Method for catalytically synthesizing dabigatran intermediate based on immobilized lipase

Abstract

The invention discloses a method for catalytically synthesizing a dabigatran intermediate based on immobilized lipase. The method comprises the following steps: firstly, immobilizing optimized CALB lipase with an amino acid sequence as shown in SEQ ID NO: 2 by taking a dimethyl sulfoxide-resistant carrier comprising silanization modified mesoporous SiO2, polyvinylidene fluoride, polymethyl methacrylate and the like as carriers to obtain immobilized lipase, and then, carrying out a reaction on a compound II serving as a substrate in a DMSO organic phase, so as to obtain the immobilized lipase. The immobilized lipase is utilized to catalyze a compound II and a compound III to perform an addition reaction, and a dabigatran key intermediate compound I is efficiently and highly selectively generated; the immobilized carrier adopted by the invention can effectively tolerate a DMSO solvent, is firm in enzyme binding, can be repeatedly used for more than 10 times, has good activity retention, solves the problem that a biocatalyst is easy to inactivate and difficult to recover in a strong polar solvent, has the advantages of high conversion rate, good stereoselectivity, low production cost and environmental protection, and has a good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to a method for synthesizing a dabigatran intermediate based on immobilized lipase catalysis, and belongs to the field of preparation of pharmaceutical intermediates. BACKGROUND

[0002] Dabigatran etexilate is an important direct thrombin inhibitor for preventing and treating thrombotic diseases. Its molecular structure contains a chiral center, so the stereoselective synthesis of the key intermediate chiral amine compound is crucial.

[0003] At present, the synthesis methods of the key intermediate mainly include chemical synthesis and biological catalysis. The chemical synthesis method usually uses heavy metal catalysts or chiral ligands, and has the problems of poor atom economy, low chiral selectivity, serious environmental pollution and complex post-treatment. The biological catalysis method, especially the enzyme catalysis method, has been widely concerned due to its high stereoselectivity, mild reaction conditions and environmental friendliness. Lipase (such as CALB, Candida antarctica Lipase B) has been proved to be able to catalyze the formation of C-N bond for synthesizing chiral amine compounds.

[0004] However, there are still great challenges in directly applying free lipase to industrial synthesis: 1) in the commonly used organic solvents for the reaction, especially strong polar solvents such as dimethyl sulfoxide DMSO, the enzyme is easy to denature and lose activity, resulting in a sharp decrease in catalytic efficiency; 2) the free enzyme is difficult to recover and reuse, and the production cost is high.

[0005] The immobilized enzyme technology is an effective means to solve the above problems. However, the conventional immobilized carriers such as ordinary silica gel and resin have the problems of swelling, structure collapse or enzyme shedding in strong polar solvents such as DMSO, and cannot maintain the enzyme activity and stability.

[0006] Therefore, developing a special immobilized carrier which can resist DMSO, has high mechanical strength and is firmly combined with the enzyme is the key to realizing the industrialization of lipase catalyzed synthesis of dabigatran intermediate. SUMMARY

[0007] The application provides a method for synthesizing a dabigatran intermediate based on immobilized lipase catalysis, and solves the problem of how to realize the preparation method of increasing the yield of the product.

[0008] The application aims to realize the following technical scheme, a method for synthesizing a dabigatran intermediate based on immobilized lipase catalysis, comprising the following steps: S1: Preparation of immobilized lipase: using dimethyl sulfoxide-resistant carrier as immobilized carrier, the optimized CALB lipase with amino acid sequence shown in SEQ ID NO: 2 is immobilized on the carrier by covalent binding method to obtain immobilized lipase; S2: Catalytic synthesis of dabigatran intermediate: in the presence of immobilized lipase prepared in step S1, DMSO organic phase, addition reaction of compound II and compound III is carried out to form C-N bond to generate target product compound I.

[0009] In step S1, the dimethyl sulfoxide-resistant carrier is selected from one of silanized modified mesoporous SiO2, polyvinylidene fluoride and polymethyl methacrylate. These materials themselves or after modification can effectively resist the erosion of DMSO and maintain structural integrity.

[0010] Preferably, the modifier of the silanized modified mesoporous SiO2 is methyl triethoxysilane, and the contact angle of the modified carrier surface is 60°-80°. After modification, the surface has moderate hydrophobicity, which is beneficial to maintaining stability in polar solvents and having beneficial interaction with hydrophobic enzyme pocket.

[0011] Preferably, the specific surface area of the dimethyl sulfoxide-resistant carrier is ≥100 m 2 / g, the pore size is 5-20 nm, and the particle size is 50-200 μm. This can ensure high enzyme loading, good substrate mass transfer efficiency and easy separation after reaction.

[0012] In step S1, the specific operation of the covalent binding method includes: after the dimethyl sulfoxide-resistant carrier is activated with 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), the optimized CALB is reacted in pH 7.0-7.5 phosphate buffer at 25°C for 2-4 h. After the reaction is completed, the solid is collected by centrifugation, washed and dried to obtain the immobilized lipase. The mass ratio of EDC to carrier is 1:5-10, and the molar ratio of EDC to NHS is 1:1. This method can form a stable amide bond to effectively prevent the enzyme from falling off during use.

[0013] In step S2, the molar ratio of compound II to compound III is 1:1-5.

[0014] In step S2, the mass ratio of compound II to the immobilized lipase is 1:10-50.

[0015] In step S2, the mass-volume ratio of compound II to DMSO is 1:10-100 g / mL.

[0016] In step S2, the addition reaction temperature is 20-40 DEG C, preferably 30-35 DEG C, the stirring rate is 200-300 rpm, preferably 250-280 rpm, and the reaction time is 24 h.

[0017] Compared with the prior art, the present application has the following advantages: 1. In the present application, the selected dimethyl sulfoxide-resistant carrier can effectively resist the dissolution and swelling of the strong polar solvent DMSO, providing a stable microenvironment for the immobilized enzyme, so that it can maintain activity and structural integrity in DMSO for a long time. 2. In the present application, the optimized CALB enzyme is firmly fixed on the carrier by the EDC / NHS covalent immobilization method, and the enzyme is not easy to leak, the prepared immobilized lipase can be reused for ≥10 times, and the conversion rate of compound II can still maintain a high conversion rate when used for the 10th time, greatly reducing the production cost. 3. In the present application, the method avoids the use of noble metal catalysts and toxic chemical reagents, has mild reaction conditions, high stereoselectivity, good atom economy, and meets the development direction of green chemistry. 4. In the present application, the process is simple and easy to scale up: the reaction system is simple, the catalyst can be recovered by filtration or centrifugation after treatment, and it is convenient for continuous operation and industrialized production. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The present application is a general synthetic route. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be further specifically described below through specific embodiments, but the present application is not limited to these embodiments.

[0020] The dimethyl sulfoxide-resistant carrier is used as the immobilization matrix, and the optimized CALB with an amino acid sequence as shown in SEQ ID NO: 2 is covalently combined and fixed on the carrier surface, and the specific steps include: Carrier pretreatment: if the carrier is a silanized modified mesoporous SiO2, methyltriethoxysilane is used as a modifier, and the surface of the mesoporous SiO2 is modified by a sol-gel method or an immersion method, the contact angle of the modified carrier surface is controlled to be 60-80 DEG, and the hydrophobicity and substrate mass transfer are considered at the same time. Carrier activation: mix a dimethyl sulfoxide-resistant carrier selected from one of silanized modified mesoporous SiO2, polyvinylidene fluoride, polymethyl methacrylate with 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to the carrier is 1:5-10, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1, and activate for 1-2 hours at room temperature to generate active ester groups on the surface of the carrier; Covalent immobilization: add the activated carrier to a phosphate buffer with a pH of 7.0-7.5, then add the optimized CALB, and oscillate at a constant temperature of 25°C for 2-4 hours to form an amide bond between the amino group of the enzyme molecule and the active ester group on the surface of the carrier; Post-processing: after the reaction is completed, collect the solid by centrifugation at 8000-10000 rpm, and sequentially wash with a phosphate buffer and DMSO for 3-5 times to remove the unbound free enzyme, and dry at 36°C for 4-6 hours to obtain the immobilized lipase.

[0021] The dimethyl sulfoxide-resistant carrier needs to meet the following parameters: specific surface area ≥100 m 2 / g, pore size 5-20 nm, to match the molecular size of compound II and compound III and ensure mass transfer efficiency, and particle size 50-200 μm, to avoid mechanical wear during stirring.

[0022] Catalytic synthesis of dabigatran intermediate: Addition reaction in the above immobilized lipase and DMSO organic phase with compound II as the substrate, the specific steps including: Reaction system configuration: add DMSO, immobilized lipase, compound II, and compound III in sequence in a 250 mL conical flask, and control the material ratio as follows: molar ratio of compound II to compound III 1:1-5, mass ratio of compound II to immobilized lipase 1:10-50, and mass-volume ratio of compound II to DMSO 1:10-100 g / mL; Catalytic reaction: place the conical flask in a constant-temperature water bath shaker, set the temperature to 20-40°C and the stirring rate to 200-300 rpm, and react for 24 hours; Product separation and enzyme recovery: after the reaction is completed, separate the immobilized lipase by suction filtration and reserve for reuse, detect the conversion rate of the filtrate by HPLC, and purify compound I by recrystallization in an ethanol-water system.

[0023] Repeated use of immobilized lipase: The recovered immobilized lipase was washed with DMSO for 2-3 times, and then the above-mentioned catalytic synthesis step was repeated to achieve ≥10 times of reuse, and the conversion rate of compound II remained more than 70% of the initial conversion rate in the 10th use.

[0024] Raw material preparation: Enzyme source: optimized CALB (amino acid sequence SEQ ID NO: 2, prepared according to the method of CN112342204B example 1, freeze-dried powder activity 1000 U / g); Support: mesoporous SiO2 (specific surface area 200 m 2 / g, pore size 10 nm), polyvinylidene fluoride (PVDF, specific surface area 150 m 2 / g, pore size 8 nm), polymethyl methacrylate (PMMA, specific surface area 120 m 2 / g, pore size 15 nm); Reagents: methyl triethoxysilane (analytical pure), EDC (analytical pure), NHS (analytical pure), potassium dihydrogen phosphate-potassium hydrogen phosphate buffer (0.1 mol / L, pH 7.2), DMSO (analytical pure), compound II (purity ≥98%), compound III (purity ≥98%); Equipment: constant temperature water bath shaker, high speed centrifuge, vacuum drying oven, HPLC (high performance liquid chromatograph, detection conditions: C18 column, mobile phase acetonitrile-water=60:40, flow rate 1 mL / min, detection wavelength 254 nm) Example 1 Catalytic method based on silanized modified mesoporous SiO2 immobilized CALB Preparation of silanized modified mesoporous SiO2: take 10 g of mesoporous SiO2, disperse in 200 mL of toluene, then add 2 mL of methyl triethoxysilane, reflux at 110°C for 4 h. After the reaction is completed, cool to room temperature, wash thoroughly with anhydrous ethanol, filter under suction, and dry at 60°C for 8 h to obtain the hydrophobically modified SiO2 support. The contact angle of the modified support surface is measured to be 75°; Preparation of immobilized lipase: take 5 g of modified mesoporous SiO2, disperse in 5 mL of pH=7.2 phosphate buffer (PBS). Then add 1 g of EDC and 0.6 g of NHS (molar ratio of EDC to NHS 1:1), activate at room temperature for 1.5 h; centrifuge and discard the supernatant, wash the support with PBS twice to remove excess reagents. Disperse the activated support in a PBS solution containing 2 g of optimized CALB, and react at 25°C and 200 rpm for 3 h. After the reaction is completed, collect the immobilized enzyme by centrifugation at 8000 rpm, wash with deionized water and anhydrous ethanol three times each, and freeze-dry to obtain the immobilized lipase. The enzyme loading is 380 U / g; Catalytic synthesis of compound I: 60 mL DMSO, 8 g immobilized lipase, 1 g compound II, 1.88 g compound III (mole ratio 1:2) were added, and the reaction was carried out at 30°C and 280 rpm for 24 h; HPLC detection showed that the conversion rate was ≥69.3%, and the recrystallization purity was ≥94.5%; Repeated use test: the conversion rate was ≥67% after the 10th reaction.

[0025] Example 2 Catalytic method based on PVDF immobilized CALB Preparation of immobilized lipase: 5 g of PVDF carrier was added to 20 mL of phosphate buffer, 0.8 g of EDC and 0.48 g of NHS (EDC to carrier mass ratio 1:6.25) were added, and the mixture was activated at room temperature for 1 h; 1.5 g of optimized CALB was added, and the mixture was shaken at 25°C for 2.5 h; the solid was collected by centrifugation at 10,000 rpm, washed, and dried under vacuum to obtain the immobilized lipase, with an enzyme loading of 320 U / g; Catalytic synthesis of compound I: 60 mL DMSO, 8 g immobilized lipase, 1 g compound II, 1.88 g compound III (mole ratio 1:2) were added, and the reaction was carried out at 30°C and 280 rpm for 24 h; HPLC detection showed that the conversion rate was ≥69.3%, and the recrystallization purity was ≥94.5%; Repeated use test: the conversion rate was ≥67% after the 10th reaction.

[0026] Example 3 Catalytic method based on PMMA immobilized CALB Preparation of immobilized lipase: 5 g of PMMA carrier was added to 20 mL of phosphate buffer, 1 g of EDC and 0.6 g of NHS were added, and the mixture was activated at room temperature for 2 h; 2 g of optimized CALB was added, and the mixture was shaken at 25°C for 4 h; after washing and drying, the immobilized lipase was obtained, with an enzyme loading of 350 U / g; Catalytic synthesis of compound I: 60 mL DMSO, 8 g immobilized lipase, 1 g compound II, 1.88 g compound III (mole ratio 1:2) were added, and the reaction was carried out at 30°C and 280 rpm for 24 h; HPLC detection showed that the conversion rate was ≥69.3%, and the recrystallization purity was ≥94.5%; Repeated use test: the conversion rate was ≥68% after the 10th reaction.

[0027] Example 4 Method for immobilizing CALB based on unmodified mesoporous SiO2 Immobilization step: 5 g of unmodified mesoporous SiO2 (contact angle 30°) was activated and immobilized according to the method of Example 1 to obtain immobilized lipase with an enzyme loading of 300 U / g; Catalysis and reuse: the initial conversion rate was > 65%, but when used for the third time, the enzyme shedding rate exceeded 30% due to slight swelling of the carrier in DMSO, and the conversion rate was ≤ 48%, so it could not be reused continuously.

[0028] Example 5 Method for immobilizing CALB based on adsorption Immobilization step: 5 g of PVDF carrier was directly added to 2 g of phosphoric acid buffer of the optimized CALB, without using the EDC / NHS system for activation, and was adsorbed for 2 h at 25°C, and then was washed and dried to obtain the adsorbed immobilized enzyme; Catalysis and reuse: the initial conversion rate was ≥ 62%, the enzyme shedding rate exceeded 25% when used for the second time, the conversion rate was ≤ 50%, and the conversion rate was only 35% when used for the fifth time.

[0029] Example 6 This example is a comparative example of Example 2 in CN112342204B Step: in a 250 ml conical flask, 60 ml of DMSO was added, followed by the addition of 10 g of CALB optimized by gene, 1 g of compound II, and 0.94 g of compound III, and then the reactor was added, stirred at 200 rpm, and reacted at 20°C for 24 h to obtain compound I. The reaction result was detected by HPLC, and the conversion rate was 70%, and the recrystallization purity was 90%.

[0030] Result: the conversion rate was 70%, but the enzyme could not be recovered, the single-enzyme cost was too high; and when the second batch of reaction was carried out, the conversion rate decreased to 65% due to the activity attenuation of the free enzyme during storage, and the batch difference was not less than 5%.

[0031] The embodiments of the present application are not limited to the above examples, and those skilled in the art can make various changes and improvements in form and details without departing from the spirit and scope of the present application, and these are considered to fall within the protection scope of the present application.

Claims

1. A method for the synthesis of a dabigatran intermediate catalyzed by immobilized lipase, characterized in that, The method comprises the following steps: S1: preparation of immobilized lipase: using dimethyl sulfoxide-resistant carrier as immobilized carrier, using covalent binding method, fixing the amino acid sequence shown in SEQ ID NO: 2 optimized CALB on the dimethyl sulfoxide-resistant carrier to obtain the immobilized lipase; S2: catalytic synthesis of dabigatran intermediate: using compound II as substrate, in the immobilized lipase prepared in step S1, DMSO organic phase, carrying out addition reaction of compound II and compound III to form C-N bond to generate compound I; In step S1, the dimethyl sulfoxide-resistant carrier is selected from one of silanized modified mesoporous SiO2, polyvinylidene fluoride and polymethyl methacrylate; In step S2, the molar ratio of compound II to compound III is 1:1-5; In step S2, the mass ratio of compound II to immobilized lipase is 1:10-50; In step S2, the mass-volume ratio of compound II to DMSO is 1:10-100 g / mL; In step S2, the addition reaction temperature is 20-40℃, the stirring rate is 200-300 rpm, and the reaction time is 24 h.

2. The method for catalytic synthesis of intermediate of dabigatran based on immobilized lipase according to claim 1, characterized in that: In step S1, the modifier of the silanized modified mesoporous SiO2 is methyl triethoxysilane, and the contact angle of the modified carrier surface is 60°-80°.

3. The method for synthesizing dabigatran intermediates based on immobilized lipase catalysis according to claim 1, characterized in that: The specific surface area of the dimethyl sulfoxide-resistant carrier in step S1 is ≥ 100 m 2 / g, with a pore size of 5-20 nm and a particle size of 50-200 μm.

4. The method for catalytic synthesis of intermediate of dabigatran based on immobilized lipase according to claim 1, characterized in that: In step S1, the specific operation of the covalent binding method comprises: after the dimethyl sulfoxide-resistant carrier is activated with 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, the optimized CALB is reacted in pH 7.0-7.5 phosphate buffer at 25℃ for 2-4 h, after the reaction is completed, the solid is collected by centrifugation, washed and dried to obtain the immobilized lipase; The mass ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to the carrier is 1:5-10; The molar ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to N-hydroxysuccinimide is 1:

1.

5. The method for catalytic synthesis of intermediate of dabigatran based on immobilized lipase according to claim 1, characterized in that: In step S2, the addition reaction temperature is 30-35℃, and the stirring rate is 250-280 rpm.

6. The method for catalytic synthesis of intermediate of dabigatran based on immobilized lipase according to claim 1, characterized in that: The immobilized lipase prepared in step S1 can be reused for ≥10 times, and the conversion rate of compound II remains more than 70% of the initial conversion rate when used for the 10th time.

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