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Synthesis of Phenoxyundecyl Diphosphate Disaccharides for Studies of the Biosynthesis of O Antigenic Polysaccharides in Enteric Bacteria

  • Vladimir Torgov
  • Leonid Danilov
  • Natalia Utkina
  • Vladimir Veselovsky
  • Alexander Kocev
  • Inka Brockhausen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)

Abstract

The biosynthesis of O antigenic polysaccharides in enteric bacteria from nucleoside diphosphate sugars (donor substrates) is catalyzed by the corresponding glycosyltransferases and proceeds through the intermediate formation of undecaprenyl diphosphate sugars (acceptor substrates). To study this process, a chemical synthesis of the compounds having the natural structure or their modified analogs is necessary. The phosphoroimidazolidate method is a universal method for synthesis of lipid diphosphate disaccharides containing 2-acetamido-2-deoxyglycosyl residue at the reducing end of the disaccharide moiety and 11-phenoxyundecyl residue as lipid fragment of the molecule. We report here protocols to synthesize the disaccharides P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-d-rhamnopyranosyl-α-d-glucopyranosyl) diphosphate [d-Rha(α1-3)-d-GlcNAcα-PP-PhU, Compound 1] and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-d-galactopyranosyl-α-d-galactopyranosyl) diphosphate [d-Gal(β1-3)-d-GalNAcα-PP-PhU, Compound 6]. We describe the procedures for identification and structure estimation of compounds by TLC, NMR, and MS. We also include the biochemical testing of Compound 6 with α2,3-sialyltransferase WbwA from Escherichia coli O104.

Key words

Chemical synthesis Phosphoroimidazolidate method d-Rha(α1-3)-d-GlcNAcα-PP-PhU d-Gal(β1-3)-d-GalNAcα-PP-PhU TLC NMR MS Sialyltransferase WbwA 

Notes

Acknowledgments

This work was supported by the Russian Foundation for Basic Research, Project 16-04-00372 and the Canadian Institutes of Health Research (GlycoNet) (to IB).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Vladimir Torgov
    • 1
  • Leonid Danilov
    • 1
  • Natalia Utkina
    • 1
  • Vladimir Veselovsky
    • 1
  • Alexander Kocev
    • 2
  • Inka Brockhausen
    • 2
  1. 1.N.D. Zelinsky Institute of Organic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Department of Biomedical and Molecular SciencesQueen’s UniversityKingstonCanada

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