Peroxynitrite-Mediated Dimerization of 3-Nitrotyrosine: Unique Chemistry along the Spectrum of Peroxynitrite-Mediated Nitration of Tyrosine

Abstract

Peroxynitrite (ONOO^-, PN) has long been considered a potent nitrating agent implicated in numerous inflammation-mediated diseases. The current work highlights an unexplored oxidation chemistry initiated under conditions of sustained PN exposure. Impetus for this investigation developed from mass spectral results that suggested dimerization of a model peptide with a single tyrosine residue that was first nitrated following extended exposure to PN generated <i>in situ</i>. In attempts to substantiate this dimerization event and divulge the possible mode of linkage between the tyrosine derivatives of the peptide monomers, 3-nitrotyrosine (3-NT) was exposed to sustained fluxes of PN in a two-component PN-generating platform developed in this laboratory. Such exposure afforded products with tandem mass spectrometry and fluorescence spectroscopy profiles indicative of C-O coupling between 3-NT moieties. Synthesis and comparative analysis of the C-C coupled 3-NT isomer corroborated these findings. Most notably, the mass spectral data of the C-C coupled 3-NT dimer displayed a 226.80 <i>m/z</i> peak following exposure to high collision energy, corresponding to symmetric cleavage of the parent dimer peak (<i>m/z</i> = 453) along with a fragmentation product at <i>m/z</i> = 180.04 (-NO₂ species). This fragmentation profile was distinct from the C-O coupled 3-NT dimer that exhibited a predominant 209.14 <i>m/z</i> peak with a small secondary 226.15 <i>m/z</i> peak indicative of asymmetric cleavage of the parent dimer. Results of this study indicate that formation of C-O coupled 3-NT dimer is promoted by elevated levels of 3-NT formed under high and sustained flux of PN.