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 (O 2 •– ), 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 (H 2 O 2 ), 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 ortho position to the fluorophore (CP-Bz- o -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- o -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- o -BA probe reacts with H 2 O 2 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.