Radical acylation of L-lysine derivatives and L-lysine-containing peptides by peroxynitrite-treated diacetyl and methylglyoxal.

Highly electrophilic α-dicarbonyls such as diacetyl, methylglyoxal, 3-deoxyglucosone, and4,5-dioxovaleric acid have been characterized as secondary catabolites that can aggregate proteins and form DNA nucleobase adducts in several human maladies, including Alzheimer's disease, rheumatoid arthritis, diabetes, sepsis, renal failure, and respiratory distress syndrome. In vitro, diacetyl and methylglyoxal have also been shown to rapidly add up the peroxynitrite anion (k2 ~ 10(4)-10(5) M(-1) s(-1)), a potent biological nucleophile, oxidant and nitrosating agent, followed by carbon chain cleavage to carboxylic acids via acetyl radical intermediate that can modify amino acids. In this study, we used the amino acid derivatives Ac-Lys-OMe and Z-Lys-OMe and synthesized the tetrapeptides H-KALA-OH, Ac-KALA-OH, and H-K(Boc)ALA-OH to reveal the preferential Lys amino group targeted by acyl radical generated by the α-dicarbonyl/peroxynitrite system. The pH profiles of the reactions are bell-shaped, peaking at approximately 7.5; hence, they are close to the pKa values of ONOOH and of the catalytic H2PO4(-) anion. RP-HPLC and ESI-MS analyses of reaction products confirmed (α)N- and (ϵ)N-acetylation of Lys by diacetyl as well as acetylation and formylation by methylglyoxal, with preference for the α-amino group. These data suggest the possibility of radical acylation of proteins in epigenetic processes, where enzymatic acetylation of these biomolecules is a well-documented event, recently reported to be as critical to the cell cycle as phosphorylation. Also noteworthy is the observed formylation of L-Lys containing peptides by methylglyoxal never reported to occur in amino acid residues of peptides and proteins.