TIME-RESOLVED SPECTROSCOPIC STUDIES OF THE PHOTOCHEMISTRY OF RIBOFLAVIN, AROMATIC N-OXIDES AND THE ABSOLUTE REACTIVITY OF HYDROXYL RADICAL DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaofeng Shi, M.S. ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Matthew S. Platz, Advisor Professor Christopher M. Hadad _________________________________ Professor Dennis E. T. Bong Advisor Graduate Program in Chemistry Professor Thomas R. Lemberger ABSTRACT Both nanosecond and ultrafast laser flash photolysis with UV-visible and infrared detection were used to observe the transient species generated photochemically from a number of photosensitizers. The reactions of these transient species were monitored spectroscopically with the aid of theoretical computation. In the study of photochemical reactions of riboflavin and nucleosides, it was found that triplet riboflavin can be quenched by a silylated guanosine derivative with a rate constant of 1.0 × 108 M-1 s-1. TRIR spectroscopy demonstrated that a hydroflavin radical is formed by an electron transfer-proton transfer mechanism. This sequential electron transfer-proton transfer between triplet riboflavin and guanosine derivative provides the direct observation of the photoinduced oxidative damage of riboflavin to the DNA nucleobase. The triplet states of isoquinoline N-oxide and benzocinnoline N-oxide react sluggishly with electron, proton and hydrogen atom donors. These triplets will react with hydroquinone by hydrogen atom transfer (proton coupled electron transfer). Triplet 4- nitroquinoline N-oxide reacts readily with electron donors to from the radical anions as previously reported. The radical anion is protonated on the oxygen atom of the N-oxide ii group to from a neutral radical. The three N-oxides of this study are not expected to serve as photochemical sources of hydroxyl radical. Singlet states of tirapazamine and desoxytirapazamine were identified by picosecond time-resolved absorption spectroscopy. The lifetimes of the S1 states and fluorescence quantum yields of aromatic N-oxides were found to be controlled by reversible cyclization to an oxaziridine. The S1 states of TPZ and dTPZ are reduced to radical anions by KSCN, KI and NaN3. Using LFP-based methodology, we have determined the rate coefficients for the reaction of hydroxyl radical with a number of monocyclic and polycyclic aromatic hydrocarbons in acetonitrile. We observed the reactivities of hydroxyl radical in acetonitrile. For simple aromatic hydrocarbons, the predominant reaction pathway in acetonitrile is the addition of the hydroxyl radical to the aromatic ring, rather than hydrogen-atom abstraction from the phenyl or benzylic C-H positions. Structure- reactivity analysis, based upon frontier molecular orbital and state correlation models indicate that charge-transfer interactions between hydroxyl radical and a given arene play an important role in the stabilization of the transition state for the reaction α-Alkoxy and hydroxy radicals were generated through thermal and photochemical reactions. Both the product analysis of the thermal reaction and the direct observation of the transient species involved suggest that α-hydroxy radical can very efficiently react with TPZ to form the TPZ-H radical, probably through a direct hydrogen atom exchange between TPZ and ketyl radicals. α-Alkoxy radicals can not proceed through this mechanism. iii Dedicated to My Family iv ACKNOWLEDGMENTS First of all, I would like to thank my advisor, Professor Matthew Platz, for his guidance, support and encouragement. He has always been inspiring and thoughtful in science, patient and progressive as a mentor, as well as caring and considerate as a friend. He has been truly a role-model for me, in many different aspects. I want to thank Professor Christopher Hadad for teaching me computational chemistry techniques. He has always been available to answer my questions inside and outside science. I am also grateful to the professors and staff members in this department who helped me in various ways. Many of the research projects in this dissertation are collaborations with current and formal members in the Platz and Hadad group, from which I learned team work and effective communication. I worked with Drs. Christopher Martin and Meng-Lin Tsao in the riboflavin project, Dr. James Poole and Matthew DeMatteo in the hydroxyl radical project, Drs. Jin Liu and James Poole in the tirapazamine project. The Ohio Laboratory of Kinetic Spectrometry in Bowling Green State University and The Center for Chemical and Biophysical Dynamics provided ultrafast laser spectroscopic instruments for this work. I would like to thank Professor Michael A. J. Rodgers and Dr. Alex Gusev in BGSU, Professor Terry Gustafson and Dr. Gotard Burdzinski of CCBD at OSU, and v Professor Burda Clemens in Case Western Reserve University for their help in the ultrafast spectroscopic measurements. I also thank Ohio Supercomputer Center for providing the resources of my computational work. Without the help and hands-on teaching of the members in the Platz group from the very first day, this work would not have been possible. I thank all of them, especially Dr. Meng-Lin Tsao, for teaching every thing about instrumentation and sharing experimental skills with me. I have experienced friendship and family-like warmth from the people in the Platz group. This has been great five years and will always be in the best of my memory. Finally, I want to thank my family: my wife and my parents, for their utmost love, concern, understanding and support. They are the source of my power for every step. vi VITA September 13, 1975. Born – Zhangjiagang, Jiangsu Province, China 1997. B.S., Chemistry, Nanjing University, China 2000. .. .M.S., Chemistry, Nanjing University, China 2000- 2005. Graduate Teaching and Research Associate The Ohio State University PUBLICATIONS Research Publications 1. DeMatteo, Matthew P.; Poole, James S.; Shi, Xiaofeng; Sachdeva, Rakesh; Hatcher, Patrick G.; Hadad, Christopher M.; Platz, Matthew S., “On the Electrophilicity of Hydroxyl Radical: A Laser Flash Photolysis and Computational Study”, J. Am. Chem. Soc. 2005, 127, 7094-7109 2. Poole, James S.; Shi, Xiaofeng,; Hadad, Christopher M.; Platz, Matthew S., “Reaction of Hydroxyl Radical with Aromatic Hydrocarbons in Non-Aqueous Solutions – a Laser Flash Photolysis Study in Acetonitrile”, J.Phy. Chem. A 2005 109, 2547-2554 3. Shi, Xiaofeng, Poole, James S.; Emenike, Ejeoma; Burdzinski, Gotard; Platz, Matthew S., “Time Resolved Spectroscopy of the Excited Singlet States of Tirapazamine and Desoxytirapazamine”, J. Phy. Chem. A, 2005 109, 1491-1496 4. Shi, Xiaofeng, Platz, Matthew S., “Time Resolved Spectroscopy of Some Aromatic N-Oxide Triplets, Radical Anions and Related Radicals”, J. Phys. Chem. B 2004, 108, 4385-4391 vii 5. Martin, Christopher B.; Shi, Xiaofeng; Tsao, Meng-Lin; Karweik, Dale; Brooke, James; Hadad, Christopher M.; Platz, Matthew S., “The Photochemistry of Riboflavin Tetraacetate and Nucleosides. A Study Using Density Functional Theory, Laser Flash Photolysis, Fluorescence, UV-Vis, and Time Resolved Infrared Spectroscopy”, J. Phy. Chem. B 2002, 106, 10263-10271 6. Wan, Shuang-Yi; Shi, Xiao-Feng; Xia, Jiang; Sun, Wei-Yin, “Studies on the Hydrolysis of p-Nitrophenyl Acetate Catalyzed by Zinc(II) Complexes with an S2N2 Binding Set”, Main Group Metal Chemistry 2001, 24, 107-110 7. Shi, Xiaofeng; Sun, Weiyin; Zhang, Li; Li, Chongde, “NH-S Hydrogen Bonding in Zinc Enzyme Model Complex with S2N2 Binding Set Studied by Normal Coordinate Analysis of Vibrational Spectra”, Spectrochimica Acta A 2000, 56A(3), 603-613 8. Sun, Wei-Yin; Shi, Xiao-Feng; Zhang, Li; Hu, Jun; Wei, Jin-Hua, “Aromatic C-H-- -S Interaction in the Arenethiolate Complexes of Cadmium(II) with S2N2 Donor Set Evidenced from 113Cd NMR Spectroscopy”, J. Inorg. Biochem. 1999, 76, 259-263 FIELDS OF STUDY Major Field: Chemistry viii TABLE OF CONTENTS Page Abstract. .ii Dedication. .iv Acknowledgments . .v Vita . .vii List of Tables. xii List of Figures . xiii Chapters 1. Introduction……………………………………………………………………….1 1.1 Photosensitization…………………………………………………………….1 1.2 Reaction Oxygen Species and Hydroxyl Radical…………………………….7 1.3 Riboflavin…………………………………………………………………….9 1.4 Tirapazamine………………………………………………………………..11 1.5 Organization of This Dissertaton…………………………………………...13 2. General Experimental and Computational Methods and Protocols……………..17 2.1 Experimental Details………………………………………………………..17 2.1.1 Nanosecond LFP System with UV-vis Detection…………………..17 2.1.2 Nano-second Time Resolved Infrared Spectroscopy……………….18 2.1.3 Ultrafast Transient UV-Vis Spectroscopy………………………….19 2.1.4 Fluorescence Measurement…………………………………………20 2.2 Computational Details………………………………………………………22 ix 3. Photochemical Reaction of Riboflavin Tetraacetate and Nucleosides………..…23 3.1 Introduction……………………………………………………..…………..23 3.2 Instrumental…………………………………………………………………26 3.3 Results and Discussion……………………………………………………...29 3.4 Conclusion…………………………………………………………………..42 4 Transient UV-vis and Time Resolved Infrared Studies of Some Triplet Aromatic N-oxides …………………………………………………………………………44 4.1 Introduction…………………………………………………………………44 4.2 Experimental………………………………………………………………..49 4.3 Results and Discussion……………………………………………………...50 4.3.1 Isoquinoline N-oxide………………………………………………..50 4.3.2 Benzo[c]cinnoline N-oxide…………………………………………54 4.3.2 4-nitroquinoline N-oxide……………………………………………56