Isolation and Characterization of Lipopolysaccharides Containing 6-O-Methyl-d-glucose from Mycobacterium Species

Abstract

Abstract A polysaccharide, composed of 6-O-methyl-d-glucose and d-glucose in a molar ratio of 6:4, was isolated from the watersoluble portion of the deacylated crude lipids of Mycobacterium phlei and M. tuberculosis. The polysaccharide was purified by gel filtration, ion exchange chromatography, and borate complex formation, and was shown to have a molecular weight of about 3000 and [α]d22 +160° (c, 0.1; water). Methylation analysis of the polysaccharide showed that it has a branched structure with an average chain length of 8 to 9 hexose units. The major type of glycosidic linkage is α-(1 → 4), and the branching involves position 3 of one of the 6-O-methyl-d-glucose residues. Oligosaccharides were prepared by acetolysis of the polysaccharide, and were purified by paper chromatography. Di-, tri-, tetra-, penta-, hexa-, and heptasaccharides of α-(1 → 4)-linked 6-O-methyl-d-glucose, in addition to maltose and maltotriose, were isolated. The only heterooligosaccharide found was O-α-d-glucopyranosyl-(1 → 3)-6-O-methyl-d-glucose. β-Amylase had no action on the polysaccharide, whereas α-amylase liberated about 20% of the total carbohydrate, mainly as d-glucose and maltose. Limited amylolysis yielded d-glucose, maltose, and unidentified oligosaccharides containing d-glucose. Intact lipopolysaccharide, containing 2 to 3 moles of ester-linked fatty acid, was prepared from a 70% ethanol extract of M. phlei. The lipopolysaccharide was soluble in water and in a 2:1 chloroform-methanol mixture, but was only slightly soluble in methanol or ethanol. Proton magnetic resonance and infrared and ultraviolet spectra were consistent with the results obtained by chemical analysis.