Isolation and characterization of Glyoxal–Arginine modifications

Abstract

Glyoxal is generated in foods and in vivo by autoxidation of unsaturated fatty acids and by the Maillard reaction of reducing sugars and amines. Due to its high reactivity, the α-dicarbonyl easily reacts with protein-bound lysine and arginine residues. The quantitatively most important modification of the ε-amino group of lysine is Nε-carboxymethyllysine [1]. Reaction with glyoxal also leads to cross-linking of proteins by formation of amides (glyoxal–amide cross-link [2]), imidazole (MODIC [3]) and imidazolium structures (GOLD [4]).Concerning the reaction of glyoxal with the guanidino group of arginine, inconsistent results have been published in the literature. We could now verify 1-(4-amino-4-carboxybutyl)-2-imino-4,5-dihydroxy-4,5-dihydroimidazolidine (dihydroxyimidazolidine) as the single primary product in incubations of Nα-t-BOC-arginine with glyoxal under physiological conditions [5]. The structure was identified as a cis/trans-isomer mixture of ca. 1:4. In a much slower reaction, dihydroxyimidazolidine is degraded to N7-carboxymethylarginine (CMA), representing a novel stable endproduct. Both structures were unequivocally established by 1H-, 13C-, HMBC-NMR and high-resolution mass spectra. Other structures could not be found under these reaction conditions. Synthesis of 1-(4-amino-4-carboxybutyl)-2-imino-5-oxo-imidazolidine (imidazolinone), which has been published earlier as the only modification so far , could not be verified [6]. However, both arginine modifications, dihydroxyimidazolidine and CMA can be transformed to the imidazolinone under acid conditions. Therefore, the detection of the imidazolinone in incubations under physiological conditions must be evaluated as an artifact of the acid sample workup procedures applied.The mechanistic relationship of all three structures was clarified in incubations at various temperatures, pH values and in the presence of o-phenylenediamine and aminoguanidine. The latter experiment using α-dicarbonyl trapping reagents proved that the reaction to the dihydroxyimidazolidine can be reversed to regenerate arginine and a glyoxal derivative. As trapping reagents are commonly used to measure ‘free’ glyoxal in foods and in vivo samples, the published data has to be evaluated very critically. In addition, it was possible to elucidate the mechanism leading from the dihydroxyimidazolidine to CMA and to exclude any participation of the imidazolinone.