Toward Specific Detection of Peroxynitrite─Intramolecular Cyclization of Boronobenzylated Pyridinium Probe in Chemical and Cellular Systems

Detecting and quantifying peroxynitrite (ONOO^-) under physiological conditions is challenging due to its low steady-state levels, resulting from typically low fluxes of nitric oxide (^•NO) and superoxide (O₂^•-), as well as its short half-life caused by rapid reactions with cellular and extracellular targets. Low-molecular-weight boronate probes that react rapidly with ONOO^- to form stable and characteristic products represent the most promising class of redox probes for ONOO^- detection and quantitative analyses in chemical and biological systems. Because boronates also respond to other nucleophilic oxidants, including hydrogen peroxide (H₂O₂), novel approaches to selectively detect ONOO^- are needed. Here, we report our studies on the application of boronobenzyl (Bz-BA) derivatives of the fluorescent coumarin-pyridine (CP) construct for specific detection of ONOO^-. We demonstrate that the probe containing a boronate moiety in the <i>ortho</i> position to the fluorophore (CP-Bz-<i>o</i>-BA) reacts rapidly with ONOO^- and forms the corresponding cyclization product on the minor, ONOO^--specific pathway. We characterized the reaction kinetics and stoichiometry of CP-Bz-<i>o</i>-BA with ONOO^- and identified the hydroxybenzyl derivative as the major and stable product, with cyclic and nitrated derivatives as the minor, but ONOO^--specific products. Liquid chromatography-mass spectrometry analyses of cell extracts confirmed the formation of the same products in RAW 264.7 macrophages activated to produce ONOO^-, demonstrating the occurrence of the same chemistry and its applicability for specific detection of ONOO^- in biological settings. Interestingly, the CP-Bz-<i>o</i>-BA probe reacts with H₂O₂ significantly faster (∼5-fold) than the previously reported aromatic boronates (e.g., coumarin-7-boronic acid, CBA), which we attribute to the presence of the positive charge of the pyridinium cation in proximity to the boronic acid moiety.