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

We 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.