Model studies of the maillard reaction of arg-lys with D-glucose

Possible routes leading from Amadori product precursors to glucose-derived protein crosslinks has been suggested by model studies examining the fate of the Amadori products in vitro. For instance, the Amadori product can undergo dehydration to give 1,4-dideoxy-1-alkylamino-2,3-hexodiulose (AP-dione), which has been isolated by trapping with aminoguanidine. In a model reaction, we selected as an AGE target the dipeptide N^α-Z-arg-lys. The proximity of the arginine and lysine residues to each other promotes stable intramolecular crosslink formation. Incubation N^α-Z-arg-lys with 10 equivalents of glucose in 0.2 M phosphate buffer (pH 7.4) at 37°C for five weeks produces at least 25 distinct reaction products upon fractionation of this mixture by HPLC. Each fraction was isolated, concentrated, and analyzed for its reactivity with a polyclonal anti-AGE antibody (RU) that has been shown previously to recognize a class of AGEs that increase as a consequence of hyperglycemia and which are inhibited from forming in human subjects by treatment with the advanced glycation inhibitor amino-guanidine. The products present within one fraction (1.5% yield) were found to block antibody binding in a dose-dependent fashion. Further purification of this fraction by HPLC revealed the presence of one major (0.6% yield) immunoreactive compound. Characterization of this adduct by UV, ESMS and ¹H-NMR spectra revealed the presence of intramolecular arg-lys-imidazole crosslink. This crosslink is non-fluorescent and acid labile and may represent an important class of immunoreactive AGE-crosslinks that form in vivo.