Organic Chemistry Option II: Chemical Biology

Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford
Organic Chemistry Option II: Chemical Biology

Dr Stuart Conway
Department of Chemistry, Chemistry Research Laboratory, University of
Oxford
email: stuart.conway@chem.ox.ac.uk
Teaching webpage (to download hand-‐outs):
http://conway.chem.ox.ac.uk/Teaching.html

Recommended books:

Biochemistry 4th Edition by Voet and Voet, published by Wiley, ISBN: 978-‐0-‐470-‐57095-‐1.

Foundations of Chemical Biology by Dobson, Gerrard and Pratt, published by OUP (primer) ISBN:
0-‐19-‐924899-‐0

1

Information flow in cells slide 7

• How does cellular information encoded in gene flow, via RNA, to form proteins?

• We must understand this process in order to harness it for exploration of biological
problems.

The central dogma of molecular biology slide 8

• How does DNA in genes direct the synthesis of RNA and protein?

• How is DNA replicated?

• Francis Crick encapsulated the outlines of this process in the “central dogma of molecular
biology”

2

The central dogma of molecular biology slide 9

• DNA directs its own replication and transcription to yield RNA, which is translated to form
proteins.

• Solid lines indicate the genetic information transfers that occur in all cells.

• Dotted lines indicate special transfers.

• The missing lines indicate transfers that the central dogma postulates never occur.

The structure of DNA and RNA slide 10

• DNA and RNA comprise a polymeric phosphate-‐sugar backbone attached to a nucleic acid
base.
3


• Nucleotides are phosphate esters of pentose (furanose) sugars.

• Deoxynucleotides lack the hydroxyl group at the 2’ position of the sugar ring.

• A nitrogen-‐containing base is linked to the 1’-‐position of the sugar.


• RNA, but NOT DNA, is susceptible to base-‐catalysed
hydrolysis.

• DNA lacks the 2’-‐hydroxyl group, which makes it
resistant to base-‐catalysed hydrolysis.

• It is possible that this chemical stability is why DNA
has evolved to be the store of genetic information.

4


• The nitrogen bases are planar, aromatic and heterocyclic.

• They are usually either purine or pyrimidine derivatives.


H H O
N
H N
N N
N
H N
N N N
N
H H
H
adenine guanine

• The major purine components of nucleic acids are adenine and guanine.

• The purines form glycosidic bonds to ribose via their N9 atoms.


• The major pyrimidine components of
nucleic acids are cytosine, uracil and
thymine (5-‐methyluracil).

• Uracil occurs mainly in RNA whereas
thymine occurs mainly in DNA.

• The pyrimidinesform glycosidic bonds to
ribose via their N1 atoms.

5


• Some DNAs contain bases that are derivatives of the standard set.

• For example N6-‐methylation of adenine and 5-‐methylation of cytosine can occur.


6


Nucleotide: adenosine monophosphate NH2
N
N
O
(R = OH in RNA and H in DNA)
HO P O N N
O
HO
HO R
Nucleoside: adenosine NH2
N
N
(R = OH in RNA and H in DNA)
HO N N
O
HO R
Base: adenine NH2
N
N
N N
H


• Nucleic acids are usually linear polymers
of nucleotides.

• The phosphate groups bridge the 3’-‐ and
5’-‐positions of successive sugar residues.

• The phosphate groups are deprotonated
at physiological pH, hence nucleic acids
are polyanions in the cell.

• Polynucelotides have directionality:
each has a 3’ end and a 5’ end.

7


• Nucleic acids were first isolated in 1869 and the presence of these molecules in cells was
demonstrated a few years later.

• In the 1930s and 1940s it was widely believed that nucleic acids had a monotonously
repeating sequence of all four bases = the so called “tetranucleotide hypothesis”.

• It was generally assumed that genes, known to be carriers of genetic information, were
proteins.

• See Biochemistry pages 85-‐89 to see the experiments that proved DNA is the carrier of
genetic information.


• Erwin Chargaff was the first to show that NH2 O
DNA contains equal numbers of adenine N H
N
CH3
N =
and thymine residues (A = T) and equal
numbers of cytosine and guanine N N O N
residues (C = G). X X
adenine thymine
(A, X=H) (T, X=H)
• These relationships are known as
“Chargaff’s rules”. NH2
O

H N
• Although not specifically stated by N =
N
Chargaff, this observation suggests some H2N N N O N
form of base pairing in the (then X X
unknown) structure of DNA. guanine cytosine
(G, X=H) (C, X=H)

8


• The Watson-‐Crick structure of B-‐DNA consists of two strands that wind about a common
axis with a right-‐handed twist to form a ~20 Å diameter double helix. The two strands are
anti parallel (run in opposite directions).

• The planes of the bases are nearly perpendicular to the helix axis.

• Each base is hydrogen bonded to a base on the opposite strand to form a planar base pair.

Complementary base pairing slide 26

• The most remarkable feature of the Watson and Crick structure is that it can accommodate
only two types of base pairs.

• Each adenine residue must pair with a thymine residue and vice versa.

9


• Each guanine residue must pair with a cytosine residue and vice versa.

• The geometries of these A:T and G:C pairs , the so-‐called Watson-‐Crick base pairs, mean
that these base pairs are interchangeable in the double helix.

10

Hydrogen bonding slide 28

• Hydrogen bonds are one of the most important non-‐covalent interactions in biological
systems.

• They take place between an electron-‐rich heteroatom (acceptor) and an electron-‐deficient

hydrogen atom (donor).

• The hydrogen atom is usually covalently linked to an electronegative atom, such as O or N.

• There is a significant electrostatic component to H-‐bonding.


• However, orbital interactions are also an important component of H-‐bonding.

• H-‐bonds can be viewed as having a σ-‐bonding component.

• Consequently, there is an optimum orientation for H-‐ bonding.


• The optimum angle for H-‐bonding is where the X-‐H bond points directly to the lone pair,
such that the angle is 180°.

• H-‐bond strength can vary between 16 and 60 kJmol-‐1.

• H-‐bonds are typically 1.5-‐2.2 Å compared to 1.0-‐1.5 Å for covalent bonds.

11


H
H
acceptor O N O acceptor donor H N
N N H donor CH3
N N H donor acceptor N
N N acceptor donor H N X
X N N
N N
N H donor acceptor O X
H O X
H
adenine thymine guanine cytosine

H H
N N H donor donor H N
N N acceptor acceptor N
X
N N
H acceptor O X
adenine cytosine

• The H-‐bond donor and acceptor patterns are such that A can only bind to T and G can only
bind to C.

• As A can only bind to T and G can only bind to C, we can immediately understand
Chargaff’s rules.

• In addition, the Watson-‐Crick structure allows for any sequences of bases on one
polynucleotide strand if the opposite strand has the complementary sequence.

• This structure also suggests that hereditary information is encoded in the sequence of
bases on either strand.

12

DNA structure advanced slide 32 & 33

• DNA has three major helical forms, B-‐DNA, A-‐DNA and Z-‐DNA.

• B-‐DNA is the biologically predominant form of DNA it forms a right-‐handed helix with
major and minor grooves.

• When relative humidity is reduced to 75%, B-‐DNA undergoes a reversible conformational
change to A-‐DNA.

• A-‐DNA forms a wide, flatter helix than B-‐DNA.

• The base pairs of A-‐DNA are tilted 20 ° with respect to the helix axis.

• Certain DNA sequences can form a left-‐handed helix that has been called Z-‐DNA.

• It is not clear whether Z-‐DNA has any biological significance -‐ it may play a role in
regulating DNA transcription.

13

RNA structure slide 34

• RNA can also adopt defined conformations.

• Transfer RNA (see later) resembles an “L” shape,
being made up of two short helical regions
connected by a hinge.

• Each helical segment comprises two portions of
the single RNA chain running in opposite
directions.

RNA structure slide 35

H
N N H O
N N H N
X
N N
H O X
adenine uracil

• Hydrogen bonding in helical RNA occurs between cytosine and guanine as in DNA.

• Cytosine is replaced by uracil, which forms complementary hydrogen bonds with adenine.

14

DNA replication slide 36

“It has not escaped our notice that the specific pairing we have
postulated immediately suggests a possible copying mechanism for
genetic material.”

• The division of cells must be accompanied by the replication of

DNA.

• In this process, mediates by DNA polymerase enzymes, each DNA

strand acts as a template for the formation of its complementary
strand.

• Consequently, every progeny cell contains a complete copy of

the DNA from the parent cell.

• Mutations arise when, through rare copying errors, one or more
wrong bases are incorporated into a daughter strand.

• DNA replication is a highly complex process.

• This complexity, when compared to the chemically similar

transcription process, arises from the need for extreme accuracy
in DNA replication so as to preserve the integrity of the genome
from generation to generation.

Translation and transcription slide 37 & 38

• DNA directs its own replication and
transcription to yield RNA, which is
translated to form proteins.

• “Transcription” indicates that the
“language” of the encoding information
remains the same.

• “Translation” indicates that the “language”
changes from that of the base sequence to
that of the amino acid sequence.

• Individual portions of a DNA molecule provide the information for the construction of
various RNA molecules and proteins.

• RNA corresponding to the region of interest is produced by transcription (the synthesis of
an RNA strand from a DNA template). The RNA produced in this case is called messenger
RNA or mRNA.

• This mRNA is then translated when molecules of transfer RNA (tRNA) align with the mRNA
via complementary base pairing between segments of three consecutive nucleotides
(codon).

• Each type of tRNA carries a specific amino acid, which are covalently joined by the
ribosome to form a protein.
15

RNA synthesis: Transcription slide 39

• The enzyme that synthesises RNA is called RNA polymerase.

• It catalyses the DNA-‐directed coupling of nucleotide triphosphates to synthesise new RNA.

• The newly synthesised RNA is complementary to the template DNA.

Transcription slide 40

• RNA synthesis proceeds in a stepwise manner in the 5’→3’ direction.

• Hence, the incoming nucleotide is added to the free 3’-‐OH of the growing RNA chain.

• RNA polymerase selects the nucleotide it incorporates into the growing RNA chain based
on the requirement that it forms a Watson-‐Crick base pair with the DNA strand that is
being transcribed (the template strand -‐ only one strand of DNA is transcribed at a time).

• The RNA polymerase moves along the DNA duplex that it is transcribing and separates a
short (~14 base pairs) segment of the DNA helix to form a transcription bubble.

• The DNA in the transcription bubble forms a short DNA-‐RNA helix with the newly
synthesised RNA.

• The DNA-‐RNA hybrid helix consists of antiparallel strands, hence the DNA’s template
strand is read in its 3’→5’ direction.
16

RNA polymerase slide 41

• RNA polymerase has the overall structure of a crab claw with two “pincers”.

• The outer surface of the protein is almost uniformly negatively charges, whereas the
surfaces that interact with nucleic acids are positively charged.

• The DNA occupies the main channel, which directs the template strand to the active site.

• There the DNA base-‐pairs with the incoming nucleotide triphosphate (not in structure).

Translation slide 42

• Translation is the RNA-‐directed synthesis of polypeptides.

• Although the formation of a peptide bond is relatively simple, the translational process in
highly complicated.

• This complexity arises from the need to link 20 different amino acids residues accurately in
the order specified by a particular mRNA.

• According to the one gene-‐one polypeptide hypothesis, the genetic message dictates the
amino acid sequences of proteins.

• As the base sequence of DNA is the only variable element in this otherwise monotonously
repeating polymer, the base sequence and the protein sequence must be linked.
17


“The problem of how a sequence of four things can determine a sequence of twenty things is
known as the coding problem.”


• With only 4 bases in DNA to code for 20 amino acids, a group of several bases (a codon) is
necessary to specify a single amino acid.

• A triplet code (3 bases per codon) is minimally required since there are 43 = 64 different
triplets of four bases.

• A doublet code would only allow 42 = 16 codons, which is insufficient to specify 20 amino
acids.

• In a triplet code as many as 44 codons might not code for amino acids.

• Alternatively, some amino acids might be specified by more than one codon -‐ a degenerate
code.

18

The genetic code slide 45

• How is DNA’s continuous sequence grouped into codons?

• Is the code overlapping? E.g. ABC codes for the first amino acids and BDC codes for the
second etc.


• Or is the code non-‐overlapping?

• E.g. ABC specifies the first amino acid and DEF the second etc.


• The genetic code (right) is a non-‐
overlapping, comma free, degenerate,
triplet code.

• The genetic code is highly degenerate:
Three amino acids (L, R, S) are each
specified by six codons.

• Only Met and Trp, two of the least
common amino acids in proteins, are
specified by a single codon.

19


• Sydney Brenner and Francis Crick formed the following hypotheses on the genetic code:

1. The code is a triplet code.

2. The code is read in a sequential manner starting from a fixed point in the gene. The
insertion or deletion of a nucleotide shifts the frame (grouping) in which in which the
succeeding nucleotides are read as codons. Thus the code has no internal punctuation that
indicates the reading frame -‐ the code is comma free.

3. All (or nearly all) of the 64 triplet codons code for an amino acid. Therefore some amino
acids are specified by more than one codon -‐ the code is degenerate.


• The sentence represents a gene in which the words (codons) each contain three letters
(bases).

• The spaces have no physical significance; they only present to indicate the reading frame.

• The deletion of the fourth letter (B) shifts the reading frame so that all of the words after
the deletion are meaningless -‐ specify the wrong amino acids.


• Insertion of a letter (X) passed the point of the original mutation restores the original
reading frame.

• Hence on the words (codons) between the two changes (mutations) are altered.

• Therefore the sentence may still be intelligible (the gene could still specify a functional
protein), particularly if the changes are close together.

20


• The major breakthrough in deciphering
the genetic code came in 1961 when
Nirenberg and Matthaei established that
UUU is the codon specifying Phe.

• They added poly(U) to a cell-‐free protein

synthesising system and showed that
this stimulated synthesis of only
poly(Phe).

• In similar experiments, poly(A) was

shown to specify poly(Lys) and poly(C)
was found to specify poly(Pro).

• UAG, UAA and UGA are “stop” codons,

which act as a signal to the ribosome to
terminate protein synthesis.

• These stop codons are also known

(somewhat inappropriately) as
nonsense codons as they are the only
codons that do not specify amino acids.

• UAG, UAA and UGA are sometimes

referred to as amber, ochre and opal
codons.

• AUG and (less frequently) GUG codons form part of the chain initiation sequence.

• These codons also specify amino acids, Met and Val, respectively.

• The arrangement of the genetic code is not random.

• Most synonyms (codons that only differ in their third nucleotide) occupy the same box in
the table.

• XYU and XYC always specify the same amino acids; XYA and XYG do so in all by two cases.

• Changes in the first codon position tend to specify the same or similar amino acids.

• Codons with second position pyrimidines (C AND U) tend to specify hydrophobic amino
acids.

• Codons with second position purines (A and G) encode mostly polar amino acids.

• It seems that the genetic code has evolved so as to minimise the deleterious effects of
mutations.

21


• How does the information in DNA actually translate into polypeptide sequences?

• In 1955 Francis Crick proposed the adaptor hypothesis stating that translation occurs
through the mediation of adaptor molecules.

• Crick suggested that the adaptors might contain RNA as codon recognition could occur by
complementary base pairing.

• Each adaptor was postulated to carry a specific amino acid and to recognise the
corresponding codon.

• At a similar time it was shown that in the course of protein synthesis 14C labelled amino
acids become bound to low molecular mass fractions of RNA.

• This RNA is known as transfer RNA or tRNA and is Crick’s putative adaptor molecule.


• All tRNAs contain:

• A 5’-‐terminal phosphate.

• A 7-‐base pair step that includes the 5’-‐

terminal nucleotide and may include
non-‐Watson-‐Crick base pairs, such as G ⋅
U. This assembly is known as the
acceptor stem as the amino acid is
appended to the 3’-‐OH group.

• A 3-‐ or 4-‐base stem ending in a loop that

that frequently contains the modified
base dihydrouridine (D), known as the D
arm.

• A 5-‐base-‐pair stem ending in a loop that

usually contains the sequence TΨC (Ψ =
pseudouridine).

• All tRNAs terminate in the sequence

CCA, with a free 3’-‐OH group.

• There are 15 invariant positions and 8

semi-‐invariant (only a purine or only a
pyrimidine) positions.
22

Modified nucleotides that occur in tRNA slide 54

The structure of yeast tRNAPhe slide 55

23

Synthesis of tRNA slide 56

• The amino acid is activated by reaction
with ATP to form aminoacyl-‐adenylate.

• This mixed anhydride then reacts with
tRNA to form aminoacyl-‐tRNA and AMP.

Ribosome slide 57

• For translation to occur, mRNA and tRNA must bind to each other, and the amino acids
carried by the tRNA must react to form the polypetide chain.

• This process is mediated by the ribosome.

• The ribosome is a ribozyme, comprising mainly ribosomal RNA (rRNA).

• Elucidating the molecular structure of the ribosome has been extremely challenging.

24

Ribosome slide 59

25



26


• The ribosomal peptidyl transfer reaction occurs ~107-‐fold faster than the uncatalysed
reaction.

• The rate enhancement does NOT occur via general acid or general base catalysis.


• The ribosome enhances the rate of peptide bond formation by properly positioning and
orienting its substrates.

• The ribosome may also play a role in excluding water from the preorganised electrostatic
environment of the active site.

27


• Ribosomes act very fast -‐ a rate of 6-‐9 amino acids per second in eukaryotic cells and 17-‐21
amino acids per second in prokaryotic cells.

28

Naturally occurring amino acids slide 66

Protein structure –α-‐helices slide 67

• Only one helical polypeptide conformation has simultaneously allowed conformational
angles and a favourable hydrogen-‐binding pattern.

• This striking element of secondary structure is known as the α-‐helix.

29

Protein structure – anti-‐parallel β-‐sheets slide 68

O H Phe O H Phe O H
N N N
N N
Lys O H Val O H Gln
Trp H O Thr H O Gln

N N
N N N
H O Ala H O Ile H

• Anti-‐parallel β-‐sheets are an important type of protein secondary structure.

• This arrangement results in a strong hydrogen bond with a near optimal N-‐O distance.

Protein structure – parallel β-‐sheets slide 69

O H Phe O H Asp
N N
N N N
H Leu O H Ile O H
O H Asp O H Trp O H Ala

N N N
N N N
Leu O H Ile O H Ile O H

• β-‐sheets can also have a parallel arrangement.

• This results in a staggered pattern of hydrogen-‐bonding.

Protein structure – β-‐turns slide 70

• There are two types of β-‐turns, Type I and Type II.

• Each comprises four key amino acids.
30

Enzymes slide 71

• Almost all chemical reactions that comprise life are catalysed by enzymes.

• The rates of enzymatically catalysed reactions are typically 106 to 1012 greater than the
corresponding uncatalysed reactions.

• The catalysis occurs under relatively mild conditions.

• Enzymes often catalyse their reactions with a high degree of substrate selectivity.

• As enzymes are chiral the active site is “chiral space” allowing differentiation between pro-‐
chiral groups.

Enzymes slide 72

• Types of enzyme catalysis:

1. Acid-‐base catalysis.

2. Covalent catalysis.

3. Metal ion catalysis.

4. Electrostatic catalysis.

5. Proximity and orientation effects.

6. Preferential binding (stabilisation) of the transition state complex.

31

Egg white lysozyme (retaining glycosidase) slide 74

O O
HO OR1 retaining glycosidase HO OR2
+ +

R2 OH R1 OH
• Lysozyme enzymes are glycoside hydrolase or glycosidase enzymes.

• These enzymes catalyse hydrolysis or transacetylation of glycosidic linkage in sugars.

• Egg white lysozyme is a retaining glycosidase, meaning that the configuration of the
anomeric centre is the same in substrate and product.

• The retaining glycosidases employ both acid-‐base catalysis and covalent catalysis in their
mechanism.

• Chemical tools and crystallography have helped to determine the proposed mechanism of
this enzyme.


• Lysozyme enzymes are involved in the destruction of bacterial cell walls.

• These enzymes work by hydrolysing β (1→4) glycosidic linkages from N-‐acetylmuramic acid
to N-‐acetylglucosamine.

• Lysozyme occurs mainly in the cells and secretions of vertebrate, where it may function as
an antibacterial agent.

• However, few pathogenic bacteria are susceptible to lysozyme alone, suggesting that this
enzyme may help dispose of bacteria after they have been killed by other means.

• Hen egg white lysozyme is the most widely studied species of lysosyme, mainly as it is
readily available -‐ one egg contains about 5 g.


32



retaining glycosidase retaining glycosidase retaining glycosidase
E35 E35 E35
O O O O O O
H H
OH OH OH
HO HO H HO
O O O R' O
HO O HO HO O
HO R HO HO R'
O O O O O O
D52 D52 D52


33



• The proposed covalent intermediate in the mechanism of lysozyme had never been
observed, as the breakdown of this intermediate must be much faster than the rate of
formation, in order for the enzyme to function efficiently.

• A sugar containing a fluoride at the anomeric position should react rapidly with the enzyme
to form a covalent intermediate.

• The additional fluorine at the C2 position of the ring will reduce the rate of the covalent
intermediate breaking down.

• Mutation of glutamate 35 to glutamine (E35Q) slows the rate of the reaction further,
meaning that the covalent intermediate with the fluorosugar accumulates and can be
observed by X-‐ray crystallography.

34

Inverting glycosidase slide 82

O
O HO
HO OR1 inverting glycosidase
OR2
+
+
R2 OH

R1 OH
• Inverting glycosidases catalyse the same overall transformation as retaining glycosidases,
but the resulting effect on the stereochemistry of the anomeric centre is different.

• The mechanism of inverting glycosidases means that the configuration of the anomeric
centre is inverted during the reaction.

• Inverting glycosidases employ acid-‐base catalysis in their mechanism.

• Glycosidases are important in all forms of life, mainly in the metabolism of complex
carbohydrates.



35


inverting glycosidase inverting glycosidase inverting glycosidase
E95 E95 E95
O O O O O O
H
OH OH OH
O O O O
R
O
H H
O
H
H
O O O O O O
D278 D278 D278

inverting glycosidase inverting glycosidase inverting glycosidase


36

Serine protease slide 88

H serine protease HO R2
N R2
R1 R1 NH2 +
O
O

• Serine proteases (e.g. trypsin, chymotrypsin, elastase) are digestive enzymes that are
synthesised in pancreatic acinar cells and secreted into the small intestine.

• These enzymes all catalyse the hydrolysis of amide bonds.

• Different enzymes have different selectivities for the amino acid side chains that flank the
amide bond to be cleaved.

• Serine proteases employ both acid-‐base catalysis and covalent catalysis in their mechanism.

• Additionally, the intermediate oxyanion is stabilised in the “oxyanion hole”.


37



serine protease H57 serine protease H57
S195 S195
O N N O N N H
H H
D102 O D102 O
O H O H
N R
H R' O
N R
R'
O
O H
H
R O R' N
H
serine protease H57 serine protease H57
S195 S195
O N N H O N N
H H
D102 O O D102 H O O
O O
O O
R H R
H

38



• The charged carbonyl carbon of the tetrahedral intermediate is stabilised by the “oxyanion
hole”.

• The negatively charged oxygen forms hydrogen bonds with the backbone NH groups of
Gly193 and Ser195.


39

Sulfatase slide 96

O O sulfatase O O
R S R OH + S
O OH HO OH

• Sulfatase enzymes cleave sulfate esters in biological systems.

• These enzymes are involved in regulating the sulfation states that determine the function
of may physiologically important molecules.

• The mechanism of sulfatase enzymes involves covalent catalysis, metal (calcium) ion
catalysis, acid-‐base catalysis.

• In addition, one of the catalytic residues is post-‐translationally modified to aid the catalytic
function of these enzymes.

Sulfatase slide 97

Sulfatase slide 98

40

Sulfatase slide 99

sulfatase sulfatase
H H
N H211 N H211
N N
H H
R R
O O
S S
O O O O
O O
H Ca2+ H Ca2+
N N
N H O N O
O H O O
H D317 H H D317
FGly51 O O FGly51 O
115H 115H
sulfatase H sulfatase
R O
H
sulfatase sulfatase
H H
N H211 N H211
N N
SO42-
O
O
S
O
H Ca2+ H Ca2+
N N
N O N O
O O O H
H D317 D317
FGly51 HO FGly51 HO
115H 115H
sulfatase sulfatase

Sulfatase slide 100

41

Phospholipase C slide 102

• Phospholipase C catalyses the hydrolysis of the minor membrane phospholipid,
phosphatidylinositol 4,5-‐bisphosphate, to give inositol 1,4,5-‐trisphosphate and diacyl
glycerol, both of which are intracellular second messengers.

• PLC uses acid-‐base catalysis and metal (calcium) ion catalysis in its mechanism.


42



PLC! H311 PLC! H311
H H
N N
Ca2+ Ca2+
N N
H H
H356 H B H356
H B
O O
O N H O N H
O P O N O P O N
H H
O O
R R
R OH
PLC! H311 PLC! H311

H H
N N
Ca2+ Ca2+
N N
H H
B H356 H B H356
H
O O
O N H O N H
O P O N O P N
H
O O
H
H H
O

43


44


• There are few potent and selective inhibitors of PLC.

• In order to develop such compounds, an effective assay for PLC (enzyme) activity is
required.

• Huang et al. have developed a compound, based on PtdIns(4,5)P2 that produce a
fluorescent molecule when PLC is functioning.

• This compound allows accurate monitoring of PLC activity inside cells and hence will enable
the discovery of new PLC inhibitors.


OH lipid OH
HO O O HO O
H2O3PO H2O3PO
H2O3PO O P O O H2O3PO O P OH
HO lipid HO
OH O
O
O
OC8H17 HN N

45


OH
HO O
H2O3PO O
H2O3PO O P OH OC8H17
HO O OH
OC8H17 HO O
phospholipase C H2O3PO O
H2O3PO O P OH
O HO O O N N
H
O N N
H
O
OC8H17
O
H2N
O N N
N H H
fluorescent
CO2


OH
HO O
H2O3PO
H2O3PO O P OH
HO OH O
O
OC8H17 HO O OC8H17 H2N
phospholipase C H2O3PO
H2O3PO O P OH
O HO O N
fluorescent
O N N
H

46

Affinity chromatography slide 114

• Many proteins possess the ability to selectively bind small molecules tightly but non-‐
covalently.

• This property can be used to assist the purification of proteins using a technique called
affinity chromatography.

• The molecule that binds to the protein (ligand) is covalently attached to a solid support.

• When an impure protein solution is passed through this chromatographic material the
desired protein binds to the immobilised material.

• Other substances and proteins are washed through the column with the buffer.

• The desired protein can be recovered (in a highly purified form) by changing the elution
conditions.

• An affinity matrix can also be used to identify which proteins bind to the immobilised
ligand.

• Proteins that bind to the matrix and that can be competed off by the presence of the
soluble ligand, demonstrate selective binding to the ligand.

• This technique has been used to understand cellular signalling pathways.

47


OR1
O
P O OR2
HO O OH
OH
R3O OR5
OR4


48


O
OCOC15H31
O
P O OCOC11H22NH
HO O ONa = solid support

OH
(NaO)2OPO OPO(ONa)2
OPO(ONa)2

• The various PIPs were synthesised and immobilised onto a solid phase via a terminal amino
group.

• The immobilised PIPs formed an affinity matrix that was used to identify novel PIP-‐binding
proteins and to determine their binding selectivity.


• Cell lysates were passed through affinity columns made of the immobilised PIPs.

• The proteins that bound to the matrix were then passed down the column having been
pre-‐incubated with soluble ligand.

• Proteins that did not bind in the second case are the proteins that bind to the ligand
selectively.

49


O
OCOC15H31
O
P O OCOC11H22NH
HO O ONa = solid support

OH
(NaO)2OPO OPO(ONa)2
OPO(ONa)2

• Protein kinase B (PKB) is known to bind to PtdIns(3,4,5)P3.

• PKB binds most strongly to the natural D-‐form of PtdIns(3,4,5)P3.

• The protein does not bind to unfunctionalised, control, beads.


50

Transition state mimics slide 122

• One method that enzymes catalyse reactions is by binding preferentially to the reaction
transition state rather than the substrate or product.

• Therefore molecules that mimic the transition state could potentially act as competitive
inhibitors of the enzyme.


E35 E35 E35
O O O O O O
H H
OH OH OH
HO HO H HO
O O O R' O
HO O HO HO O
HO R HO HO R'
O O O O O O
D52 D52 D52


51


• The δ-‐lactone above is a mimic of the lysozyme transition state.

• The ester unit (red) is coplanar, similar to the SN2 transition state.


HO OH neuraminidase HO OH HO OH
CO2H (retaining glycosidase) CO2H OH
O O glycoconjugate O OH O CO2H
HO R HO R HO R
HO HO HO

• Neuraminidases, which are involved in virus proliferation are retaining glycosidases.

• Neuraminidases cleave the budding virus particle from the host cell.

• Inhibition of neuraminidases is potentially of therapeutic value.


HO OH
HO OH LG
HO OH
O CO2H
O CO2H O CO2H O CO2H
HO R
HO R HO R HN R
NH H2N
HO HO
Nu
H2N
!!"#$%&'()*+'#($&$, -./. 0,1,'2& 3&4)56

• Both Relenza and Tamiflu, which are marketed as treatments for influenza, are
neuraminidase transition state mimics and competitive inhibitors.

• Relenza was one of the first examples of crystal structures being employed in rational drug
design.
52


Caged compounds slide 129

• Caged compounds are biologically active molecules that are rendered biologically inert by
the addition of a photolabile protecting group to an important functional group.

• Removal of the protecting group releases (uncages) the active compound.

• This technique is often applicable to the non-‐invasive control of biologically important
compounds inside cells.

• A high degree of temporal and spatial control is obtained by using caged compounds.

53



NO2 NO H
OR light O
ROH
MeO MeO
OMe OMe
inactive compounds active compound

• The dimethoxynitrobenzyl moiety is a commonly used caging group.

• Irradiation with light of wavelength ~355 nm gives efficient release of the caged compound.

54


OH
O O O O O H
N H O OH N O
N H N
R R R
O R O
O O
H3CO H3CO H3CO
H3CO
OCH3 OCH3 OCH3
OCH3
NO O
H
ROH
H3CO
OCH3

• The first step of photocleavage is the n → π* excitation of the nitro group to the 1st singlet
excited state.

• The singlet state then decay, via intersystem crossing, to the triplet state, which behaves as
a diradical in the subsequent “dark” reactions.

Photoaffinity labelling slide 137

• Photoaffinity labelling employs a photoactivatable but chemically inert ligand analogue.

• Once the ligand is bound to the receptor, activation by light forms a highly reactive species
that binds covalently to the protein at the site of interaction.

• Subsequent analysis of the protein can provide information about the site of ligand
interaction.

55


light
azide R N nitrene (singlet or triplet)
R N N N
R light R
diazo compounds
N N carbene (singlet or triplet)
R R
light
diazonium salts carbocation
R N N X R
R light R
N
diazirines carbene
N
R R
O O
light carbonyl excited state
benzophenones
(triplet or singlet)
R R


light
R N N N R N N N R N + N2


Nu
H+ Nu
N1 2
1 3
2 N HN
3
7 7 4
6 4
6 5
5

56


57

Organic Chemistry Option II: Chemical Biology

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Organic Chemistry Option II: Chemical Biology

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Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford

Organic Chemistry Option II: Chemical Biology

Information flow in cells slide 7

The central dogma of molecular biology slide 9

The structure of DNA and RNA slide 12

The structure of DNA and RNA slide 14

• They are usually either purine or pyrimidine derivatives.

The structure of DNA and RNA slide 17

The structure of DNA and RNA slide 19

The structure of DNA and RNA slide 20

The structure of DNA and RNA slide 21

The structure of DNA and RNA slide 25

Complementary base pairing slide 27

Hydrogen bonding slide 28

• They take place between an electron-­‐rich heteroatom (acceptor) and an electron-­‐deficient

• There is a significant electrostatic component to H-­‐bonding.

Hydrogen bonding slide 29

• H-­‐bonds can be viewed as having a σ-­‐bonding component.

• Consequently, there is an optimum orientation for H-­‐ bonding.

Hydrogen bonding slide 30

• H-­‐bond strength can vary between 16 and 60 kJmol-­‐1.

Complementary base pairing slide 31

DNA structure advanced slide 32 & 33

RNA structure slide 34

DNA replication slide 36

• The division of cells must be accompanied by the replication of

• In this process, mediates by DNA polymerase enzymes, each DNA

• Consequently, every progeny cell contains a complete copy of

• DNA replication is a highly complex process.

• This complexity, when compared to the chemically similar

Translation and transcription slide 37 & 38

RNA synthesis: Transcription slide 39

Transcription slide 40

RNA polymerase slide 41

Translation slide 42

Translation slide 43

The genetic code slide 45

The genetic code slide 46

The genetic code slide 47

The genetic code slide 48

The genetic code slide 51

• They added poly(U) to a cell-­‐free protein

• In similar experiments, poly(A) was

• UAG, UAA and UGA are “stop” codons,

• These stop codons are also known

• UAG, UAA and UGA are sometimes

The genetic code slide 52

Translation slide 53

• A 7-­‐base pair step that includes the 5’-­‐

• A 3-­‐ or 4-­‐base stem ending in a loop that

• A 5-­‐base-­‐pair stem ending in a loop that

• All tRNAs terminate in the sequence

• There are 15 invariant positions and 8

Modified nucleotides that occur in tRNA slide 54

The structure of yeast tRNAPhe slide 55

Synthesis of tRNA slide 56

• The ribosome is a ribozyme, comprising mainly ribosomal RNA (rRNA).

Ribosome slide 59

Translation slide 60

Translation slide 62

Translation slide 64

Naturally occurring amino acids slide 66

• They take place between an electron-‐rich heteroatom (acceptor) and an electron-‐deficient

• There is a significant electrostatic component to H-‐bonding.

• H-‐bonds can be viewed as having a σ-‐bonding component.

• Consequently, there is an optimum orientation for H-‐ bonding.

• H-‐bond strength can vary between 16 and 60 kJmol-‐1.

• They added poly(U) to a cell-‐free protein

• A 7-‐base pair step that includes the 5’-‐

• A 3-‐ or 4-‐base stem ending in a loop that

• A 5-‐base-‐pair stem ending in a loop that

Protein structure – anti-‐parallel β-‐sheets slide 68

• This results in a staggered pattern of hydrogen-‐bonding.

• Inverting glycosidases employ acid-‐base catalysis in their mechanism.