Photocontrollable Peroxynitrite Generator Based on <i>N</i>-Methyl-<i>N</i>-nitrosoaminophenol for Cellular Application

Abstract

We designed and synthesized a photocontrollable peroxynitrite (ONOO^–) generator, P-NAP, which has <i>N</i>-methyl-<i>N</i>-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 (O₂^•–), which reacts with NO to afford ONOO^– under diffusion control (<i>k</i> = 6.7 × 10⁹ 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 <i>N</i>-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 O₂^•– 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