Synthetic Mannosides Act as Acceptors for Mycobacterial 1-6 Mannosyltransferase Jillian R. Brown, a, * Robert A. Field, b Adam Barker, a Mark Guy, c Ravinder Grewal, a Kay-Hooi Khoo, d Patrick J. Brennan, a Gurdyal S. Besra c,y and Delphi Chatterjee a,y a Department of Microbiology, Mycobacterial Research Laboratories, Colorado State University, Fort Collins, CO, 80523, USA b School of Chemistry, University of St. Andrews, Purdie Building, St. Andrews, Fife KY16 9ST, UK c Department of Microbiology and Immunology, University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK d Institute of Biological Chemistry, Academica Sinica, Taipei, Taiwan, Republic of China Received 3 July 2000; accepted 4 October 2000 AbstractÐA series of synthetic mannosides was screened in a cell-free system for their ability to act as acceptor substrates for mycobacterial mannosyltransferases. Evaluation of these compounds demonstrated the incorporation of [ 14 C]Man from GDP- [ 14 C]Man into a radiolabeled organic-soluble fraction and analysis by thin layer chromatography and autoradiography revealed the formation of two radiolabeled products. Each synthetic acceptor was capable of accepting one or two mannose residues, resulting in a major and a minor mannosylated product. Both products from each acceptor were isolated and their mass was con®rmed by fast-atom bombardment±mass spectrometry (FABMS). Characterization of each mannosylated product by exo-glycosidase diges- tion, acetolysis and linkage analysis by gas chromatography±mass spectrometry of partially per-O-methylated alditols, revealed only a1-6-linked products. In addition, the antibiotic amphomycin selectively inhibited the formation of mannosylated products suggesting polyprenolmonophosphate-mannose (C 35/50 -P-Man) was the immediate mannose donor in all mannosylation reactions observed. The ability of synthetic disaccharides to act as acceptor substrates in this system, is most likely due to the action of a mycobacterial polyprenol-P-Man:mannan a1-6 mannosyltransferase involved in the biosynthesis of linear a1-6-linked lipomannan. # 2001 Elsevier Science Ltd. All rights reserved. Introduction Mycobacterial diseases, such as tuberculosis and leprosy, remain serious human health concerns. One critical fea- ture that contributes to the problematic pathogenicity of mycobacteria is the unique and intricate structure of the mycobacterial cell wall, which results in low permeability to most chemotherapeutic agents and thus promotes resistance. The cell wall structure, which is made up of polysaccharides, proteins and lipids, has been shown 1 4 to contain two major polysaccharide components, ara- binogalactan (AG) and lipoarabinomannan (LAM). Both AG and LAM contain approximately 70 arabino- syl residues and 30 hexosyl residues (galactosyl in AG and mannosyl in LAM). The synthesis of these cell wall components requires the concerted action of a large number of glycosyltransferases. 5,6 Biological studies have implicated LAM as an important cell-surface molecule involved in host-pathogen interactions 5 and agents that interfere with the biogenesis of either AG or LAM are expected to have serious consequences for cell pathogenicity. Indeed, the biological importance of the mycobacterial lipoglycans, LAM, lipomannan (LM) and the phosphatidylinositol mannosides (PIMs), has led to a preliminary investigation into their biosynthesis. 4 Brie¯y, LAM and LM originate from a phosphatidyl- inositol (PI) core which is elaborated to give Ac 1 PIM 2 , linear a1-6-linked LM, mature LM and ®nally LAM 7,8 (Fig. 1). Three families of mannosyltransferases have been suggested to be involved in the biosynthesis of linear a1-6-linked LM. The ®rst family of enzymes catalyzes the transferofManresiduesfromGDP-Man to PI and other short PIM intermediates. 8 A second family catalyzes the transfer of Man residues from GDP-Man to a variety of polyprenol monophosphates, while a third 0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(00)00300-X Bioorganic & Medicinal Chemistry 9 (2001) 815±824 *Corresponding author at current address: Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, Uni- versity of California, San Diego, La Jolla, CA, 92093-0687, USA. Tel.: +1-858-822-1102;fax:+1-858-534-5611; e-mail: jibrown@ucsd.edu y Both authors shared responsibility for this work.