Synthesis of hexose-related imidazolidinones: Novel glycation products in the Maillard reaction

Carbohydrate-peptide esters which mimic the reactivity of sugar 6-phosphates in nonenzymatic glycations were used as model compounds for the study of the Maillard reaction in vitro. We found that intramolecular cyclization of the monosaccharide ester in which the sugar moiety (D-glucose or D-galactose) is linked, through the C-6 hydroxy group, to the C-terminal carboxy group of the endogenous opioid pentapeptide leucine-enkephalin, in methanol as the solvent, resulted in the formation of imidazolidinone diastereoisomers having cis or trans relative geometry of the substituents at the imidazolidinone ring moiety. The diastereoisomeric imidazolidinones were separated and each transformed by hydrolysis into the corresponding D-gluco- and D-galacto-related imidazolidinone products of leucine-enkephalin. Along with the previous evidence that, from the same sugar-peptide esters by changing the reaction conditions Amadori rearrangement products could be obtained [Horvat et al. (1998) J Chem Soc Perkin Trans 1:99–13], the presented results point to the possibility that similar carbohydrate-related imidazolidinones may also be generated in the early stage of the Maillard reaction in vivo.     

Peptide and amino acid glycation: new insights into the Maillard reaction.

Nonenzymatic glycation of proteins, peptides and other macromolecules (the Maillard reaction) has been implicated in a number of pathologies, most clearly in diabetes mellitus. but also in the normal processes of aging and neurodegenerative amyloid diseases such as Alzheimer's. In the early stage, glycation results in the formation of Amadori-modified proteins. In the later stages, advanced glycation end products (AGE) are irreversibly formed from Amadori products leading to the formation of reactive intermediates, crosslinking of proteins, and the formation of brown and fluorescent polymeric materials. Although, the glycation of structural proteins has been attributed a key role in the complications of diabetes, recent attention has been devoted to the physiological significance of glycated peptide hormones. This review focuses on the physico-chemical properties of the Amadori compounds of bioactive peptides of endogenous and exogenous origin, such as Leu-enkephalin and morphiceptin, investigated under different conditions as well as on novel pathways in the Maillard reaction observed from investigating intramolecular events in ester-linked glycopeptides.     

Fructose-induced N-terminal glycation of enkephalins and related peptides

The formation of glycation products in model systems consisting of fructose and the endogenous opioid peptides not containing lysine residue, such as Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) and Met-enkephalin (Tyr-Gly-Gly-Phe-Met), or of their fragments, Tyr-Gly-Gly-Phe and Tyr-Gly-Gly, was examined. N-(2-Deoxy-aldos-2-yl)-peptides (Heyns compounds) as well as diastereoisomeric imidazolidinone compounds were identified as reaction products of N-terminal amino group glycation for each of the peptides studied. The structure of the glycation products and relative configuration of C-2 substituents on the imidazolidinone ring in diastereoisomers were determined by NMR experiments. The chemical and enzymatic stability of the fructose-derived glycated products of Leu- and Met-enkephalin was studied in phosphate-buffered saline (pH 7.4) and in human serum at 37 degrees C. The obtained results revealed that glycation increases the stability of the parent peptide to enzymatic degradation. As a result of different configuration at the newly formed stereogenic center, large stability differences in the 2S* and 2R* isomers of the imidazolidinone compounds were observed.     

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

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.     

A conserved interaction with the chromophore of fluorescent proteins

The chromophore of fluorescent proteins, including the green fluorescent protein (GFP), contains a highly conjugated imidazolidinone ring. In many fluorescent proteins, the carbonyl group of the imidazolidinone ring engages in a hydrogen bond with the side chain of an arginine residue. Prior studies have indicated that such an electrophilic carbonyl group in a protein often accepts electron density from a main-chain oxygen. A survey of high-resolution structures of fluorescent proteins indicates that electron lone pairs of a main-chain oxygen-Thr62 in GFP-donate electron density into an antibonding orbital of the imidazolidinone carbonyl group. This n→π* electron delocalization prevents structural distortion during chromophore excitation that could otherwise lead to fluorescence quenching. In addition, this interaction is present in on-pathway intermediates leading to the chromophore, and thus could direct its biogenesis. Accordingly, this n→π* interaction merits inclusion in computational and photophysical analyses of the chromophore, and in speculations about the molecular evolution of fluorescent proteins.