Oligosaccharides methylation method

The methylation method has been the principal means for determining the type of ring of the monosaccharide constituents and their points of attachment to one another in the oligosaccharide molecule. The usual procedure involves (1) complete methylation of the disaccharide (2) hydrolysis of the methylated disaccharide to partially methylated monosaccharide units, and their identification by comparison of their properties with those of known methylated compounds (3) determination of the unmethylated positions in each unit, these being the positions which were available for ring formation and for linkage between units and (4) determination of the ring type (pyranose or furanose) by supplementary evidence, such as determination of the rate of hydrolysis of the partially methylated lactone (31). 

The methods used to elucidate the linkage positions in an oligosaccharide (methylation, oxidative cleavage of glycols) were outlined under monosaccharides. 

In a study completed during the early development of f.a.b.-m.s., both f.d. and f.a.b. were used to characterize 101 fractions containing neutral oligosaccharides isolated from human milk. Samples were examined as their peracetylated alditols. In subsequent work, the structures of two minor acidic oligosaccharides from human milk were investigated. The per-methylated derivatives were analyzed by f.a.b.-m.s., and their compositions and sequences were defined by the f.a.b. data. Methylation analysis and partial formolysis were the other principal methods used. 

The structures of oligosaccharides and polysaccharides are usually determined by a combination of methods specific enzymatic hydrolysis to determine stereochemistry and produce smaller fragments for further analysis methylation analysis to locate glycosidic bonds and stepwise degradation to determine sequence and configuration of anomeric carbons. 

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. 

In order to try to overcome some of the problems associated with chemical synthesis of oligosaccharides containing N-acetylneuraminic acid, Sabesan and Paulson [277] have used a combination of chemical and enzymatic methods using purified sialyl-transferases in the presence of CMP-iV-acetylneuraminic acid and synthetic acceptor molecules to give sialyl derivatives of oligosaccharides which were characterised by NMR. Thus, methyl P-D-galactopyranoside, methyl P-D-lactoside and iV-acetyl-... 

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