Synthesis of alfa-N-linked glycopeptides

It’s now well established that glycosylation of proteins and peptides plays a very important role in biological processes, as it introduces enormous structural diversity and modulates proteins’activity. Glycopeptides are involved in cell-cell communication, receptor-mediated endocytosis, in the interaction and subsequent invasion of pathogens and immune response. Besides the influence carbohydrate residues have on the physiochemical and biological properties of proteins, glycans appear to stabilize tertiary structure and also aid folding and transport by protecting proteins from proteolysis. In the case of N-linked glycoprotein or glycopeptides, the natural linkage is a β-N-glycosidic bond. Replacement of this natural linkage with unnatural ones can lead to modifications tolerated by most biological systems and yet less susceptible to chemical or enzyme-mediated hydrolysis. Hence unnatural glycopeptides can help to mimic and/or interfere with molecular recognition events in which glycoproteins participate. One of such unnatural linkage could be a alpha-N-glycosidic bond between the sugar and the aminoacid chain. Previous studies on the conformation of both α and β chitobiose-peptides showed that the stereochemistry at the anomeric centre of the N-linked carbohydrate dramatically affects the backbone conformation of glycopeptides. : , but, on the contrary of alpha-N-glycosyl peptides which are abundantly synthetized, unnatural glycopeptides with alpha-N-glycosidic bond, are essentially unexplored molecules because currently one of the major limitations is synthetic accessibility. Recently our laboratory developed a methodoly for the synthesis of unprotected alpha-N-linked glycosylaminoacids trought the traceless Staudinger ligation, using functionalized phosphines with glutamic acid derivatives, while a route to obtain alpha-N-linked glucosyl aspartic derivatives was introduced by DeShong and coworkers. Starting from this strategy we were able to optimize and scale up the stereoselective synthesis of alpha-N-glycosilated building blocks of glucosyl and galactosyl asparagine derivative with suitable protecting groups for solid phase synthesis (Figure 1). We further investigated the methods in solution for the coupling with other aminoacids, both from the amine and carboxylic termini, in order to identify the best condition applicable in future solid phase synthesis. During the synthesis of such dipeptides we had to face some problems as the generation of by-products derived from cyclization of the aparagine residue resulting in a stable five member ring, that is a well known drawback in peptide synthesis of residues containing asparagine derivatives. Finally we find out the conditions of couplings and with these informations in hands we decided to constuct glycopeptides in solid phase that could be mimic of Antifreeze glycopeptides (AFGPs). These molecules allow survival of many marine fishes that live in polar waters where temperatures decline below the colligative freezing points of their body fluids. Essentially their function is binding to the surface of embryonic ice crystals and inhibit their growth. The AFGPs isolated from fish blood plasma consist of repeating tripeptide units (Ala-Thr-Ala)n with a disaccharide moiety (Galβ1-3GalNAcα1-) attached to each threonyl residue. Our idea is to modify this structure by the substitution of the sugar part with the galactose unit and binding it trought a N-α-link, instead of a O-link. This will enable us to begin to study the structural features and properties of such unnatural glycopeptides.