Oligosaccharide structural studies

Price, N. P. J. 2006. Oligosaccharide structures studied by hydrogen-deuterium exchange and MALDI-TOF mass spectrometry. Anal. Chem. 78 5302—5308. 

Analysis of the deuterium isotopic shifts in the C-NMR spectrum has been particularly useful for sugar and oligosaccharide structural studies and spectral assignments (18, 151). Application of the differential isotope shift technique was used to study the tautomers of psicose, (33a-33d), in solution (Fig. 2.37a) (384). Signal assignments were made by comparison of calculated and observed cumulative isotope shifts. This method distinguished the C-1 and C-6 resonances. 

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

Field strengths currently available have been used, but it is obvious that, for structural studies of the oligosaccharides of glycoproteins, a large field-strength is desirable. 

Although this technique has been extensively used in the past for structural studies of monosaccharides and small oligosaccharides, its usefulness for study of the structure of oligosaccharides of glycoproteins has only begun. 

Takahashi N, Hitotsuya H, Hanzawa H, Arata Y, Kurihara Y, Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin,... 

CH3C02CH0 would affotd fragment 79. This structural analysis demonstrates the potential of the technique for structural studies of oligosaccharides. 

For polysaccharides composed of oligosaccharide repeating units, the structural studies may ultimately lead to a complete structure. Other polysaccharides have less-ordered structures, and the aim of the structural studies should be to define the factors governing these structures. Polysaccharide methodology has been summarized2 in two review articles. 

Structural studies on the oligosaccharide derivatives obtained by partial, acid hydrolysis of fully methylated polysaccharides often furnish valuable information on the positions at which the oligosaccharides were linked in the original polysaccharide. When the folly methylated Klebsiella O group 9 lipopolysaccharide,27 which contains D-galactopyranose and D-galactoforanose residues, was subjected to mild, acid hydrolysis, and the product reduced with lithium aluminum deuteride and remethylated with trideuteriomethyl iodide, a good yield of the disaccharide derivative 10 was obtained. 

Even though /8-elimination from the reducing terminal may be a useful tool in structural studies of oligosaccharides it is, in the authors opinion, of only limited value when applied directly to polysaccharides. Side reactions may completely obscure the results after the first three or four residues have been eliminated from a polysaccharide, and, in some instances, the degradation may stop after only a few residues have been eliminated. For polysaccharides of irregular structure, the structural composition in the vicinity of the reducing terminal may not be the same in the different molecules. As a result of the biosynthesis, or the isolation procedure, there may not even be any reducing terminals in the polysaccharide molecules or, at most, only in some of them. 

Baddiley and coworkers used V-deacetylation-deamination in structural studies on two oligosaccharides containing 2-amino-2-deoxy-D-galactose that had been obtained by dephosphorylation of the capsular substances from Pneumococcus types 10A (Ref. 177) and 29 (Ref. 13), respectively. The former (110) yielded 2-O-a-D-galac-... 

Structural studies of glycosphingolipids involves determination of the structure of the oligosaccharide chain and of the lipid moiety. For the oligosaccharide chain, it is necessary to determine the composition, molar ratio, and sequence of the monosaccharides, their pyranose or furanose nature, and the position of glycosidic bonds and their configuration for the lipid moiety, the composition of the fatty acids and sphingosine bases must be determined. Used for these purposes are the classical, chemical methods, conventionally accepted in the chemistry of carbohydrates and lipids and based on the degradation of compounds, enzymic, and physicochemical methods, primarily mass spectrometry and n.m.r. spectroscopy. 

It had been realized from the beginning that the final goal was the application of the new methods to structural studies in the wider fields of oligosaccharides and polysaccharides. The important reference substance, 2,3,6-trimethyl-D-glucose was isolated by Denham in the course of work at St. Andrews on the methylation of cellulose in which he made use of dimethyl sulfate as the methylating agent. 

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