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Title: Synthesis of Glycoproteins and C-linked Glycopeptides
Author: Kramer, Holger
ISNI:       0000 0001 3602 3886
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2006
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Protein glycosylation is of great importance in nature due to its significance towards protein folding and stability, cell-cell communication and immunology. This thesis·reports strategies that allow the synthesis of glycoprotein mimics as single glycoforms. This has been achieved through a combination of site-directed mutagenesis with unnatural amino acid incorporation and subsequent orthogonal chemical modification. The method also extends to the preparation of differentially modified glycoprotein mimics by means of two mutually orthogonal modification reactions. In addition to that, the direct synthesis of unprotected C-linked glycopeptides by olefin cross metathesis was investigated. Unnatural functionality which can be modified in an orthogonal manner to all other functional groups occurring in proteins has been introduced using methionine analogue incorporation. By combining residue specific replacement with site-directed mutagenesis alkene, alkyne and azide functionalities were site-selectively installed on the protein surface. The chemical modification was exemplified using Cu(!) catalysed triazole formation on Azido homoalanine (Aha) and Homopropargyl glycine (Hpg) residues with glycosyl azides and. alkynyl glycosides as reaction partners. The formed protein conjugates are attractive mimics of naturally occurring glycoproteins. The unnatural triazole linkage has the advantages of high chemical stability and it is not expected to be susceptible towards enzymatic degradation. The natural enzymatic activity of the protein was found to be retained in both the mutant proteins containing unnatural amino acids as well as in the modified protein conjugates. In addition to retaining the natural function of the enzyme, new lectin-binding properties were conferred upon the glycoconjugates. Specificity and strength of lectin-binding were shown to be dependent on the nature of the conjugated glycan. A combination of disulfide formation using glyco-MTS reagents and triazole synthesis allowed the creation of the first artifical glycoprotein bearing two different carbohydrate structures. Differential modification using the abovemethods also allowed the synthesis of a mimic of P-selectin glycoprotein ligand as a biological probe. This demonstrated the utility of this approach not only towards conjugation of glycosides, but also in the mimicry of other posttranslational modifications such as tyrosine sulfation. The synthesis of vinyl glycine (vGly) from selenomethionine (SeMet) was investigated as a means of incorporation of this ~,'Y-unsaturated amino acid into proteins and peptides. Facile oxidation to the selenoxide was achieved using hydrogen peroxide. The selenoxide elimination step required thermal activation and was shown to depend on the reaction medium and on the addition of seleninic acid scavengers. Furthermore, the utility of olefin cross metathesis (CM) for the direct synthesis of unprotected C-linked glycopeptides was investigated. To this end a number of alkene containing model peptides and olefinic C-glycosides were synthesized.' For the peptide systems both vinyl glycine (vGly) and homoalyll glycine (Hag) were utilized. The CM reaction was tested with a view towards modification of biologically important macromolecules. Therefore polar protic reaction media were investigated. It could be shown that the phosphine-free 2nd generation Hoveyda-Grubbs catalyst is stable over extended periods of time in methanol as reaction solvent. Nevertheless, catalyst decomposition occurs in the presence of terminal alkenes. As a result, repeated addition of the catalyst is required to achieve conversion in the metathesis reaction. At the same time olefin isomerisation processes take place with increasing catalyst loading. A variety of reaction conditions were investigated in order to limit the undesired side reactions and promote the productive CM process.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available