Epimeric and amino disaccharide analogs as probes of an α-(1→6)-mannosyltransferase involved in mycobacterial lipoarabinomannanbiosynthesis

Abstract

Mycobacterial lipoarabinomannan (LAM) is an important, immunologically active glycan found in the cell wall of mycobacteria, including the human pathogen Mycobacterium tuberculosis. At the core of LAM is a mannan domain comprised of α-(1→6)-linked-mannopyranose (Manp) residues. Previously, we and others have demonstrated that α-Manp-(1→6)-α-Manp disaccharides (e.g., Manp-(1→6)-α-ManpOctyl, 1) are the minimum acceptor substrates for enzymes involved in the assembly of the LAM mannan core. We report here the synthesis five epimeric and three amino analogs of 1, and their subsequent biochemical evaluation against an α-(1→6)-ManT activity present in a membrane preparation from M. smegmatis. Changing the manno- configuration of either residue of 1 to talo- or gluco- led to a reduction or loss of activity, thus confirming earlier work showing that the C-2 and C-4 hydroxyl groups of each monosaccharide were important for enzymatic recognition. Characterization of the products formed from these analogs was done using a combination of mass spectrometry and glycosidase digestion, and full substrate kinetics were also performed. The analogs in which the acceptor hydroxyl group had been replaced with an amino group were, as expected, not substrates for the enzyme, but were weak inhibitors.