The highly specific fluorescence visualization of mitochondrial peroxynitrite <i>via</i> a naphthalimide probe

Peroxynitrite (ONOO^-), a reactive nitrogen species with potent oxidative capacity, induces cell death through chemical modifications of biomolecules including proteins, lipids and nucleic acids, while being implicated in the occurrence of diseases, such as Alzheimer's disease and cancer. The development of precise monitoring tools is therefore critical for elucidating its pathophysiological mechanisms. Given the mitochondrial predominance in ONOO^- biosynthesis, we engineered Mito-NAP-ONOO, a mitochondria-targeted fluorescent probe featuring (4-methoxyphenylthiophenyl)carbonyl as the recognition moiety, pyridine for mitochondrial localization and naphthalimide as the fluorogenic core. The electron-deficient thiocarbonate group suppresses intramolecular charge transfer (ICT), maintaining the probe in a quenched state. Upon ONOO^- interaction, cleavage of the thiocarbonate moiety restores ICT-mediated fluorescence (quantum yield enhancement: <i>Φ</i> = 1.39% to 28.8%). Comprehensive photophysical characterization revealed exceptional selectivity, submicromolar sensitivity (LOD = 722 nM), robust photostability, and broad pH tolerance. Live-cell imaging demonstrated precise mitochondrial localization (Pearson's coefficient = 0.86) and dynamic ONOO^- detection during exogenous/endogenous stimulation. This innovative probe design establishes a valuable platform for investigating mitochondrial ONOO^- dynamics in pathophysiological contexts.