Cluster Sialoside Inhibitors for Influenza Virus: Synthesis, NMR, and Biological Studies

Two alphaDNeuAc(2-->6)betaDGal(1-->4)betaDGlcNAc units which are the receptor determinants for the influenza virus hemagglutinin have been anchored on a galactose in order to design structures capable of bimodal viral binding. To determine the optimum alphaDNeuAc distance for the best intramolecular binding to the hemagglutinin trimer, the attachment sites of the two alphaDNeuAc(2-->6)betaDGal(1-->4)betaDGlcNAc units have been systematically varied by proper choice of the galactose glycosylation sites. Thus, we have chemoenzymatically prepared five heptasaccharides of the general formula alphaDNeuAc(2-->6)betaDGal(1-->4)betaDGlcNAc(1-->x)[alphaDNeuAc(2-->6)betaDGal(1-->4)betaDGlcNAc(1-->y)]betaDGalOR, where x and y are 2 and 3, 2 and 4, 2 and 6, 3 and 6, and 4 and 6, respectively. The structural identity and the complete proton and carbon chemical shift assignments of the tri-, penta-, and heptasaccharides have been established by a combination of 1D TOCSY, 1D and 2D NOESY, and H-1-C-13 correlation techniques. One-dimensional and two-dimensional NOESY experiments have been used to assess the conformational properties of these molecules. These, together with Monte Carlo simulations, suggested that the end C-2 to C-2 atoms of the sialic acid residues in these bivalent receptor structures are separated by approximately 19 angstrom in 2,4-heptasaccharides to approximately 9 angstrom in 3,6- and 4,6-heptasaccharides, with the others falling between these two distances. The five heptasaccharides, when evaluated as inhibitors of the influenza virus adsorption to resialylated human erythrocytes, showed that relative to the methyl alpha-sialoside the 3,6- and 4,6-disialosides with a 9-angstrom end distance exhibited 10- and 8.4-fold better inhibitory activity, respectively, whereas the 2,4-disialoside with a approximately 19-angstrom sialic acid end distance had no increased inhibitory activity. Conjugation of the 3,6-heptasaccharide to bovine serum albumin increased the inhibitory potency even more, suggesting the therapeutic potential of a macromolecule containing the 3,6-heptasaccharides for influenza viral inhibition.