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Chiral Separation of Drugs and Drug Intermediates by Immobilized Biocatalyst

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Chiral Separation of Drugs and Drug Intermediates by Immobilized Biocatalyst CHIRAL SEPARATION OF DRUGS AND DRUG INTERMEDIATES BY IMMOBILIZED BIOCATALYST A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY BHALCHANDRA K. VAIDYA UNDER THE GUIDANCE OF DR. B. D. KULKARNI CHEMICAL ENGINEERING AND PROCESS DEVELOPMENT DIVISION, NATIONAL CHEMICAL LABORATORY PUNE - 411008, INDIA DECEMBER 2009 Chiral Separation of Drugs and Drug Intermediates by Immobilized Biocatalyst A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY BHALCHANDRA K. VAIDYA UNDER THE GUIDANCE OF DR. B. D. KULKARNI CHEMICAL ENGINEERING AND PROCESS DEVELOPMENT DIVISION, NATIONAL CHEMICAL LABORATORY PUNE - 411008, INDIA DECEMBER 2009 CERTIFICATE This is to certify that the work incorporated in the thesis entitled ‘Chiral Separation of Drugs and Drug Intermediates by Immobilized Biocatalyst’ submitted by Mr. Bhalchandra K. Vaidya was carried out by the candidate under my supervision/guidance at Chemical Engineering and Process Development Division, National Chemical Laboratory, Pune (India). Such material as has been obtained from other sources, has been duly acknowledged in the thesis. Dr. Bhaskar D. Kulkarni December 2009 Deputy Director & Head, Chemical Engineering & Process Development Division, National Chemical Laboratory, Pune - 411008 (India). DECLARATION BY THE CANDIDATE This is to certify that the work incorporated in the thesis entitled ‘Chiral Separation of Drugs and Drug Intermediates by Immobilized Biocatalyst’ is my own work conducted under the supervision/guidance of Dr. B. D. Kulkarni, at Chemical Engineering and Process Development Division, National Chemical Laboratory, Pune (India). I further declare that to the best of my knowledge, this thesis does not contain any part of work, which has been submitted for the award of any degree either of this University or any other University without proper citation. Mr. Bhalchandra K. Vaidya Dr. Bhaskar D. Kulkarni (Research student) (Research guide) Dedicated to my parents & teachers ... CONTENTS ● Acknowledgement iii ● List of Abbreviations v ● List of Figures x ● List of Schemes xiv ● List of Tables xvi Chapter 1 Introduction 1 - 46 1.1 Role of chirality in pharmaceuticals 2 1.2 Methods of enantioselective synthesis 12 1.3 Biocatalysts for enantioselective synthesis 18 1.4 Scope of the thesis 25 1.5 Research objectives 28 1.6 Outline of the thesis 33 Chapter 2 Enantioselective synthesis of unnatural amino acids using 47 - 86 covalently immobilized lipase on porous beaded polymers 2.1 Introduction 48 2.2 Materials and methods 50 2.3 Results and discussion 60 2.4 Conclusions 81 Chapter 3 Chiral resolution of unnatural amino acid esters using 87 - 119 immobilized lipase in membrane bioreactor 3.1 Introduction 88 3.2 Materials and methods 91 3.3 Results and discussion 98 3.4 Conclusions 114 Chapter 4 Chiral resolution of unnatural amino acid amides using 120 - 150 immobilized resting cells of Rhodococcus erythropolis MTCC 1526 4.1 Introduction 121 4.2 Materials and methods 124 i 4.3 Results and discussion 130 4.4 Conclusions 144 Chapter 5 Use of immobilized Aspergillus melleus aminoacylase for 151 - 185 enantioselective synthesis of unnatural amino acids 5.1 Introduction 152 5.2 Materials and methods 153 5.3 Results and discussion 164 5.4 Conclusions 180 Chapter 6 Preparation of cross-linked enzyme aggregates of Aspergillus 186 - 217 melleus aminoacylase for enantioselective synthesis of unnatural amino acids 6.1 Introduction 187 6.2 Materials and methods 189 6.3 Results and discussion 196 6.4 Conclusions 213 Chapter 7 Preparative scale enantioselective synthesis of vicinal diols 218 - 251 using immobilized Solanum tuberosum epoxide hydrolase 7.1 Introduction 219 7.2 Materials and methods 221 7.3 Results and discussion 231 7.4 Conclusions 248 Chapter 8 Conclusions 252 - 256 8.1 Chapter-wise conclusions 253 8.2 Enantioselectivity of biocatalysts towards synthesis of unnatural 255 amino acids ● Publications, Conference Presentations and Awards 257 ii Acknowledgements This gives me immense pleasure to express a deep sense of respect and gratitude to Dr. B. D. Kulkarni, Deputy Director and Head of Chemical Engineering & Process Development Division, National Chemical Laboratory (NCL) Pune, for the responsibilities taken as my research guide. His rational approach, invaluable advice and constant support helped me to rise up to this level. I really consider myself honoured for having him as my guide. I am equally obliged to my co-guide Dr. Sanjay Nene, Head of Biochemical Engineering Department, NCL-Pune who has put countless efforts for making this work successful. I express my earnest gratitude to Dr. Sanjay Nene for his keen interest, excellent guidance, valuable suggestions and parental care during my stay in NCL-Pune. Without his intellectual inputs, motivation and consistent support the objective would not have been accomplished. His creative thinking, administrative skills, unbiased nature and gracious personality inspired me a lot. Working with him was an enriching and enlivening experience. I am extremely grateful to Dr. Alain Archelas, Directeur de Recherches au CNRS, Biosciences, Université Paul Cézanne, Marseille, France for allowing me to work in his laboratory for about six months. The work regarding epoxide hydrolase was successful solely because of his efforts, guidance and help. Starting form hostel arrangements up to getting a student residential card, he helped me in all my difficulties during my stay in Marseille, France. I would also like to thank Dr. Jose M. Guisán and Dr. Cesar Mateo, Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Madrid, Spain for their assistance in epoxide hydrolase immobilization. It is my pleasure to thank Dr. Surendra Ponrathnam for his valuable guidance regarding enzyme immobilization on functional polymers and membranes. His ideas, supervision and assistance gave a proper direction to the present research. The assistance from Dr. C. R. Rajan is gratefully acknowledged. I am sincerely indebted to Dr. R. V. Gadre and Mr. V. V. Jogdand for their technical suggestions and constructive discussions which indeed helped me to plan and execute my experiments. I thank all other scientists and staff members of Biochemical Engineering Group especially Dr. H. V. Adhikane and Dr. (Mrs.) Sangita Kasture for their timely help. I take this opportunity to extend my heartfelt gratitude to my dear friends Anirudhha Deshpande, Ganesh Ingavale, Jahnvi Shah, Kiran Desai, Laxman Savargave, Narahari Pujari, Renuka Joshi, Sandeep Golegaonkar, Snehal Mutalik and Suyog Kuwar for helping me in my experimental work, for their valuable suggestions and moral support. I would like to thank all my friends and colleagues especially Abhijeet Karale, Amol Dive, D. M. Thakar, Gayatri Iyer, Geetanjali Lale, Hitesh Suthar, Kumar Babu, Mayura Dange-Deshpande, Santosh Dhule and Tushar Deshpande for their co-operation and providing a friendly working ambiance. I wish to thank Dr. S. Shivram, the Director, National Chemical Laboratory for allowing me to work in this laboratory and to submit this work in the form of Ph. D. thesis. The present research would not have been feasible without financial assistance from the Council of Scientific and Industrial Research (CSIR), New Delhi, India. The Senior Research Fellowship from CSIR-India is gratefully acknowledged. I would also like to acknowledge the funding from Department of Science and Technology (SERC Programme), New Delhi, India for part of the iii work. I am grateful to the Science and Technology Service, Embassy of France in India, New Delhi, India for offering me a ‘Sandwich Ph.D. Fellowship’ to carry out part of my Ph.D. research in University of Paul Cézanne, Marseille, France. Last but not the least, I would like to dedicate this moment of joy to my parents and dear sister, Vidya without their moral support the goal would not have been accomplished. – B. K. Vaidya iv LIST OF ABBRIVIATIONS Prefixes: d- or (+)- Dextrorotatory enantiomer l or (-) Levorotatory enantiomer dl- or (±)- Equi molar mixture of dextrorotatory and levorotatory enantiomers (i.e. racemic mixture) D- Dexter enantiomer L- Laevus enantiomer DL- Equi molar mixture of Dexter and Laevus enantiomers (i.e. racemic mixture) R- or (R)- Rectus enantiomer S- or (S)- Sinister enantiomer RS- or (RS)- Equi molar mixture of Rectus and Sinister enantiomers (i.e. racemic mixture) Rac- Racemic mixture Acronyms and abbreviations: AGE Allyl glycidyl ether AGE-(C) polymer Polymers synthesized using AGE (as monomer units) and cyclohexanol (as porogen) AGE-(H) polymer Polymers synthesized using AGE (as monomer units) and hexanol (as porogen) AGE-(L) polymer Polymers synthesized using AGE (as monomer units) and lauryl alcohol (as porogen) AGE-Re Agar entrapped Rhodococcus erythropolis cells AIBN 2,20-Azobis(isobutyronitrile) AL Alcaligens lipase ALG-Re Alginate entrapped Rhodococcus erythropolis cells ANOVA Analysis of Variance CLD Cross-link density CLE Cross-linked enzyme CLEA Cross-linked enzyme aggregates CLEC Cross-linked enzyme crystal CRL Candida rugosa lipase DKR Dynamic kinetic resolution v DMSO Dimethyl sofoxide DoE Design of Experiments DOPA 3,4-Dihydroxy phenylalanine DOPA-ester 3,4-Dihydroxy phenylalanine ethyl ester DPC Differential photocalorimeter DVB Divinyl benzene EGDM Ethylene glycol dimethacrylate EH Epoxide hydrolase EMB Enzyme membrane bioreactor Enz Native enzyme moiety * * * Enz , Enz1 , Enz2 Partially deactivated enzyme moieties EnzD Totally
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