Mechanism of formation of the putative advanced glycosylation end product and protein cross-link 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole

2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) is a fluorescent molecule which was originally discovered in chloroform extract of ammoniacal solution of acid-hydrolyzed glycated proteins and proposed to represent a protein cross-link. The absence of a lysyl residue side chain and other observations promoted a detailed study of its mechanism of formation. Glycated alpha-t-Butoxycarbonyllysine was incubated for 29 days and periodically assayed for FFI and FFI-like fluorescence. Whereas fluorescence increased over time, FFI recovery was unexpectedly highest on day 0 and lowest on day 29, suggesting that FFI was directly derived from Amadori products. FFI was also recovered from hydrolysates of glycated neopentylamine, furosine, and browned poly-L-lysine but was virtually undetectable in similar solutions basified with NaOH, triethylamine, or pyridine instead of ammonia. Gas chromatography-mass spectrometry analysis of FFI from similar hydrolysates basified in the presence of 15N-enriched NH4Cl revealed for all precursors a parent ion peak at 230 instead of 228 m/e units, suggesting that the two imidazole nitrogen atoms had been incorporated from free ammonia into FFI. Spontaneous FFI synthesis occurred when furosine was reacted with aqueous ammonia at room temperature. These results do not support the proposition that FFI is an advanced glycosylation end product or a protein cross-link. They suggest that FFI is formed from ammonia and furosine which are by-products of acid-hydrolyzed glycated proteins.