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.
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Acknowledgements
Generous support by the Danish National Research Foundation (grant no. DNRF79) and by the Villum Foundation (grant no. VKR022710) is gratefully acknowledged. The authors thank C.W. Ronson and J.T. Sullivan for bacterial production of the EPS octa-saccharide and M. loti R7A Nod factors.
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M.B.T., J. Sauer and K.J.J. designed the study. C.T.H., N.N.M., J. Sauer and M.B.T. performed the experiments. C.T.H., N.N.M., J. Stougaard, M.B.T. and K.J.J. wrote the manuscript.
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Hjuler, C., Maolanon, N., Sauer, J. et al. Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies. Nat Protoc 12, 2411–2422 (2017). https://doi.org/10.1038/nprot.2017.109
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DOI: https://doi.org/10.1038/nprot.2017.109
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