Small molecule probes for peroxynitrite detection

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).