Synthesis of thrombin-inhibiting heparin mimetics without side effects

Abstract

Unwanted side effects of pharmacologically active compounds can usually be eliminated by structural modifications. But the complex heterogeneous structure of the polysaccharide heparin has limited this approach to fragmentation, leading to slightly better-tolerated heparin preparations of low molecular mass. Despite this improvement, heparin-induced thrombocytopaenia (HIT), related to an interaction with platelet factor 4 (PF4) and, to a lesser extent, haemorrhages, remain significant side effects of heparinotherapy. Breakthroughs in oligosaccharide chemistry made possible the total synthesis of the pentasaccharide antithrombin-binding site of heparin. This pentasaccharide represents a new family of potential antithrombotic drugs, devoid of thrombin inhibitory properties, and free of undesired interactions with blood and vessel components. To obtain more potent and well-tolerated antithrombotic drugs, we wished to synthesize heparin mimetics able to inhibit thrombin, that is, longer oligosaccharides. Like thrombin inhibition, undesired interactions are directly correlated to the charge and the size of the molecules, so we had to design structures that were able to discriminate between thrombin and other proteins, particularly PF4. Here we describe the use of multistep converging synthesis to obtain sulphated oligosaccharides that meet these requirements.

Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

“…A comprehensive study of the relationship of the saccharide sequences and gDbinding affinity remains to be investigated when a series of structurally defined HS oligosaccharides are available. The structural information from this study will serve as a lead compound for the chemical synthesis of HS oligosaccharides for further investigation (45).…”

Mechanism by Which Exosites Promote the Inhibition of Blood Coagulation Proteases by Heparin-activated Antithrombin

“…Heparins-Synthetic pentasaccharide and hexadecasaccharide heparins containing the antithrombin binding sequence (5,37) were provided by Maurice Petitou of Sanofi-Aventis Recherche (Toulouse, France). A full-length heparin containing the pentasaccharide binding sequence and ϳ50 saccharides in length was isolated from commercial heparin by size and affinity fractionation as described previously (30).…”

“…Reaction with factor Xa Binding of S195A Factor Xa to the Labeled AntithrombinsThe ϳ40% fluorescence increase accompanying the addition of S195A factor Xa to P7-NBD-antithrombin-heparin complex allowed quantitation of the binding affinity of the inactive factor Xa for antithrombin in the absence and presence of the heparin pentasaccharide and a hexadecasaccharide heparin mimetic capable of bridging antithrombin and proteases (37). Titrations of S195A factor Xa into P7-NBD-antithrombin complexed with the natural pentasaccharide, a higher affinity pentasaccharide, (5) or the hexadecasaccharide heparin resulted in saturable increases in fluorescence of comparable magnitude ( Fig.…”

Anticoagulant heparan sulfate: structural specificity and biosynthesis

“…The structural requirement for binding of this oligosaccharide to thrombin, a cluster of sulfated saccharide units, appears to be less selective than for AT. Petitou and colleagues proved that the order of the three domains is essential for thrombin inhibition (19). Namely, the ATbinding domain is positioned at the reducing end of the nonsulfated linker, and the thrombinbinding domain at the nonreducing end of the linker.…”

“…However, the pentasaccharide unit only inhibits the activity of factor Xa mediated by AT. A much larger oligosaccharide is required to exhibit complete anti-thrombin activity (18,19). Selectivity for HS by protein binding partners stems from a number of different structural variables that affect the overall charge and conformation.…”