NMR Spectroscopy of Carbohydrates
Purpose:
Elucidation of the structure and sequence of oligo- and polysaccharides, including glycosides, glycolipids, etc.
Methods used:
NMR spectroscopy takes advantage of the magnetic moment of the atomic nuclei, which align themselves parallel or antiparallel in an applied, strong magnetic field. These two states are slightly different in energy, and the nuclei can jump from one state to the other by irradiation of the sample with a radiofrequency pulse. The energy required to cause this transition depends on the electron density around each individual nucleus and is reflected in what is known as “chemical shift.” The chemical shift is inversely proportional to the electron density around the nucleus and provides useful information about the chemical structure of a molecule. Additional structural information is gained from observing the so-called “coupling constants” of different NMR signals. Nuclei that are separated by 1, 2, or 3 bonds show a characteristic signal splitting in multiple peaks depending on the number of neighboring nuclei. The distance between the split parts of a signal is called coupling constant and depends on the geometry of the bonds between the coupled nuclei. The nuclei most commonly detected by NMR spectroscopy are hydrogen and carbon, but nitrogen and phosphorus are frequently detected as well.
The simplest and most common NMR spectra are 1-dimensional, i.e. the intensity of the signal is shown as a function of the chemical shift. 1-D spectra can be used to identify pure glycans and glycolipids if they are known structures or similar to known structures. Other uses are the determination of anomeric configuration, estimation of sample purity and major components in a mixture. However, complete structural elucidation of an unknown sample requires 2-dimensional (2-D) NMR spectra, in which correlations between different signals of the same spectrum are visualized.
COSY (correlation spectroscopy) spectra, for instance, show which hydrogen nuclei (protons) are coupled to each other through 2 or 3 bonds.
TOCSY (total correlation spectroscopy) spectra show which protons belong to the same “spin system.” A spin system is a molecular substructure containing a “chain” of proton-proton couplings. These are usually isolated either by hetero atoms like oxygen or nitrogen or by quaternary carbon atoms (carbons without attached hydrogens). A typical spin system would be, for example, a monosaccharide residue in a polysaccharide.
COSY and TOCSY spectra of the same compound, when taken together, can supply the information necessary to assign the signals of a known compound and, in some simple cases, can enable the spectroscopist to elucidate the structure of a molecule. However, in most cases, additional experiments are necessary to determine the structure.
NOESY (nuclear Overhauser effect spectroscopy) and ROESY (rotating frame nuclear Overhauser effect spectroscopy) show correlations of protons that are close in space. These experiments are used in carbohydrate NMR to determine the monosaccharide sequence of oligo- or polysaccharides.
HSQC (heteronuclear single quantum coherence) spectra display the correlations between protons and the carbons to which they are attached. This information is helpful when assigning the proton chemical shifts and also shows which positions in each constituent monosaccharide are glycosylated, as glycosylation causes a significant downfield shift in the carbon dimension.
HMBC (heteronuclear multiple bond correlation) spectra correlate carbons and protons linked over more than one bond. This method is more reliable in determining the monosaccharide sequence than NOESY or ROESY, because the conformation of the saccharide has only a minor influence on the HMBC spectrum. However, this heteronuclear technique is less sensitive than the homonuclear NOESY, and therefore requires much more sample.
All of the 2-D spectra are displayed with two chemical shift axes, whereby correlations show up as signals with different values on each axis (“cross peaks”) connecting two signals with different chemical shifts.
General Criteria for the sample:
The sample should be at least 90 % pure. If the sample is a mixture, the information that can be obtained by NMR is limited to an estimation of major components.
The time required to obtain a spectrum is inversely proportional to the square of the sample amount. Practical minimum sample requirements are 50 nanomoles for a 1-D proton or COSY spectrum, 100 nanomoles for a TOCSY or NOESY, 1 micromole for a 2-D HSQC or a 1-D carbon spectrum, and 10 micromoles for an HMBC.
Example Results:
