Peroxynitrite (ONOO^-) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. Here, the development of a new water-soluble and cationic boronate probe based on a coumarin-imidazolium scaffold (CI-Bz-BA) for the fluorescent detection of ONOO^- in cells is reported. The chemical reactivity of the CI-Bz-BA probe toward selected oxidants known to react with the boronate moiety was characterized, and the suitability of the probe for the direct detection of ONOO^- in cell-free and cellular system is reported. Oxidation of the probe results in the formation of the primary hydroxybenzyl product (CI-Bz-OH), followed by the spontaneous elimination of the quinone methide moiety to produce the secondary phenol (CI-OH), which is accompanied by a red shift in the fluorescence emission band from 405 nm to 481 nm. CI-Bz-BA reacts with ONOO^- stoichiometrically with a rate constant of ∼1 × 10⁶ M^-1s^-1 to form, in addition to the major phenolic product CI-OH, the minor nitrated product CI-Bz-NO₂, which is not formed by other oxidants tested or via myeloperoxidase-catalyzed oxidation/nitration. Both CI-OH and CI-Bz-NO₂ products were also formed in the presence of cogenerated fluxes of nitric oxide and superoxide radical anion produced during decomposition of a SIN-1 donor. Using RAW 264.7 cells, we demonstrate the ability of the probe to report endogenously produced ONOO^-via fluorescence measurements, including plate reader real time monitoring and two-photon fluorescence imaging. Liquid chromatography/mass spectrometry analyses of cell extracts and media confirmed the formation of both CI-OH and CI-Bz-NO₂ in macrophages activated to produce ONOO^-. We propose the use of a combination of real-time monitoring of probe oxidation using fluorimetry and fluorescence microscopy with liquid chromatography/mass spectrometry-based product identification for rigorous detection and quantitative analyses of ONOO^- in biological systems.

Derivatives of coumarin, containing oxidant-sensitive boronate group, were recently developed for fluorescent detection of inflammatory oxidants. Here, we report the synthesis and the characterization of 3-(2-benzothiazolyl)-7-coumarin boronic acid pinacol ester (BC-BE) as a fluorescent probe for the detection of peroxynitrite (ONOO^–), with high stability and a fast response time. The BC-BE probe hydrolyzes in phosphate buffer to 3-(2-benzothiazolyl)-7-coumarin boronic acid (BC-BA) which is stable in the solution even after a prolonged incubation time (24 h). BC-BA is slowly oxidized by H₂O₂ to form the phenolic product, 3-benzothiazol-2-yl-7-hydroxy-chromen-2-one (BC-OH). On the other hand, the BC-BA probe reacts rapidly with ONOO⁻. The ability of the BC-BA probe to detect ONOO^– was measured using both authentic ONOO^– and the system co-generating steady-state fluxes of O₂^•– and ^•NO. BC-BA is oxidized by ONOO^– to BC-OH. However, in this reaction 3-benzothiazol-2-yl-chromen-2-one (BC-H) is formed in the minor pathway, as a peroxynitrite-specific product. BC-OH is also formed in the reaction of BC-BA with HOCl, and subsequent reaction of BC-OH with HOCl leads to the formation of a chlorinated phenolic product, which could be used as a specific product for HOCl. We conclude that BC-BA shows potential as an improved fluorescent probe for the detection of peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of oxidant-specific products will help to identify the oxidants detected.