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University Micrcffilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8526145 Bhattacharjee, Mrinal Kanti THE STUDY OF DONOR AND ACCEPTOR SUBSTRATE ANALOGS OF DEXTRANSUCRASE The Ohio State University Ph.D. 1985 University Microfilms International 300 N. Zeeb Road, Ann Arbor, Ml 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages 2. Colored illustrations, paper or print 3. Photographs with dark background ^ 4. Illustrations are poor copy 5. Pages with black marks, not original copy 6. Print shows through as there is text on both sides of page 7. Indistinct, broken or small print on several pages S 8. Print exceeds margin requirements 9. Tightly bound copy with print lost in spine 10. Computer printout pages with indistinct print 11. Page(s) lacking when material received, and not available from school or author. 12. Page(s) seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkled pages 15. Other University Microfilms International THE STUDY OF DONOR AND ACCEPTOR SUBSTRATE ANALOGS OF DEXTRANSUCRASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mrinal K. Bhattacharjee, B.Sc., M. Sc. The Ohio State University 1985 Reading Committee : jroved^by Prof. M. M. King Prof. R. M. Mayer Advi ser Prof. M-D. Tsai Department of Cheml DEDICATION To my parents ii ACKNOWLEDGEMENT I wish to express my deepest gratitude to my adviser, Dr. Robert M. Mayer, for his guidance and support throughout this study. I also like to thank my colleagues in the research group for their helpful suggestions. I am grateful to Dr.Irwin Goldstein, Dr. Derek Horton and Dr. Howard Sloan for providing some of the sugars and Dr. David A. Grahame for providing highly purified dextransucrase. Thanks are due to Dr. C. R. Weisenberger for obtaining the mass spectra. I wish to acknowledge Robert Daiber, Rebecca Jezerinac, Vincent Leber, Stella Sung and Robert Wheatley for their assistance in certain technical aspects of the research. i i i VITA June 04, 1958 Born - Dhanbad, India 1978 B.Sc., Indian Institute of Technology, Kharagpur 1980 M.Sc., Indian Institute of Technology, Kharagpur 1980-1982 Teaching Associate, Chemistry Department, The Ohio State University, Columbus, Ohio 1982-1985 Research Associate, Chemistry Department, The Ohio State University, Columbus, Ohio PUBLICATION "Interaction of Deoxy Halo Derivatives of Sucrose with Dextransucrase", Bhattacharjee, M. K. and Mayer, R. M., Carbohydr. Res., in press. FIELD OF STUDY Major Field : Biochemistry i v TABLE OF CONTENTS Page DEDICATION ii ACKNOWLEDGEMENTS iii VITA iv LIST OF TABLES viii LIST OF FIGURES ' ix LIST OF ABBREVIATIONS x CHAPTERS I. INTRODUCTION 1 A. Structure of Dextran 3 B. Purification and Properties of Dextransucrase ... 7 C. Mechanism of Dextransucrase Reaction 9 1. Chain Initiation 9 2. Chain Propagation 12 a. Mode of Elongation 12 b. Direction of Chain Growth 13 c. Formation of Branches 20 d. Mechanism of Catalysis 21 i. Carbonium Ion Mechanism 22 ii. Covalent Ion Mechanism 24 iii. Hydrolysis 26 3. Chain Termination 27 D. Reaction Pathways of Dextransucrase 28 E. Substrate Specificity 29 1. Donor Substrate Specificity 31 2. Acceptor Substrate Specificity 33 F. Purpose of Investigation 34 II. MATERIALS AND METHODS 35 A. Materials 35 1. Organisms 35 2. Enzymes 35 v CONTENTS (continued) Page 3. Saccharides and Derivatives 36 4. Chromatography Materials 37 5. Chemicals and Reagents 37 B. Methods 38 1. Measurement of Dextransucrase Activity .... 38 a. Coupled Enzyme Assay 38 b. Radioactive Assay 39 2. Reactivation of SDS Inhibited Enzyme .... 40 3. Assay of Acceptor Activity 41 4. Inactivation of Enzyme . 42 a. Chemical Inactivation 42 b. Photochemical Inactivation 42 5. Chromatographic Methods 42 a. Thin Layer Chromatography 43 b. Paper Chromatography 43 c. Column Chromatography 45 i. Silica Gel 45 ii. Ion Exchange 45 iii. Gel Filtration 46 d. High Performance Liquid Chromatography . 47 6. Radioactive Analysis 47 7. Spectroscopic Methods 48 a. *-H NMR Spectroscopy 48 b. C NMR Spectroscopy 48 c. UV/VIS Spaectroscopy 48 d. IR Spectroscopy 49 e. Mass Spectroscopy 49 8. Saccharide Analysis 49 9. SDS El ectrophoresis 49 10. General Synthetic Procedures 51 a. Preparation of Dry Solvents and Reagents . 51 b. Halo Derivatives of Sugars 52 c. Methyl Glycosides 54 d. Acetyl ation Reactions 56 e. Deacetyl ation Reactions 56 11. Chemical Synthetic Procedures 56 a. 6,6'-dichloro-6,6'-dideoxy-sucrose .... 56 b. 6,6'-dibromo-6,6'-dideoxy-sucrose .... 58 c. Monobromosucroses 59 d. 6,1',6'-tribronio-6,r,6,-trideoxy-sucrose . 59 e. Methyl,6-chloro-6-deoxy-a-(D)-glucopyranoside 62 f. Methyl,6-bromo-6-deoxy-a-(D)-glucopyranoside 65 g. Methyl,6-iodo-6-deoxy-a-(D)-gl ucopyranoside 65 h. Methyl,6-bromo-6-deoxy-a-(D)-galactopyranoside 65 i. Methyl.6-bromo-6-deoxy-a-(D)-mannopyranoside 66 j. Methyl ,6-deoxy-a-(D)-gal actosides .... 69 k. Methyl ,6-deoxy-a-(L)-mannosides .... 72 1. Methyl-(D)-al 1 osides 72 vi CONTENTS (Continued) Page m. Methyl,6-azido-6-deoxy-a-(D)-gl ucopyranoside 73 n. Methyl,3,6-anhydro-a-(D)-glucopyranoside . 76 o. Methyl,2-deoxy-a-(D)-gl ucopyranoside ... 77 p. (D)-glucal 78 q. 6-deoxy-(L)-gl ucal 78 r. Methyl,2,3,6-trideoxy-a-(L)-erythro- hex-2-enopyranoside ... 79 s. 6'-bromo-6'-deoxy-maltose 79 12. Enzymatic Synthetic Procedures 87 III. RESULTS AND DISCUSSIONS 91 A. Interaction of Halosucrose Derivatives with Dextransucrase 91 B. Acceptor Substrate Specificity : Analogs of Methyl,a-(D)-glucopyranoside . 115 1. Acceptor Reactions 115 2. Unsaturated Derivatives 138 3. Photochemical Inactivation 139 C. Acceptor Substrate Specificity : Oligosaccharides . 146 D. Acceptor Substrate Specificity : Branch Formation . 167 1. Preparative Synthesis of Products with Methyl,a-(D)-gl ucopyranoside as Acceptor . 167 2. Preparative Synthesis of Products with Maltose as Acceptor .... 175 3. Preparative Synthesis of Products with 6'-bromo-6'-deoxy-maltose as Acceptor .... 178 4. Methyl,6-bromo-6-deoxy-a-(D)-glucopyranoside as Acceptor . 183 5. Preparative Synthesis of Products with Methyl, 6-bromo-6-deoxy-a-(D)-glucopyranoside as Acceptor 191 6. Preparative Synthesis of Products with Nigerose as Acceptor .... 207 IV. CONCLUSION 219 V. BIBLIOGRAPHY 221 vii LIST OF TABLES Table Page 1. Chemical shifts of synthesized sugars Ill 2. Comparison of acceptor activities of analogs of a-methyl glucoside 120 3. Effect of unsaturated derivatives of a-methyl glucoside on dextransucrase 141 4. Comparison of acceptor activities of maltooligosaccharides 152 5. Comparison of acceptor activities of disaccharides 166 6. Chemical shifts of acceptor products 218 vii i LIST OF FIGURES Figure Page 1. Postulated structure of dextran synthesized by Streptococcus sanguis ..... 5 2. Model for chain growth from the non-reducing end as proposed by Neely 15 3. Model for chain growth from the reducing end as proposed by Ebert and Schenk 15 4. Model for chain growth at the reducing end for Dextransucrase as proposed by Robyt et.al. .... 18 5. Proposed pathway for reactions catalyzed by dextransucrase 29 1 O 6. C NMR spectra of halo derivatives of sucrose 60 7. Mass spectrum of 5,6'-dichloro-6,6'-dideoxy- sucrose peracetate 63 1 ^ 8. C NMR spectra of halo derivatives of monosaccharides . 67 9. 13C NMR spectra of methyl glycosides of (D)-fucose ... 70 10. 13C NMR of methyl glycosides of (L)-rhamnose and allose . 74 11. 13C NMR of other analogs of a-methyl glucoside .... 80 12. HPLC elution profiles 82 13. IR spectrum of methyl,6-azido-6-deoxy-a-(D)- glucopyranoside 84 14.