| Glycosylation is the most common post-translational modification of proteins and modulates their biological properties in several crucial ways. In order to engage in detailed studies of the effects of protein glycosylation, glycoscientists seek to obtain samples of glycoproteins in high purity and abundance. The nature of glycoprotein and oligosaccharide synthesis makes this acquisition difficult. The development and application of methodology to obtain glycoproteins are described herein.;The hypothesis that CD2, a glycoprotein that requires glycosylation for folding, could be made to fold by bioorthogonal chemistry was investigated by applying glycosyl iodoacetamides to CD2 variants bearing a thiol handle install by cysteine mutation at the glycosylation site. Unfortunately, folding could not be induced by chemical glycosylation, suggesting that the linkage and proximity of the sugar are crucial to achieving stabilization.;Finally, an on-going investigation of the enhanced aromatic sequon is described. Several glycosylated building blocks with non-natural linkages and functional groups and their incorporation into a glycosylated Pin WW protein is described. Enzymatic elaboration of Pin WW using Endoglycosidase A and the resulting increase in thermal stability is described. Preliminary studies of the effect of incorporation of Asn(glucose) rather than Asn(GlcNAc) into the enhanced aromatic sequon is described as well. In short, glucose was found to be nearly as stabilizing as GlcNAc suggesting that interactions between the 2-acetamido functional group of GlcNAc and a threonine side chain in the sequon is less important for achieving stability in this context.;Specifically, the scope and limitations of the recently-developed sugar-assisted ligation (SAL) with complex oligosaccharides were investigated. We found that glycopeptides bearing a thiol handle on the 2-acetamido functional group are amenable to enzymatic elaboration at the C-3 and C-4 positions of O-linked glycopeptides. In most cases, glycan elaboration at C-4 does not have deleterious effect on SAL. Glycosylation of C-3, however, renders SAL completely ineffective. C-6 elaboration was probed using a chemically synthesized amino acid and it was found that such elaboration has a small negative effect on ligation efficiency. N-linked glycans were successfully elaborated by treatment with Endoglycosidase A and this elaboration had only a small negative effect on ligation efficiency. |
