Nuclear magnetic resonance spectroscopy of carbohydrates

Carbohydrate NMR Spectroscopy is the application of nuclear magnetic resonance (NMR) spectroscopy to structural and conformational analysis of carbohydrates. This tool allows the carbohydrate chemist to determine the structure of monosaccharides and oligosaccharides from synthetic and natural sources. It is also a useful tool for determining sugar conformations. Modern high field strength NMR instruments used for carbohydrate samples, typically 500 MHz or greater, are able to run a suite of 1D and 2D experiments to determine primary structure and conformation of carbohydrate compounds.

Main article: Nuclear magnetic resonance

Sensitivity and Sample Size

In addition to ever increasing magnet size several other factors determine the sensitivity of an NMR instrument. Shigemi tubes are ideal for small sample sizes and are available matched to a variety of deuterated NMR solvents. Cold probes are available that cryogenically cool the NMR systems radio frequency (rf) receiver coils and pre-amplifier to provide increased sensitivity, reducing thermal noise.

Carbohydrate chemical shift

Main article: chemical shift

Common chemical shift ranges for nuclei within carbohydrate residues are:

• Typical ¹H NMR chemical shifts of carbohydrate ring protons are 3 – 6 ppm.
• Typical ¹³C NMR chemical shifts of carbohydrate ring carbons are 60 – 110 ppm

In the case of simple monosaccharide molecules, all protons are typically separated at a high enough field strength (usually > 500 MHz).

Anomeric center

Main article: anomer

Typically the chemical shifts for anomeric protons found further downfield (higher ppm) on the NMR spectrum than other ring protons, generally 4-6 ppm. Because of their distinctive shifts, these signals are generally the most diagnostic component of the spectrum.

The equatorial anomeric proton of an α-pyranoside is found downfield of the axial anomeric proton of a β-pyranoside. This difference is related the deshielding effect of the ring oxygen which is closer to an equatorial anomeric proton. Additionally, the anisotropic effects of circulating σ electrons form a deshielded region around the equatorial proton.

The anomeric carbon is typically found at 90-110 ppm, typical of any acetal carbon. The use of chemical shift for determining C-1 stereochemistry is unreliable, but the carbon of a β-anomer is usually found downfield relative to the α-anomer.

Non-anomeric centers

Typically the non-anomeric protons are found from 3 - 4 ppm. As expected some spectral overlap is seen, but with today’s high field instruments individual resonances can usually be resolved for monosaccharides. Typically the non-anomeric carbons are found from 60 – 85 ppm. As expected the secondary carbons will be found further downfield from a primary carbon.

Oligosaccharide NMR

Oligosaccharide ¹H NMR spectra tend to be a mess in the region of the spectra from 3 – 4 ppm. Quite often the individual signals are overlapped, and cannot be distinguished in the simple 1-D experiment. It is therefore advantageous to utilize 2D experiments

Correlation spectroscopy

Main article: correlation spectroscopy

COSY, or Correlation SpectroscopY, is often useful for carbohydrate structures due to the extended spin systems found in these molecules. The cross peaks in a 2D - COSY spectrum indicate couplings (2 and 3 bond) between two protons. Using this coupling information we can “walk” around a monosaccharide ring determining each proton, provided there is an obvious entry point, usually the anomeric proton.

The 2D - TOCSY experiment is similar to the 2D - COSY experiment, in that cross peaks of coupled protons are observed. However, the additional information obtained in a TOCSY is correlations for all the protons in the spin system. In the case of oligosaccharides, each sugar residue is an isolated spin system, so it is possible to differentiate all the protons of a specific sugar residue. A 1D version of TOCSY is also available and by irradiating a single proton the rest of the spin system can be revealed.

Recent advances in this technique include the 1D - CSSF - TOCSY (Chemical Shift Selective Filter - TOCSY) experiment. This produces higher quality spectra and allows coupling constants to be reliably extracted and used to help determine stereochemistry.

References

• Silverstein, Robert M. (2005). Spectrometric Identification of Organic Compounds. Wiley. 
• Robinson, Philip T.; et al. (2004). "In phase selective excitation of overlapping multiplets by gradient-enhanced chemical shift selective filters.". Journal of Magnetic Resonance 170 (1): 97–103. doi:10.1016/j.jmr.2004.06.004. PMID 15324762.