A Dissertation entitled Ribonucleic Acids in Disease Etiology and Drug Discovery by Immaculate Sappy Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry ________________________________________ Amanda C. Bryant-Friedrich, Ph.D., Committee Chair ________________________________________ Zahoor A Shah, Ph.D., Committee Member ________________________________________ Steven M Peseckis, Ph.D., Committee Member ________________________________________ Caren Steinmiller, Ph.D., Committee Member ________________________________________ Amanda Bryant-Friedrich, PhD, Dean College of Graduate Studies The University of Toledo December 2019 Copyright 2019, Immaculate Sappy This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Ribonucleic Acids in Disease Etiology and Drug Discovery by Immaculate Sappy Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry The University of Toledo December 2019 Pseudouridine (Ψ), the 5-ribosyl isomer of uridine (U) is the most abundant nucleic acid modification found in all domains of life and all types of RNA. Studies have shown that, urinary levels of pseudouridine are higher in Alzheimer’s Disease (AD) patients and that RNA oxidation is a major component in the pathogenesis of Alzheimer’s Disease (AD) and other neurodegenerative disorders. Therefore, there is a potential correlation between higher urinary levels of pseudouridine in AD patients and oxidative stress. Hence, subjecting pseudouridine to oxidative conditions may provide some key information about the role of this nucleoside in RNA related processes and its role in disease etiology. Besides neurodegenerative disorders, antibiotic resistance is an additional threat to human health. Analogous to the development and use of nucleoside-analogue inhibitors (NAIs) of viral nucleotide polymerases for treatment of viruses, nucleotide analog inhibitors of bacterial RNA polymerase such as pseudouridimycin are also being investigated. These NAIs can limit bacterial resistance by mimicking the RNAP nucleoside triphosphate (NTP) binding site. iii To investigate these possibilities, the design, synthesis and characterization of pseudouridine analogs will be performed and these nucleosides will be evaluated for their antibacterial properties and their oxidative fate in RNA. iv I dedicate my dissertation to the spirit of my beloved dad. I also, dedicate this work to my family and most importantly to my loving and adored children for their patience and tolerance. A special dedication to my best friend turned husband for always being supportive, enduring, encouraging, loving and caring. Acknowledgements First, I am grateful to God Almighty for establishing me to complete this program. My sincere gratitude to Dr. Amanda C. Bryant-Friedrich, who believed in me when I did not believe in myself, invested in me and allowed me to enjoy working in her research group and for her valuable scientific and personal experience. Your continued guidance helped me to grow as an individual and also as an outstanding chemist. Thank you for your unceasing mentorship! You created so many opportunities for me and designed tools for me to grow not only as a woman but a mother and a scientist. I will be forever grateful. To my committee members Dr. Peseckis, Dr. Shah and Dr. Steinmiller thank you for your support and involvement. To Dr. Bedi, I am truly thankful for all your help and valuable advice. Thank you for walking with me spiritually during this Journey. To my lab members past and present, thank you for making the Amanda Bryant-Friedrich Lab home and maintaining the sense of family. To my Husband, thank you for being so patient with me. May God bless you. To my kids, not all heroes wear capes. Thank you for all your encouragement, reassurances and support. May God Almighty richly bless and reward you. To my family and friends, may God bless you for all your prayers and support. v Table of Contents ACKNOWLEDGEMENTS ............................................................................................ V LIST OF TABLES .......................................................................................................... IX LIST OF FIGURES ......................................................................................................... X LIST OF SCHEMES ................................................................................................... XIII LIST OF ABBREVIATIONS ..................................................................................... XIV LIST OF SYMBOLS ................................................................................................... XVI 1 INTRODUCTION ..................................................................................................... 1 1.1 OVERVIEW ............................................................................................................ 1 1.2 RNA DAMAGE IN DISEASE ETIOLOGY ........................................................................ 3 1.3 RIBONUCLEOSIDES IN DRUG DISCOVERY ................................................................... 4 2 BACKGROUND ....................................................................................................... 5 2.1 ROLE OF PSEUDOURIDINE IN DISEASE ETIOLOGY ....................................................... 5 2.2 RNA OCCURRENCE IN LIVING ORGANISMS ................................................................ 6 2.2.1 Bacteria ............................................................................................................... 6 2.2.2 Viruses ................................................................................................................ 7 2.2.3 Mammalian cells ................................................................................................ 8 2.3 FUNCTIONS ................................................................................................................. 8 2.4 MODIFIED RIBONUCLEIC ACIDS ................................................................................. 9 2.4.1 Pseudouridine Occurrence and Disease Relevance .......................................... 12 2.5. RNA OXIDATION IN HUMAN CELLS ........................................................................ 14 2.5.1 Oxidative Stress ................................................................................................ 14 2.5.2 Mechanisms of Oxidative Damage to RNA ..................................................... 17 2.5.3 Role of RNA Oxidation in Disease Processes .................................................. 19 3 SYNTHESIS OF A C5´-PSEUDOURIDINYLRADICAL PRECURSOR ....... 22 3.1 SYNTHESIS OF MODIFIED RIBONUCLEOSIDES AS C5´-RADICAL PRECURSORS .......... 22 3.3 PHOTOCHEMICAL GENERATION AND STUDY OF THE C5´-PSEUDOURIDINYL RADICAL. ....................................................................................................................................... 47 3.4 CONCLUSION AND FUTURE DIRECTION ..................................................................... 48 4 EXPERIMENTAL PROCEDURE SYNTHESIS OF C5´-RADICAL PRECURSOR 13 ............................................................................................................. 50 4.1 METHODS AND MATERIALS ...................................................................................... 50 Method ....................................................................................................................... 50 Material ...................................................................................................................... 50 4.2 SYNTHESIS OF PSEUDOURIDINE ................................................................................ 51 4.2.1 Synthesis of (3aR,6R,6aR)-6-(((tert-butyldiphenylsilyl) oxy)methyl)-2,2- dimethyldihydrofuro[3,4-d][1,3]dioxol-4(3aH)-on (62) ........................................... 51 vi 4.2.2 Synthesis of (3aR,6R,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-4-(2,4-di- tert-butoxypyrimidin-5-yl)- 2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol (80) 34 ................................................................................................................................. 53 4.2.3 Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-1-((4S,5R)-5-((R)-(2,4-di-tert- butoxypyrimidin-5-yl)(hydroxy)methyl)-2,2-dimethyl-1,3-dioxolan-4-yl)ethan-1-ol (81) 34 .......................................................................................................................... 54 4.2.4 Synthesis of 2,4-di-tert-butoxy-5-((3aS,4S,6R,6aR)-6-(((tert- butyldiphenylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4- yl)pyrimidine (82) 34 ................................................................................................... 55 4.3 SYNTHESIS OF C5´- PSEUDOURIDINYLRADICAL PRECURSOR .................................... 56 4.3.1 Synthesis of ((3aR,4R,6S,6aS)-6-(2,4-di-tert-butoxypyrimidin-5-yl)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol (60) 34 .............................. 56 4.3.2 Synthesis of (3aS,4S,6S,6aS)-6-(2,4-di-tert-butoxypyrimidin-5-yl)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbaldehyde (86) ............................. 57 4.3.3 Synthesis of 2-((tert-butyldimethylsilyl)oxy)-2-((3aR,4S,6S,6aS)-6-(2,4-di-tert- butoxypyrimidin-5-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-