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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell Information and Leaming 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMT SYNTHESIS OF MODIFIED MANNOSE OLIGOSACCHARIDES AS POTENTIAL INHIBITORS OF MYCOBACTERIAL LIFO ARAB INOMANN AN BIOSYNTHESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Vinodhkumar Subramaniam, M.S. ***** The Ohio State University 2000 Dissertation Committee: Approvedyby Dr. Todd L. Lowary. Adviser Dr. T V . RajanBabu Adviser Dr. Christopher M. Hadad Department of Chemistry UMI Number; 9994944 UMI UMI Microform 9994944 Copyright 2001 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Infections by Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). have reemerged as a public health threat in recent years. The resurgence in TB has sparked renewed interest in identifying new antibiotics that can be used to treat this disease, which claims nearly three million lives worldwide each year. This disease has been difficult to treat and can be attributed in part to the unusual structure of the cell wall of the organism, which presents a formidable barrier to the passage of drugs into the organism. In order to identify new anti-tuberculosis agents, our research goals are of two-fold: The first objective is to synthesize oligosaccharides which are potential substrates for the enzymes (mannosyltransferases) involved in the biosynthesis of one of the polysaccharides (mannosylated lipoarabinomannan, Man-LAM) found in the cell wall complex that surrounds the organism. The second objective is to prepare mannose-based disaccharides (type A and B, Page 38) which are potential inhibitors of the LAM biosynthesis. Such compounds are believed to be excellent lead candidates for new anti­ tuberculosis agents. During the synthesis of oligosaccharide substrates, a key step is the stereoselective synthesis of octyl P-arabinofuranoside via a 5^2 type displacement reaction of octanol with a benzyl-substituted arabinofuranosyl chloride. Selective deprotection followed by sequential addition of thioglycosides, afforded the oligosaccharide fragments of Man-LAM. In the synthesis of the disaccharide inhibitors, our strategy was to proceed via a common intermediate leading to more than one target. We tlrst developed a methodology for the synthesis of type A mannose disaccharides, which are modified at C-2' of the mannose ring. We then made use of the intermediates used in the preparation of type A disaccharides in the synthesis of type B disaccharides, which contain an additional modification at C-6' of the ring. These compounds will play a significant role in understanding the carbohydrate-enzyme interactions and in turn, can be useful as lead compounds for drug design. Additionally, we also synthesized potential suicide inhibitors, which could function by forming a covalent bond with the active site of the enzyme. We characterized the target oligosaccharides by 800 MHz NMR spectroscopy to ensure purity of such compounds for biological studies. Biological investigations on type A disaccharides were performed in collaboration, which suggested that 4 out of 5 compounds in type A class are substrates of the enzyme a(l-^6)-ManT, but none inhibited the enzyme. Biological studies on type B disaccharides are in progress, as are studies with a ( I—>2)-ManT. Ill This dissertation is dedicated to my parents, Subramaniam and Lalitha, for upbringing me with Godly values, my best friend Juanda, for her love and support, my late sister, Vidhya, for her respect and care toward me, all my friends, extended family and the one whom I agapee (love) the most. IV ACKNOWLEDGMENT I want to take this opportunity to begin by thanking Dr. Todd Lowary for his project ideas, guidance and financial support. My sincere thanks to Dr. RajanBabu and Dr. Hart, for their encouragement and motivation. I also, want to thank ail the professors who taught me courses at OSU, I IT, and at Loyola. Especially, to Dr. S. Govindarajan, Dr. V. Sreenivasan, Dr. D. Loganathan and Dr. M.N.S. Rao for their personal attention and input toward my academic as well as personal growth. My heart-felt gratitude to Dr. Charles Cottrell, Dr. Karl Vermillion, Susan Hatcher, Dr. Kari Green-Church and others for their help toward NMR and MS experiments. I also, want to thank Dr. G. Besra and coworkers (from UK) for conducting biological experiments on my final compounds. My appreciation to Dr. Robert Field and Phil, for their helpful suggestions toward my research. I want to thank Dr. Hart’s Group, Dr. Babu’s Group, Dr. Parquette s Group and Dr. Paquette’s Group for letting me borrow chemicals under unavoidable circumstances. I take this opportunity to convey my best wishes and thanks to all the Lowary group members (present and the past) for their help, support and encouragement. Matt, I thank God for you and fc. our friendship, words cannot be expressed to describe your love toward me. My gratitude to Joe, Grace, Wallace, Oana, Charla, Lori and Haifeng for their comradeship, to Prakxiti for her sincerity in lab maintenance and in sending elemental analysis samples, to Lori, Oana, Chris and Justin for ordering chemicals, to Doug for helping with NMR and with resume writing. Special thanks to Raj for his brotherly love and helping me with synthesis of building blocks. 1 am grateful to the department of chemistry for their financial support as a GTA and instrumentation facility. 1 appreciate Bobbie Cassity, Kathie, Martha and others in the chemistry office for their sincerity and compassionate service toward me. I want to thank all my friends, brothers and sisters in faith for being there for me and helped me to have a family type atmosphere. The pursuit of ones Ph.D. can be a challenging event; you work hard, teach hard, and sleep little. When the experiments are going well, you feel as if you are on top of the world, and when they are not. you may feel and experience isolation. It was during both of these times that God taught me some invaluable lessons: “Truly there is one that sticks closer than a brother." During these difficult times I learned to persevere and to trust God to help me with those situations that were beyond my control, whether it was a compound or a conflict. Being so close to finishing and having to deal with my sister's death, I remember Him comforting me and saying "Just show up, and I will help you with your work." God also, taught me to be compassionate and helpful to my colleagues, especially when their work was not going well. I know now that the good times are victories of the difficult. To the reader I want to leave with you this message: your belief in God and His love will take you far beyond your comprehension. vi VITA May, 28 1973 .......................................................Born - Chennai. India 1990 - 1993 ........................................................... B.S., Loyola College, India 1993 - 1995 ........................................................... M.S. Chemistry, Indian Institute of Technology, Chennai, India 1995 - present ..................................................... Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Subramaniam, V.; Lowary, T. L. "Synthesis of Oligosaccharide Fragments of Mannosylated Lipoarabinomannan from Mvcobacteriiim tuberculosis ", Tetrahedron. 1999, 55, 5965-76. 2. Kannan, T.; Subram aniam , V.: Loganathan, D. “Syn th esis of Glycosyl phosphoramidates: Novel Isosteric Analogs of Glycosyl Phosphates ”, Molecules, Third International Electronic Conference on synthetic organic chemistry, 1999, ECSOC-3. 3. Subramaniam, V.; Kannan, T.; Loganathan, D. “Synthesis of Glycosyl phosphoramidates: Novel Isosteric Analogs of Glycosyl Phosphates ”, Paper presented in the National Carbohydrate Conference, 1997, Kanpur, India. 4. Subramaniam, V.: Pathak, T.; Ganesh, K. N. “Synthesis of New Class of Acyclonucleosides Containing Tertiary Nitrogen Atom”, Project report submitted to Jawaharlal Nehru Center for Advanced