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

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 in situ. 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 m/z peak following exposure to high collision energy, corresponding to symmetric cleavage of the parent dimer peak (m/z = 453) along with a fragmentation product at m/z = 180.04 (–NO(2) species). This fragmentation profile was distinct from the C–O coupled 3-NT dimer that exhibited a predominant 209.14 m/z peak with a small secondary 226.15 m/z 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.