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Content archived on 2024-05-29

Native chemical glycosylation: a novel and efficient method for the synthesis of complex glycoconjugates

Final Activity Report Summary - NCG (Native chemical glycosylation: A novel and efficient method for the synthesis of complex glycoconjugates)

The glycosylation of proteins is one of the most complex modifications within eukaryotes. An evident complication of the study of naturally occuring glycoproteins and their role in biological processes is their occurence as glycoforms: because glycosylation is a post-translational process (and thus not under direct genetic control) each given protein bears different oligosaccharides at varying positions resulting in hundreds or even thousands of protein variants with different properties. Therefore for the benefit of any investigation and therapeutic applications, the development of synthetic assembly approaches toward well-defined glycoproteins has always been of prime importance. The common classes of oligosaccharides found on eukaryotic cells are primarily defined according to the nature of the linkage to the peptide backbone. The first major class of glycoproteins are the N-linked glycopeptides, where a sugar chain is covalently linked by an amide bond to an asparagine (Asn) residue of a polypeptide chain. The second major class of glycoproteins are of the O-linked type. In this case the oligosaccharide is typically linked to the polypeptide via a serine (Ser) or threonine (Thr) residue. The development of synthetic strategies towards N-linked glycopeptides is relatively well established as methods to synthesise the amide linkage are well developed. This has lead to the convergent (non-lineair) and divergent (lineair) approach towards N-linked glycopeptides. During a convergent synthesis a glycan is coupled directly to a peptide whereas the lineair approach uses a glycoamino acid to stepwise constrict the glycopeptide. Synthetic strategies to O-linked glycopeptides, however, are to date based on the lineair strategy, thus incorporating a pre-formed O-linked glycoamino acid. The basis for this is that in contrast to the hydroxyl group of individual Ser/Thr residues, hydroxylamino acids in a peptide sequence are highly inactive glycosyl acceptors. In addition, there is the generally poor solubility of peptides under general glycosylation conditions (i.e. relatively non-polar solvents). Indeed, to date, there has been no successful convergent strategy towards O-linked glycopeptides/proteins.

We envisioned that the introduction of a glycosyl donor moiety in the proximity of a hydroxylated amino acid side-chain might lead to a novel and more efficient method for the convergent construction of O-linked glycopeptides. The rationale behind this strategy is based on the combination of two key features. First the ability to chemo- and siteselectively modify cysteine residues with electrophilic thiol-specific carbohydrate reagents yielding disulfide linked glycoconjugates. Secondly, that these glycosyl disulfides can be utilised as effective glycosyl donors in O-glycoside formation. Using this methodology, we were able to synthesise, in one-pot, O-linked glycopeptides in good overall yields from their corresponding disulfide linked glycopeptides. The retained cysteine residue can then be used for additional functionalisations or be reduced to an alanine residue. By emplyoing the acid-labile para-methoxybenzyl group as protective group, we were also able to access unprotected disulfide and O-linked glycopeptides.

During the proximity based glycan transfer outlined above, both inter- and intramolecular glycosylation can take place. We reasoned that forming a (di)sulfide linkage directly onto a hydroxylamino acid residue should lead to a sole intramolecular glycan transfer, which is highly beneficial in terms of stereochemistry and efficiency (yield). After some prelimiary results, this approach is being investigated in a collaboration with Dr H. H. Jensen (Aarhus University). It is believed that this methodology, along with the one outlined above, holds great potential in convergently constructing O-linked (in this case threonine) glycopeptides in a site- and chemoselective manner.