CONFORMATIONAL STUDIES ON THE SELECTIN AND NATURAL-KILLER-CELL RECEPTOR LIGANDS SULFO-LACTO-N-FUCOPENTAOSES AND SIALYL-LACTO-N-FUCOPENTAOSES (SULNFPII AND SLNFPII) USING NMR-SPECTROSCOPY AND MOLECULAR-DYNAMICSSIMULATIONS - COMPARISONS WITH THE NONACIDIC PARENT MOLECULE LNFPII

This investigation is focused on the conformational behavior of the bl ood group Lewis(a) (Le(a))-active pentasaccharide lacto-N-fucopentaose II (LNFPII) and its sulfated and sialylated analogs, SuLNFPII and SLN FPII. The latter two are more potent oligosaccharide ligands for the a nimal lectins, E- and L-selectin, and the natural killer cell receptor , NKR-P1, than are the shorter chain analogs based on the trisaccharid e Le(a) domain. We report here that the three oligosaccharides based o n the fucopentasaccharide have very similar average solution conformat ions as determined from NMR spectroscopical parameters, in particular C-13 chemical shift differences. From restrained simulated annealing a nd restrained molecular dynamics (MD) simulations performed in order t o determine the most probable conformational distributions around the glycosidic linkages we derive models for these oligosaccharides that a re in good agreement with experimental parameters, such as rotating-fr ame Overhauser effects (ROE's) and long-range H-1, C-13 coupling const ants across the glycosidic linkages. In these model structures the Le( a) domain at the nonreducing end of the longer chain oligosaccharides approximates the same rigid structure as in the shorter analogs. The G al beta 1-4Glc linkage at the reducing end is also rather rigid, showi ng only little more flexibility than the Le(a) domain. However, the Ne uAc alpha 2-3Gal linkage in SLNFPII, and the GlcNAc beta 1-3Gal linkag e in all three oligosaccharides are flexible, in each case fluctuating mainly between two minimum energy structures: (phi = -81 degrees, psi = 8 degrees) and (phi = -160 degrees, psi = 20 degrees) for the NeuAc alpha 2-3Gal linkage, as reported previously for the isomeric sequenc e 3'-sialyl Le(X), and (phi -25 degrees, psi = -26 degrees) and (phi = 20 degrees, psi = 24 degrees) for the GlcNAc beta 1-3Gal linkage. The flexibility of the latter linkage may allow the lactosyl ii domain at the reducing end to fit with little strain into extended carbohydrate binding sites on the recognition proteins, and, for the purposes of d rug designs, it will be important to establish which conformational di stribution is assumed for the GlcNAc beta 1-3Gal linkage in these long er chain oligosaccharides in the bound state.