Phenyl Radical-Mediated Fluorogenic Cyclization for Specific Detection of Peroxynitrite

Peroxynitrite (ONOO^-), a biological oxidizing and nitrating species responsible for post-translational modification of cellular proteins, has been implicated in numerous pathologies carrying an inflammatory component. Specific detection of ONOO^- in biological systems remains a challenge, and boronates are regarded as the most promising class of probes for the detection and quantitation of ONOO^-. Oxidation of boronate probes by ONOO^- results in the formation of minor ONOO^--specific products via a pathway involving a phenyl radical-type intermediate, in addition to the major phenolic product. Here, we report fluorogenic cyclization of the phenyl-type radical formed during oxidation of a boronate probe by ONOO^-, with the production of a fluorescent product, and we propose a new approach for the specific detection of ONOO^- based on this observation. We characterized the kinetics and stoichiometry of the reaction of benzophenone-2-boronic acid with ONOO^- and identified 2-hydroxybenzophenone as the major product and fluorenone (FLN) and 2-nitrobenzophenone as the minor ONOO^--specific products. Hydrogen peroxide neither alone nor in the presence of myeloperoxidase and nitrite produces FLN or 2-nitrobenzophenone. FLN can be selectively detected using fluorescence spectroscopy, providing a chemical principle for the development of next-generation probes for ONOO^-, with noninvasive, fluorescence-based detection of ONOO^--specific products. Fluorescence-based monitoring of FLN was successfully applied for the detection of ONOO^- generated in situ from the decomposition of SIN-1, a thermal source of the superoxide radical anion and nitric oxide.