Mechanistic Studies on the Lysine-Induced N-Formylation of 2,5-Dimethyl-p-benzoquinonediimine

2,5-Dimethyl-p-benzoquinonediimine was used as a model to study the reactivity of p-benzoquinonediimines, the first oxidation intermediates of allergenic p-amino aromatic compounds, toward lysine, as it has been suggested that this amino acid could play a key role in the induction mechanism of allergic contact dermatitis for a number of chemicals. The use of 6-[C-13]lysine and N-acetyl-6-[C-13]lysine, in association with C-13 NMR and HPLC in tandem with mass spectrometry techniques, allowed the identification of 4-amino-2,5-dimethylformanilide, 4-amino-2,5-dimethyl[C-13]formanilide, and the derivative containing the amino acid covalently bound at the para position. While enzymatic N-acetylation of p-phenylenediamine (PPD) has been described in the literature, in human skin for example, to our knowledge this was the first time that N-formylation of a PPD derivative induced by the reaction with an amino acid such as lysine was observed in solution, together with the formation of an adduct with the amino acid. To afford an explanation for the lysine-induced N-formylation,we undertook mechanistic studies, and they showed that 2,5-dimethyl-p-benzoquinonediimine was involved in an oxido reduction process that is capable of deaminating the alpha-NH2 group, even when N-acetylated, and the epsilon-NH2 groups of lysine in an oxidative way, forming the real reactive intermediates for N-formylation. This initially unexpected behavior should be considered when investigating the reactivity of such compounds with lysine-containing peptides or proteins in the context of haptenprotein binding studies.