NOVEL CHEMISTRY AND CHEMICAL TOOLS FOR HYDROGEN SULFIDE RESEARCH By BO PENG A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Department of Chemistry MAY 2016 © Copyright by BO PENG, 2016 All Rights Reserved © Copyright by BO PENG, 2016 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of BO PENG find it satisfactory and recommend that it be accepted. Ming Xian, Ph.D., Chair Cliff Berkman, Ph.D. Jeff Jones, Ph.D. Rob Ronald, Ph. D. ii ACKNOWLEDGEMENT I would like to thank Dr. Ming Xian for supervising me during my Ph.D study and research at WSU. He provided guidance in person on both my experimental techniques and scientific writing. Dr. Xian led me to the research fields of hydrogen sulfide, from where I developed my research interest in bioorganic chemistry. I learned not only various research skills from him, but also critical thinking which is important for my entire research career. Dr. Xian also encouraged me to broaden my horizons in research fields by providing opportunities attending to conferences and symposiums. To my committee members, Dr. Cliff Berkman, Dr. Rob Ronald, and Dr. Jeff Jones, I will always be thankful for teaching me important core courses and giving all the suggestions and comments on my seminars, research and proposals. I would also like to thank Dr. Hector Aguilar-Carreno for his help in developing my cell culture skills and advising in the cell experiments I have done in his laboratory. I also want to thank Dr. Chulhee Kang for teaching me chemical biology and helping me with my grant applications. I would like to thank Dr. Phil Garner for teaching me synthetic methods. During my Ph.D. study, I received the assistance and support from every members of Dr. Xian’s group including Dr. Chunrong Liu, Dr. Chung-Min Park, Dr. Dehui Zhang, Dr. Pei Zhang, Dr. Nelmi Devarie, Dr. Jia Pan, Dr. Wei Chen Dr. Yu Zhao, Tyler Biggs, Armando Pacheco, Jianming Kang, Shi Xu, and Jake Day. iii I would like to thank Nikki Clark, Michael Finnegan, Scot Wherland, and Ryan Rice for their help in my teaching assistance work. I also want to thank Trent Amonett, Debbie Arrasmith, Lori Bruce, Stacie Olsen-Wilkes, Molly Spain, Yoshi Kodama and all the other staffs in the chemistry department who offered me help during these years. Most importantly, I want to thank my parents for encouraging me to study abroad from the beginning and for offering spiritual support whenever I need. I also want to thank my best friend, my beloved partner Tianjiao for her unconditional love, and support. Lastly, I would like to thank the Chemistry Department, Graduate School, and College of Science and Art at WSU for giving me the opportunity to study here and providing all the support these years. Also, I would like to thank all the research funding and scholarship I have received for their financial support. iv NOVEL CHEMISTRY AND CHEMICAL TOOLS FOR HYDROGEN SULFIDE RESEARCH Abstract by Bo Peng, Ph.D. Washington State University May 2016 Chair: Ming Xian Hydrogen sulfide (H2S) is a critical cell signaling molecule which has attracted attention recently for its contributions to human health and diseases. H2S has been considered as a cytoprotectant and gasotransmitter in many tissue types, including mediating vascular tone in blood vessels as well as neuromodulation in the brain. It is important, therefore, to understand the fundamental chemistry of H2S and to develop effective and convenient methods for H2S detection. The thesis will discuss three aspects of H2S: first, the development of fluorescent probes for the detection of H2S; second, the study on the generation of H2S from NADH (reduced nicotinamide adenine dinucleotide) model compounds and sulfane sulfurs; lastly, development of a new method for persulfide generation. Development of such chemical tools will help study the mechanisms by which H2S modulates signaling pathways and maintains cellular functions. v TABLE OF CONTENTS Page ACKNOWLEDMENT..………………………………..…….……………….iii ABSTRACT………………………………………………….………..……….v LIST OF TABLES...…………………………………………….………...…....x LIST OF FIGURES...…………………………………………..…………......xi LIST OF SCHEMES………………………………………………………...xiii ABBREVIATIONS………………...…………………………………………xv CHAPTER 1. INTRODUCTION…………………..…………………………..1 1.1 HYDROGEN SULFIDE…………………………………………...........1 1.2 TRADITIONAL DETECTION OF HYDROGEN SULFIDE…...….….3 1.3 FLUORESCENT PROBES FOR HYDROGEN SULFIDE….......……..4 1.3.1 REDUCTION-BASED H2S FLUORESCENT PROBES………...4 1.3.2 NUCLEOPHILIC REACTION-BASED H2S FLUORESCENT PROBES………………………………………………………………6 1.3.3 METAL SULFIDE FORMATION-BASED H2S FLUORESCENT PROBES………………………………………….……………………..9 1.4 ENDOGENOUS GENERATION OF H2S…………………..………….9 1.5 CHEMISTRY OF S-SULFHYDRATION………………..……………11 1.6 REFERENCES…………………..…………………………………….14 CHAPTER 2. DISULFIDE-BASED FLUORESCENT PROBES FOR HYDROGEN SULFIDE……………………………………………………….26 vi 2.1 ABSTRACT……………………………………………………….…...26 2.2 DESIGN AND SYNTHESIS OF PROBES…..………………………..26 2.3 FLUORESCENCE PROPERTIES AND RESPONSES OF PROBES WSP1-5 TO H2S………………………………..………………………….29 2.4 LIVING CELL IMAGING STUDIES………..……………………..…39 2.5 CONCLUSION…………………………………………..…………….42 2.6 EXPERIMENTAL SECTION…………………………………………42 2.6.1 SYNTHESIS……………………………………………..………43 2.6.2 FLUORESCENCE ANALYSIS…………………….…………...47 2.7 REFERENCE……….…………………………………………..…....52 CHAPTER 3. DISELENIDE-BASED FLUORESCENT PROBES FOR HYDROGEN SULFIDE……………………………………………………..54 3.1 ABSTRACT………………..………………………………………..…54 3.2 DESIGN AND SYNTHESIS……………………………………..…54 3.3 FLUORESCENCE ANALYSIS……………………………………….58 3.4 CONCLUSION……………………………….………………………..63 3.5 EXPERIMENTAL SECTION………………………………………....64 3.5.1 CHEMICAL SYNTHESIS…………………………………........64 3.5.2 MODEL REACTIONS AND STABILITY STUDIES………......67 3.5.3 FLUORESCENCE ANALYSIS…………………………………68 3.6 REFERENCES………………………………….…………………..…71 vii CHAPTER 4. STUDY OF THE REACTIONS BETWEEN SULFANE SULFURS AND NAD(P)H AS A NON-ENZYMATIC H2S GENERATION PATHWAY...........................................................................................................73 4.1 ABSTRACT…………………………………………………………....73 4.2 REACTIONS BETWEEN NADH MODEL COMPOUNDS AND SULFANE SULFUR....................................................................................73 4.3 DETECTION OF H2S GENERATION…………..……………………78 4.4 CONCLUSION………………………………………………………...80 4.5 EXPERIMENTAL SECTION………………………………………....81 4.5.1 CHEMICAL SYNTHESIS…………...………………………….81 4.5.2 REACTIONS BETWEEN NADH MODEL COMPOUNDS...…84 4.5.3 DETECTION OF H2S GENERATION……...…………………..84 4.6 REFERENCES……………………………………………………...…86 CHAPTER 5. DEVELOPMENT OF A NEW METHOD FOR PERSULFIDE GENERATION…………………………….....................................................88 5.1 ABSTRACT……………………………………………………………88 5.2 DESIGN OF THE NEW METHOD…………………………………...88 5.3 TEST THE NEW STRATEGY WITH THIOLS……………………....89 5.4 CONCLUSION………………………………………………………...92 5.5 EXPERIMENTAL SECTION……………………...………………….93 5.6 REFERENCES……………………………………………….………..95 viii APPENDIX……..............................................................................................96 ix LIST OF TABLES Table 2.1 Fluorescent properties of probes WSP1-5………………………....30 Table 2.2 Turn-on fold changes and detection limits of probes WSP1-5…….33 Table 3.1 Fluorescent properties of SeP probes……………………………58 x LIST OF FIGURES Figure 2.1 Time-dependent fluorescence changes of WSP1……….................31 Figure 2.2 Time-dependent fluorescence changes of WSP probes…………....32 Figure 2.3. Fluorescence spectra changes of WSP probes……...…..……….33 Figure 2.4 Fluorescence emission spectra of WSP probes with varied concentrations of NaHS……………………………………………..…………34 Figure 2.5 Fluorescence intensity changes of WSP probes at different pH………………………………………………………………………………36 Figure 2.6 Fluorescence intensity of WSP probes in the presence of various reactive sulfur species……………………………….…………………...…….37 Figure 2.7 Fluorescence intensity of WSP probes with esterase…...……….39 Figure 2.8 Fluorescence images of H2S in HeLa cells.......................................40 Figure 2.9 Fluorescence images of H2S production from a H2S donor...........42 Figure 3.1 Time-dependent fluorescence changes of SeP probes……………..59 Figure 3.2 Fluorescence emission spectra of SeP2 in the presence of varied concentrations of Na 2 S……………….……...……………………….60 Figure 3.3 Fluorescence intensity of SeP probes in the presence of various reactive sulfur species………………………………………………………….61 Figure 3.4 Fluorescence images of H2S in HeLa cells using SeP2………..….62 Figure 3.5 Fluorescent detection of in-situ generated H 2 S in human neuroblastoma cell……………………………………………………………63 xi Figure 4.1 UV absorption spectra of methylene blue which indicate the production of H2S……………………………………………………………..80 Figure 4.2 Fluorescence spectra of the samples which indicate the production of H2S……………………………………………………………………………..80 xii LIST OF SCHEMES Scheme 1.1 Structures and fluorescence turn-on mechanisms of SF1, SF2, and DNS-Az……………………………………………………………...………….6 Scheme 1.2 Structures and fluorescence turn-on mechanisms of WSP1, SFP-1 and SFP-2…………………………………………………………….…………8 Scheme 1.3 Structure and mechanism of HSip-1…………….…………………9 Scheme 2.1 General design of the nucleophilic substitution/cyclization based fluorescent probes……………………………………………………...………27 Scheme 2.2 Model reaction between compound 2.1 and H2S………….……...28 Scheme 2.3 Preparation of fluorescent probes WSP1-5……………………….29 Scheme 3.1 Design of diselenide-based probes