Abstract
Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP–GlcNAc/GalNAc. Other UDP–GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from l-fucose, GDP–fucose is constructed by one bifunctional enzyme l-fucose pyrophosphorylase (FKP) via two reactions.
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Acknowledgements
P.G.W. acknowledges National Cancer Institute (R01 CA118208), NSF (CHE-0616892) and NIH (R01 AI083754, R01 HD061935 and R01 GM085267) for financial support. W.G. acknowledges China Scholarship Council for financial support. We thank Robert Woodward for proofreading the manuscript.
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G.Z., W.G. and L.C. contributed equally to this work. P.G.W. supervised the project; L.C., W.G. and P.G.W. designed and carried out enzymatic synthesis of UDP–GlcNAc/GalNAc experiments and analyzed data; G.Z. and P.G.W. designed and carried out enzymatic synthesis of GDP–fucose experiments and analyzed data; L.C., W.G. and G.Z. wrote the paper; and P.G.W. revised the manuscript; all authors discussed the results and implications and commented on the manuscript at all stages.
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Supplementary Data: NMR, MS data and spectra (PDF 703 kb)
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Zhao, G., Guan, W., Cai, L. et al. Enzymatic route to preparative-scale synthesis of UDP–GlcNAc/GalNAc, their analogues and GDP–fucose. Nat Protoc 5, 636–646 (2010). https://doi.org/10.1038/nprot.2010.3
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DOI: https://doi.org/10.1038/nprot.2010.3
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