Carbohydrate permethylation

Three types of carbohydrate sample have been reported to give data above mass 4000, namely, permethylated polysaccharides, permethylated glycosphingolipids, and naturally acylated forms of a mycobacterial O-methyl-D-glucose polysaccharide. All are hydrophobic, and desorption is probably facilitated by their inability to form strongly hydrogen-bonded aggregates, either with themselves or with the matrix. 

III. Analysis of Permethylated Carbohydrates without Depolymerization of the Sample... 

Depolymerization of the permethylated carbohydrate is achieved by hydrolysis with acid. Under these conditions, the amino sugar residues are N-deacetylated, and the aminohexosidic linkages become resistant to hydrolysis. Stellner and coworkers29 showed that, when the acid degradation is conducted in 95% acetic acid, the amino sugar residues are also liberated, and can be analyzed by the methylation technique.29 Therefore, acetolysis followed by acid hydrolysis is now commonly used, as it allows the analysis both of hexose and hexosamine residues. 

Polymers of D-fructose are important carbohydrate reserves in a number of plants. Inulins and levans are two major types that differ in structure. D-Fructans require only relatively mild conditions for their hydrolysis, for example, levan was qualitatively hydrolyzed by hot, dilute, aqueous oxalic acid. Permethylated fructans could be hydrolyzed with 2 M CF3CO2H for 30 min at 60°. Fructan oligosaccharides were hydrolyzed in dilute sulfuric acid (pH 2) at 70 (see Ref. 53) or 95° (0.1 M). D-Fructans from timothy haplocorm (where they comprise 63% of the water-soluble carbohydrates) could be hydrolyzed with 0.01 M hydrochloric acid at 98°. 

The distribution and structural elucidation of saponins have been reviewed. Glucuronides with a free carboxy-group and carbohydrate residues at C-2 and C-4 are readily cleaved with acetic anhydride in refluxing pyridine to give the genuine aglycone (see Vol. 7, p. 145). Details of the mass spectra of a series of permethylated oleanane saponins have been discussed. 

The analysis of monosaccharide mixtures as the permethylated derivatives was proposed early in the application of gas-liquid chromatography to carbohydrates, but the method has now been superseded by more convenient procedures.230,231 There are, however, situations in which this method is useful, such as during a structural study of a polysaccharide by the methylation technique. The mixture of partially methylated monosaccharides obtained by methanolysis may then be fully methylated, and the proportions of the various monosaccharides determined. This approach has been used, for example, in studies on a galactomannan392 and on tamarind-kernel polysaccharide.393 Such an analysis also constitutes a useful check to ensure that no significant change in the composition of the polysaccharide occurred during methylation. 

Permethylation of carbohydrates.1 Permethylation of carbohydrates in a solid-liquid system is not possible because of lack of solubility in benzene or methylene chloride. The difficulty can be circumvented by reaction of peracetylated sugars with methyl bromide in C6H6-aqueous NaOH with tetrabutylammonium hydrogen sulfate as catalyst. 

Permethyiation of carbohydrates. One widely used method for this reaction employs dimsylsodium in DMSO and methyl iodide. The. purity of the permethylated products is significantly improved if dimsylpotassium is used as the base.2... 

An important general method in characterization of carbohydrates is the classical exhaustive methylation (permethylation). Repeated treatment with a methylating agent such as methyl iodide converts all free OH groups to OCH3 groups. Then, complete acid... 

The development of chemical, physical, and enzymic methods for investigating the primary structure of the carbohydrate fractions of glycoconjugates has, during the past ten years, allowed the determination of the structures of numerous glycans. In this regard, the combination of the techniques of permethylation and 360-MHz, proton-n.m.r. spectroscopy has afforded real progress in the rapid determination of the primary structures of oligosaccharide chains. 

The carbohydrate sequence of the major tetrasaccharlde was determined by examining the nitrous acid deamination products (25) as permethylated disaccharides by chemical ionization mode of gas chromatography/mass spectrometry. The products were identified as hexosyl-2,5-anhydromannitol and hexosyl-myoinositol,indicating that the major tetrasaccharide had the sequence Galp(l->4)GlcNAcp(l->4)Glcp(l- -2)Inos (Figure 5). 

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