WO2024032575A1 - 一种含双抗凝血酶结合序列的肝素十二糖及其制备方法与应用 - Google Patents

一种含双抗凝血酶结合序列的肝素十二糖及其制备方法与应用 Download PDF

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WO2024032575A1
WO2024032575A1 PCT/CN2023/111623 CN2023111623W WO2024032575A1 WO 2024032575 A1 WO2024032575 A1 WO 2024032575A1 CN 2023111623 W CN2023111623 W CN 2023111623W WO 2024032575 A1 WO2024032575 A1 WO 2024032575A1
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heparin
idoa2s
sulfated
glca
catalysis
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French (fr)
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刘纯慧
张桂姣
仇亚琪
王琳
李婧茹
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山东大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to a heparin dodecose containing a double antithrombin binding sequence and its preparation method and application, and belongs to the technical field of biomedicine.
  • UHF unfractionated heparin
  • LMWH low-molecular-weight heparins
  • enoxaparin, dalteparin, nadroparin, and tinzaparin are complex low-molecular-weight mixtures obtained by chemically or enzymatically controlled partial depolymerization of UFH.
  • the weight-average molecular weight is usually 3500 to 6000 Da. They have gradually replaced UFH as the first choice for clinical practice. Anticoagulant drugs. Although animal-derived UFH and LMWH have the advantages of relatively low cost and mature production technology, their structural heterogeneity leads to insurmountable clinical limitations, and there are problems such as impurity contamination and fragile raw material supply chains. Studies have confirmed that the anticoagulant effect of animal-derived heparin is highly dependent on the unique pentasaccharide sequence (abbreviated as: GlcNS/Ac6S-GlcA-GlcNS6S3S-IdoA2S) that is randomly distributed in the sugar chain and specifically binds to antithrombin (AT).
  • GlcNS/Ac6S-GlcA-GlcNS6S3S-IdoA2S unique pentasaccharide sequence
  • Protamine is the earliest antidote for animal-derived heparin approved by the FDA. It can eliminate the anticoagulant activity of animal-derived heparin and restore the body's normal coagulation. Among them, the anticoagulant activity of UFH can be completely neutralized by protamine, and LMWH can Partially neutralized. This "neutralizable" property of heparin facilitates the termination of anticoagulation therapy according to the treatment process, and can effectively avoid adverse reactions such as bleeding. Unfortunately, the anticoagulant activity of fully chemically synthesized fondaparinux cannot be neutralized by protamine, resulting in great limitations in its clinical application. Therefore, anticoagulant heparins that can be neutralized by protamine are developed. New molecules are imminent.
  • the present invention provides a new heparin dodecose molecule containing dual antithrombin binding sequences and its preparation method and application.
  • UDP-GlcNTFA Uridine diphosphate-N-trifluoroacetylglucosamine
  • UDP-GlcNAc Uridine diphosphate-N-acetylglucosamine
  • UDP-GlcA Uridine diphosphate-glucuronic acid
  • PAPS 3'-adenosine phosphate-5'-phosphosulfate.
  • KfiA Escherichia coli K5N-acetylglucosaminyltransferase
  • PmHS2 Pasteurella multocida Heparosan synthase 2
  • NST N-sulfatyltransferase
  • the first object of the present invention is to provide a new heparin dodecose molecule containing a double AT binding sequence and without consecutive multiple trisulfate disaccharides (IdoA2S-GlcNS6S), or a pharmaceutically acceptable salt thereof, which has the following properties:
  • R 1 and R 3 are sulfonyl group (-SO 3 H) or acetyl group (-COCH 3 );
  • R 2 is sulfonyl group or hydrogen (-H);
  • R 4 is selected from phenyl or substituted phenyl, aromatic heterocycle or substituted aromatic heterocycle with characteristic UV absorption;
  • Sugar residue G is glucuronic acid (GlcA) or iduronic acid (IdoA).
  • the substituent of the substituted phenyl or substituted aromatic heterocycle is nitro, halogen, hydroxyl or trifluoromethyl.
  • the new heparin dodecose molecule containing a double AT binding sequence and without multiple consecutive trisulfate disaccharides is one of the following:
  • the new heparin dodecose molecule containing a double AT binding sequence and without multiple consecutive trisulfate disaccharides has significant anti-factor Xa activity and no obvious anti-factor IIa activity.
  • anti-factor Xa activity can be effectively neutralized by protamine, and the neutralization rate of anti-factor Xa activity by protamine is >80%.
  • the second object of the present invention is to provide a method for preparing heparin dodecose containing a double AT-binding sequence and without multiple consecutive trisulfate disaccharides (IdoA2S-GlcNS6S), using a chemical enzymatic synthesis strategy.
  • a method for preparing heparin dodecose containing a double AT binding sequence and without multiple consecutive trisulfate disaccharides uses a glucuronic acid (GlcA) derivative with a reducing end covalently connected to the R 4 group as the starting substrate.
  • GlcA glucuronic acid
  • a method in which the glycosyltransferase catalyzed reaction of steps a and b is repeated at least once and the chemical enzymatic modification reaction of steps c, d, e, f, g is combined with four or five steps. Law;
  • Step a under the catalysis of N-acetylglucosaminyltransferase (KfiA) or Heparosan synthase 2 (PmHS2), UDP-GlcNTFA or UDP-GlcNAc is used as the glycosyl donor, and the GlcNTFA residue of the glycosyl donor or The GlcNAc residue is transferred to GlcA at the non-reducing end of the substrate via an ⁇ -1,4 glycosidic bond to obtain an intermediate compound;
  • KfiA N-acetylglucosaminyltransferase
  • PmHS2 Heparosan synthase 2
  • Step b under the catalysis of PmHS2 enzyme, UDP-GlcA is used as the glycosyl donor, and the GlcA residue of the glycosyl donor is connected to the glucosamine (GlcNTFA or GlcNAc) at the non-reducing end of the substrate through a ⁇ -1,4 glycosidic bond. ) to obtain an intermediate compound;
  • step c the heparin intermediate is placed on ice in a mild alkaline aqueous solution, and all GlcNTFA residues of the sugar chain are detrifluoroacetyl (TFA) and converted into GlcNH 2 , and then activated by N-sulfatyl transferase (NST). It is converted into GlcNS under catalysis to obtain N-sulfated intermediate;
  • TFA detrifluoroacetyl
  • NST N-sulfatyl transferase
  • Step d under the co-catalysis of C 5 -isomerase (C 5 -epi) and 2-O-sulfotransferase (2OST), the N-sulfation product sugar chain is between two GlcNS or GlcNS (non-GlcNS).
  • the specific GlcA residue between the reducing end) and GlcNAc is converted into 2-O-sulfated iduronic acid (IdoA2S) to obtain an intermediate containing the IdoA2S residue;
  • Step e under the sole catalysis of 2OST, the specific GlcA residue between the two GlcNS of the substrate or between GlcNS (non-reducing end) and GlcNAc is converted into 2-O-sulfated gluconic acid (GlcA2S) to obtain Intermediates of GlcA2S;
  • Step f under the joint catalysis of 6-O-sulfatyltransferases 1 and 3 (6OST1, 6-OST3), the 6-OH of all GlcNS or GlcNAc residues in the substrate sugar chain is sulfated and modified into GlcNS6S or GlcNAc6S, obtaining 6-O-sulfated intermediate;
  • step g under the catalysis of 3-O-sulfotransferase 1 (3OST1), the 3-OH of GlcNS6S between GlcA and IdoA2S in the substrate sugar chain is sulfated (GlcNS6S3S) to obtain the final target compound.
  • 3-O-sulfotransferase 1 3-O-sulfotransferase 1 (3OST1)
  • the starting substrate is p-nitrophenyl- ⁇ -D-glucuronide (GlcA-PNP).
  • N-acetylglucosaminyltransferase (KfiA) and Heparosan synthase 2 (PmHS2) are recombinantly expressed in Escherichia coli, and the source of N-acetylglucosaminyltransferase (KfiA) In E. coli K5, Heparosan synthase 2 (PmHS2) is derived from Pasteurella multocida.
  • the amount of glycosyl donor added is more than 1.2 times the equivalent of the substrate.
  • heparin modifying enzymes are recombinantly expressed using E.
  • NST, 2OST, 6OST1, 6-OST3, and 3OST1 heparin modifying enzymes all use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfate group donor;
  • MES N-morpholino)ethanesulfonic acid
  • the addition amount of enzyme and heparin intermediate substrate, and the reaction time are not limited, and the obtained reaction
  • the liquid was purified by anion exchange column chromatography to obtain the product.
  • the amount of sulfate group donor added is 1.5-10 times the equivalent of the substrate.
  • the preparation method of the heparin dodecose containing dual antithrombin binding sequences of the present invention is based on repeated experiments to study the catalytic activity and substrate specificity of each glycosyltransferase and heparin modifying enzyme on different heparin intermediate molecules. Established.
  • the preparation method is selected from one of the following synthetic routes:
  • the preparation method of heparin dodecose containing double AT binding sequences and without multiple consecutive trisulfate disaccharides the steps are as follows:
  • the pentasaccharide skeleton intermediate is placed on ice in a mild alkaline aqueous solution. All GlcNTFA residues of the sugar chain are detrifluoroacetyl (TFA) and converted into GlcNH 2 , and then N-sulfate transferase (NST) ) to convert it into GlcNS under the catalysis to obtain N-sulfated pentasaccharide intermediate;
  • TFA detrifluoroacetyl
  • NST N-sulfate transferase
  • step 1) replace the glycosyl donor with UDP-GlcNAc, and extend the sugar chain catalyzed by KfiA or PmHS2 to obtain a hexasaccharide intermediate; use the hexasaccharide intermediate as the substrate, refer to step 2) to extend the sugar chain as Heptasaccharide, to obtain a heptasaccharide intermediate; use the heptasaccharide intermediate as the substrate and alternately repeat steps 1) and 2) to continue extending the sugar chain to obtain an undecaose intermediate;
  • the undecanose intermediate is placed on ice in a mild alkaline aqueous solution, and all GlcNTFA residues of the sugar chain are detrifluoroacetyl (TFA) and converted into GlcNH 2 , and then N-sulfate transferase (NST) ) to convert it into GlcNS under the catalysis to obtain N-sulfated undecose;
  • TFA detrifluoroacetyl
  • NST N-sulfate transferase
  • step 8 for heparin undecaose containing two or three IdoA2S residues, repeat step 1) to extend the sugar chain to obtain heparin dodecaose containing two or three IdoA2S residues;
  • Heparin dodecose containing two or three IdoA2S residues is placed on ice in a mild alkaline aqueous solution, and a newly introduced GlcNTFA residue in the sugar chain is detrifluoroacetyl (TFA) and converted into GlcNH 2 , and then converted into GlcNS under the catalysis of N-sulfatyltransferase (NST) to obtain N-sulfated heparin dodecose containing two or three IdoA2S residues respectively;
  • TFA detrifluoroacetyl
  • NST N-sulfatyltransferase
  • the third object of the present invention is to provide the application of heparin dodecaose containing double AT binding sequences and without multiple consecutive trisulfate disaccharides for the preparation of anticoagulant and antithrombotic drugs.
  • An anticoagulant and antithrombotic drug including the above-mentioned heparin dodecose containing a double AT-binding sequence and without multiple consecutive trisulfate disaccharides and one or more pharmaceutically acceptable carriers or excipients, and the heparin dodecose and The proportion of carriers or excipients is not limited.
  • the novel heparin dodecaose of the present invention containing a double AT binding sequence and without multiple consecutive trisulfate disaccharides has significant anti-factor Xa activity and no obvious anti-factor IIa activity.
  • this The invention demonstrates for the first time that the aldose GlcA/IdoA-linked double AT-binding pentasaccharide sequence can activate AT alone and exhibit potent inactivation of factor Xa activity.
  • the anti-factor Xa activity of the disaccharide is significantly better than the heparin dodecaccharide previously reported by Professor Jian Liu.
  • the anti-Xa factor activity of the heparin dodecose of the present invention can be effectively neutralized by protamine.
  • the neutralization rate of protamine on the anti-Xa factor activity of the target compounds I-2 and I-10 is >80%.
  • the heparin dodecaccharide structure of the present invention does not have four consecutive trisulfate disaccharides, the anticoagulant activity is still Efficiently neutralized by protamine.
  • the heparin dodecose of the present invention can be effectively neutralized by protamine and can be used to prepare cost-effective and safer potent anticoagulant and antithrombotic drugs.
  • the double AT-binding sequence contained in the heparin dodecose of the present invention can activate AT alone, so its anti-Xa activity is better than fondaparinux sodium and the reported dodecose containing only a single AT-binding sequence; its anti-Xa activity The activity of Such as short half-life) and adverse reactions (such as HIT, etc.).
  • the minimum number of synthesis steps of the heparin dodecose of the present invention is 18 steps and the maximum is 21 steps, which is less than the currently reported dodecose (22-23 steps), the cost is lower, and it can be used to prepare low-cost tools Advantages and safer powerful anticoagulant and antithrombotic drugs.
  • Figure 1 is the high performance liquid chromatography (A) and mass spectrum (B) of the new heparin dodecose molecule I-2 prepared in Example 3 and the high performance liquid chromatography (C) and mass spectrum (D) of I-10. ;
  • Figure 2 is the 1 H NMR (A) and HSQC (B) spectra of the new heparin dodecose molecule I-2 prepared in Example 3;
  • Figure 3 is the in vitro anti-Xa factor of the new heparin dodecose molecules I-2 and I-10 prepared in Example 3;
  • Figure 4 shows the neutralizing effect of protamine on the anticoagulant activity of the new heparin dodecose molecules I-2 and I-10 in Example 3 in vitro.
  • Example 1 Chemoenzymatic synthesis of heparin pentasaccharide intermediate containing a single IdoA2S residue
  • the chromatographic conditions were 0 ⁇ 100% KH 2 PO 4 gradient elution within 45 min, and the flow rate was 0.5 mL/min. , the detection wavelength is 310nm, and the yield rate is ⁇ 95%.
  • TFA trifluoroacetic acid
  • Backbone intermediate 3mer-1 using 3mer-1 as the substrate, repeat the above reaction of KfiA and PmHS2 to obtain pentasaccharide backbone intermediate 5mer-1. Its purity is >82.5% as measured by PAMN-HPLC, and its component is measured by ESI-MS. 1181.09Da, consistent with the theoretical value.
  • PAMN-HPLC measured its purity to be >95%
  • ESI-MS measured its molecular weight to be 1129.27Da.
  • the product 5mer-3 has one more sulfate group than 5mer-2.
  • Compound 5mer-3 is a heparin pentasaccharide containing one IdoA2S residue.
  • Example 2 Chemical enzymatic synthesis of heparin undecose intermediates containing two and three IdoA2S residues
  • heparin pentasaccharide 5mer-3 containing an IdoA2S residue refer to Example 1, replace the glycosyl donor with UDP-GlcNAc and extend the sugar chain catalyzed by KfiA, then use UDP-GlcA as the glycosyl donor and PmHS2 enzyme Catalyze further Steps to extend the sugar chain, and then alternately perform KfiA (glycosyl donor UDP-GlcNTFA) and PmHS2 (glycosyl donor UDP-GlcA) enzymatic sugar chain elongation until heparin undecaose 11mer-1 is formed, Q Sepharose chromatography column (1 ⁇ 20cm) purification; then LiOH treatment to remove trifluoroacetyl and NST catalysis for N-sulfation modification to obtain heparin undecose 11mer-2. Its purity was >99% as measured by PAMN-HPLC and its purity as measured by ESI-MS
  • heparin undecose 11mer-2 is catalyzed by C 5 -isomerase (C 5 -epi) and 2-O-sulfate transferase (2OST) to N-sulfate the undecose sugar chain.
  • C 5 -epi C 5 -isomerase
  • 2OST 2-O-sulfate transferase
  • the specific GlcA between the two GlcNS is converted into 2-O-sulfated iduronic acid (IdoA2S).
  • the reaction solution is purified by Q Sepharose chromatography column (1 ⁇ 20cm) to obtain 11mer-3 ( Heparin undecanose containing two IdoA2S residues); if the reaction solution is supplemented with an appropriate amount of enzyme and PAPS, continue the reaction until a new product is completely generated, and purify with Q Sepharose chromatography column (1 ⁇ 20cm) to obtain 11mer-4 (containing three heparin undecanose of IdoA2S residues).
  • the molecular weight of 11mer-3 was measured by ESI-MS to be 2522.21Da, which has one more sulfate group than 11mer-2, indicating that it has a new IdoA2S;
  • the molecular weight of 11mer-4 was measured by ESI-MS to be 2602.40 Da, which has two more sulfate groups than 11mer-2, indicating that it has two new IdoA2S, which is consistent with expectations.
  • the KfiA enzymatic sugar chain extension was carried out according to the method of Reference Example 1, and the dodecaccharide was purified with a Q-Sepharose strong anion column (1cm ⁇ 20cm).
  • the intermediate 12mer-1/2 is then subjected to chemical trifluoroacetyl removal and enzymatic N-sulfation modification according to the above method to obtain the dodecaccharide intermediate 12mer-3/4.
  • the molecular weights measured by ESI-MS were 2763.56Da and 2843.39Da respectively, which were consistent with the theoretical values.
  • Place dodecose substrate 12mer-3/4 in MES buffer with pH 7.0 ⁇ 7.5 and 50mmol/L, add 7 times the equivalent of PAPS, 4mL of 6-OST-1 and 4mL of 6-OST-3 enzyme , adjust the reaction volume to 140 mL, and react in a water bath at 37°C overnight.
  • the reaction rate of 12mer-5/6 is >99%, adjust the pH of the reaction solution to 4-5 with dilute acetic acid to terminate the reaction. Freeze and thaw in a -20°C refrigerator to remove the enzyme without purification.
  • the product I-2 was purified using a Q-Sepharose strong anion column (1cm ⁇ 10cm) with a purity of More than 92%, the molecular weight was 3403.12Da measured by ESI-MS, consistent with the theoretical value; the product I-10 was obtained, the purity of which reached more than 97%, the molecular weight was 3482.97Da measured by ESI-MS, consistent with the theoretical value.
  • the NMR (600MHz, D 2 O) spectrum of I-2 is shown in Figure 2, and the structure is consistent with expectations.
  • Example 4 Determination of in vitro anticoagulant activity of heparin dodecose I-2 and I-10
  • the IC 50 values of the anti-FXa activity of the novel heparin dodecaose I-2 and I-10 prepared in the present invention were 16.77, 18.03ng/mL (4.34, 4.54nmol /L), the IC 50 values of unfractionated heparin (UFH) and fondaparinux (Arixtra) measured under the same conditions were 139ng/mL and 12.63ng/mL (7.3nmol/L) respectively, based on molar concentration The IC 50 values of the anti-FXa activity of the new heparin dodecaccharides I-2 and I-10 are much smaller than fondaparinux sodium.
  • novel heparin dodecaose I-2 and I-10 prepared by the present invention have no significant anti-factor IIa activity (omitted). Therefore, the novel heparin dodecaose I-2 and I-10 prepared by the present invention are specific inhibitors of factor Xa.
  • the heparin dodecaose I-2 and I-10 prepared by the present invention are new heparin molecules whose anticoagulant activity can be neutralized by protamine.
  • the IC 50 values of the anti-FXa activities of I-2 and I-10 are 4.34 and 4.54 nmol/L respectively, with little difference; as shown in Example 5 and Figure 4, protamine
  • the neutralization rate for I-2 anticoagulant activity is >80%, while the neutralization rate for I-10 anticoagulant activity is >90%.
  • the difference is obvious. Therefore, the sugar residue G is replaced by GlcA with IdoA2S on the effect of heparin dodecose
  • the effect on anti-Xa activity is small, but the effect on its protamine neutralization efficiency is greater.

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Abstract

一种含双抗凝血酶结合序列的肝素十二糖及其制备方法与应用，具有式I所示的结构。还提供含有通式I结构化合物的化学酶法制备方法以及应用。其中的肝素十二糖合成步数明显少、总产率显著高；具有强效特异抗Xa因子活性，且其抗Xa因子活性可以被鱼精蛋白有效地中和，中和率大于80％；无连续多个三硫酸双糖，不易引起连续多个三硫酸双糖（IdoA2S-GlcNS6S）依赖的药代动力学缺陷（如半衰期短）和不良反应（如HIT等）。适合用于制备更加安全、具显著成本优势的新型抗凝血抗血栓药物。

Description

一种含双抗凝血酶结合序列的肝素十二糖及其制备方法与应用 技术领域
本发明涉及一种含双抗凝血酶结合序列的肝素十二糖及其制备方法与应用，属于生物医药技术领域。
背景技术
肝素类药物用作临床抗凝剂已超过90年，至今仍广泛应用于血栓栓塞性疾病、外科手术、血液透析等，全球市场规模超过80亿美元。目前市售普通肝素即未分级肝素(unfractionated heparin，UFH)是主要从猪小肠黏膜提取得到的多分散的多糖组分，重均分子量～14000Da；低分子量肝素(low-molecular-weight heparins,LMWH)如依诺肝素、达肝素、那屈肝素、亭扎肝素则是UFH经化学或酶法控制部分解聚得到的复杂低分子量混合物，重均分子量通常为3500～6000Da，已逐渐替代UFH成为首选临床抗凝药物。动物源UFH及LMWH虽然具有成本相对低廉、生产工艺成熟等优点，但结构异质性导致其无法克服的临床局限性，并存在杂质污染、原料供应链脆弱问题。研究已经证实，动物源肝素的抗凝作用高度依赖糖链中随机分布、与抗凝血酶(antithrombin，AT)特异性结合的独特五糖序列(简写为：GlcNS/Ac6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S)，其约占整个肝素链的1/3。2001年批准上市的磺达肝癸钠(商品名Arixtra)为该五糖序列的甲基糖苷衍生物，是首个全合成的、结构确定的肝素类单一化合物，为开发新一代抗凝肝素药物提供了思路。
鱼精蛋白是最早由FDA批准的动物源肝素的解毒剂，能够消除动物源肝素的抗凝活性而恢复机体正常的凝血作用，其中UFH的抗凝活性能够完全被鱼精蛋白中和，LMWH能被部分中和。肝素的这种“可中和”特性便于根据治疗进程结束抗凝治疗，能够有效避免出血等不良反应。遗憾的是，全化学合成的磺达肝癸钠的抗凝活性完全不能被鱼精蛋白中和，导致其在临床应用中受到较大限制，因此研发可被鱼精蛋白中和的抗凝肝素新分子迫在眉睫。
据报道，美国北卡大学教堂山分校Jian Liu教授采用化学酶法合成了一种含单一AT结合五糖、4个连续三硫酸双糖(IdoA2S-GlcNS6S)的肝素十二糖，并证实该分子的抗凝活 性能够被鱼精蛋白有效地中和，不足之处是其合成需22～23步，步骤繁琐，总产率偏低，且化合物存在4个连续三硫酸双糖，容易导致体内半衰期短等药代动力学缺陷、潜在的肝素诱导的血小板减少症(HIT)等不良反应。
因此，研发抗凝作用更强、药代动力学更佳、副作用更低的“可中和”肝素新分子迫在眉睫。
发明内容
针对现有技术的不足，本发明提供一种含双抗凝血酶结合序列的肝素十二糖新分子及其制备方法与应用。
术语说明：
AT：抗凝血酶
IdoA：艾杜糖醛酸
GlcA：葡糖醛酸
UDP-GlcNTFA：尿苷二磷酸-N-三氟乙酰葡糖胺
UDP-GlcNAc：尿苷二磷酸-N-乙酰葡糖胺
UDP-GlcA：尿苷二磷酸-葡糖醛酸
PAPS：3'-磷酸腺苷-5'-磷酸硫酸。
KfiA：大肠杆菌K5N-乙酰氨基葡糖基转移酶
PmHS2：多杀巴斯德菌Heparosan合酶2
NST：N-硫酸基转移酶
C5-epi：C5-异构化酶
2OST：2-O-硫酸基转移酶
6OST：6-O-硫酸基转移酶
3OST：3-O-硫酸基转移酶
本发明是通过如下技术方案实现的：
本发明的第一个目的是提供一种含双AT结合序列、无连续多个三硫酸双糖(IdoA2S-GlcNS6S)的肝素十二糖新分子，或其药学上可接受的盐，其具有如下式I所示结构：
R1、R3为磺酰基(-SO3H)或乙酰基(-COCH3)；R2为磺酰基或氢(-H)；
R4选自具有特征紫外吸收的苯基或取代苯基，芳杂环或取代芳杂环；
糖残基G为葡糖醛酸(GlcA)或艾杜糖醛酸(IdoA)。
根据本发明优选的，取代苯基或取代芳杂环的取代基为硝基、卤素、羟基或三氟甲基。
根据本发明优选的，含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖新分子为下列之一：
根据本发明优选的，所述的含双AT结合序列、无连续多个三硫酸双糖(IdoA2S-GlcNS6S)的肝素十二糖新分子具有显著的抗Xa因子活性，无明显的抗IIa因子活性，抗Xa因子活性可以被鱼精蛋白有效地中和，抗Xa因子活性被鱼精蛋白中和的中和率>80％。
本发明的第二个目的是提供含双AT结合序列、无连续多个三硫酸双糖(IdoA2S-GlcNS6S)的肝素十二糖的制备方法，采用化学酶法合成策略进行。
含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖的制备方法，该方法以还原末端共价连接R4基团的葡糖醛酸(GlcA)衍生物为起始底物，为如下步骤a、b糖基转移酶催化反应至少重复一次与步骤c、d、e、f、g化学酶法修饰反应中其中四步或五步组合的方 法；
步骤a，在N-乙酰氨基葡糖基转移酶(KfiA)或Heparosan合酶2(PmHS2)催化下，以UDP-GlcNTFA或UDP-GlcNAc为糖基供体，糖基供体的GlcNTFA残基或GlcNAc残基以α-1,4糖苷键被转移至底物非还原末端的GlcA上，得到中间体化合物；
步骤b，在PmHS2酶催化下，以UDP-GlcA为糖基供体，糖基供体的GlcA残基以β-1,4糖苷键连接至底物非还原末端的葡糖胺(GlcNTFA或GlcNAc)，得到中间体化合物；
步骤c，肝素中间体在温和的碱性水溶液中静置于冰上，糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化中间体；
步骤d，在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，N-硫酸化产物糖链中两个GlcNS之间或GlcNS(非还原端)与GlcNAc之间的特定GlcA残基被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)，得到含IdoA2S残基的中间体；
步骤e，在2OST的单独催化下，底物两个GlcNS之间或GlcNS(非还原端)与GlcNAc之间的特定GlcA残基被转变为2-O-硫酸化葡糖酸(GlcA2S)，得到含GlcA2S的中间体；
步骤f，在6-O-硫酸基转移酶1和3(6OST1、6-OST3)的共同催化作用下，底物糖链的全部GlcNS或GlcNAc残基的6-OH发生硫酸化修饰成为GlcNS6S或GlcNAc6S，得到6-O-硫酸化中间体；
步骤g，在3-O-硫酸基转移酶1(3OST1)的催化作用下，底物糖链中GlcA与IdoA2S之间的GlcNS6S的3-OH发生硫酸化(GlcNS6S3S)，得到最终目标化合物。
根据本发明优选的，起始底物为对硝基苯基-β-D-葡糖醛酸苷(GlcA-PNP)。
根据本发明优选的，步骤a中，N-乙酰氨基葡糖基转移酶(KfiA)、Heparosan合酶2(PmHS2)是以大肠杆菌重组表达，N-乙酰氨基葡糖基转移酶(KfiA)来源于大肠杆菌K5，Heparosan合酶2(PmHS2)来源于多杀巴斯德菌(Pasteurella multocida)。
根据本发明优选的，步骤a、b中，酶催化反应所用的缓冲液为50mmol/L Tris-HCl，Tris-HCl中含6mmol/L MnCl2，pH＝7.0-7.5，反应温度20℃～37℃，酶与底物的加入量、反应时间不受限定；得到的酶促反应液利用反相C18或阴离子交换柱层析纯化得中间体化合物。
根据本发明优选的，步骤a、b中，糖基供体的加入量为底物的1.2倍当量以上。
根据本发明优选的，步骤c、d、e、f、g中，NST、C5-epi、2OST、6OST1、6-OST3、3OST1肝素修饰酶是利用大肠杆菌、酵母或昆虫细胞重组表达得到；NST、2OST、6OST1、6-OST3、3OST1肝素修饰酶均以3'-磷酸腺苷-5'-磷酸硫酸(PAPS)为硫酸基供体；各修饰酶催化反应的缓冲液为50mmol/L 2-(N-吗啉代)乙烷磺酸(MES)，pH＝7.0～7.5，反应温度20℃～37℃，酶与肝素中间体底物的加入量、反应时间不受限定，得到的反应液利用阴离子交换柱层析纯化得产物。
根据本发明优选的，步骤c、d、e、f、g中，硫酸基供体的加入量为底物的1.5-10倍当量。
本发明的含双抗凝血酶结合序列的肝素十二糖的制备方法是通过反复试验研究各糖基转移酶、肝素修饰酶对不同肝素中间体分子的催化活性和底物特异性的基础上建立的。
根据本发明优选的，所述的制备方法选自如下合成路线之一：
最为优选的，含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖的制备方法，合成路线如下：
a→b→a→b→c→d→a→b→a→b→a→b→c→d→a→c→f→g。
具体的，含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖的制备方法，步骤如下：
1)在N-乙酰氨基葡糖基转移酶(KfiA)或Heparosan合酶2(PmHS2)催化下，以UDP-GlcNTFA为糖基供体，糖基供体的GlcNTFA残基以α-1,4糖苷键被转移至底物非还原末端的GlcA上，得到二糖骨架中间体；
2)在PmHS2酶催化下，以UDP-GlcA为糖基供体，糖基供体的GlcA残基以β-1,4糖苷键被连接至二糖骨架非还原末端的GlcNTFA上，得到三糖骨架中间体；
3)重复步骤1)、步骤2)将糖链延长，得到五糖骨架中间体；
4)五糖骨架中间体在温和的碱性水溶液中静置于冰上，糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化五糖中间体；
5)在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，N-硫酸化五糖中间体糖链中两个GlcNS之间的特定GlcA残基被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)；得到含一个IdoA2S残基的肝素五糖；
6)参照步骤1)，糖基供体更换为UDP-GlcNAc，由KfiA或PmHS2催化延长糖链，得六糖中间体；以六糖中间体为底物，参照步骤2)将糖链延长为七糖，得七糖中间体；以七糖中间体为底物交替重复步骤1)、步骤2)继续将糖链延长，得到十一糖中间体；
7)十一糖中间体在温和的碱性水溶液中静置于冰上，糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化十一糖；
8)在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，N-硫酸化十一糖糖链中两个GlcNS之间的特定GlcA被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)，得到含两个IdoA2S残基的肝素十一糖；补加适量酶与底物并延长反应时间，进一步使十一糖中GlcNS(非还原端)与GlcNAc之间的特定GlcA残基转化为IdoA2S，得到含三个IdoA2S残基的肝素十一糖；
9)步骤8)中，含两个或三个IdoA2S残基的肝素十一糖分别重复步骤1)将糖链延长，得到含两个或三个IdoA2S残基的肝素十二糖；
10)含两个或三个IdoA2S残基的肝素十二糖在温和的碱性水溶液中静置于冰上，糖链新引入的一个GlcNTFA残基脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，分别得到含两个或三个IdoA2S残基的N-硫酸化肝素十二糖；
11)在6-O-硫酸基转移酶1和3(6OST1、6-OST3)的共同催化作用下，含两个或三个IdoA2S残基的N-硫酸化肝素十二糖糖链的全部GlcNS或GlcNAc残基的6-OH发生硫酸化修饰成为GlcNS6S或GlcNAc6S，得到两种6-O-硫酸化肝素十二糖；
12)在3-O-硫酸基转移酶1(3OST1)的催化作用下，两种6-O-硫酸化肝素十二糖底物糖链中GlcA与IdoA2S之间的GlcNS6S的3-OH发生硫酸化(GlcNS6S3S)，分别得到含2个IdoA2S的目标化合物I-2或含3个IdoA2S的目标化合物I-10。
本发明的第三个目的是提供含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖的应用，用于制备抗凝抗血栓药物。
一种抗凝抗血栓药物，包括上述含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖和一种或多种药学上可接受载体或赋形剂，肝素十二糖与载体或赋形剂的比例不受限定。
本发明的技术特点及优点：
1、经生色底物法测定，本发明含双AT结合序列、无连续多个三硫酸双糖的新型肝素十二糖具有显著的抗Xa因子活性，无明显的抗IIa因子活性，同时本发明首次证实糖醛糖GlcA/IdoA连接的双AT结合五糖序列能够单独激活AT而表现出强效灭活Xa因子活性。肝素十二糖抗Xa因子的半数抑制摩尔浓度(IC50)显著低于阳性市售磺达肝癸钠，因此，本发明含双AT结合序列、无连续多个三硫酸双糖的新型肝素十二糖的抗Xa因子活性显著优于之前Jian Liu教授报道的肝素十二糖。
2、本发明的肝素十二糖的抗Xa因子活性可以被鱼精蛋白有效地中和，例如鱼精蛋白对目标化合物I-2、I-10的抗Xa因子活性的中和率>80％，与未分级肝素接近，而市售磺达肝癸钠的活性几乎完全不能被中和，尽管本发明的肝素十二糖结构中不具有4个连续的三硫酸双糖，但抗凝活性仍可被鱼精蛋白高效中和。
3、本发明的肝素十二糖的结构(糖残基G、R1～R4)的轻微改变对其抗Xa活性影响较小，对其鱼精蛋白中和效率、药代动力学特征影响较大。
4、本发明的肝素十二糖可以被鱼精蛋白有效地中和，可用于制备具成本优势的、更加安全的强效抗凝血抗血栓药物。
5、本发明的肝素十二糖所含的双AT结合序列能够单独激活AT，故其抗Xa活性优于仅含单一AT结合序列的磺达肝癸钠和已报道的十二糖；其抗Xa活性可以被鱼精蛋白有效地中和，中和率>80％；无连续多个三硫酸双糖，不易引起连续多个三硫酸双糖(IdoA2S-GlcNS6S)依赖的药代动力学缺陷(如半衰期短)和不良反应(如HIT等)。
6、本发明的肝素十二糖合成步数最少步数为18步，最多为21步，少于现有已报道的十二糖(22～23步)，成本更低，可用于制备具成本优势的、更加安全的强效抗凝血抗血栓药物。
附图说明
图1是实施例3制备的肝素十二糖新分子I-2的高效液相色谱图(A)、质谱图(B)以及I-10高效液相色谱图(C)、质谱图(D)；
图2是实施例3制备的肝素十二糖新分子I-2的1H NMR(A)和HSQC(B)谱图；
图3是实施例3制备的新型肝素十二糖新分子I-2、I-10的体外抗Xa因子；
图4是鱼精蛋白体外对实施例3的肝素十二糖新分子I-2、I-10抗凝活性的中和作用。
具体实施方式
下面结合具体实施例对本发明进行进一步描述和理解，但不能限制本发明的保护范围，下列实例中的目标化合物的编号与表1相同。实施例中涉及的药品及试剂，若无特殊说明，均为普通市售产品。
实施例1：含单一IdoA2S残基的肝素五糖中间体的化学酶法合成
称取500mg硝基苯基-β-D-葡糖醛酸苷(GlcA-PNP，1)溶于200mL 50mmol/L Tris-HCl缓冲液(含6mmol/L MnCl2，pH＝7.2)，同时加入底物1.2倍当量的UDP-GlcNTFA及5mL KfiA酶，室温搅拌过夜，反应用PAMN-HPLC检测，色谱条件为在45min内以0→100％KH2PO4梯度洗脱，流速为0.5mL/min，检测波长为310nm，待产率≥95％，用三氟乙酸(TFA)调pH至2-3中止反应，反应液用C18层析柱(3.0×50cm)进行纯化，以含0.1％TFA的甲醇-水洗脱，收到目标组分，为二糖骨架中间体，将得到的二糖骨架中间体置于与200mL 50mmol/L Tris-HCl缓冲液(含6mmol/L MnCl2，pH＝7.2)中，同时加入1.2倍当量的UDP-GlcA、5mL PmHS2酶，室温搅拌过夜，PAMN-HPLC检测反应至产率≥97％，以C18层析柱纯化得三糖 骨架中间体3mer-1，以3mer-1为底物，重复上述KfiA、PmHS2反应，得到五糖骨架中间体5mer-1，PAMN-HPLC测得其纯度>82.5％，ESI-MS测得其分量1181.09Da，与理论值相符。
取400mg五糖骨架中间体5mer-1溶于100mL去离子水，置于冰上，逐滴加入0.5mol/L LiOH溶液至pH＝12，继续置于冰浴中2h，PAMN-HPLC检测反应进程；反应结束后，以冰醋酸调节pH至中性，加入1mol/L MES溶液(pH＝7.5)使其终浓度为50mmol/L，同时加入2.5倍当量的PAPS、3mL NST酶，室温搅拌过夜，利用PAMN-HPLC检测反应；反应产率>95％时醋酸调pH至4-5终止反应，用Q Sepharose层析柱(30×1.6cm)纯化，流速为3mL/min，以0→100％含1mol/LNaCl、50mmol/LNaAc缓冲液(pH＝5)梯度洗脱，检测波长为260nm和310nm，收集目标组分、脱盐、干燥得N-硫酸化的肝素五糖5mer-2；PAMN-HPLC测得其纯度>78％，ESI-MS测得其分量1149.17Da，与理论值相符。
取肝素五糖5mer-2于pH＝7.0～7.5、50mmol/L的MES缓冲液中加入2mmol/L CaCl2及酶C5-epi，调整反应体积为100mL，于37℃水浴反应2h。然后加入约1.5倍当量的PAPS、额外C5-epi和足量2-OST酶，室温反应过夜；PAMN-HPLC检测反应，并根据需要补加酶或PAPS，至反应结束，反应液用Q-Sepharose强阴离子柱(30×1.6cm)纯化得到产物5mer-3。PAMN-HPLC测得其纯度>95％，ESI-MS测定其分子量为1129.27Da，产物5mer-3较5mer-2增加一个硫酸酸基团。化合物5mer-3为含一个IdoA2S残基的肝素五糖。
5mer-3的合成路线如下式Ⅱ：
实施例2：含两个及三个IdoA2S残基的肝素十一糖中间体的化学酶法合成
取220mg含一个IdoA2S残基的肝素五糖5mer-3，参照实施例1，糖基供体更换为UDP-GlcNAc并由KfiA催化延长糖链，接着以UDP-GlcA为糖基供体、PmHS2酶催化进一 步延长糖链，然后交替进行KfiA(糖基供体UDP-GlcNTFA)、PmHS2(糖基供体UDP-GlcA)酶促糖链延长，直至形成肝素十一糖11mer-1，Q Sepharose层析柱(1×20cm)纯化；然后LiOH处理脱三氟乙酰基、NST催化进行N-硫酸化修饰得肝素十一糖11mer-2，PAMN-HPLC测得其纯度>99％，ESI-MS测得其分子量为2442.16Da，与理论值相符。
得到的肝素十一糖11mer-2在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，N-硫酸化十一糖糖链中两个GlcNS之间的特定GlcA被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)，反应进行一定时间后，反应液Q Sepharose层析柱(1×20cm)纯化得11mer-3(含两个IdoA2S残基的肝素十一糖)；反应液如补加适量酶与PAPS后，继续反应至完全生成新产物，Q Sepharose层析柱(1×20cm)纯化得11mer-4(含三个IdoA2S残基的肝素十一糖)。
11mer-3经ESI-MS测得其分子量为2522.21Da，较11mer-2增加1个硫酸基团，表明其新增一个IdoA2S；
11mer-4经ESI-MS测得其分子量为2602.40Da，较11mer-2增加2个硫酸基团，表明其新增两个IdoA2S，与预期相符。
11mer-3/4的合成路线如下式Ⅲ：
实施例3：含双AT结合序列的肝素十二糖I-2、I-10的制备及表征
分别以实施例2的十一糖11mer-3/4为底物，参照参照实施例1的方法进行KfiA酶法糖链延长，以Q-Sepharose强阴离子柱(1cm×20cm)纯化得十二糖中间体12mer-1/2，然后参照上述方法依次进行化学三氟乙酰基脱除、酶法N-硫酸化修饰得十二糖中间体12mer-3/4。ESI-MS测定其分子量分别为2763.56Da以及2843.39Da，与理论值相符。
将十二糖底物12mer-3/4置于pH＝7.0～7.5、50mmol/L的MES缓冲液中，加入7倍当量PAPS，4mL的6-OST-1和4mL的6-OST-3酶，调整反应体积为140mL，37℃水浴反应过夜。利用SAX-HPLC检测反应进程，根据需要补加酶或PAPS。色谱条件为：流速为1mL/min，以0→100％洗脱液B(50mmol/L NaAc+2mol/L NaCl，pH＝5)梯度洗脱为，检测波长为260nm和310nm。待12mer-5/6反应率>99％，反应液用稀醋酸调pH＝4-5终止反应，-20℃冰箱冻融除酶，无需纯化。
将上述反应液调节pH＝7.0～7.5，并加入约2.5倍当量PAPS、5mL的3-OST-1酶，调 整反应体积为200mL，37℃水浴反应过夜。PAMN-HPLC检测反应至底物修饰率为99％以上，反应液用稀醋酸调pH＝4-5后，用Q-Sepharose强阴离子柱(1cm×10cm)纯化得到产物I-2，其纯度达92％以上，ESI-MS测定其分子量为3403.12Da，与理论值相符；得到产物I-10，其纯度达97％以上，ESI-MS测定其分子量为3482.97Da，与理论值相符。I-2的NMR(600MHz,D2O)谱图见图2，结构与预期相符。
其合成路线如下式Ⅳ：
实施例4：肝素十二糖I-2、I-10的体外抗凝活性测定
利用商品化的试剂盒采用生色底物法测得本发明制备得到的新型肝素十二糖I-2、I-10抗FⅩa活性的IC50值为16.77、18.03ng/mL(4.34、4.54nmol/L)，同样条件下测得未分级肝素(UFH)、和磺达肝癸钠(Arixtra)的IC50值分别为139ng/mL、12.63ng/mL(7.3nmol/L)，以摩尔浓度计新型肝素十二糖I-2、I-10抗FⅩa活性的IC50值远小于磺达肝癸钠。经生色底物法测定，测试结果见图3所示，本发明制备得到的新型肝素十二糖I-2、I-10无显著的抗IIa因子活性(略)。因此，本发明制备得到的新型肝素十二糖I-2、I-10均为Xa因子的特异抑制剂。
实验例5：鱼精蛋白对肝素十二糖I-2、I-10抗凝活性的中和作用测定
采用生色底物法，加入不同浓度的鱼精蛋白对新型肝素十二糖I-2、I-10抗FⅩa活性的 影响，由测定结果可知，与UFH类似，新型肝素十二糖I-10的体外抗FⅩa活性可以完全被鱼精蛋白逆转；新型肝素十二糖I-2的体外抗FⅩa活性可以被鱼精蛋白逆转80％以上，见图4。因此，本发明制备得到的肝素十二糖I-2、I-10为抗凝活性可被鱼精蛋白中和的新型肝素分子。
如实施例4和图3所示，I-2、I-10抗FⅩa活性的IC50值分别为4.34、4.54nmol/L，差别不大；如实施例5和图4所示，鱼精蛋白对I-2抗凝活性的中和率>80％，而对I-10抗凝活性的中和率>90％，差别明显，因此糖残基G由GlcA替换为IdoA2S对肝素十二糖的抗Xa活性影响较小，但对其鱼精蛋白中和效率影响较大。

Claims (10)

  1. 一种含双AT结合序列、无连续多个三硫酸双糖(IdoA2S-GlcNS6S)的肝素十二糖新分子，或其药学上可接受的盐，其具有如下式I所示结构：
    R1、R3为磺酰基(-SO3H)或乙酰基(-COCH3)；R2为磺酰基或氢(-H)；
    R4选自具有特征紫外吸收的苯基或取代苯基，芳杂环或取代芳杂环；
    糖残基G为葡糖醛酸(GlcA)或艾杜糖醛酸(IdoA)。
  2. 根据权利要求1所述的肝素十二糖新分子或其药学上可接受的盐，其特征在于，取代苯基或取代芳杂环的取代基为硝基、卤素、羟基或三氟甲基。
  3. 根据权利要求1所述的肝素十二糖新分子，其特征在于，为下列之一：
  4. 权利要求1所述的含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖的制备 方法，该方法以还原末端共价连接R4基团的葡糖醛酸(GlcA)衍生物为起始底物，为如下步骤a、b糖基转移酶催化反应至少重复一次与步骤c、d、e、f、g化学酶法修饰反应中其中四步或五步组合的方法；
    步骤a，在N-乙酰氨基葡糖基转移酶(KfiA)或Heparosan合酶2(PmHS2)催化下，以UDP-GlcNTFA或UDP-GlcNAc为糖基供体，糖基供体的GlcNTFA残基或GlcNAc残基以α-1,4糖苷键被转移至底物非还原末端的GlcA上，得到中间体化合物；
    步骤b，在PmHS2酶催化下，以UDP-GlcA为糖基供体，糖基供体的GlcA残基以β-1,4糖苷键连接至底物非还原末端的葡糖胺(GlcNTFA或GlcNAc)，得到中间体化合物；
    步骤c，肝素中间体在温和的碱性水溶液中静置于冰上，糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化中间体；
    步骤d，在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，N-硫酸化产物糖链中两个GlcNS之间或GlcNS(非还原端)与GlcNAc之间的特定GlcA残基被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)，得到含IdoA2S残基的中间体；
    步骤e，在2OST的单独催化下，底物两个GlcNS之间或GlcNS(非还原端)与GlcNAc之间的特定GlcA残基被转变为2-O-硫酸化葡糖酸(GlcA2S)，得到含GlcA2S的中间体；
    步骤f，在6-O-硫酸基转移酶1和3(6OST1、6-OST3)的共同催化作用下，底物糖链的全部GlcNS或GlcNAc残基的6-OH发生硫酸化修饰成为GlcNS6S或GlcNAc6S，得到6-O-硫酸化中间体；
    步骤g，在3-O-硫酸基转移酶1(3OST1)的催化作用下，底物糖链中GlcA与IdoA2S之间的GlcNS6S的3-OH发生硫酸化(GlcNS6S3S)，得到最终目标化合物。
  5. 根据权利要求4所述的制备方法，其特征在于，起始底物为对硝基苯基-β-D-葡糖醛酸苷(GlcA-PNP)，步骤a中，N-乙酰氨基葡糖基转移酶(KfiA)、Heparosan合酶2(PmHS2)是以大肠杆菌重组表达，N-乙酰氨基葡糖基转移酶(KfiA)来源于大肠杆菌K5，Heparosan合酶2(PmHS2)来源于多杀巴斯德菌(Pasteurella multocida)；
    步骤a、b中，酶催化反应所用的缓冲液为50mmol/L Tris-HCl，Tris-HCl中含6mmol/L MnCl2，pH＝7.0-7.5，反应温度20℃～37℃，酶与底物的加入量、反应时间不受限定；得到 的酶促反应液利用反相C18或阴离子交换柱层析纯化得中间体化合物，糖基供体的加入量为底物的1.2倍当量以上。
  6. 根据权利要求4所述的制备方法，其特征在于，步骤c、d、e、f、g中，NST、C5-epi、2OST、6OST1、6-OST3、3OST1肝素修饰酶是利用大肠杆菌、酵母或昆虫细胞重组表达得到；NST、2OST、6OST1、6-OST3、3OST1肝素修饰酶均以3'-磷酸腺苷-5'-磷酸硫酸(PAPS)为硫酸基供体；各修饰酶催化反应的缓冲液为50mmol/L 2-(N-吗啉代)乙烷磺酸(MES)，pH＝7.0～7.5，反应温度20℃～37℃，酶与肝素中间体底物的加入量、反应时间不受限定，得到的反应液利用阴离子交换柱层析纯化得产物，步骤c、d、e、f、g中，硫酸基供体的加入量为底物的1.5-10倍当量。
  7. 根据权利要求4所述的制备方法，其特征在于，所述的制备方法选自如下合成路线之一：
  8. 根据权利要求4所述的制备方法，其特征在于，合成路线如下：
    a→b→a→b→c→d→a→b→a→b→a→b→c→d→a→c→f→g。
  9. 含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖的制备方法，步骤如下：
    1)在N-乙酰氨基葡糖基转移酶(KfiA)或Heparosan合酶2(PmHS2)催化下，以UDP-GlcNTFA为糖基供体，糖基供体的GlcNTFA残基以α-1,4糖苷键被转移至底物非还原末端的GlcA上，得到二糖骨架中间体；
    2)在PmHS2酶催化下，以UDP-GlcA为糖基供体，糖基供体的GlcA残基以β-1,4糖苷键被连接至二糖骨架非还原末端的GlcNTFA上，得到三糖骨架中间体；
    3)重复步骤1)、步骤2)将糖链延长，得到五糖骨架中间体；
    4)五糖骨架中间体在温和的碱性水溶液中静置于冰上，糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化五糖中间体；
    5)在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，N-硫酸化五糖中间体糖链中两个GlcNS之间的特定GlcA残基被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)；得到含一个IdoA2S残基的肝素五糖；
    6)参照步骤1)，糖基供体更换为UDP-GlcNAc，由KfiA或PmHS2催化延长糖链，得六糖中间体；以六糖中间体为底物，参照步骤2)将糖链延长为七糖，得七糖中间体；以七糖中间体为底物交替重复步骤1)、步骤2)继续将糖链延长，得到十一糖中间体；
    7)十一糖中间体在温和的碱性水溶液中静置于冰上，糖链的GlcNTFA残基全部脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，得到N-硫酸化十一糖；
    8)在C5-异构化酶(C5-epi)、2-O-硫酸基转移酶(2OST)的共同催化下，N-硫酸化十一糖糖链中两个GlcNS之间的特定GlcA被转变为2-O-硫酸化艾杜糖醛酸(IdoA2S)，得到含两个IdoA2S残基的肝素十一糖；补加适量酶与底物并延长反应时间，进一步使十一糖中GlcNS(非还原端)与GlcNAc之间的特定GlcA残基转化为IdoA2S，得到含三个IdoA2S残基的肝素十一糖；
    9)步骤8)中，含两个或三个IdoA2S残基的肝素十一糖分别重复步骤1)将糖链延长，得到含两个或三个IdoA2S残基的肝素十二糖；
    10)含两个或三个IdoA2S残基的肝素十二糖在温和的碱性水溶液中静置于冰上，糖链新引入的一个GlcNTFA残基脱三氟乙酰基(TFA)转变为GlcNH2，然后在N-硫酸基转移酶(NST)催化下使之转变为GlcNS，分别得到含两个或三个IdoA2S残基的N-硫酸化肝素 十二糖；
    11)在6-O-硫酸基转移酶1和3(6OST1、6-OST3)的共同催化作用下，含两个或三个IdoA2S残基的N-硫酸化肝素十二糖糖链的全部GlcNS或GlcNAc残基的6-OH发生硫酸化修饰成为GlcNS6S或GlcNAc6S，得到两种6-O-硫酸化肝素十二糖；
    12)在3-O-硫酸基转移酶1(3OST1)的催化作用下，两种6-O-硫酸化肝素十二糖底物糖链中GlcA与IdoA2S之间的GlcNS6S的3-OH发生硫酸化(GlcNS6S3S)，分别得到含2个IdoA2S的目标化合物I-2或含3个IdoA2S的目标化合物I-10。
  10. 含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖的应用，用于制备抗凝抗血栓药物；
    抗凝抗血栓药物，包括权利要求1所述的含双AT结合序列、无连续多个三硫酸双糖的肝素十二糖和一种或多种药学上可接受载体或赋形剂。
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