Reaction between Peroxynitrite and Triphenylphosphonium-Substituted Arylboronic Acid Isomers: Identification of Diagnostic Marker Products and Biological Implications

Aromatic boronic acids react rapidly with peroxynitrite (ONOO-) to yield phenols as major products. This reaction was used to monitor ONOO- formation in cellular systems. Previously, we proposed that the reaction between ONOO(-) and arylboronates (PhB(OH)(2)) yields a phenolic product (major pathway) and a radical pair PhB(OH)(2)O center dot-center dot center dot center dot(NO2)-N-center dot (minor pathway). [Sikora, A. et al. (2011 ) Chem. Res. Toxicol. 24 , 687 - 697]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO- and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)(2)). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO(2) formed from the reaction between o-MitoPhB(OH)(2) and ONOO- was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)(2)O center dot- radical anion with subsequent reaction of the resulting phenyl radical with center dot NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of the o-MitoPhB(OH)(2)O center dot- radical anion is significantly lower than that of m-MitoPhB(OH)(2)O center dot- and p-MitoPhB(OH)(2)O center dot- radical anions. The nitrated product, o-MitoPhNO(2), is not formed by the nitrogen dioxide radical generated by myeloperoxidase in the presence of the nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO-. Incubation of o-MitoPhB(OH)(2) with RAW 264.7 macrophages activated to produce ONOO- yielded the corresponding phenol o-MitoPh(OH)(2) as well as the diagnostic nitrated product, o-MitoPhNO(2). We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO- in biological systems