Chiral Separations: A TutorialPharmaceutical AnalysisOctober 13, 2012

OutlineStereochemistry RefresherRelationships of StereoisomersTerminologyChiral SeparationsWhy do we need chiral separations?Different approaches to enantiopure productsChromatographic Chiral SeparationsWhat is Chiral Recognition? 3-point ruleSFC vs. HPLCTypes of CSPsScreening optionProblem solvingAbsolute Stereochemistry

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

Chiral vs Achiral CompoundsAchiral Molecule: Has no stereogenic center; the carbon atom has less than 4 non-equivalent substituents attachedhas a plane of symmetryone that is superimposable on its mirror image (the two are identical) i.e. nail, ball, a baseball bat Not optically activeChiral Molecule: Has one stereogenic center (typically C, but can be N, P, etc.), which is attached to 4 different substituents asymmetricone that is not superimposable on its mirror image (the two are not identical) i.e. hands, keys, shoesthe two mirror image forms are called enantiomers Optically active

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 outwhere = observed rotation, l = cell length in dm, c = concentration in g/mL, and D is the 589nm light from a sodium lamp1999 William Reusch, All rights reserved (most recent revision 7/14/2006) whreusch@msu.edu

Stereochemistry TermsIsomers: Compounds with the different chemical structures and the same molecular formulaStereoisomers: compounds made up of the same atoms but have different arrangement of atoms in spaceEnantiomers are the 2 mirror image forms of a chiral moleculecan contain any number of chiral centers, as long as each center is the exact mirror image of the corresponding center in the other moleculeIdentical 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 techniquesDiastereomers: stereoisomers that are not enantiomersHave 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

OutlineStereochemistry RefresherRelationships of Stereoisomers TerminologyChiral SeparationsWhy do we need chiral separations?Different approaches to enantiopure productsChromatographic Chiral SeparationsWhat is Chiral Recognition? 3-point ruleHPLC vs. SFCTypes of CSPsScreening optionProblem solving

Racemate vs. Single Enantiomerhttp://www.fda.gov/cder/guidance/stereo.htm;C&EN, May 5, 2003, pg. 56

Chiral Blockbuster DrugsNote: Sales figures from IMS HealthCourtesy of C&EN, September 5, 2005, Volume 83, Number 36, pp. 49-53Nine of the top 10 drugs have chiral active ingredients

Examples Albuterol (anti-asthmatic inhalant)D-albuterol may actually cause airway constrictionLevalbuterol (L-albuterol) avoids side effectsAllegra (allergy medication)Single enantiomer of Seldane that avoids life-threatening heart disorders of SeldaneFluoxetine (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

Approaches to Pure Enantiomers Courtesy of Christina Kraml, Wyeth

OutlineStereochemistry RefresherRelationships of Stereoisomers TerminologyChiral SeparationsWhy do we need chiral separations?Different approaches to enantiopure productsChromatographic Chiral SeparationsWhat is Chiral Recognition? 3-point ruleHPLC vs. SFCTypes of CSPsScreening optionProblem solving

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- interactionsDipole stackingInclusion complexingSteric bulk

Five general types of CSPs used in chromatography:Polymer-based carbohydratesPirkle or brush-type phasesCyclodextrinsChirobiotic phases Protein-based

Chiral Chromatographyhttp://www.chemhelper.com/enantiomersep.html

Classification of Chiral Stationary Phases (CSP)Polymer-based CarbohydratesChiral polysaccharide derivatives, i.e. amylose and cellulose, coated on a silica supportEnantiomers form H-bonds with carbamate links between side chains and polysaccharide backboneSteric restrictions at polysaccharide backbone may prevent access of one of enantiomers to H-bonding siteCan be used with normal phase HPLC, SFC, RP-HPLCLimitations: Not compatible with a wide range of solvents other than alcoholsAvailable 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 compatibilitiesCourtesy of Chiral Technologies, Inc.

Naproxen examples using polymer-based CSPsConditions:Chiralpak AD-HHexane/IPA/TFA, 80:20:0.1Flow: 1.0 mL/minConditions:Chiralpak AS-RHaq. H3PO4 (pH2)/ACN, 60:40Flow: 0.7mL/minConditions:Chiralpak AD-H, 100x4.6mmCO2/MeOH, 80:20Flow: 5.0 mL/minConditions:Chiralpak AD-H, 100x4.6mmCO2/MeOH, 90/10Flow: 2.0 mL/minCourtesy of Chiral Technologies, Inc.

Classification of Chiral Stationary Phases (CSP)Pirkle or Brush-type Phases: (Donor-Acceptor)Small chiral molecules bonded to silicaMore specific applications; strong 3-point interactions through 3 classes:-donor phases-acceptor phasesMixed donor-acceptor phasesBinding sites are -basic or -acidic aromatic rings (- interactions), acidic and basic sites (H-bonding), and steric interactionSeparation occurs through preferential binding of one enantiomer to CSPMostly used with normal phase HPLC, SFC. May get less resolution with RP-HPLC; compatible with a broad range of solventsLimitations: only works with aromatic compoundsAvailable 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 phasesCourtesy of Regis Technologies, Inc.

Naproxen examples using Pirkle-type CSP(Normal phase)(Reversed phase)Courtesy of Regis Technologies, Inc.

Classification of Chiral Stationary Phases (CSP)Cyclodextrin CSPsAlpha, beta and gamma-cyclodextrins bond to silica and form chiral cavities3-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 otherUsed in RP-HPLC and polar organic modeLimitations: analyte must have hydrophobic or aromatic group to fit into cavityAvailable columns:Cyclobond (-, -, and -cyclodextrins) from Astec, Inc.ORpak CDA (), ORpak CDB (), ORpak CDC () from JM Scienceshttp://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Brush

Conditions Results 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):23C Chart Speed (cm/min):0.4cm/min. Detection (nm):254nm Injection Volume (L):2.0L Sample Concentration (mg/mL):5.0mg/mL

Chlorpheniramine example using Cyclodextrin-type CSPhttp://www.astecusa.com/applications/result_Mod.aspchlorpheniramine

Peak1 16.1 Peak2 18.1

Classification of Chiral Stationary Phases (CSP)Chirobiotic PhasesMacrocyclic glycopeptides linked to silica Contain a large number of chiral centers together with cavities for analytes to enter and interactPotential interactions:- complexes, H-bonding, ionic interactionsInclusion complexation, steric interactions Capable of running in RP-HPLC, normal phase, polar organic, and polar ionic modesAvailable columns:Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2 (Teicoplanin), Chirobiotic R (Ristocetin A) from Astechttp://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Macrocyclic

Conditions Results 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):25C Chart Speed (cm/min):0.5 Detection (nm):254 Injection Volume (L):2 Sample Concentration (mg/mL):5 Naproxen example using Chirobiotic-type CSPhttp://www.astecusa.com/applications/result_Mod.aspNaproxen

Peak1 8.78 Peak2 10.48

Classification of Chiral Stationary Phases (CSP)Protein-based CSPsNatural proteins bonded to a silica matrixProteins contain large numbers of chiral centers and interact strongly with small chiral analytes through:Hydrophobic and electrostatic interactions, H-bondingLimitations: 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 capacityAvailable columns:Chiral AGP (-glycoprotein) from ChromTechHSA (human serum albumin) from ChromTechBSA (bovine serum albumin) from Regis Technologies

Naproxen examples using Protein-based type CSPhttp://www.chromtech.se/nap-2x.htmHuman Serum Albumin CSPAcid glycoprotein CSP

Selecting a CSPGeneral use column with no solubility issues Polymer-based phases Specific applications; solubility issuesPirkle-typeChirobiotic phasesSFC onlyPolymer-based, Pirkle-type, ChirobioticBiological SamplesProtein-based phases

Suggested Applications of CSPsCompiled from Snyder, et. al, Practical HPLC Method Development, 2nd ed., John Wiley and Sons, Inc. 1997, p. 549

Chiral SFC vs. HPLC AdvantagesReduced solvent Amounts (CO2 reduces liquid waste)Reduced toxicitySolvent types (alkanes, chlorinated, etc)CO2 has a net zero environmental impactSafetyReduce flammabilitySeparation speed/efficiency

DisadvantagesEquipment costsMaintenance/robustnessSolubility

= 1.76Run time = 20.5 minutesFlow rate = 1.5 mL/min = 1.35Run time = 10 minutesFlow rate = 0.4 mL/minHPLC (normal phase)SFC (normal phase)Flurbiprofen examples using HPLC and SFChttp://www.registech.com/chiral/sfcappguide2006.pdf

Chiral ScreenMobile phases: CO2 + methanol or isopropanolColumns: Chiralpak AD-H, AS-HChiralcel OD-H, OJ-HChiralpak IA (immobilized AD)

Daicel Chiralcel OD-H25% MeOH, 140 barDaicel Chiralpak AD-H30% MeOH, 140 bar> LOADABILITYChanging Stationary Phase

Problem Solving Approaches Derivatization of final products and intermediatesUse 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 injectionsEnhances chiral recognition and improves 3-point interactions; improves baseline separation ability by either HPLC or SFCCBZ protecting group easily attached and removed during synthetic processesKraml, Christina et. al ,Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromatoGraphy, J. of Chrom A, 1100 (2005) 108-115..

July 24-27, 2006, San Diego, CACBZ-DerivatizationunderivatizedPurify: ~2g/hrLow S/N ratioPoor separationAurigemma, 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 enantioseparationCourtesy of Roger Stringham, Chiral Technologies, Inc

Use of Basic Additives to Mobile Phase and Sample solvent *Trans()-2-PhenylcyclopropanamineHCl, CAS No. 1986-47-6Aurigemma, C., BSAT 2005, Boston, MA

Analytical SFC*(S,S) Whelk-O 1, 250x4.6mm, 10u i.d. (Regis Technologies, Inc.) 40% IPA w/ 0.1% IPAm 2.5 mL/min @ 140 barIsopropylamine inMobile phase onlyPreparative

NO residual base in collected sampleNo IPAm in sample solventIPAm added to sample solventIPAm Use of Basic Additives to Sample Solvent only Aurigemma, C., BSAT 2005, Boston, MA

SummaryDirect separations of enantiomers achieved by changing CSPsSolubility issues can be resolved by adding CBZ or another protecting groupPoor peak shapes can be overcome by addition of additives to MP, MP + sample solvent, or sample solvent only

Questions??

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