Chiral Separations: A

Tutorial

Christine Aurigemma
Pfizer Global Research & Development,
La Jolla, CA
July 24, 2006

July 24-27, 2006, San Diego, CA

 Outline

I. Stereochemistry Refresher
a. Relationships of Stereoisomers
b. Terminology
II. Chiral Separations
a. Why do we need chiral separations?
b. Different approaches to enantiopure products
III. Chromatographic Chiral Separations
a. What is Chiral Recognition? 3-point rule
b. SFC vs. HPLC
c. Types of CSP’s
d. Screening option
e. Problem solving
IV. Absolute Stereochemistry (Oliver McConnell)

July 24-27, 2006, San Diego, CA

 Relationships of Stereoisomers
Isomers: Compounds with the
same molecular formula
Same atom Different atom
connectivity connectivity

Stereoisomers Constitutional (or structural)

Interconvert through Not readily
isomers
rotation about a Interconvertible
single bond

Conformational Configurational
isomers or rotamers isomers
Constitutional (structural)
isomers Configurational isomers
w/o w/
chiral centers (opt. inactive) chiral centers (optically active)
mirror images at this carbon
Enantiomeric
Conformational isomers
Achiral Chiral
Not mirror images at this carbon
Diastereomeric
Geometric isomers
Diastereomers Enantiomers
Not mirror images
Diastereomers Cis, Trans
(E,Z) isomers
cis and trans isomers
mirror images
Courtesy of Brown/Foote, Organic Chemistry, 3/e, Figure 1
Enantiomers
Harcourt, Inc. items and derived items copyright 2002 by Harcourt, Inc. and
http://www.chem.uic.edu/web1/OCOL-II/WIN/STEREO/ISOMER.HTM July 24-27, 2006, San Diego, CA
 Chiral vs Achiral Compounds
Chiral Molecule: Achiral Molecule:
• Has one stereogenic center • Has no stereogenic center; the
(typically C, but can be N, P, carbon atom has less than 4
etc.), which is attached to 4 non-equivalent substituents
different substituents  attached
asymmetric • has a plane of symmetry
• one that is not superimposable • one that is superimposable on
on its mirror image (the two are its mirror image (the two are
not identical) identical)
– i.e. hands, keys, shoes – i.e. nail, ball, a baseball bat
• the two mirror image forms are • Not optically active
called enantiomers
• Optically active

http://wps.prenhall.com/wps/media/objects/724/741576/Instructor_Resources/Chapter_05/Text_Images/FG05_01-10UN.JPG July 24-27, 2006, San Diego, CA

 Determination of Optical
Activity
• Each enantiomer has an equal but opposite optical rotation; can
be measured using optical rotation polarimeter
• One enantiomer rotates polarized light in a clockwise direction
and is then designed as (+), or dextrorotatory
• The other enantiomer rotates polarized light in counter-clockwise
direction and is the (-) enantiomer, or levorotatory
• Racemates (1:1 mixture of enantiomers) have no observable
optical rotation; they cancel each other out


Specific Rotation = []D = l*c
where  = observed rotation, l = cell length in dm,
c = concentration in g/mL, and D is the 589nm light from a sodium lamp

©1999 William Reusch, All rights reserved   (most recent revision 7/14/2006) whreusch@msu.edu July 24-27, 2006, San Diego, CA
 Stereochemistry Terms
• Isomers: Compounds with the different chemical structures and the same
molecular formula
• Stereoisomers: compounds made up of the same atoms but have
different arrangement of atoms in space
• Enantiomers are the 2 mirror image forms of a chiral molecule
– can contain any number of chiral centers, as long as each center is the
exact mirror image of the corresponding center in the other molecule
– Identical physical and chemical properties, but may have different
biological profiles. Need chiral recognition to be separated.
– Different optical rotations (One enantiomer is (+) or dextrorotatory
(clockwise), while the other is (-) or levorotatory (counter clockwise))
• Racemate: a 1:1 mixture of enantiomers.
– Separation of enantiomers occurs when mixture is reacted with a chiral
stationary phase to form 2 diastereomeric complexes that can be
separated by chromatographic techniques
• Diastereomers: stereoisomers that are not enantiomers
– Have different chemical and physical characteristics, and can be
separated by non-chiral methods.
– Has at least 2 chiral centers; the number of potential diastereomers for
each chiral center is determined by the equation 2n, where n=the
number of chiral centers

July 24-27, 2006, San Diego, CA

 Outline
I. Stereochemistry Refresher
a. Relationships of Stereoisomers
b. Terminology
II. Chiral Separations
a. Why do we need chiral separations?
b. Different approaches to enantiopure products
III. Chromatographic Chiral Separations
a. What is Chiral Recognition? 3-point rule
b. HPLC vs. SFC
c. Types of CSP’s
d. Screening option
e. Problem solving

July 24-27, 2006, San Diego, CA

 Racemate vs. Single Enantiomer
• Single enantiomers of chiral active pharmaceutical
ingredients (APIs) may have different:
– Pharmacokinetic properties in animal models
• Absorption, distribution, metabolism and excretion
– Pharmacological or toxicological effects
• Biologically “active” isomer may have desirable effects
• Biologically “inactive” isomer may have undesirable side effects (i.e.
increased toxicity)
• Increased pressures by regulatory authorities to
switch from racemic to single enantiomer APIs
• Development of chiral APIs raises issues regarding:
– acceptable manufacturing control of synthesis and impurities
– pharmacological and toxicological assessment of both
enantiomers
– proper assessment of metabolism and distribution
– proper clinical evaluation of these drugs

http://www.fda.gov/cder/guidance/stereo.htm;
C&EN, May 5, 2003, pg. 56 July 24-27, 2006, San Diego, CA
 Chiral Blockbuster Drugs
Nine of the top 10 drugs have chiral active ingredients

GLOBAL 2004 ACTIVE FORM OF ACTIVE

THERAPY CLASS
SALES ($ BILLIONS) INGREDIENT(S) INGREDIENT(S)

Lipitor $12.0 Atorvastatin Single enantiomer Cholesterol reducer

Zocor 5.9 Simvastatin Single enantiomer Cholesterol reducer
Plavix 5.0 Clopidogrel Single enantiomer Antithrombotic
Nexium 4.8 Esomeprazole Single enantiomer Antiulcerant
Zyprexa 4.8 Olanzapine Achiral Antipsychotic
Norvasc 4.8 Amlodipine Racemate Antihypertensive
Seretide/Advair 4.7 Salmeterol Racemate Bronchodilator

Fluticasone Single enantiomer Anti-inflammatory

Red blood cell

Erypo 4.0 Epoetin alpha Protein
stimulant
Ogastro 3.8 Lansoprazole Racemate Antiulcerant
Effexor 3.7 Venlafaxine Racemate Antidepressant

TOTAL $53.5

Note: Sales figures from IMS Health

Courtesy of C&EN, September 5, 2005, Volume 83, Number 36, pp. 49-53 July 24-27, 2006, San Diego, CA
 Examples
• Albuterol (anti-asthmatic inhalant)
– D-albuterol may actually cause airway constriction
– Levalbuterol (L-albuterol) avoids side effects
• Allegra (allergy medication)
– Single enantiomer of Seldane that avoids life-
threatening heart disorders of Seldane
• Fluoxetine (generic name for Prozac,
depression medication)
– R-Fluoxetine – improved efficacy; minimizes side
effects, i.e. anxiety and sexual dysfunction. Other
indications (eating disorders)
– S-Fluoxetine – use for treatment of migraines

July 24-27, 2006, San Diego, CA

 Approaches to Pure Enantiomers

• Chiral Synthetic Approach Many Samples

– Stereoselective or asymmetric syntheses Small Scale

– Biotransformation or Enzymatic resolution

– Catalytic enantioselective processes
• Racemic Approach
– Crystallization
– Chiral salt resolution
– CE (capillary electrophoresis)
– SMB (simulated moving bed technology)
Few Samples
– Chromatography (HPLC, SFC)
Large Scale

Courtesy of Christina Kraml, Wyeth July 24-27, 2006, San Diego, CA

 Outline
I. Stereochemistry Refresher
a. Relationships of Stereoisomers
b. Terminology
II. Chiral Separations
a. Why do we need chiral separations?
b. Different approaches to enantiopure products
III. Chromatographic Chiral Separations
a. What is Chiral Recognition? 3-point rule
b. HPLC vs. SFC
c. Types of CSP’s
d. Screening option
e. Problem solving

July 24-27, 2006, San Diego, CA

 Chiral Chromatography
• Chiral Recognition: Ability of chiral stationary phase, CSP, to
interact differently with each enantiomer to form transient-
diastereomeric complexes; requires a minimum of 3 interactions
through:
– H-bonding
– π-π interactions
– Dipole stacking
– Inclusion complexing
CSP Biphenyl derivative
– Steric bulk

• Five general types of CSPs used in chromatography:

1. Polymer-based carbohydrates
2. Pirkle or brush-type phases
3. Cyclodextrins
4. Chirobiotic phases
5. Protein-based

http://www.chemhelper.com/enantiomersep.html July 24-27, 2006, San Diego, CA

 Classification of Chiral
Stationary Phases (CSP)
1) Polymer-based Carbohydrates
– Chiral polysaccharide derivatives, i.e. amylose and cellulose,
coated on a silica support
– Enantiomers form H-bonds with carbamate links between
side chains and polysaccharide backbone
– Steric restrictions at polysaccharide backbone may prevent
access of one of enantiomers to H-bonding site
– Can be used with normal phase HPLC, SFC, RP-HPLC
– Limitations: Not compatible with a wide range of solvents
other than alcohols

• Available columns:
– i.e. Chiralpak AD, AD-RH, AS, AS-RH, and Chiralcel OD, OD-RH, OJ, OJ-RH,
etc. from Chiral Technologies, Inc.
– Chiralpak IA and IB…same chiral selectors as AD and OD, respectively, but
these are immobilized on the silica; more robust and has much greater
solvent compatibilities

Courtesy of Chiral Technologies, Inc. July 24-27, 2006, San Diego, CA

 CH3
OH
Naproxen examples using
MeO
O polymer-based CSPs

Conditions: Conditions:
Chiralpak AD-H Chiralpak AS-RH
Hexane/IPA/TFA, 80:20:0.1 aq. H3PO4 (pH2)/ACN, 60:40
Flow: 1.0 mL/min Flow: 0.7mL/min

Conditions: Conditions:
Chiralpak AD-H, 100x4.6mm Chiralpak AD-H, 100x4.6mm
CO2/MeOH, 80:20 CO2/MeOH, 90/10
Flow: 5.0 mL/min Flow: 2.0 mL/min
Courtesy of Chiral Technologies, Inc. July 24-27, 2006, San Diego, CA
 Classification of Chiral
Stationary Phases (CSP)
2) Pirkle or Brush-type Phases: (Donor-Acceptor)
– Small chiral molecules bonded to silica
– More specific applications; strong 3-point interactions through 3 classes:
• π-donor phases
• π-acceptor phases
• Mixed donor-acceptor phases
– Binding sites are π-basic or π-acidic aromatic rings (π-π interactions), acidic
and basic sites (H-bonding), and steric interaction
– Separation occurs through preferential binding of one enantiomer to CSP
– Mostly used with normal phase HPLC, SFC. May get less resolution with RP-
HPLC; compatible with a broad range of solvents
– Limitations: only works with aromatic compounds

• Available columns:
• Whelk-O 1, Whelk-O 2, ULMO, DACH-DNB (mixed phases), -Burke 2,
β-Gem 1 (π-acceptor phases), Naphthylleucine (π-donor phases), from
Regis Technologies, Inc.
• Phenomenex Chirex phases

Courtesy of Regis Technologies, Inc. July 24-27, 2006, San Diego, CA

 Naproxen examples using
Pirkle-type CSP
(Reversed phase) (Normal phase)

Courtesy of Regis Technologies, Inc. July 24-27, 2006, San Diego, CA

 Classification of Chiral
Stationary Phases (CSP)
3) Cyclodextrin CSPs
– Alpha, beta and gamma-cyclodextrins bond to silica and
form chiral cavities
– 3-point interactions by:
• Opening of cyclodextrin cavity contains hydroxyls for H-bonding
with polar groups of analyte
• Hydrophobic portion of analyte fits into non-polar cavity
(inclusion complexes)
– One enantiomer will be able to better fit in the cavity than
the other
– Used in RP-HPLC and polar organic mode
– Limitations: analyte must have hydrophobic or aromatic
group to “fit” into cavity

• Available columns:
– Cyclobond (-, -, and -cyclodextrins) from Astec, Inc.
– ORpak CDA (), ORpak CDB (), ORpak CDC () from JM
Sciences
http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Brush July 24-27, 2006, San Diego, CA
 Chlorpheniramine example
using
Cyclodextrin-type CSP
Conditions Results
chlorpheniramine
Column: CYCLOBOND I 2000

Dimensions (mm): 250x4.6mm

Catalog Number: 20024

Mobile Phase: 10/90: CH3CN/1% TEAA, pH 4.1

Flow Rate (mL/min): 1.0 mL/min.

Temp (oC): 23°C

Chart Speed (cm/min): 0.4cm/min.

Detection (nm): 254nm

Injection Volume (µL): 2.0µL Peak1 16.1

Sample Concentration (mg/mL): 5.0mg/mL Peak2 18.1

http://www.astecusa.com/applications/result_Mod.as July 24-27, 2006, San Diego, CA

 Classification of Chiral
Stationary Phases (CSP)
4) Chirobiotic Phases
– Macrocyclic glycopeptides linked to silica
– Contain a large number of chiral centers
together with cavities for analytes to enter and
interact
– Potential interactions:
• π-π complexes, H-bonding, ionic interactions
• Inclusion complexation, steric interactions
– Capable of running in RP-HPLC, normal phase,
polar organic, and polar ionic modes

• Available columns:
– Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2
(Teicoplanin), Chirobiotic R (Ristocetin A) from Astec

http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Macrocyclic July 24-27, 2006, San Diego, CA

 Naproxen example using
Chirobiotic-type CSP

Conditions Results
Naproxen
Column: CHIROBIOTIC V

Dimensions (mm): 250x4.6

Catalog Number: 11024

Mobile Phase: 10/90:THF/0.1% TEAA, pH7

Flow Rate (mL/min): 1.0 mL/min.

Temp (oC): 25°C

Chart Speed (cm/min): 0.5

Detection (nm): 254

Injection Volume (µL): 2

Peak1 8.78
Sample Concentration (mg/mL): 5 Peak2 10.48

http://www.astecusa.com/applications/result_Mod.asp July 24-27, 2006, San Diego, CA

 Classification of Chiral
Stationary Phases (CSP)
5) Protein-based CSPs
– Natural proteins bonded to a silica matrix
– Proteins contain large numbers of chiral centers and interact
strongly with small chiral analytes through:
• Hydrophobic and electrostatic interactions, H-bonding
– Limitations:
• Requires aqueous based conditions in RP-HPLC
• Analyte must have ionizable groups such as amine or acid.
• Not suited for preparative applications due to low sample capacity

• Available columns:
– Chiral AGP (-glycoprotein) from ChromTech
– HSA (human serum albumin) from ChromTech
– BSA (bovine serum albumin) from Regis Technologies

July 24-27, 2006, San Diego, CA

 Naproxen examples using
Protein-based type CSP

Human Serum Albumin CSP Acid glycoprotein CSP

http://www.chromtech.se/nap-2x.htm July 24-27, 2006, San Diego, CA

 Selecting a CSP
• General use column with no solubility issues
Polymer-based phases
• Specific applications; solubility issues
Pirkle-type
Chirobiotic phases
• SFC only
Polymer-based, Pirkle-type, Chirobiotic
• Biological Samples
Protein-based phases

July 24-27, 2006, San Diego, CA

 Suggested Applications of CSPs
Class of Compound Type(s) of CSP
Acids Protein, cellulose/amylose, Pirkle, Chirobiotic
Amino Acids Cyclodextrins, protein, Chirobiotic
Protein, cellulose/amylose, Pirkle, cyclodextrins,
Amines Chirobiotic
Alcohols Protein, cellulose/amylose, Pirkle, cyclodextrins
Protein, cellulose/amylose, Pirkle, cyclodextrins,
Amides Chirobiotic
Esters Pirkle, cellulose, Chirobiotic
Sulfoxides Pirkle, cellulose, protein, Chirobiotic
Carbamates Pirkle
Ureas Pirkle
Crown ethers Cyclodextrins
Metallocenes Cyclodextrins
Thiols Pirkle
Amino alcohols Pirkle, Chirobiotic
Succinamides Pirkle
Hydantoins Pirkle, Chirobiotic
Binaphtols Pirkle
β-Lactams Pirkle, cellulose
Polycyclic aromatic
hydrocarbons Cyclodextrins
Cyclic drugs Protein
Aromatic drugs Protein, Pirkle
Lactones Cellulose, Pirkle
Cyclic ketones Pirkle, cellulose
Alkaloids Cellulose, Pirkle
Dihydropyridines Cellulose, Pirkle
NSAIDS Pirkle, cellulose, protein
Oxazolindones Pirkle
Peptides Chirobiotic

Compiled from Snyder, et. al, “Practical HPLC Method Development”, 2 nd ed., John Wiley and Sons, Inc. 1997, p. 549 July 24-27, 2006, San Diego, CA
 Chiral SFC vs. HPLC
• Advantages
– Reduced solvent
• Amounts (CO2 reduces liquid waste)
– Reduced toxicity
• Solvent types (alkanes, chlorinated, etc)
• CO2 has a net zero environmental impact
– Safety
• Reduce flammability
– Separation speed/efficiency

• Disadvantages
– Equipment costs
– Maintenance/robustness
– Solubility

July 24-27, 2006, San Diego, CA

 Flurbiprofen examples using
HPLC and SFC
HPLC (normal phase) SFC (normal phase)

 = 1.76  = 1.35
Run time = 20.5 minutes Run time = 10 minutes
Flow rate = 1.5 mL/min Flow rate = 0.4 mL/min

http://www.registech.com/chiral/sfcappguide2006.pdf July 24-27, 2006, San Diego, CA

 Chiral Screen
Column 1

Column 2

Column 3
SFC Detector
Column 4

Column 5
Solvent selector
valve Column selector
valve
Solvents

• Mobile phases: CO2 + methanol or isopropanol

• Columns:
– Chiralpak AD-H, AS-H
– Chiralcel OD-H, OJ-H
– Chiralpak IA (immobilized AD)

July 24-27, 2006, San Diego, CA

Changing Stationary Phase

Daicel Chiralcel OD-H

25% MeOH, 140 bar

Daicel Chiralpak AD-H

30% MeOH, 140 bar
> LOADABILITY

July 24-27, 2006, San Diego, CA

 Problem Solving Approaches
• Derivatization of final products and
intermediates
– Use of protecting groups such as t-BOC and CBZ (carbobenzyloxy)
– CBZ derivatization of chiral primary and secondary amines
(common intermediates or final products of enantioselective
synthesis)
– Adding CBZ can improve compound solubility, enables high
efficiency purifications through repetitive, stacked injections
– Enhances chiral recognition and improves 3-point interactions;
improves baseline separation ability by either HPLC or SFC
– CBZ protecting group easily attached and removed during synthetic
processes

H PhCH2OCOCl H
N O O Pd/C N
iPr2NEt H2
N
+ CO2 + toluene

• Acylation of amine with benzyl chloroformate

• Amine is regenerated by catalytic hydrogenolysis using palladium on carbon
• Product is isolated by simple filtration and evaporation of the solvent

Kraml, Christina et. al ,“Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromato
Graphy”, J. of Chrom A, 1100 (2005) 108-115.. July 24-27, 2006, San Diego, CA
 CBZ-Derivatization

• 47.5 g per day

• Isolated 70 g
• 35.4 hrs.
purification time

mAU

50
Low S/N ratio
Poor separation
40

30

20

10

0 0.5 1 1.5 2 2.5 3 3.5 min

underivatized Purify: ~2g/hr

Aurigemma, C., BSAT 2005, Boston, MA July 24-27, 2006, San Diego, CA
 • Addition of strong acid additives to mobile
phase
– Especially useful for separation of chiral amines
– 0.1% ethanesulfonic acid (ESA) added to ethanol, or
0.1% methanesulfonic acid (MSA) added to methanol
will cause formation of ion pairs with the amine to
increase chances of successful enantioseparation

Courtesy of Roger Stringham, Chiral Technologies, Inc July 24-27, 2006, San Diego, CA
 •Use of Basic Additives to Mobile Phase and
Sample solvent

Analytical
mAU
700
H2N*
600 (S,S) Whelk-O 1,
SFC 500

400
250x4.6mm, 10u i.d.
(Regis Technologies, Inc.)
300

Isopropylamine in 200
40% IPA w/ 0.1%
Mobile phase only 100
IPAm 2.5
0
mL/min @ 140 bar
0 1 2 3 4 5 6 7 8 9 min

360
Preparative
350
340
330
350 340
320 330
340
310
330 320
300

No additive in
320 310

IPAm in sample
290
310 300
280
300 290
270
290 280
260
280 270
250
270 260
240
260 250

sample solvent
230

solvent
250 240
220
240 230
210
230 220
200
220 190 210
210 180 200
200
170 190
190 160 180
180 150 170
170 140 160
160 130 150
150 120 140
140 110 130
130 100
120
120 90
110
110 80
100
100 70
90
90 60
80 80
50
70 70
40
60 30 60
50 20 50
40 10 40
30 0 30
20 -10 20
10 10
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
0 0
-10 -10

0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9

Result: better peak shapes, allowing for high throughput purifications

through stacked injections and yielding pure enantiomers
*Trans()-2-Phenylcyclopropanamine•HCl, CAS No. 1986-47-
6 July 24-27, 2006, San Diego, CA
 •Use of Basic Additives to Sample Solvent only

No IPAm in
sample solvent

IPAm added to
sample solvent

NO residual base in

collected sample

IPAm

Aurigemma, C., BSAT 2005, Boston, MA

July 24-27, 2006, San Diego, CA
 Summary

• Direct separations of enantiomers

achieved by changing CSP’s
• Solubility issues can be resolved by
adding CBZ or another protecting
group
• Poor peak shapes can be overcome by
addition of additives to MP, MP +
sample solvent, or sample solvent only

July 24-27, 2006, San Diego, CA

Questions??

July 24-27, 2006, San Diego, CA