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Development of organoboron-based probes and platforms for tracking oxidative cellular metabolites
Haggett, Jack G.
Haggett, Jack G.
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2023
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Abstract
Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), alkyl hydroperoxides (ROOH), and peroxynitrite (ONOO-) are oxidative metabolites involved in homeostasis maintenance and disease states. Although the canonical understanding of these metabolites has largely focused on their contribution to disease and inflammation states1,2, emerging evidence has revealed that ROS also play critical roles in signaling pathways, maintaining biological homeostasis, and coordinating the wound healing process.3–5 These findings justify a need for further studying ROS speciation and elucidating their roles in biological systems.
To understand the roles of ROS in biological systems, it is necessary to track them with high spatial and chemical selectivity. This is most commonly accomplished with reactivity-based fluorescent probes, a class of chemical tools that change their fluorescence intensity/emission after reaction with an analyte of interest.6,7 Boronic acids and pinacolboronate esters are commonly used reactive motifs in fluorescent probes. However, the chemoselectivity of these compounds is limited, as boronate functionalities can oxidize rapidly with both H2O2 and ONOO-.8 Better tools to discriminate between these two metabolites will help untangle the roles that each oxidative species plays in disease and homeostasis.
Diazaborines (DABs) are a family of pseudo aromatic aryl boronic acids formed upon cyclization with a neighboring imine group.9 In aqueous solution, the formation of these compounds is rapid and spontaneous, motivating their use in bioorthogonal strategies.10–12 In this dissertation, we will first show that diazaborines oxidize slowly with H2O2 but rapidly with ONOO-.13 Next, we will show how the merger of a diazaborine motif into a fluorophore scaffold yields a fluorescent probe that is selective for ONOO- even in the presence of high concentrations of H2O2. This new chemical tool, Peroxynitrite Probe-1 (PNP-1), was used in live cell imaging experiments to track exogenously added and endogenously produced ONOO-.14 Finally, we will discuss the development of a new hydrogel scaffold with covalently-linked, ROS-activated fluorescent probes. This material, ROSmap, enhances the detection and spatial visualization of exogenously applied ROS. Overall, this work describes a fundamental physical-organic study motivating the development of a novel ONOO--selective chemical switch, the development of this functionality into a ONOO--selective fluorescent probe, and finally, the expansion and adaptation of related technologies into a new fluorescent hydrogel matrix able to detect exogenous ROS with spatial fidelity.
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