Synthesis, Kinetics and Mechanisms of Designer and Natural Product Antioxidants: From Solution to Cells By Bo Li Thesis submitted to the Department of Chemistry and Biomolecular Sciences in conformity with the requirements for the Degree of Doctor of Philosophy in Chemistry University of Ottawa Ottawa, Ontario, Canada (March 2016) © Bo Li, Ottawa, Canada, 2016 Abstract Lipid peroxidation has been implicated in the onset and progression of many degenerative diseases, including cardiovascular disease, Alzheimer’s disease and cancer. Accordingly, for more than 50 years, considerable effort has been devoted to the design of synthetic compounds or the discovery of natural products that can slow lipid peroxidation. Despite the enormous investments made to date, no clear antioxidant strategies have emerged for the treatment and/or prevention of degenerative disease. We argue that this is because of a lack of fundamental understanding of the chemical reactivity of these compounds in relevant contexts. Herein, we describe studies of our optimized synthetic radical-trapping antioxidant (RTA) – the tetrahydronaphthyridinols (THNs). We first present the synthesis of a series of THN analogs of α-tocopherol (Nature’s premier lipid-soluble radical-trapping antioxidant) with varying sidechain substitution and then demonstrate how systematic changes in the lipophilicity of these potent antioxidants impact their peroxyl radical-trapping activities in lipid bilayers and mammalian cell culture. Their regenerability by water-soluble reductants in lipid bilayers, binding to human tocopherol transport protein (hTTP), and cytotoxicity were also evaluated to provide insight on whether this type of antioxidant can be potentially pushed toward animal studies. We also describe analogous studies of natural products such as the garlic-derived thiosulfinate allicin and the grape-derived polyphenol resveratrol. These compounds have attracted significant attention in the past 20 years due to their purported health benefits, which are often ascribed to their purported radical-trapping activities. To date, systematic studies on their radical-trapping activities in solution, lipid bilayers and mammalian cells have been lacking. We have determined that allicin and petivericin, while effective RTAs in solution, are not so in lipid bilayers. Moreover, the compounds are not antioxidants in cell culture, but instead kill the cells. Similarly, resveratrol and its dimers pallidol and quadrangularin A, are found to be inefficient RTAs in lipid bilayers. Our studies to date rather suggest that they autoxidize readily to produce hydrogen peroxide, which may induce expression of phase 2 antioxidant enzymes, affording cytoprotection. Our insights underscore the need for systematic studies of antioxidant activity in multiple contexts. II Abstrait La peroxydation des lipides a été impliquée au début et pendant la progression de plusieurs maladies dégénératives, incluant les maladies cardiovasculaires, l’Alzheimer et le cancer. Par conséquent, pendant plus de 50 ans, des efforts considérables ont été consacrés à la conception de produits synthétiques et à la découverte de produits naturels pouvant ralentir la peroxydation lipidique. Malgré les investissements considérables jusqu’à présent, il n’y a aucune stratégie impliquant des antioxydants pour le traitement des maladies dégénératives. Nous argumentons que cela est causé par un manque fondamental de compréhension sur la réactivité chimique de ces composés dans des contextes spécifiques. Nous décrivons, ici, les études de nos antioxydants synthétiques optimisés pouvant piéger des radicaux, les tetrahydronaphthyridinols (THNs). Nous présentons, en premier, la synthèse d’une série de THN analogue à l’α-tocophérol (l’un des meilleurs antioxydant naturel solubles dans les lipides pouvant piéger des radicaux) avec différentes substitutions de chaine sur le côté de la molécule; par la suite, nous démontrons comment des changements systématiques de la lipophilicité de ces antioxydants ont un impact sur leurs réactivités avec les radicaux peroxyles dans les bicouches lipidiques et dans les cellules de mammifères. Leur régénération dans les bicouches lipidiques par des réducteurs solubles dans l’eau, leurs fixations à la protéine transporteuse de tocophérol (hTTP), ainsi que leurs cytotoxicité ont aussi été évalués afin de déterminer si ce type d’antioxydant pourrait mener à des études animales. Nous décrivons aussi des études analogues de produits naturels telles que l’allicin, le thiolsulfinate retrouvé dans l’ail et le polyphénol resvératrol retrouvé dans les grappes. Ces composés ont attirés beaucoup d’attention au cours des dernières 20 années due à leurs bénéfices pour la santé, souvent attribué à leurs activités alléguées en tant qu’antioxydant capable de piéger des radicaux (APR). Jusqu’à présent, il y a très peu d’études systématiques sur leur capacité à piéger des radicaux en solutions, en bicouche lipidiques et dans des cellules de mammifères. Nous avons déterminé que les thiosulfinates allicin et petivericin, même s’ils sont efficaces en solution, ne le sont pas dans les bicouches lipidiques. De plus, ces composée ne sont pas des antioxydants dans les cultures cellulaires, tuant plutôt les cellules. De manière semblable, le resvératrol et ses dimères pallidol et quadrangularin A, III sont inefficacse comme APR dans les bicouches lipidiques. Jusqu’à date, nos études suggèrent plutôt qu’ils s’auto-oxydent rapidement pour produire du peroxyde d’hydrogène, ce qui pourrait induire l’expression d’enzyme antioxydante de phase 2, procurant ainsi une cytoprotection. Nos recherches et les connaissances approfondies que nous avons recueillies soulignent la nécessité d’études systématiques pour déterminer l’activé des antioxydants dans des contextes multiples. IV Acknowledgements First and foremost, I would like to thank my supervisor Professor Dr. Derek A. Pratt for accepting me as his student and introducing me into the amazing “free radicals and antioxidants” world. Huge thanks for giving me the opportunity to carry out research under his guidance over the past few years. It is his attitude, enthusiasm for chemistry and broad chemical knowledge which made me feel confident during challenging times. Without his support, it would not have been possible for me to progress to such a level. I would also like to thank all the members in the Pratt group for their support, discussion, and advise on the project in the past few years. Thanks for their patience in answering all the questions I had. It has been a great time working with all you guys. I would like to thank all the collaborators that involved in these projects. Special thanks go to Dr. Eric Ye for the NMR spectroscopy and Peter Andrew Ochalski for flow cytometry assistance. Huge thanks to my friends and my family members. Being with all of you helped me not feel alone in Canada and fight through the hardship in my research and hardtime in the past few years. V Statement of Originality I hereby certify that all of the work described within this thesis is the original work of the author, with those exceptions noted in the thesis that are carried out by the collaborators listed in the preface in each chapter. Any published (or unpublished) ideas and/or techniques from the work of others are fully acknowledged in accordance with the references. Bo Li March 2016 VI Table of Contents Abstracts……………………...……………………………………………………………..II Abstrait………………………………………………………………………………...…...III Acknowledgements………………………………………………………………………...V Statement of Originality…………………………………………………………………...VI Table of Contents………………………………………………………………………….VII List of Schemes…………………………………………………………………………XII List of Figures …………………………………………………………………………XV List of Tables……………………………………………………………………….....XXVII List of Charts…………………………………………………………………………XXIX List of Abbreviations…………………………………………………………………XXX Chapter 1: Background and Significance……………………………………………...…1 1.1 Reactive Oxygen Species, Autoxidation and Its Inhibition by Radical-Trapping Antioxidants…………………………………………………………………………………1 1.1.1 Reactive Oxygen Species………………………………………………………....1 1.1.2 (Lipid) Autoxidation………………………………………………………….......4 1.1.3 Antioxidants………………………………………………………………………6 1.1.3.1 Preventive Antioxidants………………………………………………........6 1.1.3.2 Radical-Trapping Antioxidants…………………………………………….7 1.2 Factors Affecting Radical-Trapping Activities of Phenolic and Aminic Antioxidants……............................................................................................................10 1.3 Mechanism of H-atom Transfer………………………….……………………….........12 1.4 Methods to Determine the Efficacy of RTAs…………………………………..………14 1.4.1 Measurements of RTA Activity in Solution…………………...………………...15 1.4.1.1 Indirect Methods………………………………………………………..16 1.4.1.1.1 Inhibited Autoxidation……………………………...……………16 1.4.1.1.2 Peroxyl Radical Clocks……………………………………............21 1.4.1.2 Direct Methods…………………………..……………………………….26 1.4.1.2.1 (Laser) Flash Photolysis…………………………………...………26 VII 1.4.1.2.2 Pulse Radiolysis……………………………...……………………28 1.4.2 Measurements of RTA Activity in Biphasic Media……………………………..29 1.4.2.1 Inhibited Autoxidation ……………………………………………………31 1.4.2.2 Peroxyl Radical Clocks…………………………………………………...33 1.4.2.3 Competition with Fluorogenic Substrates………………………………...34 1.4.3 Determining RTA Activity in Cell Culture……………………………………...38 1.5 Research Objectives……………………………………………………………………40 1.5.1 Besting Vitamin E: Exploring the Reactivity of Naphthyridinol Antioxidants from Solution