ENANTIOSEPARATION OF CHIRAL DRUGS IN LIQUID CHROMATOGRAPHY WITH ANTIBIOTIC CAPPED MACROCYCLE BONDED SILICA PARTICLES AS CHIRAL STATIONARY PHASES

ENANTIOSEPARATION OF CHIRAL DRUGS IN LIQUID CHROMATOGRAPHY WITH ANTIBIOTIC-CAPPED MACROCYCLE-BONDED SILICA PARTICLES AS CHIRAL STATIONARY PHASES ZHAO JIA (M.Sc., NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2015 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Zhao Jia 22 Jan 2015 I I ACKNOWLEDGEMENT A quote from Jacob Riis always spurs me on to greater effort and may be the best footnote to illuminate the time of my Ph.D study: When nothing seems to help, I go and look at a stonecutter hammering away at his rock perhaps a hundred times without as much as a crack showing in it Yet at the hundred and first blow it will split in two, and I know it was not that blow that did it, but all that had gone before A true Ph.D means far more than the degree title The core of Ph.D career is not to pursue nice experiment data for publication, but a journey to transcend oneself, to perfect the philosophy of life, to own the wisdom of thinking Beyond the overwhelming experience of reading journals, staying up for experiments, giving presentations and writing papers, it is truly a training process to learn how to consider, analyze and solve problems in aspects of life Many years later, I may forget the great mass of details mentioned in this dissertation But I will remember the bittersweet journey and the wonderful time with you forever Firstly, I am deeply grateful to my supervisor Dr Gong Yinhan To be your student makes me lifelong benefits I learnt a lot from you about life, family and research Your technical excellence and the ability to deal with problems set a good example for me to follow In addition, it is my real pleasure to be oriented and supported with your care and patience through my student career Meanwhile, I have been very privileged to get to know and II to collaborate with the “bond of sisters in chromatography”— Dr S.K Thamarai Chelvi, Dr Wang Chong, Dr Soh Shu Fang, Ms Tan Huey Min, Ms Tan Daphane, Ms Tan Sharon, Ms Pang Shu Hui and Ms Shan Yu I really appreciate your sincere help in life and study, and the enjoyable discussions with you about life, politics, culture, food, dialect, Ph.D and all that make my student life colourful, with no time for boredom I also want to thank all other colleagues who helped me during last four years in particular Prof Yong Eu Leong, Dr Li Jun, Dr Tong Yoke Yin, Dr Hong Xin, Mr Zhang Zhiwei, Ms Chua Seok Eng, Ms Mok Poh Pheng and Ms Tang Sing Kwang Last but not least, I wish to express my sincere appreciation to my parents, grandparents and other family members for their unconditionally infinite support and selfless giving throughout everything in my life Special thank to my wife for her kind-hearted support, understanding and encouragement from our initial encounter III TABLE OF CONTENTS Declaration page I Acknowledgements II Table of contents IV Summary VIII List of publications IX List of tables XI List of figures XIII Abbreviation XVI Chapter Introduction 1.1 Chirality and enantiomers 1.2 Enantioseparation 1.3 Chromatography 1.4 Chiral chromatography 12 1.5 Chiral stationary phase 15 1.6 Research objectives and hypothesis 18 Chapter Synthesis of novel macrocycle-bonded silica stationary phases 2.1 Introduction 21 2.1.1 Cyclodextrins and calixarenes 21 2.1.2 Vancomycin and rifamycin 24 2.1.3 Novel CSPs with multiple recognition sties 27 2.2 Experimental 28 2.2.1 Reagents and materials 28 2.2.2 Apparatus 30 IV 2.2.3 Synthesis of modified ß-CD-bonded silica particles 31 2.2.4 Synthesis of modified MCR-bonded silica particles 40 2.3 Results and discussion 47 2.3.1 SEM/EDS analysis 47 2.3.2 Mass spectrometry analysis 50 2.3.3 Elemental analysis 50 2.3.4 Fourier-transform infrared spectroscopy analysis 51 2.3.5 Summary 53 Chapter Application of VCD-HPS and RCD-HPS as CSPs in HPLC 3.1 Introduction 55 3.2 Experimental 55 3.2.1 Reagents and materials 55 3.2.2 Apparatus 55 3.2.3 Preparation of stationary phase materials 56 3.2.4 Preparation of novel CSP-packed HPLC columns 56 3.2.5 Chromatographic procedures 59 3.3 Results and discussion 60 3.3.1 Evaluation of VCD-HPS and RCD-HPS 60 3.3.2 Enantioseparation on VCD-HPS in HPLC 63 3.3.3 Enantioseparation on RCD-HPS in HPLC 70 3.3.4 Summary 78 Chapter Application of C[4]CD-HPS and MCRCD-HPS as CSPs in CEC 4.1 Introduction 80 4.2 Experimental 84 4.2.1 Reagents and materials 84 4.2.2 Apparatus 85 4.2.3 Preparation of stationary phase materials 85 4.2.4 Preparation of novel CSP-packed CEC Columns 86 4.2.5 Chromatographic procedures 87 V 4.3 Results and discussion 88 4.3.1 Evaluation of C[4]CD-HPS and MCRCD-HPS 88 4.3.2 Enantioseparation on C[4]CD-HPS in CEC 89 4.3.3 Enantioseparation on MCRCD-HPS in CEC 92 4.3.4 Summary 98 Chapter Application of MCR-HPS and BAMCR-HPS as CSPs in HPLC 5.1 Introduction 100 5.2 Experimentl 100 5.2.1 Reagents and materials 100 5.2.2 Apparatus 100 5.2.3 Preparation of stationary phase materials 101 5.2.4 Preparation of novel CSP-packed HPLC columns 101 5.2.5 Chromatographic procedures 101 5.3 Results and discussion 102 5.3.1 Evaluation of MCR-HPS and BAMCR-HPS 102 5.3.2 Enantioseparation on MCR-HPS in HPLC 107 5.3.3 Enantioseparation on BAMCR-HPS in HPLC 108 5.3.4 Summary 111 Chapter Application of RMCR-HPS and 15C5-MCR-HPS as stationary phases in liquid chromatography 6.1 Introduction 114 6.2 Experimental 114 6.2.1 Reagents and materials 114 6.2.2 Apparatus 114 6.2.3 Preparation of stationary phase materials 115 6.2.4 Preparation of novel CSP-packed CEC and HPLC columns 116 6.2.5 Chromatographic procedures 116 6.3 Results and discussion 117 6.3.1 Evaluation of RMCR-HPS and 15C5-MCR-HPS VI 117 6.3.2 Enantioseparation on RMCR-HPS in HPLC and CEC 120 6.3.3 Enantioseparation on 15C5-MCR-HPS in HPLC 124 6.3.4 Summary 126 Chapter Conclusions and future work 7.1 Conclusions 129 7.2 Limitation and prospects 129 Bibliography 134 VII Compared to the BAMCR-HPS packed column previously reported (Tan et al., 2011), better enantioselectivity and higher resolution were achieved on 15c5-MCR-HPS packed column for a broad range of chiral compounds including benzyl mandelate (no separation on BAMCR-HPS versus α = 3.1 and Rs = 4.1 on 15C5-MCR-HPS), ibuprofen (no separation on BAMCR-HPS versus α = 12.3 and Rs = 5.9 on 15C5-MCR-HPS), indapamide (α = 1.5 and Rs = 2,1 on BAMCR-HPS versus α = 1.8 and Rs = 2.3 on RMCR-HPS), methyl mandelate (no separation on BAMCR-HPS versus α = 2.6 and Rs = 2.2 on 15C5-MCR-HPS), 1-(1-naphthyl)ethylamine (α = 1.3 and Rs = 1.3 on BAMCR-HPS versus α = 1.8 and Rs = 1.7 on 15C5-MCR-HPS), metoprolol (no separation on BAMCR-HPS versus α = 4.8 and Rs = 1.3 on 15C5-MCR-HPS), promethazine (no separation on BAMCR-HPS versus α = 7.0 and Rs = 7.4 on 15C5-MCR-HPS) and warfarin (no separation on BAMCR-HPS versus α = 2.0 and Rs = 4.5 on 15C5-MCR-HPS) under similar mobile phase conditions These results indicate that 15C5 plays an important role in 15C5-MCR-HPS to improve chiral recognition for higher coverage of chiral compounds, and also indirectly prove the successful synthesis of 15C5-MCR-HPS 6.3.4 Summary Two novel modified MCR-bonded silica particles, RMCR-HPS and 15C5-MCR-HPS, were successfully applied as CSPs for HPLC to separate 126 aromatic positional isomers and chiral compounds Their chromatographic performance suggests they are of notable advantages and potential in enantioseparation for a wide range of chiral drugs Meanwhile, there is no significant change in the chromatographic performance of RMCR-HPS and 15C5-MCR-HPS packed HPLC columns observed after continuous usage over half year under different type of mobile phase conditions 127 CHAPTER CONCLUSIONS AND FUTURE WORK 128 7.1 Conclusions A series of novel antibiotic-capped macrocycle-bonded silica particles, RCD-HPS, VCD-HPS, C[4]CD-HPS, MCRCD-HPS, RMCR-HPS and 15C5-MCR-HPS, were successfully synthesized by successive multiple step liquid solid phase reaction, and applied as CSPs in HPLC and CEC These CSPs have multiple chiral recognition sites with many different functional moieties including vancomycin, rifamycin, β-CD, crown ether and types of calixarenes Their structures were characterized by means of analysis methods including SEM/EDX, MS, elemental analysis and FTIR Owing to the cooperative function of antibiotics and anchored macrocycle moieties, these novel CSPs exhibited excellent chromatographic selectivity for separation of positional isomers of disubstituted benzenes and enantiomers of a wide range of chiral drug compounds in HPLC and CEC under varied mobile phase conditions 7.2 Limitation and prospects The novel CSPs in this dissertation were prepared by multiple step liquid solid phase reactions The synthesis method can be improved by optimization of reaction parameters like time and temperature, isolation and purification of intermediate product synthesized like MCR-HP 129 Based on the van Deemter equation, there is a significant improvement in separation efficiency when the particle size decreased to 2.5 μm or less Meanwhile, column efficiency is not diminished at increased flow rate or linear velocity Thus, separation speed and peak capacity will extend to the new limitations as smaller particles used This method takes full advantage of chromatographic principles for separations by using the column packed smaller particles with higher flow rates to achieve superior resolution and sensitivity In particular, preparing novel CSPs based on sub 2-μm and core-shell (solid core) particles will significant stimulate the development The gain in separation efficiency achievable will definitely be beneficial also for chiral separations Since the first sub-2 μm CSP reported (Cancelliere, 2010), it can be expected that an outstanding production of HPLC columns packed with already known or new CSPs would be marketed soon For HPLC, fast separation is always a challenges which often leads to high operation pressure Therefore, ultra high pressure liquid chromatography is developed to fulfil the much stricter requirements on pumping and flow system for high-pressure operation (Hayes et al., 2014) Compared to HPLC, the preparation of CEC column is always a challenge though many manufacture methods reported For the packed-column CEC, the most serious obstacle is bubble formation within the packed column arising from the on-column frits during the operation, which often leads to a poor baseline, irreproducible retention times and even current breakdown or no peak Meanwhile, packed columns suffer from time-consuming fabrication 130 procedures, poor permeability and fragility These limitations associated with packed columns have stimulated the development of alternative column technologies Among the various approaches, monolithic columns or continuous-bed columns has recently attracted increasing interest A monolithic column is typically a continuous porous solid anchored to the capillary wall, which is prepared through in situ polymerization or consolidation inside the column The stationary phase could be attached on the monolithic surface or embedded in the monolithic matrix The attraction of monolithic CEC columns is partially due to the fact that no frits are necessary to keep the stationary phase in place Therefore the problem of bubble formation arising from the frits in packed columns could be avoided Compared with open-tubular columns, the monolithic columns have much higher surface areas and adsorption capacities and thus the sample loading could be significantly improved Owing to high permeability, the separation speed is enhanced in monolithic columns and the requirement of high-pressure conditioning with an HPLC pump is eliminated Currently, various procedures and a wide variety of precursors offer valuable flexibility of the surface chemistry for monolithic columns (Subramanian, 2007) The preparative separation of chiral drugs and intermediates on CSPs is increasingly becoming an alternative to enantioselective synthetic routes, mainly due to advantages with regard to time and reliability of scale-up Advances in chromatographic techniques, such as recycling/peak shaving, automated repetitive injection mode and simulated moving bed (SMB) chromatography are further reasons Recycling/peak shaving and SMB 131 chromatography require less stationary phase and solvent and, therefore, offer higher specific productivity Novel CSPs with wide application area will stimulate the development and production of chiral drugs For example, paclitaxel is a medication used to treat ovarian cancer, breast cancer and many other types of cancer It is the crude extract from the bark of Pacific yew with relatively low but highly variable yields Total synthesis method coupled with necessary purification by chromatographic methods with novel CSPs has been studied for decades to instead of current production methods like plant cell fermentation technology or semi-synthesis Beside vancomycin and rifamycin, there are several macrocylic molecules which are capable to be used for developing novel CSPs with multiple chiral recognition sites Same as vancomycin, eremomycin is a macrocylic glycopeptides antibiotic The eremomycin molecule has two amino sugar residues while vancomycin has one There are three cavities, three sugar moieties, five aromatic rings, nine hydroxyl groups, seven amido groups, three amino groups, one carboxylic group with totally twenty two chiral centers in the eremomcyci molecule Different from vancomycin, eremomycin has different structure with trinucleus amino acid fragment, in which eremomycin has one chloro-substituted aromatic ring while vancomycin has two (Staroverov et al., 2006) Similar to CDs, cyclofructans is cyclic oligosaccharides and the newest class of chiral stationary phases for LC Currently, there are commercial derivatized cyclofructans-based columns available It was reported that this 132 kind column works more effectively with organic solvents and supercritical fluids with great capability for preparative-scale separations Therefore, eremomycin and cyclofructans are interested us to employ them as new chiral recognition sites in novel CSPs for future work Meanwhile, our group attempted a new type of mixed octadecylsilyl- and (3-(C-methylcalix[4]resorcinarene)-2-hydroxypropoxy)-propylsilyl-bonded silica particles (ODS-MCR-HPS) which was used as stationary phase for HPLC (Tan et al., 2012) The octadecylsilyl (ODS) columns is widely used in reversed-phase HPLC mode owing to its excellent reproducibility and versatility, but limited in certain separations, for example, poor selectivity of positional isomers of disubstituted benzenes (Liu et al, 2005) Interestingly, the ODS-MCR-HPS column 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