Synthesis and enzymatic and NMR studies of novel sialoside probes: Unprecedented, selective neuraminidase hydrolysis of and inhibition by C-6-(methyl)-Gal sialosides

Article abstract of DOI:10.1021/ja00084a003

We report here the synthesis of sialoside analogs, namely, αDNeuAc(2-6)(6-Me,R)βDGal-OR¹ (R isomer, tg rotamer analog) and αDNeuAc(2-6)(6-Me,S)βDGal-OR¹ (S isomer, gt rotamer analog, R¹ = CH₂CH₂SiMe₃ or H) and the corresponding sulfur linked thiosialosides useful for the determination of carbohydrate structural requirements 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 the galactose more rigid, (b) to maintain the C-6-O-6 arm of the galactose predominantly in 'tg' or 'gt' rotamer orientation, and (c) to evaluate the importance of these two rotamer orientations in neuraminidase catalyzed hydrolysis. Compared to the natural disaccharide αDNeuAc(2-6)βDGal-OR, the gt rotamer analogs are very poorly hydrolyzed by neuraminidases from the influenza A virus, Arthrobacter ureafaciens(A.U.), Vibria cholerae (V.C.), and Clostridium perfringens (C.P.) In contrast, the tg rotamer analogs are hydrolyzed by all four neuraminidases 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 smaller 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 investigations of these sialosides show that in tg rotamer analogs there is a preferential anti orientation of the sialoside aglycon as compared to the natural or the 'gt sialosides'. Computer assisted docking of these analogs into the binding pocket of the influenza A neuraminidase-sialic acid crystal structure shows that the tg rotamer analog fits favorably into the neuraminidase binding pocket, whereas the natural isomer in the gt rotamer orientation or the gt rotamer analog encounters severe repulsive 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 important implications in the rational design of neuraminidase inhibitors.