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Title: Chemical tools to probe carbohydrate recognition and glycoconjugate biosynthesis
Author: Ivanova, Irina
ISNI:       0000 0004 5919 6503
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2015
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In eukaryotes the assembly of complex glycoconjugate structures takes place in the endoplasmic reticulum (ER) and Golgi apparatus, where a number of specific membrane-bound glycosyltransferases transfer sugar moiety from soluble or lipid linked activated sugar nucleotides to variety of acceptors with correct stereo and regioselectivity. Many glycoconjugates structures have been elucidated; however, most glycosyltransferases involved in their biosynthesis have not been identified and characterised. Currently, cell-free radiolabelled assays are widely used to detect their activities. These types of assays are sensitive and broadly applicable, but can be complicated by well-known factors related to the application of radioactive substances. The purpose of this study was the development of simple and sensitive fluorescence-based methodology to detect membrane-bound glycosyltransferases activities, which could replace traditional radiolabelled methods used in the field. Fluorescent acceptors, such as 4-(1-(7-hydroxy-coumarin-3-yl)-1H-1,2,3-triazol-4-yl)-)propyl α-D-mannopyranoside (α-Man-HTC) (2.12) and 4-(1-(7-hydroxy-coumarin-3-yl)-1H-1,2,3-triazol-4-yl)-)propyl α-D-mannopyranosyl-(1→6)-α-D-mannopyranoside (α-Man-1,6-α-Man-HTC) (2.13) were designed to have linkages that mimic the authentic acceptors. The fluorescence-based assays were benchmarked against established radiolabelled assays to detect mannosyltransferase activities involved in the biosynthesis of lipoarabinomannan in Mycobacterium smegmatis and galactosyltransferase activities involved in the decoration of GPI anchor in Trypanosoma brucei. Further application of this methodology allowed detection and characterisation of mannosyltransferase activities responsible for biosynthesis of N-glycans in Euglena gracilis, along with detection of N-acetylglucosamine-1-phosphate transferase activity. In a separate study, a series of linear and cyclic 1,4/1,5-triazole-linked-pseudo-galctooligomers were synthesised from 2-(2-(2-azidoethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-β-D-galactopyranoside (4.07) utilising Cu(I)-catalysed click reactions. These compounds were used to mimic authentic substrates for Trypanosoma cruzi trans-sialidases in order to block Trypanosoma cruzi macrophage invasion.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available