Glycotentacles: Synthesis of Cyclic Glycopeptides, Toward a Tailored Blocker of Influenza Virus Hemagglutinin

Abstract

Influenza virus infection is initiated by the binding of hemagglutinin (HA), a viral carbohydrate-binding membrane protein, to sialic acid-containing oligosaccharides, such as GM3 trisaccharide [Neu5Aca(2,3)Galb(1,4)Glc], on the host cell surfaces.[1] Since this molecular-recognition process leads to the host cell-virus adhesion stage, molecules with a high affinity for the viral HA would be potent candidates for blockers of the influenza virus. Various types of HA blockers carrying sialooligosaccharides have been synthesized over the last decade.[2] Enhanced blocking effects by the multidentate (multivalent) glycoligands were often found by clustering [3] the sialooligosaccharides on macromolecular scaffolds, such as synthetic polymers, [4] cyclodextrins, [5] and dendrimers, [6] although the mechanism of this multiple interaction has not yet been fully investigated. In these macromolecular approaches, structural information on the spatial arrangement of the multiple binding sites of the protein is not usually accounted for in the design of ligands. Recently, it was found that ligands containing small oligodentate carbohydrate units, that are designed topologically to bind simultaneously to several binding sites of a single lectin, are very effective blockers. [7,8] The syntheses of the STARFISH dendrimer used to inhibit the adherence of verotoxins, is an excellent example of how detailed structure analysis has led to the creation of a strongly effective "multidentate" ligand. [7, 2e] Thus, on the basis of the HA structure, we created cyclic peptide scaffolds containing tridentate carbohydrate units as a potential HA blockers by using a convenient chemoenzymatic method. HA is a trimeric protein and each subunit contains one sialooligosaccharide binding site on the outer portion. From the three-dimensional structure of HA, as revealed by X-ray analysis, [9] these three binding sites are located on the apexes of an equilateral triangle, in which each binding pocket is separated by approximately 40-50 . To achieve a rational molecular design for an HA blocker, we focused on the use of cyclic peptides [10] as a scaffold because of the flexibility of the synthetic strategy and their biological compatibility in the human body.Herein, we report a chemoenzymatic synthesis of a series of cyclic peptides presenting three sialotrisaccharide units [Neu5Aca(2,3)Galb(1,4)Glc] by using transglutaminase and glycosyltransferase (Scheme 1). We also discuss the effect of peptide sequence on their biological activities.Scheme 1 shows the synthetic route to cyclic glycopeptides by means of a combined chemical and enzymatic strategy. In this case, a cyclic peptide 1 prepared by employing the Fmoc-Asp-ODmab method [11] on a chlorotrityl resin [12] was enzymatically coupled with 6-aminohexyl lactoside (4) using transglutaminase from guinea pig liver.[13] The transamination reaction between three Gln residues of cyclic peptide 1 and 4 (large excess) gave a mixture of mono-(1-m), bi-(1-b), and tri-substituted (1-t) derivatives. After...

Pathogen Inhibition by Multivalent Ligand Architectures

“…They observed that the tridentate sialyl lactose ligand on a particular cyclic peptide template, where all the three ligands were directed outward from the cyclic peptide ring, exhibited highest affinity (K d = 0.65 mM) for HA protein on the SPR sensor surface (see Figure 4e). 98 Here, the distance between binding sites on the hemagglutinin structures needed to be known in advance in order to simultaneously bridge and reinforce the bond formation between the tripod units and binding pockets at the HA head. Using longer PEG segments not only raised the probability of the ligand and receptor binding, but also greatly increased the entropic cost through molecular conformations of the tethered chains.…”

Sialobiology of Influenza: Molecular Mechanism of Host Range Variation of Influenza Viruses

“…The shape of the glycopeptide protein scaforld, which is determined by the amino acid sequences employed, is also found to be significant in determining the inhibitory activity. 87) We also developed glycopolymers containing multivalent sialyloligosaccharides (Neu5Aca2-3Galb1-4GlcNAcb1-, Neu5Aca2-6Galb1-4GlcNAcb1-, Neu5Aca2-3Galb1-3Gal-NAca1-, Neu5Aca2-3Galb1-3GalNAcb1-) with a poly (Lglutamic acid) backbone, 88) triantennary oligosaccharide branches (Neu5Aca2-3Galb1-4GlcNAcb1-, Neu5Aca2-6Galb1-4GlcNAcb1-) with a polyacrylamide backbone, 89) and sialyllactose (Neu5Aca2-3Galb1-4Glcb1-; Neu5Aca2-6Galb1-4Glcb1-) with a polystyrene backbone 90) that inhibit the receptor binding function of the hemagglutinin of influenza viruses. The glycopolymer inhibited the hemagglutination of influenza A virus, and its activity level was 10 3 -times higher than that of the oligosaccharide itself, indicating that clustering of the sugar chain in the polymer backbone may be important for inhibitory activity of the viral hemagglutinin.…”

Recent Anti-Influenza Strategies in Multivalent Sialyloligosaccharides and Sialylmimetics Approaches

“…A cyclic peptide presenting three 3-sialyllactoside units ( 1 0 ) was prepared by chemoenzymatic method using transglutaminase and glycosyltransferase [52] by S.-I. Nishimura's group.…”