Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies

Nat Protoc. 2017 Nov;12(11):2411-2422. doi: 10.1038/nprot.2017.109. Epub 2017 Oct 26.

Abstract

Glycobiology, in particular the study of carbohydrate-protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments. The first stage is to add an oligo(ethylene glycol) linker (OEG-linker) that has a terminal triphenylmethanethiol group to the reducing end of the oligosaccharide by oxime formation catalyzed by aniline. The triphenylmethyl (trityl) tag is then removed from the linker to expose a thiol (stage 2) to allow a conjugation reaction at the thiol group (stage 3). There are many possible conjugation reactions, depending on the desired application. Examples shown in this protocol are as follows: (i) coupling of the oligosaccharide to a support for surface plasmon resonance (SPR) studies, (ii) fluorescence labeling for microscale thermophoresis (MST) or bioimaging, and (iii) biotinylation for biolayer interferometry (BLI) studies. This protocol starts from unprotected carbohydrates and provides glycoconjugates in milligram amounts in just 2 d.

MeSH terms

  • Aniline Compounds / chemistry
  • Biotinylation
  • Catalysis
  • Chemistry Techniques, Synthetic*
  • Glycoconjugates / chemical synthesis*
  • Glycomics / methods*
  • Interferometry
  • Lipopolysaccharides / chemistry*
  • Optical Imaging
  • Oximes / chemistry
  • Polyethylene Glycols / chemistry
  • Protein Binding
  • Sulfhydryl Compounds / chemistry*
  • Surface Plasmon Resonance
  • Trityl Compounds / chemistry*

Substances

  • Aniline Compounds
  • Glycoconjugates
  • Lipopolysaccharides
  • Nod factor IV, Rhizobium meliloti
  • Oximes
  • Sulfhydryl Compounds
  • Trityl Compounds
  • Polyethylene Glycols
  • aniline