SYNTHESIS AND CHARACTERIZATION OF C8 ANALOGS OF C-DI-GMP; NEW SYNTHETIC METHOD FOR 5’-CAPPED OLIGORIBONUCLEOTIDES by ELIZABETH VELIATH A Dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Chemistry written under the direction of Roger A. Jones and approved by ________________________ ________________________ ________________________ ________________________ New Brunswick, New Jersey January, 2011 ABSTRACT OF THE DISSERTATION Synthesis and Characterization of C8 Analogs of c-di-GMP; New Synthetic Method for 5’-Capped Oligoribonucleotides By ELIZABETH VELIATH Dissertation Director: Roger A. Jones This research is centered on two projects of biological importance composed of RNA –based compounds: C8 analogs of cyclic diguanosine monophosphate (c-di-GMP) and 5’-capped oligoribonucleotides. c-di-GMP is an important bacterial second messenger molecule that is critical for the transition between a biofilm-protected sessile state and a virulent, single cellular motile state in species like V. Cholerae and S.Typhimurium. The synthesis and solution phase structural analysis of a family of C8-modified analogs is described. Starting from unmodified c-di-GMP, elaboration at the C8 position of both guanine moieties of c-di- GMP resulted in the bromo, thio, methylthio, phenyl, and meta-acetylphenyl analogs. Biophysical studies of all five compounds was performed using 1D and (1H, 31P, 13C) and 2D (DOSY, HMBC/HMQC) NMR to ascertain the level of G-quadruplex formation. It was found that only c-di-Br-GMP as the K+ salt form adopts the formation of higher order complexes containing guanine quartet structures. All analogs have an NMR-visible amino resonance that is most prominent at low temperatures due to protection from exchange by the self-stacking, and that disappears at elevated ii temperatures, which does not occur in with the 8-bromo-GMP monomer illustrating the special structural characteristics of the symmetric dimer. Capped RNA has a unique 5’-end structure containing a terminal N7-methylated guanosine that is joined via a triphosphate bridge to the 5’-OH of all eukaryotic mRNAs. This structure plays a vital role in regulating RNA maturation, processing, transport and translation, but capped RNA is extremely difficult to synthesize chemically due to its instability. A new protection strategy was devised that uses a lipophilic dimethoxytrityl (DMT) group on the amino group of the N7-methylated guanine activated capping reagent. This allows efficient purification of the final capped RNA on reverse-phase HPLC. Additionally, the DMT group increases the capping reagent solubility in organic solvents which improves the final coupling step. The synthetic method is general to include a variety of mixed sequence oligonucleotides, and is compatible with reverse-phase HPLC. This method has been used to synthesize and purify the unmodified cap structure m7GpppG, the individual 7 7 diastereomers of the α-thiophosphate analog m Gppp(s)G, m GpppT8 and mixed 7 7 7 sequences of m Gppp-(2’-O-Me-GAUGC), m Gppp-(2’-O-Me-GAUGC)2, m Gppp-(2’- 7 O-Me-GUAUC)4 and m Gppp-(GUAUC)4. iii DEDICATION This work is dedicated to my husband Aaron, whose continued support, encouragment and understanding, has been absolutely invaluable to me throughout my graduate career; to my brother, Andrew, for his friendship and generosity; and to my parents, George Veliath and Ginger Cyr, for always believing in me. I also wish to extend my deepest thanks to my extended family and friends for their encouragement and support as well. iv ACKNOWLEDGEMENTS I am extremely grateful to my advisor, Professor Roger A. Jones, for his guidance, inspiration and continuous support, throughout my graduate research in the field of nucleic acid chemistry. I thank him for the countless conversations that have helped hone my skills in becoming a better scientist. I am extremely grateful to Professor Barbara L. Gaffney for many useful conversations pertaining to chemistry, for her daily role as a mentor and for her friendship and encouragment. I would like to thank the members of my thesis committee, Professor Jeehiun K. Lee, Professor Lawrence Williams from the Department of Chemistry at Rutgers and Professor Ed LaVoie from the Department of Pharmacy at Rutgers for their advice, time and helpful suggestions. I would like to thank Professor Kenneth Breslauer for help with spectrophotometric instrumentation; and Dr. Jens Volker for many useful conversations pertaining to spectroscopy of oligonucleotides. I would like to thank the past Jones group lab members, as well as the friends I have forged strong friendships with over the years at Rutgers, for making my graduate study a very enriching experience. v I am grateful for the significant financial support provided by the Rutgers Excellence Fellowship, the GAANN Fellowship, NIH and the Department of Chemistry and Chemical Biology at Rutgers, the State University of New Jersey. vi TABLE OF CONTENTS Abstract ..……………………………………………………………………………. ii Dedication ………………………………………………………………………….. iv Acknowledgements ………………………………………………………………….. v Table of Contents ………………………………………………………………….. viii List of Figures ………………………………………………………………………. xii List of Schemes ……………………………………………………………………. xiv List of Tables ……………………………………………………………………….. xvii List of Abbreviations …………………………………………………………...…. xviii Chapter 1 A Brief Overview of Synthetic Ribonucleoside Chemistry ………………………....1 References …………………………………………………………………………. 2 Chapter 2 Synthesis and Characterization of C8 Analogs of c-di-GMP ………………………3 1. Biological Background of c-di-GMP ………………………………………….. 3 1.1 c-di-GMP as Bacterial Second Messenger Signaling Molecule ………….. 3 1.2 Discovery of c-di-GMP …………………………………………………... 4 1.3 c-di-GMP Regulates Biofilm Production ………………………………… 4 1.4 c-di-GMP Regulates Virulence and Pathogenesis …………………………6 1.5 Enzymes that Synthesize and Degrade c-di-GMP …………………………8 1.6 c-di-GMP Target Receptors ………………………………………………..8 1.7 Temporal and Spatial Regulation of c-di-GMP Levels …………………...12 1.8 Effects of c-di-GMP on Mammalian Cells ………………………………. 14 vii 1.9 References ………………………………………………………………..15 2. Synthetic Background of c-di-GMP and Analogs ……………………………18 2.1 c-di-GMP Synthetic Introduction ………………………………………..18 2.2 Phosphotriester Chemistry ……………………………………………….19 2.3 H-Phosphonate Chemistry ……………………………………………….21 2.4 Combination of Phosphotriester, H-Phosphonate, and Phosphoramidite Chemistry ………………………………………………………………...23 2.5 Other Methods ……………………………………………………………28 2.6 References ………………………………………………………………..29 3. Synthesis of C8 Analogs of c-di-GMP …………………………….………….31 3.1 Introduction ……………………………………………………………....31 3.2 Synthesis of c-di-Br-GMP ………………………………………………..31 3.3 Synthesis of c-di-thio-GMP ………………………………………………34 3.4 Synthesis of c-di-methylthio-GMP ……………………………………….38 3.5 Synthesis of c-di-phenyl-GMP and c-di-acetylphenyl-GMP …….……….39 3.6 Conclusions ……………………………………………………….………45 3.7 Experimental Procedures …………………………………………………46 3.8 References ……………………………………………………….………..52 3.9 Appendix …………………………………………………………………54 4. Biophysical Studies of C8 Analogs of c-di-GMP …………………………….78 4.1 Introduction ………………………………………………………………78 4.2 Previous Work on the Biophysical Studies of c-di-GMP ………………..78 4.3 Biophysical Studies of c-di-Br-GMP Salt Forms ………………………..81 viii 4.3.1 NMR Studies of c-di-Br-GMP ………………………………..81 4.3.1.1 Introduction …………………………………………...81 4.3.1.2 K+ form of c-di-Br-GMP ……………………………...82 4.3.1.3 Na+ form of c-di-Br-GMP …………………………….86 4.3.1.4 Li+ form of c-di-Br-GMP ……………………………..90 4.3.1.5 TEA+ form of c-di-Br-GMP ……………………….….91 4.3.1.6 Salt Forms of 8-Br-GMP Monomer ……………….….93 4.3.1.7 Conclusions for NMR Studies of c-di-Br-GMP ….…..96 4.3.2 UV Studies of c-di-Br-GMP ……………………………….…96 4.3.2.1 UV Melting Study for K+ and Na+ Forms of c-di-Br-GMP ………………………………………….96 4.3.2.2 Results ………………………………………………..97 4.3.3 Further Investigation of NMR-Visible N2-Amino Resonance .98 4.3.3.1 Heteronuclear 2D NMR of K+ and Na+ Forms of c-di-Br-GMP …………………………………………99 4.3.3.2 Effect of pH on UV Profile of Na+ Form of c-di-Br-GMP ………………………………….……..101 4.4 Biophysical Studies of c-di-thio-GMP: K+, Na+, and TEA+ forms …..102 4.5 Biophysical Studies of c-di-methylthio-GMP: K+, Na+, and TEA+ forms ………………………………………………………105 4.6 Biophysical Studies of c-di-phenyl-GMP: K+, Na+, and TEA+ forms ..107 4.7 Biophysical Studies of c-di-acetylphenyl-GMP: K+ and Na+ forms ….110 4.8 NMR Re-examination of c-IMP-GMP: K+ form ……………………..112 ix 4.9 Conclusions ……………………………………………………………114 4.10 References ……………………………………………………………..116 Chapter 3 New Synthetic Method for 5’-Capped Oligoribonucleotides ……………………..118 1. Capped RNA Biological Background …………………………………………….118 1.1 Introduction ……………………………………………………………...118 1.2 Biological Function of Capped RNA ……………………………………118 1.3 Chemical Structure of Capped RNA …………………………………….119 1.4 Role of Capped RNA in Regulating RNA Decay ……………………….122 1.4.1 Deadenylation-Independent Decay Mechanisms ………………122 1.4.2 Deadenylation-Dependant Decay Mechanisms ………………..124 1.5 References ………………………………………………………………..126 2. Synthetic Background of Capped RNA ……………………………….………….129 2.1 Introduction ………………………………………………….…………..129 2.2 Enzymatic Methods ……………………………………………………..129 2.3 Combination of Enzymatic and Chemical Methods …………………….130 2.4 Chemical Methods ………………………………………………………132 2.5 References ……………………………………………………………….145 3. Synthesis of 5’-Capped Oligoribonucleotides