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Journal of Biomolecular NMR

, Volume 59, Issue 2, pp 95–110 | Cite as

NMR structure analysis of uniformly 13C-labeled carbohydrates

  • Carolina Fontana
  • Helena Kovacs
  • Göran Widmalm
Article

Abstract

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of 13C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly 13C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-13C)-sucrose, 342 Da] and one compound of medium molecular weight (13C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The 13C resonances are traced using 13C–13C correlations from homonuclear experiments, such as (H)CC–CT–COSY, (H)CC–NOESY, CC–CT–TOCSY and/or virtually decoupled (H)CC–TOCSY. Based on the assignment of the 13C resonances, the 1H chemical shifts are derived in a straightforward manner using one-bond 1H–13C correlations from heteronuclear experiments (HC–CT–HSQC). In order to avoid the 1 J CC splitting of the 13C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either 13C or 1H detected experiments, namely CC–CT–COSY, band-selective (H)CC–TOCSY, HC–CT–HSQC–NOESY or long-range HC–CT–HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the 1H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the 13C-enriched polysaccharide were assigned by using HC–H2BC spectra. The assignment of the N-acetyl groups with 15N at natural abundance was completed by using HN–SOFAST–HMQC, HNCA, HNCO and 13C-detected (H)CACO spectra.

Keywords

Carbohydrates 13C-uniform labeling NMR Structure determination 

Notes

Acknowledgments

This work was supported by grants from the Swedish Research Council and the Knut and Alice Wallenberg Foundation. The research that has led to these results has received funding from the European Commission’s Seventh Framework Programme FP7/2007–2013 under grant agreement no. 215536.

Supplementary material

10858_2014_9830_MOESM1_ESM.pdf (587 kb)
Supplementary material 1 (PDF 587 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Carolina Fontana
    • 1
  • Helena Kovacs
    • 2
  • Göran Widmalm
    • 1
  1. 1.Department of Organic Chemistry, Arrhenius LaboratoryStockholm UniversityStockholmSweden
  2. 2.Bruker BioSpin AGFällandenSwitzerland

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