Mass spectrometric study on the reaction of glyoxylic acid with chemically protected lysine at the α-amine group

Abstract

Glyoxylic acid (GA) is a human metabolite potentially involved in glycation processes and is considered a precursor of N^ε-carboxymethyl lysine (CML). Depending on the reaction conditions, different mechanisms of the reaction of GA with chemically protected lysine at the α-amine group or unprotected lysine have been reported. The objective of this study was to shed light on the reaction that occurs at physiological pH, using mass spectrometry and some complementary tools. In the first approach, six GA species were found in water, methanol and their mixtures with relative distribution depending on the solvent composition. The product of GA reaction with Z-Lys (N^α-(carbobenzyloxy)-L-lysine) was Z-CML, and two participating GA species were assigned to those preferentially formed in methanol. The reaction mixture in an HEPES buffer at pH 7.2 was analyzed using high-resolution MS at different times over 100 days. Protonated molecules [M + H]⁺ and respective sodium adducts ([M + Na]⁺), changes in their abundance over time and MS/MS data were used to assign the reaction intermediates. Additional experiments using liquid chromatography with MS were performed for the confirmation of key intermediates. At physiological pH, Z-CML was detected in the reaction mixture after 40 days. A plausible reaction mechanism was proposed, which involved the formation of adducts between GA species and the ε-amine group of Z-Lys, followed either by two decarboxylation steps and oxidation of the aldehyde group to carboxylic group or by decarboxylation and hydrolytic degradation. The slow formation of Z-CML was attributed to the accumulation of intermediates preceding the final product in the proposed reaction scheme, indicating that oxidative conditions would favor Z-CML production, which was in agreement with the well-known accelerated formation of AGEs under oxidative stress.