Amides Are Novel Protein Modifications Formed by Physiological Sugars

The Maillard reaction, or nonenzymatic browning, proceeds in vivo, and the resulting protein modifications (advanced glycation end products) have been associated with various pathologies. Despite intensive research only very few structures have been established in vivo. We report here for the first time N6-{2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl}lysine (GOLA) and N6-glycoloyllysine (GALA) as prototypes for novel amide protein modifications produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with Nα-t-butoxycarbonyl-lysine showed that glyoxal and glycolaldehyde are immediate precursors, and reaction pathways are directly linked to Nε-carboxymethyllysine via glyoxal-imine structures. GOLA, the amide cross-link, and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), the imidazolium cross-link, share a common intermediate. The ratio of GOLA to GOLD is greater when glyoxal levels are low at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose and lysine depends directly upon oxidation. With the advanced glycation end product inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidative fragmentation of the Amadori product as a second mechanism of GOLA formation exactly coinciding with Nε-carboxymethyllysine synthesis. In contrast, the formation of GALA appears to depend solely upon glyoxal-imines. After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent lens protein. This suggests amide protein modifications as important markers of pathophysiological processes.