Jean Choay

Structure-activity relationship in heparin: A synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity

The structures of the tetrasaccharide (beta-D-glucuronic acid)1 leads to 4 (N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine)1 leads to 4(2-0-sulfate-alpha-L-iduronic acid)1 leads to 4(N-sulfate-6-0-sulfate-D-glucosamine) and of the pentasaccharide (N-sulfate-6-0-sulfate-alpha-D-glucosamine)1 leads to 4(beta-D-glucuronic acid)1 leads to 4(N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine)1 leads to 4(2-0-sulfate-alpha-L-iduronic acid)1 leads to 4(N-sulfate-6-0-sulfate-D-glucosamine), both prepared for the first time, by chemical synthesis from D-glucose and D-glucosamine, have been confirmed by nuclear magnetic resonance. The synthetic tetrasaccharide neither binds to AT-III nor induces anti-factor Xa activity enhancement of this inhibitor. In contrast, the synthetic pentasaccharide strongly binds to AT-III (Ka: 7.10(6)M-1) forming an equimolar complex and also enhances the AT-III inhibitory activity towards factor Xa. These results confirm that the synthetic pentasaccharide with the above structure corresponds to the actual minimal sequence required in heparin for binding to AT-III.

Conformer populations of L-iduronic acid residues in glycosaminoglycan sequences

The 1H-n.m.r. 3J values for the L-iduronic acid (IdoA) residues for solutions in D2O of natural and synthetic oligosaccharides that represent the biologically important sequences of dermatan sulfate, heparan sulfate, and heparin have been rationalized by force-field calculations. The relative proportions of the low-energy conformers 1C4, 2S0, and 4C1 vary widely as a function of sequence and of pattern of sulfation. When IdoA or IdoA-2-sulfate units are present inside saccharide sequences, only 1C4 and 2S0 conformations contribute significantly to the equilibrium. This equilibrium is displaced towards the 2S0 form when IdoA-2-sulfate is preceded by a 3-O-sulfated amino sugar residue, and towards the 1C4 form when it is a non-reducing terminal. For terminal non-sulfated IdoA, the 4C1 form also contributes to the equilibrium. N.O.e. data confirm these conclusions. Possible biological implications of the conformational flexibility and the counter-ion induced changes in conformer populations are discussed.

Synthesis of heparin fragments: A methyl α-pentaoside with high affinity for antithrombin III☆

Abstract The synthesis is described of the methyl α-glycoside of the pentasaccharide which represents the sequence in heparin responsible for binding and activation of the anticoagulant protein Antithrombin II. It was obtained in a yield much better than that of the previously synthesised pentasaccharide and exhibited the same biological properties.

Distinctive adjuvanticity of synthetic analogs of mycobacterial water-soluble components

Abstract Synthetic N -acetyl-muramyl- l -alanyl- d -isoglutamine represents the minimal structure capable of duplicating the activity of mycobacteria in Freunds complete adjuvant. In contrast to mycobacterial adjuvant preparations, that function only in the form of water-in-oil emulsions, this compound and a second synthetic analog ( N -acetyl-muramyl- l -alanyl- d -isoglutamic acid) augment the humoral immune responses of mice equally as well as aqueous solutions. Whereas N -acetyl-muramyl- l -alanyl- d -isoglutamic acid administered to guinea pigs in water-in-oil emulsion has no effect, N -acetyl-muramyl- l -alanyl- d -isoglutamine induces delayed hypersensitivity to ovalbumin and azobenezene-arsonate N -acetyl- l -tyrosine and increases the level of antibody against ovalbumin. Under these conditions, challenge with the synthetic adjuvants themselves evokes no skin responses. Moreover, Freunds complete adjuvant sensitizes guinea pigs to tuberculin and to native mycobacterial water-soluble adjuvant but not to the synthetic analogs.

Synthesis of heparin fragments. A chemical synthesis of the trisaccharide O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-α-d-glucopyranosyl)-(1→4)-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-(1→4)-2-deoxy-2-sulfamido-6-O-sulfo-d-glucopyranose heptasodium salt

Abstract Known 3- O -benzyl-1,2- O -isopropylidene-α- d -glucofuranose was first converted into methyl 3- O -benzyl-1,2- O -isopropylidene-β- l -idofuranuronate. Acid hydrolysis, followed by acetylation and treatment with titanium tetrabromide, gave methyl (2,4-di- O -acetyl-3- O -benzyl-α- l -idopyranosyl bromide)uronate, which was immediately transformed into methyl 4- O -acetyl-3- O -benzyl-β- l -idopyranuronate 1,2-( tert -butyl orthoacetate). A two-step replacement of the 4- O -acetyl by a 4- O -chloroacetyl group gave the key derivative, crystalline methyl 3- O -benzyl-4- O -chloroacetyl-β- l -idopyranuronate 1,2-( tert -butyl orthoacetate). Condensation of this orthoester with an excess of crystalline benzyl 6- O -acetyl-3- O -benzyl-2-(benzyloxycarbonyl)amino-2-deoxy-α- d -glucopyranoside in chlorobenzene in the presence of 2,6-dimethylpyridinium perchlorate gave crystalline benzyl 6- O -acetyl-3- O -benzyl-2-(benzyloxycarbonyl)amino-2-deoxy-4- O -(methyl 2- O -acetyl-3- O -benzyl-4- O -chloroacetyl-α- l -idopyranosyluronate)-α- d -glucopyranoside in 40% yield. O -Demonochloroacetylation, followed by condensation with known 3,6-di- O -acetyl-2-azido-4- O -benzyl-2-deoxy-α- d -glucopyranosyl bromide in di-chloromethane in the presence of 2,4,6-trimethylpyridine, silver triflate, and molecular sieve provided benzyl O -(3,6-di- O -acetyl-2-azido-4- O -benzyl-2-deoxy-α- d -glucopyranosyl)-(1→4)- O -(methyl 2- O -acetyl-3- O -benzyl-α- l -idopyranosyluronate)-(1→4)-6- O -acetyl-3- O -benzyl-2-(benzyloxycarbonyl)amino-2-deoxy-α- d - glucopyranoside in 88% yield. O -Deacetylation with sodium hydroxide, followed successively by O -sulfation in N,N -dimethylformamide in the presence of sulfur trioxide-trimethylamine complex, catalytic hydrogenolysis, and N -sulfation in water with the same sulfating agent, gave the heptasodium salt of O -(2-deoxy-2-sulfamido-3,6-di- O -sulfo-α- d -glucopyranosyl)-(1→4)- O -(2- O -sulfo-α- l -idopyranosyluronic acid)-(1→4)-2-deoxy-2-sulfamido-6- O -sulfo- d -glucopyranose. This trisaccharide, which is a fragment of the minimal antithrombin III-binding region in heparin, neither binds to antithrombin III nor induces anti-Xa activity.

Antithrombotic activity of a synthetic heparin pentasaccharide in a rabbit stasis thrombosis model using different thrombogenic challenges

A synthetic pentasaccharide, representing the critical sequence required in heparin for binding to antithrombin III (AT III), produces strong anti-factor Xa activity in vitro in the presence of AT III and is devoid of any activity directed towards thrombin. This pentasaccharide provides a unique tool to study the question of whether an agent capable of inhibiting factor Xa but devoid of anti-factor IIa activity in vitro, has the capacity to produce an antithrombotic effect in vivo. We have previously demonstrated in a rabbit stasis thrombosis model using a human serum challenge, a significant antithrombotic effect of the pentasaccharide. This finding and discrepancies with some earlier reports on the antithrombotic actions of other oligosaccharide fragments, led us to extend these studies. Four modifications of the stasis thrombosis model were developed using the following thrombogenic challenges selected for their specified induction sites of thrombosis, thromboplastin, an activated prothrombin complex concentrate, a non-activated prothrombin complex concentrate administered simultaneously with Russells viper venom, and factor Xa. Dose-dependent antithrombotic responses were obtained in all four systems with ED50 values between 25-43 ug/kg for pentasaccharide as compared to 16-47 ug/kg for heparin. Complete inhibition of induced thrombosis was obtained in all four systems for pentasaccharide. Ex vivo analysis revealed expected anti-factor Xa levels but no anti-factor IIa activity. It is concluded that an oligosaccharide with high anti-factor Xa activity and devoid of anti-factor IIa activity is capable of inhibiting thrombosis induced in rabbit stasis models, but that higher dosages than heparin are required for this effect.

Conformation of the pentasaccharide corresponding to the binding site of heparin for antithrombin III.

The conformation in solution of the pentasaccharide methyl glycoside (As-G-A*-Is-AM; 1), which represents the binding site of heparin for Antithrombin III, has been investigated using molecular mechanics and 1H-n.m.r. spectroscopy. The pentasaccharide has a rather rigid (As-G-A*) and a more flexible (Is-AM) region. A simplified model of 1, comprising two conformations, corresponding to the 1C4 and the 2S0 forms of the iduronate residue, and modified at the G-A* glycosidic linkage with respect to the energy minimum, reproduces most of the observed 3J values and n.O.e. enhancements. The possible role in the binding to Antithrombin III of a low-energy conformer, not observed in solution, is discussed.

Structure-function relationships in the inhibitory effect of heparin on complement activation: Independency of the anti-coagulant and anti-complementary sites on the heparin molecule

Fluid phase heparin inhibits formation of the classical and alternative pathway C3 convertase of complement in assays performed either with purified complement proteins or in whole serum. Experiments using oligosaccharides of homogeneous mol. wt obtained by mild nitrous hydrolysis of heparin, demonstrated that the inhibitory activity of heparin increased exponentially with mol. wt for fragments containing between 4 and 14 saccharidic units and that fragments of mol. wt above 4700 (greater than 14 saccharidic units) had a similar anti-complementary activity to that of native heparin. Fragments of homogeneous mol. wt (octasaccharides) separated by ion exchange chromatography on the basis of negative charges, exhibited increasing inhibitory activity with increasing sulfate content. Over-sulfation of fragments of defined mol. wt resulted in a constant enhancement of the relative capacity of each fragment species to inhibit formation of the classical and alternative pathway C3 convertases. A synthetic pentasaccharide representing the minimal critical sequence responsible for the binding of heparin to anti-thrombin III exhibited a similar inhibitory capacity on formation of the C3 convertases as another synthetic pentasaccharide that was devoid of anti-Xa activity. These studies contribute to define a minimal structure of the heparin molecule with C3b- and C4b-binding capacity and definitively establish the independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

The inhibition of the generation of thrombin and the antithrombotic effect of a pentasaccharide with sole anti-factor Xa activity

A chemically synthesized heparin pentasaccharide (Institut Choay, Paris, France) has been shown to exhibit an antithrombotic action in a rabbit stasis induced thrombosis model, in an IV dose range of 25 to 200 micrograms/kg (0.5 to 3.5 micrograms/ml plasma circulating concentrations). Ex vivo plasma analysis from treated animals revealed expected anti-factor Xa activity but no direct inhibitory effect against thrombin. Global anticoagulant activities were not found by PT and APTT methods. Platelet activation remained unaffected at the antithrombotic dosages of pentasaccharide. To more specifically elucidate the anti-factor Xa mediated antithrombotic mechanism of action of this pentasaccharide, it was studied in several thrombin generation assays. Pentasaccharide added to human and rabbit plasmas in vitro from 0 to 5.0 micrograms/ml produced a concentration dependent effect up to a 35 to 50% inhibition of generated thrombin. In ex vivo studies similar concentration dependent inhibition of thrombin generation was observed. Analysis of plasma obtained from animals in which a complete antithrombotic effect was observed in vivo demonstrated an approximate 45 to 55% inhibition of thrombin generation. These results indicate that a relationship exists between the pentasaccharide induced inhibition of experimental venous stasis thrombosis and the inhibition of thrombin generation.

Correlation of structure and adjuvant activity of N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), its derivatives and analogues. Anti-adjuvant and competition properties of stereoisomers.

Abstract The synthetic N-acetyl muramyl-dipeptide (MDP) 1 has been shown to be fully adjuvant active in a water-in-oil emulsion; we now report a study on the adjuvant activity of 1O derivatives and analogues of MDP under similar conditions. NaBH 4 reduction of MDP 1 leads to the inactive muramicitol dipeptide 2 ; β-elimination gives the lactyl-dipeptide 3 , which seems to inhibit adjuvant activity. Shortening of the lactyl side chain of MDP gives nor-MDP, 4 , which is less active. Amongst the analogues in which one amino acid of the dipeptide moiety is replaced by another one, the L-Ser analogue 5 is fully active, whereas replacement of L-Ala by Gly or of D-iso-Gln by D-Glu-OH, or D-Glu (OMe) 2 , or D-Glu (OMe)-NH 2 gives less active compounds ( 6 , 8 , 9 , 1O ). The diastereoisomer 7 where L-Ala is replaced by D-Ala, shows an anti-adjuvant activity.