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. II.
a. b. a. b. a. b. c. d. e.

Stereochemistry Refresher Chiral Separations

Relationships of Stereoisomers Terminology Why do we need chiral separations? Different approaches to enantiopure products What is Chiral Recognition? 3-point rule SFC vs. HPLC Types of CSPs Screening option Problem solving

III.

Chromatographic Chiral Separations

IV.

Absolute Stereochemistry (Oliver McConnell)

July 24-27, 2006, San Diego, CA

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

Stereoisomers
Interconvert through rotation about a single bond Not readily Interconvertible

Constitutional (or structural) isomers

Conformational isomers or rotamers

Configurational isomers

w/o chiral centers (opt. inactive)

Conformational isomers mirror images at this carbon Enantiomeric Not mirror images at this carbon Diastereomeric

w/ chiral centers (optically active)

Constitutional (structural) isomers Configurational isomers

Achiral

Chiral Diastereomers Enantiomers

Geometric isomers
Not mirror images Diastereomers

Cis, Trans (E,Z) isomers

Courtesy of Brown/Foote, Organic Chemistry, 3/e, Figure 1 Harcourt, Inc. items and derived items copyright 2002 by Harcourt, Inc. and http://www.chem.uic.edu/web1/OCOL-II/WIN/STEREO/ISOMER.HTM

cis and trans isomers mirror images Enantiomers July 24-27, 2006, San Diego, CA

Chiral vs Achiral Compounds

Chiral Molecule: Has one stereogenic center (typically C, but can be N, P, etc.), which is attached to 4 different substituents asymmetric one that is not superimposable on its mirror image (the two are not identical) the two mirror image forms are called enantiomers Optically active
i.e. hands, keys, shoes

Achiral Molecule: Has no stereogenic center; the carbon atom has less than 4 non-equivalent substituents attached has a plane of symmetry one that is superimposable on its mirror image (the two are identical) Not optically active
i.e. nail, ball, a baseball bat

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.
a. b.

Stereochemistry Refresher
Relationships of Stereoisomers Terminology

II.
a. b.

Chiral Separations
Why do we need chiral separations? Different approaches to enantiopure products

III.
a. b. c. d. e.

Chromatographic Chiral Separations

What is Chiral Recognition? 3-point rule HPLC vs. SFC Types of CSPs Screening option 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 SALES ($ BILLIONS) Lipitor Zocor Plavix Nexium Zyprexa Norvasc Seretide/Advair $12.0 5.9 5.0 4.8 4.8 4.8 4.7 ACTIVE INGREDIENT(S) Atorvastatin Simvastatin Clopidogrel Esomeprazole Olanzapine Amlodipine Salmeterol Fluticasone Erypo Ogastro Effexor TOTAL 4.0 3.8 3.7 $53.5 Epoetin alpha Lansoprazole Venlafaxine FORM OF ACTIVE INGREDIENT(S) Single enantiomer Single enantiomer Single enantiomer Single enantiomer Achiral Racemate Racemate Single enantiomer Protein Racemate Racemate THERAPY CLASS Cholesterol reducer Cholesterol reducer Antithrombotic Antiulcerant Antipsychotic Antihypertensive Bronchodilator Anti-inflammatory Red blood cell stimulant Antiulcerant Antidepressant

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 lifethreatening 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
Stereoselective or asymmetric syntheses Biotransformation or Enzymatic resolution Catalytic enantioselective processes
Many Samples Small Scale

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.
a. b.

Stereochemistry Refresher
Relationships of Stereoisomers Terminology

II.
a. b.

Chiral Separations
Why do we need chiral separations? Different approaches to enantiopure products

III.
a. b. c. d. e.

Chromatographic Chiral Separations

What is Chiral Recognition? 3-point rule HPLC vs. SFC Types of CSPs Screening option 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 transientdiastereomeric complexes; requires a minimum of 3 interactions through:
H-bonding - interactions Dipole stacking Inclusion complexing Steric bulk

CSP

Biphenyl derivative

Five general types of CSPs used in chromatography:

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

Naproxen examples using polymer-based CSPs

Conditions: Chiralpak AD-H Hexane/IPA/TFA, 80:20:0.1 Flow: 1.0 mL/min

Conditions: Chiralpak AS-RH aq. H3PO4 (pH2)/ACN, 60:40 Flow: 0.7mL/min

Conditions: Chiralpak AD-H, 100x4.6mm CO2/MeOH, 80:20 Flow: 5.0 mL/min

Courtesy of Chiral Technologies, Inc.

Conditions: Chiralpak AD-H, 100x4.6mm CO2/MeOH, 90/10 Flow: 2.0 mL/min 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: -donor phases -acceptor phases Mixed donor-acceptor phases Binding sites are -basic or -acidic aromatic rings (- 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 RPHPLC; 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, -Gem 1 (-acceptor phases), Naphthylleucine (-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:

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

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)

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 Column: Dimensions (mm): Catalog Number: CYCLOBOND I 2000 250x4.6mm 20024

chlorpheniramine

Mobile Phase:
Flow Rate (mL/min): Temp (oC): Chart Speed (cm/min): Detection (nm):

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

1.0 mL/min. 23C 0.4cm/min. 254nm

Injection Volume (L):

2.0L
5.0mg/mL

Sample Concentration (mg/mL):

Peak1 16.1 Peak2 18.1

http://www.astecusa.com/applications/result_Mod.asp

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:
Capable of running in RP-HPLC, normal phase, polar organic, and polar ionic modes
- complexes, H-bonding, ionic interactions Inclusion complexation, steric interactions

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 Column: Dimensions (mm): Catalog Number: CHIROBIOTIC V 250x4.6 11024

Naproxen

Mobile Phase:
Flow Rate (mL/min): Temp (oC): Chart Speed (cm/min): Detection (nm):

10/90:THF/0.1% TEAA, pH7

1.0 mL/min. 25C 0.5 254

Injection Volume (L):

2
5

Sample Concentration (mg/mL):

Peak1 8.78 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 Specific applications; solubility issues SFC only
Pirkle-type Chirobiotic phases

Polymer-based phases

Polymer-based, Pirkle-type, Chirobiotic

Biological Samples

Protein-based phases

July 24-27, 2006, San Diego, CA

Suggested Applications of CSPs

Class of Compound
Acids Amino Acids Amines Alcohols Amides Esters Sulfoxides Carbamates Ureas Crown ethers Metallocenes Thiols Amino alcohols Succinamides Hydantoins Binaphtols -Lactams Polycyclic aromatic hydrocarbons Cyclic drugs Aromatic drugs Lactones Cyclic ketones Alkaloids Dihydropyridines NSAIDS Oxazolindones Peptides
Compiled from Snyder, et. al, Practical HPLC Method Development, 2nd ed., John Wiley and Sons, Inc. 1997, p. 549

Type(s) of CSP
Protein, cellulose/amylose, Pirkle, Chirobiotic Cyclodextrins, protein, Chirobiotic Protein, cellulose/amylose, Pirkle, cyclodextrins, Chirobiotic Protein, cellulose/amylose, Pirkle, cyclodextrins Protein, cellulose/amylose, Pirkle, cyclodextrins, Chirobiotic Pirkle, cellulose, Chirobiotic Pirkle, cellulose, protein, Chirobiotic Pirkle Pirkle Cyclodextrins Cyclodextrins Pirkle Pirkle, Chirobiotic Pirkle Pirkle, Chirobiotic Pirkle Pirkle, cellulose Cyclodextrins Protein Protein, Pirkle Cellulose, Pirkle Pirkle, cellulose Cellulose, Pirkle Cellulose, Pirkle Pirkle, cellulose, protein Pirkle Chirobiotic

July 24-27, 2006, San Diego, CA

Chiral SFC vs. HPLC

Advantages
Reduced solvent
Amounts (CO2 reduces liquid waste) Solvent types (alkanes, chlorinated, etc) CO2 has a net zero environmental impact Reduce flammability

Reduced toxicity Safety

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 Run time = 20.5 minutes Flow rate = 1.5 mL/min

= 1.35 Run time = 10 minutes 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

SFC
Solvent selector valve Solvents

Column 3 Column 4 Column 5 Column selector valve

Detector

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 N

PhCH2OCOCl iPr2NEt

O N

Pd/C H2

H 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 0.5 1 1.5 2 2.5 3 3.5 min

underivatized
Aurigemma, C., BSAT 2005, Boston, MA

Purify: ~2g/hr
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 SFC
Isopropylamine in Mobile phase only
mAU 700

H2N*

600 500

400

300

200

100 0

(S,S) Whelk-O 1, 250x4.6mm, 10u i.d. (Regis Technologies, Inc.) 40% IPA w/ 0.1% IPAm 2.5 mL/min @ 140 bar
2 3 4 5 6 7 8 9 min

Preparative
350 340 330 320 310 300 290 280 270 260 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 0 1 2
360 350 340 330 320 310 300 290 280 270 260 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10

340 330 320 310 300 290 280 270 260 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20
5 10 15 20 25 30 35 40 45

No additive in sample solvent

IPAm in sample solvent

10 50 0 -10

55

60

65

70

75

80

85

90

95

10

11

Result: better peak shapes, allowing for high throughput purifications through stacked injections and yielding pure enantiomers
*Trans()-2-PhenylcyclopropanamineHCl, CAS No. 1986-47-6
Aurigemma, C., BSAT 2005, Boston, MA

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 CSPs 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