-
istry contain a mixture of potassium osmate (K2Os2(OH)4), as a source of osmium
in its OsO4 oxidation state, potassium ferricyanide (K3Fe(CN)6) to reoxidize the
reduced form of osmium, potassium carbonate (K2CO3), and one of two differ-
ent alkaloid-based chiral ligand catalysts, (DHQ)2PHAL for the so-called AD-
mix-α and (DHQD)2PHAL for AD-mix-β. In each catalyst, two equivalents of
a chiral alkaloid (dihydroquinine for (DHQ)2PHAL and dihydroquinidine for
(DHQD)2PHAL) are connected to a central phthalizine structure, hence the
PHAL in the names. As shown in Fig. 8.33, these two catalyst systems allow for
complementary alkene face selectivity in the dihydroxylation reaction (RL for large,
RM for medium, and RS for small substituents in the alkene structure) making this
a valuable catalytic process for the generation of vicinal diol products with a high
level of optical purity.
Like the asymmetric epoxidation of allylic alcohols, Sharpless asymmetric dihy-
droxylation has been used extensively in a variety of asymmetric synthesis schemes
since its introduction. A useful example is synthesis of α-hydroxy-β-benzamido-
substituted 3-phenylpropanoic acid side chain of the anticancer agent taxol (see
Fig. 4.33 and associated text for details) from methyl cinnamate shown in Fig. 8.34.
There are many other examples of catalytic asymmetric reactions including
asymmetric aminohydroxylations (similar to the ADH reaction), asymmetric ep-
oxidations and cyclopropanations of simple alkenes, asymmetric alkylations, halo-
genations, Michael additions, and many others too numerous to list here. It is fair to
say that this is an ever-expanding area of chemical research that will continue to be
of immense importance both academically and industrially for many years to come.


N N N N
O O
H H
O O
N N
(DHQ)2PHAL
(dihydroquinine)
OsO4
HO OH RS RM
RS RM AD-mix-β AD-mix-α RL H
RM (DHQ)2PHAL
RL (DHQD)2PHAL
H RS HO OH
K2OsO2(OH)4 H K2OsO2(OH)4
70–90% yield RL 70–90% yield
high K2CO3 K2CO3 high
enantioselectivity K3Fe(CN)6 K3Fe(CN)6 enantioselectivity
OsO4
RL R
N N N N O S
O
O O H Os
H H O
N O H RM
O O
N N
O O
N N
(DHQD)2PHAL
(dihydroquinidine)
simplified catalyst complex
FIGURE 8.33
Structural prediction model for Sharpless alkene asymmetric dihydroxylation (ADH).
O OH O
AD-mix-α OCH3
OCH3
OH > 99%
enantioselctivity
methyl cinnamate (2R,3S)-methyl 2,3-dihydroxy-
3-phenylpropanoate
steps
O
O
OAc
Ph NH O O
Ph NH O
O baccatin III
OH
OH ester
formation OH
HO
taxol O
Ph AcO (2R,3S)-3-benzamido-2-hydroxy-
O
3-phenylpropanoic acid
O (Taxol side-chain)
FIGURE 8.34
Use of Sharpless AD-mix-α for synthesis of taxol side chain from methyl cinnamate.
USE OF ENZYMES FOR SYNTHESIS OF OPTICALLY

PURE COMPOUNDS: BIOCATALYSIS
The use of enzymes or whole cell systems for biocatalysis in organic synthesis has
been known for many years, but with the incorporation of modern tools for pro-
tein discovery, genetic engineering, and bioprocess design, exciting new frontiers

continue to emerge at this intersection of organic chemistry and biology. The ability
Bioorganic Synthesis 400 of enzymes to convert simple and readily available compounds into useful high-
value products results from three unique features: i) their ability to selectively
react with specific functional groups in the presence of others (chemoselectivity);
ii) the ability to catalyze transformations at specific sites within a molecular structure
(regioselectivity): and iii) their ability to transform prochiral compounds into
single enantiomer chiral compounds (enantioselectivity). These features make en-
zymes especially attractive as potentially recyclable catalysts that can function in
ways difficult or impossible to achieve by conventional chemical means, while doing
so under relatively mild and environmentally sustainable conditions. By some esti-
mates, as much as 30% of the world’s chemical manufacturing will incorporate some
form of biocatalysis by midcentury.
Earlier in Chapter 2 we broadly grouped enzymes into six main classes:
• Oxidoreductases: catalyze oxidation–reduction reactions.

• Transferases: catalyze the transfer of a functional group from one molecule to
another.
• Hydrolases: catalyze cleavage of C–X bonds by addition of H2O (X = O, N, S).
• Lyases: catalyze cleavage of C–X bonds without hydrolysis or oxidation (X = C,
O, N, S).
• Isomerases: catalyze rearrangement (isomerization) of existing atoms within a
molecule.
• Ligases: catalyze formation of new C–X bonds (X = C, S, O, and N).
Within each class are a variety of subclasses, each of which usually describes
some specific characteristic related to the reaction catalyzed by the enzyme. Thus a
ketoreductase and a dioxygenase are both a type of oxidoreductase, a dehydratase and
an aldolase are both a type of lyase, an esterase and a peptidase are both a type of hy-
drolase and so on. With these various enzyme functions in mind, we will spend the
remainder of this final section highlighting just a few of the more important types
of enzyme systems most commonly used in organic synthesis with an emphasis on
applications of a medicinal nature.
Among the most widely used enzyme systems for simple organic synthesis ap-
plications are those found in common baker’s yeast. These whole cell applications
are simple and inexpensive to use and yeasts can often tolerate certain organic cosol-
vents to improve water solubility. Nutrient feeding with sugars is required through-
out these fermentations to replace inactivated enzymes and cofactors and since the
organisms are sometimes sensitive to poisoning by reactants or products, high dilu-
tion is often required to keep concentrations below toxic levels. Asymmetric ketone
reductions are the most common type of reaction, making use of ketoreductase
enzymes present in the yeast fermentation. Figure 8.35 shows several examples of
these sorts of baker’s yeast reductions as well as a general model for prediction of

O baker's yeast

HO H general model
reduction
for enantioselection
RS RL H2O, sucrose RS RL (S = small, L = large)
92–99%
enantioselectivity
O O HO H O O O H OH O
i) ii)
OEt OEt OCH3 OCH3
O O H OH O O O HO H O
iii) iv)
Cl3C OEt Cl3C OEt BrH2C OC8H17 BrH2C OC8H17
FIGURE 8.35
Enantioselective enzymatic reduction of ketones using baker’s yeast.
FIGURE 8.36
Enantioselective baker’s yeast reduction for synthesis of the calcium channel blocker diltiazem.
the stereochemical outcome based on steric factors present in the structure of the
ketone being reduced.
These reductions have found wide application in organic synthesis. An especially
interesting example is shown in Fig. 8.36 in the synthesis of the calcium channel
blocker diltiazem, a benzothiazepine used in the treatment of hypertension, angina,
certain arrhythmias, and also as a migraine preventative.
In this scheme, compound 8.36a is present as a racemic mixture due to the
ready interconversion of one enantiomer into the other by way of the intermedi-
ate enol form. The baker’s yeast enzyme specifically catalyzes reduction only of the
(S)-enantiomer as shown; thus, all of the material is eventually reduced to (S,S)-
8.36b by way of reversible conversion of inert (R)-8.36a into the reactive (S) form.
Subsequent chemical steps convert 8.36b into the product diltiazem.

Microbial oxidations have also played a significant role in the manufacture of

Bioorganic Synthesis 402 various compounds of medicinal use. A particularly important example is illustrated
in Fig. 8.37. After initial isolation of the HMG-CoA reductase inhibiting compound
ML236B from the fungus Penicillium citrinum in the early 1970s, it was determined
that certain of its hydroxylated derivatives had superior cholesterol-lowering activ-
ity and pravastatin was eventually targeted for potential development and clinical
trials. Since regio-and stereospecific hydroxylation of ML236B by chemical means
was not economically feasible, microbial oxidation via Streptomyces carbophilus was
developed, leading to eventual commercial production of pravastatin, a drug with
annual sales now in excess of $3 billion worldwide.
Soil bacteria have long been known to oxidatively produce interesting chiral me-
tabolites of aromatic compounds and in some cases this can be accomplished on a
multigram scale. Subsequent chemical modification of such metabolites has led to
stereoselective total syntheses of a variety of high-value target products over the
years. Of particular note are the arene-cis-diols produced by enzymatic dioxygen-
ation of certain aromatics by various bacterial strains. Figure 8.38 shows the biosyn-
thesis of a chiral arene-cis-diol from toluene by the action of a toluene dioxygenase
enzyme (TDO) found in P. putida.
The resulting chiral metabolite (1S,2R)-3-methylcyclohexa-3,5-diene-1,2-diol
is then converted to the corresponding acetonide acetal by acid-catalyzed reaction
HO ONa HO ONa
O O
OH O OH O
O O
H Streptomyces
H
carbophilus
P450 enzyme
HO
ML236B
pravastatin (Pravachol)
(compactin sodium salt)
FIGURE 8.37
Microbial allylic hydroxylation of ML236B to give the cholesterol-lowering drug pravastatin.
CH3 CH3 CH3

toluene
dioxygenase H3CO OCH3
OH O
(TDO) O3
(R)
(P. putida) (S) H+ then
OH O (CH3)2S
toluene
(1S,2R)-3-methylcyclohexa- 8.38a
3,5-diene-1,2-diol
O O CH3
CO2H steps Al2O3 O

O O
benzene O
HO O heat O
PGE2α (aldol)
OH 8.38c 8.38b
FIGURE 8.38
Use of a toluene dioxygenase-derived diol in a formal synthesis of the prostaglandin PGE2α.

with acetone dimethylacetal as shown. The protected diol is next subjected to double

ozonolysis, yielding the corresponding ketoaldehyde which then undergoes an Al2O3-
catalyzed aldol condensation to produce the chiral cyclopentenone derivative (4S,5S)-
4,5-dihydroxycyclopent-2-eneone acetonide. Many subsequent synthetic steps
eventually convert this key intermediate to the prostaglandin PGE2α. Synthetic elabo-
ration of similar arene-cis-diols has led to stereoselective total syntheses of morphine
as well as pancratistatin, a cytotoxic agent with anticancer potential isolated from
spider lily.
Extensive use has also been made of the ability of certain enzymes to distinguish
between enantiotopic functional groups present in prochiral compounds. One ex-
ample is the use of esterase enzymes such as pig liver esterase (PLE), as illustrated
in Fig. 8.39. Here, PLE catalyzes hydrolysis of only the pro-(S) ester function present
in prochiral 8.39a to produce the resulting monoester 8.39b with excellent enanti-
oselectivity. The chirality present in 8.39b is then exploited in subsequent synthetic
steps to eventually produce enantiomerically pure virantmycin, an antifungal, anti-
viral, antibiotic originally isolated from Streptomyces nitrosporeus.
Enzymatic C–C bond-forming reactions are of obvious interest since the forma-
tion of such bonds in a stereoselective manner is fundamental to the synthesis of
many complex organic structures. One of the simplest of all C–C bond-forming re-
actions is the formation of cyanohydrins from aldehydes or ketones via addition of
HCN. In this context, enzymes known as oxynitrilases have been used extensively
for the enantioselective addition of HCN to various carbonyls. The resulting opti-
cally active cyanohydrins make excellent starting materials for the stereoselective
synthesis of a variety pharmacologically significant products.
One such use of enzymatic cyanohydrin formation is found in the synthesis of
clopidogrel (marketed as Plavix), a potent anticoagulant used for the prevention of
blood clots and heart attack associated with various diseases of the vascular system.
The basic approach is outlined in Fig. 8.40 and begins with enantiospecific conversion
of 2-chlorobenzaldehyde into the corresponding chiral cyanohydrin by the action
of KCN in the presence of the enzyme mandelonitrile lyase. Subsequent chemical
hydrolysis of the nitrile function yields (R)-2-(2-chlorophenyl)-2-hydroxyacetic
acid. Further synthetic steps transform this key intermediate into clopidogrel. With
current worldwide sales in excess of $6 billion, clopodogrel is among the top-selling
pharmaceuticals of the 21st century.
HO2C Cl
H3CO2C CO2CH3 HO2C CO2CH3
pig liver steps OCH3
esterase (PLE) (S) N
pH 8, 7 days H
OCH3 OCH3
8.39a 8.39b (–)-virantmycin
(prochiral) > 97% S
FIGURE 8.39
Desymmetrization of a prochiral diester by pig liver esterase in synthesis of (−)-virantmycin.

Cl O

Cl H OH
mandelonitrile
lyase H3O +
H (R)
CN
KCN
H2O/iPrOH
2-chlorobenzaldehyde (R)-2-(2-chlorophenyl)-
2-hydroxyacetonitrile
Cl H CO2CH3 Cl H OH
steps
(R)
N CO2H
S
clopidogrel (Plavix) (R)-2-(2-chlorophenyl)-
2-hydroxyacetic acid
FIGURE 8.40
Enantioselective enzymatic cyanohydrin formation for synthesis of the anticoagulant clopidogrel.
O
O 2-deoxyribose- H
O 5-phosphate aldolase OH O OH
+ (DERA) DERA
2 H
H H OH
Cl
Cl 8.41a Cl 8.41b
HO OH
OH
OH O
steps O steps O
F O O
N
OtBu OH
H Cl 8.41c
N Ph CN 8.41d
Ph
O atorvastatin (Lipitor) > 99% enantioselectivity
FIGURE 8.41
Use of sequential enantioselective enzymatic aldol additions in synthesis of atorvastatin.
The aldol reaction is perhaps the most fundamental of all C–C bond-forming
reactions and our concluding example makes use an aldolase enzyme for con-
struction of a key intermediate in the manufacture of the cholesterol-lowering
drug atorvastatin (marketed as Lipitor) via sequential enantioselective aldol ad-
dition reactions. As shown in Fig. 8.41, the process involves initial addition of the
enolate equivalent derived from acetaldehyde to 2-chloroacetaldehyde, catalyzed
by 2-deoxyribose-5-phosphate aldolase (DERA) to give the initial aldol addition
product 8.41a. Addition of a second enolate equivalent from acetaldehyde to 8.41a
leads to hydroxyaldehyde 8.41b which cyclizes to the hemiacetal 8.41c. Subsequent
synthetic elaboration gives the key intermediate 8.41d which is eventually converted
via many steps to atorvastatin.
There are many additional examples of industrial application of enzymes that
are too numerous to treat here in any detail, including synthetic processes such as

alkene reduction (enoate reductases), oxidative insertion reactions (Baeyer–Villiger

oxygenases), nitrile hydrolysis (nitrile hydrolases) and C–N bond formation (trans-
aminases) among others.
We mentioned earlier that the use of biocatalysis has many benefits, including
efficiency, environmental compatibility, and broad reaction spectrum in addition
to their crucial stereo-and regioselectivity. Nevertheless, there are drawbacks to
the use of biocatalysis: i) enzymes are usually only available in one (natural) en-
antiomeric form, so one cannot ordinarily control which enantiomeric form of a
desired compound will be produced; ii) enzymes often have reduced activity in
solvents other than water (substrate water solubility is always a potential problem);
iii) enzymes usually have narrow operating parameters (temperature, pH, etc.);
iv) enzymes are sometimes subject to inhibition phenomena (reduction of reaction
rate caused by substrate or product concentration factors); and v) many enzymes
require specific cofactors such as flavin, heme, NADH, ATP in order to function
(cofactors are expensive, relatively unstable, and are required in stoichiometric
amounts).
SOME FINAL THOUGHTS
If biocatalysis seems like the last word for the synthetic production of pharmaceuti-
cal agents and other commodity organic chemicals, further advances in molecular
biology, protein engineering, directed evolution, and synthetic biology may seem
likely to eventually relegate organic synthesis to books dealing mainly with the his-
tory of science. Indeed, the field of organic synthesis has often been described in
recent times as a “mature” science that is unlikely to expand much beyond its cur-
rent well-defined boundaries. However, with so much attention being focused on
the growing influence of biotechnology, it is surprisingly easy to overlook the in-
credible power of synthesis to produce or modify organic structures in ways that
are currently unavailable via biological means. Our ability to synthesize novel com-
pounds or to structurally alter existing ones is still one of the most important ways
to discover new medicinal agents and to modify existing therapeutic activity pro-
files. Achieving these results by biological means can be difficult or economically
impractical due in part to some of the constraints inherent in the basic chemistry
employed by biosynthetic pathways. From an industrial perspective, the use of bio-
catalysis usually comes early in a process and is used mainly to introduce chirality
that is further exploited by subsequent synthetic organic transformations. It is worth
remembering that the domain of reactions and pathways available in synthetic or-
ganic chemistry is remarkably vast when compared to nature’s relatively limited set
and that an understanding of how biological reaction systems work requires a deep
understanding of fundamental organic chemistry. For these reasons alone it seems
likely that these two fields of knowledge will continue to inform and support one
another for many years to come.

STUDY PROBLEMS
1. In 1853, Louis Pasteur made various attempts to synthesize quinine. During
these investigations, he found that treatment of quinine with aqueous sulphuric
acid led to an isomerization that afforded a new compound called quinotoxine.
Propose a mechanism for this reaction.
HN
OH
H2SO4 O
H3CO H2O
H3CO
N
N
quinine quinotoxine
2. The one-pot enzymatic transformation shown involves use of an enzyme called

tyrosinase. Determine what the enzyme is actually doing in this process and also
account mechanistically for the trace byproduct shown (hint: a small amount of
catechol can be isolated from the reaction mix if a sample is pulled immediately
after adding the enzyme).
O
OH O H
tyrosinase H O
enzyme O +
+
OEt CHCl3, 3 hr OEt
H
70% yield (trace)
O O
3. Use Cram’s rule to predict the major diastereomeric product from each of the
reactions shown:

CH3

H PhMgBr
Ph
(then H2O)
O
CH3
LiAlH4
O
Ph (then H2O)
Ph
O
EtLi
CH3 (then H2O)
CH3
4. Enzymatic resolution involves the use of various enzymes acting on a racemic

mixture of substrates and taking advantage of the enzyme’s ability to catalyze
the reaction of a functional group present in one enantiomer much more rapidly
than in the other enantiomer (a process generally referred to as kinetic resolu-
tion). Given a lipase enzyme that catalyzes ester hydrolysis as shown, explain
(using words and structures) how a racemic mixture of 2-methyl-3-pentanol ace-
tate could be enzymatically resolved to yield both pure enantiomers of 2-methyl-
3-pentanol given the information shown.
lipase enzyme
H2O (fast)
OAc
racemic
mixture +
lipase enzyme
H2O (slow)
OAc
5. Provide the missing structures in the reaction sequence shown for synthesis of
5-methoxyflavone.

Bioorganic Synthesis 408 OH KOtBu

CH3I (1.0 equiv.)
CH3 K2CO3, then PhCOCl
acetone
OH O
C9H10O3 C16H14O4 (a ketoester)
KOtBu
(1.0 equiv.) heat
O
H2SO4
OCH3 O H2O
5-methoxyflavone
C16H14O4 (a β-diketone)
6. What is the product of the reaction shown? Is this reaction stereoselective or

stereospecific? Explain.
1. BH3-THF
?
2. H2O2, NaOH
single product,
α-pinene 100%
7. Give the final product expected from the sequence of reactions shown:
O O
OCH3 1. LDA, THF
O N
2. CH3I
3. LiAlH4, ether

8. Provide a reasonable mechanism for each of these reactions:

O O
O NaOCH3, CH3OH
OCH3
+
OCH3 then H3O+
O
O
O
KOtBu
O O O
tBuOH
OH
CN H3CO OCH3 O OCH3
+ CH3OH
O +
+ CN
O O O OH
9. Provide the intermediates and mechanisms involved in the following asymmet-

ric synthesis of (+)-cannabispirenone-A. What is the absolute configuration of
this product?
N
O
H3CO H3CO
CO2CH3 1.
2. H3O+
3. NaOEt,
OCH3 then H3O+ OCH3
(+)-cannabispirenone A
10. Provide a reasonable mechanism for formation of the indicated products

(begin with an N-alkylation followed by a reaction similar to the Claisen
rearrangement).
I O
N
OCH2OCH3 +
O N
CH3CN O CH2OCH3
H
(solvent) I
O O
O
O

11. Provide the expected products from each of these ketone reductions.
O
BH
O
catechol borane
Ph
H Ph
O
O
N
B
(R)-CBS H
catalyst
BH3-THF
Ph
H Ph
O O
O
N
B
(S)-CBS H
catalyst
12. Baker’s yeast reductions have also been used in asymmetric reduction of α,β-
unsaturated ketones and aldehydes. An example is shown here. Based on what
you know about how such biological reductions are carried out, draw a likely
structure for an intermediate product that is involved in this reaction. Explain.
baker's
O yeast H3CO2C OH
H3CO2C
> 98% (S)

Suggested Further Readings
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Index
abietic acid, 159 alkaloids

absinthe, 145, 147 from anthranilic acid, 341
absolute configuration, 3 from histidine, 341–â•‰342
acetaldehyde, 5, 98, 319, 404 from lysine, 312–â•‰316
acetals, 28 from nicotinic acid, 338–â•‰340
acetate hypothesis, 209, 213, 254, 372 from ornithine, 306–â•‰311
acetate pathway, 184–â•‰252 from phenylalanine, 317
acetoacetyl-â•‰CoA, 135 from tryptophan, 327–â•‰337
acetyl-â•‰CoA, 62–â•‰65 from tyrosine, 318–â•‰326
acetylenase enzymes, 200 from xanthosine, 343–â•‰344
acetylsalutaridinol, 323, 324 alkylation, 26, 27, 44, 65, 194, 219
acidity constants (pKa, Table 1.3), 17 asymmetric, 383
acidity trends, 17–â•‰18 alternariol, 213, 215, 372
aconitase, 101 methyl ether of, 372
aconitic acid, 101 Amadori rearrangement, 126
acoradiene, 156 amide resonance, 121
acoryl cation, 155 amikhelline, 220
acronycine, 341 α-â•‰a mino acids
acyl carrier protein (ACP), 190 D,L-â•‰designation, 112
acyl phosphates, 36 essential, 112
acyl transfer, 192 flow chart for biosynthesis, 114
adenine, 86 list of, 113
adenosine, 86 C-â•‰terminal, 121
adenosine triphosphate (ATP), 44–â•‰45 N-â•‰terminal, 121
S-â•‰adenosylmethionine (SAM), 65–â•‰66 amino sugar, 81, 86
adrenaline, 50, 318, 319 p-â•‰aminobenzoic acid, 265, 266
aglycone, 86–â•‰87 7-â•‰a minocephalosporanic acid (7-â•‰ACA),
agroclavine, 329 352–â•‰354
ajmalicine, 333 aminoglycosides, 88
alanine, 57, 113, 117, 120, 338, 381 6-â•‰a minopenicillanic acid (6-â•‰APA),
aldol addition, 23, 32, 64, 100, 136, 209 350–â•‰352
enantioselective, 389, 404 amorpha-â•‰4,11-â•‰diene, 155
aldol condensation, 23, 32, 368, 387, amphetamine, 317
394, 403 amphidinolide F, 363
aldolase enzyme, 93, 400, 404 amphotericin B, 185, 250, 291, 363
aldose carbohydrate, 77, 80 amylose, 85
aldosterone, 174 anabasine, 339
417

anacardic acid, 232 biotin

Index 418
androstenedione, 172, 173 as a CO2 carrier, 63, 64, 100, 190
anethole, 268, 270, 271 structure of, 64
anhydrotetracycline, 230, 231, 253 Birch, A. J., 184
anileridine, 325, 326 bisabolene, 155
anisomorphal, 151, 152 bisabolyl cation, 155, 156
anomeric carbon, 78 bond dissociation energies, 22
anomers, 78 bornyl cation, 145–147
anthocyanidins, 287 brevianamide F, 345
anthocyanins, 284, 286, 287 bromocryptine, 330
anthracyclines, 229, 230, 246 Brønsted acid-base, 15, 19
anthranilic acid, 129, 265, 266, 303
alkaloids from, 341 cabergoline, 330
anthraquinone, 225, 228 cadaverine, 312, 313, 315, 316, 340
apigenin, 285, 286 cafestol, 133, 134
apigenin chalcone, 285 caffeic acid, 269, 271
apigetrin, 286 caffeine, 343, 344
arabinose, 77, 81, 82 calcidiol, 176
arachidonic acid, 199–206, 257 calcitriol, 176
arene oxide, 51, 52 calicheamicin, 70
arogenic acid, 264 camphor, 50, 51, 133, 134, 146, 147
artemisinin, 155 camphothecin, 333
aryltetralins, 276 carbapenums, 350, 352, 353, 355
asparagine, 113, 118, 125, 347, 349 carbinolamine, 28, 29
aspartame, 345 carbocations
aspartic acid, 63, 113, 117, 339, 345 cyclopropylmethyl-c yclobutyl-
aspergione B, 373 homoallyl, 24, 165
aspidospermine, 333, 336 fates of, 24
aspirin, 86, 203, 205, 279 means of formation, 20
asymmetric synthesis, 383, 384, 387 protosteryl, 165
atorvastatin, 237, 404 rearrangements of, 24
ATP. See adenosine triphosphate stability trends, 20
atropine. See hyoscyamine carbohydrates, 75–105, 113
autotroph, xvi disaccharides, 83–85
avermectin PKS, 248 furanose forms, 30, 77–78
monosaccharides, 76–83
bacitracin A, 346 polysaccharides, 86
Baeyer-Villiger monoxygenase, 52 pyranose forms, 30, 77–78
Baeyer-Villiger oxidation, 171, 200, carbon cycle, xv
220, 257 carbonyl addition reactions, 27, 30, 31
baker’s yeast, 400, 401, 410 carboprost, 204, 205
beeswax, 189 N-carboxybiotin, 65, 100
benzofuran, 13, 219, 224, 291 carnauba wax, 189
benzopyran, 221, 291 carotenes, 133, 135, 178
benzopyrones, 279 carotenoids, 133, 149, 176–178, 413
benzylpenicillin, 350, 351 carpanone, 371, 372
benzyltetrahydroisoquinolines, 319–321 carveol, 147
BH4. See tetrahydrobiopterin carvone, 148
biocatalysis, 399, 400, 405 caryophyllene, 134
biogenetic isoprene rule, 131, 149, 184 casmiroin, 341, 342
bioprene, 140 castanospermine, 314, 315

catabolism, xvii, 112, 190 chrysanthemic acid. See pyrethric acid
419 Index
catalysis chrysanthemyl PP, 150
enzymatic, 20 chrysomelidial, 151, 152
heterogeneous, 394 cicaprost, 205, 206
homogeneous, 392 cinchona alkaloids, 336
by Lewis acids, 19 cinchonaminal, 336
catalytic domains, 236 cinchonidine, 336
dehydratase (DH), 242 cinchonidinone, 336
enoylreductase (ER), 242 cinnamaldehyde, 270, 271
ketoreductase (KR), 240 cinnamic acid, 269
ketosynthase (KS), 240 cinnamyl alcohol, 270, 271
thioesterase (TE), 243 citral. See geranial
catechin, 12, 13 citric acid cycle, 100–104
catecholamine, 319 citronellal, 143, 144
catharanthine, 333, 334, 335 citronellol, 143, 144
cathenamine, 334 citryl-CoA, 100
13
cathinone, 317, 318 C-labeled acetate, 210, 215–218
α-cedrene, 156 cladinose, 89–91, 245
β-cedrene, 156 Claisen condensation, 34, 59, 63, 135,
cedryl cation, 156 191, 306
cefdinir, 352, 354 Claisen rearrangement, 367
cefixime, 352, 354 aromatic, 371
ceftaroline, 352, 354 chair transition state, 262
cefuroxime, 352, 354 of chorismic acid, 263
celecoxib, 174, 175 clopidogrel, 403, 404
cellobiose, 84, 85 CoASH. See coenzyme A
cellulose, 84, 85, 86, 120, 274 cocaine, 301, 309–310
cephalexin, 352, 353 codeine, 323, 324, 326, 327
cephalosporins, 349–352 codeinone, 323, 324, 357
cephalothin, 352, 353 coenzyme A, 61, 62
chain folding patterns coenzymes (cofactors), 44, 61,
in flavone biosynthesis, 285 236, 369
in polyketide biosynthesis, 212–214, cofactors. See coenzymes
218, 230, 372 colchicine, 301, 357, 376, 377
in sesquiterpene biosynthesis, 154 Collie, J. N., 184
in sesterterpene biosynthesis, γ-coniceine, 302, 303
162, 163 coniferyl acetate, 271
in steroid biosynthesis, 166 coniferyl alcohol, 271–275
chalcone synthase, 285 coniine, 300–304, 312
cheilanthadiol, 162, 163 conjugate addition, 30, 31
chemoselectivity, 400 conjugate reduction, 47, 191–192
chiral auxiliaries, 386–390 constitutional isomers, 2
chiral catalysis, 390–391 copalyl PP, 160
chiral pool compounds, 380 corticosteroid, 173, 174
chiral reagents, 381–383 cortisol, 369
chitin, 83, 86 corydaline, 321
chlorotetracycline, 230 corylifol A, 289, 290
cholecalciferol, 175 corynantheal, 336, 337
cholesterol, xix, 68, 133, 166, 167, 170–173 corytuberine, 320–322
chorismate mutase, 263 coumadin. See warfarin
chorismic acid, 261, 263, 265 2-coumaric acid, 279–281

4-coumaric acid, 269, 280–283 6-deoxyerythronolide B synthase (DEBS),

Index 420
coumarins, xxi, 268, 269, 279, 280 240, 241, 242–248
coumestans, 290 deoxynivalenol, 156, 157
coumestrol, 289, 290 deoxypodophyllotoxin, 276, 277, 294
COX-1/COX-2 inhibitors, 203 deoxyribose, 82
Cram’s rule, 384, 385 2-deoxyribose-5 -phosphate aldolase, 404
crepenynic acid, 200 deoxyxylulose phosphate pathway,
crotonyl-ACP, 191 136–137
18-crown-6, 252 depsipeptides, 346
cubane, 363 desoamine, 245, 247
cuprenyl cation, 157 diamine oxidase enzymes, 56, 57, 306,
curved arrow notation, 14 307, 311, 312, 313
cycloartenol, 165, 167 diastereoselectivity, 384, 386, 387,
cyclobutane conformations, 8 388, 389
cyclohexane conformations, 9 diastereotopic atoms, 7, 8
cyclooxygenase, 203, 207 dicoumarol, 281, 282, 295
cyclopentane conformations, 8 dictamnine, 341, 342
cyclopropane conformation, 8 didehydroshikimic acid, 259, 260
CYP enzymes. See cytochrome P450 Dieckmann condensation, 34
cystathionine, 116 Diels-A lder reaction, xix, 221, 238,
cytochrome P450, 50, 224, 227 335, 386
digoxigenin, 169, 170
dactinomycin, 347, 348 digoxin, 169, 170
daidzein, 268, 285, 288, 289, 290 dihomo-γ-linolenic acid, 199, 202, 203
dammarenyl cation, 166 dihydrobenzofuran, 273, 291, 292
dammerenediol, 166 dihydrokaempferol, 287
danaidone, 311, 312 dihydrokavain, 282, 283
daunorubicin, 86, 87, 185, 186, 229, dihydromonacolin L, 239
246, 379 dihydrooropheic acid, 201
daunosamine, 86, 228, 229 1,2-dihydropyridine, 339, 340
Davis, B., 258 dihydroxyacetone phosphate, 94
10-deacetylbaccatin III, 68, 161, 162 dihydroxynaphthoic acid, 159
decalin, 12, 238 dihydroxypterocarpene, 289, 290
decarboxylation, 44, 53, 65, 136, 191, 229 diketopiperazines, 344, 345
of α-a mino acids, 57, 58 diltiazem, 401
of o- or p-hydroxybenzoic acids, 59, 60, dimethylallyl diphosphate (DMAPP), 65,
220, 223, 232, 233 67, 68, 138–150
of α-ketoacids, 60–63, 98, 99 in alkylation reactions, 66
of β-ketoacids, 59, 103, 171 13
C-labeled, 156, 157
dehydrocholesterol, 172, 175, 176 in formation of benzopyrans and
dehydrodiconiferyl alcohol, 273, 274 benzofurans, 292, 342
dehydrogeissoschizine, 334, 335 structure of, 66
dehydroquinic acid, 259, 260, 261 dimethylthujaplicatin, 276, 277
dehydroreductones, 126, 127 dinoprost, 204, 205
dehydroshikimic acid, 259, 261 dinoprostone, 204, 205
dehydrosterculic acid, 195, 197 diosgenin, 167, 170, 172
dehydrotetracycline, 230, 231 dioxygenase enzymes, 43, 222
demethylepipodophyllotoxin, 277, 278 catechol type, 53
6-deoxyerythronolide B, 240, 241, intermolecular, 53
245, 346

intramolecular, 53, 338, 339 epoprostenol, 205
421 Index
α-ketoglutarate-dependent, 54 equilenin, 363
diphosphoglycerate, 95, 96 ergostine, 330
diprenorphine, 327 ergot alkaloids, 327, 329, 331
disaccharide, 76, 83, 84, 85, 125 ergotamine, 330
disparlure, 397, 398 ertapenum, 354
disulfide linkage, 2, 103, 121, 122, 123 erypoegin H, 67, 289, 290
diterpenes, 133, 134, 158–161 erythromycin, 89, 186, 240–247
DMAPP. See dimethylallyl diphosphate erythrose-4-phosphate, 105, 107, 111,
dodecahedrane, 363 114, 259
dolichodial, 151, 152 eryvarin S, 289, 290
dolichotheline, 342, 343 eserethole, 375, 376
dopamine, 50, 286, 301, 318–321 eseroline, 375, 376
doripenem, 354, 355 estradiol, 172, 173, 285, 289
doronenine, 311, 312 estragole, 270, 271
doxorubicin, 227, 228, 229, 246 estrone, 172, 173
doxycycline, 231, 232 etoposide, 68, 69, 276, 277, 278
duegelin, 292 etorphine, 326, 327
eugenol, 258, 259, 271, 299
E1 reaction, 23 eugenone, 219, 221
E1cB reaction, 22, 23 euphol, 166, 167
E2 reaction, 15, 16, 22, 23
eicosanoids, 199, 202 FAD/FADH2. See flavin adenine
eicosapentaenoic acid, 202 dinucleotide
electrophiles, 13, 14, 25, 118 farnesene, 153
electrophilic addition, 25, 386 farnesol, 133, 134, 153
ellipticine, 333, 336 farnesyl cation, 154
emodin, 225, 226, 254 farnesyl diphosphate (FPP), 153, 159,
enamines, 28, 29 164, 181
enantiomers, 2–4, 112, 132, 144 FAS. See fatty acid synthase
resolution of, 376, 378–380, 391 fatty acid synthase (FAS), 190–193,
enantioselectivity, 384, 388, 391, 394–399 210, 234
in biocatalysis, 400 fatty acids
enantiotopic atoms, 5, 6, 7, 403 acetylenic, 200, 201
enediol, 80–82, 92–94, 106, 107, 126 biosynthesis, 189–201
enolase, 96 monounsaturated, 187, 198, 232
enolate ions, 31–34, 63–65 polyunsaturated, 187–189, 195, 198–201
stereochemistry, 35, 36 saturated, 49, 187, 210, 234
of thioesters, 34, 192, 212 trans-fats, 187, 188
use in asymmetric synthesis, 384, fatty alcohols, 188
388–389, 394, 404 fenchol, 145, 146
ent-kaurene, 160 fenchone, 145, 146
enzymes, 5–8, 20 fenchyl cation, 145, 146
definition and nomenclature, 42–43 fentanyl, 325, 326
use in asymmetric synthesis, 399, fermentation, 98, 161, 237, 350, 400
404, 405 ferrulic acid, 269
ephedrine, 300, 317, 318 Fischer projections, 5, 76
epimerase enzyme, 44, 106 flavin adenine dinucleotide (FAD), 48–49
epimerization, 81, 82, 148, 178, 243, 277 flavin mononucleotide (FMN), 48
epimers (C-2), 80, 81 flavonoids, 12, 268, 284–287

flavoprotein, 48 glyceraldehyde phosphate

Index 422
FMN. See flavin mononucleotide dehydrogenase, 94
folic acid, 266 glyceraldehyde-3-phosphate, 93, 94,
formononetin, 289, 290, 292 105–110, 137
FPP. See farnesyl diphosphate glycerophospholipids, 185, 187
frontalin, 74, 390 glycolysis, 92–98
fructose, 76, 79, 80, 81, 85 glycoproteins, 124, 314
fructose diphosphate aldolase, 93 in blood group antigens, 125–126
fructose-1,6-diphosphate, 92, 93, 94 glycosides, 83–88, 151, 169, 170
fructose-6 -phosphate, 92, 93, 107, 109 GPP. See geranyl diphosphate
fumarase, 6, 7, 104 grandisol, 181, 364, 365, 366
fumaric acid, 6, 7, 103, 104 griseofulvin, 7, 185, 186, 224, 227, 253
functional groups (Table 1.1), 1 guaiacylglycerol-β-coniferyl ether, 268,
furanose carbohydrates, 30, 77 274, 275
gutta-percha, 132, 133, 134, 135
galanthamine, 370
gallic acid, 259, 261 harmalan, 331, 332
gemeprost, 204, 205 harmine, 331, 332
genistein, 285, 288 Haworth projections, 79, 80
gentisic acid, 268 hedysarimcoumestan D, 290, 291
geranial, 143, 144 hemiacetals
geraniol, 134, 143–144, 151, 152, 181, cyclic, 30, 77, 83, 224, 276, 289, 332
332, 366 formation of, 28
geranyl diphosphate (GPP), 140–144, hemiterpenes, 133, 134, 139
149, 152 heterocycles (Table 1.2), 12
geranylfarnesyl diphosphate (GFPP), 162, heterotrophs, xvii
163, 164 hexokinase, 92
geranylgeranyl diphosphate (GGPP), 158– histamine, 341, 342, 343
162, 176–177 histidine, 113, 303
germacrene, 154 alkaloids from, 341–343
germacrone, 154 HMG-CoA. See
germacryl cation, 154 β-hydroxymethylglutarylCoA
GFPP. See geranylfarnesyl diphosphate HMG-CoA reductase, 236, 402
GGPP. See geranylgeranyl diphosphate hordenine, 318, 319
gibberellic acid, 159, 160 1,3-hydride shifts, 24
glabroisoflavanone A, 289, 291 hydrindane, 11, 12
glucocorticoids, 173 hydrocortisone, 174
gluconeogenesis, 98, 100, 111, 189 hydrolase enzymes, 44, 400
glucosamine, 81, 82, 83, 125 p-hydroxycinnamic acid. See
glucose, xvi, 30, 75–80, 82, 173, 349, 363 4-coumaric acid
in cellulose and celloboise, 85, 86 4-hydroxycoumarin, 280, 281, 282
metabolism of, 92–98, 105–109 hydroxydihydrodaidzein, 289, 290
glucose-6 -phosphate, 92, 93, 105 hydroxyformononetin, 289, 290
glucose-6 -phosphate dehydrogenase, 105 β-hydroxymethylglutarylCoA (HMG-
glutamic acid, 57, 103, 113–115, CoA), 136, 237
117–118, 380 p-hydroxyphenylethanal, 320, 321
glutamine, 113, 118, 230, 266 4-hydroxyphenylpyruvic acid, 263, 264
glutamine synthetase, 118 17-α-hydroxyprogesterone, 172, 173, 174
glutathione, 204, 205, 208 hyoscine, 306, 308, 309
glycation, 126, 127 hyoscyamine, 306, 308, 309
glyceraldehyde, 76, 77, 381 hypericin, 225, 226, 228

ibuprofen, 174, 175, 203, 392 α-ketol rearrangement, 119, 137, 138
423 Index
iloprost, 205, 206 β-ketoreductase, 191, 192, 234, 400
imines, 27–29, 55, 379 ketose carbohydrate, 76, 79, 81, 93
in alkaloid biosynthesis, 302, 305–307, khellin, 219, 220, 222
311–320 Knoevenagel condensation, 32, 33,
in PLP reactions, 115–117 233, 285
iminoaspartic acid, 338, 339 kynurenine, 338, 339
imipenem, 354, 355
indole, 13, 266, 267 β-lactams, 302, 349–354
indole alkaloids, 327, 329 lactic acid, 98, 111, 346
indole terpene alkaloids, 331–333 lactose, 85
indole-C2N fragment, 69, 70 ladderanes, 195
indolizidine alkaloids, 303, 314, 315 lanosterol, 165, 166, 167, 170, 171,
insulin, 121 172, 175
intricatin, 373, 375 lavender oil, 144
iodolactonization, 386 L-DOPA, 319
ionophores, 252 asymmetric synthesis of, 392
IPP. See isopentenyl diphosphate lemon grass oil, 143
iridoids, 151, 152 leucine, 113, 118, 119, 347, 348
irododial, 151, 152, 332 leucopelargonidin, 287
isocitrate dehydrogenase, 101 leukotrienes (LT), 206
isocitric acid, 101, 102, 103, 104 LTA4, 207
isoeugenol, 271, 299 LTB 4, 207
isoflavonoids, 268, 269, 285, 288–291 LTC 4, 208
isoleucine, 112, 113, 118, 119 LTD4, 208
isomenthol, 148, 149, 180 LTE 4, 208
isomenthone, 148, 149 Lewis Acid-Base theory, 19
isomerase enzymes, 44, 136, 400 lignans, xxi, 111, 268, 272–276, 297, 299
isopenicillin N, 350, 351, 353 lignin, 268, 269, 270, 274, 275, 276
isopenicillin N synthase, 350 limonene, 131, 132, 144, 145, 147, 148
isopentenyl alcohol, 134, 139 linalool, 25, 26, 134, 143, 144
isopentenyl diphosphate (IPP), 67, 68, linalyl acetate, 143, 144
135–141, 163 linalyl diphosphate, 142, 143, 144,
isopiperitenol, 148 145, 149
isopiperitenone, 148 lipids, 185, 188, 189
isoprene, 67, 68, 70, 131, 145, 176, 184 lipoic acid, 12, 13, 98, 99
isoprene rule, 131 use with TPP, 61–63, 103, 104
isopulegone, 148, 149 lipoxygenase, 207
isovitexin, 286 liquiritigenin, 288, 297
littorine, 306, 308
japonilure, 39, 380, 381 loganin, 152, 332, 333
Julian, P., 373 lophocerine, 319, 320
lovastatin, xix, xx, 185, 186, 236, 237, 239
kava, 282 lovastatin nonaketide synthase B
kavain, 282, 283 (LovB), 237
kavalactones, 282, 283 lovastatin nonaketide synthase C
kavapyrones. See kavalactones (LovC), 237
α-ketobutyric acid, 118, 119 LPP. See linalyl diphosphate
α-ketoglutarate-dependent LSD, 330
dioxygenase, 53, 54 lupanine, 315, 316, 317
α-ketoglutaric acid, 57, 102–104, 113–115

lupin alkaloids. See quinolizidine N-methyl-Δ1-pyrrolinium cation, 306,

Index 424
alkaloids 309, 339
lupinine, 315, 316, 317 methysticin, 282
lutein, 178 mevaldic acid, 136
lyase enzymes, 44, 400 mevalonic acid, 135–136, 237
lycopene, 176, 177, 178 mineralocorticoid, 173
lysergic acid, 70, 329, 330 minocycline, 231
lysine, 68, 303, 350, 351 modular PKS, 234
alkaloids from, 312–314 monacolin J, 239
lyxose, 77, 81, 82 monacolin L, 239
monensin A, 185, 251
macrolides, 185, 240, 250
monoamine oxidase enzymes, 56
Maillard reaction, 126, 127
monoamine oxidase inhibitor (MAOI),
malabaricol, 166, 169
318, 332
malate dehydrogenase, 104
monocrotaline, 311–312
malic acid, 6, 7, 102, 104, 105, 381
monolignols, 270, 275
malonamyl-CoA, 230
monooxygenase enzymes, 49–52
malonyl-CoA, 135, 190, 191, 210
monosaccharides, 76
formation of, 63–65
monoterpenes, 140–149
maltose, 84–85
acyclic, 143
mandelonitrile lyase, 403
irregular, 149–151
mangiferin, 86
mono-and bicyclic, 144–147
Mannich base, 34
morphine, 12, 300–301, 309, 322–324
Mannich reaction, 33–34
morphine rule, 325
in alkaloid biosynthesis, 304–307
mutarotation, 78
mannose, 77, 81
MVA. See mevalonic acid
MAO. See monoamine oxidase
mycarose, 245
Markovnikov orientation, 25
mycosamine, 250
matairesinol, 276–277
myrcene, 143–144
matricaria acid, 200
myristicin, 270–271
medicarpin, 289–290
melatonin, 327–328 NAD+/NADP+. See nicotinamide adenine
melissyl alcohol, 189 dinucleotide: oxidized form
menthol, 133, 147–149 NADH/NADPH. See
menthone, 148 nicotinamide adenine
meropenem, 354–355 dinucleotide: reduced form
mescaline, 69, 319 naloxone, 323, 326
meso compounds, 4 naproxen, 174, 203
methadone, 325–326 asymmetric synthesis of, 392
methamphetamine, 317 naringenin, 285–288
methoxybonducellin, 373 narwedine, 370
methyl salicylate, 279, 280 natural rubber, 134–135
N-methylconiine, 303 necic acids, 312
methylecgonine, 309, 310 necines, 312
methylenedioxy group, 69 neoisomenthol, 148
methylmalonyl-CoA, 240 neolignans, 273
methylmanuifolin K, 291 neomenthol, 148–149
methylorsellinic acid, 222, 224 neomycin, 87, 347
methylphloroacetophenone, 219, 223, 226 neopinone, 323–324
N-methylpseudoephedrine, 317 nepetalactol, 332

nepetalactone, 151–152 intermolecular, 224, 226, 228,
425 Index
neral, 143, 144 273–275
nerol, 143, 144 intramolecular, 227, 228, 322, 323,
nerolidol, 153 349, 370
nerolidyl diphosphate (NLPP), 153, 155 oxidosqualene, 165–167
neryl diphosphate (NPP), 143, 144, oxycodone, 323, 326–327
153, 156 oxynitrilase, 403
nicotinamide adenine dinucleotide oxytetratcycline, 230
oxidized form (NAD+/NADP+)
as hydride acceptor, 47 palmitic acid, 188, 193
in oxidation reactions, 47 palmitoleic acid, 232–233
nicotinamide adenine dinucleotide pancratistatin, 403
reduced form (NADH/NADPH) parthenolide, 154
as hydride donor, 47 pelargonidin, 287
in reduction reactions, 46 pelletierine, 312–313
nicotine, 301, 339 penicillin G. See benzylpenicillin
nicotinic acid, 303 penicillinase, 350
alkaloids from, 338–340 penicillin-cephalosporin rearrangement,
nightshade alkaloid. See hyoscine; 352–353
hyoscyamine penicillins, 349–353
NIH shift, 51–52 pentacycloanammoxic acid, 195, 197
NLPP. See nerolidyl diphosphate pentose phosphate pathway, 105–109
noradrenaline, 319 peptides, 120–124
norcoclaurine, 320–321 pergolide, 330
norephedrine, 317, 388 pethidine, 325
norpseudoephedrine, 317 peucenin, 219, 221
noscapine, 321 PGH2 synthase, 203
NPP. See neryl diphosphate phellandrenes, 145
NSAID, 174, 203, 205, 392 phenoxy radicals, 54, 113, 272, 371
nucleophile, 13 phenylalanine, 68, 258, 264
nucleophilic acyl substitution, 36 alkaloids from, 317–318
nystatin A, 250 biosynthesis of, 263–264
phenylalanine ammonia lyase (PAL),
ophiobolin A, 133, 135, 162 7, 8, 269
ophiobolin F, 162–163 phenylmenthol, 389
Oppenauer oxidation, 368 phenylpropanoids, 269–272
opsin, 179 phenylpropanolamine (PPA), 318
optical activity, 2 phenylpropenes, 270–271
orcinol, 213 phenylpyruvic acid, 263
ornithine, 68, 69, 71 phloroacetophenone, 212, 213, 219
alkaloids from, 306–310 phosphatidic acid, 187
orsellinic acid, 209–213 phosphatidyl choline, 187
oubain, 169–170 phosphoenolpyruvate, 96, 259
oxaloacetic acid, 63, 100–104 phosphofructokinase, 92
oxalosuccinic acid, 101–103 phosphogluconate dehydrogenase, 105
oxazaborolidines, 394 6-phosphogluconic acid, 105
oxazolidinones, 388–389 6-phosphogluconolactone, 105
β-oxidation, 49, 190, 193, 278 phosphoglucose isomerase, 92
oxidative phenolic coupling, 54, 55 3-phosphoglyceraldehyde, 267, 338
2-phosphoglycerate, 96, 250

3-phosphoglycerate, 95, 113, 114 presqualene diphosphate, 164

Index 426
phosphoglycerate kinase, 95 pretetramide, 230
phosphoglycerate mutase, 96 primary metabolism, xvii
5-phosphoribose diphosphate, 266 primary metabolites, xvii
photosynthesis, xvi prochiral π-systems, 6
physostigmine, 328, 375–376 prochirality, 5
phytoalexins, 289 progesterone, 167
phytoene, 176–177 biosynthesis of, 172–174
phytoestrogens, 272, 285 partial synthesis of, 368
phytol, 158–159 total synthesis of, 366
phytyl diphosphate (phytyl PP), 159 proline, 113, 345, 391, 394
Pictet-Spengler reaction, 318–320, 331–333 propionyl-CoA, 194, 240
pig liver esterase (PLE), 403 prostacyclin, 202, 205
pilocarpine, 342–343 prostaglandins (PG), xxi, 111, 199,
pilosine, 342–343 202, 203
α-pinene, 131, 145, 384 PGE2, 204, 205, 403
β-pinene, 145 PGF2, 204, 205
Pinnatoxin A, 363 PGG2, 204
pinoresinol, 258, 273, 276 PGH2, 203, 204, 205, 256
pinyl cation, 145 prostanoids, 203, 206, 207
pipecolic acid, 312, 314 proteinogenic, 112
piperidine alkaloids, 312–315 proteins, 120–124
Δ1-piperidinium cation, 312, 315, 317, 339 protocatechuic acid, 259
pKa values. See acidity constants protohypericin, 225, 228
PKS. See polyketide synthase protopine, 321
Plavix. See clopidogrel pseudodeflectusin, 373
PLP. See pyridoxal-5’-phosphate pseudoephedrine, 317, 391
podophyllotoxin, xviii, 258, 273, 276, pseudopelletierine, 312
294, 334 psicose, 81
polyketide synthase (PKS), 210, 236, 240, pterocarpans, 289
245, 248 pulegone, 148
type I, 235 purine alkaloids, 343
type II, 234 putrescine, 56, 306, 310
type III, 234 pyrethric acid, 150
polyketides, 208–249 pyrethrins, 150
aromatic, 211–227 pyridine-3-carboxylic acid. See
biosynthesis, 208–210 nicotinic acid
chain folding, 212–214, 218, 230, 372 pyridoxal-5’-phosphate (PLP)
isotopic labeling, 213–218 in amino acid decarboxylations, 58
macrolide, 240–251 in transamination, 57
polymyxin B, 347 pyridoxamine-5’-phosphate (PMP), 57, 115
polysaccharides, 84–86 pyrogallol, 260
polyterpenes, 133 pyrrolizidine alkaloids, 303, 310–312
PPA. See phenylpropanolamine pyruvate carboxylase, 100
pravastatin, 237, 402 pyruvic acid, 62, 96, 97–105
precholicalciferol, 175
pregnenelone, 172 quinine, 301, 333, 336
Prelog-Djerassi lactonic acid, 388 quinolinic acid, 338
prenyl transferase, 141 quinolizidine alkaloids, 315
prephenic acid, 261, 263–265 quinone methide, 271, 273, 274

radicals (carbon), 20 serine, 113–117, 187, 267
427 Index
formation and stability of, 21 serotonin, 50, 286, 327
in mechanisms, 22 sesquarterpenes, 176
rapamycin, 312 sesquiterpenes, 133, 152–159
rapamycin PKS, 248 sesterterpenes, 133, 162–163
rautandiol B, 290 Sharpless asymmetric
regioselectivity, 400 dihydroxylation, 398
resonance forms, 14 Sharpless epoxidation, 394–398
resveratrol, 233, 281, 285 shikimate pathway, 258–292
(R)-reticuline, 322 shikimic acid, 111, 258–260
(S)-reticuline, 320–322 sinalbin, 87
retinal, 67, 139, 179 sinapic acid, 269
retinol, 133, 179 β-sitosterol, 167
retro-a ldol, 93, 126 SN1 reaction, 27, 170
retro-Claisen condensation, 194, 278, 279 SN2 reaction, 14, 27, 89
retronecine, 68, 310 solenopsins, 304
rhodopsin, 179 sorbose, 81
riboflavin-5’-phosphate. See flavin sparteine, 315
mononucleotide spearmint, 148
ribose, 77, 81 spermacetti, 189
ribose-5-phosphate, 105, 107, 111, 114, 137 squalene, 133, 164, 165
ribulose, 81 squalene monooxygenase, 165
ribulose-5-phosphate, 105 St. John’s wort, 225
Robinson annulation, 32, 387 stemmadenine, 335
rose oil, 143 stereoisomers, 2–5
rotenoic acid, 292 steroids, 165–176
rotenoids, 292 stigmasterol, 167, 368
rotenone, 292 stipitatic acid, 222, 224
rubber, 139, 140 stipitatonic acid, 222, 224
rubrofusarin, 213, 218 streptomycin, 87
Ruzicka, L., 131 strictosidine, 333, 336
strychnine, 70, 300, 333, 363
sabinone, 147 styrylpyrones, 282
salicin, 86 substrate control, 384–386
salicyl alcohol, 86 succinate dehydrogenase, 104
salicylic acid, 86, 265, 279 succinic acid, 103
salsolinol, 319 succinyl-CoA, 102–104
salutaridine, 323 succinyl-CoA synthetase, 103
salutaridinol, 323 sucrose, 76, 85
SAM. See S-adenosylmethionine sufentanyl, 325, 326
Schiff’s bases. See imines swainsonine, 68, 314
scopolamine. See hyoscine synthesis
secoisolariciresinol, 276 asymmetric, 383
secologanin, 152, 332–333 biomimetic, 369
secondary metabolism, xvii formal, 373
secondary metabolites, xviii partial, 366
sedoheptulose-7-phosphate, 107, 137 for structural confirmation, 373
semi-synthesis. See synthesis: partial
senecionine, 312 total, 364
senkirkine, 312 T-2 toxin, 156

tabersonine, 335 trichothecenes, 156
Index 428
tacrolimus, 185 triglycerides. See triacylglycerols
tagatose, 81 trihydroxyisoflavanone, 289
tajixanthone, 220–221 triose phosphate isomerase, 94
tannic acid, 259 triquetrumone A, 291
taxadiene, 133, 161 tristearin, 187
taxol, 68, 133, 161, 334, 398 triterpenes, 133, 135, 164, 167
TDP-glucose, 89 tropine, 306, 308
teniposide, 276 tropinone, 306, 312, 369
terpenes tryprostatins A and B, 345
classifications of (Table 4.1), 133 tryptophan, 69, 113, 258, 268
history of, 131 alkaloids from, 303, 327–331
regular and irregular, 132 biosynthesis of, 265–267
α-terpenine, 145 tyramine, 65, 318
α-terpineol, 25, 145 tyrosine, 68, 113, 258, 264
4-terpineol, 145 alkaloids from, 303, 318–324
terpinyl cation, 144 biosynthesis of, 51–52, 263–264
testosterone, 172
tetraacetic acid lactone, 212 UDP. See uridine diphosphate
tetracycline, 185, 230, 231 UDP-glucaronic acid, 89
tetrahedral intermediate, 34, 36, 61, UDP-glucose, 88
191, 211 umbelliferone, 268, 280
tetrahydrobiopterin (BH4), 50 uridine diphosphate (UDP), 88
tetrahydroxyisoflavanone, 288 urushiol, 232
tetraterpenes, 133, 135, 176, 177–180 usnic acid, 219, 224, 226
thebaine, 323, 326 valine, 113, 118–119
theobromine, 343 valinomycin, 346
theophylline, 343 vancomycin, 347–349
thiamine diphosphate (TPP), 60–63 vancosamine, 348
thienamycin, 353–355 vanillic acid, 278
thioglycosides, 87 vanillin, 258, 278
thromboxanes (TX), 202 vinblastine, 301, 334–335
TXA 2, 205 vincamine, 335
TXB2, 206 vincristine, 334–335
thujaplicatin, 276 vindoline, 334, 335
thujene, 147 violaxanthin, 178
thujone, 147 virantmycin, 403
thujyl cation, 147 visnagin, 219, 222
tigecycline, 232 vitamin A. See retinol
toluene dioxygenase, 43, 402 vitamin A aldehyde. See retinal
topoisomerase II, 277 vitamin D3. See cholecalciferol
TPP. See thiamine diphosphate vitamin K1, 158, 159
TPP ylide, 61–63 vitexin, 286
tramadol, 325, 326
transaldolase, 44, 107 Wagner-Meerwein shifts, 24
transamination, 57–58 Wallach, O., 131
transferase, 44, 400 warfarin, 281, 282
transketolase, 44, 107, 109 waxes, 188–189
triacylglycerols, 185, 187 Wieland-Miescher ketone, 394, 396
trichodiene, 156–158 wyerone, 201

xanthine, 343 yangonin, 282
429 Index
xanthofusin, 222 yatein, 276
xanthophylls, 177
xanthosine, 343, 344 zeazanthin, 178
xanthoxylin, 219 zingiberene, 133
xylose, 77, 81 zwitterion, 113
xylulose, 81
xylulose-5-phosphate, 106

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Library of Congress Cataloging-in-Publication Data

1 1. Brief Organic Review

30 Nucleophilic 1,4-​(Conjugate) Addition Reactions: Remote Attack on

75 3. Biosynthesis of Carbohydrates and Amino Acids

97 What Happens to the Pyruvic Acid from Glycolysis

131 4. The Terpenoid Pathway: Products from Mevalonic

170 Conversion of Lanosterol (C30) to Cholesterol (C27): Where Did

184 5. The Acetate Pathway: Biosynthesis of Polyketides and Related Compounds

258 6. The Shikimate Pathway: Biosynthesis of Phenolic Products

263 Conversion of Chorismic Acid to Prephenic Acid

300 7. Biosynthesis of Alkaloids and Related Compounds

312 Piperidine-​Type Alkaloids Derived from Lysine

361 8. Organic Synthesis in the Laboratory

399 Use of Enzymes for Synthesis of Optically Pure Compounds: Biocatalysis

411 Suggested Further Readings

THE UNIQUE ROLE OF CARBON

CARBON STORAGE AND THE ROLE OF METABOLISM

combustion respiration decomposer

fossil fuel ocean organic compounds death dead organic

fossilization feeding death

sunlight or other sources to synthesize complex organic molecules from simpler

DISTINGUISHING PRIMARY VERSUS

SECONDARY METABOLITES AND NATURAL PRODUCTS

Alternatively, secondary metabolism in organisms employs metabolic pathways

tryptophan adenine glucopyranose

and therefore unnecessary for an organism’s growth, development, or reproduction.

NATURAL PRODUCTS IN ORGANIC

THE ORGANIC CHEMISTRY OF SECONDARY

Introduction xx Enzyme-S O Enzyme-S O O

to introduce undergraduate students to the elegantly simple organic chemistry used

GOALS AND STRUCTURE OF THIS BOOK

My hope is that Bioorganic Synthesis: An Introduction will prove useful in support-

Chapter 3 covers basic carbohydrate chemistry and the biosynthesis of amino

In addition to simple hydrocarbon structures (alkanes, alkenes, alkynes, and aro-

Table 1.1 Common Organic Functional Groups

STEREOCHEMISTRY REVIEW: THE SHAPES

Constitutional Isomers Stereoisomers

diastereomers: (2S,3S)- vs. (2S,3R)-3-phenylserine and (E)- vs. (Z)-3-methyl-2-pentene

of absolute configuration assignment. Compounds with more than one stereocenter

(1S,2S) (1R,2R) (1R,2S) (1R,2S)

staggered eclipsed vertical Fischer Projection

A convenient way to represent the configuration of certain acyclic compounds

PROCHIRAL RELATIONSHIPS: ONE STEP

By prochiral carbon we mean an achiral carbon atom in a molecule which, through

PROCHIRAL Π-​S YSTEMS: “TWO-​FACED”

prochiral attack on attack on

prochiral carbon addition to "Si" face only

DIASTEREOTOPIC ATOMS AND GROUPS: ONE

If replacement of one of two identical atoms (or groups) at an achiral carbon by an

H NH2 ammonia lyase

RING SYSTEM CONFORMATIONS: FROM LYING

The more stable of two chair conformers for a monosubstituted cyclohexane

11 Brief Organic Review

BICYCLIC SYSTEMS: JOINING OF RINGS

HETEROCYCLIC RING SYSTEMS: ONE ATOM

13 Brief Organic Review

Table 1.2 Some Common Heterocyclic Ring Systems

NUCLEOPHILES AND ELECTROPHILES: GIVE

HO: Br: HS: H 2 O: :NH 3 H 2 S:

30 Nucleophilic 1,4-(Conjugate) Addition Reactions: Remote Attack on

312 Piperidine-Type Alkaloids Derived from Lysine

PROCHIRAL Π-S YSTEMS: “TWO-FACED”

BRØNSTED ACID–B ASE REACTIONS: PROTON

mechanistic terms is necessary. Unlike more common two-electron bond-making

Recall that the E1 reaction is a first-order process with carbocation formation as

ELECTROPHILIC ADDITIONS: Π-B ONDS

NUCLEOPHILIC 1,4-( CONJUGATE) ADDITION

form alkoxide intermediates which after protonation, afford β-hydroxyaldehydes,

7. A simple bond-line structure for the compound (+)-juvabione is shown.

namely the conversion of the –OH group of alcohols (R–OH) or carboxylic acids

NADH/N ADPH: NATURE’S VERSION OF SODIUM