Photocontrollable Peroxynitrite Generator Based on N-Methyl-N-nitrosoaminophenol for Cellular Application

Abstract

We designed and synthesized a photocontrollable peroxynitrite (ONOO(-)) generator, P-NAP, which has N-methyl-N-nitrosoaminophenol structure with four methyl groups introduced onto the benzene ring to block reaction of the photodecomposition product with ONOO(-) and to lower the semiquinoneimine's redox potential. The semiquinoneimine intermediate generated by photoinduced release of nitric oxide (NO) reduces dissolved molecular oxygen to generate superoxide radical anion (O(2)(•-)), which reacts with NO to afford ONOO(-) under diffusion control (k = 6.7 × 10(9) M(-1) s(-1)). NO release from P-NAP under UV-A (330-380 nm) irradiation was confirmed by ESR spin trapping. Tyrosine nitration, characteristic of ONOO(-), was demonstrated by HPLC analysis of a photoirradiated aqueous solution of P-NAP and N-acetyl-l-tyrosine ethyl ester. ONOO(-) formation was confirmed with a ONOO(-)-specific fluorogenic probe, HKGreen-3, and compared with that from 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), which is the most widely used ONOO(-) generator at present. The photoreaction of P-NAP was influenced by superoxide dismutase, indicating that generation of O(2)(•-) occurs before ONOO(-) formation. The quantum yield for formation of duroquinone, the main P-NAP photodecomposition product, was measured as 0.86 ± 0.07 at 334 nm with a potassium ferrioxalate actinometer. Generation of ONOO(-) from P-NAP in HCT-116 cells upon photoirradiation was successfully imaged with HKGreen-3A. This is the first example of a photocontrollable ONOO(-) donor applicable to cultured cells.