Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Synthesis of non-natural ManNAc analogs for the expression of thiols on cell-surface sialic acids

Nature Protocols volume 1, pages 2377–2385 (2006)Cite this article

Abstract

The sialic acid biosynthetic pathway in mammalian cells utilizes N-acetyl-D-mannosamine (ManNAc) as a natural metabolic precursor and has the remarkable ability to biosynthetically process non-natural ManNAc analogs. Herein, we describe a recipe-style protocol for the synthesis of the novel peracetylated analog Ac5ManNTGc (1) that contains a pendant acetylthio- group and enables incorporation of thiol functionalities into the glycocalyx of living cells. We also describe the synthesis of the oxygen analog Ac5ManNGc (2), which serves as an appropriate control compound for biological experiments with 1. Both 1 and 2 were prepared from a reported, common intermediate 8, which is selectively acetylated at the hydroxyl groups. In contrast to previous methods, this synthetic approach introduces O-acetyl groups first, followed by N-acylation. Starting from the commercially available D-mannosamine hydrochloride (5), gram quantities of both 1 and 2 can be prepared over five steps in about 2–3 weeks.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Synthesis of non-natural ManNAc analogs.
Figure 2: Uptake and metabolic response to peracetylated ManNAc.

Similar content being viewed by others

References

  1. Sampathkumar, S.-G., Li, A.V., Jones, M.B., Sun, Z. & Yarema, K.J. Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat. Chem. Biol. 2, 149–152 (2006).

    Article  CAS  Google Scholar 

  2. Sampathkumar, S.-G., Jones, M.B. & Yarema, K.J. Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry. Nat. Protocols 1, 1840–1851 (2006).

    Article  CAS  Google Scholar 

  3. Angelino, N.J., Bernacki, R.J., Sharma, M., Doson-Simmons, O. & Korytnyk, W. Versatile intermediates in the selective modification of the amino function of 2-amino-2-dexoy-D-mannopyranose and the 3-position of 2-acetamido-2-deoxy-D-mannose: potential membrane modification in neoplastic control. Carbohyd. Res. 276, 99–115 (1995).

    Article  CAS  Google Scholar 

  4. Gooch, J. & Hawtrey, A.O. Synthesis of thiol-containing analogues of puromycin and a study of their interaction with N-acetylphenylalanyl-transfer ribonucleic acid on ribosomes to form thioesters. Biochem. J. 149, 209–220 (1975).

    Article  CAS  Google Scholar 

  5. Collins, B.E., Fralich, T.J., Itonori, S., Ichikawa, Y. & Schnaar, R.L. Conversion of cellular sialic acid expression from N-acetyl- to N-glycolylneuraminic acid using a synthetic precursor, N-glycolylmannosamine pentaacetate: inhibition of myelin-associated glycoprotein binding to neural cells. Glycobiology 10, 11–20 (2000).

    Article  CAS  Google Scholar 

  6. Jacobs, C.L. et al. Substrate specificity of the sialic acid biosynthetic pathway. Biochemistry 40, 12864–12874 (2001).

    Article  CAS  Google Scholar 

  7. Saxon, E. et al. Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. J. Am. Chem. Soc. 124, 14893–14902 (2002).

    Article  CAS  Google Scholar 

  8. Luchansky, S.J. et al. Constructing azide-labeled cell surfaces using polysaccharide biosynthetic pathways. Methods Enzymol. 362, 249–272 (2003).

    Article  CAS  Google Scholar 

  9. Keppler, O.T., Horstkorte, R., Pawlita, M., Schmidt, C. & Reutter, W. Biochemical engineering of the N-acyl side chain of sialic acid: biological implications. Glycobiology 11, 11R–18R (2001).

    Article  CAS  Google Scholar 

  10. Kim, E.J. et al. Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acetylmannosamine (ManNAc) analogs in Jurkat (human T-lymphoma-derived) cells. J. Biol. Chem. 279, 18342–18352 (2004).

    Article  CAS  Google Scholar 

  11. Hinderlich, S., Oetke, C. & Pawlita, M. in Handbook of Carbohydrate Engineering (ed. Yarema, K.J.) Ch. 13, 387–405 (Francis & Taylor/CRC Press, Boca Raton, Florida, 2005).

    Book  Google Scholar 

  12. Jacobs, C.L. et al. Metabolic labeling of glycoproteins with chemical tags through unnatural sialic acid biosynthesis. Methods Enzymol. 327, 260–275 (2000).

    Article  CAS  Google Scholar 

  13. Biology and brimstone (editorial). Nat. Chem. Biol. 2, 169 (2006).

  14. Villavicencio-Lorini, P., Laabs, S., Danker, K., Reutter, W. & Horstkorte, R. Biochemical engineering of the acyl side chain of sialic acids stimulates integrin-dependent adhesion of HL60 cells to fibronectin. J. Mol. Med. 80, 671–677 (2002).

    Article  CAS  Google Scholar 

  15. Jones, M.B. et al. Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids. Biotechnol. Bioeng. 85, 394–405 (2004).

    Article  CAS  Google Scholar 

  16. Sarkar, A.K., Fritz, T.A., Taylor, W.H. & Esko, J.D. Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal β1,4GalcNAc β-O-naphthalenemethanol. Proc. Natl. Acad. Sci. USA 92, 3323–3327 (1995).

    Article  CAS  Google Scholar 

  17. Fishman, P.H. & Brady, R.O. Biosynthesis and function of gangliosides. Science 194, 906–915 (1976).

    Article  CAS  Google Scholar 

  18. Wang, Z., Sun, Z., Li, A.V. & Yarema, K.J. Roles for GNE outside of sialic acid biosynthesis: modulation of sialyltransferase and BiP expression, GM3 and GD3 biosynthesis, proliferation and apoptosis, and ERK1/2 phosphorylation. J. Biol. Chem. 281, 27016–27028 (2006).

    Article  CAS  Google Scholar 

  19. Kim, E.J., Jones, M.B., Rhee, J.K., Sampathkumar, S.-G. & Yarema, K.J. Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications. Biotechnol. Prog. 20, 1674–1682 (2004).

    Article  Google Scholar 

  20. Menozzi, G. et al. Reaction of 2-dimethylaminomethylene-1,3-diones with nucleophiles. 13. 4H-Thieno[3,4-c]pyrazole derivatives with antiinflammatory, analgesic, antipyretic and platelet antiaggregating activities. Farmaco 47 (1992).

  21. Harwood, L.M., Mooday, C.J. & Percy, J.M. Experimental Organic Chemistry: Standard and Microscale (Blackwell Science, Malden, MA, 1999).

    Google Scholar 

  22. Tanner, M.E. The enzymes of sialic acid biosynthesis. Bioorg. Chem. 33, 216–228 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the funding from the Whitaker Biomedical Engineering Institute, Arnold and Mabel Beckman Foundation, the National Institutes of Health (1R01CA112314-01A1) and the National Science Foundation (QSB-0425668).

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, The Johns Hopkins University, 3400 N Charles Street, Clark Hall Rm 106A, Baltimore, 21218, Maryland, USA

    Srinivasa-Gopalan Sampathkumar, Adrienne V Li & Kevin J Yarema

Authors
  1. Srinivasa-Gopalan Sampathkumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adrienne V Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kevin J Yarema
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kevin J Yarema.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sampathkumar, SG., Li, A. & Yarema, K. Synthesis of non-natural ManNAc analogs for the expression of thiols on cell-surface sialic acids. Nat Protoc 1, 2377–2385 (2006). https://doi.org/10.1038/nprot.2006.319

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.319

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing