Glycoside substrate for oligosaccharide synthesis, its preparation method and application in synthesis of heparin anticoagulant oligosaccharide

Abstract

The present application relates to a kind of glycoside substrates for oligosaccharide synthesis and its preparation method and application in the synthesis of heparin anticoagulant oligosaccharide, belong to chemical enzyme synthesis and pharmaceutical synthesis technical field.The glycoside substrate for oligosaccharide synthesis chemical structure is as shown in formula (1) as follows.The present application further provides the preparation method of the glycoside substrate, the application of the glycoside substrate in the preparation of heparin anticoagulant oligosaccharide, the method for preparing heparin anticoagulant hexasaccharide using the glycoside substrate, and the anticoagulant hexasaccharide prepared using the glycoside substrate.The glycoside substrate uses p-hydroxybenzoic acid compound as aglycone structure, can be decomposed into naturally occurring or easily metabolized fragment in vivo, avoids the risk of toxic pNP glycoside metabolism to produce nitrophenol, significantly improves drug safety.Formula (1)

Description

Technical Field

[0001] This invention relates to a glycoside substrate that can be used for oligosaccharide synthesis, its preparation method, and its application in the synthesis of heparin anticoagulant oligosaccharides, belonging to the fields of chemical enzymatic synthesis and drug synthesis technology. Background Technology

[0002] Heparin is a linear polysaccharide composed of repeating disaccharide units of glucosamine and iduronic acid. Heparin-based drugs can enhance the activity of antithrombin, thereby inhibiting various coagulation factors and exerting an anticoagulant effect. As a key drug for anticoagulation and antithrombotic therapy, heparin has been used clinically for over 50 years and is widely used for clinical indications such as thromboembolic diseases, myocardial infarction, hemodialysis, and extracorporeal circulation anticoagulation.

[0003] Traditional heparin production primarily relies on extraction from animal tissues, which carries risks of structural inhomogeneity and contamination. Chemical enzymatic synthesis, as a more precise and safer alternative, has made significant progress in recent years. The synthesis of heparin using p-nitrophenyl glucuronic acid (pNP-GlcA) as the initiating acceptor is a classic strategy in chemical enzymatic synthesis. The synthesis involves using pNP-GlcA as the initiating acceptor, elongating the glycan backbone under the catalysis of specific glycosyltransferases, and then undergoing sulfation and isomerization modifications under the catalysis of various sulfate transferases and isomerases, ultimately yielding heparin-like compounds with anticoagulant activity. The introduction of the p-nitrophenyl (pNP) group makes the synthesis process easier to monitor, greatly reducing the difficulty of synthesis. However, pNP, as a non-natural, potentially toxic aromatic nitro compound, may trigger unwanted immune responses during use and may affect drug metabolism or produce toxic p-nitrophenol, posing safety risks.

[0004] Therefore, there is an urgent need to develop a glycoside substrate with a novel structure that can be used for oligosaccharide synthesis and its preparation method, in order to address the potential safety risks associated with the current technology that requires the introduction of p-nitrophenyl (pNP) groups to prepare heparin anticoagulant oligosaccharides. Summary of the Invention

[0005] To address the safety concerns of heparin enzymatic synthesis of initiation receptors in existing technologies, this invention provides a glycoside substrate for oligosaccharide synthesis, its preparation method, and its application in the synthesis of heparin anticoagulant oligosaccharides.

[0006] The present invention mainly designs and synthesizes a novel glycoside compound with a new structure that can be used for heparin synthesis, and uses this compound as an initiator and acceptor substrate to synthesize a novel heparin anticoagulant oligosaccharide with anti-factor Xa activity.

[0007] The technical solution of this invention is as follows:

[0008] A novel glycoside substrate for oligosaccharide synthesis and its pharmaceutically acceptable salt are shown in the following formula (1):

[0009]

[0010] Equation (1)

[0011] Where R is -H, -OH or -CH3.

[0012] The above-mentioned method for preparing novel glycoside substrates that can be used for oligosaccharide synthesis includes the following steps:

[0013] (1) Add p-hydroxybenzoic acid compounds to thionyl chloride, then add N,N-dimethylformamide dropwise, stir the reaction at 20~30℃ and 180~220rpm for 1.5~2.5h, and after rotary evaporation and drying, p-hydroxybenzoyl chloride compounds are obtained;

[0014] (2) Add the p-hydroxybenzoyl chloride compound obtained in step (1) to the NaOH solution, then add 8-aminooctanoic acid dropwise, and stir the reaction at 70~80℃ and 180~220rpm for 2~3h to obtain a reaction solution containing sodium octanoate of 8-(4-hydroxybenzoamide)octanoate.

[0015] (3) Adjust the pH of the reaction solution containing sodium octanoate of 8-(4-hydroxybenzamide) obtained in step (2) to 3~4, and after filtration and drying, obtain sodium octanoate of 8-(4-hydroxybenzamide);

[0016] (4) Add the 8-(4-hydroxybenzamide) octanoic acid compound obtained in step (3), MgCl2, uridine diphosphate-glucuronic acid (UDP-GlcA) and uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L to Tris-HCl buffer to construct the reaction system; incubate the reaction system overnight at 35~40℃ in a water bath for 12~16h, and after purification, obtain a novel glycoside substrate that can be used for oligosaccharide synthesis.

[0017] According to a preferred embodiment of the present invention, in step (1), the reaction is carried out by stirring at 25°C and 200 rpm for 2 hours.

[0018] According to a preferred embodiment of the present invention, in step (1), the p-hydroxybenzoic acid compound is p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid or 4-hydroxy-2-methylbenzoic acid.

[0019] According to a preferred embodiment of the present invention, in step (1), the mass-to-volume ratio of the p-hydroxybenzoic acid compound to thionyl chloride is (1~3):(2~4), unit: g / mL.

[0020] According to a preferred embodiment of the present invention, in step (1), the amount of N,N-dimethylformamide added is 2 to 4% of the mass of the p-hydroxybenzoic acid compound.

[0021] According to a preferred embodiment of the present invention, in step (2), the concentration of the NaOH solution is 1.5~2.5M, and the amount used is 4.5~5.5 times the equivalent of the p-hydroxybenzoyl chloride compound; the molar ratio of the p-hydroxybenzoyl chloride compound to 8-aminooctanoic acid is 1:(1~2).

[0022] More preferably, the concentration of the NaOH solution is 2M, and the amount used is 5 times the equivalent of the p-hydroxybenzoyl chloride compound; the molar ratio of the p-hydroxybenzoyl chloride compound to 8-aminooctanoic acid is 1:1.5.

[0023] According to a preferred embodiment of the present invention, in step (2), the reaction is carried out by stirring at 75°C and 200 rpm for 2.5 h.

[0024] According to a preferred embodiment of the present invention, in step (3), the pH of the reaction solution containing sodium octanoate (4-hydroxybenzamide) is adjusted using 1M hydrochloric acid.

[0025] According to a preferred embodiment of the present invention, in step (4), the uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L is an existing enzyme that has been disclosed in Chinese patent document CN121109339A.

[0026] According to a preferred embodiment of the present invention, in step (4), the concentration of Tris-HCl buffer in the reaction system is 40-60 mM, the concentration of 8-(4-hydroxybenzamide)octanoic acid compound is 5-15 mM, the concentration of MgCl2 is 5-15 mM, the concentration of uridine diphosphate-glucuronic acid (UDP-GlcA) is 10-20 mM, and the concentration of uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L is 0.01-0.2 mg / mL.

[0027] According to a preferred embodiment of the present invention, in step (4), the reaction is carried out overnight at 37°C for 16 hours in a water bath, followed by purification by C18 column chromatography.

[0028] According to the present invention, the flowchart of the method for preparing the novel glycoside substrate that can be used for oligosaccharide synthesis is as follows:

[0029] .

[0030] Application of the above-mentioned novel glycoside substrates in the preparation of heparin anticoagulant oligosaccharides.

[0031] A method for preparing a novel heparin anticoagulant hexasaccharide using the above-mentioned novel glycoside substrate includes the following steps:

[0032] a. Add a novel glycoside substrate, MgCl2, heparin backbone synthase 2 (PmHS2) and uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) to Tris-HCl buffer to construct heparin oligosaccharide backbone synthesis reaction system-1; then react heparin oligosaccharide backbone synthesis reaction system-1 in a water bath at 30℃ overnight for 12-16 h, and after purification, obtain the disaccharide intermediate;

[0033] b. Add disaccharide intermediate, MgCl2, heparin backbone synthase 2 (PmHS2) and uridine diphosphate-glucuronic acid (UDP-GlcA) to Tris-HCl buffer to construct heparin oligosaccharide backbone synthesis reaction system-2; then react heparin oligosaccharide backbone synthesis reaction system-2 in a water bath at 30℃ overnight for 12-16 h, and after purification, obtain trisaccharide intermediate;

[0034] c. Continue using the trisaccharide intermediate as a substrate and repeat steps a to b to extend the sugar chain until the heparin hexasaccharide backbone intermediate is obtained.

[0035] d. Add the heparin hexasaccharide backbone to deionized water, then add LiOH solution dropwise until the pH reaches 12. React at 20-30℃ for 2-3 hours, adjust the pH to 7.0-7.2, and continue to add MES buffer, 3'-adenosine-5'-phosphate sulfate (PAPS) and N-sulfate transferase (NST) to the reaction solution to construct heparin oligosaccharide sulfated reaction system-1. Then, react heparin oligosaccharide sulfated reaction system-1 overnight in a water bath at 37℃ for 12-16 hours. After purification, N-sulfated heparin hexasaccharide is obtained.

[0036] e. Dissolve N-sulfated heparin hexasaccharide in MES buffer, add CaCl2 and C5-epimerase (C5-epi), react at 37°C for 1.5-2.5 h, then add 3'-adenosine-5'-phosphate sulfate (PAPS) and 2-O-sulfotransferase (2OST) to construct heparin oligosaccharide sulfation reaction system-2; react heparin oligosaccharide sulfation reaction system-2 overnight at 37°C in a water bath for 12-16 h, and after purification, obtain heparin hexasaccharide containing IdoA2S;

[0037] f. Heparin hexasaccharide containing IdoA2S was dissolved in MES buffer, and then CaCl2, 3'-adenosine-5'-phosphate sulfate (PAPS) and 6-O-sulfotransferase (6OST) were added to construct heparin oligosaccharide sulfation reaction system-3. Heparin oligosaccharide sulfation reaction system-3 was reacted overnight at 37°C in a water bath for 12-16 h. After purification, heparin oligosaccharide containing GlcNS6S was obtained.

[0038] g. Heparin oligosaccharide containing GlcNS6S was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate (PAPS) and 3-O-sulfatyltransferase (3OST) were added to construct heparin oligosaccharide sulfate reaction system-4. Heparin oligosaccharide sulfate reaction system-4 was reacted overnight at 37°C in a water bath for 12-16 h. After purification, a novel heparin anticoagulant hexasaccharide was obtained.

[0039] According to a preferred embodiment of the present invention, in step a, in the heparin oligosaccharide backbone synthesis reaction system-1, the concentration of Tris-HCl buffer is 40-60 mM, the concentration of the novel glycoside substrate is 1-5 mM, the concentration of MgCl2 is 8-12 mM, the concentration of heparin backbone synthase 2 (PmHS2) is 0.05-0.2 mg / mL, and the molar ratio of uridine diphosphate-N-difluoroacetylglucosamine (UDP-GlcNDFA) to the novel glycoside substrate is 1.5:1.

[0040] According to a preferred embodiment of the present invention, in step b, in the heparin oligosaccharide backbone synthesis reaction system-2, the concentration of Tris-HCl buffer is 40-60 mM, the concentration of disaccharide intermediate is 1-5 mM, the concentration of MgCl2 is 8-12 mM, the concentration of heparin backbone synthase 2 (PmHS2) is 0.05-0.2 mg / mL, and the molar ratio of uridine diphosphate-glucuronic acid (UDP-GlcA) to disaccharide intermediate is 1.5:1.

[0041] According to a preferred embodiment of the present invention, in step d, in the heparin oligosaccharide sulfation reaction system-1, the concentration of the heparin hexasaccharide backbone is 1~5mM, the concentration of the MES buffer is 40~60mM, the concentration of N-sulfotransferase (NST) is 0.05~0.2mg / mL, and the molar ratio of 3'-adenosine-5'-phosphate sulfate (PAPS) to the heparin hexasaccharide backbone is 6:1.

[0042] According to a preferred embodiment of the present invention, in step e, in the heparin oligosaccharide sulfation reaction system-2, the concentration of N-sulfated heparin hexasaccharide is 0.1~1mM, the concentration of MES buffer is 40~60mM, the concentration of CaCl2 is 1~5mM, the concentration of C5-epimerase is 0.05~0.2mg / ml, the molar ratio of 3'-adenosine-5'-phosphate sulfate (PAPS) to N-sulfated heparin hexasaccharide is 2:1, and the concentration of 2-O-sulfotransferase (2OST) is 0.1~0.3mg / mL.

[0043] According to a preferred embodiment of the present invention, in step f, the concentration of heparin hexasaccharide containing IdoA2S in the heparin oligosaccharide sulfation reaction system-3 is 0.1~1mM, the concentration of MES buffer is 40~60mM, the concentration of CaCl2 is 1~5mM, the concentration of 6-O-sulfotransferase (6OST) is 0.05~0.2mg / ml, and the molar ratio of 3'-adenosine-5'-phosphate sulfate (PAPS) to heparin hexasaccharide containing IdoA2S is 6:1.

[0044] According to a preferred embodiment of the present invention, in step g, the concentration of heparin oligosaccharide containing GlcNS6S in the heparin oligosaccharide sulfation reaction system-4 is 0.1~1mM, the concentration of MES buffer is 40~60mM, the concentration of 3-O-sulfotransferase (3OST) is 0.05~0.2mg / ml, and the molar ratio of 3'-adenosine-5'-phosphate sulfate (PAPS) to heparin oligosaccharide containing GlcNS6S is 2:1.

[0045] According to the present invention, the flowchart of the preparation method of the above-mentioned novel heparin anticoagulant hexasaccharide is as follows:

[0046] .

[0047] A novel heparin anticoagulant hexasaccharide was prepared according to the above method.

[0048] This invention also provides the application of the above-mentioned novel heparin anticoagulant hexasaccharide in the preparation of safe and efficient anticoagulant drugs and antithrombotic drugs.

[0049] Experimental procedures not described in detail in this invention can be performed according to conventional experimental procedures in this technical field.

[0050] The beneficial effects of this invention are as follows:

[0051] 1. This invention provides a novel glycoside substrate for the enzymatic synthesis of heparin. This glycoside substrate uses a p-hydroxybenzoic acid-like compound as its aglycone structure, with a well-defined metabolic pathway. In vivo, it can be broken down into naturally occurring or easily metabolized fragments such as p-hydroxybenzoic acid and 8-aminooctanoic acid, avoiding the risk of toxic p-nitrophenol production associated with traditional pNP glycoside metabolism and significantly improving drug safety. Simultaneously, it provides a new option besides pNP initiation receptors for the enzymatic synthesis of heparin-like compounds, enriching the heparin synthesis toolkit.

[0052] 2. This invention provides a method for preparing a novel glycoside substrate that can be used for the chemical enzymatic synthesis of heparin. In this preparation method, the glycoside substrate is constructed by an enzymatically catalyzed glycosylation reaction. The reaction conditions are mild, requiring no high temperature, high pressure, or strong acid / base conditions, reducing the formation of byproducts, improving the purity and yield of the target product, and simultaneously reducing production energy consumption and costs.

[0053] 3. This invention provides a method for preparing a novel heparin hexasaccharide using a novel glycoside substrate, and the novel heparin hexasaccharide prepared using this method. This novel heparin anticoagulant hexasaccharide retains the characteristic anti-factor Xa activity structure of heparin compounds, and its anticoagulant activity mechanism is clearly defined, providing an important candidate compound for the development of a new generation of low-immunogenicity, high-safety anticoagulant drugs.

[0054] 4. This invention demonstrates that structural changes at the reducing end of heparin oligosaccharides do not affect their anticoagulant activity, providing more options for enriching the molecular library of heparin drugs and expanding the design space for structural optimization of heparin compounds. Attached Figure Description

[0055] Figure 1 The results are from the mass spectrometry analysis of 1-O-[8-(4-hydroxybenzamido)octanoic acid]-β-D-glucuronic acid prepared in Example 1.

[0056] Figure 2 The mass spectrometry results are those of 1-O-[8-(2,4-dihydroxybenzamido)octanoic acid]-β-D-glucuronic acid prepared in Example 2.

[0057] Figure 3 The results are from liquid chromatography and mass spectrometry analysis of the novel heparin anticoagulant hexasaccharide prepared in Example 4; in the figure, A is the result of liquid chromatography analysis and B is the result of mass spectrometry analysis.

[0058] Figure 4 The results are the in vitro anti-Xa factor activity assay results of the novel heparin anticoagulant hexasaccharide prepared in Example 4. Detailed Implementation

[0059] 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.

[0060] The uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L, heparin backbone synthase 2 (PmHS2), N-sulfate transferase (NST), C5-isomerase (C5-epi), 2-O-sulfate transferase (2OST), 3-O-sulfate transferase (3OST), and 6-O-sulfate transferase (6OST) described in the following examples are all existing enzymes with publicly available amino acid sequences, and can be prepared by commercially available enzymes or by recombinant expression in hosts such as Escherichia coli, yeast, and insect cells.

[0061] Example 1

[0062] A method for preparing a novel glycoside substrate, 1-O-[8-(4-hydroxybenzamido)octanoic acid]-β-D-glucuronic acid, which can be used for oligosaccharide synthesis, includes the following steps:

[0063] (1) Add 2g of p-hydroxybenzoic acid to 3mL of thionyl chloride, then add 0.06g of N,N-dimethylformamide dropwise, stir at 25℃ and 200rpm for 2h, then remove excess thionyl chloride by rotary evaporation, and then dry to recover 2.09g of p-hydroxybenzoyl chloride, with a yield of 92.3%;

[0064] (2) Add 1g of p-hydroxybenzoyl chloride to 6.5mL of 2M NaOH solution, then add 1.1g of 8-aminooctanoic acid dropwise, and stir the reaction at 75℃ and 200rpm for 2.5h to obtain a reaction solution containing sodium 8-(4-hydroxybenzoamide)octanoate.

[0065] (3) After the reaction reached equilibrium, the pH of the reaction solution containing sodium 8-(4-hydroxybenzamido)octanoate was adjusted to 3.5 using 1M hydrochloric acid. After filtration and drying, 1.10 g of 8-(4-hydroxybenzamido)octanoic acid was obtained, with a yield of 61.4%.

[0066] (4) Add 8-(4-hydroxybenzamido)octanoic acid, MgCl2, uridine diphosphate-glucuronic acid (UDP-GlcA) and uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L to Tris-HCl buffer to construct a reaction system; incubate the reaction system overnight at 37°C in a water bath for 16 h, and then use HPLC to detect the reaction progress. When the substrate conversion rate is >95%, add formic acid to adjust the pH to 4~5 to terminate the reaction. Then purify by C18 column chromatography to obtain a novel glycoside substrate that can be used for oligosaccharide synthesis.

[0067] In the reaction system (volume 45 mL), the concentration of Tris-HCl buffer (pH 7.5) was 50 mM, the concentration of 8-(4-hydroxybenzamido)octanoic acid was 10 mM, the concentration of MgCl2 was 10 mM, the concentration of UDP-GlcA was 15 mM, and the concentration of uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L was 0.1 mg / mL.

[0068] HPLC detection was performed using a Shim-pack GIS C18 column (4.6×250mm, 5μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The time program was as follows: 0~15min, B phase concentration 20%~100%; 15~20min, B phase concentration 100%; 20~25min, B phase concentration 20%. The detection wavelength was 254nm.

[0069] The novel glycoside substrate prepared in this embodiment for oligosaccharide synthesis is 1-O-[8-(4-hydroxybenzamido)octanoic acid]-β-D-glucuronic acid, and its structural formula is shown below:

[0070] .

[0071] Mass spectrometry analysis was performed on it, and the results were as follows: Figure 1 As shown.

[0072] Depend on Figure 1 It is known that the molecular weight of the novel glycoside substrate 1-O-[8-(4-hydroxybenzamido)octanoic acid]-β-D-glucuronic acid, which can be used for oligosaccharide synthesis, is 455.59, which is consistent with the theory.

[0073] Example 2

[0074] A method for preparing a novel glycoside substrate, 1-O-[8-(2,4-dihydroxybenzamido)octanoic acid]-β-D-glucuronic acid, which can be used for oligosaccharide synthesis, includes the following steps:

[0075] (1) 2.2 g of 2,4-dihydroxybenzoic acid was added to 3 mL of thionyl chloride, and then 0.06 g of N,N-dimethylformamide was added dropwise. The mixture was stirred at 25 °C and 200 rpm for 2 h. The excess thionyl chloride was then removed by rotary evaporation and dried to obtain 2.24 g of 2,4-dihydroxybenzoyl chloride, with a yield of 91.3%.

[0076] (2) Add 1g of 2,4-dihydroxybenzoyl chloride to 6.5mL of 2M NaOH solution, then add 1.1g of 8-aminooctanoic acid dropwise, and stir the reaction at 75℃ and 200rpm for 2.5h to obtain a reaction solution containing sodium octanoate of 8-(2,4-dihydroxybenzoamide)octanoate.

[0077] (3) After the reaction reached equilibrium, the pH of the reaction solution containing sodium 8-(2,4-dihydroxybenzamido)octanoate was adjusted to 3.5 using 1M hydrochloric acid. After filtration and drying, 1.12 g of 8-(2,4-dihydroxybenzamido)octanoic acid was obtained, with a yield of 65.4%.

[0078] (4) Add 8-(2,4-dihydroxybenzamido)octanoic acid, MgCl2, uridine diphosphate-glucuronic acid (UDP-GlcA) and uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L to Tris-HCl buffer to construct a reaction system; incubate the reaction system overnight at 37°C in a water bath for 16 h, and then use HPLC to detect the reaction progress. When the substrate conversion rate is >95%, add formic acid to adjust the pH to 4~5 to terminate the reaction. Then purify by C18 column chromatography to obtain a novel glycoside substrate that can be used for oligosaccharide synthesis.

[0079] In the reaction system (volume 45 mL), the concentration of Tris-HCl buffer (pH 7.5) was 50 mM, the concentration of 8-(2,4-dihydroxybenzamido)octanoic acid was 10 mM, the concentration of MgCl2 was 10 mM, the concentration of UDP-GlcA was 15 mM, and the concentration of uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L was 0.1 mg / mL.

[0080] HPLC detection was performed using a Shim-pack GIS C18 column (4.6×250mm, 5μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The time program was as follows: 0~15min, B phase concentration 20%~100%; 15~20min, B phase concentration 100%; 20~25min, B phase concentration 20%. The detection wavelength was 254nm.

[0081] The novel glycoside substrate prepared in this embodiment for oligosaccharide synthesis is 1-O-[8-(2,4-dihydroxybenzamido)octanoic acid]-β-D-glucuronic acid, the structural formula of which is shown below:

[0082] .

[0083] Mass spectrometry analysis was performed on it, and the results were as follows: Figure 2 As shown.

[0084] Depend on Figure 2 It is known that the molecular weight of the novel glycoside substrate 1-O-[8-(2,4-dihydroxybenzamido)octanoic acid]-β-D-glucuronic acid, which can be used for oligosaccharide synthesis, is 471.56, consistent with the theory.

[0085] Example 3

[0086] A method for preparing a novel heparin hexasaccharide backbone intermediate using a novel glycoside substrate includes the following steps:

[0087] a. Add 1-O-[8-(4-hydroxybenzamido)octanoic acid]-β-D-glucuronic acid, MgCl2, PmHS2 enzyme and UDP-GlcNDFA prepared in Example 1 to Tris-HCl buffer to construct heparin oligosaccharide backbone synthesis reaction system-1; then react heparin oligosaccharide backbone synthesis reaction system-1 in a water bath at 30°C overnight for 12 h, and then use HPLC to detect the reaction progress. When the substrate conversion rate is >99%, formic acid is added to adjust the pH to 4~5 to terminate the reaction. Then, the reaction is purified by C18 column chromatography, eluted with methanol-water containing 0.1% TFA, and the target fraction is obtained. After rotary evaporation under reduced pressure, the disaccharide intermediate is obtained.

[0088] HPLC detection was performed using a Shim-pack GIS C18 column (4.6×250mm, 5μm). The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was acetonitrile. The chromatographic conditions were 0-100% gradient elution of phase B from 0 to 15 min, with a flow rate of 1 mL / min and a detection wavelength of 254 nm.

[0089] In the heparin oligosaccharide backbone synthesis reaction system-1 (volume 45 mL), the concentration of Tris-HCl buffer (pH 7.5) was 50 mM, the concentration of 1-O-[8-(4-hydroxybenzamido)octanoic acid]-β-D-glucuronic acid was 5 mM, the concentration of MgCl2 was 10 mM, the concentration of PmHS2 enzyme was 0.1 mg / mL, and the concentration of UDP-GlcNDFA was 7.5 mM.

[0090] b. Add disaccharide intermediate, MgCl2, PmHS2 enzyme and UDP-GlcA to Tris-HCl buffer to construct heparin oligosaccharide backbone synthesis reaction system-2; then react heparin oligosaccharide backbone synthesis reaction system-2 overnight at 30℃ in a water bath for 12 h, and then use HPLC to detect the reaction progress. When the substrate conversion rate is >99%, formic acid is added to adjust the pH to 4~5 to terminate the reaction. Then, the reaction is purified by C18 column chromatography, eluted with methanol-water containing 0.1% TFA, and the target fraction is obtained. After rotary evaporation under reduced pressure, the trisaccharide intermediate is obtained.

[0091] The HPLC detection system is the same as in step a;

[0092] In the heparin oligosaccharide backbone synthesis reaction system 2 (volume 45 mL), the concentration of Tris-HCl buffer (pH 7.5) was 50 mM, the concentration of disaccharide intermediate was 5 mM, the concentration of MgCl2 was 10 mM, the concentration of PmHS2 enzyme was 0.1 mg / mL, and the concentration of UDP-GlcA was 7.5 mM.

[0093] c. Continue using the trisaccharide intermediate as a substrate and repeat step a to extend the sugar chain to obtain the tetrasaccharide intermediate;

[0094] Using the tetrasaccharide intermediate as a substrate, repeat step b to extend the sugar chain and obtain the pentasaccharide intermediate;

[0095] Using the pentasaccharide intermediate as a substrate, step a was repeated to extend the sugar chain, resulting in a novel heparin hexasaccharide backbone intermediate.

[0096] The structural formula of the novel heparin hexasaccharide backbone intermediate prepared using the novel glycoside substrate in this embodiment is shown below:

[0097] .

[0098] Example 4

[0099] A method for preparing a novel heparin anticoagulant hexasaccharide using a novel hexasaccharide backbone intermediate includes the following steps:

[0100] 1) The novel heparin hexasaccharide backbone prepared in Example 2 was added to deionized water, and then 1 mol / L LiOH solution was added dropwise until the pH reached 12. The reaction was carried out at 25°C for 2 h. The pH was adjusted to 7.0, and MES buffer, PAPS and NST enzyme were added to the reaction solution to construct heparin oligosaccharide sulfation reaction system-1. Then, heparin oligosaccharide sulfation reaction system-1 was reacted overnight at 37°C in a water bath for 12 h. The reaction progress was then detected by HPLC. When the substrate conversion rate was >99%, formic acid was added to adjust the pH to 4~5 to terminate the reaction. The reaction was then purified by Q Sepharose strong anion exchange column, eluted with a gradient of 0→100% 2 mol / L NH4HCO3 solution, and the target component was obtained. After rotary evaporation under reduced pressure, N-sulfated heparin hexasaccharide was obtained.

[0101] HPLC detection was performed using a YMC-Pack Polyamine column (4.6×250mm, 5μm). Mobile phase A was deionized water, and mobile phase B was 1M KH2PO4 solution. The chromatographic conditions were gradient elution of 100% phase A for 0–6 min and 0–100% phase B for 6–40 min, with a flow rate of 0.5 mL / min and a detection wavelength of 254 nm.

[0102] The flow rate for purification using the Q Sepharose strong anion exchange column was 6 mL / min, and the detection wavelength was 254 nm.

[0103] In the heparin oligosaccharide sulfation reaction system-1 (volume 1000mL), the concentration of the novel heparin hexasaccharide backbone is 1mM, the concentration of MES buffer (pH=6.5) is 50mM, the concentration of NST enzyme is 0.1mg / mL, and the concentration of PAPS is 6mM.

[0104] 2) N-sulfated heparin hexasaccharide was dissolved in MES buffer, CaCl2 and C5-epi enzyme were added, and the reaction was carried out at 37°C for 2 h. PAPS and 2OST enzyme were then added to construct heparin oligosaccharide sulfation reaction system-2. Heparin oligosaccharide sulfation reaction system-2 was reacted overnight at 37°C in a water bath for 12 h. The reaction progress was then monitored by HPLC. When the substrate conversion rate was >99%, formic acid was added to adjust the pH to 4-5 to terminate the reaction. The product was then purified by Q Sepharose strong anion exchange column with a gradient elution of 0→100% 2 mol / L NH4HCO3 solution. The target fraction was obtained and obtained by rotary evaporation under reduced pressure to obtain heparin hexasaccharide containing IdoA2S.

[0105] The HPLC detection system and the purification parameters of the Q Sepharose strong anion exchange column are the same as in step 1).

[0106] In the heparin oligosaccharide sulfation reaction system-2 (volume 1000mL), the concentration of N-sulfated heparin hexasaccharide is 0.2mM, the concentration of MES buffer (pH=6.5) is 50mM, the concentration of CaCl2 is 2mM, the concentration of C5-epi enzyme is 0.1mg / ml, the concentration of PAPS is 0.4mM, and the concentration of 2OST enzyme is 0.2mg / mL.

[0107] 3) Heparin hexasaccharide containing IdoA2S was dissolved in MES buffer, and then CaCl2, PAPS and 6OST enzyme were added to construct heparin oligosaccharide sulfation reaction system-3. Heparin oligosaccharide sulfation reaction system-3 was reacted overnight at 37℃ in a water bath for 12 h. The reaction progress was then monitored by HPLC. When the substrate conversion rate was >99%, formic acid was added to adjust the pH to 2~3 to terminate the reaction. The product was then purified by QSepharose strong anion exchange column, eluted with a gradient of 0→100% 2mol / L NH4HCO3 solution. The target fraction was collected and obtained by rotary evaporation under reduced pressure to obtain heparin oligosaccharide containing GlcNS6S.

[0108] The HPLC detection system and the purification parameters of the Q Sepharose strong anion exchange column are the same as in step 1).

[0109] In the heparin oligosaccharide sulfation reaction system-3 (volume 1000mL), the concentration of heparin hexasaccharide containing IdoA2S is 0.2mM, the concentration of MES buffer (pH=6.5) is 50mM, the concentration of CaCl2 is 2mM, the concentration of PAPS is 1.2mM, and the concentration of 6OST enzyme is 0.1mg / mL.

[0110] 4) Heparin oligosaccharides containing GlcNS6S were dissolved in MES buffer, and then PAPS and 3OST enzyme were added to construct heparin oligosaccharide sulfation reaction system-4. Heparin oligosaccharide sulfation reaction system-4 was reacted overnight at 37℃ in a water bath for 12 h. The reaction progress was then monitored by HPLC. When the substrate conversion rate was >99%, formic acid was added to adjust the pH to 4~5 to terminate the reaction. The product was then purified by QSepharose strong anion exchange column, eluted with a gradient of 0→100% 2mol / L NH4HCO3 solution. The target fraction was collected and obtained by rotary evaporation under reduced pressure to obtain the novel heparin anticoagulant hexasaccharide.

[0111] HPLC detection was performed using a YMC-Pack Polyamine column (4.6×250mm, 5μm). Mobile phase A was deionized water, and mobile phase B was 1M KH2PO4 solution. The chromatographic conditions were elution with 60-100% B phase for 0-30 min and 100% B phase for 30-50 min, with a flow rate of 0.5 mL / min and a detection wavelength of 254 nm.

[0112] Q Sepharose strong anion exchange column purification parameters are the same as in step 1).

[0113] In the heparin oligosaccharide sulfation reaction system-4 (volume 1000mL), the concentration of heparin oligosaccharide containing GlcNS6S is 0.2mM, the concentration of MES buffer is 50mM, the concentration of CaCl2 is 2mM, the concentration of PAPS is 0.4mM, and the concentration of 3OST enzyme is 0.1mg / mL.

[0114] The structural formula of the novel heparin anticoagulant hexasaccharide prepared in this embodiment is shown below:

[0115] .

[0116] First, perform liquid chromatography analysis (using the same method as in step 4); then perform mass spectrometry analysis. The results of the liquid chromatography and mass spectrometry analyses are as follows: Figure 3 As shown.

[0117] Depend on Figure 3 It can be seen that the purity of the novel heparin anticoagulant hexasaccharide is over 93%, and the molecular weight is 1931.24 Da, which is consistent with the theory.

[0118] Test case

[0119] The anticoagulant hexasaccharide and antifactor Xa activity of the novel heparin prepared in Example 3 with unfractionated heparin (UFH) were determined using a chromogenic substrate method. The results are as follows: Figure 4 As shown.

[0120] Depend on Figure 4 It can be seen that the novel heparin anticoagulant hexasaccharide of this invention has an IC50 value.50 The value was 10.9 ng / mL, while the IC50 for unfractionated heparin (UFH) was... 50 The value was 118.2 ng / mL. This indicates that the novel heparin anticoagulant hexasaccharide of this invention is a potent Xa inhibitor, and its activity is higher than that of unfractionated heparin (UFH). It can be used to prepare anticoagulant drugs and has broad application prospects.

[0121] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A glycoside substrate for oligosaccharide synthesis and its pharmaceutically acceptable salt, the chemical structure of which is shown in formula (1): Equation (1) in, R can be -H, -OH, or -CH3.

2. The method for preparing the glycoside substrate for oligosaccharide synthesis as described in claim 1, characterized in that, The steps include the following: (1) Add p-hydroxybenzoic acid compounds to thionyl chloride, then add N,N-dimethylformamide dropwise, stir the reaction at 20~30℃ and 180~220rpm for 1.5~2.5h, and after rotary evaporation and drying, p-hydroxybenzoyl chloride compounds are obtained; (2) Add the p-hydroxybenzoyl chloride compound obtained in step (1) to the NaOH solution, then add 8-aminooctanoic acid dropwise, and stir the reaction at 70~80℃ and 180~220rpm for 2~3h to obtain a reaction solution containing sodium octanoate of 8-(4-hydroxybenzoamide)octanoate. (3) Adjust the pH of the reaction solution containing sodium octanoate of 8-(4-hydroxybenzamide) obtained in step (2) to 3~4, and after filtration and drying, obtain sodium octanoate of 8-(4-hydroxybenzamide); (4) Add the 8-(4-hydroxybenzamide)octanoic acid compound obtained in step (3), MgCl2, uridine diphosphate-glucuronic acid and uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L to Tris-HCl buffer to construct the reaction system; incubate the reaction system overnight at 35~40℃ in a water bath for 12~16h, and after purification, obtain the glycoside substrate that can be used for oligosaccharide synthesis.

3. The preparation method according to claim 2, characterized in that, In step (1), the p-hydroxybenzoic acid compound is p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, or 4-hydroxy-2-methylbenzoic acid; the mass-volume ratio of the p-hydroxybenzoic acid compound to thionyl chloride is (1~3):(2~4), unit: g / mL; the amount of N,N-dimethylformamide added is 2~4% of the mass of the p-hydroxybenzoic acid compound.

4. The preparation method according to claim 2, characterized in that, In step (2), the concentration of the NaOH solution is 1.5~2.5M, and the amount used is 4.5~5.5 times the equivalent of the p-hydroxybenzoyl chloride compound; the molar ratio of the p-hydroxybenzoyl chloride compound to 8-aminooctanoic acid is 1:(1~2). More preferably, the concentration of the NaOH solution is 2M, and the amount used is 5 times the equivalent of the p-hydroxybenzoyl chloride compound; the molar ratio of the p-hydroxybenzoyl chloride compound to 8-aminooctanoic acid is 1:1.

5.

5. The preparation method according to claim 2, characterized in that, In step (4), the concentration of Tris-HCl buffer in the reaction system is 40-60 mM, the concentration of 8-(4-hydroxybenzamide)octanoic acid compound is 5-15 mM, the concentration of MgCl2 is 5-15 mM, the concentration of uridine diphosphate-glucuronic acid is 10-20 mM, and the concentration of uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L is 0.01-0.2 mg / mL.

6. The use of the glycoside substrate of claim 1 in the preparation of heparin anticoagulant oligosaccharides.

7. A method for preparing heparin anticoagulant hexasaccharide using the glycoside substrate of claim 1, characterized in that, The steps include the following: a. Add the glycoside substrate described in claim 1, MgCl2, heparin backbone synthase 2 and uridine diphosphate-N-difluoroacetylglucosamine to Tris-HCl buffer to construct heparin oligosaccharide backbone synthesis reaction system-1; then react heparin oligosaccharide backbone synthesis reaction system-1 in a water bath at 30°C overnight for 12-16 h, and after purification, obtain the disaccharide intermediate; b. Add disaccharide intermediate, MgCl2, heparin backbone synthase 2 and uridine diphosphate-glucuronic acid to Tris-HCl buffer to construct heparin oligosaccharide backbone synthesis reaction system-2; then react heparin oligosaccharide backbone synthesis reaction system-2 in a water bath at 30℃ overnight for 12-16 h, and after purification, obtain trisaccharide intermediate; c. Continue using the trisaccharide intermediate as a substrate and repeat steps a to b to extend the sugar chain until the heparin hexasaccharide backbone intermediate is obtained. d. Add the heparin hexasaccharide backbone to deionized water, then add LiOH solution dropwise until the pH reaches 12. React at 20-30℃ for 2-3 hours, adjust the pH to 7.0-7.2, and continue to add MES buffer, 3'-adenosine-5'-phosphate sulfate and N-sulfate transferase to the reaction solution to construct heparin oligosaccharide sulfated reaction system-1; then react heparin oligosaccharide sulfated reaction system-1 in a water bath at 37℃ overnight for 12-16 hours. After purification, N-sulfated heparin hexasaccharide is obtained. e. Dissolve N-sulfated heparin hexasaccharide in MES buffer, add CaCl2 and C5-epimerase, react at 37°C for 1.5-2.5 h, then add 3'-adenosine-5'-phosphate sulfate and 2-O-sulfotransferase to construct heparin oligosaccharide sulfation reaction system-2; react heparin oligosaccharide sulfation reaction system-2 in a water bath at 37°C overnight for 12-16 h, and after purification, obtain heparin hexasaccharide containing IdoA2S; f. Heparin hexasaccharide containing IdoA2S was dissolved in MES buffer, and then CaCl2, 3'-adenosine-5'-phosphate sulfate and 6-O-sulfotransferase were added to construct heparin oligosaccharide sulfate reaction system-3. Heparin oligosaccharide sulfate reaction system-3 was reacted overnight at 37°C in a water bath for 12-16 h. After purification, heparin oligosaccharide containing GlcNS6S was obtained. g. Heparin oligosaccharide containing GlcNS6S was dissolved in MES buffer, and then 3'-adenosine-5'-phosphate sulfate and 3-O-sulfotransferase were added to construct heparin oligosaccharide sulfate reaction system-4. Heparin oligosaccharide sulfate reaction system-4 was reacted in a water bath at 37°C overnight for 12-16 h. After purification, heparin anticoagulant hexasaccharide was obtained.

8. The preparation method according to claim 7, characterized in that, In step a, in the heparin oligosaccharide backbone synthesis reaction system-1, the concentration of Tris-HCl buffer is 40-60 mM, the concentration of glycoside substrate is 1-5 mM, the concentration of MgCl2 is 8-12 mM, the concentration of heparin backbone synthase 2 is 0.05-0.2 mg / mL, and the molar ratio of uridine diphosphate-N-difluoroacetylglucosamine to glycoside substrate is 1.5:1; In step b, in the heparin oligosaccharide backbone synthesis reaction system-2, the concentration of Tris-HCl buffer is 40-60 mM, the concentration of disaccharide intermediate is 1-5 mM, the concentration of MgCl2 is 8-12 mM, the concentration of heparin backbone synthase 2 is 0.05-0.2 mg / mL, and the molar ratio of uridine diphosphate-glucuronic acid to disaccharide intermediate is 1.5:1; In step d, in the heparin oligosaccharide sulfation reaction system-1, the concentration of the heparin hexasaccharide backbone is 1~5mM, the concentration of the MES buffer is 40~60mM, the concentration of N-sulfotransferase is 0.05~0.2mg / mL, and the molar ratio of 3'-adenosine-5'-phosphate sulfate to the heparin hexasaccharide backbone is 6:

1. In step e, in the heparin oligosaccharide sulfation reaction system-2, the concentration of N-sulfated heparin hexasaccharide is 0.1~1mM, the concentration of MES buffer is 40~60mM, the concentration of CaCl2 is 1~5mM, the concentration of C5-epimerase is 0.05~0.2mg / ml, the molar ratio of 3'-adenosine-5'-phosphate sulfate to N-sulfated heparin hexasaccharide is 2:1, and the concentration of 2-O-sulfotransferase is 0.1~0.3mg / mL; In step f, in the heparin oligosaccharide sulfation reaction system-3, the concentration of heparin hexasaccharide containing IdoA2S is 0.1~1mM, the concentration of MES buffer is 40~60mM, the concentration of CaCl2 is 1~5mM, the concentration of 6-O-sulfotransferase is 0.05~0.2mg / ml, and the molar ratio of 3'-adenosine-5'-phosphate sulfate to heparin hexasaccharide containing IdoA2S is 6:

1. In step g, in the heparin oligosaccharide sulfation reaction system-4, the concentration of heparin oligosaccharide containing GlcNS6S is 0.1~1mM, the concentration of MES buffer is 40~60mM, the concentration of 3-O-sulfotransferase is 0.05~0.2mg / ml, and the molar ratio of 3'-adenosine-5'-phosphate sulfate to heparin oligosaccharide containing GlcNS6S is 2:

1.

9. A heparin anticoagulant hexasaccharide, characterized in that, Prepared according to the method described in claim 7 or claim 8.

10. The use of the heparin anticoagulant hexasaccharide according to claim 9 in the preparation of safe and efficient anticoagulant drugs and antithrombotic drugs.

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