Monitoring Oxidation Reactions by Photochemical Techniques by Hamdy Saad Sadek El–Sheshtawy A thesis submitted in partial fulfillment for the degree of Doctor of Philosophy in Chemistry Approved Thesis Committee: Prof. Dr. Werner M. Nau Jacobs University Bremen, Bremen Prof. Dr. Nikolai Kuhnert Jacobs University Bremen, Bremen Dr. Aurica Farcas “Petru Poni” Institute of Macromolecular Date of defense: November 7, 2011 Chemistry, Romania School of Engineering and Science Declaration of Authorship I, Hamdy El–Sheshtawy, declare that this thesis entitled, “ Monitoring Oxidation Reactions by Photochemical Techniques" and the work presented in it are my own. I confirm that: • This work was done wholly or mainly while in candidature for a doctoral degree in chemistry at Jacobs University. • Where I have consulted the published work of others, this is always clearly attributed. • Where I have quoted from the work of others, the source is always given with the exception of such quotations; this thesis is entirely my own work. • I have acknowledged all main sources of help. • Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself. Signed: Date: ii Yesterday we obeyed kings and bent our necks before emperors. But today we kneel only to truth, follow only beauty, and obey only love. Khalil Gibran iii To my Parents iv Abstract PhD Thesis by Hamdy Saad El–Sheshtawy This doctoral thesis is devoted to monitor oxidation processes by photochemical techniques. Oxidation reactions play a crucial role in both environmental and biological systems. Therefore, my thesis started with the study of oxygen scavenging of aryl- substituted benzyl radicals. In this part of my work, a combination of experimental techniques (laser-flash photolysis) and theoretical calculations (DFT) was used to elucidate the mechanism of an elementary step in an oxidation process. The effect of substituents on the scavenging rate constants was studied in detail. Solvent effects on the reaction progress, as well as temperature effects were investigated. For the first time, a negative activation energy was observed: the scavenging rate constants of aryl–benzyl radicals in acetonitrile decreased at higher temperature. In the second part of my thesis, the effect of the solvent on H atom abstractions from C–H donors by oxygen-centered radicals (the cumoxyl radical) and n–π* excited molecules (2,3-diazabicyclo[2.2.2]oct–2–ene) was investigated. For this, theoretical calculations and experimental results were again combined to rationalize the solvent effects on the H atom abstraction from several C–H donors by the oxygen–centered radicals. The results are compared to those previously reported in the literature for H atom abstraction from O–H donors. In the last part, I developed a fluorescence-based supramolecular tandem assay to monitor a biologically important oxidation process, namely an enzymatic oxidation. When searching for a macrocyclic receptor (required for tandem assays) for the substrate of peroxidases, namely iodide, I found a novel interaction (halogen bonding) in v supramolecular host–guest complexes associated with cucurbit[n]urils (CBs), which is based on the encapsulation of molecular iodine (I2) inside the nanocavity of CB6. Halogen bonds have been found to play a crucial role in the complex stability in the case of CB6•I2, while for the CB7•I2 association complex the hydrophobic effect is dominant. Based on the encapsulation of I2 into the CB7 cavity, we developed a supramolecular tandem assay for monitoring the activity of peroxidases, namely myeloperoxidase (MPO), lactoperoxidase (LPO), and horseradish peroxidase (HRP). This assay has the advantage of using a small inorganic substrate (I–), in contrast to the previously employed organic compounds. In addition, the supramolecular tandem assay requires only µM concentrations of the substrate (I–), such that it overcomes the use of high substrate concentration (needed for other methods), which is known to adversely affect (inhibit) peroxidase activity. vi Acknowledgments I would like to thankfully acknowledge the enormous support, encouragement and guidance of my supervisor, Prof. Dr. Werner M. Nau, who provided me with a diverse playground of projects to develop upon. I want to thank Prof. Dr. Nikolai Kuhnert and Dr. Aurica Farcas for being kind enough to be committee members for my PhD thesis. I would also like to thank all members of the Nau research group for providing a stimulating environment, both socially as well as professionally. In particular, I thank Dr. Apurba L. Koner, Indrajit Ghosh for their fruitful discussions regards the wide variety of subjects and instruments in the laboratory. Special thanks go to Dr. Bassem Bassil for solving extremely complex supramolecular structures and being patient until good results were obtained. I want to also thank Dr. Vanya Uzunova for the ITC measurements, and Dr. Patrice Donfack for Raman spectroscopy measurements. Also I would like to extend my deepest thankful to Dr. Xiangyang Zhang for the preparation of the substituted dibenzylketones, which have been used during this study. I acknowledge the kind and generous financial support of the Egyptian Government, DFG, and NanoFun center at Jacobs University. Finally, to my wife, and our children Ahmed and Ayah, all I can say that I want to express my deep love for you. Your patience, love and encouragement have upheld me, particularly in those many days in which I spent more time with my lab than with you. vii Contents Abstract v Acknowledgements vii List of Figures x List of Tables xii 1 Introduction 2 1.1 Scope of the Thesis and Overview…………………………………………… 2 1.2 Environmental Oxidation Reactions ………………………….……………… 5 1.2.1 Oxidation Reactions in Combustion Chemistry……….……………….. 5 1.2.2 Oxidation Reactions in Atmospheric Chemistry……………………….. 7 1.2.3 The Benzyl Radical as a Model for Combustion and Atmospheric Radical Reactions………………………………………………………. 9 1.3 Oxidation Reactions in Biological Systems………………………………….. 11 1.3.1 An Overview…………………………………………………………… 11 1.3.2 Mechanism of Compound I Formation in Heme Peroxidases…………... 12 1.3.3 Halide Oxidation by Heme Peroxidases………………………………... 13 1.3.4 Defense Mechanisms Based on Halogen Oxidation…………………… 14 References…………………………………………………………………… 16 2 Oxygen Scavenging of Aryl–Substituted Benzyl Radicals 20 2.1 Introduction…………………………………………………………………... 20 2.2 Oxygen Scavenging Rate Constants…………………………………............. 22 2.3 Solvent Effect on the Scavenging Rate Constants…………………………… 28 2.4 Temperature Effect on the Scavenging Rate Constants……………………… 30 References………………………...………………………………….............. 34 viii 3 Solvent Polarity Affects H–Atom Abstractions from C–H Donors 36 3.1 Introduction…………………………………………………………………… 36 3.1.1 Intramolecular H–Atom Transfer (Isomerization)……………….......... 37 3.1.2 Solvent Effect on Intermolecular H–Atom Transfer…………………… 37 References…………………………………………………………………… 46 4 Catalytic Oxidation of Iodide by Peroxidases: A Supramolecular Tandem Assay for Monitoring Peroxidase Activity 49 4.1 Supramolecular Host–Guest Assemblies……………………………………… 49 4.2 Driving Forces for Host–Guest Interactions…………………………………... 49 4.2.1 Hydrogen Bond………………………………………………………….. 49 4.2.2 Halogen Bond………………………….………………………………... 50 4.3 Cucurbiturils…………………………………………………………………… 52 4.4 Iodine by the Barrel–On the Macrocyclic Recognition of an Element………... 55 4.4.1 Molecular Iodine Binding with Cucurbit[n]urils……………………….. 55 4.4.2 Halogen Bond in CB6•I2 Crystal Structure…………………………….. 59 4.4.3 Iodine as Solvatochromic Probe for Polarizability Inside Cucurbit[n]uril Cavity……………………………………………............. 61 4.4.4 Raman Spectroscopy of Iodine Encapsulated Inside Cucurbit[n]urils Cavity…………………………………………………... 66 4.5 Supramolecular Tandem Assays for Monitoring Peroxidase Activity……… 72 4.5.1 Supramolecular Tandem Assay……………………………………….. 72 4.5.2 Monitoring Heme Peroxidases Activity by Supramolecular Tandem Assays……………………………………………………….. 73 4.5.3 Peroxidase Inhibition by Melatonin…………………………………… 77 4.6 Storage of CBs•I2 in Gel Matrix…………………………………….............. 79 References…………………………………………………………………… 81 5 Appendices 85 Appendix I …………………………………………………………………….. 86 Appendix II …………………………………………………………………… 101 Appendix III……………………………………………………………………. 123 Appendix IV …………………………………………………………………… 164 ix List of Figures: 1.1 Mono–and–di substituted benzyl–type triplet diradicals used for the ΔD scale. 10 2.1 Transient absorption decay traces observed for benzyl radical………………... 23 2.2 Optimized transition state geometries of the substituted benzyl radicals ……... 25 2.3 Hammet relationship for the aryl–substituted benzyl radicals in n–hexane….. 27 2.4 Transient decay traces of benzyl radical in acetonitrile and in n–hexane……… 28 2.5 Transient absorption decay traces of benzyl radical at different temperatures… 30 2.6 Potential energy profile for unsubstituted benzyl radical………………………. 33 3.1 Transient absorption decays of cumyloxyl radicals in aerated CH3CN, EtOAc/CH3CN (50/50), and in EtOAc in the presence of hexamethylbenzene... 41 3.2 Fluorescence lifetime decays of DBO in deaerated CH3CN, EtOAc/CH3CN (50/50), and in EtOAc in the presence of 1,3–cyclohexadiene…………………. 41 3.3 Plots of log k versus the composition of a binary EtOAc/CH3CN mixture…….. 3.4 Optimized transition–state structures and solvation energies of the cumyloxyl radical and tert–butoxyl radical………………………………………………… 44 4.1 UV-Vis titrations of iodine