A Diazaborine Activity‐Based Sensing Fluorescent Probe Reports Endogenously Produced Mitochondrial Peroxynitrite in Live Macrophages

• DOI: 10.1002/cbic.202400972
• Publication Date: 2025-03-12T10:12:28Z
• Authors: Jack G. Haggett; G. Dinesh Kumar; Michael J. Melville; Chelsea G. Johansen; Luke D. Knudson; Nikki L. Farnsworth; Dylan W. Domaille

AbstractWe present the synthesis, properties, and imaging applications of a new class of diazaborine‐based probes (Peroxynitrite Probe‐1, PNP‐1) for selective peroxynitrite (ONOO−) imaging in live cells. PNP‐1 features a diazaborine‐based reaction motif that provides excellent discrimination between H2O2 and ONOO−, solving a persistent challenge of organoboron‐based fluorescent probes for oxidative metabolite imaging. We demonstrate the utility of PNP‐1 to detect endogenously produced ONOO− in live RAW 264.7 macrophages by fluorescence microscopy, with probe selectivity confirmed with inhibition of NADPH oxidases and nitric oxide synthase, the requisite enzymatic machinery for ONOO− production. Co‐localization studies unexpectedly reveal preferential mitochondrial localization, which we show is dependent on the naphthalimide scaffold. Taken together, our results show that diazaborines are a novel motif for selective ONOO− reactivity, positioning them for incorporation into other ONOO−‐specific chemical biology tools.