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.

In this study, a new fluorescent probe containing dicyanovinyl moiety has been designed and synthesized. Fluorescent probe based on styrylbenzimidazolium derivative was reported for the effective detection of bisulfite and distinguish it from biothiols by exploiting dicyanovinyl group as the recognition site. The photophysical properties of the novel styrylbenzimidazolium derivative were assessed by determination of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime. Its spectroscopic behavior towards various analytes has been evaluated in aqueous media at a pH of 7.4. The highest increase in fluorescence intensity of compound 5 in the presence of different analytes was observed for sodium bisulfite and the limit of detection was estimated to be 0.25 μM. The styrylbenzimidazolium dye was applied to detect bisulfite in various wine sample using fluorimetry. Finally, the ability of this novel probe to detect HSO3- in red wine samples was evaluated.

Peroxynitrite (ONOO^‒/ONOOH) is a short-lived but highly reactive species that is formed in the diffusion-controlled reaction between nitric oxide and the superoxide radical anion. It can oxidize certain biomolecules and has been considered as a key cellular oxidant formed under various pathophysiological conditions. It is crucial to selectively detect and quantify ONOO^– to determine its role in biological processes. In this review, we discuss various approaches used to detect ONOO^‒ in cell-free and cellular systems with the major emphasis on small-molecule chemical probes. We review the chemical principles and mechanisms responsible for the formation of the detectable products, and plausible limitations of the probes. We recommend the use of boronate-based chemical probes for ONOO^‒, as they react directly and rapidly with ONOO^–, they produce minor but ONOO^‒‒specific products, and the reaction kinetics and mechanism have been rigorously characterized. Specific experimental approaches and protocols for the detection of ONOO^– in cell-free, cellular, and in vivo systems using boronate-based molecular probes are provided (as shown in Boxes 1–6).