Novel chiral stationary phase for the enantioseparation of racemic drugs 1

Enantioseparation of selected racemic compounds was evaluated on phenylcarbamate CSP ETHE-3PC-L which was packed into a HPLC analytical column [∅ 4.6 x 250 mm] under normal and reversed

NOVEL CHIRAL STATIONARY PHASE FOR THE ENANTIOSEPARATION OF RACEMIC DRUGS

LO MEE YOON

(MSc)

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE

2005

Acknowledgements

I would like to express my immense gratitude to my supervisor, A/P Ng Siu Choon for his guidance and supervision throughout the project I would also like to thank

my co-supervisor, Prof Ching Chi Bun for his support

I would like to thank my parents and sisters for their advice and encouragement during my studies I would also like to thank my boyfriend, kinseng for his understanding and continual support

I would like to thank all my friends in the functional polymer groups, Dr Chen Lei, Dr I Wayan, Yeang Chyn, Yi Fei, Teck Chia, Yin Fun, Xiang Hua, Zou Yong, Wei Hua and Daming for their kind support Especially thanks to Frances for her kind assistance Last but not the least; I would like to thank National University of Singapore for the award of the Research Scholarship

Table of Contents

Acknowledgements……….i

Table of Contents……….ii

Summary………vii

Abbreviations and Symbols……….x

1.4 High Performance Liquid Chromatographic (HPLC) 14

1.6 Cyclodextrin and Chiral Stationary Phase 26

1.7 Three-Point Interaction − Origin of CSP 33

2.2.1 Preparation of 5-Iodo-hex-1-ene and 11-Iodo-undec-1-ene 52

2.2.2 Synthesis of 2a, 2b and 2c 53 2.3 Synthesis of Perfunctionalized Carbamolylated β-CD 3a-3f 55

6.5 Organic Modifier under Reversed Phase 128

6.6 Concentration of TEAA Buffer under Reversed Phase 129

7.3 Synthesis of Perfunctionalized Carbamolylated β-CD 147

7.4 Hydrosilylation and Immobilization 150

Summary

The importance of chirality is attracting more attention from researchers nowadays Purification of enantiomers is always preferred for safer clinical applications particularly in biomedical and pharmaceutical areas Chiral drugs are generally administered either as enantiomers or as racemates, and very often two enantiomers of the same racemate possess different pharmacological effects In order to avoid side effects that could be caused by the presence of an undesirable component in a racemic drug, it is important to design a method for effective separation of racemates

The separation of enantiomers is widely studied in analytical chemistry, especially in the area of high performance liquid chromatography (HPLC) due to its versatility In this work, series of ether-linkage β-cyclodextrin (β-CD) bonded chiral stationary phases (CSPs) were synthesized The CSPs were prepared by hydrosilylation and immobilization via a stable ether-linkage onto the surface of silica gel The well-

defined CSPs were characterized and packed into HPLC analytical (with –L) or microbore (without –L) columns for the analysis of racemic compounds or drugs

The synthesized CSPs were evaluated under normal and reversed phase HPLC Selected chiral compounds and drugs were chromatographed on the CSPs for the investigation of possible separation mechanisms Nonpolar solvents such as hexane and isopropanol (IPA) were used as mobile phase under normal phase H-bonding and π-π interactions dominate under normal phase Nonpolar solvents tend to occupy the CD

cavity and preventing inclusion complexation from taking place Under reversed phase, polar solvents such as water, triethylammonium acetate (TEAA) buffer, methanol (MeOH) and acetonitrile (ACN or CH3CN) were used Under reversed phase, inclusion complexation takes place

In this work, six β-CD bonded CSPs (ETHE-3PC, ETHE-6PC, ETHE-11PC,

ETHE-3NC, ETHE-3pCPC and ETHE-3pMPC) were conveniently prepared and

characterized Enantioseparation of selected racemic compounds was evaluated on

phenylcarbamate CSP ETHE-3PC-L which was packed into a HPLC analytical column

[∅ 4.6 x 250 mm] under normal and reversed phase The CSP was proven to be a generic CSP to resolve a wide range of structurally diverse racemic compounds

Three CSPs with the same spacer length, ETHE-3NC, ETHE-3pCPC and

ETHE-3pMPC were prepared The CSPs were modified with different perfunctionalized

substituents on the remaining C2-, C3- and C6- CD hydroxyl groups Chromatographic

results for the three CSPs were compared with ETHE-3PC for the investigation of the

effects of substituents on enantioseparations All four CSPs were packed into HPLC microbore column [∅ 2.1 x 150 mm] Naphthylcarbamate CSP ETHE-3NC showed good discriminating abilities towards amines and β-blockers; 4-methoxyphenylcarbamte

CSP 3pMPC showed good enantioselectivity particularly for β-blockers

ETHE-3pCPC on the other hand, showed high enantioselectivity for weak acids, carbonyls,

alcohols, flavanones and other electron-rich analytes However, the three CSPs failed to perform satisfactory enantioseparation under normal phase

Phenylacarbamte CSP ETHE-3PC, ETHE-6PC and ETHE-11PC were prepared

in different spacer lengths for the study of the effect of spacer length on the enantioseparation abilities of the CSPs Different spacer lengths afforded different chiral discrimination behaviors, particularly on solute retentivity and selectivity There appears

an optimum spacer length which seems to be close to 6 methylene groups under normal and reversed phase

Lastly, study on the optimization of chromatographic conditions such as mobile phase composition, flow rate, buffer concentration and pH, and selection of polar organic modifiers were included Thermodynamics and surface loading studies were also investigated

Abbreviations and Symbols

Chapter 1 Introduction

1.1 Overview

Chirality1 is formally defined as the geometric property of a rigid object of not being superimposable with its mirror image Molecules that can be superimposed on their mirror images are achiral or not chiral Chirality is found throughout biological systems, from the basic building blocks of life such as DNA, amino acids, carbohydrates and lipids

to the layout of the human body

D

C

Figure 1.1 A chiral molecule with carbon attached to

four different substituents

Chirality is often illustrated with the idea of left- and right-handedness: 2, 3 a left hand and right hand are mirror images of each other but are not superimposable The two mirror images of a chiral molecule in Figure 1.2 are termed enantiomers Like hands, enantiomers come in pairs.4 Enantiomers are stereoisomers that are related as nonsuperimposable mirror images Stereoisomers are molecules with the same constitution that differ with respect to spatial arrangement of certain atoms or groups Stereoisomerism can result from a variety of sources aside from the single asymmetric carbon (chiral or stereogenic center, a carbon with four different substituents) A

molecule with a stereogenic axis can also be chiral Enantionmers have identical physical properties except for the rotation of the plane of polarized light.3 Rotation of light will not

be observed if the light is passed through an equimolar mixture of a racemic mixture or racemate The difference in the interaction of the chiral selector with the two enantiomers

is called enantioselectivity This difference can be of a thermodynamic or kinetic nature

A diastereomer is an optical isomer that is not related as an object and its mirror image, it has more than one chiral center

Figure 1.2 Mirror images of a chiral molecule

As the number of carbons with chirality increases in a molecule, the number of possible enantiomers also increases Two isomers (one pair of enantiomers) can be found for a chiral molecule having one chiral centre; four isomers (two pairs of enantiomers) for

a molecule with two chiral atoms and so on That is, the number of stereoisomers is 2n, where n = number of chiral atom(s) A chiral molecule has at least one pair of enantiomers

Optically active compounds are of immense importance on account of their widespread occurrence in nature.3 Most optically active drugs are chiral as a result of the presence of an asymmetrically substituted tetrahedral carbon atom.5-7 However, chirality can also result from other types of molecular asymmetry For example, the presence of any asymmetrically substituted atom of tetrahedral geometry, such as silicon, quaternary nitrogen, and metals such as manganese, copper etc that form tetrahedral coordination complexes can also result in chirality Similarly, compounds containing asymmetrically substituted tetrahedral phosphorus atoms are also chiral The antineoplastic agent cyclophosphamide is one example of a compound with a chiral phosphorus moiety (Figure 1.3)

O P N

H O N Cl

Cl

O P N

H O N Cl Cl

Figure 1.3 Optical isomers of cyclophophamide

Chirality is also a property of compounds containing an asymmetrically substituted atom of pyramidal geometry For example, secondary and tertiary amines bearing four different substituents, with one "substituent" being the lone pair of electrons

on nitrogen are chiral However, due to rapid pyramidal inversion in Figure 1.4, the individual enantiomers are not usually separable or resolvable On the other hand, amines that contain an asymmetrically substituted nitrogen atom in a ring system, particularly at

a bridgehead position for example Troger’s base, may not undergo facile inversion, and thus it is easier to be resolved

N

CH3

H CH2 CH3

N N

Figure 1.4 (A) Pyrimidal inversion of a chiral

nitrogen; (B) Troger’s base

The same is true for asymmetrically substituted pyramidal sulfur derivatives such

as sulfoxides, sulfonic esters, sulfonium salts and sulfites For example, the sulfur atom

of the nonsteroidal anti-inflammatory sulindac in Figure 1.5 bears four different substituents (one being a pair of electrons) and hence is chiral

H3

O

CH3HOOC

Figure 1.5 Chiral sulfoxide sulindac

As we all know, many biologically active compounds exist as a mixture of stereoisomers Administration of a single enantiomer is associated with improved potency and selectivity of the drug and diminished side events as a result of the activity

of the unwanted enantiomer.8-10 This is due to the fact that binding sites of enzymes and receptors preferentially interact with only one of the stereoisomers.11 In pharmacology, chirality is an important factor in drug efficacy Enantiomers of a drug may differ in potency, pharmacological action, metabolism, toxicity, plasma disposition and urine excretion kinetics.12 As summarized by Ariens10, “often only one isomer is

therapeutically active, but this does not mean that the other is really inactive It may very well contribute to the side effects The therapeutically non-active isomer in a racemate should be regarded as an impurity (50% or more) It is emphasized how in clinical pharmacology, and particularly in pharmacokinetics, neglect of stereoselectivity in action leads to the performance of expensive, highly sophisticated scientific nonsense.”

Ariens’s critical review of “Sophisticated Nonsense in Pharmacokinetics and Clinical

Pharmacology” in 1984 shows that neglect of stereochemistry in drug development was

widespread and only in the last decade or so has it achieved a prominent place in drug design It is therefore important to determine the stereoisomeric composition of chemical compounds, especially of pharmaceutical significance.13 Unfortunately, there are many racemic drugs where the stereospecificity of the metabolism and/or the pharmacodynamic effects of the enantiomers are still unknown.14-16

Today, about 56 % of the synthetic drugs14, 17 currently in use are chiral compounds, out of these chiral pharmaceuticals, 36 % have a single chiral centre and 88

% of these chiral synthetic drugs are used therapeutically as racemates The remaining 64

% of the organic pharmaceuticals contain more than one chiral centre and from these substances only 20 % is administered as a racemic mixture According to analysts from Technological Catalyst International (TCI) in 1997, among the top 500 drugs sold worldwide, 269 are single enantiomers and their sales accounted for 52 % (US$71.1 billion) of the total US $135.9 billion.3, 18-19 Since chiral compounds represent more than

50 % of the worldwide most frequently prescribed drugs, the interest in the preparation and isolation of chiral drugs has increased dramatically However, in spite of the knowledge that the specific effect of a drug is often caused by just one enantiomer, racemic mixtures are still frequently applied as it is still impossible for researchers to separate pure enantiomers out of all marketed racemates The differences in physiologic properties between the enantiomers of these drugs have not yet been examined in many cases, mainly because of the difficulties in obtaining optically pure enantiomers

Therefore, it is not surprising that intensive efforts have been directed worldwide towards the economic production of enantiomerically pure drugs.20-21 The conventional method of separating the optical isomers of racemic compounds has always been difficult and expensive.4, 22-25 It involves the preparation of diastereomeric intermediates which can be separated from each other by differential crystallization, hydrolysis and purification Furthermore, it is difficult to develop such procedures into an industrial scale process This has prevented pharmaceutical companies from exploiting the true clinical potential of pure drugs A more promising preparative scale separation technique which has attracted attention recently is to resolve such substances by chromatographic

techniques using CSPs.26-30 There has been tremendous impetus to develop separation methods based on chiral HPLC, both on analytical and preparative scales.31-37

In the process development aspect of research, characterization study of commercial and self-synthesized novel CSPs has been going on for the past few years with emphasis on evaluating adsorbents’ suitability for large-scale separations.38 Recent development in CSP design often comes with considerable understanding in the separation process Such understanding helps to enhance improvement in CSP design and

to assign absolute configuration from observed elution orders Column dynamics study is also being conducted to seek proper flow parameters for continuous operations However, the nature of a chiral recognition process employed is still not very clear

1.2 Importance of Chirality

Pharmacological activities in drugs are generally ascribed to only one enantiomeric form while the other is either without effect at best or even toxic.39 In the late 1950's and early 60's, a drug called thalidomide was marketed as a tranquilizer in Europe.13, 40-43 In late 1961, thalidomide was found to be a dangerous drug for pregnant

women, where the d-form is a safe sedative but the l-form causes severe birth defects and

deformities (Figure 1.6) Arising from the increasing pressure exerted by the scientific community towards restricting the use of chiral drugs in their racemic form, the Food & Drug Administration of the U.S (FDA) began to regulate the marketing of racemates in new drug submissions.24, 44-45

N H N O

O

H N O

O

O O

Figure 1.6 Two enantiomers of thalidomide

Chirality is also important in the bioactivity of a compound Most chemists are familiar with the role of chirality in odorants46 such as (4S)-(+)-carvone, which has a distinct caraway odor, as compared to (4R)-(-)-carvone, which has a characteristically sweet spearmint odor (Figure 1.7) Although the role of chirality in odor perception is still a rather modern area of interest, it should be noted that more than 285 enantiomeric pairs (570 enantiomers) are known to exhibit either differing odors or odor intensities.47

(4R)-(-)-carvoneSpearmint odor

*

*

Figure 1.7 Enantiomers of carvone

Awareness of the stereoselectivity of drug action has intensified since the thalidomide tragedies in the 1960s.48 Racemic drugs can cause problems because of the differences not only in the biological effects but also in the pharmacokinetics of the

enantiomers as stated by Caldwell in the 1980s, “The racemate killed a number of people

who had accumulated gram quantities of the enantiomer that was more slowly metabolized”

Table 1.1 Examples of Pharmaceutical and Food and Drink products

that show the effect of chirality

Thalidomide S-Isomer

R-Isomer

Teratogenic Sleep inducing, antinausea Barbiturates S-Isomer

R-Isomer

Depressant Convulsant Labetalol S,R-Isomer

R,S-Isomer

Alpha-blocker Beta-blocker Carvone R-Isomer

S-Isomer

Spearmint odor Caraway odor Limonene R-Isomer

S-Isomer

Orange odor Lemon odor

Aspartame R,R-Isomer

S,R-Isomer Sweet odor Bitter odor

Table 1.1 lists the activities of some stereoisomers in both Pharmaceutical and Food and Drink industries The marketing of drugs in active pure enantiomeric form helps to increase production capacity and reduce waste of starting materials and time It also helps to halve drug dosage and prevents side effects resulted from unwanted

enantiomers In addition, it provides opportunities for “Racemic Switching” in which

racemic compounds are re-developed as single enantiomers.49-50

In 1848 at the age of 26, Pasteur52 did his first work on molecular asymmetry, bringing together the principles of crystallography, chemistry and optics Because of the hemihedral facets on the crystals of racemic sodium ammonium tartrate, he was able to separate the mirror image crystals of the isomers by the use of a magnifying glass and tweezers Characterization of the physical properties of individual enantiomers, in which the only difference lies in the opposite rotation of a plane of polarized light led Pasteur to postulate that the enantiomers have different three-dimensional arrangements and on the macroscopic and microscopic levels they are mirror images of each other Furthermore,

he advanced the field by studying the influence of one chiral compound upon another and introduced the technique of resolution via diastereoisomer formation This separation of enantiomers by diastereomeric formation is the basis of many modern chromatographic

separations He formulated a fundamental law: Asymmetry differentiates the organic

world from the mineral world In other words, asymmetric molecules are always the

product of life forces His work became the basis of a new science – Stereochemistry

The optical resolution of synthetic racemates has remained as a challenging task since the historical resolution of racemic tartaric acid by Louis Pasteur in 1848 In 1874, the Dutch physical chemist Jacobus Hendricus Van’t Hoff53 and the French chemist Achille Le Bel54 independently theorized that the molecular basis of chirality that was first observed by Pasteur was an asymmetric carbon The asymmetric carbon proposed by Van’t Hoff was in the correct tetrahedral shape, whereas Le Bel proposed a square

pyramid In 1951 Kotake et al55 studied the influence of a chiral mobile phase on the resolution of several amino acids on paper chromatography and found that the separation could be ascribed to the chirality of the support (cellulose) Later, Dalgliesh56 suggested a necessary three-point simultaneous interaction between the resolvable amino acids and the cellulose surface

From the 1950s till the 1960s, development in the separation techniques was

dominated by Gas Chromatography (GC) In 1966, Gil-Av et al57 reported the first

successful direct enantiomeric separation in GC, a step that has a large echo in the

chromatographic field However, GC selectivity was later found to be limited In 1979, the first successful HPLC CSP was developed by William Pirkle.58, 63-64 In 1984, silica-bonded CDs were first developed by Armstrong and DeMond.59-62 It was found that the silica-bonded CDs are the most convenient, efficient and economic separation medium

for chiral drugs Hence, its importance in industrial area attracted more and more

attention, and many researchers started to synthesize more novel silica-bonded CD-based CSPs with better enantioselectivity A brief historical account of chiral separations by

chromatography is summarized in Table 1.2

Table 1.2 Historical accounts of chiral separations by chromatography methods

1848 Manual separation of sodium ammonium tartrate crystals.52

1936 Use of D- or L-quartz in chiral resolution 65

1939 Chromatographic resolution of camphor derivatized on lactose

Henderson and Rule: Resolution of racemic camphor derivatives 66

1952 Dalgliesh: Three-point rule in paper chromatography of amino acids.56

1966 Gil-Av et al: Direct resolution of enantiomers by GC.57

1971 Davankov and Rogozhin: Chiral ligand exchange chromatography 67

1972 Wuff and Sarhan: Enzyme analogue polymers for chiral LC 68

Hesse and Hagel: Cellulose triacetate used for chiral resolution 69 Stewart and Doherty: Agarose-bonded bovine serum albumin (BSA) for chiral resolution 70

1974 Blaschke: Synthesis of chiral polymers 71

1975 Cram et al: Chromatography with chiral crown ethers.

1977 Use of chiral chemical shifts reagent for chiral LC

1979 Pirkle and House: Synthesis of first silica-bonded CSP 58

Okamoto et al: Synthesis of helical polymer for chiral LC.72

1981 First commercially available chiral HPLC phase 64

1982 Allenmark: Use of agarose-bonded BSA in chiral LC.73

1984 Armstrong and DeMond: Use of silica-bonded CDs 59

1987 Classification of CSPs 74

1989 Development of silica-bonded CD phases

1.4 High Performance Liquid Chromatography

The history of chromatography began with the work of Tswett in Russia He was able to separate various plant pigments with a simple column filled with fine calcium carbonate With time, theory developed and chromatography took many forms from liquids passing through a stationary phase to gases passing through coated glass capillaries These methods have been used for a long time in various fields of chemistry Chromatographic methods that have a different principle for separation have also been proposed, such as partition, ion-exchange, and size-exclusion chromatography In the 1960s, the high speed of LC was realized as a result of new developments such as surface coated stationary phase packings and smaller diameter spherical packings; all of these

contributed to the phrase, "High Performance Liquid Chromatography” or “HPLC”, a

term which today is the most common name for modern LC instrumentation

There are various methods for enantiomeric analysis that do not require the separation of enantiomers These include polarimetry, nuclear magnetic resonance, isotopic dilution, calorimetry and enzyme techniques.75-77 The disadvantages of all these techniques, however, are the need for pure chiral samples and their relative slowness A typical analytical problem requires separation and quantitation of enantiomers and sometimes identification of the levorotatory or dextrorotatory enantiomer HPLC provides fast and accurate methods for enantiomeric separation and quantitation Chromatographic methods are still considered as the most useful means for chiral separation.78-80

In HPLC, the characteristics of both the stationary phase and the mobile phase can affect the selectivity and performance The variety of these selective interactions can be increased by suitable chemical modification of the silica surface Therefore one can say that HPLC is a more versatile and powerful method than other chromatographic methods.81-84 HPLC can often easily achieve separations and analyses that would be difficult or impossible with other chromatographic techniques However, there are some disadvantages that can go wrong with such separations; there are probably more difficult parameters to be optimized in HPLC than in any other form of chromatography

1.4.1 Separation Modes in HPLC

In HPLC, there are two separation modes that can be performed by choosing different combinations of mobile and stationary phase polarities, namely normal phase and reversed phase separation.85-88

1.4.1.a Normal Phase Chromatography

Normal phase chromatography uses a separation system that consists of a nonpolar mobile phase and a polar stationary phase A typical example of this type of separation mode is adsorption chromatography using silica gel as the stationary phase Polar silanol groups exist on the silica gel surface, and the solutes are retained by adsorption onto the silanol sites and desorption from the sites Typical mobile phases in

this mode should be nonpolar, such as n-hexane or IPA Often a stationary phase can be

used in both the normal and reversed phase separation modes

1.4.1.b Reversed Phase Chromatography

In contrast to the normal phase mode, the reversed phase mode requires the combination of a polar mobile phase and a nonpolar stationary phase Although this is the most popular separation mode in HPLC, the retention mechanism is not yet thoroughly understood It is common knowledge that the basic separation is controlled by hydrophobic interaction between solutes and stationary phase, although there is also evidence showing partition, adsorption and electrostatic mechanisms contributing to the retention of certain types of solutes Typical mobile phase solvents in this separation mode are MeOH or ACN; addition of water to both solvents is the usual way to control retention and selectivity Adding water to these solvents increases the polarity of the mobile phase, which increases the retention of the solutes Other solvents such as tetrahydrofuran (THF), dichloromethane (CH2Cl2) and chloroform (CHCl3) can also be used

1.5 Chromatographic Methods

There are two approaches for chiral HPLC: Indirect, which utilizes derivatizing agents; and Direct, which uses CSPs or chiral mobile phase additives

1.5.1 Indirect Chromatographic Method

Indirect methods involve derivatization of a given enantiomeric mixture with a chiral reagent, leading to a pair of diastereomers that can be resolved

chromatographically using an achiral column Because diastereomers possess different physiochemical properties, they can be separated in an achiral environment The main disadvantage of this method is the need to synthesize non-commercially available pure derivatizing reagents The indirect method of separating enantiomers in HPLC is frequently used in the bioanalytical field

1.5.2 Direct Chromatographic Method

The direct method involves use of various CSPs, namely, brush-type, cellulosic, CDs, ligand-exchange and protein-based.89-90 CSPs are stationary phases that are designed to separate enantiomeric compounds They can be bonded to solid supports or

created in situ on the surface of the solid adsorbent where their surface cavities can allow

specific interaction with one enantiomeric form of a chiral molecule The direct methods employ CSPs or mobile-phase systems The enantiomers of their derivatives are allowed

to pass through a column containing a CSP; or the derivatives are passed through an achiral column using a chiral solvent or a mobile phase containing a chiral additive

The mechanism of separation in a CSP is dependent on the given mode of separation used Enantiomers can be resolved by the formation of a diastereomeric complex between the solute and a chiral molecule that is bound to the stationary phase The separation is due to differences in energy between temporary diastereomeric complexes formed between the solute isomers and the CSP; the larger the difference, the greater the separation The observed retention and efficiency of a CSP is the total of all the interactions between the solutes and the CSP, including achiral interactions

1.5.2.a Chiral Mobile Phase Additives

Direct separation of enantiomers on an achiral column using a chiral mobile phase additive91-92 is applied only in HPLC Chiral discrimination is due to differences in the stabilities of the diastereomeric complexes, solvation in the mobile phase, and/or binding

of the complexes to the solid support Though less expensive conventional LC columns can be used, it is inconvenient for preparative applications because the chiral additive must be removed from the enantiomeric solutes

1.5.2.b Chiral Stationary Phases

At present, there are over a hundred of CSPs for HPLC that are commercially available According to Wainer33, there are five major classes of HPLC CSPs based on

the type of CSP complexes formed The Type 1 or “Pirkle” phase forms

analyte-CSP complexes by attractive-repulsive interactions, mainly by electron donor-acceptor

mechanisms The Type 2, exemplified by derivatized cellulose, involves attractive interactions followed by inclusion into chiral cavities The Type 3 CSPs, such as CDs and crown ethers, form inclusion complexes In the Type 4 CSP, the analyte is a part of a diastereomeric metal complex (chiral ligand-exchange chromatography) The Type 5 CSP

is a protein, e.g., bovine serum albumin The analyte-CSP complexes are based on the combination of hydrophobic and polar interactions Table 1.3 summarizes some examples

of commercially available CSPs

Table 1.3 Classification of commercial CSPs

1 Pirkle-type (π-donors or π-acceptors) phenylglycine,

DNB-leucine

2 Derivatized cellulose (attractive

interactions followed by inclusion)

Chiralcel OA, OB, OD, OF, OJ

3 CDs, polyacrylates, polyacrylamides,

crown ethers (inclusion complexation)

Cyclobond I, II, III; Chiralpak

OP, OT; Chiralcel CR

Type 1.Donor-Acceptor (Pirkle) CSPs

Pirkle’s group58, 63 is generally acknowledged as the inventor of Type 1 CSP

Pirkle has done extensive mechanistic studies utilizing computer modelling of diastereomeric complex interaction and various homologous series studies to elucidate the chiral recognition mechanism of the CSP Enantiomeric separation is achieved primarily by π-π interactions between the π-donor and π-acceptor aromatic rings of the racemic analyte and CSP and vice versa; secondly, through H-bonding involving secondary amines and carbonyl groups on the CSP with acidic proton, hydroxyl, and amino groups on the analyte; and finally, dipolar interactions as in dipole stacking and steric interactions arising from the bulky nonpolar groups attached near the chiral center

of the CSP that provide conformational control Type 1 CSPs can separate a wide

spectrum of racemic compounds, such as alkyl and aryl carbinols, aryl substituted hydantoins, lactams, succinamides, phthalides, sulfoxides, sulfides, amides and imides

The mobile phase used for Type 1 CSPs usually contains a nonpolar organic solvent with

various amounts of polar modifier, e.g., hexane and IPA

Type 2 Derivatized Cellulose and Related CSPs

Okamoto et al72 developed the first commercially successful series of cellulose triacetates initially coated and later bonded to a silica gel support Unmodified cellulose was reported to resolve amino acids and derivatives, di-aminocarboxylic acids and synthetic alkaloids However, poor resolution and broad peaks were obtained due to slow mass transfer and slow diffusion The highly polar hydroxyl groups characteristic of cellulose often lead to non-stereoselective binding between the analyte and cellulose In addition, cellulose is unable to withstand normal HPLC pressures Derivatization of cellulose brings about practically useful CSPs with high chiral recognition mechanisms

that can separate a wide range of racemic compounds Wainer et al in 1986 concluded that the chiral recognition mechanism generally accepted for most of the Type 2 CSPs

involves attractive interactions followed by the steric fit of the molecules into the chiral surface The attractive interactions include H-bonding, dipole-dipole and π-π interactions

Some derivatized amyloses are also classified as Type 2 CSPs

Type 2 CSPs that are commercially available from the Daicel Chemical Industries

have been developed by Okamoto and co-workers CSPs from Daicel are mainly divided into 4 series including cellulose and its derivatives, amylose and its derivatives, crown ethers and polymethylmethacrylate (Figure 1.8) Among the four series, the first three were used on HPLC while the fourth was used in ligand chromatography These CSPs are

mostly used in normal phase separation with low polarity mobile phases; typically

alcohol/n-hexane mixtures Chlorinated solvents must be avoided since they may strip the

cellulose from the silica support Moreover, since starch is water-soluble, in such columns the mobile phases must have a zero water content to ensure column durability These CSPs are widely used and reported to separate a variety of alkaloids and pharmaceutical compounds.93-94

OOR

amino acids.100-101 The separation is relatively good under normal phase, polar organic phase and reversed phase

R R

R R

R R

Silica Gel

SUFFIX OMe DM (dimethylated)

COCH3 AC (acetylated)

CH2CHCH3OH

CH3

CH3CONH

DMP (3,5-dimethylphenyl

carbamte)

Figure 1.9 Derivatives of CYCLOBOND I 2000

Armstrong has studied extensively the CDs and popularized them as both CSPs and mobile phase additives CDs possess excellent selectivities for a wide range of racemic compounds including amines, alcohols, carboxylic acids, epoxides and others At

present, CD is most often used for chiral separation as a CSP in both GC and LC, and as

mobile phase additives in LC, GC, and CE Crown ethers are also classified as Type 3

CSPs

ChiraDex® from Merck is a versatile HPLC column characterised by broad enantioselectivity and can be used for the separation of enantiomers of numerous different classes of substances ChiraDex® is based on β-CD covalently linked to spherical particles of silica and is well suited for the chiral separation of hydrocarbons, steroids, phenol esters and derivatives, aromatic amines, heterocycles with 5-membered ring to 7-membered ring The CSP is mostly used under reversed phase mode

There are also commercially available CSPs from Chirosep such as CYCLOSE®, CHIROSE® and CHIRALOSE®, CHIRALCROWN® and CHIROGEL® They are based

on pure mono-CD derivatives, CD polymers and other types of polymeric networks The main difference of CD-based CSPs CYCLOSE® from the ether-linkage CSPs prepared in this work is the application of KOH as base and the thioether or sulfone-linkage used in the CYCLOSE® CSPs as illustrated in Figure 1.10 (synthetic procedure for ether-linkage CD-based CSP will be discussed in Chapter 2 and 7) The spacer is linked not only at C-

6 but also at C-2 of the CD moiety The CSPs are heat and chemically stable

O H O H O H H

O OH H H

O H

H

O H H

O O H H H OH

O

H H OH

H O O H H

H OH

O H

H

OHOH

O

H

H OH H

O OH H H

O H

O

H

H OH H

O OH H H

O

O

H H O H

H OH H

H OH

S Si

O

O

O H O H O H H

O OH H H OH

O H

H

O H H

O O H H H OH

O

H H OH

H O O H H

H OH

O H

H

OHOH

O

H

H OH H

O OH H H

O H

O

H

H OH H

O OH H H

O

O

H H O H

H OH H

H OH

S

beta CD

Type 4 Ligand Exchange CSPs

Ligand exchange chromatography (LEC) involves the formation of reversible coordination complexes between a bidentate analyte, a divalent metal ion and a chiral ligand immobilized in the stationary phase The metal ion is a transition metal (usually

Cu2+) and the chiral ligand is an amino acid (e.g., proline) LEC was first reported by Davankov’s group in Moscow in 1971

Type 5 Protein CSPs

Proteins are polymers composed of chiral units (L-amino acids) and are known to

bind small molecules They can be immobilized on a solid support by a variety of

bonding chemistry The retention mechanism for Type 5 CSPs is most likely based on

H-bonding, ionic interactions or other interactions The mobile phases are generally aqueous buffers that are used in limited pH ranges with organic modifiers Although the capacity

of a protein column is limited, they offer a broad range of applicability for separating

neutral, cationic, and anionic racemic species.102

There are several factors supporting the reason why chiral chromatography is selected for chiral enantioseparation It involves low production cost (US$50-100 per kg

of CSP) and also performs good reproducibility enantiomeric separation Among the separation techniques used in modern science and technology, chiral chromatography

stands out in that it is a “multi-step” separation method No irreversible chemical

reactions or phase transitions are involved; the solutes to be resolved appear from the chromatographic column in the same, unmodified form and in the same mobile phase but

in different volume fractions The overall resolution of the solutes is the sum of a large number of solute discrimination contributions from elementary adsorption-desorption cycles, which slow down movement of the solute having higher affinity for the stationary phase Due to the cumulative nature of the chromatographic separation, it is trivial practice to completely resolve a mixture of two components that differ in the free energy

of interaction with the stationary phase by as little as 0.025 kJ/mol, which corresponds to the column selectivity value of α = 1.01

In addition, as a “multi-step” technique, the selector can be incorporated into the

CSP This diminishes requirements to the acceptable level of enantioselectivity of the

chiral selector by several orders of magnitude (from ca 10 kJ/mol to ca 0.03 kJ/mol) without sacrificing the purity or the yield of the enantiomers solved Furthermore, it is much less sensitive to the optical purity of the chiral selector, since stronger retention of the erroneous enantiomer at an accidentally defective sorption site of the CSP will be superseded by the combined action of most of the active sorption sites Therefore, it is possible to obtain, quantitatively and in the optically pure state, both enantiomers under resolution, even when the chiral selector has an optical purity lower than 100%

1.6 Cyclodextrin Chiral Stationary Phases

CDs have been used in chromatography since the 1960s, but a rapid development

of the studies on CD chromatographic applications occurred in the 1980s This resulted not only from the better recognition of their advantageous properties but also from the fact that they have become commercially available

Table 1.4 Physical properties of CDs

formed by partial degradation action of Bacillus Macerans amylase on starch and the

enzymatic coupling of cleaved glucose units into crystalline, homogeneous toroidal structures of different molecular size They are composed of α-D-glucose units linked via the 1, 4 position.103-108 Table 1.4 shows the physical properties of the CDs.109-119

H

H O H H

O OH H H OH

O H

H

O H

H

O O H H

H OH

O H

H

O HOH

H

OH O

O H

O

H

H

OH H

O

OH

H H

O H

O

H

H O H

H OH

H H

O H

O H

O

H O H H

O OH H H OH

O H

H

O H H

O O H H

H OH

O

H

H OH

H

O O H H

H OH

O H

H O O H H

H O H O

H

H

OH H

O

OH H H

O H

O

H H

OH H

O

OH

H H

O H

O

H H O H

H OH H

H

OH

O H O H O H O OH

H H OH

O H H O H O OH H H OH

O

H H

OH

H O O H H OH

O H

H OH H

O O H

O H

H OH H

O

O H H

OH

O H

H OH H

O OH H H O

O

O

H O OH H H O

O H H O

H OH H

H O

α-Cyclodextrin β-Cyclodextrin

Figure 1.11 Three common forms of CD

O H

OH

O

OH O

O

O H

1 - 6 numbering of glucose unit

Figure 1.12 Chiral centers and numbering of carbon in glucose unit

β-CD has 35 stereogenic centers as illustrated in Figure 1.12 The toroidal

structure has a hydrophilic surface resulting from the C-2, C-3, and C-6 hydroxyl groups making them water-soluble The interior of CD cavity is composed of glycoside oxygen and methylene hydrogens, giving it an apolar character Therefore, CDs can include polar molecules of appropriate dimensions in their cavities and bind them through dipole-dipole interactions, H-bonding or π-π interactions The α-CD cavity can hold a 5- or 6-membered ring The internal diameter of β-CD varies from 6.0-8.0 Å and can easily accommodate molecules that are the size of biphenyl or naphthalene γ-CD can bind to molecules as large as substituted pyrenes

15.4 A 6.0-6.5 A

7.9 A

Apolar Cavity

Secondary Hydroxyl Groups

Primary Hydroxyl Groups

Figure 1.13 Interior and exterior of a β-CD

It is proven that water solubility of CD increases greater than twenty-fold if hydroxyl groups in position C-2 or C-3 are functionalized It is important to note that not all of the fourteen secondary and seven primary hydroxyls of β-CD are substituted This

is because since the CDs are attached to the silica through one or more of the primary hydroxyls, substitution on the primary side of the CD is somewhat sterically restricted The C-2 hydroxyl groups become more reactive than the C-3 hydroxyl groups Therefore, the substitution occurs mainly at the C-2 position The number of substitutions per CD, or

“Degree of Substitution” typically falls between 3 and 10 In general, a higher degree of

substitution is obtained for small substituents presumably due to the steric hindrance imposed by bulky substituents

The structure of the CDs gives rise to their remarkable ability to form inclusion complexes with various molecules of a polar or nonpolar nature, and even with ions These CD complexation processes are highly stereoselective and can be considered as the method of choice for resolution of various isomers namely, structural, geometrical, diastereomeric and enantiomeric isomers.118-119 The main factors of crucial importance