A heparin anticoagulant oligosaccharide with low platelet reduction induction activity, and a preparation method and application thereof

Abstract

This invention relates to a heparin anticoagulant oligosaccharide with low thrombocytopenia-inducing activity, its preparation method, and its application, belonging to the field of biomedical technology. The heparin anticoagulant oligosaccharide with low thrombocytopenia-inducing activity comprises an AT-binding sequence and a -GlcNS(6S)-GlcA2S- or -GlcNS(6S)-IdoA2S- linker sequence, specifically heparin anticoagulant dodecanosaccharide 12I-2, heparin anticoagulant dodecanosaccharide 12G-1, or heparin anticoagulant dodecanosaccharide 12G-2. This invention also provides methods for preparing heparin anticoagulant dodecanosaccharides 12I-2, 12G-1, and 12G-2, and their applications in the preparation of safe and effective anticoagulant and antithrombotic drugs. The heparin anticoagulant oligosaccharide designed in this invention is a specific dodecanoic acid molecule. By optimizing its sugar chain length, key structural domains and spatial conformation, its ability to form pathogenic H / PF4 complexes with platelet factor 4 (PF4) is weakened to the greatest extent. In clinical anticoagulation applications, it has a significant safety advantage in reducing the incidence of HIT and related fatal thrombosis (HITT).

Description

Technical Field

[0001] This invention relates to a heparin anticoagulant oligosaccharide with low thrombocytopenia induction activity, its preparation method and application, belonging to the field of biomedical technology. Background Technology

[0002] Heparin-induced thrombocytopenic purpura (HIT) is a potentially fatal adverse reaction to heparin-based immunotherapies. Its core pathological mechanism involves the binding of heparin molecules to platelet factor 4 (PF4) released by platelets, forming a "heparin-PF4 complex" (H / PF4). This complex induces the body to produce IgG antibodies against H / PF4. After binding to the complex, these antibodies further activate Fcγ receptors on the platelet surface, leading to platelet activation, aggregation, decreased platelet consumption, and a hypercoagulable state, ultimately causing arterial and venous thrombosis, clinically manifesting as severe thrombocytopenia accompanied by thromboembolic events. Epidemiological studies indicate that approximately 1-5% of patients receiving unfractionated heparin therapy will develop HIT, with 30-50% potentially developing thrombosis, and a mortality rate as high as 10-20%. Although the risk of HIT is slightly lower with low molecular weight heparin, it cannot be completely avoided.

[0003] Currently, the main strategy for avoiding HIT in clinical practice is to replace heparin anticoagulants (such as direct thrombin inhibitors and factor Xa inhibitors). However, these drugs have problems such as high cost, lack of effective antagonists, and limited routes of administration (e.g., oral administration or dose adjustment), making them difficult to replace heparin, especially in scenarios requiring local anticoagulation such as extracorporeal circulation and extracorporeal membrane oxygenation. Another strategy is to develop heparin derivatives with lower HIT risk through chemical modification or screening. For example, fondaparinux, as a synthetic pentose, has an extremely low HIT risk because it specifically targets FXa and hardly binds to PF4; however, it cannot inhibit thrombin and has slow clearance in vivo, limiting its application in acute hypercoagulable states. At the same time, existing clinical heparin preparations (including low molecular weight heparin) generally suffer from high structural heterogeneity, large batch-to-batch variability, and incomplete elimination of potential HIT triggers, posing technical challenges to improving safety.

[0004] Therefore, developing a novel heparin anticoagulant that combines highly efficient anticoagulant activity with a significant reduction in the risk of HIT has become an important technical challenge in clinical anticoagulation therapy, and has significant clinical value for optimizing the treatment safety of high-risk groups of cardiovascular disease. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a heparin anticoagulant oligosaccharide with low thrombocytopenia-inducing activity, its preparation method, and its application. This invention, through precise chemical structure design, prepares a highly homogeneous heparin oligosaccharide with optimized glycan chain length, selective key sulfation sites, and a specific spatial conformation. This oligosaccharide can maximally disrupt the ability to form pathogenic H / PF4 complexes while retaining its core anticoagulant efficacy, thereby inhibiting HIT from its root cause.

[0006] Terminology Explanation:

[0007] AT: Antithrombin;

[0008] GlcA-pNP: p-nitrophenyl-β-D-glucuronic acid;

[0009] UDP-GlcNTFA: Uridine diphosphate-N-trifluoroacetylglucosamine;

[0010] UDP-GlcNDFA: Uridine diphosphate-N-difluoroacetylglucosamine;

[0011] UDP-GlcA: Uridine diphosphate-glucuronic acid;

[0012] PAPS: 3'-Adenosine-5'-phosphate sulfate;

[0013] NaKfiA: N-acetylglucanyltransferase;

[0014] PmHS2: Heparin backbone synthase 2;

[0015] NST: N-sulfate transferase;

[0016] C5-epi: C5-isomerase;

[0017] 2OST: 2-O-sulfate transferase;

[0018] 6OST: 6-O-sulfate transferase;

[0019] 3OST: 3-O-sulfate transferase.

[0020] The technical solution of this invention is as follows:

[0021] A heparin anticoagulant oligosaccharide with low thrombocytopenia-inducing activity and its pharmaceutically acceptable salt, the chemical structure of which is shown in formula (1):

[0022]

[0023] Equation (1)

[0024] Wherein, R1 is a phenyl or substituted phenyl, aromatic heterocyclic or substituted aromatic heterocyclic, or hydrogen (-H), or alkyl and alkylamine derivative groups with characteristic ultraviolet absorption; R2 is hydrogen (-H) or -SO3. - .

[0025] According to a preferred embodiment of the present invention, the heparin anticoagulation oligosaccharide with low thrombocytopenia induction activity comprises an AT binding sequence and a -GlcNS(6S)-GlcA2S- or -GlcNS(6S)-IdoA2S- linker sequence, specifically heparin anticoagulation dodecanose 12I-2, heparin anticoagulation dodecanose 12G-1 or heparin anticoagulation dodecanose 12G-2;

[0026] The chemical structure of heparin anticoagulant dodecanose 12I-2 is shown in formula (2) below:

[0027]

[0028] Equation (2)

[0029] The chemical structure of heparin anticoagulant dodecanose 12G-1 is shown in formula (3) below:

[0030]

[0031] Equation (3)

[0032] The chemical structure of heparin anticoagulant dodecanose 12G-2 is shown in the following formula (4):

[0033]

[0034] Equation (4).

[0035] The preparation method of the above-mentioned heparin anticoagulant dodecanose 12I-2 includes the following steps:

[0036] (1) First, heparin nonaglycone backbone, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) were added to Tris-HCl buffer to construct reaction system 1; reaction system 1 was reacted overnight at 35~40℃ in a water bath for 12~16h, and after purification, intermediate product 1 was obtained;

[0037] Then, intermediate product 1, uridine diphosphate-glucuronic acid and heparin backbone synthase 2 (PmHS2) were added to Tris-HCl buffer to construct reaction system 2; reaction system 2 was reacted overnight at 35~40℃ in a water bath for 12~16h, and after purification, intermediate product 2 was obtained.

[0038] Next, intermediate product 2, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) were added to Tris-HCl buffer to construct reaction system 3; reaction system 3 was reacted overnight in a water bath at 35~40℃ for 12~16h, and then the reaction solution was transferred to an equal volume of 0.1mol / L lithium hydroxide aqueous solution and reacted at 4℃ for 25~35min to obtain a solution containing intermediate product 3;

[0039] Finally, the solution containing intermediate product 3 was transferred to MES buffer, and 3'-adenosine-5'-phosphate sulfate (PAPS) and N-sulfate transferase (NST) were added to construct reaction system 4. Reaction system 4 was reacted overnight at 35~40℃ in a water bath for 12~16h. After purification, N-sulfated heparin dodecanose was obtained.

[0040] (2) Dissolve the N-sulfated heparin dodecanose obtained in step (1) in MES buffer, then add CaCl2 and C5-epimerase, react at 35~40℃ for 1.5~2.5h, then add 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfate transferase (2OST) to construct reaction system 5; react reaction system 5 overnight in a water bath at 25~30℃ for 12~16h, and after purification, obtain heparin dodecanose modified with iduronic acid-2-O-sulfate.

[0041] (3) The heparin dodecanose modified with iduronic acid-2-O-sulfate described in step (2) was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate (PAPS) and recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) were added to construct reaction system 6. The reaction system 6 was reacted overnight at 35~40℃ in a water bath for 12~16h, and 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1) were added. The reaction was then reacted overnight at 20~30℃ in a water bath for 12~16h. After purification, heparin anticoagulant dodecanose 12I-2 was obtained.

[0042] The preparation method of the above-mentioned heparin anticoagulant dodecanose 12G-1 or heparin anticoagulant dodecanose 12G-2 includes the following steps:

[0043] 1) The heparin nonaglycone backbone was dissolved in a 0.1 mol / L lithium hydroxide aqueous solution and reacted at 4 °C for 25-35 min. The pH was adjusted to 7.0, and then the reaction solution was added to MES buffer. 3'-adenosine-5'-phosphate sulfate (PAPS) and N-sulfate transferase (NST) were added to construct reaction system 7. Reaction system 7 was reacted overnight at 35-40 °C in a water bath for 12-16 h. After purification, N-sulfated heparin nonaglycone was obtained.

[0044] 2) Dissolve the N-sulfated heparin nonose obtained in step 1) in MES buffer, add 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfotransferase (2OST) to construct reaction system 8; react reaction system 8 overnight at 25~30℃ for 12~16h, and after purification, GlcA2S modified heparin nonose is obtained;

[0045] 3) First, add the GlcA2S-modified heparin nonose obtained in step 2), uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) to Tris-HCl buffer to construct reaction system 9; react system 9 overnight at 35~40℃ for 12~16h, and after purification, obtain intermediate product 4;

[0046] Then, intermediate product 4, uridine diphosphate-glucuronic acid and heparin backbone synthase 2 (PmHS2) were added to Tris-HCl buffer to construct reaction system 10; reaction system 10 was reacted overnight at 35~40℃ in a water bath for 12~16h, and after purification, intermediate product 5 was obtained.

[0047] Next, intermediate product 5, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) were added to Tris-HCl buffer to construct reaction system 11; reaction system 11 was reacted overnight in a water bath at 35~40℃ for 12~16h, and then the reaction solution was transferred to an equal volume of 0.1mol / L lithium hydroxide aqueous solution and reacted at 4℃ for 25~35min to obtain a solution containing intermediate product 6;

[0048] Finally, the solution containing intermediate product 6 was transferred to MES buffer, and 3'-adenosine-5'-phosphate sulfate (PAPS) and N-sulfate transferase (NST) were added to construct reaction system 12. Reaction system 12 was reacted overnight at 35~40℃ in a water bath for 12~16h. After purification, N-sulfated heparin dodecanose was obtained.

[0049] 4) Dissolve the N-sulfated heparin dodecanose obtained in step 3) in MES buffer, add CaCl2 and C5-epimerase, react at 35~40℃ for 1.5~2.5h, then add 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfate transferase (2OST) to construct reaction system 13; react reaction system 13 overnight in a water bath at 25~30℃ for 12~16h, and after purification, obtain heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S);

[0050] 5) The heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) obtained in step 4) was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate (PAPS) and 6-O-sulfate transferase-1 (6OST-1) were added to construct reaction system 14. Reaction system 14 was reacted overnight at 35~40℃ in a water bath for 12~16h, and 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1) were added. The reaction was continued overnight at 25~30℃ in a water bath for 12~16h. After purification, heparin anticoagulant dodecanose 12G-1 was obtained.

[0051] 6) The heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) obtained in step 4) was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate (PAPS) and recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) were added to construct reaction system 15. Reaction system 15 was incubated overnight at 35~40℃ for 12~16h, and 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1) were added. The reaction was continued overnight at 20~30℃ for 12~16h. After purification, heparin anticoagulant dodecanose 12G-2 was obtained.

[0052] According to a preferred embodiment of the present invention, the heparin nine-saccharide skeleton described above is prepared according to the following method:

[0053] S1: Place p-nitrophenyl-β-D-glucuronic acid, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase in Tris-HCl buffer, with a total reaction volume of 500 mL; react the reaction solution overnight at 37 °C for 12-16 h, adjust the pH to 3, and then purify by C18 column chromatography to obtain the heparin disaccharide backbone;

[0054] S2: Take the heparin disaccharide backbone, uridine diphosphate-glucuronic acid and heparin backbone synthase 2 obtained in step S1 and place them in Tris-HCl buffer. The total volume of the reaction solution is 500 mL. The reaction solution is reacted overnight at 37°C. The pH of the reaction solution is adjusted to 3 with trifluoroacetic acid. After purification by C18 column chromatography, the heparin trisaccharide backbone is obtained.

[0055] S3: Repeat the operations described in steps S1 to S2 three times consecutively to obtain the heparin nine-saccharide skeleton;

[0056] In the repeated process, the oligosaccharide products obtained in the previous step are used as glycosyl acceptor substrates for the new round of reaction, and step S1 is performed using uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase, respectively, followed by step S2 using uridine diphosphate-glucuronic acid and heparin backbone synthase 2.

[0057] According to a preferred embodiment of the present invention, the Tris-HCl buffer is a Tris-HCl buffer containing 10 mmol / L MnCl2, with a concentration of 50 mmol / L and a pH of 7.5; the MES buffer is a MES buffer with a concentration of 50 mmol / L and a pH of 7.5.

[0058] According to a preferred embodiment of the present invention, in step (1), the concentration of heparin nonaglycone backbone in reaction system 1 is 1~5mM, the molar ratio of heparin nonaglycone backbone to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.2, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml;

[0059] In the reaction system 2, the concentration of intermediate product 1 is 1~5mM, the molar ratio of intermediate product 1 to uridine diphosphate-glucuronic acid (UDP-GlcA) is 1:1.2, and the concentration of heparin backbone synthase 2 (PmHS2) is 0.1~0.3mg / ml.

[0060] In the reaction system 3, the concentration of intermediate product 2 is 1~5mM, the molar ratio of intermediate product 2 to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.5, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml;

[0061] In the reaction system 4, the concentration of intermediate product 3 is 1~5mM, the molar ratio of intermediate product 2 to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of N-sulfate transferase (NST) is 0.1~0.3mg / ml.

[0062] In step (2), in the reaction system 5, the concentration of N-sulfated heparin dodecanose is 1~5mM, the concentration of CaCl2 is 1~10mM, the concentration of C5-epimerase is 0.1~0.3mg / ml; the molar ratio of N-sulfated heparin dodecanose to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of 2-O-sulfotransferase (2OST) is 0.4~0.6mg / mL;

[0063] In step (3), in the reaction system 6, the concentration of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) is 1~5mM, the molar ratio of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:3, and the concentration of recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) is 0.4~0.6mg / ml;

[0064] After adding 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1), the concentration of 3'-adenosine-5'-phosphate sulfate (PAPS) in reaction system 6 was 1~8 mM, and the concentration of 3-O-sulfate transferase-1 (3OST-1) was 0.1~0.6 mg / ml.

[0065] According to a preferred embodiment of the present invention, in step 1), the concentration of heparin nonaglycone backbone in the reaction system 7 is 1~5mM, the molar ratio of heparin nonaglycone backbone to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of N-sulfate transferase (NST) is 0.1~0.3mg / ml.

[0066] In step 2), in the reaction system 8, the concentration of N-sulfated heparin nonose is 1~5mM, the molar ratio of N-sulfated heparin nonose to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:9, and the concentration of 2-O-sulfotransferase (2OST) is 0.1~0.3mg / ml;

[0067] In step 3), in the reaction system 9, the concentration of GlcA2S-modified heparin nonaose is 1~5mM, the molar ratio of GlcA2S-modified heparin nonaose to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.2, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml;

[0068] In the reaction system 10, the concentration of intermediate product 4 is 1~5mM, the molar ratio of intermediate product 4 to uridine diphosphate-glucuronic acid (UDP-GlcA) is 1:1.2, and the concentration of heparin backbone synthase 2 (PmHS2) is 0.1~0.3mg / ml.

[0069] In the reaction system 11, the concentration of intermediate product 5 is 1~5mM, the molar ratio of intermediate product 5 to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.5, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml.

[0070] In the reaction system 12, the concentration of intermediate product 6 is 1~5mM, the molar ratio of intermediate product 6 to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of N-sulfate transferase (NST) is 0.1~0.3mg / ml.

[0071] In step 4), in the reaction system 13, the concentration of N-sulfated heparin dodecanose is 1~5mM, the concentration of CaCl2 is 1~10mM, the concentration of C5-epimerase is 0.1~0.3mg / ml; the molar ratio of N-sulfated heparin dodecanose to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of 2-O-sulfotransferase (2OST) is 0.4~0.6mg / mL;

[0072] In step 5), in the reaction system 14, the concentration of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) is 1~5mM, the molar ratio of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:3, and the concentration of 6-O-sulfotransferase-1 (6OST-1) is 0.4~0.6mg / ml;

[0073] After adding 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1), the concentration of 3'-adenosine-5'-phosphate sulfate (PAPS) in reaction system 14 was 1~8 mM, and the concentration of 3-O-sulfate transferase-1 (3OST-1) was 0.1~0.6 mg / ml.

[0074] In step 6), in the reaction system 15, the concentration of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) is 1~5mM, the molar ratio of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:3, and the concentration of recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) is 0.4~0.6mg / ml;

[0075] After adding 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1), the concentration of 3'-adenosine-5'-phosphate sulfate (PAPS) in reaction system 14 was 1~8 mM, and the concentration of 3-O-sulfate transferase-1 (3OST-1) was 0.1~0.6 mg / ml.

[0076] This invention also provides the application of the above-mentioned heparin anticoagulant oligosaccharide with low thrombocytopenia induction activity in the preparation of safe and efficient anticoagulant drugs and antithrombotic drugs.

[0077] This invention significantly reduces the risk of heparin-induced thrombosis (HIT) through molecular structure optimization, thereby overcoming common treatment bottlenecks of traditional heparin drugs. Regarding its antithrombotic mechanism, this molecule effectively inhibits thrombus formation by interacting with coagulation factors, making it suitable for the prevention and treatment of deep vein thrombosis, pulmonary embolism, and postoperative thromboembolism.

[0078] Experimental procedures not described in detail in this invention can be performed according to conventional experimental procedures in this technical field.

[0079] The technical features of this invention are as follows:

[0080] Compared to conventional heparin, the unique advantage of the heparin anticoagulation oligosaccharide with low thrombocytopenia induction activity provided by this invention lies in its extremely low HIT induction activity: this property stems from the molecular design that weakens the binding ability with PF4 (platelet factor 4), resulting in a reduced probability of PF4-antibody complex formation, thereby avoiding HIT-mediated thrombocytopenia and thrombotic complications, and significantly improving clinical safety.

[0081] In a preferred embodiment of the invention, the molecule exhibits excellent reversibility in anticoagulation applications and can be efficiently neutralized by protamine. This key characteristic ensures that in the event of unexpected bleeding or the need for emergency surgery (such as coronary artery bypass grafting or interventional treatment) during medication, the anticoagulation effect can be rapidly antagonized by protamine, preventing the risk of major bleeding and significantly improving the risk control capability of the treatment plan.

[0082] The beneficial effects of this invention are as follows:

[0083] 1. The heparin anticoagulant oligosaccharide with low thrombocytopenia-inducing activity designed in this invention is a specific dodecaglycoside molecule (structures shown in Formulas 1-4). By optimizing its glycan chain length, key structural domains, and spatial conformation, its ability to form a pathogenic H / PF4 complex with platelet factor 4 (PF4) is weakened to the greatest extent. Since the H / PF4 complex is the core initiating step in triggering the HIT immune cascade, this structural design reduces the risk of antibody production and subsequent platelet activation, consumption, and thrombosis from the source. This gives it a significant safety advantage in clinical anticoagulation applications compared to animal-derived unfractionated heparin (UFH) and conventional low molecular weight heparin (LMWH), significantly reducing the incidence of HIT and related fatal thrombosis (HITT), and providing a new candidate heparin drug for achieving the requirement of significantly reducing the risk of heparin-induced thrombocytopenia (HIT).

[0084] 2. The heparin anticoagulant oligosaccharide with low thrombocytopenia-inducing activity provided by this invention retains key anti-factor Xa activity, effectively blocking the coagulation cascade and meeting the basic efficacy requirements of anticoagulation therapy. More importantly, its anticoagulant effect maintains reliable reversibility, and can be effectively neutralized by readily available clinical antagonists—protamine sulfate. This characteristic is crucial in high-risk situations such as sudden bleeding events or emergency surgery, enabling rapid restoration of coagulation function, greatly improving risk control and treatment flexibility in clinical applications, solving the clinical dilemma of lacking rapid and effective antagonistic methods for some non-heparin anticoagulants, and effectively improving the core anticoagulant activity and target interventionability of heparin drugs.

[0085] 3. The method for preparing heparin anticoagulant oligosaccharides with low thrombocytopenia-inducing activity provided by this invention utilizes multiple recombinantly expressed glycosyltransferases and modifying enzymes, combined with specific reaction sequences and controlled conditions (such as selective deprotection, regio / stereospecific sulfation, and epimerization), to achieve high-precision, stepwise, and controllable synthesis of the target dodecanesaccharide molecule. This method completely avoids the inherent high structural heterogeneity and large batch-to-batch variations of traditional heparin drugs extracted from animal tissues, thus ensuring the stability and uniformity of drug quality from the source. This not only directly affects the reliability and predictability of anticoagulant effects but also enhances the certainty of reducing HIT risk at the molecular structure level, achieving uniformity in the prepared heparin drug and overcoming the drawbacks of natural extracts.

[0086] 4. The heparin-based anticoagulant oligosaccharide with low thrombocytopenia-inducing activity provided by this invention offers a better option for high-risk surgeries and in vitro anticoagulation scenarios. Given the current clinical need for drugs with high anticoagulant efficacy and extremely low HIT risk, such as in extracorporeal circulation and extracorporeal membrane oxygenation, existing non-heparin anticoagulants are expensive, have limited administration, or lack effective antagonists. In particular, while fondaparinux sodium products have low HIT risk, their pharmacological properties restrict their application in these scenarios. The dodecanosaccharide molecule of this invention combines good anticoagulant activity with extremely low HIT induction and protamine reversibility through design, making it a potentially safer, more reliable, and practical alternative to heparin in such high-risk clinical scenarios requiring rapid modulation of anticoagulation effects. Attached Figure Description

[0087] Figure 1 The results of electrospray ionization high-resolution mass spectrometry (ESI-HRMS) of heparin anticoagulation dodecaose 12I-2;

[0088] In the figure, the horizontal axis represents the mass-to-charge ratio (m / z), and the vertical axis represents the signal strength.

[0089] Figure 2 The results of electrospray ionization high-resolution mass spectrometry (ESI-HRMS) of heparin anticoagulant dodecaose 12G-1;

[0090] In the figure, the horizontal axis represents the mass-to-charge ratio (m / z), and the vertical axis represents the signal strength.

[0091] Figure 3 The results of electrospray ionization high-resolution mass spectrometry (ESI-HRMS) for heparin anticoagulation dodecaose 12G-2;

[0092] In the figure, the horizontal axis represents the mass-to-charge ratio (m / z), and the vertical axis represents the signal strength.

[0093] Figure 4 The results are for the proton nuclear magnetic resonance (NMR) spectrum of heparin anticoagulation dodecaose 12I-2;

[0094] In the figure, the horizontal axis represents the chemical shift value (δ), and the vertical axis represents the signal intensity.

[0095] Figure 5 The results are for the heparin anticoagulation dodecaose 12G-1 NMR 1H spectrum.

[0096] In the figure, the horizontal axis represents the chemical shift value (δ), and the vertical axis represents the signal intensity.

[0097] Figure 6 The results are for the proton nuclear magnetic resonance (NMR) spectrum of heparin anticoagulation dodecaose 12G-2;

[0098] In the figure, the horizontal axis represents the chemical shift value (δ), and the vertical axis represents the signal intensity.

[0099] Figure 7 The graph shows the results of the assay of anticoagulant dodecanosaccharides 12I-2 and 12G-2 and the anti-factor Xa activity of enoxaparin.

[0100] In the figure, the horizontal axis represents the sample concentration, and the vertical axis represents the absorbance at a wavelength of 405 nm.

[0101] Figure 8 The figure shows the results of an in vitro protamine neutralization experiment between heparin anticoagulation dodecanosaccharides 12I-2 and 12G-2 and enoxaparin;

[0102] In the figure, the horizontal axis represents the sample concentration, and the vertical axis represents the absorbance at a wavelength of 405 nm.

[0103] Figure 9 The figure shows the results of the isothermal titration calorimetric (ITC) titration experiment of heparin anticoagulation dodecanose 12G-2 and platelet factor 4 (PF4);

[0104] In the figure, the horizontal axis represents the molar ratio of 12G-2 to PF4, and the vertical axis represents the instantaneous or cumulative heat change (enthalpy change, ΔH) and the corresponding signal strength.

[0105] Figure 10 The image shows the results of an enzyme-linked immunosorbent assay (ELISA) for heparin anticoagulation dodecaose 12G-2, unfractionated heparin (UFH), and the complex formed by enoxaparin and PF4 / platelet factor 4 antibody (PF4 / Ab).

[0106] In the figure, the horizontal axis represents different heparin samples, and the vertical axis represents the measured reaction values.

[0107] Figure 11 Figure 1 shows the results of mouse immunization experiments using heparin anticoagulation dodecaose 12G-2 and enoxaparin.

[0108] In the figure, the horizontal axis represents the injected heparin sample, and the vertical axis represents the HIT antibody obtained from the assay. Detailed Implementation

[0109] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art.

[0110] The N-acetylglucosyltransferase (NaKfiA), heparin backbone synthase 2 (PmHS2), N-sulfotransferase (NST), C5-isomerase (C5-epi), 2-O-sulfotransferase (2OST), 3-O-sulfotransferase-1 (3OST-1), 6-O-sulfotransferase-1 (6OST-1), and HepIII described in the following examples are all existing enzymes with publicly available amino acid sequences, which can be prepared by commercially available enzymes or by recombinant expression in hosts such as Escherichia coli, yeast, and insect cells.

[0111] The recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) was constructed and prepared according to the report of Lin Yi (Org. Biomol.Chem., 2020, 18, 8094-8102) et al.

[0112] In the embodiments, the Tris-HCl buffer is a Tris-HCl buffer containing 10 mmol / L MnCl2, with a concentration of 50 mmol / L and pH=7.5; the MES buffer is a MES buffer with a concentration of 50 mmol / L and pH=7.5.

[0113] Example 1: Chemical enzymatic synthesis of the heparin nonaglycone backbone

[0114] A method for synthesizing the heparin nonaglycone backbone, the specific steps of which are as follows:

[0115] S1: 500 mg p-nitrophenyl-β-D-glucuronic acid (GlcA-pNP, as substrate), uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA, 1.5 equivalents of substrate, as glycosyl donor), and N-acetylglucosyltransferase (NaKfiA, 0.2 mg / mL) were placed in a pre-prepared Tris-HCl buffer solution, with a total reaction volume of 500 mL. The reaction solution was incubated overnight at 37 °C for 16 h, and the reaction progress was monitored using high-performance liquid chromatography (PAMN-HPLC, detection wavelength: 310 nm) with UV-Vis detection. When the substrate conversion reached over 90%, the reaction was terminated, and the pH was adjusted to 3 with trifluoroacetic acid. The product was then purified by octadecyl-bonded silica gel (C18) reversed-phase column chromatography to obtain the heparin disaccharide backbone product (GlcNTFA-GlcA-pNP).

[0116] S2: Take the heparin disaccharide backbone (GlcNTFA-GlcA-pNP, 500 mg, as substrate), uridine diphosphate-glucuronic acid (UDP-GlcA, 1.2 equivalents of substrate, as glycosyl donor), and heparin backbone synthase 2 (PmHS2, 0.2 mg / mL) obtained in step S1 and place them in a pre-prepared Tris-HCl buffer solution, with a total reaction volume of 500 mL. Incubate the reaction solution overnight at 37 °C for 16 h, and monitor the reaction progress using high-performance liquid chromatography with UV-Vis detection (PAMN-HPLC, detection wavelength: 310 nm). When the substrate conversion reaches more than 95%, terminate the reaction, adjust the pH to 3 with trifluoroacetic acid, and then purify by octadecyl bonded silica gel (C18) reversed-phase column chromatography to obtain the heparin trisaccharide backbone product (GlcA-GlcNTFA-GlcA-pNP).

[0117] S3: Repeat the operations described in steps S1 to S2 three times to obtain the heparin nine-sugar backbone (GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-pNP).

[0118] In the repeated process, the oligosaccharide products obtained in the previous step are used as the glycosyl acceptor substrates for the new round of reaction, and step S1 is performed using uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase, respectively, followed by step S2 using uridine diphosphate-glucuronic acid and heparin backbone synthase 2.

[0119] The specific reaction process is shown in the following diagram:

[0120] .

[0121] Example 2: Preparation of Heparin Anticoagulant Dodecanose 12I-2

[0122] A method for preparing heparin anticoagulant dodecanose 12I-2 includes the following steps:

[0123] (1) The heparin nonaglycone skeleton (200 mg, as substrate) prepared in Example 1, uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA, 1.2 equivalents of substrate) and N-acetylglucosyltransferase (NaKfiA, 0.2 mg / mL) were added to Tris-HCl buffer to construct a 100 mL reaction system 1. The reaction system 1 was reacted overnight at 37 °C in a water bath for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion rate exceeded 90%, the reaction was terminated and purified by Q-Sepharose strong anion exchange chromatography column to obtain intermediate product 1.

[0124] Intermediate product 1 (200 mg, as substrate), uridine diphosphate-glucuronic acid (UDP-GlcA, 1.2 equivalents of substrate), and heparin backbone synthase 2 (PmHS2, 0.2 mg / mL) were then added to Tris-HCl buffer to construct a 100 mL reaction system 2. The reaction system 2 was incubated overnight at 37 °C in a water bath for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 95%, the reaction was terminated, the pH was adjusted to 3 using trifluoroacetic acid, and the intermediate product 2 was purified using a Q-Sepharose strong anion exchange column.

[0125] Intermediate product 2 (200 mg, as substrate), uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA, 1.5 equivalents of substrate), and N-acetylglucosyltransferase (NaKfiA, 0.2 mg / mL) were added to Tris-HCl buffer to construct a 100 mL reaction system 3. The reaction system 3 was incubated overnight at 37 °C for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion was higher than 90%, the reaction solution was transferred to an equal volume of 0.1 mol / L lithium hydroxide aqueous solution and reacted at 4 °C for 30 min to obtain a solution containing intermediate product 3.

[0126] Finally, the pH of the solution containing intermediate 3 was adjusted to 7.0, and the solution was transferred to MES buffer. Using intermediate 3 as a substrate, 3'-adenosine-5'-phosphate sulfate (PAPS, 12 equivalents of substrate) and N-sulfate transferase (NST, 0.2 mg / mL) were added to construct a 50 mL reaction system 4. The reaction system 4 was incubated overnight at 37 °C for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 99%, the reaction was terminated, and the solution was purified using a Q-Sepharose strong anion exchange column to obtain N-sulfated heparin dodecanose (GlcNS-GlcA-GlcNS-GlcA-GlcNS-GlcNS-GlcA-GlcNS-GlcNS-GlcA-pNP).

[0127] The specific reaction process is shown in the following diagram:

[0128]

[0129] (2) The N-sulfated heparin dodecanose (150 mg, as substrate) obtained in step (1) was dissolved in MES buffer, and then CaCl2 (2 mM) and C5-epimerase (C5-epi, 0.2 mg / mL) were added. After reacting at 37°C for 2 h, 3'-adenosine-5'-phosphate sulfate (PAPS, 12 equivalents of substrate) and 2-O-sulfate transferase (2OST, 0.5 mg / mL) were added to construct a 50 mL reaction system 5. The reaction system 5 was reacted overnight at 37°C in a water bath for 16 h. The reaction process was continuously monitored by PAMN-HPLC, and the corresponding enzyme 2OST or PAPS was added in time according to the reaction process. When the substrate conversion rate exceeded 99%, the reaction was terminated, and the product was purified by Q-Sepharose strong anion exchange chromatography column to obtain heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S).

[0130] The specific reaction process is shown in the following diagram:

[0131]

[0132] (3) Dissolve the heparin dodecanose (100 mg, as substrate) modified with iduronic acid-2-O-sulfate as described in step (2) in MES buffer, then add 3'-adenosine-5'-phosphate sulfate (PAPS, 1.5 equivalents of substrate) and 0.5 mg / ml of recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) to construct a 50 mL reaction system 6; incubate reaction system 6 overnight at 37°C in a water bath for 16 minutes. h, 3'-adenosine-5'-phosphate sulfate (PAPS, 2 equivalents) and 3-O-sulfate transferase-1 (3OST-1, concentration 0.2 mg / ml after addition) were added, and the reaction was carried out overnight at 37°C in a water bath for 12 h. The reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 99%, the reaction was terminated, and the product was purified using a Q-Sepharose strong anion exchange column to obtain heparin anticoagulant dodecanediosaccharide 12I-2.

[0133] The specific reaction process is shown in the following diagram:

[0134] .

[0135] The heparin anticoagulant dodecanose 12I-2 prepared in this embodiment was analyzed by electrospray ionization high-resolution mass spectrometry (ESI-HRMS) and nuclear magnetic resonance hydrogen spectroscopy (NMR 1H spectroscopy). The results are as follows: Figure 1 and Figure 4 As shown.

[0136] Depend on Figure 1 and Figure 4It can be seen that this embodiment successfully prepared heparin anticoagulant dodecanose 12I-2, the specific structure of which is shown in the following formula:

[0137] .

[0138] Example 3: Preparation of Heparin Anticoagulant Dodecansaccharides 12G-1 and 12G-2

[0139] A method for preparing heparin anticoagulant dodecanosaccharides 12G-1 and 12G-2 includes the following steps:

[0140] 1) The heparin nonaglycone backbone (100 mg, as substrate) prepared in Example 1 was dissolved in a 0.1 mol / L lithium hydroxide aqueous solution and reacted at 4 °C for 30 min. The pH was adjusted to 7.0, and then the reaction solution was added to MES buffer. 3'-adenosine-5'-phosphate sulfate (PAPS, 12 equivalents of substrate) and N-sulfate transferase (NST, 0.2 mg / ml) were added to construct a 50 mL reaction system 7. The reaction system 7 was incubated overnight at 37 °C for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 90%, the reaction was terminated and purified using a Q-Sepharose strong anion exchange column to obtain N-sulfated heparin nonaglycone.

[0141] The specific reaction process is as follows:

[0142]

[0143] 2) The N-sulfated heparin nonaglycone obtained in step 1) (100 mg, as substrate) was dissolved in MES buffer, and 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfate transferase (2OST, 0.4 mg / ml) were added to construct a 50 mL reaction system 8. The reaction system 8 was incubated overnight at 37 °C for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 99%, the reaction was terminated and purified using a Q-Sepharose strong anion exchange chromatography column to obtain GlcA2S modified heparin nonaglycone (GlcA-GlcNS-GlcA2S-GlcNS-GlcA2S-GlcNS-GlcA2S-GlcNS-GlcA-pNP).

[0144] The specific reaction process is as follows:

[0145]

[0146] 3) First, add the GlcA2S-modified heparin nonaose (100 mg, as substrate) obtained in step 2), uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA, 1.2 equivalents of substrate) and N-acetylglucosyltransferase (NaKfiA, 0.2 mg / mL) to Tris-HCl buffer to construct a 50 mL reaction system 9; react system 9 overnight at 37 °C for 12 h, and continuously monitor the reaction progress by PAMN-HPLC; when the substrate conversion exceeds 90%, the reaction is terminated and purified by Q-Sepharose strong anion exchange chromatography column to obtain intermediate product 4;

[0147] Intermediate product 4 (100 mg, as substrate), uridine diphosphate-glucuronic acid (UDP-GlcA, 1.2 equivalents of substrate), and heparin backbone synthase 2 (PmHS2, 0.2 mg / mL) were then added to Tris-HCl buffer to construct a 50 mL reaction system 10. The reaction system 10 was incubated overnight at 37 °C in a water bath for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 95%, the reaction was terminated, the pH was adjusted to 3 with trifluoroacetic acid, and the intermediate product 5 was purified using a Q-Sepharose strong anion exchange column.

[0148] Intermediate product 5 (100 mg, as substrate), uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA, 1.5 equivalents of substrate), and N-acetylglucosyltransferase (NaKfiA, 0.2 mg / mL) were added to Tris-HCl buffer to construct a 50 mL reaction system 11. The reaction system 11 was incubated overnight at 37 °C in a water bath for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion rate was higher than 90%, the reaction solution was transferred to an equal volume of 0.1 mol / L lithium hydroxide aqueous solution and reacted at 4 °C for 30 min to obtain a solution containing intermediate product 6.

[0149] Finally, the pH of the solution containing intermediate 6 was adjusted to 7.0, and the solution was transferred to MES buffer. Using intermediate 6 as the substrate, 3'-adenosine-5'-phosphate sulfate (PAPS, 12 equivalents of substrate) and N-sulfate transferase (NST, 0.2 mg / mL) were added to construct a 50 mL reaction system 12. The reaction system 12 was incubated overnight at 37 °C in a water bath for 12 h, and the reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 99%, the reaction was terminated, and the solution was purified using a Q-Sepharose strong anion exchange column to obtain N-sulfated heparin dodecanose.

[0150] The specific reaction process is as follows:

[0151]

[0152] 4) Dissolve the N-sulfated heparin dodecanose (50 mg, as substrate) obtained in step 3) in MES buffer, add CaCl2 (2 mM) and C5-epimerase (C5-epi, 0.2 mg / mL), and react at 37 °C for 2 h. Then add 3'-adenosine-5'-phosphate sulfate (PAPS, 12 equivalents of substrate) and 2-O-sulfate transferase (2OST, 0.5 mg / mL) to construct a 20 mL reaction system 13. Incubate reaction system 13 overnight at 37 °C for 16 h. Monitor the reaction progress continuously using PAMN-HPLC and add the corresponding enzyme 2OST or PAPS as needed according to the reaction progress. When the substrate conversion exceeds 99%, terminate the reaction and purify using a Q-Sepharose strong anion exchange column to obtain heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S).

[0153] The specific reaction process is as follows:

[0154]

[0155] 5) Dissolve the heparin dodecanose (50 mg, as substrate) modified with iduronic acid-2-O-sulfate (IdoA2S) obtained in step 4) in MES buffer, then add 3'-adenosine-5'-phosphate sulfate (PAPS, 1.5 equivalents of substrate) and 6-O-sulfate transferase-1 (6OST-1, 0.5 mg / ml) to construct a 20 mL reaction system 14; incubate reaction system 14 overnight at 37°C for 16 h, and continue to add 3'-adenosine sulfate... Glycoside-5'-phosphate sulfate (PAPS, supplemented with 12 equivalents) and 3-O-sulfate transferase-1 (3OST-1, concentration after supplementation 0.2 mg / ml) were added and reacted overnight at 37°C for 12 h in a water bath, followed by overnight at 25°C for 12 h. The reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 99%, the reaction was terminated, and the product was purified using a Q-Sepharose strong anion exchange column to obtain heparin anticoagulant dodecanedioic acid 12G-1.

[0156] The specific reaction process is as follows:

[0157]

[0158] 6) Dissolve the heparin dodecanose modified with iduronic acid-2-O-sulfate obtained in step 4) (50 mg, as substrate) in MES buffer, then add 3'-adenosine-5'-phosphate sulfate (PAPS, 1.5 equivalents of substrate) and 0.5 mg / ml recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) to construct a 25 mL reaction system 15; incubate reaction system 15 overnight at 37°C in a water bath. After 12 hours, 3'-adenosine-5'-phosphate sulfate (PAPS, 2 equivalents) and 3-O-sulfate transferase-1 (3OST-1, 2 equivalents) were added, and the reaction was continued overnight at 37°C in a water bath for 12 hours. The reaction progress was continuously monitored by PAMN-HPLC. When the substrate conversion exceeded 99%, the reaction was terminated, and the product was purified using a Q-Sepharose strong anion exchange column to obtain heparin anticoagulant dodecanediosaccharide 12G-2.

[0159] The specific reaction process is as follows:

[0160] .

[0161] The heparin anticoagulant dodecanose 12G-1 and heparin anticoagulant dodecanose 12G-2 prepared in this embodiment were analyzed by electrospray ionization high-resolution mass spectrometry (ESI-HRMS) and nuclear magnetic resonance hydrogen spectroscopy (NMR 1H spectroscopy). The results are as follows: Figures 2-3 and Figures 5-6 As shown.

[0162] Depend on Figures 2-3 and Figures 5-6 It can be seen that heparin anticoagulation dodecaose 12G-1 and heparin anticoagulation dodecaose 12G-2 were successfully prepared in this embodiment.

[0163] The structure of heparin anticoagulant dodecanose 12G-1 is shown in the following formula:

[0164] .

[0165] The structure of heparin anticoagulant dodecaose 12G-2 is shown in the following formula:

[0166] .

[0167] Example 4: Determination of the in vitro anticoagulant activity of heparin dodecaose

[0168] The anti-Xa activity assay of the heparin anticoagulant oligosaccharide with hypothrombocytopenia-inducing activity of this invention was performed in accordance with the Chinese Pharmacopoeia, as follows:

[0169] First, the heparin anticoagulant dodecanose 12I-2 prepared in Example 2, the heparin anticoagulant dodecanose 12G-2 prepared in Example 3, and commercially available unfractionated heparin and enoxaparin were dissolved in sterile water, respectively. These solutions were then gradually diluted to prepare a series of test solutions with concentrations of 0, 20, 40, 60, 80, 100, and 120 nM. In a 96-well microplate, 40 μl of each concentration of the test solution was added; then 40 μl of antithrombin solution was added to each well, and the reaction was carried out at 37°C for 2 min; next, 40 μl of thrombin solution was added, and the reaction was carried out at 37°C for 2 min; then 40 μl of chromogenic substrate solution was added, and the reaction was carried out at 37°C for 2 min; finally, 80 μl of 20% glacial acetic acid was added to terminate the reaction, and the absorbance at 405 nm was immediately measured using a microplate reader. Nonlinear regressions were performed with absorbance as the ordinate and the concentration of the test sample series solutions as the abscissa to calculate the IC50 of the synthetic anticoagulant pentasaccharide and the commercially available fondaparinux sodium on factor Xa inhibition. 50 Value, result as Figure 7 As shown.

[0170] Depend on Figure 7 It is known that the IC50 of the heparin anticoagulant dodecanosaccharides 12I-2 and 12G-2 synthesized in this invention is... 50 The values ​​were 28 ng / ml and 20 ng / ml, respectively, and the IC50 values ​​for commercially available ungraded heparin and enoxaparin were... 50 The values ​​were 136.56 ng / ml and 109 ng / ml, indicating that the heparin anticoagulant pentasaccharide synthesized in this invention has potent anti-factor Xa activity and higher activity than commercially available products. It can be used to prepare anticoagulant drugs and has broad application prospects.

[0171] Example 5: Heparin dodecanose FXa factor activity protamine inhibition experiment

[0172] The heparin anticoagulant dodecanose 12I-2 prepared in Example 2, the heparin anticoagulant dodecanose 12G-2 prepared in Example 3, and enoxaparin were dissolved in sterile phosphate-buffered saline (PBS) to prepare 1 μg / mL solutions of 12I-2, 12G-2, and enoxaparin. Protamine was dissolved in sterile phosphate-buffered saline (PBS) to prepare protamine solutions with gradient concentrations of 4, 8, 12, and 16 μg / mL.

[0173] Add 40 μL of enoxaparin solution, 12I-1 solution, or 12G-2 solution to the reaction vessel, followed by 20 μL of protamine sulfate solution at a gradient concentration, and incubate at 37±0.5℃ for 2 minutes. Then add 40 μL of FXa factor solution (8 μg / mL) and incubate at 37±0.5℃ for 2 minutes. Next, add 40 μL of substrate S-2765 solution (0.8 mg / mL) and incubate at 37±0.5℃ for 2 minutes. Terminate the reaction by adding 80 μL of 20% acetic acid solution, and immediately measure the absorbance at 405 nm using a microplate reader. Using the absorbance of a blank sample without heparin and protamine sulfate as the baseline value for 100% FXa factor activity, calculate the in vitro neutralizing efficacy of different concentrations of protamine sulfate against each heparin-based reagent. The results are as follows: Figure 8 As shown.

[0174] Depend on Figure 8 The results show that heparin anticoagulant dodecaose 12I-2 and 12G-2 exhibit a neutralization efficiency of up to approximately 80% against the therapeutic neutralizer protamine. This clearly demonstrates that when patients receiving heparin anticoagulant dodecaose 12I-2 or 12G-2 experience emergencies requiring rapid reversal of the anticoagulant effect, standard doses of protamine can efficiently and reliably neutralize its anticoagulant activity. This figure is significantly superior to the reference drug enoxaparin (approximately 60%). This provides a stronger guarantee for safe clinical use and greatly reduces the risk of uncontrollable bleeding due to difficulties in neutralization.

[0175] Example 6: ITC Experiment Using Heparin / PF4 Isothermal Titration Microcalorimetry

[0176] Unfractionated heparin (UFH), enoxaparin, fondaparinux, the heparin anticoagulant dodecanose 12G-2 prepared in Example 3, and platelet factor 4 (PF4) were dissolved in Hank's balanced salt solution (HBSS) to prepare homogeneous stock solutions. All solutions were sterilized by filtration through a 0.22 μm filter and equilibrated at 25 ± 0.5 °C for 30 minutes before the experiment. Measurements were performed using a MicroCal PEAQ-ITC isothermal titration microcalorimeter (Malvern, UK) at 25 ± 0.5 °C. PF4 solution was injected into the sample cell (200 μL), while enoxaparin and 12G-2 solutions were injected into the titration syringe (40 μL). The titration program was set to 10 μL per injection with an injection interval of 200 seconds, and a continuous stirring rate of 750 rpm was maintained during the experiment. Raw data were collected and baseline corrected. After fitting, thermodynamic parameters such as dissociation constant (Kd), binding enthalpy change (ΔH), and binding stoichiometry (n) were calculated. The results are as follows: Figure 9 As shown.

[0177] Depend on Figure 9 It can be seen that the ΔH of the heparin anticoagulation dodecaose 12G-2 synthesized in this invention is significantly lower, confirming that its binding affinity is reduced. This result indicates that the binding strength of the heparin anticoagulation dodecaose 12G-2 with PF4 is weaker, and it has a lower risk.

[0178] Example 7: Enzyme-linked immunosorbent assay (ELISA) to determine the binding activity of PF4 / H complex with antibody.

[0179] The binding affinity of platelet factor 4 / heparin (PF4 / H) complex to anti-PF4 / heparin antibody (aPF4 / H Ab) was assessed using an indirect enzyme-linked immunosorbent assay (ELISA). The specific method is as follows:

[0180] The heparin anticoagulant dodecanosaccharide 12G-2 (H) and platelet factor 4 (PF4) prepared in Example 3 were added to Hank's balanced salt solution (HBSS) and premixed to obtain a PF4 / H complex solution with a concentration of 20 μg / mL. The premixed PF4 / H complex solution (100 μL / well) was coated onto a 96-well plate and incubated overnight (16 hours) at 4°C. The coating solution was discarded, and the plate was washed three times (5 minutes each) with PBS solution containing 0.05% Tween-20. Then, the plate was blocked with PBST blocking buffer (200 μL / well) containing 5% skim milk powder at 37±0.5°C for 1 hour. After blocking and washing again (PBST, 3 times), biotinylated anti-PF4 monoclonal antibody (KKO antibody, Thermo Fisher, 0.2 μg / mL, 100 μL / well) was added, and the plate was incubated at 37±0.5°C for 0.5 hours. After washing, horseradish peroxidase (HRP)-labeled streptavidin (Thermo Fisher Scientific, 1 μg / mL, 100 μL / well) was added and incubated at 37 ± 0.5 °C for 0.5 h. After five washes, 100 μL of tetramethylbenzidine (TMB) substrate was added to each well and incubated at 37 ± 0.5 °C in the dark for 15 min. Finally, the absorbance was measured at 450 nm using a microplate reader, with unfractionated heparin (UFH) and enoxaparin as controls. The results are as follows. Figure 10 As shown.

[0181] Depend on Figure 10 It is evident that the heparin anticoagulant dodecanosaccharide 12G-2 synthesized in this invention exhibits a significantly reduced EIA signal compared to enoxaparin, and the molecule causes minimal perturbation to the PF4 protein structure, confirming that 12G-2 is an ideal candidate molecule for antithrombotic therapy with both safety and efficacy.

[0182] Example 8: Mouse model of heparin-induced thrombocytopenia (HIT) induced by active immunization

[0183] A mouse active immunization model was established: Eight SPF (specific pathogen-free) mice were used in each group. During the immunization induction phase (days 1 to 5), mice in each group were administered the drug via tail vein injection at fixed time points daily to obtain the mouse active immunization model. The immunizing agents included platelet factor 4 (PF4, 1 mg / kg / mouse) and 0.1 mg / kg of unfractionated heparin, enoxaparin, heparin anticoagulant dodecanediosaccharide 12G-2 prepared in Example 3, or fondaparinux sodium.

[0184] Mice were maintained under standard feeding conditions until day 15 post-immunization. Peripheral blood was collected from mice into EDTA-K2 anticoagulant centrifuge tubes (anticoagulant:blood volume ratio 1:9), centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant plasma was collected and stored at -80°C for later use. The levels of relevant HIT antibodies in the plasma were measured using a heparin-induced thrombocytopenia (HIT) antibody detection kit. Figure 11 As shown.

[0185] Depend on Figure 11 It was found that the HIT antibody level induced by heparin anticoagulation dodecaose 12G-2 (0.11±0.04 ng / ml) was significantly lower than that induced by unfractionated heparin (0.83±0.40 ng / ml) or enoxaparin (0.58±0.34 ng / ml). This indicates that the heparin anticoagulation dodecaose 12G-2 synthesized in this invention has lower immunogenicity compared to UFH and enoxaparin, and is comparable to fondaparinux sodium, suggesting its potential clinical value in reducing the risk of HIT.

Claims

1. A heparin anticoagulant oligosaccharide with low thrombocytopenia-inducing activity and its pharmaceutically acceptable salt, characterized in that, The chemical structure is shown in formula (1): Equation (1) Wherein, R1 is a phenyl or substituted phenyl, aromatic heterocyclic or substituted aromatic heterocyclic, or hydrogen (-H), or alkyl and alkylamine derivative groups with characteristic ultraviolet absorption; R2 is hydrogen (-H) or -SO3. - .

2. The heparin anticoagulant oligosaccharide as described in claim 1, characterized in that, The heparin anticoagulation oligosaccharide contains an AT binding sequence and a -GlcNS(6S)-GlcA2S- or -GlcNS(6S)-IdoA2S- linking sequence, specifically heparin anticoagulation dodecanose 12I-2, heparin anticoagulation dodecanose 12G-1 or heparin anticoagulation dodecanose 12G-2; The chemical structure of heparin anticoagulant dodecanose 12I-2 is shown in formula (2) below: Equation (2) The chemical structure of heparin anticoagulant dodecanose 12G-1 is shown in formula (3) below: Equation (3) The chemical structure of heparin anticoagulant dodecanose 12G-2 is shown in the following formula (4): Equation (4).

3. The method for preparing heparin anticoagulant dodecanose 12I-2 as described in claim 2, characterized in that, The steps include the following: (1) First, heparin nonaglycone backbone, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) were added to Tris-HCl buffer to construct reaction system 1; reaction system 1 was reacted overnight at 35~40℃ in a water bath for 12~16h, and after purification, intermediate product 1 was obtained; Then, intermediate product 1, uridine diphosphate-glucuronic acid and heparin backbone synthase 2 (PmHS2) were added to Tris-HCl buffer to construct reaction system 2; reaction system 2 was reacted overnight at 35~40℃ in a water bath for 12~16h, and after purification, intermediate product 2 was obtained. Next, intermediate product 2, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) were added to Tris-HCl buffer to construct reaction system 3; reaction system 3 was reacted overnight in a water bath at 35~40℃ for 12~16h, and then the reaction solution was transferred to an equal volume of 0.1mol / L lithium hydroxide aqueous solution and reacted at 4℃ for 25~35min to obtain a solution containing intermediate product 3; Finally, the solution containing intermediate product 3 was transferred to MES buffer, and 3'-adenosine-5'-phosphate sulfate (PAPS) and N-sulfate transferase (NST) were added to construct reaction system 4. Reaction system 4 was reacted overnight at 35~40℃ in a water bath for 12~16h. After purification, N-sulfated heparin dodecanose was obtained. (2) Dissolve the N-sulfated heparin dodecanose obtained in step (1) in MES buffer, then add CaCl2 and C5-epimerase, react at 35~40℃ for 1.5~2.5h, then add 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfate transferase (2OST) to construct reaction system 5; react reaction system 5 overnight in a water bath at 25~30℃ for 12~16h, and after purification, obtain heparin dodecanose modified with iduronic acid-2-O-sulfate. (3) The heparin dodecanose modified with iduronic acid-2-O-sulfate described in step (2) was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate (PAPS) and recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) were added to construct reaction system 6. The reaction system 6 was reacted overnight at 35~40℃ in a water bath for 12~16h, and 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1) were added. The reaction was then reacted overnight at 20~30℃ in a water bath for 12~16h. After purification, heparin anticoagulant dodecanose 12I-2 was obtained.

4. The method for preparing heparin anticoagulant dodecaose 12G-1 or heparin anticoagulant dodecaose 12G-2 as described in claim 2, characterized in that, The steps include the following: 1) The heparin nonaglycone backbone was dissolved in a 0.1 mol / L lithium hydroxide aqueous solution and reacted at 4 °C for 25-35 min. The pH was adjusted to 7.0, and then the reaction solution was added to MES buffer. 3'-adenosine-5'-phosphate sulfate (PAPS) and N-sulfate transferase (NST) were added to construct reaction system 7. Reaction system 7 was reacted overnight at 35-40 °C in a water bath for 12-16 h. After purification, N-sulfated heparin nonaglycone was obtained. 2) Dissolve the N-sulfated heparin nonose obtained in step 1) in MES buffer, add 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfotransferase (2OST) to construct reaction system 8; react reaction system 8 overnight at 25~30℃ for 12~16h, and after purification, GlcA2S modified heparin nonose is obtained; 3) First, add the GlcA2S-modified heparin nonose obtained in step 2), uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) to Tris-HCl buffer to construct reaction system 9; react system 9 overnight at 35~40℃ for 12~16h, and after purification, obtain intermediate product 4; Then, intermediate product 4, uridine diphosphate-glucuronic acid and heparin backbone synthase 2 (PmHS2) were added to Tris-HCl buffer to construct reaction system 10; reaction system 10 was reacted overnight at 35~40℃ in a water bath for 12~16h, and after purification, intermediate product 5 was obtained. Next, intermediate product 5, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase (NaKfiA) were added to Tris-HCl buffer to construct reaction system 11; reaction system 11 was reacted overnight in a water bath at 35~40℃ for 12~16h, and then the reaction solution was transferred to an equal volume of 0.1mol / L lithium hydroxide aqueous solution and reacted at 4℃ for 25~35min to obtain a solution containing intermediate product 6; Finally, the solution containing intermediate product 6 was transferred to MES buffer, and 3'-adenosine-5'-phosphate sulfate (PAPS) and N-sulfate transferase (NST) were added to construct reaction system 12. Reaction system 12 was reacted overnight at 35~40℃ in a water bath for 12~16h. After purification, N-sulfated heparin dodecanose was obtained. 4) Dissolve the N-sulfated heparin dodecanose obtained in step 3) in MES buffer, add CaCl2 and C5-epimerase, react at 35~40℃ for 1.5~2.5h, then add 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfate transferase (2OST) to construct reaction system 13; react reaction system 13 overnight in a water bath at 25~30℃ for 12~16h, and after purification, obtain heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S); 5) The heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) obtained in step 4) was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate (PAPS) and 6-O-sulfate transferase-1 (6OST-1) were added to construct reaction system 14. Reaction system 14 was reacted overnight at 35~40℃ in a water bath for 12~16h, and 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1) were added. The reaction was continued overnight at 25~30℃ in a water bath for 12~16h. After purification, heparin anticoagulant dodecanose 12G-1 was obtained. 6) The heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) obtained in step 4) was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate (PAPS) and recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) were added to construct reaction system 15. Reaction system 15 was incubated overnight at 35~40℃ for 12~16h, and 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1) were added. The reaction was continued overnight at 20~30℃ for 12~16h. After purification, heparin anticoagulant dodecanose 12G-2 was obtained.

5. The preparation method according to claim 3 or claim 4, characterized in that, The heparin nonaglycone skeleton is prepared according to the following method: S1: Place p-nitrophenyl-β-D-glucuronic acid, uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase in Tris-HCl buffer, with a total reaction volume of 500 mL; react the reaction solution overnight at 37 °C for 12-16 h, adjust the pH to 3, and then purify by C18 column chromatography to obtain the heparin disaccharide backbone; S2: Take the heparin disaccharide backbone, uridine diphosphate-glucuronic acid and heparin backbone synthase 2 obtained in step S1 and place them in Tris-HCl buffer. The total volume of the reaction solution is 500 mL. The reaction solution is reacted overnight at 37°C. The pH of the reaction solution is adjusted to 3 with trifluoroacetic acid. After purification by C18 column chromatography, the heparin trisaccharide backbone is obtained. S3: Repeat the operations described in steps S1 to S2 three times consecutively to obtain the heparin nine-saccharide skeleton; In the repeated process, the oligosaccharide products obtained in the previous step are used as glycosyl acceptor substrates for the new round of reaction, and step S1 is performed using uridine diphosphate-N-trifluoroacetylglucosamine and N-acetylglucosyltransferase, respectively, followed by step S2 using uridine diphosphate-glucuronic acid and heparin backbone synthase 2.

6. The preparation method according to claim 3, characterized in that, In step (1), in the reaction system 1, the concentration of heparin nonaglycone backbone is 1~5mM, the molar ratio of heparin nonaglycone backbone to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.2, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml; In the reaction system 2, the concentration of intermediate product 1 is 1~5mM, the molar ratio of intermediate product 1 to uridine diphosphate-glucuronic acid (UDP-GlcA) is 1:1.2, and the concentration of heparin backbone synthase 2 (PmHS2) is 0.1~0.3mg / ml. In the reaction system 3, the concentration of intermediate product 2 is 1~5mM, the molar ratio of intermediate product 2 to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.5, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml; In the reaction system 4, the concentration of intermediate product 3 is 1~5mM, the molar ratio of intermediate product 2 to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of N-sulfate transferase (NST) is 0.1~0.3mg / ml.

7. The preparation method according to claim 3, characterized in that, In step (2), in the reaction system 5, the concentration of N-sulfated heparin dodecanose is 1~5mM, the concentration of CaCl2 is 1~10mM, the concentration of C5-epimerase is 0.1~0.3mg / ml; the molar ratio of N-sulfated heparin dodecanose to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of 2-O-sulfotransferase (2OST) is 0.4~0.6mg / mL; In step (3), in the reaction system 6, the concentration of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) is 1~5mM, the molar ratio of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:3, and the concentration of recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) is 0.4~0.6mg / ml; After adding 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1), the concentration of 3'-adenosine-5'-phosphate sulfate (PAPS) in reaction system 6 was 1~8 mM, and the concentration of 3-O-sulfate transferase-1 (3OST-1) was 0.1~0.6 mg / ml.

8. The preparation method according to claim 4, characterized in that, In step 1), in the reaction system 7, the concentration of heparin nonaglycone backbone is 1~5mM, the molar ratio of heparin nonaglycone backbone to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of N-sulfate transferase (NST) is 0.1~0.3mg / ml; In step 2), in the reaction system 8, the concentration of N-sulfated heparin nonose is 1~5mM, the molar ratio of N-sulfated heparin nonose to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:9, and the concentration of 2-O-sulfotransferase (2OST) is 0.1~0.3mg / ml; In step 3), in the reaction system 9, the concentration of GlcA2S-modified heparin nonaose is 1~5mM, the molar ratio of GlcA2S-modified heparin nonaose to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.2, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml; In the reaction system 10, the concentration of intermediate product 4 is 1~5mM, the molar ratio of intermediate product 4 to uridine diphosphate-glucuronic acid (UDP-GlcA) is 1:1.2, and the concentration of heparin backbone synthase 2 (PmHS2) is 0.1~0.3mg / ml. In the reaction system 11, the concentration of intermediate product 5 is 1~5mM, the molar ratio of intermediate product 5 to uridine diphosphate-N-trifluoroacetylglucosamine (UDP-GlcNTFA) is 1:1.5, and the concentration of N-acetylglucosyltransferase (NaKfiA) is 0.1~0.3mg / ml. In the reaction system 12, the concentration of intermediate product 6 is 1~5mM, the molar ratio of intermediate product 6 to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of N-sulfate transferase (NST) is 0.1~0.3mg / ml.

9. The preparation method according to claim 4, characterized in that, In step 4), in the reaction system 13, the concentration of N-sulfated heparin dodecanose is 1~5mM, the concentration of CaCl2 is 1~10mM, the concentration of C5-epimerase is 0.1~0.3mg / ml; the molar ratio of N-sulfated heparin dodecanose to 3'-phosphoadenosine-5'-phosphate sulfate (PAPS) is 1:12, and the concentration of 2-O-sulfotransferase (2OST) is 0.4~0.6mg / mL; In step 5), in the reaction system 14, the concentration of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) is 1~5mM, the molar ratio of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:3, and the concentration of 6-O-sulfotransferase-1 (6OST-1) is 0.4~0.6mg / ml; After adding 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1), the concentration of 3'-adenosine-5'-phosphate sulfate (PAPS) in reaction system 14 was 1~8 mM, and the concentration of 3-O-sulfate transferase-1 (3OST-1) was 0.1~0.6 mg / ml. In step 6), in the reaction system 15, the concentration of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) is 1~5mM, the molar ratio of heparin dodecanose modified with iduronic acid-2-O-sulfate (IdoA2S) to 3'-adenosine-5'-phosphate sulfate (PAPS) is 1:3, and the concentration of recombinant mutant enzyme Dr-6OST-3 (R112E / R206E / R329E) is 0.4~0.6mg / ml; After adding 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfate transferase-1 (3OST-1), the concentration of 3'-adenosine-5'-phosphate sulfate (PAPS) in reaction system 14 was 1~8 mM, and the concentration of 3-O-sulfate transferase-1 (3OST-1) was 0.1~0.6 mg / ml.

10. The application of the heparin anticoagulant oligosaccharide with low thrombocytopenia induction activity as described in claim 1 in the preparation of safe and effective anticoagulant drugs and antithrombotic drugs.

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