Characterization of 3-O-methyl-D-mannose polysaccharide precursors in Mycobacterium smegmatis.

Mycobacterium smegmatis, when incubated under appropriate conditions with L-[methyl-Tlmethionine, accumulates significant amounts of small [methyl-T]-labeled oligosaccharides that are related to the known 3O-methyl-D-mannose polysaccharides (Maitra, S. K., and Ballou, C. E. (1977) J. Biol. Chem. 252, 2459-2469). We have fractionated the water-soluble material from the 70% ethanol extract of such cells by Sephadex G-50 column chromatography, high pressure liquid chromatography, and finally by Bio-Gel P-4 column chromatography. An homologous series of compounds with the structures Man-(MeMan)r-OCHs up to Man(MeMan)g-OCHs, and a parallel series with the structures (MeMan)S-OCHs up to (MeMan)g-OCHa were obtained and characterized. All hexoses are al +4-linked, the methyl aglycon has the (Y configuration, and the mannose is methylated in position 3. All of the compounds are structurally related to each other as though they were biosynthetic precursors of the larger 3-0methylmannose polysaccharides. We also detected small amounts of a methylmannobiose, Man-ManOCH3, that may represent the earliest unmethylated intermediate in the pathway. As an example of the methodology, the substance [methyZ-T]Man-(MeMan)G-OCH3 was methylated with [DJmethyl iodide and the product was hydrolyzed to give 2,3,4,6-tetra-O-[D3]methylmannose and 2,6,di-O[DJmethyl-3-O-[T]methylmannose.TheMan-(MeMan)BOCH, consumed 2 mol of periodate and released 1 mol of formic acid/m01 of oligosaccharide. Reduction and mild hydrolysis of the oxidized product yielded (MeMan)e-OCHJ. These results establish that the unmethylated mannose is in a terminal position. The proton magnetic resonance snectrum showed an anomeric protonsignal for the terkinal mannose (8 = 5.17 ppm) that was lost on Smith degradation: it showed a signal (6 = 4.80 ppm) for the 3-Gmethylmannose attache: to the methyl aglycon; and a signal (8 = 5.21 ppm) for the other hexoses in the chain. The a-methyl aglycon gave a signal at 3.41 ppm, and the 3-0-methylmannose methyl signals were at lower field (3.44 to 3.47 ppm). Oligosaccharides that lacked the unmethylated mannose did not show the anomeric proton signal at 5.17 ppm, whereas the absence of the methyl aglycon was