Affordable Access

deepdyve-link
Publisher Website

Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies.

Authors
  • Hjuler, Christian T1, 2
  • Maolanon, Nicolai N1, 2
  • Sauer, Jørgen1, 2
  • Stougaard, Jens3, 4
  • Thygesen, Mikkel B1, 2
  • Jensen, Knud J1, 2
  • 1 Centre for Carbohydrate Recognition and Signaling, Copenhagen University, Frederiksberg, Denmark. , (Denmark)
  • 2 Department of Chemistry, University of Copenhagen, Frederiksberg, Denmark. , (Denmark)
  • 3 Centre for Carbohydrate Recognition and Signaling, Aarhus University, Aarhus, Denmark. , (Denmark)
  • 4 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. , (Denmark)
Type
Published Article
Journal
Nature protocols
Publication Date
Nov 01, 2017
Volume
12
Issue
11
Pages
2411–2422
Identifiers
DOI: 10.1038/nprot.2017.109
PMID: 29072708
Source
Medline
License
Unknown

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.

Report this publication

Statistics

Seen <100 times