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.

光化学硝化剂 5-甲基-1,4-二硝基-1H-咪唑(DNI) 最近被描述为在 390 nm 照射下硝化蛋白质中酪氨酸残基的有效工具 (Long T. et al., 2021)。在此，我们描述了从前体 4-甲基-5-硝基-1 H-咪唑一步合成 DNI，具有良好的收率 (66%) 和高纯度 (>99%)。DNI 的光谱分析显示两个最大峰（228 和 290 nm），最大硝化产率和动力学发生在 290 nm。电子顺磁共振（EPR）和质谱（MS）自旋捕获分析证明在DNI 照射下形成二氧化氮（ ^• NO ₂），这意味着DNI 分子中N-N 键的均裂。DNI 在 290、390 nm 或 UVA 光（315-400 nm）的照射下，产生酪氨酸硝化，产率接近约 10% 。相对于 290 nm 曝光下的 DNI，为 30%。事实上，使用 α-突触核蛋白作为模型蛋白，DNI 触发的主要蛋白质翻译后修饰是 3-硝基酪氨酸的生成，如 MS 分析所示。此外，还观察到二酪氨酸的形成。最后，通过酪氨酸类似物探针对羟基苯乙酸( PHPA)的硝化和细胞蛋白酪氨酸硝化证明了牛主动脉内皮细胞中DNI 光解后细胞内^NO ₂的产生。