Shuhei Koshida

Synthesis and biological activity of oligomer-model compounds containing units of a key platelet-binding disaccharide of heparin

Abstract A key disaccharide unit in heparin, O -(2-deoxy-2-sulfamido-6- O -sulfo-α- d -glucopyranosyl)-(1→4)-2- O -sulfo-α- l -idopyranosyluronic acid, was previously found to be responsible for the binding interaction of heparin to platelets. A clustering effect to enhance the binding was found to be dependent on the number and frequency of the disaccharide units in a heparin molecule. To systematically examine the clustering effect, three oligomer-model compounds containing two or three units of the disaccharide were synthesized. These compounds inhibited 3 H-labelled heparin binding to human platelets more strongly than a compound containing only one unit of the disaccharide.

Synthesis of oligomeric assemblies of a platelet-binding key disaccharide in heparin and their biological activities

Abstract Heparin, highly sulfated glycosaminoglycan, binds to platelets. A key disaccharide unit in heparin was previously found to be responsible for the binding, and the frequency of the disaccharide unit was important for the binding potency. A newly developed method based on the reductive amination was effectively applied to prepare structurally defined oligomeric assemblies possessing multiple units of the key disaccharide. From their platelet-binding activities measured by the competitive binding assay, the enhancement of the activity was clearly observed with increasing number of the key disaccharide.

Synthesis of heparin partial structures and their binding activities to platelets.

A synthetic pentasaccharide corresponding to the antithrombin III-binding region in heparin was also found to bind to human platelets. To identify the platelet-binding site in the pentasaccharide which is expected to be a novel sequence in heparin responsible for its platelet-binding, five partial structures of this particular pentasaccharide were synthesized. In a competitive assay using [3H]-heparin, a trisaccharide, O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glucopyranosyl)-1--> 4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranose, was concluded to be a high-affinity site for heparins binding to platelets.

Synthesis and biological activity of a model disaccharide containing a key unit in heparin for binding to platelets

Abstract To determine the specific site(s) in heparin necessary for binding to platelets, synthesis of a model compound containing the disaccharide sequence, O-(2-deoxy-2-sulfamido-6-O-sulfo-α-D-glucopyranosyl)-(1→4)-2-O-sulfo-α-L-idopyranuronic acid, found in heparin was performed by α-selective glycosidation using a phenyl thioglycoside as a donor. The compound inhibited 125 I-labelled heparin binding to human platelets to a greater extent than a heparin-derived disaccharide, obtained by the heparinase I digestion, yet contained the same number of sulfate groups per molecule.

An efficient method for the assembly of sulfated oligosaccharides using reductive amination

Abstract A generally applicable efficient method was developed for assembly of structurally defined sulfated oligosaccharides. Using a reductive amination reaction at low pH with newly designed linkers possessing multiple aromatic amino groups, facile preparations of oligosaccharide assemblies were achieved without any alteration in the sulfated oligosaccharide parts.

Novel photo affinity cross-linking resin for the isolation of heparin binding proteins

A new photo cross-linking reagent, 2-(4-azidophenylamino)-4-(1-ammonio-4-azabicyclo[2,2,2]oct-1-yl)-6-morpho-lino-1,3,5-triazine chloride (named AA-D) was developed, which was used for the identification of several heparin-binding proteins on the surface of intact platelets. Also a functional resin for the isolation of heparin-binding proteins from a protein mixture was prepared. Heparin was first immobilized onto a polystyrene Merrifield resin or Argo-gel™ using a reductive amination reaction. Then the immobilized heparin was coupled with the AA-D reagent to give a functional resin. The resin was incubated with an individual protein, such as ovalbumin, bovine serum albumin (BSA), antithrombin III (ATIII) or a synthetic peptide corresponding to the heparin-binding domain of ATIII, or with a mixture of the above proteins. This was then photo-irradiated to induce the cross-linking between the heparin and the protein bound to it. Ovalbumin, a non-heparin-binding protein, was recovered from the supernatant of the irradiated incubation mixture. BSA, known to bind to heparin nonspecifically, was found in the washing of a high ionic strength solution. In contrast, native ATIII and its heparin-binding domain peptide were covalently bound to the heparin on the resin and liberated from the resin only after degradation of the resin-bound heparin with nitrous acid or heparinase I treatment. The experiments demonstrated that the resin modified with heparin and the AA-D have a definite practical value in the selective isolation of heparin-binding proteins.