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Divergent synthesis of amino acid-linked O-GalNAc glycan core structures

Nature Protocols volume 20, pages 480–517 (2025)Cite this article

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Abstract

O-GalNAc glycans, also known as mucin-type O-glycans, are primary constituents of mucins on various mucosal sites of the body and also ubiquitously expressed on cell surface and secreted proteins. They have crucial roles in a wide range of physiological and pathological processes, including tumor growth and progression. In addition, altered expression of O-GalNAc glycans is frequently observed during different disease states. Research dedicated to unraveling the structure–function relationships of O-GalNAc glycans has led to the discovery of disease biomarkers and diagnostic tools and the development of O-glycopeptide-based cancer vaccines. Many of these efforts require amino acid-linked O-GalNAc core structures as building blocks to assemble complex O-glycans and glycopeptides. There are eight core structures (cores one to eight), from which all mucin-type O-glycans are derived. In this protocol, we describe the first divergent synthesis of all eight cores from a versatile precursor in practical scales. The protocol involves (i) chemical synthesis of the orthogonally protected precursor (3 days) from commercially available materials, (ii) chemical synthesis of five unique glycosyl donors (1–2 days for each donor) and (iii) selective deprotection of the precursor and assembly of the eight cores (2–4 days for each core). The procedure can be adopted to prepare O-GalNAc cores linked to serine, threonine and tyrosine, which can then be utilized directly for solid-phase glycopeptide synthesis or chemoenzymatic synthesis of complex O-glycans. The procedure empowers researchers with fundamental organic chemistry skills to prepare gram scales of any desired O-GalNAc core(s) or all eight cores concurrently.

Key points

  • O-GalNAc glycans are primary constituents of mucins and are ubiquitously expressed on cell surface and secreted proteins. Synthetic versions are used to study their role in normal and pathological physiology. Most O-GalNAc glycans can be synthesized from eight core structures.

  • In this protocol, the eight amino acid-linked O-GalNAc core structures are synthesized from a single precursor and five glycosyl donors. The precursor is Tn antigen with distinct orthogonal protecting groups.

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Fig. 1: Structures of Tn antigen and O-GalNAc glycan cores 1–8, and their biosynthetic pathway.
Fig. 2: Divergent synthetic strategy to O-GalNAc glycan cores 1–8.
Fig. 3: The complete synthetic process flowchart.
Fig. 4: Chemical synthetic routes to generate glycosyl donors 10 and 11 from commercial substrates.
Fig. 5: Chemical synthetic routes to generate glycosyl donors 12–14.
Fig. 6: Gram-scale synthesis of the versatile precursor 9 and divergent synthesis of cores 1–4.
Fig. 7: Divergent synthesis of cores 5–8 from 9.

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Data Availability

All relavent data (NMR and MS results) are available in ‘Analytical Data’ and Supplementary Information.

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Acknowledgements

This work is supported by the National Institute of Health (R44GM123820 and U54HL142019 to L.L.). T.S. thanks the support of Center for Diagnostics & Therapeutics fellowship.

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Authors and Affiliations

  1. Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA

    Madhusudhan Reddy Gadi, Jinghua Han, Tangliang Shen, Shuquan Fan, Zhongying Xiao & Lei Li

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  1. Madhusudhan Reddy Gadi
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Contributions

M.R.G., J.H. and L.L. designed the research and developed the method. M.R.G., J.H., T.S. and S.F. performed the experiments. M.R.G., J.H. and L.L. drafted the manuscript, which was revised by all authors.

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Correspondence to Lei Li.

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Key references using this protocol

Wang, S. et al. Nat. Commun. 12, 3573 (2021): https://doi.org/10.1038/s41467-021-23428-x

Gadi, M. R. et al. Chem. Sci. 14, 1837–1843 (2023): https://doi.org/10.1039/D2SC06925C

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Supplementary Information

Supplementary Figs. 1–3, Table 1 and NMR data and spectra.

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Gadi, M.R., Han, J., Shen, T. et al. Divergent synthesis of amino acid-linked O-GalNAc glycan core structures. Nat Protoc 20, 480–517 (2025). https://doi.org/10.1038/s41596-024-01051-6

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