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Development of Chemical Probes for Studying Formaldehyde Biology By Development of Chemical Probes for Studying Formaldehyde Biology By Thomas Francis Brewer A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Christopher J. Chang, Chair Professor Matthew B. Francis Professor Ming C. Hammond Professor Niren Murthy Spring 2017 Development of Chemical Probes for Studying Formaldehyde Biology © 2017 by Thomas Francis Brewer Abstract Development of Chemical Probes for Studying Formaldehyde Biology By Thomas Francis Brewer Doctor of Philosophy in Chemistry University of California, Berkeley Professor Christopher J. Chang, Chair Formaldehyde (FA) is a reactive carbonyl species (RCS) produced in living systems through a diverse set of cellular pathways that span epigenetic regulation to metabolic processing of endogenous metabolites. At the same time, however, aberrant elevations in FA contribute to pathologies ranging from cancer and diabetes to heart, liver, and neurodegenerative diseases. Traditional methods for the detection of biological FA rely on sample destruction and/or extensive processing, resulting in a loss of spatiotemporal information. Disentangling the complex interplay between FA physiology and pathology motivates the development of chemical tools that can enable selective detection of this RCS in biological environments with spatial and temporal fidelity. This dissertation describes the design and applications of a FA-responsive homoallylamine trigger which undergoes imine condensation with FA, followed by 2-aza-Cope rearrangement and hydrolysis, irreversibly forming a diagnostic product. Importantly, this 2-aza-Cope reactivity- based trigger was found to discriminate FA from other RCS such as methylglyoxal, 4- hydroxynonenal, and glucosone as well as from simple carbonyl-containing species such as acetaldehyde and pyruvate. Throughout this body of work, emphasis was placed on the design of FA probes whose responsiveness and readout could be applied to interrogate FA biology in a number of biological systems. To this end, the FA-reactive trigger was appended to a variety of useful diagnostic readouts, including intensity-based fluorescence (FA Probe-1), excitation-ratiometric fluorescence (Ratiometric FA Probe-1 and Ratiometric FA Probe-2), bioluminescence (FA Luciferin-1), and immunohistochemistry (Puromycin FA Probe-1). All of these probes were shown to be responsive and selective toward FA in vitro and subsequently characterized in various cell culture model systems. This work established the utility of the 2-aza-Cope reaction as a FA detection platform and demonstrated its versatility for generating many functional imaging probes, paving the way for future interrogation of FA biology in living, intact systems. 1 Dedication To my parents, for unfaltering love and support. i Table of Contents Acknowledgments iii Chapter 1 Detection of small-molecule analytes in living systems 1 Chapter 2 Design, synthesis, and application of a first-generation 2-aza- 38 Cope-based probe for detection of formaldehyde in living cells Chapter 3 Development of an excitation-ratiometric formaldehyde 67 imaging platform and its application in living cells Chapter 4 Toward in vivo FA detection methods: immunohistochemistry- 109 and bioluminescence-based probes for formaldehyde Appendix 1 Preparation and evaluation of cell-trappable superoxide probes 125 Appendix 2 Preparation and evaluation of dihydroethidium derivatives with 153 altered photophysical properties Appendix 3 Selectivity tests for reactive carbonyl species 178 Appendix 4 LC-MS usage and maintenance 182 ii Acknowledgments I would like to thank my family for supporting me, particularly my brother Kevin and my sister Fallon, as well as my parents Kevin and Bridget. Their love and guidance has cheered me on throughout my life, and I have always counted on their support as a foundation for all my efforts. Thank you to Professor Chris Chang for mentoring me throughout my graduate career, allowing me to rise to the challenges of synthetic chemistry and chemical biology and guiding me in my growth as a scientist. All of the members of the Chang Lab have helped to make my time in graduate school enjoyable and fruitful. Their friendship and support has been invaluable to me. I would like to thank Lakshmi Krishnamoorthy, Carl Onak, Mark Vander Wal, Marie Heffern, Joey Cotruvo, Brian Michel, Eva Nichols, Cheri Ackerman, and Ryan Walvoord for their guidance and training to me. Thank you to Ben Thuronyi, Vivian Lin, Dan Finley, Stephen Wilson, Professor Michelle Chang, Professor Matt Francis, and Professor Ming Hammond for providing me with training as an organic chemist and chemical biologist during my first year of graduate school. I thank Allegra Aron and Cameron Baker for their camaraderie and constant support throughout our time in graduate school together. I can always lean on them when times are rough, and they are the first people I turn to when I have good news to celebrate. Their friendship is dear to me and continues to brighten my life. I thank Brooke Bullock and Professor Bobby Arora for their advice and training to me as an undergraduate. They helped to shape my passion as a young scientist and their encouragement is what brought me to graduate school. Finally, I would like to thank Jesse Koehler for his unconditional love and support. iii Chapter 1 Detection of small molecule analytes in living systems Portions of this work were published in the following scientific journals: Brewer, T. F.;* Garcia, F. J.;* Onak, C. S.;* Carroll, K. S.; Chang, C. J. “Chemical Approaches to Discovery and Study of Sources and Targets of Hydrogen Peroxide Redox Signaling Through NADPH Oxidase Proteins.” Annu. Rev. Biochem. 2015, 84, 765–790. Bruemmer, K. J.;* Brewer, T. F.;* Chang, C. J. “Fluorescent probes for imaging formaldehyde in biological systems.” Curr. Opin. Chem. Biol. 2017, 39, DOI: 10.1016/j.cbpa.2017.04.010. * denotes equal contribution 1 1.1. Background and Motivation Reactive species such as hydrogen peroxide (H2O2) and formaldehyde (FA) have well- established toxicity in living systems and participate in the pathophysiology of various disease states. At the same time, however, these reactive species are produced endogenously and play diverse roles in maintaining normal physiology. Better understanding of how, when, and why these small molecules are produced in living systems will help to elucidate how cells maintain the delicate balance between signal and stress, potentially leading to new insights in human health and disease. Owing to the transient nature of reactive species, fluorescence-based probes, which generally display high sensitivity and can be used to determine spatial and temporal distributions in live specimens, are particularly appealing tools for the detection of H2O2, FA, and related species. Novel probes must meet a high bar in terms of their selectivity, kinetics, and photophysical properties. An ideal chemical probe must be inert to the conditions present in the cell and should not require the addition of other reagents. Furthermore, the probe must be selective against similar analytes, and it should exhibit a turn-on, rather than turn-off, response in order to minimize background. Ideally, a family of probes with varying wavelengths should be available so that their use can be tailored to the specific application and made compatible with probes for different species. To introduce the reader to this area of research, an overview of relevant FA and H2O2 probes is presented. 1.2. Formaldehyde (FA) Formaldehyde (FA) is an endogenously-produced reactive carbonyl species (RCS) released in biological processes ranging from epigenetics to one carbon metabolism (Figure 1.1).1- 2 Although more widely known as an environmental toxin and carcinogen,3 FA is found at relatively high concentrations intracellularly under normal physiological conditions, reaching up to 500 µM in certain organelles.4-5 FA is a one-carbon fuel involved in maintenance of cell homeostasis, and its abundance and high reactivity suggest a potential role as a physiological signaling molecule.6-8 FA is the product of demethylation events of N-methylated amino acid residues (e.g. lysine, arginine, histidine) mediated by demethylase enzymes,9 such as lysine specific demethylase 1 (KDM1)10-11 and Jumonji domain-containing proteins (Figure 1).12-14 FA is also produced through N-demethylation of DNA and RNA bases, such as m6A, by AlkB homologues (ALKBH).15 Methylation and demethylation events signal transcription factors to promote or repress transcription, and disruption of normal methylation markers could have wide ranging effects in disease states, particularly cancer progression.16-17 Similarly, metabolism of methylated amines, including the abundant endogenous metabolite methylamine, by semicarbazide-sensitive amine oxidases (SSAO) release FA.18-19 Several demethylase enzymes utilize tetrahydrofolate (THF) as a cofactor to bind FA, yielding 5,10-methylene-THF, known as “active formaldehyde.”20-21 Folate derivatives are essential for mitochondrial one-carbon metabolism, in which demethylation events release FA, which is further incorporated into important cellular building blocks such as amino acids, purines, and phospholipids.22 A canonical example is the sarcosine pathway, where dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SARDH) demethylate dimethylglycine sequentially,
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