AN ABSTRACT OF THE DISSERTATION OF Chanokporn Phaosiri for the degree of Doctor of Philosophy in Pharmacy presented on March 10, 2004. Title: Syntheses and Evaluation of Putative Enzyme Inhibitors of Isoprenoid Biosynthesis Abstract approved: Redacted for privacy Philip The discovery of the methylerythritol phosphate pathway (the MEP pathway) as an alternate pathway for isoprenoid biosynthesis in some organisms including most bacteria, malarial parasites and plants, but not in animals, has stimulated extensive studies in this area. Research has revealed the potential of finding novel antibacterials, antimalarial drugs, and herbicides from enzyme inhibitors of this pathway.The natural products fosmidomycin and FR900098 appear to be very promising antibacterial and antimalarial compounds.Both compounds have inhibition activities against the second enzyme in the MEP pathway, deoxyxylulose- 5-phosphatereductoisomerase(DXR),whichmediatestheconversionof deoxyxylulose-5-phosphate (DXP) into methylerythritol-4-phosphate (MEP). This thesis presents one aspect of the MEP pathway studies.Six different analogs of DXP were designed based on the structural features of DXP to understand the requirements of the DXR-substrate binding. Compounds with the trivialnames I -Me-DXP (containing an ethyl ketone moiety), DX-phosphonate (DXP havinga phosphonate group rather than a phosphate group), 4-epi-DXP (possessing the opposite stereochemistry at theC4position compared to DXP), 4-deoxy-DXP (lacking the hydroxyl group at theC4position), 3-deoxy-DXP (lacking the hydroxyl group at theC3position), and DXP carboxamide (having a primary amide group rather than the methyl ketone) were synthesized and tested as alternate substrates and enzyme inhibitors against DXR.The compound DX-phosphonate was the only alternate substrate among the synthesized compounds. The remaining analogs of DXP acted as weak competitive inhibitors against DXR. Kinetic studies of these compounds provided an overall picture of how the substrate DXP binds to DXR. Further studies of the compound 1-Me-DXP, using the published X-ray crystal structures of DXR and DXR mutagenesis demonstrated more detail of the DXR active site.The results present useful information for designing better enzyme inhibitors. The mechanism for the rearrangement of DXP to MEP by DXR was also studied. Two possible mechanisms for this rearrangement have been proposed, the cx-ketol rearrangement and the retroaldol/aldol rearrangement.Several approaches including the use of the potential alternate substrates, 4-deoxy-DXP and 3-deoxy- DXP were tried. Unfortunately none of the results obtained can definitively rule out either of the mechanisms. Further studies are needed to completely understand this mechanism and establish additional strategies for inhibition of DXR. Syntheses of an intermediate from the DXR reaction, methylerythrose-4- phosphate, were also attempted in order to better understand the chemistry mediated by DXR. Even though the target compound was not successfully obtained, several synthetic approaches to this compound were useful for the syntheses of the different DXP analogs mentioned above. ©Copyright by Chanokporn Phaosiri March 10, 2004 All Rights Reserved Syntheses and Evaluation of Putative Enzyme Inhibitors of Isoprenoid Biosynthesis by Chanokporn Phaosiri A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented March 10, 2004 Commencement June 2004 Doctor of Philosophy thesis of Chanokporn Phaosiri presented on March 10, 2004. APPROVED: Redacted for privacy Major Professor, representing Redacted for privacy Dean of the CoIIeg of Pharmacy Redacted for privacy Dean of Graduate I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Redacted for privacy Chanokporn Phaosiri, Author ACKNOWLEDGEMENTS First and foremost, II would like to thank my major professor, Dr. Philip Proteau for his advice, guidance, and understanding. His professional and personal support has been overwhelming thoughout my graduate career. I am also indebted to Dr. Mark Zabriskie for his unconditional help and support in every situation.I also want to thank all my committee members, Dr. William Gerwick, Dr. David Home, and my graduate representative, Dr. Daniel Rockey for their support and inspiration. Thanks to the National Institutes of Health for funding this research. Thanks to Dr. Satoshi Tabata for providing the dxr gene. I also would like to express my gratitude to Brian Arbogast and Jeff Morre for their mass spectral work. Thanks to Roger Kohnert, Dr. Thomas Williamson and Dr. Brian Marquez for their help with NMR techniques. I would like to acknowledge previous members of the Zabriskie and Proteau Labs, Dr. Michael Jackson and especially Dr. Younhi Woo for her advice and patience every single day in the past four years. Thanks to all my colleagues Dr. Xihou Yin, Dojung Kim, David Blanchard, Laura Grochowski, Roberta Fernandes, Morgan Parker, Gerwick's lab members and their families for their friendship and encouragement. The College of Pharmacy's staff, Gary Miller and Debra Peters are thanked for their enthusiastic help. I also would like to thank my parents, Cot. Sunthorn Phaosiri, Permporn Phaosiri, my brother, Ngampol Phaosiri and my friends back home in Thailand for always believing in me. Special thank to my significant other, Yingrodge Suntiwuth for his beautiful friendship and support during the past two years. CONTRIBUTION OF AUTHORS Dr. Xihou Yin conducted initial experiments for DXR expression and purification, including DXP synthesis in Chapter Two. Roberta Fernandes performed modeling experiments for DXR and DXR mutagenesis in Chapter Three. TABLE OF CONTENTS PAGE CHAPTER ONE: ISOPRENOID NATURAL PRODUCTS AND BIOSYNTHESES INTRODUCTION I CLASSIFICATION OF ISOPRENOIDS 3 BIOSYNTHESES OF ISOPRENOID PRECURSORS 6 THE MEVALONATE PATHWAY 6 THE METHYLERYTHIUTOL PHOSPHATE PATHWAY 8 DISTRIBUTION OF THE MEVALONATE PATHWAY AND THE METHYLERYTHRITOL PHOSPHATE PATHWAY 15 REFERENCES 17 CHAPTER TWO: SYNTHESES AND EVALUATION OF DXP ANALOGS INTRODUCTION 26 EXPERIMENTAL DESIGN 31 EXPERIMENTAL SYNTHESES 32 RESULTS 45 DISCUSSION 49 EXPERIMENTAL SECTION 53 REFERENCES 94 TABLE OF CONTENTS (Continued) PAGE CHAPTER THREE: ENZYMATIC STUDIES OF 1-METHYL-DXP INTRODUCTION 103 EXPERIMENTAL DESIGN 106 RESULTS 107 DISCUSSION 113 EXPERIMENTAL SECTION 115 REFERENCES 119 CHAPTER FOUR: MECHANISMS FOR THE REARRANGEMENT OF DXP TO MEl' INTRODUCTION 121 RESULTS 132 DISCUSSION 136 EXPERIMENTAL SECTION 137 REFERENCES 142 CHAPTER FIVE: METHYLERYTHROSE PHOSPHATE AS AN INTERMEDIATE IN THE DXR-MEDIATED REACTION INTRODUCTION 145 RESULTS AND DISCUSSION 146 EXPERIMENTAL SECTION 158 REFERENCES 168 TABLE OF CONTENTS (Continued) PAGE CHAPTER SIX: CONCLUSION 172 BIBLIOGRAPHY 175 LIST OF FIGURES FIGURE PAGE 1.1 Examples of Isoprenoid Natural Products 1 1.2 Example of Hemiterpenoid Formation 4 1.3 Examples of Cyclic Monoterpenes, Sesquiterpenes and Diterpenes 4 1.4 Structures of HMG-CoA Reductase Inhibitors 7 1.5 The putative role of the sidechains in the mechanism of CDP-ME synthase 11 1.6 The substrate binding site of MEcPP synthase 12 2.1 Inhibitors of deoxyxylulose phosphate reductoisomerase (DXR) 27 2.2 Examples of phosphonate drugs 29 2.3 Putative inhibitors and alternate substrates of DXR 31 2.4 A non-linear regression plot of DX-phosphonateas a substrate for DXR 45 2.5 Double reciprocal plots for inhibition of DXR byl-Me-DXP 46 2.6 Double reciprocal plots for inhibition of DXR by 1-deoxy-D-ribulose 5-phosphate 46 2.7 Double reciprocal plots for inhibition of DXR by 4-deoxy-DXP 47 2.8 Double reciprocal plots for inhibition of DXR by 3-deoxy-DXP 47 2.9 Double reciprocal plots for inhibition of DXR by DXP carboxamide 48 2.10 The alternate substrate and the inhibitors against DXR 48 2.11 The reactions of DX-phosphonate along the MEP pathway 52 3.1 The active site geometry of DXR complexed with DXP 105 LIST OF FIGURES (Continued) FIGURE PAGE 3.2 GC!MS analysis of A). Synthetic ethylerythritol triacetate. B). The putative ethylerythritol triacetate derivative from the enzymatic reaction. C). Co-injection of synthetic and enzymatically prepared triacetates 110 3.3 The predicted mass spectral fragmentation pattern of ethylerythritol triacetate 111 3.4 'H NMR spectrum of ethylerythritol triacetates A).Synthetic Standard B).The product from the enzymatic reaction 112 3.5 Stereoview of the active site geometry of DXR complexed with fosmidomycin by Steinbacher et al. 113 4.1 Inhibitors of ketol acid reductoisomerase 123 4.2 Active site of KARl complexed with 2,3-dihydroxy-3-methylvalerate 123 4.3 The pairwise sequence alignments of DXR and KARl from E.coli based on the ClustaiW (1.82) program 124 4.4 The pairwise sequence alignment of DXR and AraD (epimerase) from E. coli based on the ClustaiW (1.82) program 127 4.5 The deoxy analogs of DXP 129 LIST OF TABLES TABLE PAGE 1.1 Recombinant enzymes of the MEP pathway 15 2.1 Sensitivity of different P.falciparum strains to fosmidomycin, 29 FR900098, chioroquine, and pyrimethamine 4.1 A comparison of E.coli DXR, KARl and the epimerase AraD 128 4.2 The results from the DXR assay of acetol (27) and glycolaldehyde phosphate (12, GALP) 135 LIST OF ABBREVIATIONS Ac Acetyl aq Aqueous BnBr Benzylbromide br Broad calcd Calculated c Concentration CI Chemical Ionization COSY Correlated Spectroscopy