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

Abstract

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)2O•−…•NO2 (minor pathway). [Sikora A. et al., Chem Res Toxicol 24, 687-97, 2011]. 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-MitoPhNO2 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)2O•− radical anion with subsequent reaction of the resulting phenyl radical with •NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of o-MitoPhB(OH)2O•− radical anion is significantly lower than that of m-MitoPhB(OH)2O•− and p-MitoPhB(OH)2O•− radical anions. The nitrated product, o-MitoPhNO2, is not formed by nitrogen dioxide radical generated by myeloperoxidase in the presence of 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-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO− in biological systems.

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation

“…11). We have shown previously that stimulation of RAW 264.7 cells leads to formation of o-MitoPhOH and small amounts of o-MitoPhNO 2 (21,22). As shown in Fig.…”

“…8a) and is unaffected by glutathione or other biologically relevant reductants (21,22). As this product is not fluorescent, we used HPLC with absorption detection for quantitative analyses, and we confirmed it is formed in RAW 264.7 cells in activated macrophages (22). However, using an LC-MS/MS method, we found that this product (i.e.…”

“…These products showed up at the m/z value of 414 and were assigned to isomers of o-MitoPh(NO 2 )OH (Fig. 8a) (22). In addition, a small amount of the protonated form of triphenylphosphonium oxide (TPPϭO, m/z ϭ 279) was also detected.…”

Nitroxyl (HNO) Reacts with Molecular Oxygen and Forms Peroxynitrite at Physiological pH

“…This is, in part, due to the lack of availability of specific probes that directly react with ONOO Ϫ to form specific diagnostic products. Here, we present unambiguous evidence for ONOO Ϫ formation during the reaction between HNO and molecular oxygen using the recently developed assays for specific detection and quantification of peroxynitrite (47,48).…”

Targeting Mitochondria with Small Molecules: The Preparation of MitoB and MitoP as Exomarkers of Mitochondrial Hydrogen Peroxide

“…More precisely, it reacts with the conjugate base of hydrogen peroxide, the hydroperoxide anion, so that the higher pH of the mitochondrial matrix gives an additional four---fold acceleration relative to reaction in the cytosol. With the exception of peroxynitrite and HOCl, other ROS do not react with MitoB to give this product (51,52). Importantly, the rate constant for reaction between the mitochondrial peroxidase, peroxiredoxin III, and hydrogen peroxide is over a million times higher than that for reaction with MitoB (49).…”