Highly water-soluble, artificial glycopolypeptides with a gamma-polyglutamic acid (gamma-PGA) backbone derived from Bacillus subtilis sp. and multivalent sialyloligosaccharide units have been chemoenzymatically synthesized as potential polymeric inhibitors of infection by bird and human influenza viruses. 5-Trifluoroacetamidopentyl beta-N-acetyllactosaminide and 5-trifluoroacetamidopentyl beta-lactoside were enzymatically synthesized from LacNAc and lactose, respectively, by cellulase-mediated condensation with 5-trifluoroacetamido-1-pentanol. After deacetylation, the resulting 5-aminopentyl beta-LacNAc and beta-lactoside glycosides were coupled to the alpha-carboxyl groups of the gamma-PGA side chains. The artificial glycopolypeptides carrying LacNAc and lactose were further converted to Neu5Acalpha2-(3/6)Galbeta1-4Glcbeta and Neu5Acalpha2-(3/6)Galbeta1-4GlcNAcbeta sialyloligosaccharide units by alpha2,3- and alpha2,6-sialyltransferase, respectively. The interaction of these glycopolypeptides with various influenza virus strains has been investigated by three different methods. Glycopolypeptides carrying Neu5Acalpha2,6LacNAc inhibited hemagglutination mediated by influenza A and B viruses, and their relative binding affinities for hemagglutinin were 10(2)- to 10(4)-fold higher than that of the naturally occurring fetuin control. A glycopolypeptide carrying Neu5Acalpha2,6LacNAc inhibited infection by A/Memphis/1/71 (H3N2) 93 times more strongly than fetuin, as assessed by cytopathic effects on virus-infected MDCK cells. The avian virus [A/duck/Hong kong/4/78 (H5N3)] bound strongly to Neu5Acalpha2,3LacNAc/Lac-carrying glycopolypeptides, whereas the human virus [A/Memphis/1/71 (H3N2)] bound to Neu5Acalpha2,6LacNAc in preference to Neu5Acalpha2,6Lac. Taken together, these results indicate that the binding of viruses to terminal sialic acids is markedly affected by the structure of the asialo portion, in this case either LacNAc or lactose, in the sugar chain of glycopolypeptides.