Expanding the Utility of Triazabutadiene Chemical Probes To Enable Their Use in the Study of Protein Dynamics via 19F NMR

Abstract

Mitochondrial function is central to cellular energy production, metabolic regulation, and signaling, and its dysfunction is implicated in diverse pathologies such as neurodegeneration, cancer, and metabolic disorders. This dissertation aims to investigate and expand novel chemical biology strategies to create pathways to probe mitochondrial protein modifications and dynamics, employing bioconjugation approaches to facilitate selective protein labeling and targeted small- molecule delivery. The work is organized into four major chapters, each addressing critical aspects of mitochondrial biology, chemical probe design, and analytical methodologies.Chapter 1 provides an overview of mitochondrial structure and function, emphasizing the organelle’s roles in oxidative phosphorylation, calcium homeostasis, apoptotic signaling, and the biosynthesis of essential biomolecules. The discussion highlights how disruptions in these processes contribute to mitochondrial dysfunction (MD) and subsequent disease states, establishing the rationale for developing tools to interrogate mitochondrial biology. Additionally, strategies for targeting mitochondria in drug discovery are reviewed, with a focus on modulating mitochondrial dynamics as a promising therapeutic avenue. Chapter 2 introduces bioconjugation as a chemical technique for studying protein modifications, detailing the principles of probe design using aryl diazonium ions (ADIs) and their masked precursors, triazabutadienes (TBDs). This chapter explores the reactivity and selectivity of electrophilic probes that covalently modify nucleophilic amino acid residues—especially tyrosine—through azo coupling reactions. The advantages and limitations of native versus bioorthogonal labeling strategies are discussed, and a comprehensive comparison of analytical techniques is presented. Methods such as in-gel fluorescence, UV–Vis spectroscopy, mass spectrometry (LC–MS/MS), and nuclear magnetic resonance (NMR) spectroscopy are evaluated for their roles in confirming probe attachment and mapping modification sites at residue-level resolution. Chapter 3 focuses on the experimental application of these chemical probes. A para- trifluoromethyl-substituted aryl diazonium ion (p-CF3 ADI) is synthesized and employed as a model system to monitor protein and DNA modifications. The incorporation of trifluoromethyl groups enables sensitive detection via 19F NMR, a technique that benefits from fluorine’s 100% natural abundance and lack of endogenous background in biological systems. Studies in both cell lysates and purified protein systems, using myoglobin as a model, demonstrate the probe’s ability to report on binding interactions, conformational dynamics, and reaction kinetics, providing detailed insights into probe behavior and chemical reactivity. Chapter 4 outlines the design and synthesis of dual-functional triazabutadienes equipped with mitochondrial-targeting motifs, such as triphenyl phosphonium. This work extends the utility of ADI-based probes to spatially controlled applications, enabling the selective delivery of reactive species to the mitochondria. The synthesis and preliminary evaluation of these organelle-targeted probes lay the groundwork for future studies aimed at elucidating mitochondrial protein interactions and advancing therapeutic development. Collectively, this dissertation expands the chemical biology toolkit with novel bioconjugation strategies, offering robust platforms for investigating protein modifications, mitochondrial dynamics, and subcellular targeting. The findings promise to advance our understanding of mitochondrial biology and inform the design of next-generation therapeutics for diseases associated with mitochondrial dysfunction.