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

Chembiochem. 2025 Apr 1;26(7):e202400972. doi: 10.1002/cbic.202400972. Epub 2025 Apr 4.

Abstract

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.

Keywords: activity-based sensing; fluorescent imaging; mitochondria; oxidative stress; peroxynitrite.

MeSH terms

  • Animals
  • Boron Compounds* / chemical synthesis
  • Boron Compounds* / chemistry
  • Fluorescent Dyes* / chemical synthesis
  • Fluorescent Dyes* / chemistry
  • Fluorescent Dyes* / metabolism
  • Macrophages* / cytology
  • Macrophages* / metabolism
  • Mice
  • Microscopy, Fluorescence
  • Mitochondria* / chemistry
  • Mitochondria* / metabolism
  • Peroxynitrous Acid* / analysis
  • Peroxynitrous Acid* / metabolism
  • RAW 264.7 Cells

Substances

  • Fluorescent Dyes
  • Peroxynitrous Acid
  • Boron Compounds