Determination of glycan structure by NMR

Authors：

Category

Isolation & structural analysis of glycans

Protocol Name

Authors

Yamaguchi, Yoshiki
Structural Glycobiology Team, RIKEN

KeyWords

glycan structure NMR

Methods

Preparation of the glycan sample:

1)	Isolate the glycan at >90% homogeneity and lyophilize the sample. Since buffers or salts may prevent the NMR analyses, they should be removed in advance as much as possible.	Comment 0

Dissolve the sample with 200 or 500 μL of D₂O. Transfer the solution into a 5-mm NMR tube (Fig. 1).

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NMR measurement:

1)	Measure the one-dimensional ¹H-NMR spectrum (Fig. 2). Identify the “structural reporter group” such as anomeric protons ²⁾. Analyze the chemical shift and vicinal coupling constant (e.g. ³J_{H1, H2}) of each proton signal.	Comment 0

If necessary, measure a series of two-dimensional NMR spectra. One-dimensional ¹³C-NMR typically requires a large amount of sample (10 mg).

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Notes

*Glycolipid can be dissolved in DMSO-d₆ or a mixed solvent (DMSO-d₆:D₂O = 98:2) ³⁾.

*If a cryogenic probe is used with optimized experimental conditions, picomole amounts of oligosaccharides can be characterized by ¹H-NMR spectroscopy ⁴⁾.

Figure & Legends

Fig. 1. Standard sample tubes for NMR measurements.

Normal 5-mm NMR tube filled with 500 μL solution (bottom) and Shigemi 5-mm NMR tube (top) with 200 μL solution.

Fig. 2. 500 MHz ¹H-NMR spectrum of a pyridylaminated biantennary oligosaccharide dissolved in D₂O.

The spectrum was obtained using a DRX-500 spectrometer equipped with aTXI cryogenic probe (BrukerBioSpin). Asterisk indicates the signal from low-molecular-weight impurities. The probe temperature was set at 25°C. 128 scans are acquired and the experimental time is about 5 min.

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Date of registration：2015-02-13 11:33:02