Generation of oxygen free radicals by glycated proteins is widely believed to be one of the causes of oxidative stress in diabetes and aging. Metal ion catalysis is regarded as an essential part of the oxidative mechanism. In this work, we also considered an alternative "metal-free" superoxide radical formation by a number of fructose-amino acids (Amadori compounds) derived from glycine and lysine, which represent the simplest models for early glycated proteins. In the superoxide dismutase-dependent cytochrome c assay, 1 mM Chelex-treated aqueous solutions of monofructose-amino acids 4-6 generated 0.9-3.6 x 10(-10) M s-1 O2*- at pH 7. Surprisingly, the rates of superoxide radical formation in the solutions of difructose-amino acids 7-9 were significantly higher (0.75-5.8 x 10(-9) M s-1 O2*-). The percentage of acyclic sugar anomers (</=0. 8-85%) and their "enolization" rate constants (5 x 10(-6) to 2 x 10(-3) s-1) varied broadly for the compounds studied and positively correlated with the rates of superoxide radical formation. The presence of Cu2+ markedly increased the rate of superoxide radical formation at metal concentrations higher than 1 microM, while Fe3+ did not accelerate the reaction even at 100 microM. Therefore, in addition to the metal ion-catalyzed oxygen free radical formation, metal-free enol oxidation of fructosyl groups on glycated amino acid residues may contribute to the generation of oxygen free radicals and their subsequent oxidative damage to proteins.