SYNTHESIS AND ENZYMATIC AND NMR-STUDIES OF NOVEL SIALOSIDE PROBES - UNPRECEDENTED, SELECTIVE NEURAMINIDASE HYDROLYSIS OF AND INHIBITION BY C-6-(METHYL)-GAL SIALOSIDES

We report here the synthesis of sialoside analogs, namely, alpha DNeuA c(2-6)(6-Me,R)beta DGal-OR(1) (R isomer, tg rotamer analog) and alpha DNeuAc(2-6)(6-Me,S)beta DGal-OR(1) (S isomer, gt rotamer analog, R(1) = CH(2)CH(2)SiMe(3) or H) and the corresponding sulfur linked thiosial osides useful for the determination of carbohydrate structural require ments in neuraminidase hydrolysis and for the design of neuraminidase inhibitors. The purpose of methyl substitution at C-6 of the galactose in these analogs is (a) to render the rotation around C6-C5 bond of t he galactose more rigid, (b) to maintain the C-6-O-6 arm of the galact ose predominantly in ''tg'' or ''gt'' rotamer orientation, and (c) to evaluate the importance of these two rotamer orientations in neuramini dase catalyzed hydrolysis. Compared to the natural disaccharide alpha DNeuAc(2-6)beta DGal-OR, the gt rotamer analogs are very poorly hydrol yzed by neuraminidases from the influenza A virus, Arthrobacter ureafa ciens (A.U.), Vibrio cholerae (V.C.), and Clostridium perfringens (C.P .). In contrast, the tg rotamer analogs are hydrolyzed by all four neu raminidases at comparable rates relative to the natural disaccharide. Detailed enzyme kinetic analysis indicates that the gt rotamer analogs bind less efficiently to the neuraminidases and have 4- to 18-fold sm aller V-max, as compared to the tg rotamer analogs. Evaluation of the sulfur analogs as neuraminidase inhibitors indicates that only a ''tg rotamer'' thiosialoside analog is a good competitive inhibitor of the four neuraminidases. The inhibition constant K-i ranges from 0.3 to 1 mM. Neither the natural thiosialoside analog nor the gt thiosialoside analogs are effective inhibitors (K-i > 5 mM). Detailed NMR investigat ions of these sialosides show that in tg rotamer analogs there is a pr eferential anti orientation of the sialoside aglycon as compared to th e natural or the ''gt sialosides''. Computer assisted docking of these analogs into the binding pocket of the influenza A neuraminidase-sial ic acid crystal structure shows that the tg rotamer analog fits favora bly into the neuraminidase binding pocket, whereas the natural isomer in the gt rotamer orientation or the gt rotamer analog encounters seve re respulsive interactions with the arginine residues at the catalytic site. The perturbation of these important arginine residues appear to be responsible for the lack of neuraminidase catalyzed hydrolysis or inhibition by the gt rotamer analogs. These findings may have importan t implications in the rational design of neuraminidase inhibitors.