Polysaccharides linkage analysis

The elucidation of the structure of polysaccharides and of the carbohydrate portion of glycoconjugates involves sugar analysis, linkage analysis, and the determination of anomeric configurations and the sequences of the sugar residues. When non-sugar substituents are present, these should also be identified and located. 

Methylation analysis is the most important procedure for linkage analysis in carbohydrate-containing polymers (Lindberg, 1972). The procedure requires that all free hydroxyl in the oligo- or polysaccharide are etheri-fied. Following depolymerization of the methylated material, the monomers are separated, identified, and quantified. The free hydroxyl groups in the partially methylated monomers mark the position(s) at which the sugar residues were substituted. 

Practical applications are again stressed in the Chapter by Lonngren and Svensson (Stockholm) on Mass Spectrometry in Stmctural Analysis of Natural Carbohydrates. They build on the fundamentals of carbohydrate mass spectrometry, as laid down by Kochetkov and Chizhov in Volume 21, and demonstrate the profound analytical value of mass spectrometry for structural analysis of complex polysaccharides. In particular, this tool has dramatically increased the scope of the traditional methylation linkage-analysis procedure, especially when used in conjunction with gas-liquid chromatographic methods of separation. The latter topic is the subject of complementary Chapters by Dutton, one already published in Volume 28 and the other scheduled for publication in Volume 30. 

CWM was then extracted sequentially with water at 80 and with ammonium oxalate at 80, delignified with acidified sodium chlorite at 70 , and then further extracted with M and 4M sodium hydroxide. The crude polysaccharide extracts were likewise analyzed for constituent sugars and uronic acid (Table II). In order to obtain defined polysaccharide fractions the following separations were attempted. Polysaccharide fraction A was separated by ion-exchange chromatography on diethylaminoethyl(DEAE)-Sephadex A-50 and the neutral fraction afforded a virtually pure arablnan ( in Scheme 2) after selective precipitation with cetyltrimethylammonium hydroxide (7 ). Linkage analysis by methylation confirmed that this arablnan was of the highly branched type associated with pectins for which the representative, but not unique, structure (J ) may be advanced. 

Note The important difference between the alkylation procedures involving sodium hydride in polar apiotic solvents and those involving alkyl halides and metal salts is that 0-acyl groups are completely replaced by 0-alkyl groups [30], N-Acyl groups survive and N-ulkyl-N-acylamido derivatives are produced. This alkylation can be performed on an extremely small amount of material and, thus, the procedure provides a rapid means for methylation linkage analysis in the polysaccharide [30], lipopolysaccharide [31] and peptide [20, 32] fields. 

Uronosyl residues of the polysaccharides offer the same problems for glycosyl linkage analysis as they do for glycosyl composition analysis. Before the linkages to the uronosyl residues can be ascertained, the uronosyl residues must be reduced by the carbodiimide method (124) to their corresponding deuterium-labeled alditols. 

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