Chemoenzymatic Synthesis of Glycan-arranged Polymeric Inhibitors against Influenza Virus Infection

Abstract

Highly water-soluble, artificial glycopolypeptides with a γ-polyglutamic acid (γ-PGA) backbone derived from Bacillus subtilis and multivalent sialyloligosaccharide units have been chemoenzymatically synthesized as potential polymeric inhibitors of infection by influenza virus. 5-Trifluoroacetamidopentyl β-N-acetyllactosaminide (1) was enzymatically synthesized from N-acetyllactosamine (LacNAc: Galβ1-4GlcNAc) by cellulase-mediated condensation with 5-trifluoroacetamido-1-pentanol. Next, enzymatic sugar elongation of the LacNAc unit to 1 was carried out by consecutive use of β1,3-N-acetylglucosaminyltransferase II (β3GnTII) and β1,4-galactosyltransferase I (β4GalTI) to produce tetra- and hexasaccharide glycosides (2 and 3) with tandem and triplet LacNAc repeats. After deacetylation, the resulting 5-aminopentyl di-, tetra- and hexasaccharide glycosides (4-6) were coupled to the α-carboxy groups of the γ-PGA side chains. Next, in order to synthesize an artificial sialoglycopolypeptide, we developed a large-scale production of rat α2,6-sialyltransferases (α2,6-SiaT). The α2,6-SiaT was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV-CP^--Chi^-) bacmid. The expressed α2,6-SiaT and commercially available α2,3-SiaT were used for sialylation of asialoglycopolypeptides (7-9). The structure-activity relationship of the resulting α2,3/6-sialoglycopolypeptides (10-15) with different glycans in the array has been investigated by focus-forming and solid-phase binding assays. The avian viruses specifically bound to glycopolypeptides carrying a short sialo-glycan with higher affinity than to a long glycan. In contrast, human viruses preferentially bound to long α2,3/6 sialylated glycan with LacNAc repeats in the receptors. Our strategy provides a facile way to design strong polymeric inhibitors of infection by avian and human influenza viruses.