Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manβ1,4GlcNAc library

Tessa Keenan, Natasha e. Hatton, Jack Porter, Jean-Baptiste Vendeville, David e. Wheatley, Mattia Ghirardello, Alice. j. c. Wahart, Sanaz Ahmadipour, Julia Walton, M. carmen Galan, Bruno Linclau, Gavin j. Miller*, Martin a. Fascione*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

Abstract

β-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manβ1,4GlcNAc a constituent of the core N-glycan in eukaryotes was recently identified as an immune activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of β-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manβ1,4GlcNAc analogues using the β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer β-mannan-like glycans. We also pioneer a “reverse thiophosphorylase” enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorolysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases.
Original languageEnglish
Pages (from-to)11638-11646
Number of pages9
JournalChemical Science
Volume14
Issue number42
Early online date29 Sept 2023
DOIs
Publication statusPublished - 1 Nov 2023

Bibliographical note

Funding Information:
MAF thanks UKRI (EP/X023680/1) for project grant funding. UK Research and Innovation (UKRI, Future Leaders Fellow-ship, MR/T019522/1) and the Engineering and Physical Sciences Research Council (EP/P000762/1) are thanked for project grant funding to GJM. Keele University are thanked for PhD studentship funding to AW and JP. BL thanks EPSRC core capability funding EP/K039466/1. MCG thanks Cancer Research UK (grant number C30758/A2979) and European Research Council (ERC-COG GLYCOTOOLS 648239). MAF, BL and MCG are grateful to the Industrial Biotechnology Catalyst (Innovate UK, BBSRC, EPSRC) to support the translation, development and commercialization of innovative Industrial Biotechnology processes: BB/M028941/1, BB/M02847X/1, BB/M028976/1. We thank Dr Ed Bergstrom and The York Center of Excellence in Mass Spectrometry was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1; EP/M028127/1). We thank Dr Alex Heyam for discussions around the IPAP NMR experiments.

Publisher Copyright:
© 2023 The Royal Society of Chemistry.

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