PHOTOAFFINITY LABELING VIA NITRENIUM ION CHEMISTRY: THE PHOTOCHEMISTRY OF 4-AMINOPHENYLAZIDES. Valentyna Voskresenska A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2011 Committee: Dr. R. Marshall Wilson, Advisor Dr. Zhaohui Xu, Graduate Faculty Representative Dr. Michael Y. Ogawa Dr. Thomas H. Kinstle ii ABSTRACT Dr. R. Marshall Wilson, Advisor Phenyl azides with powerful electron-donating substituents are known to deviate from the usual photochemical behavior of other phenyl azides. They do not undergo ring expansion, but form basic nitrenes that protonate to form nitrenium ions. The photochemistry of the widely used photoaffinity labeling system 4-amino-3-nitrophenyl azide, has been studied by transient absorption spectroscopy from femtosecond to microsecond time domains and from a theoretical perspective. The nitrene generation from 4-amino-3-nitrophenyl azide occurs on the S2 surface, in violation of Kasha’s rule. The resulting nitrene is a powerful base and abstracts protons extremely rapidly from a variety of sources to form a nitrenium ion. In methanol, this protonation occurs in about 5 ps, which is the fastest intermolecular protonation observed to date. Suitable proton sources include alcohols, amine salts, and even acidic C-H bonds such as acetonitrile. The resulting nitrenium ion is stabilized by the electron-donating 4-amino group to afford a diiminoquinone-like species that collapses relatively slowly to form the ultimate cross- linked product. In some cases in which the anion is a good hydride donor, cross-linking is replaced by reduction of the nitrenium ion to the corresponding amine. However, the efficiency of 4-amino-3-nitrophenyl azide in generating the reactive nitrenium is impaired by associated photochemistry of the nitro group and a tendency for the initially formed nitrene to undergo reduction to the corresponding amine. We have examined possible alternative molecules that might yield reactive nitrenium ion more efficiently, and thus, provide more effective photoaffinity labeling agents. The 2-(N,N-diethylamino)-5-azidopyridine has been investigated as this regard and found to offer substantial advantages over 4-amino-3- iii nitrophenyl azide as a photoaffinity agent. Ultrafast transient spectroscopy confirms that 2-(N,N- diethylamino)-5-azidopyridine proceed via a reactive nitrenium ion species, and the same reactive species is formed both photochemically and thermally at relatively low temperatures. The coupling reactions of 2-(N,N-diethylamino)-5-azidopyridine proceed more rapidly than those of 4-amino-3-nitrophenyl azide in reactions with fewer side products, including reduction to the amino analogs. In addition, 2-(N,N-diethylamino)-5-azidopyridine displays fluorescence that ceases upon conversion to products, a property that might offer distinct advantages in photoaffinity labeling studies in complex biological systems. iv ACKNOWLEDGMENTS There are so very many great people to whom I owe my recognition and gratitude which has accumulated during my years of graduate study. I would like to start by thanking my adviser, Professor R. Marshall Wilson. Dr. Wilson, this work could not have been accomplished without your intellectual support and thoughtful guidance. I wish to thank you for your enthusiasm, encouragement and concern during my graduate career. I want to express my gratitude to our collaborator, Professor Alexander N. Tarnovsky for sharing his knowledge the course of discussions that proved highly useful. I thank Maxim Panov for beautiful ultrafast spectroscopic studies and calculation studies. I also thank Mike Ryazancev for help in calculation studies and useful discussions. Working on those projects together was a pleasure for me. I also would like to thank our collaborators from the Ohio State University Dr. C. M. Hadad, S. Vyas and A. H. Winter. I am very thankful to my MS thesis advisor and current committee member Professor T. H. Kinstle, who had the great impact on my scientific career. I also wish to thank Professor M. Ogawa, committee member during my MS and PhD programs, for his compassion and time. I thank all members of our group, Denis Nilov, Dmitry Komarov and Alexei Shamaev for creating friendly and inspiring atmosphere in the lab. I would also like to express my appreciation to Professor Castellano for opportunity to use instrumentation in his laboratories; Dr. Jedrzej Romanowicz, and Dr. D. Y.Chen for their help with mass spectrometry and NMR experiments. My special thank-you goes to my dear friends, especially, to Elena and Vadim Glik, Anna and Andrey Fedorov, Kate and Alex Mejeritsky, for their constant support, encouragement v and our friendship. My beloved parents and sister deserve undying gratitude for being very supportive and unconditionally loving. And finally, I want to thank my husband, Sergey, and my lovely daughter, Anastasia, who fill my life with joy and happiness. vi TABLE OF CONTENTS Page CHAPTER I. BACKGROUND ............................................................................................. 1 1.1 Photoaffinity Labeling ......................................................................................... 1 1.2 Laser Thechniques in the Study of Photoaffinity Labeling ................................. 6 1.3 Photochemistry of Aryl Azides ............................................................................ 9 Fluorosubstituted Azides ............................................................................... 14 1.4 Photochemical Formation and Reactions of Nitrenium Ions ............................... 16 1.5 References ............................................................................................................ 23 CHAPTER II. EXPERIMENTAL METHODS ..................................................................... 30 2.1 UV-VIS Absorption Spectroscopy ...................................................................... 30 2.2 Steady-State Fluorescence Spectroscopy ............................................................. 30 2.3 Nanosecond UV-VIS Time Resolved Absorption Spectroscopy ........................ 31 2.4 Femtosecond UV-VIS Time Resolved Absorption Spectroscopy ....................... 33 2.5 Computational Details ......................................................................................... 36 2.6 X-Ray Crystallographic Structure Determination of 31 ...................................... 36 2.7 References ............................................................................................................ 37 CHAPTER III. THE PHOTOCHEMISTRY OF 4-(N,N-DIETHYLAMINO)-3-NITROPHENYL AZIDE: PROTONATION OF THE NITRENE TO AFFORD REACTIVE NITRENIUM ION PAIRS EXPERIMENTAL METHODS ................................................................................ 41 3.1 Introduction .......................................................................................................... 41 3.2 Synthesis of 4-(N,N-Diethylamino)-3-nitrophenyl Azide and Study of its Photolysis Products. ............................................................................................................ 44 Alcohols as Nucleophiles ............................................................................... 49 vii Phenols as Nucleophiles ................................................................................ 50 Dimethylamine Hydrochloride as Nucleophile ............................................. 50 3.3 Photophysical Studies .......................................................................................... 55 UV-VIS Steady State ..................................................................................... 55 Ultrafast transient Absorption and Nanosecond LPF Spectra of Azide 25 in 2-Propanol ......................................................................................................... 56 Reactivity of Azide 25 in the Presence of Different Solvents ....................... 65 3.4 Discussion ............................................................................................................ 76 Theoretical Considerations ............................................................................ 76 Nitrene Electronic Configuration ................................................................... 78 3.5 Conclusions .......................................................................................................... 98 3.6 References ............................................................................................................ 99 CHAPTER IV. AN EFFECTIVE NITRENIUM ION PRECURSOR FOR PHOTOAFFINITY LABELING: 2-(N,N-DIALKYLAMINO)-5-AZIDOPYRIDINE ........................................ 105 4.1 Introduction .......................................................................................................... 105 4.2 Synthesis of 5-Azido-2-(N,N-diethylamino)pyridine (65) and Studies of the Products of the Photolysis .......................................................................................... 109 4.3 Photophysical Studies .......................................................................................... 114 Ground State Absorption ............................................................................... 114 Fluorescence Studies of 5-Azido-2-(N,N-diethylamino)pyridine 65 ............. 116 Ultrafast Transient Absorption Spectra of Azide in Protic Solvents ............. 120 Nanosecond Laser Flash Photolysis