A fucoidan-based heparin pentasaccharide, its preparation method, and its application

Abstract

This invention belongs to the fields of medicinal chemistry, glycochemistry, and biomedicine, and provides a fucoidan-based heparin pentasaccharide, its preparation method, and its applications. The preparation method involves the chemical linkage and purification of sulfated azidoethyl-α-L-pyranoside with alkyne-based heparin pentasaccharide to obtain the target product. This compound exerts anticoagulant and antithrombotic effects by potently inhibiting the intrinsic coagulation factor X enzyme complex (FXase). Its APTT prolongation and FXase inhibitory activity are significantly superior to heparin pentasaccharide, and it significantly reduces the risk of bleeding at equivalent antithrombotic doses. It can be used in the development of drugs or functional foods for thrombotic cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of medicinal chemistry and glycochemistry, specifically relating to a class of heparin pentasaccharide compounds modified with sulfated fucose, their scalable preparation process, and their application in the preparation of drugs or functional foods for the prevention and treatment of thrombotic diseases. The thrombotic diseases include venous thromboembolism, disseminated intravascular coagulation, and cardiovascular and cerebrovascular embolism; furthermore, this compound can also be used to improve microcirculatory disorders. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are the leading cause of death threatening human health, and their morbidity and mortality rates continue to rise. Thrombotic diseases (including coronary heart disease, stroke, pulmonary embolism, and deep vein thrombosis) constitute the main pathological manifestations of these diseases. Anticoagulants are currently the cornerstone of the prevention and treatment of thrombotic diseases, with significant clinical application value and a large market size. However, existing clinical anticoagulants generally suffer from a major drawback: significant bleeding adverse reactions. Taking the widely used enoxaparin sodium as an example, its clinical bleeding adverse reaction rate exceeds 10%, severely limiting the treatment window and patient compliance. Therefore, developing innovative drugs that combine potent anticoagulant activity with low bleeding tendency has become a key scientific problem urgently needing a breakthrough in the prevention and treatment of thrombotic cardiovascular diseases.

[0003] The patent applicant, as a core member, isolated and prepared LFG-53 from sea cucumber. This is a novel anticoagulant candidate drug that can potently and selectively inhibit endogenous coagulation factor X enzyme complex (FXase). Preclinical studies have shown that its bleeding tendency is significantly lower than that of enoxaparin. This drug received clinical trial approvals from the US FDA in 2021 and the China National Medical Products Administration in 2022, respectively, and has entered the development stage.

[0004] FXase is the rate-limiting enzyme in the intrinsic coagulation pathway. Selective inhibition of this target can ensure antithrombotic efficacy while avoiding interference with other coagulation processes, thus mitigating the bleeding risk associated with traditional anticoagulants. The applicant's previous study published in PNAS (Zhao et al., Proc Natl Acad Sci USA, 2015, 112 (27): 8284–8289) confirmed that the smallest active fragment of sea cucumber fucosylated glycosaminoglycan (FG) is a nonaglycone structure, and its anticoagulant and antithrombotic activity and low bleeding characteristics are comparable to LFG-53. Further structure-activity relationship studies (Xiao et al., Thromb Haemost, 2019, 119 (5):705–715) revealed that the sulfated fucose (FucS) side chain in this nonaglycone molecule is an essential pharmacophore for its specific recognition and binding of coagulation factor IXa (FIXa), thereby inhibiting the activity of the FXase complex.

[0005] However, the synthesis of this nine-saccharide structure is extremely difficult: the overall yield of its chemical synthesis route is only 1-3%, the process is complex and costly, making it difficult to achieve large-scale preparation, which seriously restricts its drug development and industrialization prospects.

[0006] In view of this, this invention proposes an innovative structural simplification strategy: retaining the core pharmacophore of FXase inhibitory activity—the sulfated fucose side chain—while using the more mature and accessible heparin pentose as the main chain backbone to construct a nonasugar analog. This strategy aims to retain or optimize the selective inhibitory activity and low bleeding advantage of the original nonasugar against FXase, while significantly reducing preparation costs and improving process feasibility through modular synthesis, providing a technical solution for developing next-generation anticoagulant drugs with low bleeding tendency. Summary of the Invention

[0007] The purpose of this invention is to provide a fucoidan pentasaccharide, its preparation method, and its application. Fucoidan pentasaccharide exhibits potent anticoagulant activity and can effectively inhibit endogenous coagulation factor X enzyme (FXase). It can be used to prepare drugs or functional foods for the treatment of thrombotic cardiovascular diseases.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a fucoidan-based heparin pentasaccharide, which is a homologous oligosaccharide compound having formula (I).

[0010] (I)

[0011] In formula (I), A is 2-deoxy-6-O-sulfonyl-2-(sulfonamido)-α-D-glucose; B is β-D-glucuronic acid; C is 2-deoxy-3,6-di-O-sulfonyl-2-(sulfonamido)-α-D-glucose; D is 2-O-sulfonyl-α-L-iduronic acid; E is methyl-O-2-deoxy-6-O-sulfonyl-2-(sulfonamido)-α-D-glucose; F is α-L-fucose; and R are independently -SO3. － Or -H; R1 is -SO3 － .

[0012] Furthermore, the weight-average molecular weight of the fucoidan pentasaccharide is 2.0~2.9kDa, and the polydispersity index is between 1.0 and 1.6; the sulfate content is 20%~50%.

[0013] Furthermore, the monosaccharide composition includes D-glucosamine, D-glucuronic acid, L-iduronic acid and fucose, wherein the molar ratio of D-glucosamine, D-glucuronic acid, L-iduronic acid and fucose is 3:(1±0.5):(1±0.5):(2±0.5).

[0014] A second aspect of the present invention provides a method for preparing the aforementioned fucoidan-heparin pentasaccharide, comprising the following steps:

[0015] S1. L-fucose and 2-azidoethanol were mixed and added to an acidic ion exchange resin. The mixture was reacted at 60-100°C for 1-8 hours. The solid-liquid mixture was separated, concentrated, purified by silica gel column chromatography, and lyophilized to obtain 1-azidoethyl α-L-pyran-fucoside. 1-azidoethyl α-L-pyran-fucoside was dissolved in N,N-dimethylformamide and a sulfur trioxide-pyridine complex was added. The mixture was reacted at 40-80°C for 2-12 hours under inert gas protection. After the reaction was completed, the mixture was cooled, neutralized to neutral by adding saturated sodium bicarbonate solution, stirred, centrifuged, concentrated, and dried to obtain 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside.

[0016] S2. Dissolve hydrogen- or sodium-form heparin pentose in MES buffer to prepare a heparin pentose solution, add 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, and react at 15-70°C for 10-120 min; then add propargylamine MES solution and react at 30-70°C for 12-48 h; after salting out, alcohol precipitation, centrifugation, desalting, and lyophilization, obtain the alkydized heparin pentose.

[0017] S3. Dissolve the alkydized heparin pentose in PBS buffer to prepare a substrate solution; prepare a catalyst solution by mixing tris(3-hydroxypropyltriazolylmethyl)amine solution and CuSO4·5H2O solution; add the catalyst solution dropwise to the substrate solution, replace the inert gas in the reaction vessel, add the reducing agent, stir for 20-40 min to obtain the reaction solution; then add 1-azidoethyl-2,3,4-tri-O-sulfonyl-α-L-pyran-fucoside to the reaction solution, and add PBS buffer to make the final concentration of alkydized heparin pentose 5-20 mg / mL, stir the reaction at 55-65℃ for 12-36 h; after the reaction is completed, salt out, precipitate with alcohol, separate the solid and liquid, wash the solid, desalt and freeze dry to obtain the fucoidolated heparin pentose.

[0018] Furthermore, in step S1, the molar ratio of L-fucose to 2-azidoethanol is 1:10-1:20;

[0019] And / or, in step S1, the amount of acidic ion exchange resin used is 0.5-5.0 times the weight of L-fucose;

[0020] And / or, in step S1, the molar ratio of 1-azidoethyl α-L-pyran fucoside to the sulfur trioxide-pyridine complex is 1:2-1:20.

[0021] Furthermore, in step S2, hydrogen- or sodium-form heparin pentasaccharide is dissolved in 0.05-0.2M MES buffer (pH 4-8) to prepare a solution with a concentration of 5-50 mg / mL;

[0022] In step S2, the molar ratio of hydrogen or sodium heparin pentasaccharide to 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:5-1:60;

[0023] And / or, in step S2, the concentration of propargylamine in the MES solution (pH 4-8) is 0.1-0.5 M, and the molar ratio of hydrogen or sodium heparin pentose to propargylamine is 1:5-1:80.

[0024] Furthermore, in step S3, the concentration of the tris(3-hydroxypropyltriazolylmethyl)amine solution is 100-300 mg / mL, and the dosage is calculated based on 5-20 μL / mg of alkynylated heparin pentasaccharide.

[0025] And / or, in step S3, the concentration of CuSO4·5H2O solution is 10-40 mg / mL, and the dosage is calculated according to 1-10 μL / mg of alkynylated heparin pentose.

[0026] And / or, in step S3, the reducing agent is Na-ascorbic acid solution with a concentration of 30-200 mg / mL, and the amount used is calculated according to 1-10 μL / mg of alkynylated heparin pentasaccharide;

[0027] And / or, in step S3, the amount of 1-azidoethyl-2,3,4-tri-O-sulfonyl-α-L-pyranfucoside is calculated based on alkynylated heparin pentose and is 2.0-6.0 equivalents.

[0028] Furthermore, in step S1, 1-azidoethyl α-L-pyranofucoside is dissolved in N,N-dimethylformamide to prepare a 0.1-0.5M solution.

[0029] In step S1 described above, the present invention provides a more preferred preparation process:

[0030] S1.1 L-fucose, 2-azidoethanol and acidic ion exchange resin are mixed in a reaction vessel, wherein the molar ratio of L-fucose to 2-azidoethanol is 1:10-1:20, and the amount of acidic ion exchange resin is 0.5-5.0 times the weight of L-fucose. The mixture is stirred at 60-100°C for 1-8 hours.

[0031] S1.2 The reaction process is monitored by thin-layer chromatography, and the reaction is terminated when the starting material spot disappears and the product spot no longer increases.

[0032] Immediately after the S1.3 reaction is complete, filter while hot and wash the resin 2-4 times with warm monohydric alcohol and / or dihydric alcohol and / or trihydric alcohol, then combine the filtrates.

[0033] S1.4 The filtrate was concentrated by rotary evaporation under reduced pressure at 50-90°C, and then treated under high vacuum for 4 hours to remove excess 2-azidoethanol. After freeze-drying, a pale yellow syrupy crude product of 1-azidoethyl α-L-pyranfucoside was obtained. The product was purified by silica gel column chromatography, and the target fraction was collected to obtain the purified product 1-azidoethyl α-L-pyranfucoside.

[0034] S1.5 Dissolve 1-azidoethyl α-L-pyranofucrose in N,N-dimethylformamide to prepare a 0.1-0.5M solution, add sulfur trioxide-pyridine complex at a molar ratio of 1:2-1:20, and react at 40-80°C for 2-12 h under inert gas protection;

[0035] After the reaction in step S1.6 is completed, cool the system to -10°C to 10°C, add saturated sodium bicarbonate solution to neutralize to pH neutral to terminate the reaction, and continue stirring for 0.2-1h.

[0036] S1.7 Centrifuge the reaction solution stirred in step S1.6 (3000-15000 rpm, 1-10 min), concentrate under reduced pressure to dryness, reconstitute with methanol, centrifuge again (3000-15000 rpm, 1-10 min), and finally concentrate and dry under reduced pressure to obtain 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside, with a yield of not less than 80%.

[0037] More preferably, in step S1.1, the molar ratio of L-fucose to 2-azidoethanol is 1:12-1:16, the reaction temperature is 75-85°C, and the reaction time is 1.5-3h.

[0038] More preferably, in step S1.1, the acidic ion exchange resin is Dowex 50-X8 resin (H⁺ type).

[0039] More preferably, in step S1.3, the monohydric alcohol and / or dihydric alcohol and / or trihydric alcohol is methanol.

[0040] More preferably, in step S1.3, each wash uses 5-15 mL of monohydric alcohol and / or dihydric alcohol and / or trihydric alcohol.

[0041] More preferably, in step S1.4, the mass ratio of silica gel to crude 1-azidoethyl α-L-pyranfucoside is 10:1-30:1.

[0042] More preferably, in step S1.4, the high vacuum treatment time is 4-8 hours.

[0043] More preferably, in step S1.5, the molar ratio of 1-azidoethyl α-L-pyran fucoside to the sulfur trioxide-pyridine complex is 1:8-1:12, the reaction temperature is 45-55°C, and the reaction time is 4-8h.

[0044] More preferably, in step S1.5, the inert gas is nitrogen.

[0045] More preferably, in step S1.6, the stirring time is 0.3-0.7 h.

[0046] Preferably, in step S1.7, the first centrifugation speed is 4000-5000 rpm and the time is 3-6 min; the second centrifugation speed is 10000-13000 rpm and the time is 2-4 min.

[0047] In step S2 described above, the present invention provides a more preferred preparation process:

[0048] S2.1 Dissolve heparin pentose in water to prepare a solution of 10-30 mg / mL, treat with a strong cation exchange resin, collect the eluent and freeze-dry to obtain hydrogen heparin pentose, with an ion exchange yield of not less than 90%;

[0049] S2.2 Prepare a 10-20 mg / mL solution of hydrogen or sodium heparin pentose in 0.05-0.2 M MES buffer (pH 4-8), add 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), with a molar ratio of hydrogen or sodium heparin pentose to 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride of 1:5-1:60, and stir the reaction at 15-70°C for 10-120 min;

[0050] S2.3 Add a MES solution (pH 4-8) of propargylamine to the reaction solution of step S2.2 at a molar ratio of hydrogen or sodium heparin pentose to propargylamine of 1:5-1:80, and continue the reaction at 30-70°C for 12-48 hours.

[0051] S2.4 After the reaction in step S2.3 is completed, cool to room temperature, add NaCl to a final concentration of 10-20% (w / v) and stir for 5-30 min. After precipitation with 70-90% ethanol, collect the precipitate by centrifugation at 3000-6000 rpm.

[0052] The S2.5 precipitate was reconstituted with deionized water, desalted by dialysis or gel column chromatography, and lyophilized to obtain a white alkydized heparin pentasaccharide powder with a total yield ≥80%.

[0053] It should be noted that the heparin pentasaccharide generally sold on the market is sodium heparin pentasaccharide.

[0054] More preferably, in step S2.1, the strong cation exchange resin used for conversion to hydrogen heparin pentasaccharide is Dowex 50W-X8 ion exchange resin (100-200 mesh, H⁺ type) or Amberlite® 732 strong acid cation exchange resin (100-200 mesh, H⁺ type) or Amberlite® IR-120 (100-200 mesh, H⁺ type).

[0055] More preferably, in step S2.2, the molar ratio of hydrogen- or sodium-form heparin pentasaccharide to DMTMM is 1:10-1:50, the reaction temperature is 20-25°C, and the reaction time is 20-40 min.

[0056] More preferably, in step S2.3, the molar ratio of hydrogen- or sodium-form heparin pentasaccharide to propargylamine is 1:10-1:70, the reaction temperature is 20-50°C, and the reaction time is 10-30h.

[0057] More preferably, in step S2.4, the final concentration of NaCl is 12-18% (w / v), and the stirring time is 10-20 min.

[0058] Preferably, in step S2.4, the centrifugation speed is 3000-5000 rpm.

[0059] In step S3 described above, the present invention provides a more preferred preparation process:

[0060] The coupling of 1-azidoethyl-2,3,4-tris-O-sulfonyl-α-L-pyranofucoside to alkynylated heparin pentasaccharide via an azido-alkynylation cycloaddition reaction specifically includes the following steps:

[0061] S3.1 Prepare an alkydated heparin pentose solution with a final concentration of 5-50 mg / mL using phosphate buffer (pH 7.2-7.6), gently vortex and sonicate until completely dissolved to obtain the substrate solution;

[0062] S3.2 Catalyst Premixing: In a separate container, a solution of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine (THPTA) and a solution of copper sulfate pentahydrate (CuSO4·5H2O) are premixed to form a catalyst solution. The concentration of the THPTA solution is 100-300 mg / mL, and the amount used is 5-20 μL per milligram of alkynylated heparin pentose. The concentration of the CuSO4·5H2O solution is 10-40 mg / mL, and the amount used is 1-10 μL per milligram of alkynylated heparin pentose. After mixing, the mixture is vortexed for 5-15 seconds and allowed to stand for 1-3 minutes.

[0063] S3.3 The catalyst solution obtained in step S3.2 is added dropwise to the substrate solution at a rate of 10-20 s / mL. After purging with an inert gas for 3-10 min, a reducing agent with a concentration of 30-200 mg / mL is added, with the amount being 1-10 μL relative to each milligram of alkynylated heparin pentose. The mixture is stirred at 150-800 rpm for 20-40 min at 20-30 °C to obtain the reaction solution.

[0064] S3.4 Add 2.0-6.0 equivalents of 1-azidoethyl-2,3,4-tri-O-sulfonyl-α-L-pyranfucoside. 1-azidoethyl-2,3,4-tri-O-sulfonyl-α-L-pyranfucoside is pre-dissolved in PBS buffer and slowly added dropwise to the reaction solution. Add PBS buffer to bring the final concentration of the acetylated heparin pentose to 5-20 mg / mL.

[0065] S3.5 Transfer the reaction system from step S3.4 to an oil bath and heat it to 55-65°C, stirring at 150-800 rpm for 12-36 hours;

[0066] After the S3.6 reaction is completed, cool to room temperature, add NaCl to a final concentration of 10-20% w / v, stir at 250-350 rpm for 20-40 min, transfer the reaction solution to a rotary evaporator and place it in an ice bath at 0-5℃ for 3-8 min, slowly add 2-5 times the volume of pre-cooled ethanol at 0℃ to -25℃ while vortexing, and let stand for 5-15 min to produce a white flocculent precipitate;

[0067] S3.7 Centrifuge the precipitate at 3500-4500 rpm for 4-8 min, discard the supernatant, wash the precipitate with 70-80% ethanol, vortex resuspend, and centrifuge again for 8-12 min. Repeat the washing 2-4 times.

[0068] S3.8 The precipitate washed in step S3.7 is re-dissolved in deionized water, desalted by dialysis or gel column chromatography, and then freeze-dried to obtain white fucoidan pentasaccharide powder with a total yield of not less than 85%.

[0069] More preferably, in step S3.2, the concentration of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine is 150-250 mg / mL, and the concentration of CuSO4·5H2O solution is 20-30 mg / mL.

[0070] More preferably, in step S3.3, the reducing agent is sodium ascorbate.

[0071] More preferably, in step S3.3, the inert gas is nitrogen, the stirring speed is 250-350 rpm, and the reaction time is 25-35 min.

[0072] More preferably, in step S3.5, the oil bath temperature is 55-65°C, the stirring speed is 150-250 rpm, and the reaction time is 18-30 h.

[0073] More preferably, in step S3.6, the final concentration of sodium chloride is 15-17% (w / v), and the stirring time is 25-35 min.

[0074] More preferably, in step S3.6, the ice bath time is 4-6 min and the pre-cooled ethanol temperature is -18°C to -22°C.

[0075] The third aspect of this invention provides the use of the above-described fucoidan-heparin pentasaccharide in the preparation of drugs or functional foods for the prevention and treatment of thrombotic diseases or for improving microcirculation.

[0076] The thrombotic cardiovascular diseases mentioned include, but are not limited to: thrombotic cardiovascular and cerebrovascular diseases, venous thromboembolism (including pulmonary venous thrombosis and peripheral venous thrombosis), peripheral arterial thrombosis, disseminated intravascular coagulation, and cardiovascular and cerebrovascular embolism.

[0077] The fucoidan heparin pentasaccharide described above can be formulated into pharmaceutically acceptable dosage forms, including but not limited to powders, granules, tablets, capsules, aerosols, dispersible tablets, pills, soft capsules, sustained-release preparations, oral liquid preparations, and injections.

[0078] Preferably, the dosage form is an injection, including lyophilized powder injection or aqueous injection.

[0079] Thrombotic cardiovascular diseases are a major cause of death and harm to human health. While existing antithrombotic drugs possess thrombolytic, anticoagulant, or antiplatelet effects, they generally pose safety risks, such as high bleeding rates. The fucoidan pentasaccharide described in this invention, as a selective inhibitor of FXase, significantly reduces bleeding tendency while maintaining significant anticoagulant activity, exhibiting high efficacy and low toxicity, thus providing an ideal candidate for the development of novel antithrombotic drugs.

[0080] The present invention has the following advantages:

[0081] (1) Novel structure: The fucoidan heparin pentose of the present invention is formed by coupling highly sulfated fucose and heparin pentose, and has both a clear sequence structure and a high degree of sulfatedity, which is a novel oligosaccharide compound that has not been reported.

[0082] (2) Strong anticoagulant efficacy and excellent safety: In vitro experiments have confirmed that the fucoidan pentasaccharide of the present invention can prolong APTT in a dose-dependent manner and selectively inhibit endogenous FXase, significantly reducing bleeding side effects, and has the dual advantages of high-efficiency anticoagulation and low bleeding risk. Attached Figure Description

[0083] Figure 1 The high-performance liquid chromatograms of 1-azidoethyl-2,3,4-tri-O-sulfonyl-α-L-pyranofucoside (AFS), alkynylated heparin pentose (AHP), and fucosylated heparin pentose (FHP) having formula (I) are shown in the embodiments of the present invention.

[0084] Figure 2 It is a 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyranfucoside 1 H NMR spectrum.

[0085] Figure 3 For the alkynylated heparin pentasaccharide in the embodiments of the present invention 1 H NMR spectrum.

[0086] Figure 4 This is a chromatogram of the monosaccharide composition of fucoidan pentasaccharide having formula (I) in an embodiment of the present invention.

[0087] Figure 5 The infrared spectrum of fucoidan pentasaccharide having formula (I) is shown in an embodiment of the present invention.

[0088] Figure 6 For the fucosylated heparin pentasaccharide of formula (I) in the embodiments of the present invention 1 H- 13 C HSQC spectrum and its classification.

[0089] Figure 7 The fucoidan pentasaccharide having formula (I) in the embodiments of the present invention 1 H- 1 H ROESY spectrum and its classification.

[0090] Figure 8 The fucoidan pentasaccharide having formula (I) in the embodiments of the present invention 1 H- 13 C HMBC spectrum and its attribution.

[0091] Figure 9 This is a graph showing the thrombosis-inhibiting activity of fucoidan-heparin pentasaccharide.

[0092] Figure 10 This is a diagram showing the bleeding tendency of fucoidan-heparin pentasaccharide. Detailed Implementation

[0093] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. The specific embodiments and drawings described herein are merely for in-depth analysis of the present invention and are not intended to limit the present invention.

[0094] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0095] Example 1: Preparation, physicochemical properties and structural analysis of fucoidan-heparin pentasaccharide

[0096] 1.1 Experimental Methods

[0097] Preparation method of 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside:

[0098] 1.1.1 In a 25 mL round-bottom flask, add 1 g (6.09 mmol) of L-fucose, 6.65 mL (87.5 mmol) of 2-azidoethanol, and Dowex 50-X8 ion exchange resin (H... + (936mg) and reacted magnetically at 80°C for 2 hours.

[0099] 1.1.2 The reaction process was monitored by thin-layer chromatography with ethyl acetate / methanol (8:3, v / v) as the developing solvent. The reaction was terminated when the starting spot disappeared and the product spot no longer increased.

[0100] 1.1.3 Immediately after the reaction was completed, hot filtration was performed using a Buchner funnel. The resin was washed three times with warm methanol (40°C, 3 × 10 mL), and the filtrates were combined.

[0101] 1.1.4 The filtrate was rotary evaporated under reduced pressure at 80°C for 6 h, purified by silica gel column chromatography, concentrated under reduced pressure, and then freeze-dried at -20°C to obtain 1-azidoethyl α-L-pyranfucoside.

[0102] 1.1.5 125 mg of 1-azidoethyl α-L-pyranofucoside (0.536 mmol) was dissolved in 1.5 mL of N,N-dimethylformamide, and 800 mg (5.03 mmol) of sulfur trioxide-pyridine complex was added. The reaction was carried out in an oil bath at 50 °C for 6 h under nitrogen protection. The reaction progress was monitored by thin-layer chromatography.

[0103] 1.1.6 After the reaction is complete, cool the system to 0°C, add 7 mL of saturated sodium bicarbonate solution to neutralize to pH neutral to terminate the reaction, and continue stirring for 0.5 h.

[0104] 1.1.7 Centrifuge the reaction solution stirred in step 1.1.6 (4500 rpm, 5 min), concentrate under reduced pressure to dryness, reconstitute with methanol, centrifuge again (12000 rpm, 3 min), and finally concentrate under reduced pressure, dry and weigh to obtain 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside.

[0105] HPGPC determination of product purity method: HPLC system: Shimadzu LC-2030C 3D; chromatographic column: Shodex OHpak SB-804 HQ (8×300mm); mobile phase: 0.1M NaCl, 0.5mL / min, 35℃; injection volume: 20µL.

[0106] NMR spectroscopy determination method: The NMR spectrum of the compound was acquired at 298 K on a Bruker AVANCE NEO 500 MHz spectrometer. (Routine...) 1 ¹H NMR: Approximately 5 mg of lyophilized sample was dissolved in 0.5 mL of MeOD and transferred to a 5 mm tube. Before the two-dimensional experiment, it was finally dissolved in 0.5 mL of MeOD containing the internal standard. The results were obtained using Bruker standard pulse sequence. 1 H– 1 H TOCSY, COSY, ROESY 1 H– 13 C HSQC and HMBC spectra; number of scans: 13 C: 1024, COSY: 4, TOCSY: 8, ROESY: 4, HSQC: 4, HMBC: 8.

[0107] 1.2 Experimental Results and Analysis

[0108] Approximately 291 mg of 1-azidoethyl-2,3,4-tris-O-sulfonyl-α-L-pyranofucoside (AFS) was prepared, with a yield of approximately 100%. HPGPC purity test results are as follows... Figure 1 As shown, the main peak is a single symmetrical peak, and the purity is greater than 90% as determined by the area normalization method. It contains a small number of impurity peaks, but these do not affect the subsequent reaction.

[0109] The 1H NMR spectrum and assignment results of this compound are shown below. Figure 2 As shown in Table 1, the data confirm that its structure is indeed 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside.

[0110] Table 1. NMR chemical shift assignments of 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside

[0111]

[0112] Note: Underlined lines represent sulfate ester linkage sites.

[0113] Example 2 Preparation of alkynylated heparin pentasaccharide

[0114] 2.1 Experimental Methods

[0115] Preparation method of alkynylated heparin pentasaccharide:

[0116] 2.1.1 Dissolve 300 mg of heparin pentasaccharide in 5 mL of water, and pass it through a Dowex 50W-X8 ion exchange resin (100-200 mesh, H...). + The solution was processed (type 1), the eluent was collected and lyophilized to obtain approximately 249 mg of hydrogen heparin pentasaccharide, with an ion exchange yield of 95%.

[0117] 2.1.2 Dissolve 200 mg (0.133 mmol) of heparin pentose in 10 mL of 0.1 M MES buffer (pH 6.5), then add 1104 mg (3.99 mmol) of DMTMM, which is equivalent to 30 moles of heparin pentose, and stir at 45 °C for 10 min.

[0118] 2.1.3 Add 426 μL (6.65 mmol) of MES solution equivalent to 50 moles of heparin pentasaccharide to the reaction solution in step 2.1.2, and heat to 45 °C and continue the reaction for 24 h.

[0119] 2.1.4 After the reaction in step 2.1.3 is completed, cool to room temperature, add NaCl to a final concentration of 15% (w / v) and stir for 10 min. After precipitation with 80% ethanol, collect the precipitate by centrifugation at 4000 rpm.

[0120] 2.1.5 The precipitate was reconstituted with 5 mL of deionized water, desalted by Sephadex G-25 gel column chromatography (1.5 × 100 cm), and lyophilized to obtain alkynylated heparin pentasaccharide.

[0121] HPGPC determination of product purity method: HPLC system: Shimadzu LC-2030C 3D; chromatographic column: Shodex OHpak SB-804 HQ (8×300mm); mobile phase: 0.1M NaCl, 0.5mL / min, 35℃; injection volume: 20µL.

[0122] NMR spectroscopy determination method: The NMR spectrum of the compound was acquired at 298 K using a Bruker AVANCE NEO 500MHz spectrometer. (Routine...) 1 ¹H NMR: Approximately 5 mg of lyophilized sample was dissolved in 0.5 mL of DMSO and transferred to a 5 mm tube. Before the two-dimensional experiment, it was finally dissolved in 0.5 mL of DMSO containing an internal standard. Acquisition was performed using Bruker standard pulse sequences. 1 H– 1 H TOCSY, COSY, ROESY 1 H– 13 C HSQC and HMBC spectra; number of scans: 13C: 1024, COSY: 4, TOCSY: 8, ROESY: 4, HSQC: 4, HMBC: 8.

[0123] 2.2 Results and Analysis

[0124] After freeze-drying, 207 mg of white alkynylated heparin pentasaccharide powder was obtained, with a yield of approximately 89%. HPGPC purity test results are as follows: Figure 1 As shown, the main peak of alkynylated heparin pentose (AHP) is a single symmetrical peak, and the purity is greater than 99% as calculated by the area normalization method.

[0125] Alkylated heparin pentasaccharide 1 The H NMR spectrum and signal attribution results are shown in the following figures. Figure 3 Spectral data confirmed that the compound's structure is an alkynylated heparin pentasaccharide, with alkynyl proton signals and anomeric hydrogen signals indicating an alkynylation degree greater than 80%.

[0126] Example 3: Preparation of Fucosyl Heparin Pentasaccharide

[0127] 3.1 Experimental Methods

[0128] Preparation method of fucoidan-heparin pentasaccharide:

[0129] 3.1.1 In a 10 mL round-bottom flask, add 30 mg of alkydized heparin pentose and 2.0 mL of PBS buffer (pH 7.38) sequentially, gently vortex and sonicate until completely dissolved to obtain the substrate solution.

[0130] 3.1.2 Catalyst premixing: Take another 1.5 mL EP tube, add 360 μL THPTA solution (200 mg / mL) and 160 μL CuSO4·5H2O solution (25 mg / mL) in sequence, vortex mix for 10 s, let stand at room temperature for 2 min to form a blue complex, and obtain the catalyst solution.

[0131] 3.1.3 Start the reaction: Slowly add the premixed catalyst solution dropwise into a round-bottom flask (about 30 seconds). Purge the air from the round-bottom flask with nitrogen for 5 minutes. Quickly add 90 μL of Na-ascorbic acid solution (100 mg / mL). The solution immediately turns light yellow. Stir at room temperature (25℃) for 30 minutes (300 rpm) to obtain the reaction solution.

[0132] 3.1.4 Dissolve 61 mg (0.113 mmol) of 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside in 300 μL of PBS, slowly add it dropwise to the reaction solution, and add PBS to a total volume of 3.0 mL to make the final concentration of alkynylated heparin pentose approximately 10 mg / mL.

[0133] 3.1.5 Heating reaction: Transfer the reaction flask to a 60°C oil bath and stir for 24 hours (200 rpm).

[0134] 3.1.6 Salting out and precipitation: After the reaction solution is cooled to room temperature, weigh 0.48 g NaCl (16% w / v) and add it directly. Continue stirring for 30 min (300 rpm) until completely dissolved. Transfer the solution to a 25 mL rotary evaporator flask, place it in an ice bath for 5 min, and slowly add 10 mL of pre-cooled ethanol (-20℃) while vortexing. Let it stand for 10 min, and a white flocculent precipitate will be visible.

[0135] 3.1.7 Centrifugation and washing: Centrifuge at 4,500 rpm for 5 min, discard the supernatant, wash the precipitate with 2 mL of 75% ethanol, vortex resuspend, and centrifuge again (10 min). Repeat the washing process twice.

[0136] 3.1.8 The precipitate was reconstituted with 2 mL of deionized water, desalted by Sephadex G-25 gel column chromatography (1.5 × 100 cm), and lyophilized to obtain fucosylated heparin pentasaccharide.

[0137] HPGPC determination of product purity method: HPLC system: Shimadzu LC-2030C 3D; chromatographic column: Shodex OHpak SB-804 HQ (8×300mm); mobile phase: 0.1M NaCl, 0.5mL / min, 35℃; injection volume: 20µL; dextran standard curve correction.

[0138] Monosaccharide composition analysis: Pre-column PMP derivatization-HPLC analysis: Agilent ZORBAX Eclipse Plus C18 (4.6×250mm, 5µm); mobile phase acetonitrile-0.1M phosphate buffer (pH 6.7) 17:83 (v / v), 1.0 mL / min, 245 nm, 30 °C.

[0139] Infrared spectroscopy analysis: Mix 1–2 mg of TPG with dry KBr, compress into tablets, and analyze on a Nicolet iS50 spectrometer at 4000–400 cm⁻¹. -1 scanning.

[0140] NMR spectroscopy determination method: NMR spectra were acquired at 298 K using a Bruker AVANCE NEO 500 / 800 MHz spectrometer. (Routine...) 1¹H NMR: Approximately 5 mg of lyophilized sample was dissolved in 0.5 mL of D₂O (99.9% D) and transferred to a 5 mm tube. Before the two-dimensional experiment, 10–20 mg of sample was thoroughly deuterated by three cycles of dissolution-lyophilization in 0.5 mL of D₂O, and finally dissolved in 0.5 mL of D₂O containing 0.05% TSP (w / v). Acquisition was performed using Bruker standard pulsed sequencing. 1 H– 1 H TOCSY, COSY, ROESY 1 H– 13 C HSQC and HMBC spectra; number of scans: 13 C: 5000, COSY: 16, TOCSY: 8, ROESY: 16, HSQC: 4, HMBC: 64.

[0141] 3.2 Experimental Results and Analysis

[0142] 3.2.1 Purity and Molecular Weight Distribution

[0143] After freeze-drying, 45 mg of white fucoidan-heparin pentasaccharide powder was obtained, with a yield of approximately 92%. HPGPC purity test results are as follows... Figure 1 As shown, the main peak of fucoidan heparin pentasaccharide (FHP) is a single symmetrical peak, and its purity is greater than 99% as calculated by the area normalization method.

[0144] 3.2.2 Monosaccharide Composition

[0145] Pre-column PMP derivatization-HPLC analysis results are as follows Figure 4 As shown, compared with the standard monosaccharide, the fucoidan pentasaccharide consists of D-glucosamine (GlcN), D-glucuronic acid (GlcA), L-iduronic acid (IdoA) and fucose (Fuc) in a molar ratio of approximately 3.4:1.0:1.2:1.7.

[0146] 3.2.3 Infrared Spectroscopy

[0147] According to the provided infrared spectrum ( Figure 5 The absorption peak analysis of fucosylated sulfaparinux sodium is as follows:

[0148] Key characteristic absorption peak assignment: 3446 cm⁻¹ -1 (Broad and strong) OH stretching vibration: originates from the hydroxyl group on the sugar ring and adsorbed water; NH stretching vibration: originates from the amino group (-NH-) of glucosamine; 1642 cm⁻¹ -1 (Medium strength) is due to C=O stretching, derived from the acetylamino group of glucosamine (-NHCOCH3), and may also include the HOH bending vibration of water of crystallization; 1235 cm⁻¹ -1 (Strong absorption) is the S=O stretching vibration of the sulfate ester group; 1046 cm⁻¹ -1(The strongest peak) is the COC stretching vibration: the CO stretching vibration of the glycosidic bond and the pyranose ring; 940 cm⁻¹ -1 This is a symmetric stretching vibration of the relatively weak COC bond, mainly originating from the ether bond vibration within the sugar ring; 888 cm⁻¹ -1 (Medium intensity) is the CH out-of-plane bending vibration of α-L-pyranofucose; 824 cm⁻¹ -1 (Medium intensity) indicates a COS stretching vibration, characteristic of the sulfate group linked to the sugar ring, further confirming the presence of a sulfate ester bond; 583 cm⁻¹ -1 The weak absorption indicates SO bending vibration: a minor vibrational mode of the sulfate group. This infrared spectrum confirms the correct structure of the target compound, fucoidanolol sodium, and its high purity (no obvious impurity peaks).

[0149] 3.2.3 NMR Spectrum Analysis

[0150] The NMR spectra and signal assignments of fucoidan-heparin pentasaccharide are shown below. Figure 6-8 As shown in Table 2, the spectral data confirms that the compound's structure is fucosylated heparin pentasaccharide. The structure is shown in formula (I).

[0151] (I)

[0152] In formula (I), A is 2-deoxy-6-O-sulfonyl-2-(sulfonamido)-α-D-glucose; B is β-D-glucuronic acid; C is 2-deoxy-3,6-di-O-sulfonyl-2-(sulfonamido)-α-D-glucose; D is 2-O-sulfonyl-α-L-iduronic acid; E is methyl-O-2-deoxy-6-O-sulfonyl-2-(sulfonamido)-α-D-glucose; F is α-L-fucose; R and R1 are both -SO3 － .

[0153] Table 2. Chemical shift assignments of fucoidan-heparin pentasaccharide

[0154]

[0155] Note: Underlined groups represent sulfate ester linkage sites, and bolded groups represent glycosidic linkage sites. a Not detected

[0156] Example 4: Evaluation of the anticoagulant activity of fucoidan-heparin pentasaccharide

[0157] 4.1 Method:

[0158] 4.1.1 In vitro anticoagulant activity assay

[0159] Anticoagulant activity was assessed using the activated partial thromboplastin time (APTT) method: Fucosylated heparin pentasaccharide (FHP), heparin pentasaccharide (Fpx), and LMWH were prepared into gradient concentration solutions of 0–640 μg / mL, 0–1280 μg / mL, and 0–320 μg / mL, respectively, using Tris-HCl buffer. 5 μL of each solution was mixed with 45 μL of normal coagulation control plasma and incubated at 37°C for 2 min. Then, 50 μL of APTT reagent (containing phospholipids and activator) preheated to 37°C was added, and incubation continued for 3 min. Subsequently, 50 μL of 0.02 mol / L CaCl2 solution preheated to 37°C was added, and the time from the addition of CaCl2 to the appearance of fibrin clots was recorded immediately. Three parallel wells were used for each concentration, and the results were averaged.

[0160] Anticoagulant activity was detected using the thrombin time (TT) method: Fucosylated heparin pentose (FHP), heparin pentose (Fpx), and LMWH were prepared into gradient concentration solutions of 0–1280 μg / mL, 0–1280 μg / mL, and 0–6.4 μg / mL, respectively, using Tris-HCl buffer. 10 μL of each solution was mixed with 90 μL of normal plasma and incubated at 37°C for 2 min. 50 μL of TT reagent (containing standardized thrombin 2.0 NIH U / mL) preheated to 37°C was added, and the coagulation time (s) was recorded immediately.

[0161] Anticoagulant activity was detected using the prothrombin time (PT) method: Fucosylated heparin pentose (FHP), heparin pentose (Fpx), and LMWH were prepared into gradient concentration solutions of 0–1280 μg / mL, 0–1280 μg / mL, and 0–1280 μg / mL, respectively, using Tris-HCl buffer. 5 μL of each solution was mixed with 45 μL of normal plasma and incubated at 37°C for 2 min. 100 μL of PT reagent (containing tissue factor and phospholipids) preheated to 37°C was added, and the coagulation time (s) was recorded immediately.

[0162] 4.1.2 Assay of Inhibitory Activity of Endogenous Coagulation Factor FXase

[0163] The inhibition assay of intrinsic coagulation factor FXase (FVIIIa-FIXa complex) was performed using the FVIII:C chromogenic substrate kit (Siemens) via a 96-well plate method. A series of gradient concentration samples (FHP, Fpx, and LMWH, 30 μL each) were prepared, along with FVIII standard (2 IU / mL, 30 μL) and R2 solution (containing FIXa, phospholipids, and Ca). 2+Mix R1 (FX substrate, 30 μL) and incubate at 37°C with shaking for 2 min to form the FXase complex; then add R1 (FX substrate, 30 μL) and continue incubation for 1 min; finally add R3 (chromogenic substrate S-2765, 30 μL) preheated to 37°C, and immediately monitor the absorbance change (ΔA / min) at 405 nm. Calculate the inhibition rate based on the OD405 change and plot the inhibition rate-concentration curve to calculate the IC50. 50 .

[0164] 4.1.3 Assay of AT-dependent anti-FIIa and anti-FXa activities

[0165] FIIa (final concentration 0.1 ng / μL) or FIXa (final concentration 0.05 ng / μL) was incubated with a series of 4-fold dilutions of the test compound (FHP, Fpx, and LMWH, 0.04–40 μM) and 0.25 μM AT in a 96-well plate at 37°C for 2 min. The corresponding chromogenic substrate (S-2238 for FIIa, Pefachrome FXa for FIXa) was added to initiate the reaction. The absorbance at 405 nm was continuously monitored using a SpectraMax Flexstation 3 microplate reader (Molecular Devices) (FIIa recorded for 2 min, FIXa for 5 min). The inhibition rate was calculated as (OD solvent – ​​OD compound) / (OD solvent – ​​OD blank) × 100%, IC50. 50 The values ​​were obtained using GraphPad Prism 9 software through nonlinear regression analysis.

[0166] 4.1.4 In vivo antithrombotic activity and bleeding tendency assay

[0167] The experiments on venous thrombosis in rats and bleeding tendency in mice were conducted according to the methods described by Zhao et al. (Proc NatlAcad Sci USA, 2015, 112 (27): 8284–8289). Male Sprague-Dawley rats (200±10g) and male Kunming mice (18±2g) were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. (Changsha, China). Animals were housed in a controlled environment (24±2°C, relative humidity 50–60%, 12h light / 12h dark cycle) with free access to standard feed and water. All experimental procedures were approved by the Animal Ethics Committee of Guangxi University of Traditional Chinese Medicine (Approval No.: GXTCMU-EC 20250326-01).

[0168] Antithrombotic activity assay: Male Sprague-Dawley rats were randomly divided into a saline control group (Control group), a fucosylated heparin pentasaccharide group (3.6 mg / kg), and an LMWH group (3.6 mg / kg), all administered subcutaneously. One hour later, the rats were anesthetized with 10% chloral hydrate (0.3 mL / kg, intraperitoneal injection), the inferior vena cava was ligated, and tissue thromboplastin was immediately injected intravenously. Twenty minutes later, the ligation was removed, the thrombus was separated, and the rats were dried at 50°C for 24 hours before being photographed and weighed. Experimental data are expressed as mean ± standard deviation (n=5).

[0169] Bleeding tendency assessment: Male Kunming mice were subcutaneously injected in the back with 36 mg / kg of fucosylated heparin pentasaccharide and LMWH (1 mL / 100 g). After 60 min, a 5 mm section of the tail was cut off, and the bleeding tail was immersed in 40 mL of distilled water at 37°C for 1 h. The hemoglobin content in the immersion solution was measured at 540 nm and converted to the amount of bleeding according to a standard curve. Data are expressed as mean ± standard deviation (n=8 per group).

[0170] 4.2 Results and Analysis

[0171] 4.2.1 Anticoagulant and antithrombotic activity

[0172] As shown in Table 3, fucosylated heparin pentasaccharide significantly prolongs the acute coagulation time (APTT) in human plasma, achieving a 2-fold increase in APTT at a concentration of 4 μg / mL, with an activity approximately three times that of heparin pentasaccharide (Fpx). This compound has a weak effect on TT (2×TT concentration of 72.37 μg / mL) and no effect on PT (concentration >128 μg / mL), indicating its selective action on the intrinsic coagulation pathway, with minimal impact on the common pathway and no interference with the extrinsic pathway. In contrast, the 2×TT concentration of LMWH is as low as 1.04 ± 0.02 μg / mL, and its TT-prolonging activity is 72 times that of fucosylated heparin pentasaccharide. Therefore, FHP exhibits potent anticoagulant activity, and its selectivity for the intrinsic coagulation pathway is significantly superior to that of LMWH.

[0173] Table 3. Effects of fucoidan-heparin pentasaccharide on APTT, TT, and endogenous FXase

[0174]

[0175] In a venous thrombosis model, fucosylated heparin pentasaccharide dose-dependently inhibited thrombus formation, with an inhibition rate of 82.5% in the 3.6 mg / kg dose group. The inhibition rate in the LMWH positive control (3.6 mg / kg) was 85.1%. Figure 9 This indicates that fucoidan-coated heparin pentasaccharide achieved antithrombotic efficacy comparable to the clinical gold standard at the same dosage.

[0176] 4.2.2 FXase inhibitory activity and AT-dependent FXa and FIIa inhibitory activities

[0177] Heparin pentasaccharide Fpx has almost no FXase inhibitory activity (IC50). 50 >2000 ng / mL), while fucosylation showed strong inhibition of this enzyme, IC50 50 The concentration was 746.61 ± 95.02 ng / mL (Table 3). This result is highly consistent with the APTT prolongation effect, confirming that fucosylated heparin pentasaccharide exerts its anticoagulant effect by targeting FXase, the rate-limiting enzyme in the intrinsic coagulation pathway.

[0178] As shown in Table 3, under AT-dependent mode: LMWH exhibits strong suppression of both FIIa and FXa, IC 50 The concentrations were 87.45±7.44 ng / mL and 116.72±55.82 ng / mL, respectively; while heparin pentasaccharide Fpx exhibited high FXa selectivity and excellent FXa inhibitory activity (IC50). 50 =37.79±6.42ng / mL), but it had almost no inhibition against FIIa (IC50). 50 >2000 ng / mL). Fucosylated heparin pentasaccharide (FHP) also showed no significant inhibition of FIIa (IC50). 50 >2000ng / mL), its FXa inhibitory activity (IC50) 50 =103.88±21.38ng / mL) is comparable to LMWH, but only about 1 / 3 of Fpx, indicating that fucosylation modification moderately reduces its inhibitory efficacy while retaining FXa selectivity, thus exhibiting stronger intrinsic coagulation pathway (FXase) inhibitory selectivity.

[0179] 4.2.3 Security Potential

[0180] Existing clinical anticoagulants (such as UFH and DOACs) generally carry a bleeding risk due to inhibition of the extrinsic pathway (prolonged PT). Fucosylated heparin pentasaccharide does not affect PT at effective anticoagulant concentrations, and its effect on prolonging TT is weaker than that of LMWH, suggesting a lower bleeding tendency. FXase is a key node in thrombosis rather than a major component of physiological hemostasis. Targeting this enzyme can preserve extrinsic hemostatic function while achieving highly effective antithrombotic effects. Therefore, this compound holds promise for development as a next-generation, safe FXase inhibitor for the prevention and treatment of venous thromboembolism and contact thrombosis-related diseases.

[0181] In a mouse hemorrhage model, subcutaneous administration of 36 mg / kg of fucosylated heparin pentasaccharide compared with equivalent doses of LMWH and Fpx showed that the blood loss in the LMWH and Fpx groups was significantly increased compared with the normal control, while there was no significant difference between the fucosylated heparin pentasaccharide group and the blank control group. Figure 10The above results suggest its potential as a low-bleeding-risk anticoagulant. Combined with its potent in vitro FXase inhibitory activity, fucoidan-derived heparin pentasaccharide is expected to become a naturally derived, highly effective, and safe FXase inhibitor, providing a unique candidate molecule for the development of new antithrombotic drugs.

[0182] In summary, this invention is the first to demonstrate that fucosylated heparin pentasaccharide can be used as a highly active and selective FXase inhibitor for the development of a new generation of low-bleeding-risk anticoagulants to prevent and treat thrombotic cardiovascular diseases.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art can still make modifications or equivalent substitutions. However, any improvements and changes that do not depart from the technical concept of the present invention should be covered within the protection scope of the present invention.

Claims

1. A fucoidan-based heparin pentasaccharide, characterized in that: The fucoidan heparin pentasaccharide is a homologous oligosaccharide compound having formula (I). （I） In formula (I), A is 2-deoxy-6-O-sulfonyl-2-(sulfonamido)-α-D-glucose; B is β-D-glucuronic acid; C is 2-deoxy-3,6-di-O-sulfonyl-2-(sulfonamido)-α-D-glucose; D is 2-O-sulfonyl-α-L-iduronic acid; E is methyl-O-2-deoxy-6-O-sulfonyl-2-(sulfonamido)-α-D-glucose; F is α-L-fucose; and R are independently -SO3. － Or -H; R1 is -SO3 － .

2. The fucoidan-heparin pentasaccharide according to claim 1, characterized in that: The weight-average molecular weight of the fucoidan pentasaccharide is 2.0~2.9kDa, and the polydispersity index is between 1.0 and 1.6; the sulfate content is 20%~50%.

3. The fucoidan-heparin pentasaccharide according to claim 1, characterized in that: The monosaccharide composition includes D-glucosamine, D-glucuronic acid, L-iduronic acid and fucose, wherein the molar ratio of D-glucosamine, D-glucuronic acid, L-iduronic acid and fucose is 3:(1±0.5):(1±0.5):(2±0.5).

4. The method for preparing fucosylated heparin pentasaccharide according to any one of claims 1-3, characterized in that... Includes the following steps: S1. L-fucose and 2-azidoethanol were mixed and added to an acidic ion exchange resin. The mixture was reacted at 60-100°C for 1-8 hours. The solid-liquid mixture was separated, concentrated, purified by silica gel column chromatography, and lyophilized to obtain 1-azidoethyl α-L-pyran-fucoside. 1-azidoethyl α-L-pyran-fucoside was dissolved in N,N-dimethylformamide and a sulfur trioxide-pyridine complex was added. The mixture was reacted at 40-80°C for 2-12 hours under inert gas protection. After the reaction was completed, the mixture was cooled, neutralized to neutral by adding saturated sodium bicarbonate solution, stirred, centrifuged, concentrated, and dried to obtain 1-azidoethyl-2,3,4-tri-O-sulfonic acid-α-L-pyran-fucoside. S2. Dissolve hydrogen- or sodium-form heparin pentose in MES buffer at pH 4-8 to prepare a heparin pentose solution. Add hydrogen- or sodium-form heparin pentose 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and react at 15-70°C for 10-120 min. Then add propargylamine MES solution and react at 30-70°C for 12-48 h. After salting out, alcohol precipitation, centrifugation, desalting, and lyophilization, obtain alkydinated heparin pentose. S3. Dissolve the alkydized heparin pentose in PBS buffer to prepare a substrate solution; prepare a catalyst solution by mixing tris(3-hydroxypropyltriazolylmethyl)amine solution and CuSO4·5H2O solution; add the catalyst solution dropwise to the substrate solution, replace the inert gas in the reaction vessel, add the reducing agent, stir for 20-40 min to obtain the reaction solution; then add 1-azidoethyl-2,3,4-tri-O-sulfonyl-α-L-pyran-fucoside to the reaction solution, and add PBS buffer to make the final concentration of alkydized heparin pentose 5-20 mg / mL, stir the reaction at 55-65℃ for 12-36 h; after the reaction is completed, salt out, precipitate with alcohol, separate the solid and liquid, wash the solid, desalt and freeze dry to obtain the fucoidized heparin pentose.

5. The method for preparing fucosylated heparin pentasaccharide according to claim 4, characterized in that: In step S1, the molar ratio of L-fucose to 2-azidoethanol is 1:10-1:20; And / or, in step S1, the amount of acidic ion exchange resin used is 0.5-5.0 times the weight of L-fucose; And / or, in step S1, the molar ratio of 1-azidoethyl α-L-pyran fucoside to the sulfur trioxide-pyridine complex is 1:2 to 1:

20.

6. The method for preparing fucosylated heparin pentasaccharide according to claim 4, characterized in that: In step S2, hydrogen- or sodium-form heparin pentasaccharide is dissolved in 0.05-0.2M MES buffer at pH 4-8 to prepare a solution with a concentration of 5-50 mg / mL. In step S2, the molar ratio of hydrogen- or sodium-form heparin pentasaccharide to 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:5 to 1:60; And / or, in step S2, in the MES solution of propargylamine with a pH of 4-8, the concentration of propargylamine is 0.1-0.5 M, and the molar ratio of hydrogen or sodium heparin pentose to propargylamine is 1:5-1:

80.

7. The method for preparing fucosylated heparin pentasaccharide according to claim 4, characterized in that: In step S3, the concentration of the tris(3-hydroxypropyltriazolylmethyl)amine solution is 100-300 mg / mL, and the dosage is calculated based on 5-20 μL / mg of alkynylated heparin pentasaccharide. And / or, in step S3, the concentration of CuSO4·5H2O solution is 10-40 mg / mL, and the dosage is calculated according to 1-10 μL / mg of alkynylated heparin pentasaccharide; And / or, in step S3, the reducing agent is Na-ascorbic acid solution with a concentration of 30-200 mg / mL, and the amount used is calculated according to 1-10 μL / mg of alkynylated heparin pentasaccharide; And / or, in step S3, the amount of 1-azidoethyl-2,3,4-tri-O-sulfonyl-α-L-pyranfucoside is calculated based on alkynylated heparin pentose and is 2.0-6.0 equivalents.

8. The method for preparing fucosylated heparin pentasaccharide according to claim 4, characterized in that: In step S1, 1-azidoethyl α-L-pyranofucoside is dissolved in N,N-dimethylformamide to prepare a 0.1-0.5M solution.

9. The use of the fucoidan heparin pentasaccharide according to any one of claims 1-3 in the preparation of a medicament or functional food for the prevention and treatment of thrombotic diseases or for improving microcirculation.

10. The fucoidan heparin pentasaccharide according to any one of claims 1-3 is formulated into a pharmaceutically acceptable dosage form.

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