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Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan-protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands. Here, we review recent progress in photo-crosslinking approaches targeting glycan-mediated interactions. We discuss two prominent emerging strategies: 1) development of photo-crosslinkable oligosaccharide ligands to identify GBP receptors; and 2) cell-surface glyco-engineering to identify glycoconjugate ligands of GBPs. Overall, photoaffinity labeling affords valuable insights into complex glycan-protein networks and is poised to help elucidate the glycan-protein interactome, providing novel targets for therapeutic intervention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1 F383A . The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc- or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc- or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.

All cells are decorated with complex carbohydrate structures called glycans that serve as ligands for glycan-binding proteins (GBPs) to mediate a wide range of biological processes. Understanding the specific functions of glycans is key to advancing an understanding of human health and disease. However, the lack of convenient and accessible tools to study glycan-based interactions has been a defining challenge in glycobiology. Thus, the development of chemical and biochemical strategies to address these limitations has been a rapidly growing area of research. In this review, we describe the use of glycosyltransferases (GTs) as versatile tools to facilitate a greater understanding of the biological roles of glycans. We highlight key examples of how GTs have streamlined the preparation of well-defined complex glycan structures through chemoenzymatic synthesis, with an emphasis on synthetic strategies allowing for site- and branch-specific display of glyco-epitopes. We also describe how GTs have facilitated expansion of glyco-engineering strategies, on both glycoproteins and cell surfaces. Coupled with advancements in bioorthogonal chemistry, GTs have enabled selective glyco-epitope editing of glycoproteins and cells, selective glycan subclass labeling, and the introduction of novel biomolecule functionalities onto cells, including defined oligosaccharides, antibodies, and other proteins. Collectively, these approaches have contributed great insight into the fundamental biological roles of glycans and are enabling their application in drug development and cellular therapies, leaving the field poised for rapid expansion., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)