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Semi-synthesis of glycoproteins by auxiliary-mediated native chemical ligation

Nyandoro, Kudakwashe; (2022) Semi-synthesis of glycoproteins by auxiliary-mediated native chemical ligation. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Nature facilitates glycosylation of proteins heterogeneously such that most biologically expressed glycoproteins are obtained as a mixture of glycoforms. This heterogeneity complicates the purification and analysis of expressed glycoproteins. It hinders understanding of how activity is mediated at molecular level. A number of chemical and biological methods have been explored to allow access to glycoproteins of a defined structure. The most efficient protocol thus far involves a chemical assembly of a polypeptide linked to N-acetyl glucosamine (GlcNAc). The monosaccharide is then extended enzymatically to the target oligosaccharide. Major limitations of the protocol are encountered in the assembly of the polypeptide-GlcNAc conjugate as a number of steps are required which often lowers the overall yield of the conjugate. In this thesis, a semi-synthetic procedure towards a protein-GlcNAc conjugate is pursued. The method utilises cleavable auxiliaries to facilitate the ligation of GlcNAc to the side chain of peptide and protein thioesters. Sugar-linked auxiliaries were synthesised and employed in native chemical ligations (NCL) with peptide thioesters at the C-terminus and on the side chains of aspartic and glutamic acids in model studies. A sugar-linked auxiliary anticipated to yield a native peptide-GlcNAc linkage failed to mediate the ligation. This was due to the inherent hemiaminal linkage which is unstable and caused its fragmentation. The hemiaminal linkage was extended with a methylene stabiliser which allowed the new sugar-linked auxiliaries to mediate ligations of GlcNAc to peptide thioesters at the C-terminus and on the side chains. Side-chain peptide thioesters were constructed by Fmoc solid phase peptide synthesis (SPPS) using Fmoc amino acid hydrazides. The hydrazides were converted to the thioesters post SPPS and cleavage. The Fmoc amino acid hydrazides were obtained by a novel regioselective ring opening of amino acid anhydrides method. A sperm whale myoglobin side-chain thioester was obtained by bacterial protein expression through incorporating an unnatural amino acid (thioD) at a defined site. An orthogonal Methanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPylRS)/ tRNACUA pair was utilised in the expression. The model protein thioester was ligated to the methylene extended sugar-linked auxiliary and approximately 30% conversion to the desired protein-GlcNAc conjugate was observed. Incomplete reaction may have been caused by slow ligation rates and side reactions such as oxidation of thiols. Slow ligations result due to the low reactivities of auxiliaries and the protein alkyl thioester (myoglobin thioD) in facilitating NCL. Alternative methods such as employing protein hydrazides which are converted to more reactive aryl thioesters in situ are currently being explored. The new method has enabled the semi-synthesis of protein- and peptide-GlcNAc neoglycoconjugates with the natural linkage extended by a methylene group.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Semi-synthesis of glycoproteins by auxiliary-mediated native chemical ligation
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10153859
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