Carbohydrate Module Method: A Simple Methodology for the Library Assembly of Oligosaccharide Mimetics

 Artificial glycopolymers and other multivalent glycoconjugates comprise a new class of bio-mimetic supramolecules. They have many applications to cell cultivation, tumor diagnosis, and detection and trapping of viruses and bacterial toxins. Their usefulness is ascribed mainly to their strong and species-specific interactions with carbohydrate receptor proteins as the result of multivalent binding and carbohydrate cluster effects. Recently, much biological interest is being directed to such glycopolymers carrying human cell surface oligosaccharides. The multivalent assembly is, however, retarded seriously by the difficulty in constructing targeting oligosaccharides.

In our synthetic study on the glycopolymers carrying the human oligosaccharides, we found that an acrylamide copolymer carrying -L-fucopyranoside and 3-sulfo--D-galactopyranoside in clusters shows potent activity to block L-selectin/sialyl LewisX tetrasaccharide adhesion. As judged from the result that none of the acrylamide copolymers carrying only one of the two glycosides showed notable activity, the observed activity was ascribable to the cooperative effects of the two key glycosides embedded in the copolymer. This finding prompted us to generalize the copolymerization approach as a practical method to assemble oligosaccharide mimetics.
Figure 1 The key concept of the carbohydrate module method
The concept of “key carbohydrate structure” and “carbohydrate module method”
For facile understanding of our carbohydrate module method, we assume a binding model between a branching oligosaccharide and the receptor protein (Figure 1a). In this model, two glycosidic residues A and B provide the key carbohydrate structures responsible for the binding with the receptor protein. Conventional mimic syntheses may target a branching trisaccharide carrying A, B, and C residues and further its multivalent models. Our approach targets a copolymer-(A+B) carrying the key glycosides (Figure 1b). The simple copolymer-(A+B) is assumed to have a certain chance to occupy both binding sites and thus to exhibit higher activity at least than the polymers carrying only one of the key glycosides. This means that the copolymerization between the appropriate glycosyl monomer-A and monomer-B provides a promising synthetic methodology to assemble the biologically active structure of the targeting oligosaccharide.

A typical experimental protocol
In practice, the present approach involves three steps: (1) segmentation of a targeting oligosaccharide into smaller glycosides, (2) preparation of the corresponding glycosyl monomers (defined as “carbohydrate modules”), and (3) the radical copolymerization of the two monomers with acrylamide. For example, a sialyl LewisX tetrasaccharide was modularized to allyl -L-fucopyranoside and p-acrylamidophenyl 3-sulfo--D-galactopyranoside (Figure 1c). We reported earlier that the derived acrylamide copolymer has potent activity to block a human L-selectin. With this methodology in either a direct or an indirect manner, we have assembled various glycopolymers carrying human oligosaccharide mimetics, including sialyl LewisX, 6-sulfo sialyl LewisX, globosyl di- and trisaccharides and heparan sulfate fragments. In our research works, we have shown that the methodology is useful not only for synthetic purposes but also for surveying the key glycoside structures for interactions with receptor proteins such as selectins, sialidases, Shiga toxins, and prions associated with infectious diseases.

Just recently, T. Lindhorst in Germany (University of Kiel) and N. Bovin in Moscow (Russian Academy of Science) reported module approaches for the synthesis of oligosaccharide mimics. Their approaches are based on the assembly of different glycoside epitopes on a dendric template and a poly-(acrylic acid), respectively. Although they differ from our copolymerization approach, all these methodologies are based on the concept of a key carbohydrate module. The module approach will clearly provide a promising chemical tool to solve the complexity of cell matrix glycoproteins and glycolipids as well as their diverse biological roles.
Yoshihiro Nishida, Kenji Sasaki and Kazukiyo Kobayashi (Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University)
References (1) Nishida Y, Uzawa H, Toba T, Sasaki K, Kondo H, Kobayashi K: A facile synthetic approach to L- and P-selectin blockers via copolymerization of vinyl monomers constructing the key carbohydrate modules of sialyl LewisX mimics. Biomacromolecules 1, 68-74, 2000
(2) Sasaki K, Nishida Y, Tsurumi T, Uzawa H, Kondo H, Kobayashi K: Facile assembly of cell surface oligosaccharide mimics by copolymerization of carbohydrate modules. Angew. Chem. Int. Ed. Engl. 41, 4463-4467, 2002
(3a) Sasaki K, Nishida Y, Uzawa H, Kobayashi K: N-Acetyl-6-sulfo-D-glucosamine as a promising mimic of N-Acetyl neuraminic acid. Bioorg. Med. Chem. Lett. 13, 2821-2823, 2003.
(3b) Sasaki K, Nishida Y, Kambara M, Uzawa H, Takahashi T, Suzuki T, Suzuki Y, Kobayashi K: Design of N-acetyl-6-sulfo--D-glucosaminide-based inhibitors of Influenza virus sialidase. Bioorg. Med. Chem. 12, 1367-1375, 2004
(4a) Dohi H, Nishida Y, Furuta Y, Uzawa H,. Yokoyama S –I, Ito S, Mori H, Kobayashi K: Molecular design and biological potential of galacto-type trehalose as a non-natural ligand of Shiga toxins. Org. Lett. 4, 355-357, 2002
(4b) Uzawa H, Kamiya S, Minoura N, Dohi H, Nishida Y, Taguchi K, Yokoyama S –I, Mori H, Shimizu T, Kobayashi K: A quartz crystal microbalance method for rapid detection and differentiation of Shiga toxins by applying a monoalkyl globobioside as the toxin ligand. Biomacromolecules, 3, 411-414, 2002
(4c) Miura Y, Sasao Y, Dohi H, Nishida Y, Kobayashi K: Self-assembled monolayers of globotriaosylceramide (Gb3) mimics: surface-specific affinity with shiga toxins. Anal. Biochem. 310, 27-35, 2002
function of glycoprotein sugar chain(GP-A00, NS-A00)/ carbohydrate-protein interaction(LE-A03, LE-B01, PG-B07, GT-C04,)/ synthesis of glycoconjugate(GP-B00, GT-A00, GT-A01, GT-A03, GT-A04, GT-A05, GT-B07)/ oligosaccharide mimetics(GP-C03, GT-A06, GT-A07, GT-D02, GT-D03)/ selectin(LE-A05, PG-B01, GD-A07)/ sialyl LewisX(GD-A07)/ proteoglycan(PG-A00, PG-A07)/ heparan sulfate(PG-A00, PG-A01, PG-A11)/ influenza virus(GL-B01, GD-A06)/ bacterial toxin(GL-B02)/ sialidase(GD-B02)
Apr. 23, 2004

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