Polysaccharides 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. 

A most useful application of methylation is for the structural analysis of polysaccharides. Permethylation, followed by hydrolysis of the product and identification of the individual methylated monosaccharides, indicates the original positions of the linkage points, as these are denoted by hydroxyl groups in the methylated monosaccharides. Considerable work has been done on the separation and identification of methylated monosaccharides by gas-liquid chromatography and mass spectrometry this topic is treated in detail in this Volume by Lbnngren and Svensson (see p. 41), Clearly, for such structural analyses to be valid, complete methyl-... 

Data from f.a.b.-m.s., and also f.d.-m.s., revealed the existence of naturally occurring, large cyclic polysaccharides. The first indication that a molecule may be cyclic comes from its precise molecular-weight determination. Cyclic molecules are 18 mass units less than their linear counterparts. Loss of water may, of course, occur in a number of ways, for example, by dehydration or lactonization, and conclusive evidence for the presence of a cyclic molecule can only be obtained from f.a.b.-m.s. of suitable derivatives, such as the permethyl derivative. Cyclic and dehydrated linear polymers are distinguishable after permethylation, as the cyclic polymer will incorporate one methyl group less than the linear molecule. 

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was proposed early in the application of gas-liquid chromatography to carbohydrates, but the method has now been superseded by more convenient procedures.230,231 There are, however, situations in which this method is useful, such as during a structural study of a polysaccharide by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. 

In the polysaccharide and the glycoprotein series, permethylated derivatives are almost exclusively used for their solvolytic decomposition and for the analysis of the mono- and oligo-saccharides formed, whereas, in the glycosphingolipid series, the investigation of the permethylated derivatives themselves by physicochemical methods has acquired great importance. They proved to be very convenient both for mass-spectrometric analysis and for n.m.r. spectroscopy (see later). 

S. Hakomori, Rapid permethylation of glycolipid + polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide, J. Biochem., 55 (1964) 1386-1388. 

J. N. C. Whyte and J. R. Englar, Polysaccharides of the red alga Rhodymenia pertusa. II. Cell-wall glucan proton magnetic resonance studies on permethylated polysaccharides, Can. J. Chem., 49 (1971) 1302-1305. 

Mishnick and co-workers have developed several techniques for determining the positions of substitutions on monosaccharide units and distribution of substituents along deriva-tized polysaccharide chains, primarily cellulose derivatives. Examples are found in references [61,62,63,64,65,66,67,68,69,70]. An important method involves perdeuteromethylation followed by partial acid-catalyzed hydrolysis, then reductive amination with propylamine, and finally permethylation to yield completely O- and A-alkylated charged oligosaccharides that are analyzed by MALDI-TOF-MS [68,70]. Statistical methods are then applied. 

Deery et al. [80] reported the on-hne coupling of SEC and ion-trap MS for the detection of sodiated polysaccharides with masses in excess of 9 kDa. Multiple-charge ions with up to five sodium ions were observed. On-line SEC-MS" allowed the determination of hnkage information in permethylated arabinogalactan oligomers. 

Permethylation of polysaccharides. Kuhn and Trischmann found that polysaccharides can be methylated very efficiently in DMSO with dimethyl sulfate and barium oxide and/or barium hydroxide. Srivastava et al. used the same method except for the substitution of sodium hydroxide as base. Sodium hydroxide pellets and dimethyl sulfate were added with stirring under nitrogen over 8 hrs. to a solution of undegraded stareh in dimethyl sulfoxide. After stirring for another 16 hrs. the mixture was heated to decompose the dimethyl sulfate, cooled, diluted, and neutralized, and the product was extracted with chloroform and precipitated from acetone with ether yield 91%, OCHa = 42.3%. Here the high solvent power of DMSO clearly contributes to the solvent effect. 

Srivastava found that a polysaccharide such as maize starch can be permethylated by ilissolving it in dimethyl sulfoxide and stirring at 25° for 48 hrs. with methyl iodiile and barium oxide. However, a later report " states that a higher degree of iiidhylation of starch is obtained by using dimethyl sulfate and sodium hydroxide in dimethyl sulfoxide. 

This compound was isolated after permethylation of the mixture resulting from the hydrolysis of methylated polysaccharides containing 2-amino-2-deoxy-D-glucose, ... 

Methylatiott. Hakomori reported that glycosides of polysaccharides can bt rapidly permethylated by treatment with dimsylsodium in DMSO followed by reaction with methyl iodide ... 

In structural studies on the capsular polysaccharide of Klebsiella K-type 9, a number of acidic and neutral oligosaccharides were isolated by ion-exchange chromatography. The homogeneities and structures of the preparations were established by g.l.c.-m.s. of the permethylated oligosaccharide alditols. The structure of the oligosaccharides was also determined by methylation analyses. The same technique was used in the structural elucidation of the capsular polysaccharide of Klebsiella K-type 47. 

An alternative procedure for the permethylation of polysaccharides is the well-known one described by Hakomori, which employs the methyl-sulfinyl carbanion in methyl sulfoxide and methyl iodide. This newer approach was based on the observation that conversion of phenols into phenoxides is greatly enhanced by virtue of the high basicity of this car-... 

The use of mass spectrometry in the structural analysis of carbohydrates, first reported in 1958 (114), was developed in detail by Kochetkov and Chizhov (115). They showed that, under electron impact, the acetylated and methyl ether derivatives of monosaccharides provided a wealth of structural information through analysis of typical fragmentation pathways of the initial molecular ion. This has proved of enormous utility in the structural elucidation of polysaccharides and complex oligosaccharides sequential permethylation, hydrolysis, reduction to the alditol, and acetylation, affords mixtures of peracetylated, partially methylated alditol acetates that can be separated and analyzed by use of a gas chromatograph coupled directly to a mass spectrometer (25). The mass spectra of stereoisomers are normally identical, while the gas chromatographic retention times readily permit differentiation of stereoisomers. 

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