Glycosylation is a prevalent form of post translational modification, believed to occur on over 50% of human proteins. Homogeneous forms of glycoproteins are essential for developing an understanding of how activity is mediated at a structural level. As biological origins of glycoproteins give rise to complex mixtures of glycoforms, homogeneous glycoprotein production has become an important goal. As chemical protein synthesis is often limited to sequences of 30-50 residues, access to large native glycoproteins is currently restricted to fragment based approaches. Protein semi-synthesis enables the preparation of larger proteins which can be difficult to obtain through chemical synthesis alone. Consequently, a general semi-synthetic strategy towards N-glycoproteins has been proposed and demonstrated on Interferon-β-1 (IFNβ), a 166 residue glycoprotein. A three fragment strategy was designed, relying on the chemical synthesis of a short glycopeptide segment and recombinant expression of the two flanking domains. Homogeneity was established through the chemical synthesis of a glycopeptide containing a natively linked N-acetylglucosamine (GlcNAc), also enabling the selective transfer of complex oligosaccharides. After cloning and expression, the recombinant fragments were functionalised to allow assembly of the protein using Native Chemical Ligation. These desired protein modifications were achieved through the application of highly chemoselective reactions. These reactions were also applied towards the generation of N-glycopeptides compatible with the ligation strategy. Further to this, existing methods enabling the direct synthesis of functionalised N-glycopeptides were also explored. After glycopeptide synthesis, endoglycosidase A enabled the transfer of oligosaccharides to the N-acetylglucosamine motif. This has allowed the preparation of the desired IFNβ glycopeptide as well as a glycosylated variant of glucagon like peptide-1. To expand the utility of endoglycosidase methodology, a novel sugar nucleotide was synthesised to facilitate the incorporation of a sialyl galactose mimic onto N-glycans. The resulting oligosaccharides may serve as novel substrates for endoglycosidases in the preparation of N-glycoprotein mimics.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:631838 |
| Date | January 2014 |
| Creators | Premdjee, B. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1434897/ |
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