Formation pathways and opioid activity data for 3-hydroxypyridinium compounds derived from glucuronic acid and opioid peptides by Maillard processes

The kinetics of formation and identity of the reaction products of the glucuronic acid with three representative opioid peptides were investigated in vitro. Peptides were conjugated with glucuronic acid either in solution or under dry-heating conditions. From the incubations performed in solution N-(1-deoxy-D-fructofuranos-1-yluronic acid)-peptide derivatives (Amadori compounds) were isolated, whereas from the dry-heated reactions products containing the 3-hydroxypyridinium moiety at the N-terminal of the peptide chain were obtained. Experiments performed under mild dry-heating conditions (40 degrees C) in model systems based on Leu-enkephalin and glucuronic acid, and in environment of either 40% or 75% relative humidity, revealed that the higher level of humidity promoted a process that enhanced 3-hydroxypyridinium compound generation. The mechanism of 3-hydroxypyridinium formation is discussed. In comparison with their respective parent peptides, the N-(1-deoxy-D-fructofuranosyl-uronic acid) derivatives of the opioid peptides showed three- to 11-fold lower mu- and delta-receptor-binding affinities and agonist potencies in the functional assays, likely as a consequence of the steric bulk introduced at the N-terminal amino group. The further decrease in opioid activity observed with the 3-hydroxypyridinium-containing peptides may be due to the lower pK(a) of the 3-hydroxypyridinium moiety and to delocalization of the positive charge in the pyridinium ring system.