Novel carbohydrate-induced modification of peptides: crystal structure and NMR analysis of ester-bridged bicyclic galactose-enkephalin adduct containing imidazolidinone moiety.

Abstract

Comparative studies based on x-ray crystallography and NMR spectroscopy were used for structural characterization of the novel minor, imidazolidinone moiety containing, product 2b of the Maillard reaction obtained in vitro by using the galactose-modified endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) 1. The x-ray analysis uniquely defined the molecular structure as cyclo-(N-(12-[-4)-D-galacto-pentitol-1-yl]-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1 --> O]acetyl]glycyl-L-phenylalanyl-L-leucyl-] (3), having an 18-membered ring with an ester bond between the secondary (C4') hydroxyl group of a D-galacto-pentitolyl residue and the C-terminal carboxy group of leucine-enkephalin. The absolute configuration of the new chiral centre at the imidazolidinone moiety was established as C2(S), indicating a cis arrangement of C2 and C4 substituents at the 5-membered heterocyclic ring. The NMR analysis of compound 2b carried out in CH3CN-d3 and DMSO-d6, indicated the existence of two isomers in solution, differing only in the position of the ester group in the molecule. NMR data for the minor isomer (13%-16%) are in agreement with structure 3. The migratory tendency of the peptidyl group from the primary (2b) to the secondary hydroxyl group (3) of a D-galacto-pentitolyl residue in methanol/water solution was confirmed by RP HPLC analysis.