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Enzymatic route to preparative-scale synthesis of UDP–GlcNAc/GalNAc, their analogues and GDP–fucose

Nature Protocols volume 5, pages 636–646 (2010)Cite this article

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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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Figure 1
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Figure 6: TLC analysis of enzymatic synthesis of N-acetylglucosamine 1-phosphate (GlcNAc-1-P) and N-acetylgalactosamine 1-phosphate (GalNAc-1-P) using N-acetylhexosamine 1-kinase (NahK).
Figure 7: TLC analysis of enzymatic synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) using N-acetylglucosamine uridyltransferase (GlmU).
Figure 8: TLC analysis of enzymatic synthesis of GDP–fucose.

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References

  1. Koeller, K.M. & Wong, C.H. Complex carbohydrate synthesis tools for glycobiologists: enzyme-based approach and programmable one-pot strategies. Glycobiology 10, 1157–1169 (2000).

    Article  CAS  Google Scholar 

  2. Varki, A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130 (1993).

    Article  CAS  Google Scholar 

  3. Chen, X., Andreana, P.R. & Wang, P.G. Carbohydrates in transplantation. Curr. Opin. Chem. Biol. 3, 650–658 (1999).

    Article  CAS  Google Scholar 

  4. Xia, C. et al. Synthesis and biological evaluation of alpha-galactosylceramide (KRN7000) and isoglobotrihexosylceramide (iGb3). Bioorg. Med. Chem. Lett. 16, 2195–2199 (2006).

    Article  CAS  Google Scholar 

  5. Yi, W. et al. Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide. J. Am. Chem. Soc. 127, 2040–2041 (2005).

    Article  CAS  Google Scholar 

  6. Mazmanian, S.K., Round, J.L. & Kasper, D.L. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620–625 (2008).

    Article  CAS  Google Scholar 

  7. Drouillard, S., Driguez, H. & Samain, E. Large-scale synthesis of H-antigen oligosaccharides by expressing Helicobacter pylori alpha1,2-fucosyltransferase in metabolically engineered Escherichia coli cells. Angew. Chem. Int. Ed. Engl. 45, 1778–1780 (2006).

    Article  CAS  Google Scholar 

  8. Mahal, L.K., Yarema, K.J. & Bertozzi, C.R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125–1128 (1997).

    Article  CAS  Google Scholar 

  9. Perugino, G., Trincone, A., Rossi, M. & Moracci, M. Oligosaccharide synthesis by glycosynthases. Trends Biotechnol. 22, 31–37 (2004).

    Article  CAS  Google Scholar 

  10. Varki, A. et al. Essentials of Glycobiology (Cold Spring Harbor Laboratory Press, New York, USA, 1999).

  11. Thibodeaux, C.J., Melancon, C.E. & Liu, H.-W. Unusual sugar biosynthesis and natural product glycodiversification. Nature 446, 1008–1016 (2007).

    Article  CAS  Google Scholar 

  12. Sim, M.M., Kondo, H. & Wong, C.H. Synthesis of dibenzyl glycosyl phosphites using dibenzyl N,N-diethylphosphoramidite as phosphitylating reagent: an effective route to glycosyl phosphates, nucleotides, and glycosides. J. Am. Chem. Soc. 115, 2260–2267 (1993).

    Article  CAS  Google Scholar 

  13. Heidlas, J.E., Lees, W.J., Pale, P. & Whitesides, G.M. Gram-scale synthesis of uridine 5′-diphospho-N-acetylglucosamine: comparison of enzymic and chemical routes. J. Org. Chem. 57, 146–151 (1992).

    Article  CAS  Google Scholar 

  14. Wagner, G.K., Pesnot, T. & Field, R.A. A survey of chemical methods for sugar-nucleotide synthesis. Nat. Prod. Rep. 26, 1172–1194 (2009).

    Article  CAS  Google Scholar 

  15. Timmons, S.C. & Jakeman, D.L. Stereoselective chemical synthesis of sugar nucleotides via direct displacement of acylated glycosyl bromides. Org. Lett. 9, 1227–1230 (2007).

    Article  CAS  Google Scholar 

  16. Nishimoto, M. & Kitaoka, M. Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum. Appl. Environ. Microbiol. 73, 6444–6449 (2007).

    Article  CAS  Google Scholar 

  17. Cai, L. et al. A chemoenzymatic route to N-acetylglucosamine-1-phosphate analogues: substrate specificity investigations of N-acetylhexosamine 1-kinase. Chem. Commun. 2944–2946 (2009).

  18. Cai, L. et al. Substrate specificity of N-acetylhexosamine kinase towards N-acetylgalactosamine derivatives. Bioorg. Med. Chem. Lett. 19, 5433–5435 (2009).

    Article  CAS  Google Scholar 

  19. Guan, W., Cai, L., Fang, J., Wu, B. & Wang, P.G. Enzymatic synthesis of UDP-GlcNAc/UDP-GalNAc analogs using N-acetylglucosamine 1-phosphate Uridyltransferase (GlmU). Chem. Commun. 6976–6978 (2009).

  20. Coyne, M.J., Reinap, B., Lee, M.M. & Comstock, L.E. Human symbionts use a host-like pathway for surface fucosylation. Science 307, 1778–1781 (2005).

    Article  CAS  Google Scholar 

  21. Yi, W. et al. Remodeling bacterial polysaccharides by metabolic pathway engineering. Proc. Natl. Acad. Sci. USA 106, 4207–4212 (2009).

    Article  CAS  Google Scholar 

  22. Wang, W. et al. Chemoenzymatic synthesis of GDP-l-fucose and the Lewis X glycan derivatives. Proc. Natl. Acad. Sci. USA 106, 16096–16101 (2009).

    Article  CAS  Google Scholar 

  23. Olsen, L.R. & Roderick, S.L. Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites. Biochemistry 40, 1913–1921 (2001).

    Article  CAS  Google Scholar 

  24. Bourgeaux, V., Piller, F. & Piller, V. Two-step enzymatic synthesis of UDP-N-acetylgalactosamine. Bioorg. Med. Chem. Lett. 15, 5459–5462 (2005).

    Article  CAS  Google Scholar 

  25. Yu, H., Chokhawala, H.A., Huang, S. & Chen, X. One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities. Nat. Protoc. 1, 2485–2492 (2006).

    Article  CAS  Google Scholar 

  26. Wu, B., Zhang, Y. & Wang, P.G. Identification and characterization of GDP-D-mannose 4,6-dehydratase and GDP-L-fucose synthetase in a GDP-L-fucose biosynthetic gene cluster from Helicobacter pylori. Biochem. Biophys. Res. Commun. 285, 364–371 (2001).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, USA

    Guohui Zhao, Wanyi Guan, Li Cai & Peng George Wang

  2. National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong, China

    Wanyi Guan

  3. College of Pharmacy, Nankai University, Tianjin, China

    Guohui Zhao

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  1. Guohui Zhao
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  4. Peng George Wang
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Contributions

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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Correspondence to Peng George Wang.

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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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