Photochemically-induced protein tyrosine nitration in vitro and in cellula by 5-methyl-1,4-dinitro-1H-imidazole (DNI): synthesis and biochemical characterization

Abstract

The photochemical nitrating agent 5-methyl-1,4-dinitro-1H-imidazole (DNI) has been recently described as an effective tool for nitrating tyrosine residues in proteins under 390 nm irradiation (Long T. et al., 2021). Herein, we describe the one-step synthesis of DNI from the precursor 4-methyl-5-nitro-1H-imidazole with good yield (66%) and high purity (>99%). Spectral analysis of DNI reveals two maximum peaks (228 and 290 nm) with maximum nitration yields and kinetics occurring at 290 nm. Electron paramagnetic resonance (EPR)- and mass spectrometry (MS)- spin trapping analysis evidenced the formation of nitrogen dioxide (^•NO₂) upon irradiation of DNI, implying the homolysis of the N-N bond in the DNI molecule. Irradiation of DNI at 290, 390 nm, or UVA light (315-400 nm), produced tyrosine nitration, with yields approaching ca. 30% with respect to DNI at 290 nm exposure. Indeed, using alpha-synuclein as a model protein, the main protein post-translational modification triggered by DNI was the generation of 3-nitrotyrosine as shown by MS analysis. Additionally, the formation of di-tyrosine was also observed. Finally, intracellular ^•NO₂ production upon DNI photolysis in bovine aortic endothelial cells was evidenced by the nitration of the tyrosine analog probe p-hydroxyphenylacetic acid (PHPA) and cellular protein tyrosine nitration.

Keywords: Free radicals; Nitrogen dioxide; Photochemical nitration; Protein nitration; Tyrosine nitration.