Two-photon fluorescent probe for cellular peroxynitrite: Fluorescence detection, imaging, and identification of peroxynitrite-specific products

A new naphthalene-based boronate probe, NAB-BE , for the fluorescence-based detection of inflammatory oxidants, including peroxynitrite, hypochlorous acid, and hydrogen peroxide, is reported. The chemical reactivity and fluorescence properties of the probe and the products are described. The major, phenolic oxidation product, NAB-OH , is formed in case of all three oxidants tested. This product shows green fluorescence, with a maximum at 512 nm, and can be excited either at 340 nm or in the near infrared region (745 nm) for two-photon fluorescence imaging. Peroxynitrite is the fastest of the oxidants tested and, in addition to the phenolic product, leads to the formation of a nitrated product, NAB-NO 2 , which can serve as a fingerprint for peroxynitrite. The probe was applied to detect peroxynitrite in activated macrophages using fluorimetry and two-photon fluorescence microscopy, and both NAB-OH and NAB-NO 2 products were detected in cell extracts by liquid chromatography-mass spectrometry. The combined use of fluorometric high-throughput analyses, fluorescence imaging, and liquid chromatography-mass spectrometry-based product identification and quantitation is proposed for most comprehensive and rigorous characterization of oxidants in biological systems. • Naphthalene-based boronate two-photon fluorescent probe for detection of peroxynitrite. • Peroxynitrite reacts with the probe almost a million times faster than H 2 O 2 and more than 30 times faster than HOCl. • The major, green fluorescent product formed during the reaction of NAB-BA with oxidants is NAB-OH . • Incubation of NAB-BA with macrophages activated to produce ONOO − yielded NAB-OH as well as the nitrated product, NAB-NO 2 . • The probe accumulates in cells and detects oxidants in the cytosolic/extranuclear compartments.