DEVELOPMENT OF NOVEL POSITIVELY CHARGED SINGLE-ISOMER CYCLODEXTRINS AND APPLICATIONS IN ENANTIOMERIC SEPARATION AND CHIRAL SYNTHESIS TANG WEI-HUA (M. Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements I would like to express my sincere gratitude to my supervisor, Associate Professor Ng, Siu Choon for his constant guidance, encouragements and beneficial discussions throughout this project. I am also indebted to Professor Chan Sze On, Hardy for his kind help in following up my graduation affairs. Special mention should be made to the contribution of Dr. Ong Teng-Teng and Dr. I M. Wayan, who offer great effort in our collaboration on this research project presented in this thesis. Their encouragements and suggestions help me very much in my research career. My acknowledgement also goes to all peers in Functional Polymer Laboratory: Mr. Lai Xiang-hua, Mr. Lee Teck-Chia, Ms. Poon Yin-Fun and Dr. Zhang Wei-Guang for their support and useful discussions; Dr. Liu Shou-Ping, Mr. Zou Yong, Mr. Zhang Sheng, Xia Hai-Bing and Cheng Da-Ming for their advices and friendship. Last but not least, I express my gratitude to National University of Singapore for the award of research scholarship and Institute of Chemical and Engineering Science Ltd for the financial support to carry out the research work reported herein. Finally, I would thank my wife, Ms Tang Jian for her continuous support, encouragement and great efforts throughout my Ph.D. study. i Table of Contents Acknowledgements i Table of contents . ii Summary vi List of Publications viii Abbreviations and Symbols . ix List of single-isomer positively charged CDs . x Chapter 1. Introduction . ………1 1. Chiral separation: need and perspective 1. Technques for chiral separation… . 1. History and development of capillary electrophoresis… 1. 3. Principle of capillary electrophoresis 1. 3. Modes of electrophoresis 10 1. Chiral selectors in capillary electrophoresis… 11 1. Cyclodextrins: the chiral selector for CE… . 15 1. 5. Natural ocurring cyclodextrins 15 1. 5. Neutral cyclodextrins 18 1. 5. Charged cyclodextrins 19 1. Research objectives and scope . 22 1. References 25 Chapter 2. Synthesis of positively charged single-isomer cyclodextrins 33 2. Approached for cyclodextrins 33 2. Synthesis of single-isomer positively charged cyclodextrins 37 2. 2. Synthesis of quaternary alkylimidazolium single-isomer β-cyclodextrins…… 37 2. 2. Synthesis of momo-alkyammonium single-isomer β-cyclodextrins . 38 2. 2. Synthesis of mono-(6A-amino-6A-deoxy)-CD hydrochlorides 39 ii 2. Summary 40 2. References 41 Chapter 3. Alkylimidazolium single-isomer β-cyclydextrins as chiral selectors in capillary electrophoresis. . 43 3. Introduction 43 3. Experimental 45 3. 2. Chemicals . 45 3. 2. Apparatus and CE Procedures . 46 3. 2. Calulations 46 3. 2. Determination of binding constants, apparent complex selectivity and theoretical optimum selector concentration . 47 3. Results and discussions 49 Part I Chiral recognition ability of BIMCDCl as chiral selector 49 3. 3. Influence of different chiral selectors on enantioseparation . 49 3. 3. Effect of pH on enantioseparation 51 3. 3. Effect of BIMCDCl concentration on enantioseparation 52 3. 3. Effect of temperature on enantioseparation 56 3. 3. Effect of organic modifier enantioseparation 59 Part II Comparison of chiral recognition ability of quaternary alkylimidazolium β-CDs as chiral selectors 62 3. 3. Influence of side chain length of the alkylimidazolium substituents on the enantioseparation ability of single-isomer imidazolium-β-CDs in CE 62 3. 3. Determination of binding constants of complex formation………………… .…64 3. Conclusions 68 3. References 69 Chapter 4. Alkylammonium single-isomer cyclodextrins as chiral selectors in capillary electrophoresis 72 4. Introduction 72 4. Experimental 74 4. 2. Chemicals . 74 4. 2. Instrumentation and operation conditions . 76 4. Results and discussions 77 Part I Chiral recognition ability of ALAMCDCl towards amino acids 77 iii 4. 3. Effect of ALAMCDCl on the electroosmotic flow . 77 4. 3. Effect of CD concentration on enantioseparation of Dns-amino acids . 80 4. 3. Effect of pH on enantioseparation of Dns-amino acids 82 4. 3. Separation with short capillary . 85 4. 3. Chiral separation of mixture of Dns-amino acids with ALAMCDCl . 86 Part II Chiral recognition ability of mono-alkylammonium cationic β-CDs 88 4. 3. Influence of BGE pH on the enantioseparation of anionic analytes with PeAMCDCl . 88 4. 3. Enantioseparation of racemic acids with PrAMCDCl, BuAMCDCl and PeAMCDCl………………… .…………………………………………… 89 4. 3. Representative separation electropherograms . 101 4. 4. Conclusions . 103 4. 5. References . 104 Chapter 5. Use of mono(6-amino-6-deoxy)-CD as chiral selectors in capillary electrophoresis 106 5. 1. Introduction . 106 5. Experimental 108 5. 2. Chemicals . 108 5. 2. Equipment and data processing 108 5. 2. Determination of binding constants, apparent complexation selectivity and theoretical optimum selector concentration 110 5. Results and discussions 110 Part I Chiral recognition ability of β-CDNH2 . 110 5. 3. Effect of pH and CD concentration on EOF . 111 5. 3. Effect of pH on migration times . 112 5. 3. Effect of pH on enantioseparation of acid analytes . …………….114 5. 3. Effect of β-CDNH2 concentration on enantioseparation . 116 5. 3. Determination of binding constants, apparent complexation selectivity and theoretical optimum selector concentration . 119 5. 3. Representative separation electropherograms . 123 Part II Comparison of chiral recognition ability of mono-amino CDs . 124 5. 3. Comparison of the resolution ability between β-CD-NH2 and β-CD-NH3Cl: Effect of BGE pH values 124 5. 3. Comparison of the resolution ability between β-CD-NH2 and β-CD-NH3Cl: Effect of CD concentration . 127 5. 3. Comparisons of the resolution ability between β-CD-NH3Cl and γ-CD-NH3Cl: Effect of CD type 130 5. Conclusions 132 iv 5. References 133 Chapter 6. Applicability of positively charged single-isomer cyclodextrin in chiral synthesis . 136 6. Introduction 136 6. Experimental 138 6. 2. Chemicals and instruments . 138 6. 2. General procedure for the NaBH4 reduction of ketones in the presence of MIMCDOTs 139 6. Results and discussions 140 6. Conclusions 146 6. References 147 Chapter 7. Experimental . 150 7. Materials 150 7. 1. Solvents . 150 7. 1. Chemicals . 150 7. Instrumentation 151 7. Synthesis 152 7. 3. Mono-(6A-(p-toluenesulfonyl)-6A-deoxy)-CDs 152 7. 3. Mono-(6A-azide-6A-deoxy)-CDs 154 7. 3. Mono-6A-(1-alkyl-3-imidazolium)-6A-deoxy-β-cyclodextrins . 156 7. 3. Mono-6A-N-alkylammonium-6A-deoxy-β-cyclodextrins 169 7. 3. Mono-6A-N-amino-6A-deoxy-cyclodextrins . 174 7. 3. Mono-6A-N-ammonium-6A-deoxy-cyclodextrin chlorides . 175 7. Chromatographic Environment . 176 7. 4. Chiral separation by use of Capillary Electrophoresis 176 7. 4. Chiral separation by use of High Performance Liquid Chromatography 180 7. References 181 Chapter 8. Conclusions and suggestions for future work . 182 8. 1. Summary of conclusions . 182 8. 2. Suggestions for future work 184 v Summary Chirality is a major concern in modern pharmaceutical industry. The increasing demand for enantiopure compounds, especially those of pharmaceutical importance, has led to the rapid development of a variety of stereoselective separation technologies. Capillary electrophoresis (CE) has become one of the most important techniques for enantiomeric separation and analysis. The overall objective of this research was to develop a family of mono-substituted, single-isomer positively charged CDs as chiral selectors for enantiomeric separation by CE and as chiral template for chiral synthesis. In achieving the overall objectives, novel methodologies were developed for the preparation of single-isomer, positively charged CDs, including mono-alkylimidazolium β-CDs, mono-alkylammonium β-CDs and mono-amino CDs. Systematic studies were conducted to investigate the chiral recognition abilities of these CDs towards anionic and ampholytic analytes. Additionally, the applicability of these CDs as chiral templates to mediate chiral synthesis was established using a quaternary alkylimidazolium β-CD for the asymmetric reduction of proachiral ketones. Alkylimidazolium single-isomer cationic CDs provided good resolution to dansyl amino acids. The length of the alkyl chain of these CDs appeared 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 recognition abilities of quaternary alkylimidazolium vi single-isomer CDs 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 was consistent with the experimental observations. Alkylammolium single-isomer cationic β-CDs demonstrated excellent chiral recognition abilities to hydroxy, carboxylic acids and modest/limited resolution to dansyl amino acids. The great enhancement in resolution is mainly attributed to the introduction of attractive electrostatic interaction between selector and analyte. 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 these CDs. Generally, the chiral recognition ability was dependent on the structure of analytes. γ-CD-NH3Cl showed better resolution to dansyl amino acids than β-CD-NH3Cl, which confirmed the “tight-fit” inclusion model. Results from chiral synthesis showed that mono-6A-(1-methyl-3-imidazolium) -6A-deoxy-β-cyclodextrin tosylate (MIMCDOTs) presented enhanced enantioselectivity for optical alcohols, as indicated by higher enantiomeric excess (ee%) values of most product alcohols. In conclusion, the newly developed single-isomer positively charged CDs are effective in enantiomeric separation of anionic analytes and in mediating the asymmetrical reduction of prochiral ketones. vii List of Publications 1. Wei-hua Tang, Teng-Teng Ong, Siu-Choon Ng, A family of single-isomer positively charged cyclodextrin as chiral selector for capillary electrophoresis: mono-6A-butylammonium-6A-deoxy-β-cyclodextrin tosylate, Electrophoresis, 2005, 26, 3125-3133. 2. Wei-hua Tang, I M. Wayan, Teng-Teng Ong, David J. Young, Siu-Choon Ng, Enantioseparation of dansyl amino acids by a novel permanently positively charged single-isomer cyclodextrin: mono-6-N-allylammonium-6-deoxy-β-cyclodextrin chloride by capillary electrophoresis, Anal. Chim. Acta, 2005, 546, 119-125. 3. Wei-hua Tang, Teng-Teng Ong, Siu-Choon Ng, Synthesis and application of single-isomer 6-mono(alkylimidazolium)-β-cyclodextrins as chiral selectors in chiral capillary electrophoresis, Electrophoresis, 2005, 26, 3839-3848. 4. Wei-hua Tang, Siu-Choon Ng, Enantioseparation of acid enantiomers in capillary electrophoresis using a novel single-isomer positively charged β-cyclodextrin: mono-6A-N-pentylammonium-6A-deoxy-β-cyclodextrin chloride, J. Chromatogr. A, 2005, 1091, 152-157. 5. Wei-hua Tang, Siu-Choon Ng, Synthesis and application of mono-6-ammonium-6-deoxy-β-cyclodextrin chloride as chiral selector for capillary electrophoresis, J. Chromatogr. A, 2005, 1094, 187-191. 6. Wei-hua Tang, Siu-Choon Ng, Enantiomeric separation of hydroxy, 10 carboxylic and dansyl amino acids by mono(6-amino-6-deoxy)-β-cyclodextrin in capillary electrophoresis, Anal. Chim. Acta, 2005, 554, 156-162. 7. Wei-hua Tang, Siu Choon Ng, Enantioselective separation in capillary electrophoresis using a novel mono-6A-propylammonium salt of β-cyclodextrin as chiral selector, Anal. Chim. Acta, 2006, 555, 63-67. 8. Wei-hua Tang, Siu-Choon Ng, Asymmetric reduction of acetophenones with NaBH4 in the presence of mono-6A-(1-methyl-3-imidazolium)-6A-deoxy-β-cyclodextrin tosylate, J. Inclus. Phenom. Macrocycl. Chem., 2006, in press. 9. I M. Wayan,Teng-Teng Ong, Wei-hua Tang, David J. Young, Siu-Choon Ng, Synthesis of ammonium substituted β-cyclodextrins for enantioseparation of anionic analytes, Tetrahedron Lett., 2005, 46, 1747-1749. 10. Wei-hua Tang, Siu-Choon Ng, Determination of apparent complex constants of alkylimidazolium substituted β-cyclodextrins with dansyl amino acids by capillary electrophoresis, in preparation. viii Abbreviations and Symbols CD CE HPLC TLC SFC GC CSP BGE MS NMR FT-IR DMF THF Ts MeOH t0 t1 t2 α Rs α cplx Cyclodextrin Capillary Electrophoresis High performance liquid chromatography Thin-layer chromatography Supercritical fluid chromatography Gas Chromatography Chiral stationary phase Background electrolyte Mass spectrometry Nuclear Magnetic Resonance Fourier Transform Infrared Spectroscopy Dimethyl formamide Tetrahydrofuran p-toluenesulfonyl Methanol Migration time of neutral marker for EOF Migration time of first enantiomer Migration time of less mobile enantiomer Separation selectivity Peak resolution Apparent complexation selectivity β EOF μeo μeff μapp K copt L l Normalized EOF mobility Electroosmotic flow Electroosmotic mobility Effective mobility Apparent mobility Binding constant Theoretical optimum selector concentration Total length of capillary Effective capillary length from injection site to detection window Applied voltage Optical rotation V [α]D ix List of single-isomer positively charged CDs + OTs N R2 N (OH) OTs: R1 R1 =H, R 2=CH 3, = = (OH) 14 O O S O + Cl N R2 N (OH) R1 CH3 (OH) 14 MIMCDOTs R =H, R 2=CH 3, R 1=H, R2 =C 2H 5, EIMCDOTs R 1=H, R2 =C 3H 7, PIMCDOTs MIMCDCl R 1=H, R =C 2H 5, EIMCDCl R 1=H, R =C 3H 7, PIMCDCl R 1=H, R2=C 4H 9, BIMCDOTs R 1=H, R2=C 6H 13 , HIMCDOTs R 1=H, R =C 4H 9, BIMCDCl R 1=H, R =C 6H 13 , HIMCDCl R 1=H, R2 =C 10 H21, DIMCDOTs R 1=CH 3, R2=CH 3, DMIMCDOTs R 1=H, R =C 10 H 21, DIMCDCl R 1=CH 3, R =CH , DMIMCDCl Series I + Cl RH N - (OH)6 (OH) 14 R=C H , ALAMCDCl R=C H , PrAM CDCl R=C H , BuAM CDCl R=C H 11 , PeAM CDCl Series II + Cl H3 N (OH)n-1 (OH)2n n=6, α-CD-NH 3Cl n=7, β-CD-NH 3Cl n=8, γ-CD-NH 3Cl Series III x [...]...List of single- isomer positively charged CDs + OTs N R2 N (OH) 6 OTs: R1 R1 =H, R 2=CH 3, = = (OH) 14 O O S O + Cl N R2 N (OH) 6 R1 CH3 (OH) 14 MIMCDOTs R 1 =H, R 2=CH 3, R 1= H, R2 =C 2H 5, EIMCDOTs R 1= H, R2 =C 3H 7, PIMCDOTs MIMCDCl R 1= H, R 2 =C 2H 5, EIMCDCl R 1= H, R 2 =C 3H 7, PIMCDCl R 1= H, R2=C 4H 9, BIMCDOTs R 1= H, R2=C 6H 13 , HIMCDOTs R 1= H, R 2 =C 4H 9, BIMCDCl R 1= H, R 2 =C 6H 13 , HIMCDCl... HIMCDOTs R 1= H, R 2 =C 4H 9, BIMCDCl R 1= H, R 2 =C 6H 13 , HIMCDCl R 1= H, R2 =C 10 H 21, DIMCDOTs R 1= CH 3, R2=CH 3, DMIMCDOTs R 1= H, R 2 =C 10 H 21, DIMCDCl R 1= CH 3, R 2 =CH 3 , DMIMCDCl Series I + Cl RH 2 N - (OH)6 (OH) 14 R=C 3 H 5 , ALAMCDCl R=C 3 H 7 , PrAM CDCl R=C 4 H 9 , BuAM CDCl R=C 5 H 11 , PeAM CDCl Series II + Cl H3 N (OH)n -1 (OH)2n n=6, α-CD-NH 3Cl n=7, β-CD-NH 3Cl n=8, γ-CD-NH 3Cl Series . 11 1. 5 Cyclodextrins: the chiral selector for CE… 15 1. 5. 1 Natural ocurring cyclodextrins 15 1. 5. 2 Neutral cyclodextrins 18 1. 5. 3 Charged cyclodextrins 19 1. 6 Research objectives and. 22 1. 7 References 25 Chapter 2. Synthesis of positively charged single- isomer cyclodextrins 33 2. 1 Approached for cyclodextrins 33 2. 2 Synthesis of single- isomer positively charged cyclodextrins. Abbreviations and Symbols ix List of single- isomer positively charged CDs x Chapter 1. Introduction …… 1 1. 1 Chiral separation: need and perspective 1 1. 2 Technques for chiral separation 5 1. 3