Acetals oligosaccharides

Oligosaccharides were isolated from PMII by weak acid hydrolysis and separation by SEC and HPAEC-PAD. The isolated oligosaccharides were desalted, reduced and methylated. GC-MS analysis of the partially methylated alditol acetates has been used to reveal the structure of the oligosaccharides. 

Plant material fractionation. The detailed steps of isolation and separation of oligosaccharide fractions were described earlier [10]. Pectin was separated by boiling the cell walls in 0.5 M ammonium-oxalate buffer, pH 5.2 at 100 C for Ih. The dialyzed solution of pectin was hydrolyzed with 0.15M HCl for 3h at 100 C. Neutralized and desalted hydrolysate was loaded to the column (lx90cm) filled with biogel TSK HW-40 (Toyo Soda, Japan) equilibrated and eluted with 50mM sodium acetate (pH 5.2) at a rate of 0.3ml/min. In all fractions (1ml) the sugars were determined by o-toluidine method (Resnikov et al., 1982) and fraction IP was collected as shown on Figure 1. 

Hydrophilic interaction chromatography on Asahipak NH2P or Excel-pak CHA-P44 with pulsed amperometric detection has been used to fractionate malto-oligosaccharides.266 The Asahipak NH2P is a polyvinyl alcohol support with a polyamine bonded phase, and the Excelpak is a sulfonated polystyrene in the Zn+2 form. Amine adsorption of sialic acid-containing oligosaccharides was performed on a Micropak AX-5 column (Varian) using acetonitrile-water-acetic acid-triethylamine.267... 

Dissolve an oligosaccharide or glycan having a reducing end to be modified in 2 1 pyri-dine/glacial acetic acid (vol/vol) with a total reaction volume of 10-100 pi. If the carbohydrate initially is insoluble in the reaction solution, a prior dissolution in a minimal amount of DMSO or water can be done and then an aliquot transferred to the reaction medium. 

The partially methylated monosaccharides obtained on depolymerization of the permethylated sample are preferably analyzed as acetates by g.l.c.-m.s., as shown by Bjomdal and coworkers.41,42 The neutral sugars and the amino sugars obtained in acetolysis-acid hydrolysis are reduced, and acetylated for the analysis, and the amino-hexitol and the neuraminic acid residues are acetylated after methanolysis. Identification with the aid of g.l.c.-m.s. has been described for all of the common components of protein- and lipid-linked glycans and oligosaccharides from animal cells, namely, the neutral sugars,41-43 hexitols,44 hexosamines,29,43,45,46 aminohexitols,31,32 and neuraminic acids.33,34,47... 

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