Chemical glycopeptide synthesis requires access to gram quantities of glyco-amino acid building blocks. An efficient and fast synthetic route has been developed for such building blocks with high yields using microwave-assisted Kochetkov amination as a key reaction. The resulting glyco-amino acid building blocks were successfully incorporated into target glycopeptides by Fmoc-solid phase peptide synthesis (SPPS) and were characterized by LC-MS and HRMS(FAB). The glycopeptides were used to investigate the effect of glycosylation on enzymatic cleavage of peptide bonds. At first, enzyme-cleavable linkers for solid-phase peptide and glycopeptide synthesis were developed. Commonly used hydroxymethyphenoxy linker (Wang Linker) in solid phase peptide synthesis was surprisingly susceptible to efficient cleavage to serine protease chymotrypsin. In order to investigate this observation further a broad range of amino acid residues were coupled to PEGA<sub>1900</sub> resin using the Wang linker and subsequent treatment with chymotrypsin gave cleavage of the amino acids of the resin in quantitative yields. A similar strategy was also applied to a glyco-tripeptide and again chymotrypsin hydrolysis gave glycopeptide in quantitative yield. It was also established that not only the acid-labile linker (HMPA) but also base labile (HMBA) and unreactive ester (HOA) linkers could be cleaved using chymotrypsin. The solid supported peptide and glycopeptides were then used to study the effect of glycosylation on proteolytic hydrolysis of peptides with two different proteases such as chymotrypsin and thermolysin. Our hydrolysis results in combination with those of others show that protection from hydrolysis is highly dependent on the individual protease and also on the position of the glycosylation site in respect to the scissile bond.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:641493 |
| Date | January 2005 |
| Creators | Bejugam, Mallesham |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13795 |
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