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 N-6-{2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl}lysine (GOLA) and N -6-glycoloyllysine (GAIA) as prototypes for novel amide protein modificatio ns produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model react ions with N-alpha-t-butoxycarbonyl-lysine showed that glyoxal and glycolald ehyde are immediate precursors, and reaction pathways are directly linked t o N-epsilon-carboxymethyllysine via glyoxal-imine structures. GOLA, the ami de 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 concentr ations. GOLA and GALA formation from the Amadori product of glucose and lys ine depends directly upon oxidation. With the advanced glycation end produc t inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidat ive fragmentation of the Amadori product as a second mechanism of GOLA form ation exactly coinciding with N-epsilon-carboxymethyllysine synthesis. In c ontrast, the formation of GALA appears to depend solely upon glyoxal-imines . After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent len s protein. This suggests amide protein modifications as important markers o f pathophysiological processes.