Development of novel positively charged single isomer cyclodextrins and applications in enatiomeric separation and chiral synthesis

In direct separation mode, the separation of optical isomers is based upon complex formation between the enantiomers and a chiral selector, resulting in the formation of labile diastereo

Chapter 1 Introduction

1 1 Chiral separation: need and perspective

Stereoisomers are isomeric molecules with an identical constitution but a different spatial arrangement of atoms The symmetry factor classifies stereoisomers as either diastereoisomers or enantiomers Diastereoisomers are stereoisomers not related as mirror images If the stereoisomers are mirror images of one another but are not superimposable, they are called enantiomers [1, 2] An example of such diasteroisomers and enantiomers are shown in Figure 1 1

C C

CH 3

H 3 C

H H

Diastereoisomers Enantiomers (● denotes the chiral center)

Figure 1 1 Stereochemical structures of the pair of diasteroisomers and enantiomers

The property of nonsuperposability is termed as chirality and the structural feature that gives rise to this asymmetry is called chiral center (also called as sterogenic center or asymmetric center) Usually, carbon is not the only atom to act as chiral center Phosphorus, sulfur and nitrogen are among the other atoms that form chiral molecules Enantiomers usually display similar chemical and physical properties at nomal conditions, except for the direction in which they rotate the plane-polarized light The respective

enantiomer will have a different sign for optical rotation, which can be a (+) or (-) An

alternative description system fro the direction of rotation is that of dextrotary (d) or laevorotatory (l) However, the terms d and l, and (+) and (-) only donate the direction in

which plane-polarized light is rotated and tell us nothing about the absorlute stereeochemical arrangement of the atoms in the molecules A nomenclature to distinguish between the three-dimensional arrangements of the atoms at a stereogenic centre has been devised by Cahn, Ingold and Prelog [3] Absolute configurations are denoted as R or S according to the sequence rule

Chirality has attracted great attention because living systems are chiral Amino acids, proteins, nucleic acids, and polysaccharides possess chiral characteristic structures that are closely related to their functions In nature, these biomolecules exist in only one of the two possible enantiomeric forms, e.g., amino acids in the L-form and sugars in the D-form Due to the fact that many of the building blocks in the body are chiral, molecules and many receptors in the body act as enantiodiscriminating processes As a consequence, metabolic and regulatory processes mediated by biological systems are sensitive to stereochemistry and different responses can be often observed when comparing the activities of a pair of enantiomers Stereoselectivity is often a characteristic feature of enzymatic reactions, messenger–receptor interactions and metabolic processes; it can vary interspecifically and even from one individual to the other [4-7] Therefore, stereochemistry has to be considered when studying xenobiotics, such as drugs, agrochemicals, food additives, flavors or fragrances

The interest in chirality and its consequences is not a new phenomenon However, increasing expectations have risen until the last decade due to scientific and economic reasons, with the pharmaceutical industry being the main contributor and driving force

Enantiomers of a racemic drug (a mixture of two enantiomers) may have different pharmacological activities, as well as different pharmacokinetic and pharmacodynamic effects Since the human body is amazingly enantioselective, it will interact with each enantiomer of a racemic drug by a separate pathway to produce different pharmacological activity Thus, one isomer may produce the desired therapeutic activities, while the other may be inactive or, in worst cases, produce unwanted effects [11, 12] A typical example for the first case is epinephrine The (-)-epinephrine, a sympathomimetic drug used for cardiac stimulation, is ten times more potent than its isomer [(+)-epinephrine] [13] A particularly tragic example attributed to chirality occurred in the early 1960’s when synthetic tranquilizer thalidomide was widely prescribed as a sedative It was used by some pregnant women who later gave birth to deformed children [14] The number of such examples is of course large, and consequently the need for enantiopure drugs is an important matter

Despite the fact that scientists have been fully aware of these differences for more than two decades, the main advances in the development of enantiomerically pure compounds have been accomplished in the last decades with the prosperity of new asymmetric synthesis methodologies, and powerful analytical and preparative separation techniques [15, 16] Many enantiomerically pure drugs have successfully reached the market Therefore, health and regulatory authorities, such as the US Food and Drug Administration (FDA), have defined more strict requirements to patent new racemic drugs, demanding a full documentation of the separate pharmacological and pharmacokinetic

profiles of the individual enantiomers, as well as their combination [5, 16-19] In addition,

a rigorous justification is required for market approval of a racemate of chiral drugs

Single enantiomer drug sales have shown a continuous growth worldwide since 1996 and many of the top-selling drugs are marketed as single enantiomer (Figure 1 2) [20, 21].The chiral drug industry soared through a major milestone in 1999, as annual sales in this rapidly growing segment of the drug market topped $100 billion for the first time [22] Worldwide sales of chiral drugs in single-enantiomer dosage forms continued growing at a rate of more than 13% annually to $133 billion in 2000 At a future growth rate estimated

by Technology Catalysts International Corporation (TCI), the figure could hit $200 billion

in 2008 In a second growth trend, according to the firm, 40% of all dosage-form drug sales in 2000 were of single enantiomers [21-23]

Figure 1 2 Worldwide Sales of single enantiomeic drugs until 2003 and expected for the

year 2005.

Researchers and economic analyzers are very confident that the chiral drug industry will continue to spur strong growth because of the efforts to improve drug efficacy and to cut development costs in the face of regulatory pressures Therefore, the importance of determining and purifying the stereoisomeric composition of chemical compounds, especially those of pharmaceutical significance, will be more clearly recognized and emphasized [24] Consequently, applicable and practical techniques to obtain the enantiopure compounds are a must

Although a number of stereoselective syntheses have been described and applied to the production of single enantiomers [25-27], relatively few are selected for large-scale preparations, especially at the early stage of developing new drugs The development of asymmetric synthesis would be expensive and time consuming and thus, analytical techniques for chiral separation of enantiomers have great potential

1 2 Techniques for chiral separation

Enantiomers can be separated either by direct or indirect separation methods The indirect

separation method is based on the formation of a covalent bond between the optical

antipodes on the one hand and a pure chiral compound, called the chiral selector, on the

other hand This chemical reaction will result in a product consisting of two isomeric

compounds which are not mirror images anymore They are known as diastereoisomers

and they can, in principle, be separated by any analytical method using an achiral separation mechanism This method is, first of all, time consuming since sample pretreatment involving a chemical reaction is necessary Secondly, the chiral selector has

to be very pure, since optical impurity will result in two more diastereomeric products

In direct separation mode, the separation of optical isomers is based upon complex formation between the enantiomers and a chiral selector, resulting in the formation of

labile diastereoisomers Separation can be accomplished if the complexes possess different

stability constants The above mentioned disadvantages of indirect separations can be avoided by direct separation mode The chiral purity of the selector only influences the resolution It has been shown that relatively good results can be obtained using a chiral selector containing up to 10% of its antipode [28]

Analytical methods used so far for the direct enantiomeric separation include high performance liquid chromatography (HPLC) [29-31], thin-layer chromatography (TLC) [32], gas chromatography (GC) [33], supercritical fluid chromatography (SFC) [34], and capillary electrophoresis (CE) [35-48] The application of gas chromatography is mainly restricted to more volatile compounds Therefore, until now, the method for the separation

of more polar compounds and most drugs is HPLC The main drawback of CE compared

to HPLC is that until now, CE has not been shown to be useful as a preparative separation tool Another advantage of HPLC over CE is the low detection limit, due to the much longer path length of the detection cell and the much higher injection volume However, the very high efficiencies usually obtained in CE, and the ease of method development, make it a good alternative for analytical separation of enantiomers Other advantages of

CE over HPLC are the low consumption of both analyte and chiral selector and the short analysis times Moreover, CE does not require the use of expensive chiral stationary phases, since the chiral selector is simply added to the buffer Alternatively, CE might be very useful for the rapid screening of novel chiral selectors, thus avoiding the waist of the

laborious synthesis of new chiral HPLC stationary phases

1 3 History and development of capillary electrophoresis

Capillary electrophoresis was evolved from eletrophoresis Electrophoresis is the separation principle in which charged particles or molecules are separated under the influence of an external electric field Already at the beginning of the last century, Von Reuss performed the first electrophoretic experiments [49] Exactly 100 years ago, Kohlrausch developed his regulating functions [50], which made it possible to theoretically describe all electrophoretic methods Electrophoresis has, since then, been mainly applied for the separation of large biomolecules like DNA and proteins, using stabilizing and sieving media such as gels The introduction of narrow bore tubes as an anti-convective medium made it possible to use free solutions instead of these gels Hjertén described the use of a rotating quartz capillary of 3 mm inner diameter (I.D.) [51] Smaller I.D capillaries were successfully applied by Everaerts [52] and Virtanen [53] The reduction of the I.D allowed the use of higher electric field strengths, resulting in

higher efficiencies and shorter times of analysis Mikkers et al [54, 55] showed that the

high efficiencies, theoretically described by Giddings [56], could be achieved Jorgenson [57] used 75 μm I.D glass capillaries, in which longitudinal diffusion was shown to be practically the only source of band broadening This important breakthrough became the milestone in the development of modern capillary electrophoresis (CE) The next important achievement was the introduction of capillary micellar electrokinetic chromatography (MEKC) by Terabe and his co-workers in 1984 [58-60] This technique owes its migration principle to electrophoresis and its separation principle to

chromatography The application range of CE techniques was expanded to neutral compounds by this outstanding innovation

Capillary electrophoresis has since then proven to be a highly efficient, analytical separation tool, not only for the separation of macromolecules but also for smaller molecules Fundamental studies as well as numerous applications have been reported in the last decade Numerous books [61-70] and a number of review papers [71-72] summarized the history and applications of capillary electrophoresis

It is without doubt that such a powerful microseparation technique as modern CE owes a lot to other analytical techniques such as slab gel electrophoresis, capillary gas chromatography and high-performance liquid chromatography

1 3 1 Principle of electrophoresis

Electrophoresis is a separation process involving in the migration of charged particles in a gel slab or buffer solution under the influence of an electric field Ionic and ionizable solutes are separated based on differences in charge, size and shape When a charged particle is placed in an electric field, it experiences a force which is proportional to its effective charge (q) and the electric field strength (E) The translational movement of the particle is opposed by a viscous drag force which is proportional to the particle velocity (V), hydrodynamic radius (r) and medium viscosity (η) When the two forces are counterbalanced, the particle moves with a steady state velocity [57, 73, 74]:

V ele =μele E Eq 1.1

where E is the applied voltage per unit column length (L), and μele is the electrophoretic

mobility given by:

Electroosmosis (Figure 1 3) in capillary tubes, on the other hand, refers to the propulsion

of the bulk solvent in the tube under the influence of an applied electric potential The silica surface consists of Si-OH groups which are ionized to SiO- in alkaline and slightly acidic media (pH>2) The negatively charged surface is counterbalanced by positive ions from the buffer and a double layer is formed Under the influence of an applied potential the positive ions in the diffusion region migrate towards the cathode and in doing so they entrain the water of hydration, resulting in electroosmotic flow The equations of electroosmotic flow are identical to those developed for electrophoretic migration since both phenomena are complementary The electroosmotic velocity (Veof) is given by:

on the electrostatic nature of the wall surface and, to a smaller extent, on the ionic nature

of the buffer

Figure 1 3. Illustration of electroosmotic flow in capillary [75]

Electroosmotic flow is directly proportional to the zeta potential and for untreated capillary walls and it generally decreases with decreasing pH, because the hydrogen ions deactivate the column surface causing a decrease in the zeta potential At moderate pH values (pH>3), the electroosmotic flow with the untreated capillary column is generary higher than the electrophoretic flow that causes all solutes (cationic, neutral and anionic)

to migrate toward the detection end of the column (usually at the cathode) Cationic and anionic solutes are separated based on differential electrophoretic migration while neutral solutes co-migrate with the electroosmotic flow velocity and are not separated

1 3 2 Modes of capillary electrophoresis [76-78]

The basic modes of capillary electrophoresis are capillary zone electrophoresis (CZE), micellar electrokinectic chromatography (MEKC), capillary gel electrophoresis (CGE), capillary electrochromatography (CEC), capillary isotachophoresis (CITP) and capillary isoelectric focusing (CIEF)

The most popular and widely applied mode is capillary zone electrophoresis (CZE) The

separation by this technique is based on the different electrophoretic mobilities of the analytes in background electrolyte (BGE) under the effect of an electric field This difference can be caused by different charges, masses or structure of chemical compounds

A high value of the EOF allows the separation of both anions and cations in one single run, using CZE in moving boundary electrophoresis

In moving boundary electrophoresis, the separation system is filled with a so-called leading electrolyte The sample is introduced at the beginning of the separation compartment After applying the voltage, the most mobile analyte will form a pure zone, followed by a mixed zone consisting of the most mobile and the second most mobile analyte According to the Kohlrausch regulation function [50], the concentration of the components in the zones is adapted to the concentration of the leading electrolyte The boundaries between the zones have self-correcting properties, due to differences in electric field strength between the zones The boundary between the leading zone and the first pure zone can be considered as an isotachophoretic boundary In a single run, moving boundary electrophoresis is only applicable to either anions or cations

1 4 Chiral selectors in capillary electrophoresis

The success of chiral separation by CE will be greatly dependant on the correct choice of chiral selector The chiral selector will, in most cases, be added to the BGE The separation of the optical isomers is based upon complex formation between the enantiomers and a chiral selector, resulting in the formation of labile diastereoisomers

Separation can be accomplished if the complexes possess different stability constants The

addition, however, of a chiral selector to an electrophoretic system does not guarantee the successful separation of all optical isomers The most important rule for chiral recognition

is that the chiral selector must be compatible in size and structure to the racemate; a minimum of three molecular interactions has to occur These interactions can be both attractive and/or repulsive Possible modes of interaction include: ion-ion bonds, dipole-dipole bonds like hydrogen bonds, Van der Waals forces and Ion-dipole bonds

Furthermore, only one of the two enantiomers needs to interact with the chiral selector via the three-point minimum mode Not all interactions between the chiral selector and the solute will meet this criterion; also achiral interactions will occur In these cases, separation optimization should be accomplished by maximizing the 3-point ‘chiral interactions’ at the expense of the non-chiral interactions [79, 80]

To achieve successful enantioseparations by CE, the chiral selector used is quite crucial

An effective chiral selector must meet several requirements, namely: (a) it should be stereoselective and form a transient diasteromeric complex with each enantiomer; (b) it should be soluble and chemically stable in the BGE; (c) it should not interfere with the detection; and, (d) it should exhibit fast complexation kinetics

The commonly used chiral selectors in CE are depicted in Figure 1.4 Cyclodextrins (CDs) are by far the most popular chiral selectors used in CE and will therefore be discussed in more detail than the other chiral selectors mentioned in this section The detailed description about cyclodextrin will be provided in the following Section 1.5

Chiral Selectors in CE

Macrocyclic antibiotics

Crown

ethers

Ligand exchange complexes

Figure 1 4. Different types of chiral selectors used in CE

Crown-ethers are macrocyclic polyethers capable of forming host-guest complexes with inorganic and organic cations Modification of the crown ether by the introduction of four carboxylic groups makes it possible to use this class of compounds as chiral selectors in

CE The crown-ether can incorporate protonated primary amino compounds by formation

of ion-dipole bonds with the oxygen atoms of the chiral selector (Figure 1.5) The chiral crown-ether (18-crown-6-ether tetracarboxylic acid) can be used for the chiral separation

of several basic compounds [81]

Figure 1 5 Structure of the complex formed between 18-crown-6 and a protonated

primary amino compound

Macrocyclic antibiotics are a new, very promising class of chiral selectors Vancomycin, rifamycin B and ristocetin A have proven to be highly selective towards the enantiomers

of a broad class of compounds [82, 83] These antibiotics are amphilytic, and are strong UV-absorbers However, in most cases, detection of the analytes is not disturbed by the high background absorption of the chiral selector since only very low concentrations of the antibiotics are needed The chiral recognition is obtained mainly by charge-charge interactions, hydrogen bonding, hydrophobic inclusion and π-π interactions These interactions can be either attractive or repulsive

Proteins also have been applied successfully as chiral selectors in CE One of the characteristics of proteins is the isoprotic and the isoelectric point, pI The protein will be positively charged if pH < pI, and negatively charged if pH > pI This indicates that the

pH will be a very important operating parameter for the optimization of chiral selectivity Similar to the charged CD-derivatives, it is possible to separate both charged and uncharged species using this chiral selector Among the many proteins used as chiral selector in CE, bovine serum albumin (BSA) is most widely applied [84, 85] The mechanism, involved in chiral recognition is comparable with that of macrocyclic

antibiotics

Chiral surfactants were first used by Terabe’s group as micellar phase for the separation of non-charged compounds [86] and optical isomers, using Micellar electrokinetic chromatography (MEKC) [58] Both natural surfactants such as bile salts, as well as optically active amino-acid derived synthetic surfactants have been used as chiral selector

in CE New chiral surfactants often have a low critical micelle concentration, are highly soluble and can be synthesized in both the L- and D-form [87] The last feature makes it possible to easily change the migration order of the optical isomers For the determination

of the optical purity of e.g drugs, it is highly favorable that the minor component migrates

in front of the major component

Many other classes of compounds have also been used as chiral selectors in CE The most important group, not mentioned so far, is probably the oligosaccharides consisting of maltodextrin, heparin and dextran sulphate Some recent comprehensive reviews give an excellent survey on the state of the art of chiral separations in CE [88, 89, 90] We do not want to repeat it here

1 5 Cyclodextrins: the chiral selector for CE

Cyclodextrins represent the most frequently used chiral selectors in CE Native CDs are cyclic oligosaccharides consisting of six (α-CD), seven (β-CD) or eight (γ-CD) glucopyranose units with a truncated cone providing a hydrophobic cavity These CDs can only exhibit modest chiral discrimination to very limited racemate pool According to the review by Gübitz [91], only 15% of the compounds listed in the review can be resolved

Such a low enantioselectivity can be ascribed to their inherent symmetry according to Easton [92]

Increased chiral discrimination can thus be expected from the use of modified CDs with

an increased asymmetry The hydroxyl groups on the rims of native CDs can be modified

to obtain CDs in variable degrees of substitution The hydroxyl substituents can be replaced at random to obtain a complex mixture of isomers where the overall effect is that

of a symmetric distribution Therefore, this type of substitution does not alter the symmetry or enantioselectivity of the CD The other type of modification has arisen from

a new trend to producing selectively substituted derivatives, usually called single-isomers This may induce changes in the asymmetry of the CD and increase enantioselectivity as a result This modification of native CDs can lead to neutral or charged CDs [93] The use

of CDs as chiral selectors is the subject of several selective reviews [46, 90, 93, 94, 95]

1 5 1 Naturally occurring cyclodextrins

Cyclodextrins (CDs) are torus shaped cyclic D-gluco-oligosaccharides produced from starch by enzymatic degradation Although CDs containing between 6 to 12 D(+)-glucopyranose units have been isolated, only those containing 6 (α-CD), 7 (β-CD) or

8 (γ-CD) residues are currently used

HO OH

O

O

HO

HO OH

O O

OH HO

OH O

OH O O HO OH OH

O HO HO OH O

O

OH HO OH O

O O

OH HO

HO O

O OH OH HO O

O

OH

OH HO

OH O

O HO

HO OH O

OH

O O

HO

HO O

O OH OH

HO

O

OH

OH HO O

O

a) Molecular structure of cyclodextrins

Rim of Secondary Hydroxyls Hydrophobic cavity

Rim of

Primary

Hydroxyls

b) Schematic representation of the cyclodextrin torus

Figure 1 6. Structure of Cyclodextrins

In general, a cyclodextrin molecule can be briefly described as a torus, but is somewhat more realistically pictured as a shallow truncated cone possessing multiple stereogenic centers with a partially blocked base, a hydrophobic interior cavity and hydrophilic edges due to the presence of hydroxyl groups All the glucose units in this toroidal structure are

in their chair-conformation The interior of the CD cavity is relatively hydrophobic, while the outside rim is more hydrophilic [96, 97] The rim on the wider side of the CD cavity contains the chiral secondary hydroxyl groups, while the opposite smaller opening is occupied by achiral primary hydroxyl groups

Figure 1.6(a) shows the structures of α-, β- and γ-CD, while the dimensions are

schematically shown in Figure 1.6(b) The size of the hydrophobic cavity is such that, in

general, the α-CD can accommodate a single phenyl ring, while β-CD and γ-CD can

accommodate substituted single and multiple ring systems This inclusion alone is not

enough for chiral recognition: interaction between substituents on the asymmetric center

of the analyte and the hydroxyl groups on the CD-rim are responsible for chiral

recognition Some physical properties of these three CDs are quite different (as shown in

Table 1.1) [98-102]

Table 1 1. Some Physical and chemical properties of native cyclodextrins

α-cyclodextrin β-cyclodextrin γ-cyclodextrin

CDs are able to be regarded as “hosts” for “guest” molecules capable of entering (in whole

or in part) the cavity and forming noncovalent host-guest inclusion complexes Almost all

applications of CDs involve complexation The mechanism of inclusion complexation in

CE is schematically shown in Figure 1.7 Inclusion complex formation and the size of

the analyte’s binding constant to the cyclodextrin are determined by several different

factors The most important factors are the ‘hydrophobic effect’, which induces the apolar

portion of the molecule to preferentially reside in the cyclodextrin cavity, and hydrogen bonding between appropriate polar segments of the guest molecule and the secondary hydroxyl groups at the mouth of the cyclodextrin cavity Other factors which can influence complex formation are Van der Waals interactions, release of high energy water from the CD cavity and a change in ring strain upon complexation

CD Cation Anion

The improvement of enantioselectivity of native CDs and their solubility in certain solvents such as water, ethanol and methanol can be achieved by modification of cyclodextrins When the hydroxyl groups on the rims of native CDs are selectively

replaced by neutral groups such as methyl, acetyl, hydroxyethyl, hydroxypropyl and cyanoethyl [37, 91, 103-105, 106], various neutral CDs can be obtained The commonly used neutral CDs are hepatakis-O-methyl-β-CD (M-β-CD), hepatakis(2, 6-di-O-methyl)-β-CD (DM-β-CD), hepatakis(2, 3, 6-tri-O-methyl)-β-CD (TM-β-CD) and hydroxypropyl-β-CD (HP-β-CD)

The first attempt to perform chiral separation using modified cyclodextrins by CE was by the group of Smolkova-Keulemansova [107] They used isotachophoresis (ITP) with the addition of DM-β-CD and TM-β-CD to the leading electrolyte for the resolution of ephedrine alkaloid enantiomers Hydroxypropylated CD’s (HP-α-CD, HP-β-CD or HP-γ-CD) were applied for the enantiomeric resolutions of some chiral pharmaceuticals containing the imidazole (1, 3-diazole) moiety [108] The above mentioned modifications

of the native cyclodextrins obviously lead to a different stereoselectivity, but also to an improved solubility As mentioned earlier, the solubility of native β-CD in water is not more than 16 mM, whereas e.g the solubility of DM-β-CD is as much as 200 mM

Depending on the magnitude of the formation constants, solubility can have a limiting effect on chiral resolution

1 5 3 Charged cyclodextrins

Charged CDs are a relatively new class of CDs, with hydroxyl groups selectively replaced

by chargeable groups They are gaining much interest because of their ability to perform fast chiral separations at low concentration and their potential to resolve neutral racemates Moreover, charged CDs are expected to give the best resolving power when the analytes are oppositely charged, because interactions between the CDs with the analytes are not

only based on inclusion complexation but also on strong electrostatic interactions Charged CDs can be either negatively or positively charged Up to date, the negatively charged CDs are by far the most thoroughly investigated and frequently used charged CDs for enantioseparation in CE A brief description of these negatively charged CDs is presented in section 1.5.3.1

1 5 3 1 Negatively charged cyclodextrins

A broad spectrum of negatively charged CDs was investigated and applied to different racemic drugs, preferentially to basic and neutral drugs The improved selectivity compared to neutral CDs is mainly attributed to the countercurrent mobility Sulfated β-CDs (S-β-CD), sulfobutyl- (SBE-β-CD) and sulfoethyl ether β-CD (SEE-β-CD) are the most frequently used negatively charged CDs [37, 39, 103, 109] However, commercially available sulfated CDs consist of numerous isomers which differ in their degree and the site of substitution Batch-batch variations in composition lead to high variations in mobility and selectivity and so poor separation reproducibility To eliminate these drawbacks, single isomer charged CDs were synthesized A family of single isomer β-CD and γ-CD derivatives was introduced in the group of Vigh [110-114] They prepared CD derivatives completely substituted in 6-position and on their larger rims with hydrophilic groups Examples are heptakis(2,3-dihydroxy-6-sulfato)-β-CD [110], heptakis(2,3-diacetyl-6-sulfato)-β-CD [111], heptakis(2,3-dimethyl-6-sulfato)-β-CD [112] and octakis(2,3-diacetyl-6-sulfato)-γ-CD [113] These CDs are able to resolve neutral, basic, zwitterionic and even acidic enantiomers As predicted by the charged resolving agent migration (CHARM) model developed by Williams and Vigh [115], the enantiomeric migration order can by reversed in several cases by increasing the selector

concentration

Other investigated negatively charged CDs include carboxymethyl-β-CD (CM-β-CD) [116, 117], carboxyethyl-β-CD (CE-β-CD) [117], and succinyl-β-CD (Succ-β-CD) [117, 118] They were employed to resolve a large variety of compounds Only a few researchers reported the use of phosphated CDs [119-120]

1 5 3 2 Positively charged cyclodextrins

Compared with negatively charged CDs, positively charged CDs, however, have been surprisingly ignored, indicated by very limited published papers concerning cationic CDs Among them, quaternary hydroxyl-propyl-β-cyclodextrin (QAHCD) and 2-hydroxy-3-trimethylammoniopropyl-β-CD (TMA-β-CD) were the only commercialized permanently positively charged CDs Since these CDs are randomly substituted at C-2, C-3 and C-6 with a degree of substitution of 3~5, batch-batch variations in composition can lead to high variations in mobility and selectivity and so poor separation reproducibility [122-127] However, these randomly multisubstituted, permanent positively charged CDs present several advantages: (i) the electrophoretic mobility is pH-independent, (ii) very low concentration is needed to resolve acidic enantiomers due to strong ionic interactions, and (iii)they can be applied for the enantioseparation of basic, neutral and acidic compounds

The disadvantages caused by random multi-substitution can be overcome by the introduction of positively charged single-isomer CDs These single-isomers cationic CDs can be either monoderivatized (only one primary hydroxy group substituted) [36, 128-132]

or persubstituted ones either only on the primary rim of CD [133-135]

Since the first reported cationic CD, i.e mono(6-aminoethylamino-6-deoxy)-β-CD [136], several monosubstituted single-isomer CDs have been developed These cationic CDs are usually amino-functionalized and potentially positively-charged β-CDs such as

6A-methylamino and 6A,D-dimethylamino-β-CD [36], 6-amino-β-CD [122], 6-ethylenediamine-β-CD [125] and 6-deoxy-6-N-histamino-β-CD (CD-hm) and 6-deoxy[4-(2-aminoethyl)imdazolyl]-6-N-histamino-β-CD (CD-mh) [130, 131] A family

of single-isomer (hydroxyl)alkylamino-β-CDs [132] was also reported recently These single-isomer cationic CDs are capable of resolving acidic and neutral compounds

1 6 Research objective and scope

Based on the above introduction, charged CDs are very advantageous to resolve oppositely charged analytes at low concentration And their applicability for the chiral separation of neutral compounds is another interest to most researchers Extensive studies have been conducted on the use of negatively charged CDs for the chiral separation of basic and neutral compounds Positively charged CDs, however, have been greatly ignored, especially single-isomer cationic CDs

The aim of this research was to design and prepare a family of mono-substituted, positively charged CDs Their application as chiral selectors for enantioseparation of acidic and neutral compounds was explored on one hand On the other hand, the applicability of these CDs as templates for chiral synthesis was also investigated

In order to fulfill the first and main goal of this study, we carried out the following investigation Firstly, we developed a novel methodology for the preparation of a new family of mono-substituted positively charged CDs Secondly, systematic studies were conducted to investigate the effects of (a) different functionalized substituents, i.e imidazolium and ammonium cations, (b) the alkyl chain length of the same series of functionalized substituents, and (c) the type of cyclodextrins on the enantioseparation abilities of these positively charged CDs Lastly, the migration behavior and the corresponding enantioselectivity obtained in this study were used to evaluate the enantioseparation performance of these CDs and to obtain a glimpse of the mechanisms involved for chiral recognition process To achieve this, the following points will be highlighted:

1 Design and develop new procedures for synthesizing a new family of mono-substituted positively charged CDs

2 Evaluate the enantioseparation abilities of these CDs as chiral selectors in CE

3 Investigate the possible optimization of separation conditions and try to find some clues about the chiral recognition mechanisms involved

Given the impetus stated above, the key aim of this project focuses on the development of

a novel family of positively charged single-isomer CDs and their application in the enantioseparation of acidic and neutral compounds All these studies will be expounded in the following chapters

The synthetic methodologies are described in Chapter 2 for the preparation of three series

of mono-substituted, positively charged single-isomer CDs, namely

mono-alkylimidazolium series, mono-alkylammonium series and mono-amino series The detailed analytical characterization of these newly prepared CDs, and instruments used for chiral separation by CE and determination of enantiomeric excess (ee%) by HPLC are summarized in Chapter 7

Chapter 3 describes the use of mono-alkylimidazolium single-isomer CDs as chiral selectors for the separation of Dns-amino acids A general model is presented to determine formation constants between chiral selectors and optical isomers Factors influencing mobility and resolution, such as concentration of the chiral selector, acidity of the background electrolyte (BGE), organic modifier content and temperature are investigated

Chapter 4 describes the use of mono-alkylammonium single-isomer CDs for chiral separation of various acidic and neutral enantiomers by CE Factors influencing mobility and resolution, such as concentration of the chiral selector, acidity of the background electrolyte (BGE), capillary length and temperature are investigated

Chapter 5 describes the use of mono-amino single-isomer CDs for chiral separation of various acidic and neutral enantiomers by CE Factors influencing mobility and resolution, such as concentration of the chiral selector, type of CD and ionic strength of the background electrolyte (BGE) are investigated

Chapter 6 illustrates the potential of alkyimidazolium single-isomer CD as a template for chiral synthesis of various ketones The effect of reduction temperature and type of CD on the enantiomeric excess of product alcohols is investigated

In Chapter 8, the main conclusions achieved in the thesis and future perspectives are summarized

1 7 References

1 I W Wainer, A L Marcotte, in Drug stereochemistry: Analytical methods and

pharmacology, I W Wainer, D E Drayer (eds); Marcel Dekker, Inc, New York, 1988

2 R Cahn, C Ingold, V Prelog, Angew Chem Int Ed Engl 1966, 5, 385

3 N M Maier, P Franco, W Linder, J Chromatogr A 2001, 906, 3

4 I.W Wainer, Drug Stereochemistry, Analytical Methods and Pharmacology, 2nd ed.,

Marcel Dekker, New York, 1993

5 H.Y Aboul-Enein, I W Wainer, The Impact of Stereochemistry on Drug Development

and Use, Wiley, New York, 1997

6 J Caldwell, J Chromatogr A 1995, 694, 39

7 J Caldwell, J Chromatogr A 1996, 719, 3

8 A N Collins, G N Sheldrake, J Crosby, Chirality in Industry; John Wiley & Sons,

Chichester, 1992

9 S Lam, G Malikin, Chirality 1992, 4: 395

10 D E Drayer, Clin Pharmacol Theor 1986, 40: 125

11 K Mori, Chem Commun 1997, 13, 1153

12 E Ariens, Med Res Rev 1986, 6, 451

13 I R Innes, M Nickersen, in The Pharmacological Basis of Therapeutics, L

S.Goodman, A Gilman (Eds.), MacMillan Publishing Co., Inc, New York, 1970, 477

14 G Blaschke, H P Kraft, K Fickentscher, F Kohler, Arneiz.-Forsch., 1979, 29: 1640

15 E.J Lee, K.M Williams, Clin Pharmacokinet 1990, 18, 339

16 J Caldwell, Chem Ind 1995, 176

17 H Shindo, J Caldwell, Chirality 1991, 3, 91

18 J.J Blumenstein, in: A.N.Collins, G.N Sheldrake, J Crosby (Eds.), Chirality and Industry II Developments in the Manufacture of Optically Active Compounds, Wiley,

New York, 1997, p 11, Chapter 2

19 J.R Brown, in: G Subramanian (Ed.), A Practical Approach to Chiral Separations by

Liquid Chromatography, VCH, Weinheim, 1994, Chapter 3, p 57

20 S.C Stinson, Chem Eng News 1997, 75, 38

21 S.C Stinson, Chem Eng News 1999, 77, 101

22 S.C Stinson, Chem Eng News 2000, 78, 55

23 S.C Stinson, Chem Eng News 2001, 79, 79

24 S Ahuja, Chiral Separation by Chromatography, Oxford: New York, 2000, p 5

25 R S Atkinson, Stereosective Synthesis, Wiley: New York, 1995

26 J Mulzer in: G Helmchen, R W Hoffmann, J Mulzer, E Schaumann (Eds.),

Stereosective synthesis, Thieme, Stuttgart, New York, 1996, vol 1, Chapter 2, p 75

27 M Kitamura, S Okada, S Suga, R Noyori, J Am Chem Soc 1989, 111: 4028

28 S Fanali, M Cristalli, R Vespalec and P Bocek, in A Chrambach, M.J Dunn and B.J Radola (Editors), Advances in Electrophoresis, VCH, Weinheim, 1994, 3

29 J Debowski, D Sybilska and J Jurczak, J Chromatogr 1983, 282, 83

30 T.J Ward and D.W Armstrong, J Liq Chromatogr 1986, 9, 407

31 G Blaschke, J Liq Chromatogr 1986, 9, 341

32 D.W Armstrong, Faulkner, Jr., and S.M Han, J Chromatogr 1988, 452, 323

33 C.P Granville, B Gehrcke, W.A Konig and I.W Wainer, J Chromatogr 1993, 622,

21

34 S Hara, A Dobashi, K Kinoshita, T Hondo, M Saito, and M Senda, J Chromatogr

1986, 371, 153

35 T Schmitt and H Engelhardt, Chromatographia 1993, 37, 247

36 A Nardi, A Eliseev, P Bocek and S Fanali, J Chromatogr 1993, 638, 247

37 S Fanali, J Chromatogr A 1997, 792, 227

38 L Valtcheva, J Mohammed, G Pettersson and S Hjerten, J Chromatogr 1993, 638,

263

39 G Gübitz, M G Schmid, Electrophoresis 2000, 21, 4112

40 R Kuhn and S Hoffstetter-Kuhn, Chromatographia 1992, 34, 505

41 A Rizzi, Electrophoresis 2001, 22, 3079

42 S Busch, J.C Kraak and H Poppe, J Chromatogr 1993, 635, 119

43 H Nishi, T Fukuyama, M Matsuo and S Terabe, J Microcol Sep 1989, 1, 234

44 S Terabe, H Shibata and Y Miyashita, J Chromatogr 1989, 480, 403

45 P Gozel, E Gassmann, H Michelsen and R.N Zare, Anal Chem 1987, 59, 44

46 T De Boer, R A De Zeeuw, K Ensing, Electrophoresis 2000, 21, 3220

47 R Ivanyi, L Jicsinszky, Z Juvancz, N Roos, K Otta, J Szejtli, Electrophoresis 2004,

25, 2675

48 B.A Ingelse, M Flieger, H A Claessens, F M Everaerts, J Chromatogr A 1996,

755, 251

49 F von Reuss, Comment Soc Phys Univ Mosquensem, 1808, 1, 141

50 F Kohlrausch, Ann Phys (Leipzig), 1897, 62, 209

51 S Hjertén, Chromatogr Rev 1967, 9, 122

52 F M Everaerts, W M L Hoving-Keulemans, Sci Tools 1970, 17, 25

53 R Virtanen, Acta Polytech Scand 1974, 123, 1

54 F E P Mikkers, F M Everaerts, Th.P.E.M Verheggen, J Chromatogr 1979, 169, 1

55 F E P Mikkers, F M Everaerts, Th.P.E.M Verheggen, J Chromatogr 1979,169, 11

56 J.C Giddings, Separ Sci 1969, 4, 181

57 J.W Jorgenson, K.D Lukacs, Anal Chem 1981, 53, 1298

58 S Terabe, K Otsuka, K Ichikawa, T Ando, Anal Chem 1984, 56, 111

59 S Terabe, K Otsuka, T Ando, Anal Chem 1985, 57, 834

60 S Terabe, K Ozaki, K Otsuka, T Ando, J Chromatogr 1985, 332, 211

61 S F Y Li, Capillary Electrophoresis: Principles, Practice and Applications, Elsevier,

Amsterdam, 1992

62 P D Grossman, J C Colburn (Eds.), Capillary Electrophoresis: Theory and Practice,

Academic Press, New York, 1992

63 N A Guzman (Ed.), Capillary Electrophoresis Technology, Chromatographic Science

Series, Vol 64, Marcel Dekker, New York, 1993

64 J Vindevogel, P Sandra, Introduction to Micellar Electrokinetic Chromatography,

Vieweg Verlag, Wiessbvaden, 1995

69 P Tandik, G Bonn, Capillary Electrophoresis of Small Molecules and Ions, VCH,

Weinheim, 1993

70 K D Altria (Ed.), Capillary Electrophoresis Guidebook, Principle and Applications,

Methods in Molecular Biology, Vol 52, Human Press, Totowa, NJ, 1996

71 W.G Kuhr, C.A Monnig, Anal Chem 1992, 64, 389R

72 C.A Monnig, R.T Kennedy, Anal Chem 1994, 66, 280R

73 J.W Jorgenson, K.D Lukacs, J Chromatogr A 1981, 218, 209

74 J.W Jorgenson, K D Lukacs, J High Resolut Chromatogr Chromatogr Commun

1981, 4, 230

75 D R Baker, Capillary electrophoresis, John Wiley & Sons, Inc, 1995, 22

76 B Chankvetadze, Capillary Electrophoresis in Chiral Analysis, John Wiley & Sons,

Inc., 1997

77 S Terabe, K Otsuka, K Ichikawa, A Tsuchiya, T Ando, Anal Chem 1984, 56, 111

78 P Muijselaar, thesis, Eindhoven University of Technology, Eindhoven, 1996

79 K B Lipkowitz, Chem.Rev 1998, 98, 1829

80 K B Lipkowitz, R Coner, M A Peterson, J Am Chem Soc 1997, 119, 11269

81 R Kuhn, F Erni, T Bereuter, J Hausler, Anal Chem 1992, 64, 2815

82 D.W Armstrong, Y.B Tang, S.S Chen, Y.W Zhou, C Bagwill, J.R Chen, Anal

Chem 1994, 66, 473

83 D.W Armstrong, K.L Rundlett, J.R Chen, Chirality 1994, 6, 496

84 D.W Armstrong, K.L Rundlett, G.L Reid, Anal Chem 1994, 66, 1690

85 S Busch, Kraak, H Poppe, J Chromatogr 1993, 635, 119

86 Y Tanaka, S Terabe, J Chromatogr A 1995, 694, 277

87 D.C Tickle, G.N Okafo, P Camilleri, R.F.D Jones, A.J Kirbu, Anal.Chem 1994, 66,

4121

88 H Nishi, J Chromatogr A 1996, 735, 57

89 H Nishi, S Terabe, J Chromatogr A 1995, 694, 245

90 S Fanali, J Chromatogr A 1996, 735, 77

91 G Gübitz, M.G Schmid, J Chromatogr A 1997, 792, 179

92 C J Easton, S.F Lincoln, Chem Soc Rev 1996, 25, 163

93 M Blanco, I Valverde, Trends Anal Chem 2003, 22, 428

94 M L Bender, M Komiyama, Cyclodextrin Chemistry, Berlin: Springer-Verlag, 1978,

p 1-24

95 O A Shpigun, I A Ananieva, N Y Budanova, E N Shapovalova, Russian Chem Rev

2003, 72, 1035

96 T J Ward, D W Armstrong, J Liq Chromatogr 1986, 9, 407

97 J Szejtli, Cyclodextrins and Their Inclusion Complexes; Academiai Kiado, Budapest,

1982

98 M J Jozwiakowski, K A Connors, Carbohydr Res 1985, 143, 51

99 J A Thoma, L Stewart, Starch: Chemistry and Technology, Whistler, R L.; Paschall,

E F (Eds.), Academic: New York, Vol I, 1965

100 L E Briggner, I Wadso, J Chem Thermodyn 1990, 22, 1067

101 R I Gelb, L M Schwartz, D A Laufer, Bioorg Chem 1982, 11, 274

102 K A Cannors, Chem Rev 1997, 97, 1325

103 S Fanali, J Chromatogr A 2000, 875, 89

104 M Miru, K Funazo, M Tanaka, Anal Chim Acta 1997, 357, 177

105 M Miru, K Kawamoto, K Funazo, M Tanaka, Anal Chim Acta 1998, 373, 47

106 Z Aturki, C Desiderio, L Mannina, S Fanali, J Chromatogr A 1998, 817, 91

107 J Snopek, I Jelinek and E Smolkova-Keulemansova, J Chromatogr 1988, 438, 211

108 B Chankvetadze, G Endresz, G Blaschke, J Chromatogr A 1995, 700, 43

109 K Verleysen, P Sandra, Electrophoresis 1998, 19, 2798

110 J B.Vincent, D M Kirby, T V Nguyen, G Vigh, Anal Chem 1997, 69, 4419

111 J B.Vincent, A D Sokolowski, T V Nguyen, G Vigh, Anal Chem 1997, 69, 4226

112 Cai, H., Nguyen, T V., Vigh, G., Anal Chem 1998, 70, 580

113 Zhu, W., Vigh, G., Electrophoresis 2003, 24, 130

114 Zhu, W., Vigh, G., Anal Chem 2000, 72, 310

115 B.A Williams, G Vigh, J Chromatogr A 1997, 777, 295

116 T Schmit, H Engelhardt, J High Resolut Chromatogr 1993, 16, 525

117 T Schmit, H Engelhardt, Chromatographia 1993, 37, 475

118 M.G Schmid, K Wimsberger, G Gübitz, Pharmazie 1996, 51, 852

119 Y Tanaka, M Yangawa, S Terabe, J High Resolut Chromatogr 1996, 19, 421

120 K Ischibuchi, S I Izumoto, H Nishi, T Sato, Electrophoresis 1997, 18,

1007

121 Z Juvancz, L Jicsinszky, K E Markides, J Microcol Sep 1997, 9, 581

122 B Chankvetadze, G Endresz, G Schulte, D Bergenthal, G Blaschke, J Chromatogr

A 1996, 732, 143

123 Y Tanaka, S Terabe, J Chromatogr A 1997, 781, 151

124 F Wang, M G Khaledi, Electrophoresis 1998, 19, 2095

125 U B Nair, D W Armstrong, Microchem J 1997, 57, 199

126 G Schulte, B Chankvetadze, G Blaschke, J Chromatogr A 1997, 771, 259

127 A Bunke, T Jira, J Chromatogr A 1998, 798, 275

128 S Terabe, Trends Anal Chem 1989, 8, 129-134

129 F Lelièvre, P Gareil, Anal Chem 1997, 69, 385

130 G Galaverna, R Corradini, A Dossena, R Marchelli, Electrophoresis 1997, 18, 905

131 G Galaverna, R Corradini, A Dossena, R Marchelli, Electrophoresis 1999, 20,

2619

132 R Iványi, L Jicsinszky, N Roos, K Otta, J Szejtli, Electrophoresis 2004, 25, 2675

133 S.K Branch, U Kolzgrabe, T M Jefferies, H F Mallwitz, J R Oxley, J

Chapter 2 Synthesis of positively charged single-isomer cylodextrins

2 1 Approaches for cyclodextrin modification

Cyclodextrins, in their native state, are rigid molecules of interest as molecular hosts Much of their utility in supramolecular chemistry derives from their modification This involves altering the shape, size and other physical properties of the CD annuli and surfaces, and introducing new chemically useful functional groups Thus, CDs can be carefully tailored to match particular guests and meet specific requirements in their host-guest complexes [1] Reasons for CD modification include (i) the need to introduce functional groups to improve selective discrimination capabilities towards various enantiomers; (ii) the necessity to introduce coordination sites for the construction of metallo-CDs; (iii) the desire to alter the physical properties of external surfaces of CDs in order to construct monolayers and micellar structures and (iv) the aspiration to build enzyme mimics and related catalysts with predetermined alignment of binding sites and catalytically active groups [2] The best method to provide cyclodextrins of any size, shape, and most importantly containing any functional groups is to selectively convert the hydroxyl groups to other desired functionalities The modification of cyclodextrins offers chemists both enormous opportunities and challenges [3]

Synthesis of chemically modified cyclodextrins has been extensively studied [2-5]

Hydroxyl groups present at the 2-, 3-, and 6-positions compete for the reagent used, which makes selective modification extremely difficult Of the three types of hydroxyl groups present in cyclodextrins, those at the 6-position are the most basic (and often most nucleophilic), those at the 2-position are the most acidic, and those at the 3-position are the most inaccessible [6, 7] These differences can be fully utilized in selective-modification of CDs

A popular method for mono-modifications at the 6-position of cyclodextrins is carried out

by a nucleophilic attack of a reagent containing the appropriate group on mono-6-sulfonylcyclodextrin [8] Mono-6-tosyl-cyclodextrins are important precursors for

a variety of modified cyclodextrins because a nucleophile can attack the electrophilic carbon atom at the 6-position to produce a corresponding functionality A nucleophilic displacement of the tosyl group may be realized by suitable nucleophiles such as iodide, azide, thioacetate, hydroxylamine, alkyl, or poly- (alkylamines) to afford monoiodo- [9], azido- [10], thio- [11, 12],hydroxylamino- [13] or (alkylamino)-cyclodextrins [14]

Monotosylation of cyclodextrin [15, 16]is often a nonselective process and produces a mixture of primary as well as secondary side tosylated products along with di- or tri-tosylated derivatives Thus, depending on the desired purity of the final product, extensive purification is usually required Despite all these problems, monotosylates have been extensively investigated [17, 18] and improvements in their preparation have been reported [17-23] In order to obtain cyclodextrin derivatives with desired properties, the remaining hydroxyl groups on the mono- or partially modified CDs may also need to be substituted [24-30] For example, permethylated cyclodextrins are more water soluble than

unmodified cyclodextrins [26] and expand their potential for further exploitation

In this work, a novel methodology for the synthesis of mono-substituted positively charged CDs was developed The synthesis methodology involved the substitution of a single hydroxyl group by alkylimidazolium, alkylammonium and amino moieties [31] The conceptual approach for preparing these positively charged single-isomer CDs involved the use of mono-6-tosyl-cyclodextrin as an important precursor for a variety of mono-modified cyclodextrins because a nucleophile can easily attack the electrophilic carbon atom at the 6 position to produce the corresponding mono-functionalized cyclodextrin A nucleophilic displacement of the tosyl group by alkylimidazoles, alkylamines and amine affords three classes of positively charged single-isomer CDs: mono-alkylimidazolium, mono-alkylammonium and mono-amino CDs

In this work, 21 novel mono-substituted single isomers cationic CDs have been prepared and used as chiral selectors in CE The chiral recognition abilities of chiral selectors consisting of different types of CD (α-, β- or γ-) and different substituents on the CD rims are being investigated The molecular structures of these 21 novel mono-substituted single isomers cationic CDs are shown in Figure 2.1

) Series I: mono-alkylimidazolium β-CDs with different alkyl side chains on the imidazole ring also with different anions The chiral recognition ability of these CDs

on the enantioseparation of dansyl amino acids was examined

) Series II: mono-alkylammonium β-CDs with different alkyl side chains These CDs

include ALAMCDCl, PrAMCDCl, BuAMCDCl and PeAMCDCl The chiral

recognition ability of these CDs on the enantioseparation of various acidic and neutral racemates was studied

) Series III: mono(6A-amino-6A-deoxy)-CD hydrochloride with different type of CDs

They are α-NH 3 Cl, β-NH 3 Cl and γ-NH 3 Cl Effect of type of cyclodextrin on the

enantioseparation of various acidic racemates was investigated and compared

O

+

R H2N Cl

(O H )14

(O H )6+ -

+-Cl

Figure 2 1. Structures of 21 positively charged single-isomer CDs

2 2 Synthesis of positively charged single-isomer cyclodextrins

All mono-substituted positively charged CDs are prepared according to the popular method for mono-modifications at the 6-position of cyclodextrins, where a nucleophilic attack of a reagent containing the appropriate group was performed on mono-6-sulfonylcyclodextrin [8]

2 2 1 Synthesis of quaternary alkylimidazolium single-isomer β-cyclodextrins

As shown in the synthetic route in Figure 2 2, mono-6-tosyl-β-cyclodextrin 2 was conveniently obtained by the reaction of β-CD 1 with tosylimidazole in water And then it

was readily converted into mono-6A-(1-alkyl-3-imidazolium)-6A-deoxy-β-CD tosylate 4

by reacting with 1-alkylimidazole at 90℃ in dry DMF for two days under nitrogen atmosphere The resultant solution was concentrated under vacuum to remove excess 1-alkyimidazole to afford yellow syrup The yellow syrup was dissolved in methanol/water (50:50 v/v) and precipitated into ethyl acetate The precipitate was vacuum-filtered and further dried under high vacuum to afford a white solid The final

product 4 was obtained after thrice crystallization of the above solid from hot water

An anion exchange process was performed to convert the -OTs anion into Cl- to reduce the

UV absorption of 4 Freshly dried 4 was dissolved in deionised water The resultant

solution was introduced into the Amberlite 900 (Cl) resin bed set in a 100ml dropping funnel stayed still for 1hour The eluant was collected and water was removed to obtain a

yellow crystalline solid The desired product 5 was obtained after being dried under vacuum over night

N

N Ts (OH)14

5a: R1=H, R2=CH3, MIMCDCl 5b: R1=H, R2=C2H5, EIMCDCl

+

R2 N N

R1

5 3

Figure 2 2. Synthetic route to mono- alkylimidazolium single-isomer β-CDs

2 2 2 Synthesis of mono- alkylammonium single-isomer β-cyclodextrins

RH 2 N TsO

-Figure 2 3. Synthetic route to mono-alkylammonium single-isomer β-CDs

According to the synthetic route in Figure 2 3, mono-6A-alkylammonium-6A-deoxy-β-CD tosylates were prepared via nucleophilic attack of various amines on mono-6-tosyl-β-cyclodextrin A solution of mono-6-tosyl-β-cyclodextrin and amine in DMF was refluxed for 5 hours under nitrogen The resultant solution was cooled to room

temperature and precipitated with analytical grade acetone The white solid was collected

by filtration and recrystallized twice from hot water, followed by drying under vacuum

over night to give 3 with high yield A further anionic exchange was performed on 3 to prepare 4 with high yield

2 2 3 Synthesis of mono-(6 A -amino-6 A -deoxy)-CD hydrochlorides

Mono-6-sulfonyl-α-cyclodextrin and mono-6-sulfonyl-γ-cyclodextrin 1 were prepared by the reaction of α- and γ-CD with p-toluenesulfonyl chloride in dry pyridine Transformation to mono-(6A-azido-6A-deoxy) α-and γ-CD 2 can be readily effected by

SN2 reaction with excess sodium azide in water under a relatively mild condition (Figure 2 4)

CD amines 3 were previously synthesized from tosylates of cyclodextrins, via ammonia

[32] or the azide [33-35] However, these routes have disadvantages that CD-NH2 is obtained in very poor yield and purity with long reaction time Upon treatment of tosylate directly with aqueous ammonia or anhydrous saturated solution of ammonia in pyridine or

N, N-dimethylformamide (DMF) at room temperature, more than 50% of the tosylate remained even after 2 weeks’ reaction time Though the reaction can be accelerated to 18 hours under high pressure, this synthetic route is undesirable for large-scale application Reduction of the azide under the catalysis of palladium, the cost of catalyst and use of hydrogen under pressure are not easy to carry out Here, we report a convenient large-scale synthesis of CD-NH2 that is easy to carry out and proceeds in satisfactory purities and yields