The 6-O-Methylglucose-containing Lipopolysaccharide of Mycobacterium phlei STRUCTURE OF THE REDUCING END OF THE POLYSACCHARIDE

Abstract The 6-O-methylglucose-containing polysaccharide (MGP), from mycobacterial species, is known to have a branched structure, with 3-O-methylglucose and glucose at the non-reducing termini. A glucose unit at the reducing terminus is linked glycosidically to the 2-hydroxyl of d-glyceric acid. This paper describes further studies which are aimed at defining the structure of the reducing end of the polysaccharide. The aglycon of MGP has been converted to the methyl ester which was reduced with sodium borotritide. α-Glucopyranosyl-(1 → 2)-glycerol-3H and α-glucopyranosyl-(1 → 6)-α-glucopyranosyl-(1 → 2)-glycerol-3H were isolated from a partial acid hydrolysate of the radioactive product, thus establishing the configuration of the glucosidic linkage to glyceric acid. The aglycon of MGP was removed by a Lossen rearrangement, and the glucose residue at the reducing terminus was reduced with sodium borotritide to yield r-MGP*. A radioactive disaccharide, which accumulated during partial acid hydrolysis of r-MGP*, was isolated in 25% yield. It was characterized as isomaltitol. Acid hydrolysis of methylated r-MGP* gave 1,2,3,4,5-penta-O-methylglucitol as the only radioactive product. Three other radioactive oligosaccharides, a tri-, tetra-, and pentasaccharide, derived from the reducing end of the polysaccharide, were isolated from a partial acid hydrolysate of r-MGP*. The trisaccharide was characterized as β-Glcp-(1 → 3)-α-Glcp-(1 → 6)-glucitol-3H; the tetrasaccharide was 6-O-methyl-α-Glcp-(1 → 4)-β-Glcp-(1 → 3)-α-Glcp-(1 → 6)-glucitol-3H; and the pentasaccharide was α-Glcp-(1 → 3)-6-O-methyl-α-Glcp-(1 → 4)-β-Glcp-(1 → 3)-α-Glcp-(1 → 6)-glucitol-3H. Coupled with the results outlined in the previous paragraph, the structures of these oligosaccharides define the configurations and linkages for the first 5 residues at the reducing end of the molecule. A procedure employing well known reactions was adapted for the selective stepwise degradation of oligosaccharides from the reducing end. This involved reduction of the reducing terminal sugar residue and selective oxidation of the resulting polyol with dilute periodate. The oxidized fragment was reduced and the small reduced aglycon was then cleaved from the remainder of the molecule by mild acetolysis. A single application of the method to isomaltotriose and the radioactive trisaccharide from r-MGP* gave isomaltose and laminaribiose, respectively. A re-examination of the methanolized products of methylated MGP by gas liquid chromatography provided direct evidence for a single (1 → 6) linkage in the polysaccharide. The sugars, in an acid hydrolysate of methylated α-amylase-digested MGP, were reduced with sodium borotritide, oxidized by sodium periodate, and reduced a second time. The products contained radioactive 2,4,6-tri-O-methylglucitol, 2,3-di-O-methylthreitol, and 2,3,4-tri-O-methylxylitol plus 2,3,4,6-tetra-O-methylglucitol in the approximate molar ratios 1:8:3. Therefore, a single (1 → 3) linkage is also present in the main chain of MGP. With the exception of this linkage, the (1 → 6) linkage mentioned above, and the branch point linkage, all remaining linkages in the polysaccharide appear to be of the (1 → 4) type. The substrate specificities of two enzymes used for structural analyses, an α-glucoamylase from Aspergillus niger and an α-glucosidase from the yeast, Debaryomyces vanryi, were investigated. At low substrate concentrations the former is highly specific for α-(1 → 4)-linked glucosyl residues, while the latter hydrolyzes a much broader spectrum of α-glucosides.