Formation of 2-nitrophenol from salicylaldehyde as a suitable test for low peroxynitrite fluxes

Abstract

There has been some dispute regarding reaction products formed at physiological peroxynitrite fluxes in the nanomolar range with phenolic molecules, when used to predict the behavior of protein-bound aromatic amino acids like tyrosine. Previous data showed that at nanomolar fluxes of peroxynitrite, nitration of these phenolic compounds was outcompeted by dimerization (e.g. biphenols or dityrosine). Using 3-morpholino sydnonimine (Sin-1), we created low fluxes of peroxynitrite in our reaction set-up to demonstrate that salicylaldehyde displays unique features in the detection of physiological fluxes of peroxynitrite, yielding detectable nitration but only minor dimerization products. By means of HPLC analysis and detection at 380 nm we could identify the expected nitration products 3- and 5-nitrosalicylaldehyde, but also novel nitrated products. Using mass spectrometry, we also identified 2-nitrophenol and a not fully characterized nitrated dimerization product. The formation of 2-nitrophenol could proceed either by primary generation of a phenoxy radical, followed by addition of the NO2-radical to the various resonance structures, or by addition of the peroxynitrite anion to the polarized carbonyl group with subsequent fragmentation of the adduct (as seen with carbon dioxide). Interestingly, we observed almost no 3- and 5-nitrosalicylic acid products and only minor dimerization reaction. Our results disagree with the previous general assumption that nitration of low molecular weight phenolic compounds is always outcompeted by dimerization at nanomolar peroxynitrite fluxes and highlight unique features of salicylaldehyde as a probe for physiological concentrations of peroxynitrite.