Communication www.rsc.org/chemcomm CHEMCOMM Efficient synthesis of the antigenic phosphoglycans of the Leishmania parasite† Dipali Ruhela and Ram A. Vishwakarma* Bio-organic Chemistry Lab, National Institute of Immunology, Aruna Asaf Ali Marg, JNU Complex, New Delhi 110067, India. E-mail: ram@nii.res.in; Fax: +91 11 6162125; Tel: +91 11 6174899 Received (in Cambridge, UK) 23rd July 2001, Accepted 21st August 2001 First published as an Advance Article on the web 12th September 2001 Antigenic phosphoglycan repeats of the Leishmania parasite can be assembled in a flexible and efficient manner without involving any glycosidation steps, and the chain can be extended either towards the non-reducing (6A-OH) or reduc- ing (1-OH) end suitable for synthesis of lipophosphoglycan, proteophosphoglycan and analogues. The protozoan parasite Leishmania causes visceral and cuta- neous leishmaniases and has a remarkable ability to survive and proliferate in extreme environments during its digenetic life cycle in the sandfly vector and the human host. At all life-cycle stages Leishmania species assemble an abundance of a unique class of glycoconjugates named phosphoglycans (reviewed in ref. 1). These include the most abundant surface molecule of infectious promastigote stage, the lipophosphoglycan (LPG), and secreted proteophosphoglycan (PPG) of the amastigote stage. There is substantial evidence 1 that the LPG and PPG are antigenic and multifunctional virulence factors essential for infectivity and survival of the parasite. The role of phosphogly- cans in parasite virulence is currently a topic of intense debate 2 in parasite biology. The intriguing structure of the LPG consists of four distinct domains: (i) alkyllysophosphatidylinositol lipid-anchor; (ii) conserved phosphosaccharide core with internal galactofur- anose residue; (iii) variable phosphoglycan repeats and (iv) neutral oligosaccharide cap. The unique feature of LPG is the variable phosphoglycan domain made of phosphodisaccharide [6Galp-b1,4-Manp-a1-phosphate] n repeats linked through phosphodiester between the anomeric-OH of mannose of one repeat and the 6-OH of galactose of the adjoining repeat. PPG molecules are made up of the phosphoglycan repeats linked to a peptide anchor. The biological, biochemical and biophysical experiments to probe the function, biosynthesis and conformation of the Leishmania phosphoglycans, and to exploit them in drug and vaccine design, require efficient chemical synthesis. Since the phosphoglycans are labile molecules, due to the presence of anomeric phosphodiester linkages, their synthesis is particularly challenging. The first synthesis of Leishmania phosphoglycans was accomplished 3 by the Dundee group from monosaccharide building blocks, using a suitably protected galactose donor and mannose acceptors. This obviously involved multiple protec- tion, deprotection, glycosidation and purification steps even before the phosphoglycan assembly began through the H- phosphonate chemistry. In our ongoing work on synthesis 4–7 and biosynthesis 8 of Leishmania glycoconjugates, an efficient route to construct phosphoglycans was required for the total synthesis of LPG and vaccine design. To circumvent the usual problems associated with glycosidation and to avoid several protection–deprotection steps required in previous synthesis, we used the readily available disaccharide lactose as starting material. Here we report a new efficient synthesis of phosphoglycans, which does not involve any glycosidation steps, and the phosphoglycan chain can be extended either towards the non-reducing (6A-OH) or reducing (1-OH) end in high yielding iterative steps. The important features of our approach include the glycal chemistry mediated gluco?manno transformation and regioselective 6A- protection to convert lactose (Galb1,4-Glu) into the suitably protected Galb1,4-Man building block, extension of PG repeats in either direction by selective deprotection at the non-reducing 6A-position or reducing 1-position and a-phosphitylation, fol- lowed by iterative PG coupling cycles. The first intermediate lactal (1) was prepared 6,7 from lactose (Scheme 1) in straightforward steps (acetylation, bromination, reductive elimination and deacetylation), and a high yield could be obtained in the reductive elimination step by the application of Zn–Vitamin-B 12 reagent. 9 The next task was to selectively protect the 6-position of the galactose residue of lactal (1) and this was achieved, after a considerable number of experiments, by dibutyltin oxide mediated silylation (Bu 2 SnO–MeOH reflux followed by TBSCl) which led exclusively to 6A-O-TBS-lactal (2). It should be mentioned here that under similar conditions most other protecting groups (benzyl, p-methoxybenzyl and allyl) led to C3A-OH protected lactals. The next step involved stereoselective gluco?manno transformation of 6A-O-TBS- lactal (2) by MCPBA under biphasic conditions which led exclusively to the manno product 6A-O-TBS-galactopyranosyl- (1?4)-b-D-mannopyranose (3). The acetylation of 3 gave the key intermediate, 1,2,3,6-tetra-O-acetyl-4-O-(2,3,4,tri-O-ace- tyl-6-O-TBS-b-D-galactopyranosyl)-a-D-mannopyranose (4), as the major isomer, which served as a central point to both † Electronic supplementary information (ESI) available: selected data for compounds 7, 8, 10 and 11. See http://www.rsc.org/suppdata/cc/b1/ b106634j/ This journal is © The Royal Society of Chemistry 2001 2024 Chem. Commun., 2001, 2024–2025 DOI: 10.1039/b106634j Published on 12 September 2001. Downloaded by Monash University on 26/10/2014 14:53:34. View Article Online / Journal Homepage / Table of Contents for this issue