Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.565248
Title: Synthesis of tools for glycoprotein remodelling
Author: Masania, J.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2011
Availability of Full Text:
Access through EThOS:
Access through Institution:
Abstract:
The production of therapeutic glycoproteins typically relies on the purification of the desired glycoprotein from engineered tissue culture systems. This process is costly and inefficient, and the isolated glycoproteins are adorned with an array of heterogeneous sugars. Expression of glycoproteins in cheaper non-mammalian host cells such as yeast produces large quantities of folded glycoproteins though the sugar chains are immunogenic to humans. This project initially aimed to develop a general strategy for the humanisation of yeast glycoproteins using the copper (I) mediated ligation between azide and acetylene functional groups, commonly known as “click” chemistry. It required that azide and acetylene groups could be efficiently incorporated into (glyco)peptides and sugars respectively, and to this end propargylic glycosides of mannose, N-acetyl glucosamine, and N-acetyl lactosamine were successfully prepared. Two different glycoprotein remodelling methods were ultimately investigated. The first method explored a chemoenzymatic strategy developed in our group. A target glycoprotein, erythropoietin (EPO), was expressed in the presence of azide bearing mannose sugars in the hope that azide groups would be incorporated during glycoprotein biosynthesis. Subsequent elaboration of the azide functionality with propargyl N-acetyl lactosamine followed by enzymatic sialylation was expected to yield a glycoprotein with human-like sialyl lactosamine antennae. However, upon purification from Pichia pastoris incorporation of azide could not be detected, limiting the strategy to in vitro glycoprotein remodelling. Several technological advances were made including optimisation of the final “click” reaction between the propargyl glycoside of N-acetyllactosamine and 4-azidomannose followed by enzymatic transfer of sialic acid. While considerable effort was directed towards the key transfer of unnatural azidosugars, from nucleotide donors to potential substrates using an α-1,2-mannosyltransferase, this step proved unreliable. The second method introduced azide groups into fully synthetic peptides using the amino acid azidohomoalanine (Aha). Click chemistry with synthetic propargyl glycosides allowed further modification to homogeneous glycopeptide analogues which were shown to be compatible with native chemical ligation, a proven tool for glycoprotein synthesis and semisynthesis. Ultimately both methods may eventually facilitate glycoprotein synthesis and remodelling such that the biological activity and immunogenicity may be modulated to suit future therapeutic requirements.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.565248  DOI: Not available
Share: