Studies on beta-D-Gal(f)-(1-->4)-alpha-L-Rha(p) octyl analogues as substrates for mycobacterial galactosyl transferase activity.

The biochemically unique structures of sugar residues in the outer cell wall of Mycobacterium tuberculosis (MTB) make the pathways for their biosynthesis and utilization attractive targets for the development of new and selective anti-tubercular agents. A cell-free assay system for galactosyltransferase activity using UDP[14C]Gal as the glycosyl donor, as well as an in vitro colorimetric broth micro-dilution assay system, were used to determine the activities of three beta-D-gal(f)(1-->4)-alpha-L-rham(p) octyl disaccharides as substrates and antimycobacterial agents respectively. The cell-free enzymatic studies using compounds 8 and 10 suggested that these disaccharides bind to and are effective substrates for a putative mycobacterial galactosyltransferase. The modified acceptor 8 was found to be a slower but prolonged binder as compared to the less substituted analogue 10 as evidenced by their Km and Vmax values. Moderate antimycobacterial activity was observed with compounds 8 and 9 against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC).

Synthetic arabinofuranosyl oligosaccharides as mycobacterial arabinosyltransferase substrates.

A series of arabinofuranosyl oligosaccharides found as constituent parts of the polysaccharide portion of the cell wall of Mycobacterium tuberculosis have been chemically synthesized. Screening of these oligosaccharides as substrates for arabinosyltransferases present in mycobacterial membrane preparations suggests that modified oligosaccharide analogs as small as disaccharides may be inhibitors of glycan biosynthesis. Such inhibitors would be of potential utility as lead compounds in the identification of new drugs for the treatment of mycobacterial infections.

Biosynthesis of mycobacterial lipoarabinomannan.

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), are potent immunomodulators in tuberculosis and leprosy. Little is known of their biosynthesis, other than being based on phosphatidylinositol (PI), and they probably originate in the phosphatidylinositol mannosides (PIMs; PIMans). A novel form of cell-free incubation involving in vitro and in situ labeling with GDP-[14C]Man of the polyprenyl-P-mannoses (C35/C50-P-Man) and the simpler PIMs of mycobacterial membranes, reisolation of the [14C]Man-labeled membranes, and in situ chase demonstrated the synthesis of a novel alpha(1-->6)-linked linear form of LM at the expense of the C35/C50-P-Man. There was little or no synthesis under these conditions of PIMan5 with its terminal alpha(1-->2)Man unit or the mature LM or LAM with copious alpha(1-->2)Man branching. Synthesis of the linear LM, but not of the simpler PIMan2, was susceptible to amphomycin, a lipopeptide antibiotic that specifically inhibits polyprenyl-P-requiring translocases. A mixture of P[3H]I and P[3H]IMan2 was incorporated into the linear LM, supporting other evidence that, like the PIMs, LM and LAM, it is a lipid-linked mannooligosaccharide and a new member of the mycobacterial glycosylphosphatidylinositol lipoglycan/glycolipid class. Hence, the simpler PIMs originate in PI and GDP-Man, but further growth of the linear backbone emanates from C35-/C50-P-Man and is amphomycin-sensitive. The origin of the alpha(1-->2)Man branches of mature PIMan5, LM, and LAM is not known at this time but is probably GDP-Man.