CHEMICAL APPROACHS TO THE STUDY OF GLYCOLIPID BIOCHEMICAL MECHANISMS OF ACTION

Introduction & Aim of work: Simplexide, a long chain secondary alcohol glycosylated by a disaccharide, is a glycolipid isolated from the marine Caribbean sponge Plakortis simplex.1 Simplexide shows immunosuppressive activity and strongly inhibits proliferation of activated T cells without cytotoxic activity.1 More recently, a very interesting and unique pattern of cytokines release from PBMC (Peripheral Blood Mononuclear Cells) was observed after stimulation with simplexide. This unprecedented profile makes simplexide a potential lead for the treatment of autoimmune diseases. Nevertheless, more detailed studies are needed in order to understand the biochemical mechanism of action and for a better comprehension of the structural requirements for activity. In the first year, a synthetic approach towards the preparation of a simplexide fluorescent probe2,3 has been developed. This compound will be functional to evaluate the subcellular distribution of the glycolipid and to clarify its mechanism of action. In particular, the analogue 1 (Fig.1), with a fluorescent group, attached to the position 6 of glucose through a spacer arm, has been designed. In order to minimize inaccurate results it is important that the addition of the fluorescent probe does not interfere in the association of the ligand with its host. The choice of position 6 of glucose should meet these requirements, since preliminary results revealed that the biological receptor of simplexide should be the CD1d protein, which recognizes glycolipids by docking their lipid part in the hydrophobic binding region while leaving the polar head group protruding from the surface of the protein binding groove for T cell recognition. Results: We have obtained the donor 2 (Fig.2), from β-D-glucose pentaacetate in seven steps, and the acceptor 3 (Fig.2), from β-D-galactose pentaacetate in four steps, in good yield. Then disaccharide 4 has been achieved by coupling of acceptor 3 and donor 2; optimization studies on this reaction have been performed allowing to obtain disaccharide 4 (Fig.2) with α anomeric linkage in satisfactory yield. Conclusion and Prospectives: Now we have in project to couple compound 4 (Fig.2) with the desired long chain alcohol; after that the target compound 1 will be obtained by means of azido reduction, conjugation with a fluorescent probe through a spacer arm, and standard removal of protecting groups. Moreover we plan to obtain different analogues in which the length of the lipid chain or/and the anomeric linkages will be varied. Finally these compounds will be tested in order to better understand the mechanism of action of simplexide. References: 1. Costantino, V., Fattorusso, E., Mangoni, A., Di Rosa, M., Ianaro, A. Bioorg. Med. Chem. Lett, 1999, 9, 271. 2. Hoang, Y. V., Micouin, L., Ronet, C., Gachelin, G., Bonin, M. ChemBioChem, 2003, 4, 27-33. 3. Im, J. S., Yu, K. O. A., Illarionov, P. A., Le Clair, K. P., Storey, J. R., Kennedy, W. M., Bersa, G. S., Porcelli S. A., J. Biol. Chem, 2004, 279, 299-310.