The Unique Antithrombin III Binding Domain of Heparin: A Lead to New Synthetic Antithrombotics

Heparin, a sulfated glycosaminoglycan, is well known for its anticoagulant effect mediated by the serine protease inhibitor antithrombin III (AT III). Heparin has been used clinically for more than half a century for the prophylaxis and treatment of venous thrombosis and thromboembolism. Up until the 1980s it was assumed that the biological activity of heparin was mainly caused by its polyanionic character. However, this paradigm was contradicted when it was discovered that part of the heparin polysaccharides contains a well-defined pentasaccharide domain that specifically binds and activates AT III. The specificity of the interaction between the characteristic pentasaccharide and AT III has become more obvious after the synthesis and biological testing of various heparin analogues. This article reviews the synthesis of the heparin pentasaccharide, some closely related counterparts, and some highly modified analogues. With the aid of molecular modeling and through “tailored” molecular modifications of the pentasaccharide, much knowledge has been gained concerning structure-activity relationships. On this basis not only have more potent and simplified derivatives been developed, but also the recognition between heparin and AT III can now be understood in greater detail at the molecular level.     

Affinity capillary electrophoresis for the determination of binding affinities for low molecular weight heparins and antithrombin‐III

The anticoagulant properties of heparin stem in part from high‐affinity binding to antithrombin‐III (AT‐III) inducing a 300‐fold increase in its inhibitory activity against the coagulation protease factor Xa. The minimal structural requirements for AT‐III binding are contained in the rare heparin pentasaccharide sequence containing a 3,6‐O‐sulfated N‐sulfoglucosamine residue. ACE is used in this work to measure the relative AT‐III binding affinities of the low molecular weight heparins (LWMHs) dalteparin, enoxaparin, and tinzaparin and the synthetic pentasaccharide drug fondaparinux (Arixtra). Determination of the AT‐III binding affinities of the LWMHs is complicated by their inherent structural heterogeneity and polydispersity. The fractional composition of 3‐O‐sulfo‐N‐sulfoglucosamine residues was determined for each drug substance using 2D NMR and used in the interpretation of the ACE results.     

A Synthetic Antithrombin III Binding Pentasaccharide Is Now a Drug! What Comes Next?

Heparin is a sulfated glycosaminoglycan isolated from animal organs that has been used clinically as an antithrombotic agent since the 1940s. In the early 1980s it was discovered that a unique pentasaccharide domain in some heparin chains activates antithrombin III (AT-III), a serine protease inhibitor that blocks thrombin and factor Xa in the coagulation cascade. Sanofi-Synthélabo and Organon developed a synthetic analogue of this pentasaccharide. The resulting antithrombotic drug arixtra, which went on the market in the USA and Europe in 2002, shows superior antithrombotic activity and brings about AT-III-mediated activity against factor Xa exclusively. Structure-based design has subsequently led to analogues with longer-lasting activity, such as idraparinux, as well as novel conjugates and long oligosaccharides with specific anti-Xa and antithrombin activities. The new drug candidates are more selective in their mode of action than heparin and less likely to induce thrombocytopenia.     

Syntheses of heparin - like pentamers containing “opened” uronic acid moieties

The syntheses of four analogues of pentasaccharide Ia, which corresponds to the minimal AT III binding region of heparin, are presented and the biological activities of these analogues will be discussed. Three of these analogues (i.e. compounds II, III and IV) contain an R-glyceric acid oxymethylene residue (i.e. B in fig.3) instead of -L-iduronic acid and in the other analogue (i.e. compound V) the β-D-glucuronic acid unit has been replaced by an s-glyceric acid oxymethylene residue (i.e. A in fig3). The R and S-glyceric acid oxymethylene residues represent an “opened” iduronic acid unit and an “opened” glucuronic acid unit, respectively, containing the essential carboxylate function in the appropriate configuration. The crucial step in the syntheses of these “opened” uronic acid pentamer analogues, was the preparation of the required glyceric acid oxymethylene residues 8a, 8b and 8c.

Analogues II and III, containing an “opened” iduronic acid moiety, display a significant AT III mediated Xa activity. Compound III contains two extra sulphate groups at unit 2. Removal of the contributing O-sulphate groups at position 3 and 6 of unit 6 of compound II (i.e. compound IV) results in a seven-fold drop in Xa activity. Replacement of the β-D-glucuronic acid unit by an S-glyceric acid oxymethylene residue (i.e. compound V) leads to almost a complete loss of Xa activity, notwithstanding the fact that all the essential and contributing charged groups are present in the molecule.     

Experimental proof for the structure of a thrombin-inhibiting heparin molecule

Kinetic studies of thrombin inhibition by antithrombin in the presence of heparin have shown that thrombin binds to heparin in a preformed heparin-antithrombin complex. To study the relative position of the thrombin binding domain and the antithrombin binding domain on a heparin molecule we have designed and synthesized heparin mimetics, which structurally are very similar to the genuine polysaccharide. Their inhibitory properties with respect to factor Xa and thrombin provide experimental evidence that in heparin the thrombin binding domain must be located at the nonreducing end of the antithrombin binding domain to observe thrombin inhibition. As expected, factor Xa inhibition is not affected by elongation of the antithrombin binding pentasaccharide sequence, regardless of the position in which this elongation takes place.     

Synthesis and Anticoagulant Activity of Bioisosteric Sulfonic‐Acid Analogues of the Antithrombin‐Binding Pentasaccharide Domain of Heparin

Two pentasaccharide sulfonic acids that were related to the antithrombin‐binding domain of heparin were prepared, in which two or three primary sulfate esters were replaced by sodium‐sulfonatomethyl moieties. The sulfonic‐acid groups were formed on a monosaccharide level and the obtained carbohydrate sulfonic‐acid esters were found to be excellent donors and acceptors in the glycosylation reactions. Throughout the synthesis, the hydroxy groups to be methylated were masked in the form of acetates and the hydroxy groups to be sulfated were masked with benzyl groups. The disulfonic‐acid analogue was prepared in a [2+3] block synthesis by using a trisaccharide disulfonic acid as an acceptor and a glucuronide disaccharide as a donor. For the synthesis of the pentasaccharide trisulfonic acid, a more‐efficient approach, which involved elongation of the trisaccharide acceptor with a non‐oxidized precursor of the glucuronic acid followed by post‐glycosidation oxidation at the tetrasaccharide level and a subsequent [1+4] coupling reaction, was elaborated. In vitro evaluation of the anticoagulant activity of these new sulfonic‐acid derivatives revealed that the disulfonate analogue inhibited the blood‐coagulation‐proteinase factor Xa with outstanding efficacy; however, the introduction of the third sulfonic‐acid moiety resulted in a notable decrease in the anti‐Xa activity. The difference in the biological activity of the disulfonic‐ and trisulfonic‐acid counterparts could be explained by the different conformation of their L ‐iduronic‐acid residues.     

Synthesis of a Pentasaccharide Corresponding to the Antithrombin III Binding Fragment of Heparin

The synthesis of a protected pentasaccharide 27b corresponding to the antithrombin III binding region of heparin is presented. This pentasaccharide was prepared from two disaccharides (12c and 23) and a monosaccharide (1). The glucuronic acid containing disaccharide 12c was prepared from easily available monomers 6 and 7. Oxidation to the uronic acid was performed in the disaccharide stage. L-Idose derivative 16, prepared via a new route, was coupled with 1,6-anhydro derivative 17, oxidized and transformed into disaccharide 23. Coupling of 12c and 23 to tetrasaccharide 24a has been investigated. Better yields were obtained without collidine, the reason for which is explained. Coupling of 24b and 1 afforded the pentasaccharide 27b, protected with acetyl at the positions to be sulphated, benzyl at the other hydroxyl functions and azide at the 2-position of the glucosamine residues. Conversion of 27b into the sulphated pentasaccharide Ib can be performed according to published procedures.     

Degeneracy of the Antithrombin Binding Sequence in Heparin: 2-O-Sulfated Iduronic Acid Can Replace the Critical Glucuronic Acid

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this “canonical” GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a ¹C₄-²S₀ equilibrium in the unbound saccharide, shift to full ²S₀ and full ¹C₄ upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.     

Chemical approaches to define the structure-activity relationship of heparin-like glycosaminoglycans

Heparin, the drug of choice for the prevention and treatment of thromboembolic disorders, has been shown to interact with many proteins. Despite its widespread medical use, little is known about the precise sequences that interact with specific proteins. The minimum heparin binding sequence for FGF1 and FGF2 necessary to promote signaling was investigated. A characteristic pentasaccharide sequence, DEFGH, is required to accelerate the inhibition of thrombin and factor Xa in the blood-coagulation cascade. The first synthetic heparin pentasaccharide drug has been approved in Europe and the US and is sold under the trade name Arixtra. Other oligosaccharides with different composition are under clinical investigation. The enormous interest in the assembly of heparin oligosaccharides will stimulate the development of new synthetic approaches. Heparin-oligosaccharide-synthesis automation similar to that of DNA or peptide synthesis will play an important role.     

Concise synthesis of a pentasaccharide related to the anti-leishmanial triterpenoid saponin isolated from Maesa balansae

Concise synthesis of the glycone part (a pentasaccharide) of the anti-leishmanial triterpenoid saponin isolated from Maesa balansae is reported. A late-stage TEMPO-mediated oxidation of a primary hydroxyl group to carboxylic acid has been achieved under phase-transfer conditions. Glycosylations were performed either by thioglycoside or glycosyl trichloroacetimidate activation using sulfuric acid immobilized on silica (H2SO4-silica) in conjunction with N-iodosuccinimide and alone, respectively. H2SO4-silica was proved to be a better choice as promoter than conventional Lewis acid promoters such as TfOH or TMSOTf.     

Differentiation of 3-O-sulfated heparin disaccharide isomers: Identification of structural aspects of the heparin CCL2 binding motif

The presence of 3-O-sulfated glucosamine residues in heparin or heparan sulfate plays a role in binding to antithrombin III and HSV infection. In this study, tandem mass spectrometry was used to differentiate between two heparin disaccharide isomers containing variable sulfate at C6 in a common disaccharide and C3 in a more rare one. The dissociation patterns shown by MS² and MS³ were clearly distinguishable between the isomers, allowing their differentiation and quantitation. Using this technique, we show that an octasaccharide with 11 sulfate groups with high affinity for inflammatory chemokine CCL2 does not contain 3-O-sulfated disaccharides.     

Preparation and photochemistry of cobalt(III) amino and amino acidato complexes containing tripodal polypyridine ligands

The photochemical reactivities of cobalt(III)-diamine and cobalt(III)-amino acid compounds have been compared using complexes that also contain polypyridyl ligands. Metallacyclic complexes result from UV-induced photodecarboxylation reactions of the amino acid complexes. UV-irradiation of closely related complexes with amine donors replacing the carboxylate donors does not lead to the production of the same metallacyclic products. The reported UV-induced fragmentation of amine donors and subsequent metallacycle formation appears not to be a general reaction. Nine cobalt(III) complexes of polypyridyl ligands have been structurally characterised, including four that also contain amino acid ligands and one that contains a three-membered metallacyclic ring.     

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma.

Novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin-PGMA-I was prepared by directly coupling amino-groups of heparin with PGMA. Heparin-PGMA-II and III were prepared by the coupling of heparin to amino-PGMA, which was obtained by amination of PGMA. Heparin-PGMA-II was prepared by coupling the carboxyl groups of heparin to amino-PGMA by using water-soluble carbodiimide as coupling reagent, and heparin-PGMA-III was prepared by the reductive amination of heparin and amino-PGMA with sodium cyanoborohydride. The heparin contents of heparin-PGMA-I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin-PGMA-I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non-specific interaction with other proteins and can be used in a wide pH range. All the three heparin-PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography.     

The influence of molecular weight of dextran forming a heparin-bonding polysaccharide layer on the chromatographic properties of sorbents for HPAC analysis of human antithrombin III

Summary Human antithrombin III (AT III) is a natural anticoagulant of blood. It is separated from blood preparations by bioselective sorption on sorbents containing heparin as a complementary ligand interacting with AT III. The paper deals with the behaviour of the chromatographic packings obtained by bonding heparin to cross-linked polysaccharide layers deposited on controlled-porosity glass. The properties of the prepared sorbents are discussed with respect to molecular weight of dextran used to form the polysaccharide layers. The results obtained show that when ligand (heparin) and AT III are used the molecular weight of cross-linked dextrans does not significantly influence the chromatographic properties of the affinity packings.     

The influence of molecular weight of dextran forming a heparin-bonding polysaccharide layer on the chromatographic properties of sorbents for HPAC analysis of human antithrombin III

Summary Human antithrombin III (AT III) is a natural anticoagulant of blood. It is separated from blood preparations by bioselective sorption on sorbents containing heparin as a complementary ligand interacting with AT III. The paper deals with the behaviour of the chromatographic packings obtained by bonding heparin to cross-linked polysaccharide layers deposited on controlled-porosity glass. The properties of the prepared sorbents are discussed with respect to molecular weight of dextran used to form the polysaccharide layers. The results obtained show that when ligand (heparin) and AT III are used the molecular weight of cross-linked dextrans does not significantly influence the chromatographic properties of the affinity packings.     

Novel Bonding of Iodine: Crystal Structure of HIS2O8

Iodine bridges two sulfate tetrahedra in the title compound and therefore participates in unusual and novel bonding. The structure of the compound (section shown on the right) can be regarded as the result of a condensation reaction between the protonated form of the hypothetical iodic(III ) acid, sulfuric acid, and hydrogensulfate.     

Synthesis of an N-Acetylated Heparin Pentasaccharide and its anticoagulant activity in comparison with the heparin pentasaccharide with high anti-factor-Xa activity

The synthesis of tri-N-acetylated heparin pentasaccharide 2 is described. It was assembled from five suitably blocked monosaccharide units ( 3 – 7 ). Glucuronic-acid building block 4 was prepared from glucose by direct Jones oxidation of the 6-O-trityl derivative 18 . The resulting acid 16 was esterified to 17 in large mounts using methyl chloroformate/base. Trimethylsilyl bromide proved to be an excellent reagent for the hydrolysis of a prop-1-enyl glycoside ( 19 →21 ). The pentasaccharide 29 was obtained by a [2 + 2] + 1 synthesis, the glycosylation reactions furnished good to very good yields. The identity of protected oligosaccharides was confirmed by ¹H-NMR spectroscopy. Sequential deblocking of the pentasaccharide, O-sulfation, and N-acetylation gave 2 which was shown to exhibit ca. 600 times lower anticoagulant activity than pentasaccharide 1 .     

Study of sulfated derivatives of polyhydroxy compounds as inhibitors of blood coagulation

Persulfated derivatives of natural polyhydroxy compounds, such as lignans secoisolariciresinol, and isolariciresinol, flavonoid dihydroquercetin, and myo-inositol, have been synthesized. The ability of these compounds to inhibit the intrinsic pathway of blood coagulation (APTT-test) and to reduce the activity of coagulation factor Xa in the presence of antithrombin(III) has been studied.     

2-氰基苄基诱导的葡萄糖醛酸β糖苷键立体选择性

以2,3-O-(2-氰基苄基)-4-O-氯乙酰基-β-对甲基苯基-D-葡萄糖醛酸硫苷等7个单糖模块作为糖基供体, 以甲醇等醇类化合物作为糖基受体, 分别在二氯甲烷和甲苯溶剂中进行了糖基化反应, 研究了葡萄糖醛酸C2位引入2-氰基苄基(BCN)以及溶剂效应对糖苷键α/β选择性的影响. 通过对糖基产物的 ¹H NMR, ¹³C NMR和HSQC等谱图分析发现, BCN可以有效提高糖苷键的β选择性, 其中部分糖基化产物的糖苷键α/β比例最高可达1/50. 为磺达肝癸钠分子中葡萄糖醛酸的β糖苷键的构建方法做出了初步探索.     

Synthesis of tri- and pentasaccharide fragments corresponding to the O-antigen of Shigella boydii type 6

A convenient synthetic strategy for the synthesis of the acidic pentasaccharide repeating unit and its trisaccharide fragment corresponding to the O-antigen of Shigella boydii type 6 has been successfully developed. A stereoselective sequential glycosylation method has been exploited to obtain the target tri- and pentasaccharide derivatives. Most of the synthetic intermediates were solid and prepared in high yields from commercially available reducing sugars following a series of protection–deprotection reactions. A late-stage TEMPO mediated selective oxidation reaction finally resulted in the pentasaccharide containing a glucuronic acid unit. A 2-(4-methoxyphenoxy) ethyl group has been chosen as the anomeric protecting group to provide trisaccharide and pentasaccharide derivatives linked to an ethylene glycol linker.