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

Chapter Introduction 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. CH3 H 3C C H C H Cis-2-butene CH3 H C mirror plane C H 3C Trans-2-butene H OH H H H 3C COOH (R)-lactic acid Diastereoisomers HO HOOC CH3 (S)-lactic acid 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 [8-10]. 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. 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. 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 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. 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]: Vele = μ ele E Eq. 1.1 where E is the applied voltage per unit column length (L), and μele is the electrophoretic mobility given by: μ ele = q Eq. 1.2 6πηr 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: Veof = μ eof E Eq. 1.3 ε ζ 4πη Eq. 1.4 μ eof = where μ eof is the electroosmotic mobility, ε is the dielectric constant of the soloution, and ζ is zeta potential. μ eof depends, to a large extent, on the magnitude of the zeta potential at the capillary wall-bulk buffer interface. The zeta potential is largely dependent 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. 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 10 chloride (28: ALAMCDOTs, 29: ALAMCDCl) A solution of Ts-β-CD (2.578 g, 2.0 mmol) and allylamine (0.343 g, 6.0 mmol) in dimethyl formamide (5 mL) was refluxed for hours under nitrogen. The resultant solution was cooled to room temperature, acetone (25 mL) was added and stirred for 30 minutes. The white solid formed was filtered and dried under vacuum overnight to give a desired product (2.48 g,) with a yield of 92.1 %. An anionic exchange was performed on 28 to prepare 29 (ALAMCDCl) with a yield of 92.1 %. Analytical data for 28: 1H NMR (300 MHz, DMSOd6): δ 7.48 (d, 2H, J = 7.85 Hz, =CHortho), 7.12 (d, 2H, J = 8.30 Hz, =CHmeta), 5.81-5.87 (m, 1H, -CH=), 5.73 (s br, 8H, OH-2 and OH-3), 5.67 (s br, 6H, OH-3), 5.16-5.38 (m, 2H, =CH2), 4.87 (d, 1H, J = 3.25 Hz, H-1), 4.83 (d, 6H, J = 3.65 Hz, H-1), 4.50 (s br, 1H, OH-6), 3.80 (t, 1H, H-3’CD), 3.60-3.63 (m, 27H, H-5CD, H-3CD and H-6CD), 3.32-3.54 (overlap with HDO, m, 14H, H-2CD, H-4CD and CH2), 3.10 (d, 1H, J = 12.05 Hz, H-4’CD), 2.85 (t, 1H, J = 8.82 Hz, H-2’CD), 2.29 (s, 3H, CH3Ts). 13C NMR (300 MHz, DMSOd6): δ 145.1 (Cipso), 137.9 (Cpara), 130.8 (-CH=), 128.1 (Cortho), 125.4 (Cmeta), 119.8 (=CH2), 101.9 (C1), 83.6 (C4’), 81.2 (C4), 73.0 (C2), 72.2 (C3), 72.0 (C5), 69.0 (C5’), 59.9 (C6), 50.6 (C6’), 40.9 (CH2), 20.7 (CH3). IR (cm-1, KBr): 3380 (O-H str), 2930 (C-H str), 1638 (C=C str), 1155 (S=O str), 1080, 1032 (C-O-C str), 756 (C-H arom op bend). ESI-MS (m/z): 1174.42 (calcd) and 1174.40 (found) for [M+]; 171.20 (calcd) and 171.30 (found) for [-OTs]. Melting point: 249~250oC. ESI-MS (m/z) for 29: 1174.42 (calcd) and 1174.50 (found) for [M+]; 172 7.3.4.2 Synthesis of mono-6A-N-propylammonium-6A-deoxy-β-cyclodextrin tosylate /chloride (30: PrAMCDOTs, 31: PrAMCDCl) Essentially the same procedure as described for compound 28 was used to afford 30 (PrAMCD) by use of propylamine. The yield was 94.6%. An anionic exchange was performed on 30 to prepare 31 (PeAMCDCl) with a yield of 86.6 %. Analytical data for 30: 1H NMR (300 MHz, DMSOd6): δ 7.48 (d, 2H, J = 7.62 Hz, =CHortho), 7.12 (d, 2H, J = 8.04 Hz, =CHmeta), 5.67-5.79 (m, 14H, OH-2 and OH-3), 4.86 (s, 1H, H-1), 4.83 (s, 6H, H-1), 4.48 (s br, 6H, OH-6), 3.78 (t, 1H, H-3’CD), 3.54-3.63 (m, 27H, H-5CD, H-3CD and H-6CD), 3.34-3.44 (overlap with HDO, m, 14H, H-2CD and H-4CD), 3.12 (d, 1H, J = 11.62 Hz, H-4’CD), 2.89 (t, 1H, J = 8.82 Hz, H-2’CD), 2.63 (t, 2H, J = 7.23 Hz, CH2), 2.29 (s, 3H, CH3Ts), 1.48 (s, 2H, J = 7.23 Hz, CH2), 1.47 (NH2), 0.85 (t, 3H, J = 7.62 Hz, CH3). 13C NMR (300 MHz, DMSOd6): δ 145.4 (Cipso), 137.7 (Cpara), 128.0 (Cortho), 125.4 (Cmeta), 101.9 (C1), 101.4 (C1’), 83.5 (C4’), 81.5 (C4), 73.0 (C2), 72.4 (C3), 72.0 (C5), 59.9 (C6), 50.2 (C6’), 34.5 (CH2), 30.6 (CH2), 20.7 (CH3Ts), 11.2 (CH3),. IR (cm-1, KBr): 3392 (O-H str), 2930 (C-H str), 1638 (arom C=C ring str), 1155 (S=O str), 1079, 1032 (C-O-C str), 756 (C-H arom op bend). Elemental analysis: calculated for C52H85SNO37.7H2O (1474.28) C: 42.36% H: 6.77% N: 0.95% S: 2.17%, determined C: 42.24% H: 6.28% N: 0.97% S: 2.11%. ESI-MS (m/z): 1177.44 (calcd) and 1177.50 found for [M+], 171.20 (calcd) and 171.30 found for [-OTs]. Melting point: 260~262oC. ESI-MS (m/z) for 31: 1177.44 (calcd) and 173 1177.60 found for [M+], 7.3.4.3 Synthesis of mono-6A-butylammonium-6A-deoxy-β-cyclodextrin tosylate/ chloride (32: BuAMCDOTs, 33: BuAMCDCl) Essentially the same procedure as described for compoumd 28 was used to prepare 32 (BuAMCDOTs) by use of butylamine. The yield was 97.3 %. An anionic exchange was performed on 32to prepare 33 (BuAMCDCl) with a yield of 84.9 %. Analytical data for 32: 1H NMR (300 MHz, DMSOd6): δ 7.48 (d, 2H, J = 8.04 Hz, =CHortho), 7.12 (d, 2H, J = 8.04 Hz, =CHmeta), 5.64-5.81 (m, 14H, OH-2 and OH-3), 4.85 (s, 1H, H-1), 4.83 (s, 6H, H-1), 4.50 (s br, 6H, OH-6), 3.75 (t, 1H, H-3’CD), 3.54-3.63 (m, 27H, H-5CD, H-3CD and H-6CD), 3.31 - 3.46 (overlap with HDO, m, 12H, H-2CD and H-4CD), 3.07 (d, 1H, J = 11.22 Hz, H-4’CD), 2.83 (t, 1H, J = 8.82 Hz, H-2’CD), 2.61 (t, 2H, J = 7.64 Hz, CH2), 2.28 (s, 3H, CH3Ts), 1.41 (s, 2H, J = 7.62 Hz, CH2), 1.27 (s, 2H, J = 7.65 Hz, CH2), 0.85 (t, 3H, J = 7.23 Hz, CH3). 13C NMR (300 MHz, DMSOd6): δ 145.4 (Cipso), 137.7 (Cpara), 128.0 (Cortho), 125.4 (Cmeta), 101.9 (C1), 101.4(C1’), 83.5 (C4’), 81.3 (C4), 73.0 (C2),72.3 (C3), 72.0 (C5),69.2 (C5’),60.0 (C6’), 48.7 (C6),46.2 (CH2), 30.6(CH2), 20.7 (CH2), 19.6 (CH3Ts), 13.7 (CH3). IR (cm-1, KBr): 3406(O-H str), 2930 (C-H str), 1641 (arom C=C ring str), 1157 (S=O str), 1079, 1032 (C-O-C str), 755 (C-H arom op bend). ESI-MS (m/z): 1190.44 (calcd) and 1190.50 found for [M+], 171.20 (calcd) and 171.30 found for [-OTs]. Melting point: 263~264oC. Elemental analysis: calculated for C53H87SNO37.6H2O (1470.32) C: 174 43.29% H: 6.78% N: 0.95% S: 2.18%, determined C: 42.95% H: 6.68% N: 0.86% S: 2.15%. ESI-MS (m/z) for 33: 1190.44 (calcd) and 1190.52 found for [M+], 7.3.4.4 Synthesis of mono-6A-pentylammonium-6A-deoxy-β-cyclodextrin tosylate/ chloride (34: PeAMCDOTs, 35: PeAMCDCl) Essentially the same procedure as described for compoumd 28 was used to prepare 34 (PeAMCDOTs) by use of pentylamine. The yield was 96.3 %. An anionic exchange was performed on 34to prepare 35 (PeAMCDCl) with a yield of 85.3 %. Analytical data for 34: 1H NMR (300 MHz, DMSOd6): δ 7.48 (d, 2H, J = 8.04 Hz, =CHortho), 7.12 (d, 2H, J = 8.43 Hz, =CHmeta), 5.66-5.81 (m, 14H, OH-2 and OH-3), 4.86 (s, 1H, H-1), 4.84 (s, 6H, H-1),4.48 (s br, 6H, OH-6), 3.75 (t, 1H, H-3’CD), 3.54-3.65 (m, 27H, H-5CD, H-3CD and H-6CD), 3.32-3.45 (overlap with HDO, m, 12H, H-2CD and H-4CD), 3.12 (d, 1H, J = 11.22 Hz, H-4’CD), 2.85 (t, 1H, H-2’CD), 2.63 (t, 2H, CH2), 2.29 (s, 3H, CH3Ts),1.47 (s, 2H, CH2), 1.25 (s, 4H, CH2), 0.86 (t, 3H, J = 6.42 Hz, CH3),. 13 C NMR (300 MHz, DMSOd6): δ 145.7 (Cipso), 137.5 (Cpara), 128.0 (Cortho), 125.4 (Cmeta), 101.9 (C1), 101.4, (C1’), 83.5 (C4’), 81.5 (C4), 73.0 (C2), 72.3 (C3), 72.0 (C5), 59.8 (C6), 48.4 (C6’), 46.4 (CH2), 30.6 (CH2), 28.5 (CH2), 21.8 (CH2), 20.7 (CH3Ts), 13.8 (CH3). IR (cm-1, KBr): 3406 (O-H str), 2929 (C-H str), 1638 (arom C=C ring str), 1155 (S=O str), 1079, 1032 (C-O-C str), 755 (C-H arom op bend). ESI-MS (m/z): 1204.44 (calcd) and 1204.40 found for [M+], 171.20 (calcd) and 171.30 found for [-OTs]. Melting point: 266~267oC. ESI-MS (m/z) for 35: 1204.44 (calcd) and 1204.50 found for [M+], 175 7. 3. Mono-6A-N-amino-6A-deoxy-cyclodextrins (OH)n (OH)n-1 (OH)n (OH)n-1 n=6 n=7 n=8 1. Ph3P/DMF 2. H2O (HO )n-1 N3 (HO )n-1 NH2 7.3.5.1 Mono-6A-N-amino-6A-deoxy-β-cyclodextrin (36: β-CD-NH2) A mixture of N3-β-CD (5.80 g, mmol) and triphenyl phosphine (1.443 g, 5.5 mmol) in DMF (10mL) was stirred at room temperature for hours. To the resultant solution was added deionized water (1.0 mL), and refluxed for hours. To the solution was added acetone to precipitate the white solid and the solid was filtered, washed with acetone and finally dried under high vacuum for overnight to give pure product 5.50 g (97.0 %), Melting point: 205-207 oC (Lit. 203 oC [2]). 7.3.5.2 Mono-6A-N-amino-6A-deoxy-α-cyclodextrin (37: α-CD-NH2) A similar procedure as described for compound 36 was used to afford 37 (α-CD-NH2). Yield: 94.43%. Melting point: 205-208℃ (Lit. 200℃ [3]). 1H NMR (300 MHz, DMSOd6): δ 5.68-5.43 (m, 12H, OH-2 and OH-3), 4.93-4.76 (m, 6H, H-1), 4.59-4.44 (m, 5H, OH-6), 3.82-3.54 (m, 24H, H-5CD, H-3CD and H-6CD), 3.48-3.24 (overlap with HDO, m, 12H, H-2CD and H-4CD); 13C NMR (400 MHz, DMSOd6): δ 101.9 (C1), 82.0 (C4), 73.2 (C3), 72.0 (C2, C5), 59.9 (C6). IR(cm-1, KBr): 3383 (O-H str), 2928 (C-H str), 1080, 1028 (C-O-C str). ESI-MS (m/z): 994 [M+Na], calcd. 972. 176 7.3.5.3 Mono-6A-N-amino-6A-deoxy-γ-cyclodextrin (38: γ-CD-NH2) Essentially the same procedure as described for compound 36 was used to afford 38 (γ-CD-NH2). Yield: 90.28%. 1H NMR (300 MHz, DMSOd6): δ5.82-5.70 (m, 16H, OH-2 and OH-3), 4.92-4.85 (m, 8H, H-1), 4.61-4.46 (m, 7H, OH-6), 3.75-3.45 (m, 32H, H-5CD, H-3CD and H-6CD), 3.43-3.24 (overlap with HDO, m, 16H, H-2CD and H-4CD); 13C NMR (300 MHz, DMSOd6): δ 101.6 (C1), 80.8 (C4), 72.8 (C3), 72.5 (C2), 72.1 (C5), 59.9 (C6). IR (cm-1, KBr): 3397 (O-H str), 2929 (C-H str), 1080, 1028 (C-O-C str). ESI-MS: 1319 [M+Na], calcd.1297. 7. 3. Mono-6A-N-ammonium-6A-deoxy-β-cyclodextrin chlorides ( OH)2n ( OH)2n n=6 n=7 n=8 HCl(aq) (HO)n NH2 (HO)n NH3Cl Mono-6A-N-ammonium-6A-deoxy-β-cyclodextrin chloride (39: β-CD-NH3Cl) To a solution of β-CD-NH2 (4.536 g, 4.0 mmol) in deionized water (20 mL) was added hydrochloric acid solution (0.1 M, 10 mL). The solution was stirred for 30 minutes and then acetone (100 mL) was added. The precipitate that was formed was collected by suction filtration, washed with acetone (25 mL) and dried under high vacuum overnight to give title compound (4.503 g, 96.2 %). IR (cm-1, KBr): 3398 (O-H str), 2927 (C-H str), 1155 (S=O str), 1081, 1029 (C-O-C str). Melting point: 203-205oC (Lit. 201 oC [2]). The analytical data were consistent with those reported in previous literature. 177 Mono-6A-N-ammonium-6A-deoxy-α-cyclodextrin chloride (40: α-CD-NH3Cl) Essentially the same procedure as described for compound 39 was used to afford 40 (α-CD-NH3Cl). Yield: 96.2%. Analytical data for 40: 1H NMR (300 MHz, DMSOd6):δ 5.68-5.43 (m, 12H, OH-2 and OH-3), 4.93-4.76 (m, 6H, H-1), 4.59-4.44 (m, 5H, OH-6), 3.82-3.54 (m, 24H, H-5CD, H-3CD and H-6CD), 3.48-3.24 (overlap with HDO, m, 12H, H-2CD and H-4CD); 13C NMR (100 MHz, DMSOd6): δ 101.9(C1), 82.0 (C4), 73.2 (C3), 72.0 (C2, C5), 59.9 (C6). ESI-MS (m/z): 1030.5 [M+Na], calcd. 1008.5; Mono-6A-N-ammonium-6A-deoxy-γ-cyclodextrin chloride (41: γ-CD-NH3Cl) Essentially the same procedure as described for compound 39 was used to afford 41 (γ-CD-NH3Cl). Yield: 93.5%. Analytical data for 41: 1H NMR (300 MHz, DMSOd6): δ 5.82-5.70 (m, 16H, OH-2 and OH-3), 4.92-4.85 (m, 8H, H-1), 4.61-4.46 (m, 7H, OH-6), 3.75-3.45 (m, 32H, H-5CD, H-3CD and H-6CD), 3.43-3.24 (overlap with HDO, m, 16H, H-2CD and H-4CD); 13 C NMR (100 MHz, DMSOd6): δ 101.6 (C1), 80.8 (C4), 72.8(C3), 72.5(C2), 72.1(C5), 59.9(C6). ESIMS (m/z): 1355.5 [M+Na], calcd.1333.5; 7. Chromatographic Environments 7. 4. Chiral separation by use of Capillary Electrophoresis 7.4.1.1 Instrument system All CE separations were carried out in the mode of capillary zone electrophoresis (CZE). Basically, the equipment consists of a high voltage power supply, a separation 178 tube, an injection module, a detector and a data collection system. Figure 7.1 schematically shows an electrophoretic separation system. Generally, for the electrophoretic experiments presented in this thesis, a Beckman P/ACE™ MDQ CE unit (Fullerton, CA, USA) was used. The Beckman instrument is fully automated, and consists of, besides the earlier mentioned features common for all electrophoretic separation systems, an autosampler and a liquid-cooled capillary cartridge. The latter allows temperature control between 20°C and 50°C. Fused silica capillaries, with an inner diameter of 50 μm and an outer diameter of 375 μm, were used as separation tubes and were obtained from Polymicro Technologies (Phoenix, AZ, USA). The required minimum length was about 27 cm. Prior to use, new capillaries were rinsed for approximately 30 minutes with a M NaOH solution. For most applications, the capillary had a total length of 59.2 cm, with an effective length of 49 cm. The high voltage power supply is capable of delivering voltages ranging from kV up to 30 kV, and currents up to 250 μA. In most experiments, electric field strengths were about 500 V/m, resulting in currents of 5-50 μA. The P/ACE™ MDQ control software allows separations to be performed at either constant voltage, constant current or constant power. Generally, the constant voltage mode was selected. Data acquisition and system control are fully controlled by 32 Karat Software (version 5). Samples were introduced into the capillary by a 0.5-psi pressure injection (typically 10 s). 179 Figure 7.1. Schematic representation of a capillary electrophoretic separation system The Beckman instrument is equipped with a variable-wavelength PDA (Photodiode Array, 190-300 nm) detector. On-line detection is made possible by removing a short section of the protective non-transparent polyimide coating from the capillary. Detection of analytes was simultaneously monitored through three channels at 214, 254 and 280 nm. 7. 4. 1. Related electrophoretic parameters 1. Migration time……Time required for the sample to elute from the injection port through the capillary to the detector. For a pair of enantiomers, t1, t2 are assigned to be the migration times of first and second eluting enantiomers and measured with seconds. 180 Absorption response t2 t1 tEOF neutral marker reversed EOF w1 w2 Migration time (t) 2. Separation selectivity (α) …. Quotient of migration times of the secondly and the firstly eluting enantiomer peaks. α = t2/t1 3. Chiral resolution (Rs) …. dependent on the separation selectivity (α) and the column efficiency (N). Rs can be easily evaluated from a chromatogram according to: Rs=2(t2-t1)/(w2+w1) where t is the migration time; w is the peak width at the baseline. All are measured with seconds. 4. Effective mobilities were calculated by the following equation: µeff= µapp-µeo = Ll/Vt – Ll/Vt0 = Ll(t0-t)/Vt0t where µeff is the effective mobility, µapp the apparent mobility, µeo the EOF, L the total length of capillary, l the effective capillary length from injection site to detection window, V the voltage applied, t the migration time of the second eluting enantiomer and t0 the migration time of the EOF. 181 5. Normalized EOF mobility (β) was calculated according to the following equation: β= μeo/μeff2. 7. 4. Chiral separation by use of High Performance Liquid Chromatography 7.4.2.1 Instrument system The employed HPLC system for determining enantiomeric excess (ee%) of the synthesized chiral alcohols comprised of a Perkin Elmer series 200 LC pump, Perkin Elmer 785A uv/vis detector, connected to a computer via Perkin Elmer Nelson 900 series interface and 600 series link. The column (Ø0.46 cm×25 cm) was packed with mono(6A-N-allylamino-6A-deoxy)perphenylcarbamoylated-β-cyclodextrin by using high-pressure slurry packing procedure, which uses an Alltech® air compression pump (Alltech, USA). A pressure of 7800 psi was employed and maintained for 20 before gradual release of the pressure. The column was conditioned with mobile phase before use. All chromatograms were obtained at ambient temperatures and at normal phase. 7.4.2.2 Preparation of the buffer solution Triethylammonium acetate buffers were prepared using 1% aqueous triethylamine, which were adjusted by addition of glacial acetic acid to the desired pH. The mobile phase, comprising of triethylammonium acetate buffer and the appropriate amount of the organic modifier, was freshly prepared, filtered, and degassed using a DEGASYS DG-2410 degasser. 7.4.2.3 Related chromatographic parameters 182 Absorption response X2 X1 A1 X0 A2 Retention time (t) The enantiomeric excess (ee%) of the synthesized racemates was calculated as follows: ee(%) = ( A2-A1 ) ( A2 +A1 ) x 100% where A1, A2 are the peak areas for a pair of enantiomers; A2 is arbitrarily assigned to the enantiomer which possesses larger peak area. 7. References 1. S. Arta, T. Yabuuchi, T. Kusumi, Chirality, 2003, 15, 609 2. R. C. Petter, J. S. Salek, C. T. Sikorski, G. Kumaravel, F. Lin, J. Am. Chem. Soc. 1990, 112, 3860 3. L. D. Melton, K. N. Slessor, Carbohydr. Res. 1971, 18, 29 183 Chapter Conclusions and Suggestions for Future Work 8. Summary of Conclusions A family of novel positively charged single-isomer CDs have been developed. More than 21 mono-functionalized CD derivatives in three series were synthesized with varied functional groups, i.e. mono-alkylimidazolium and mono-alkyammonium with different alkyl chain length, and amino with different types of CD. The obtained positively charged single-isomer CDs have well-defined structures and could be applied for the chiral separation of a large pool of anionic and amphoteric analytes under wide ranges of BGE pH values and selector concentrations. The introduction of a positive charge into the CD molecules can significantly improve their enantioselective resolution abilities to oppositely charged analytes. For alkylimidazolium single-isomer cationic CDs, they can provide good resolution to dansyl amino acids. The length of alkyl chain of these CDs appears to play an important role in their enantioseparation abilities. A suitable alkyl chain length should be shorter than 4-C as observed in this study. The main reason is attributed to the steric hindrance brought by longer alkyl chains. The steric hindrance may impede attractive electrostatic interaction between the alkylimidazolium cation and the carboxylate group of the analytes. The recognition abilities of alkylimidazolium single-isomer CDs 184 were further investigated by theoretical determination of their complex stability constants towards dansyl amino acids. Results showed that higher binding constants were obtained by CDs with an alkyl chain length lower than 4-C, which is consistent with the experiment observations. For alkylammolium single-isomer CDs, they demonstrated excellent chiral recognition abilities for hydroxyl and carboxylic acids. However, they only offered modest/limited resolution for amphoteric analytes (amino acid derivatives). The great enhancement in resolution is mainly attributed to the introduction of attractive electrostatic interaction between selector and analyte. The steric hindrance brought by the alkyl group is not as obvious as found in alkylimidazolium CDs since the imidazolium ring is more bulky than a straight alkyl chain. Therefore, ALAMCDCl displayed more powerful chiral recognition abilities than mono-alkylimidazolium CDs when resolving dansyl amino acids. ALAMCDCl was proved to be an effective chiral selector for enantioseparation of dansyl amino acids. PrAMCDCl, BuAMCDCl and PeAMCDCl were proved to be good choices as chiral selectors for the enantioseparation of various acidic compounds. Similarly, β-CD-NH2/β-CD-NH3Cl provided excellent chiral recognition abilities to carboxylic acids and dansyl amino acids since only the least bulky amino group is present on the narrow rim of the CDs. Generally, the chiral recognition ability is dependant upon the structure of analytes. γ-CD-NH3Cl showed better resolution for dansyl amino acids than β-CD-NH3Cl. This can be explained by the inclusion model. 185 The inclusion model, which can provide “tight-fit” inclusion and make the chiral center closer to the CD-moiety, will contribute more to the enantioseparation. The applicability of our designed positively charged single-isomer CDs as chiral templates to mediate asymmetric reduction was demonstrated by the use of MIMCDOTs. Results showed that MIMCDOTs presented enhanced enantioselectivity for optical alcohols, as indicated by higher enantiomeric excess (ee%) values of most product alcohols. This may be attributed to the altered shape/ asymmetry of the CD cavity by the introduction of the methylimidazolium group on the rim of CD. 8. Suggestions for future work As indicated by the above-mentioned main results obtained in the previous chapters, positively charged single-isomer CDs prepared herein are efficient chiral selectors towards a wide range of anionic and amphoteric racemates with excellent enantioseparation abilities. Thorough experimental investigation and analysis was performed to testify the chiral recognition abilities of these CDs. Also, great efforts were taken for theoretical determination of complex stability constants in order to strengthen the understanding about the separation mechanism involved. Although some fragmentary explanation for why and how the enantioselectivity is different for different CDs has been provided, it seems that the perfect mechanisms for enantioseparation are still in the dark. Further study with some advanced methodology should be carried out concerning this point. NMR and Molecular Modeling techniques 186 appear to be useful tools to achieve this goal. NMR spectroscopy has become the most important method for structural elucidation of organic compounds, particularly in the solution state. The greatest incentive to use NMR techniques for the investigation of CD complexes is the desire to understand the driving forces and binding modes in these noncovalent associations, and to make optimal use of these factors for new applications. For chiral separation performed by CZE in aqueous electrolyte, two-dimensional NMR techniques now allow the analysis of very complex structures in great detail. The inclusion model and the interaction between substituted CD and included guest analytes can be easily qualitatively analyzed. More quantitative structural data can be obtained by the combined use of Nuclear Overhauser Effects (NOEs) data, and Molecular Dynamics (MD) and Molecular Mechanics (MM) calculations. Molecular modeling, on the other hand, may also be used as a complementary and supportive tool to enhance our understanding of chiral separations. Advanced molecular modeling now allows us to reproduce and even predict intermolecular binding scenarios between relatively small molecules with high reliability. Based on the CE separation data, the explanation of where and how chiral discrimination takes place on positively charged CDs may be predicted. A clear understanding of the chiral separation process can thus be vividly obtained. Therefore, further application of NMR and MM techniques in the study of enantioseaparation mechanisms in CE seems to have a bright future. 187 [...]... 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... 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. .. 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... 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... 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... 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,... 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... 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 23 In order to fulfill the first and main goal of this study, we carried out the following investigation Firstly, we developed a novel. .. 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... 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 . 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. for the rapid screening of novel chiral selectors, thus avoiding the waist of the 7 laborious synthesis of new chiral HPLC stationary phases. 1. 3 History and development of capillary electrophoresis. 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