Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.822982
Title: Chemoenzymatic synthesis of carbohydrates and glycoconjugates
Author: Huang, Kun
ISNI:       0000 0005 0289 4578
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2018
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Abstract:
Complex carbohydrates are widespread in all living organisms, particularly on cell surfaces, and play a crucial role in various biological processes through interaction with glycan binding proteins. Accessibility of structurally defined glycans is very important for structure-function studies. Even though chemical synthesis has been improved considerably, it is still a laborious and tedious approach to obtain target glycans because of multiple protection and deprotection steps. Enzymatic synthesis has emerged as an alternative method, which can be also combined with chemical synthesis (i.e., chemoenzymatic synthesis) that significantly facilitates the manufacture of diverse complex glycans. In this project, we initially identified active glycoenzymes for glycan synthesis. One bacterial galactokinase from Leminorella grimontii was identified and characterised. Kinetic studies and evaluation of substrate specificity were conducted. Subsequently, it was used to construct a panel of substituted 6-aminogalactose-1-phosphates combined with an engineered galactose oxidase and chemical reductive amination in a one-pot system, providing a useful chemical probe toolkit for glycomics studies. Furthermore, three active ß1,4 galactosyltransferases from Neisseria meningitidis were obtained. The reversible activity of two close homologs was discovered by newly synthesised ionic liquid tagged substrates (i.e., ITag-Glc and ITag-GlcNAc), which can transfer galactose from inexpensive lactose to ITag-Glc and ITag-GlcNAc in the presence of catalytic amount of UDP. The reaction conditions were optimised and the practical application was demonstrated by preparative synthesis of pNP-ß-LacNAc. Lastly, our recently developed label-free glycoarray technique was utilised to identify specific carbohydrate ligands of C-type lectins including human (DC-SIGN R, MCL, DCIR, DC-SIGN, Langerin, MGL and DC-SIGN R-hFc) and murine (mLangerin-hFc and mDCL-hFc). MCL, DC-SIGN and MGL bound to Lewis X, Fuc, and GalNAc respectively. Langerin could recognise Man, GlcNAc, Fuc, Xyl and GalNAc. DCIR was detected on spots functionalised with LacNAc and Lewis X. However, no binding was observed for DC-SIGN R, DC-SIGN R-hFc, mLangerin-hFc and mDCL-hFc. Additionally, our glycoarray was proven to be reusable, which allowed to recycle the precious glycosides.
Supervisor: Flitsch, Sabine ; Barran, Perdita Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.822982  DOI: Not available
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