Method for continuously preparing ezetimibe intermediate through enzyme immobilization

Abstract

The invention discloses a method for continuously preparing an ezetimibe intermediate through enzyme immobilization, and belongs to the technical field of chemical synthesis. The method comprises the following steps: firstly, filling a Pickering emulsion into a fixed bed reactor, inputting a reaction solution containing a compound 2 from the upper end of a fixed bed, adding a cofactor regenerant into the reaction solution, collecting a product solution from the lower end of the fixed bed, and performing vacuum concentration to obtain a target compound 1; the Pickering emulsion is a water-in-oil type emulsion immobilized with ketoreductase and cofactors, an oil phase of the emulsion is prepared by dissolving hydrophobic SiO2 nanoparticles in an organic solvent, and a water phase of the emulsion is prepared by adding the ketoreductase, the cofactors and a coupling agent into a buffer solution. According to the present invention, the enzyme catalysis reaction is performed based on the Pkernel emulsion, such that the continuous multiple cycles can be achieved, the continuous operation time can achieve more than 400 h, the total conversion number of the cofactors can achieve more than 10000, the post-treatment is simple, the enzyme and cofactor consumption is reduced, the generated wastewater amount is reduced, the production efficiency is substantially improved, and the production cost is saved.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis, and particularly relates to a method for continuously preparing an ezetimibe intermediate by enzyme immobilization. Background Art

[0002] Ezetimibe, jointly developed by Schering-Plough and Merck, is the world's first selective cholesterol absorption inhibitor lipid-lowering drug. It was approved by the US FDA in 2002 and has become an important product in the lipid-lowering drug market. It can be used to treat hypercholesterolemia and can also be used in combination with statins. It has broad market prospects. The structural formula of ezetimibe is as follows:

[0003]

[0004] The structural formula of the key intermediate for the synthesis of ezetimibe, namely compound 1, is shown below. Its chemical name is (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxy-1-oxopentyl]-4-phenyl-2-oxazolidinone.

[0005]

[0006] The methods reported so far for synthesizing compound 1 can be mainly divided into chemical synthesis and enzyme-catalyzed asymmetric synthesis. The chemical synthesis method uses CBS chiral catalyst and borane dimethyl sulfide as reducing agent to asymmetrically reduce the carbonyl group to chiral hydroxyl group. This method requires low temperature reaction and strict requirements on reaction conditions. In addition, the chiral catalyst is expensive and the production cost is high. In addition, the reducing agent borane dimethyl sulfide will produce toxic dimethyl sulfide gas with a foul odor, which can form an explosive mixture with air, posing a high safety risk.

[0007] The enzyme-catalyzed asymmetric synthesis method uses ketoreductase, and with the participation of a cofactor, the carbonyl is reduced to a chiral hydroxyl. This method solves the cost and safety problems of the chemical synthesis method well, and the reaction conditions are mild, easy to operate, efficient, green, environmentally friendly, and has strong specificity and high stereoselectivity. However, this method relies on expensive cofactors to participate in the reaction, and the existing intermittent enzyme-catalyzed reaction system cannot realize the recycling of coenzymes, and the product and enzyme solution are difficult to separate during post-treatment, which further increases the production cost. Therefore, realizing the recycling of ketoreductase and cofactors, simplifying post-treatment operations, and improving production efficiency are key issues that need to be urgently solved in the industrial production of ezetimibe intermediate 1.

[0008] In recent years, some enzyme immobilization methods have been developed and applied to the synergistic catalytic reaction of two enzymes, such as physical adsorption, chemical bonding, sol-gel method, etc. Physical adsorption refers to the immobilization of ketone reductase and cofactors through interactions such as hydrogen bonds, van der Waals forces and electrostatic forces. However, due to the weak physical adsorption force, the enzyme and cofactor are very easy to fall off from the substrate material into the reaction system during the reaction, so it is difficult to achieve long-term enzyme recycling and cofactor recycling. Regeneration. Chemical bonding and sol-gel methods refer to the effective immobilization of enzymes by chemical bonding between the substrate material and the enzyme. However, the action of chemical bonds will cause the conformation of the enzyme to change and then cause changes in enzyme activity, thereby reducing the reaction efficiency. Existing enzyme immobilization methods are difficult to achieve the fixation of ketone reductase and cofactors without activity loss and maintain long-term recycling stability.

[0009] In addition, reducing the amount of cofactors while achieving cofactor recycling is also an important goal in the industrialization process. In the co-immobilization process of ketoreductase and cofactors, achieving the proximity of ketoreductase and cofactors at the molecular level can promote more efficient binding and reaction between ketoreductase and cofactors, thereby reducing the amount of cofactors used. Summary of the invention

[0010] In view of the above-mentioned problems, the present invention aims to provide a method for continuously preparing an ezetimibe intermediate by enzyme immobilization, in which an enzyme-catalyzed reaction is carried out in an oil-in-water Pickering emulsion that immobilizes ketoreductase and cofactors, and multiple cycles can be carried out continuously, the operating time can reach more than 400 hours, the total conversion number of the cofactor can reach more than 10,000, the post-processing is simple, the amount of enzyme and cofactor used is reduced, the production efficiency is greatly improved, and the production cost can be significantly saved.

[0011] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present invention is as follows: a method for continuously preparing an ezetimibe intermediate by enzyme immobilization, firstly, a Pickering emulsion is loaded into a fixed bed reactor, a reaction solution containing compound 2 is input from the upper end of the fixed bed, a cofactor regenerator is added to the reaction solution, a product solution is collected from the lower end of the fixed bed, and the target compound 1 is obtained by decompression concentration; the Pickering emulsion is an oil-in-water type emulsion with immobilized ketoreductase and cofactor, the oil phase of the emulsion is prepared by dissolving hydrophobic SiO2 nanoparticles in an organic solvent, and the aqueous phase is prepared by adding ketoreductase, cofactor and linker into a buffer solution;

[0012] The reaction equation for compound 2 to generate compound 1 is as follows:

[0013]

[0014] Furthermore, the cofactor regenerator is one of glucose and glucose dehydrogenase, isopropanol, ammonium formate and ammonium formate dehydrogenase, 2-octanol, and 2-heptanol.

[0015] Furthermore, the reaction temperature of the fixed bed reactor is 25-50° C., and the reaction liquid flow rate is 10-500 mL / h.

[0016] Furthermore, the preparation process of hydrophobic SiO2 nanoparticles is as follows: ultrasonically disperse the SiO2 nanoparticles in an organic solvent, add hydrophobic silane and triethylamine, and stir and reflux at 100-120°C for 3-6 hours under nitrogen protection; centrifuge after cooling, wash with toluene 3-5 times, and dry in a vacuum drying oven at 50-100°C to obtain hydrophobic SiO2 nanoparticles with interface activity.

[0017] Furthermore, the SiO2 nanoparticles are The hydrophobic silane is prepared by a synthetic method and has a size of 60 to 80 nm; the hydrophobic silane is selected from one of methyltrimethoxysilane, dichlorodimethylsilane and octyltrimethoxysilane.

[0018] Furthermore, the molar ratio of SiO2 nanoparticles to hydrophobic silane is 1:0.06-0.6, and the molar ratio of hydrophobic silane to triethylamine is 1:0.4-3.

[0019] Furthermore, the organic solvent used to prepare the oil phase is one of toluene, n-heptane, hexane, ethyl acetate, methyl tert-butyl ether or octane; the ketoreductase is selected from one or more of ES-KRED-101 to ES-KRED-287 and their mutants; the cofactor is selected from nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP + ); the linker is selected from one of polyacrylic acid, polyethyleneimine, tetraethylenepentamine, and pentaethylenehexamine; the buffer solution is PBS buffer solution with a concentration of 80 to 200 mM.

[0020] Furthermore, the ketoreductase content in the buffer solution is 1-500 μL / mL, the amount of the cofactor is 0.01-0.03 mg / mL; the amount of hydrophobic SiO2 nanoparticles added per milliliter of buffer solution is 0.04-0.08 g; and the volume ratio of the buffer solution to the organic solvent is 1:0.5-10.

[0021] Furthermore, the water-oil phases of the Pickering emulsion are mixed by pouring method, and then stirred at high speed to obtain a water-in-oil Pickering emulsion with uniform particle size for immobilizing ketoreductase and cofactor.

[0022] Furthermore, the high-speed stirring speed is 5000-10000 rpm, and the diameter of the obtained Pickering emulsion droplets is 5-300 μm.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present application loads a Pickering emulsion encapsulating ketoreductase and cofactors into a fixed bed reactor for enzyme catalysis to prepare a key intermediate compound 1 for synthesizing ezetimibe. The operation is simple, and multiple cycles can be performed continuously. The continuous operation time can reach more than 400 hours, and the total conversion number of the cofactor can reach more than 10,000.

[0025] 2. The present application uses Pickering emulsion to achieve efficient and continuous preparation of the key intermediate compound 1 of ezetimibe, which has simple post-treatment, reduced enzyme and cofactor usage, reduced wastewater generation, and greatly improved production efficiency, which can effectively save production costs;

[0026] 3. The cofactor regenerator flows into the fixed bed reactor together with the reaction liquid containing reactant 2, and flows out of the fixed bed reactor with the continuous phase after the cofactor immobilized in the emulsion is regenerated by reaction, thus realizing the recycling and utilization of the cofactor in the confined space; and after multiple continuous cycle reactions, the cofactor is still distributed inside the emulsion droplets, and the amount of the cofactor inside the emulsion droplets does not decrease significantly compared to before the reaction, thus realizing the loss-free encapsulation of the cofactor;

[0027] 4. The present application uses a water-in-oil Pickering emulsion enzyme to catalyze the preparation of compound 1. Ketoreductase and cofactors are in situ encapsulated inside the emulsion droplets, and the ketoreductase tends to aggregate and distribute at the interface. Under the electrostatic adsorption of the linker, the cofactor is enriched at the reaction interface, increasing the concentration of the cofactor at the interface. The cofactor enriched at the interface combines with the ketoreductase adsorbed at the emulsion interface, and the cofactor and ketoreductase exert efficient synergistic catalytic effects in the confined space of the emulsion droplets. When the mobile phase containing compound 2 flows through the fixed bed reactor, compound 2 will react rapidly to generate compound 1.

[0028] 5. In the preparation of Pickering emulsion, the aqueous phase of the present application contains ketoreductase, cofactor and linker. The role of the linker is to enrich the cofactor at the interface, so that the cofactor can efficiently combine with the ketoreductase at the interface, accelerate the reaction process, shorten the reaction time, and achieve more reaction cycles within the time when the ketoreductase maintains its activity; and based on the role of the linker, the amount of cofactor can be significantly reduced. It has been verified that, under the premise of the same catalytic effect, the amount of cofactor required in the emulsion without the addition of the linker is four times that of the emulsion with the addition of the linker;

[0029] 6. The structure and conformation of the ketoreductase aggregated and distributed at the interface of the emulsion droplets will not change, and its activity can be fully retained. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the related substance spectrum of the standard of compound 1;

[0031] Figure 2 The relevant material spectrum of the final product prepared in Example 1;

[0032] Figure 3 This is the chiral purity spectrum of the final product prepared in Example 1. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0034] Example 1

[0035] Step 1: Ultrasonic disperse 100g of dried SiO2 particles (particle size is about 60nm) into 1200mL of toluene, add 60g of octyltrimethoxysilane and 24g of triethylamine, stir and reflux at 110°C for 4h under nitrogen protection, centrifuge the mixed system after cooling, wash the obtained solid with toluene 3 to 5 times, and dry it in a vacuum drying oven at 60°C for 4h to obtain hydrophobic SiO2 nanoparticles with interface activity.

[0036] Step 2: 36 mL of ketoreductase (ES-KRED-101) solution, 5.6 mg of cofactor NADP + , 10 mmol polyethyleneimine was added into 320 mL 100 mM PBS buffer, and the mixture was evenly mixed by magnetic stirring to obtain an aqueous phase system; then 15.8 g of the hydrophobic SiO2 nanoparticles obtained in step 1 were ultrasonically dispersed in 800 mL n-heptane to obtain an oil phase system, and the oil-water phases were mixed by pouring method; a Pickering emulsion (the diameter of the Pickering emulsion droplets was 5 to 300 μm) of immobilized ketoreductase and cofactors was formed by high-speed stirring at 5000 rpm, and the excess oil phase was sucked out.

[0037] Step 3: The Pickering emulsion obtained in step 2 was transferred to a fixed bed reactor with an inner diameter of 20 cm, the reactor temperature was set to 30°C, and a 0.1 mol / L ethyl acetate solution of compound 2 containing 0.12 mol / L isopropanol was introduced through a constant flow pump at a flow rate of 20.0 mL / h to carry out a reduction reaction. The reaction was continued for 400 hours, the conversion rate could be maintained at more than 95%, and the total conversion number of the cofactor reached more than 10,000. The product solution was concentrated under reduced pressure to obtain compound 1 as a white solid.

[0038] The reaction equation for converting compound 2 into compound 1 is as follows:

[0039]

[0040] Figure 1 and Figure 2 The relevant material spectra of the standard product of compound 1 and the final product prepared in this example are shown respectively. After comparing the retention times of the main peaks of the two figures, it is found that the product prepared in this example is determined to be compound 1, and the purity is greater than 99%. Figure 3 This is the chiral purity spectrum of the product prepared in this example. From the figure, it can be seen that the compound 1 prepared in this example has high chiral purity and the ee value is greater than 99%.

[0041] Example 2

[0042] Step 1: Ultrasonic disperse 100 g of dried SiO2 particles (particle size is about 80 nm) into 1500 mL of toluene, add 35 g of dichlorodimethylsilane and 65 g of triethylamine, stir and reflux at 60 ° C for 4 hours under nitrogen protection, centrifuge the mixed system after cooling, wash the obtained solid with toluene 3 to 5 times, and dry it in a vacuum drying oven at 70 ° C for 2 hours to obtain hydrophobic SiO2 nanoparticles with interface activity.

[0043] Step 2: 72 mL of ketoreductase solution (ES-KRED-101), 11.2 mg of NADP + , 10 mmol of polyacrylic acid was added into 640 mL of 100 mM PBS buffer, and the mixture was evenly mixed by magnetic stirring to obtain an aqueous phase system; 31.6 g of the hydrophobic SiO2 nanoparticles obtained in step 1 were ultrasonically dispersed in 1600 mL of n-heptane to obtain an oil phase system, and the oil and water phases were mixed by pouring method; a Pickering emulsion of immobilized ketoreductase and cofactor was formed by high-speed stirring at 5000 rpm, and then the excess oil phase was sucked out.

[0044] Step 3: The Pickering emulsion obtained in step 2 is transferred to a fixed bed reactor with an inner diameter of 30 cm, the reactor temperature is set to 40°C, and a 0.2 mol / L ethyl acetate solution of compound 2 containing 0.24 mol / L 2-octanol is introduced through a constant flow pump at a flow rate of 40.0 mL / h to carry out a reduction reaction. The reaction is continued for 400 hours, the conversion rate can be maintained at more than 95%, and the total conversion number of the cofactor reaches more than 10,000. The product solution is concentrated under reduced pressure to obtain compound 1 as a white solid, with an ee value greater than 99% and a purity greater than 99%.

[0045] Example 3

[0046] Step 1: Ultrasonic disperse 300 g of dried SiO2 particles (particle size is about 80 nm) into 3600 mL of toluene, add 180 g of octyltrimethoxysilane and 36 g of triethylamine, stir and reflux at 110°C for 4 hours under nitrogen protection, centrifuge the mixed system after cooling, wash the obtained solid with toluene 3 to 5 times, and dry it in a vacuum drying oven at 80°C for 4 hours to obtain hydrophobic SiO2 nanoparticles with interface activity.

[0047] Step 2: 108 mL of ketoreductase (ES-KRED-287) solution, 16.8 mg of NADP + , 30mmol polyethyleneimine was added into 960mL 100mM PBS buffer, and the mixture was evenly mixed by magnetic stirring to obtain an aqueous phase system; then 47.4g of the hydrophobic SiO2 nanoparticles obtained in step 1 were ultrasonically dispersed in 2400mL n-heptane to obtain an oil phase system, and the oil and water phases were mixed by pouring method; a Pickering emulsion of immobilized ketoreductase and cofactor was formed by high-speed stirring at 5000rpm, and then the excess oil phase was sucked out.

[0048] Step 3: The Pickering emulsion obtained in step 2 was transferred to a fixed bed reactor with an inner diameter of 50 cm, the reactor temperature was set to 30°C, and a 0.3 mol / L ethyl acetate solution of compound 2 containing 0.36 mol / L isopropanol was introduced through a constant flow pump at a flow rate of 100.0 mL / h to carry out a reduction reaction. The reaction was continued for 400 hours, the conversion rate could be maintained at more than 95%, and the total conversion number of the cofactor reached more than 10,000. The product solution was concentrated under reduced pressure to obtain compound 1 as a white solid, with an ee value greater than 99% and a purity greater than 99%.

[0049] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any technician in the field without departing from the technical solution of the present invention should be included in the scope of the present invention.

Claims

1. A method for continuously preparing an ezetimibe intermediate by enzyme immobilization, characterized in that: The Pickering emulsion is loaded into a fixed bed reactor, and a reaction solution containing compound 2 is input from the upper end of the fixed bed. A cofactor regenerator is added to the reaction solution, and the product solution is collected from the lower end of the fixed bed and concentrated under reduced pressure to obtain the target compound 1; The Pickering emulsion is an oil-in-water type emulsion that immobilizes ketoreductase and cofactors. The oil phase of the emulsion is prepared by dissolving hydrophobic SiO2 nanoparticles in an organic solvent, and the water phase is prepared by adding ketoreductase, cofactors and linkers into a buffer solution. The reaction equation for compound 2 to generate compound 1 is as follows:

2. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 1, characterized in that: The cofactor regeneration product is one of glucose and glucose dehydrogenase, isopropanol, ammonium formate and ammonium formate dehydrogenase, 2-octanol and 2-heptanol. The molar ratio of reactant 2 to the cofactor regeneration product in the reaction solution is 1:1-1.

5.

3. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 1, characterized in that: The reaction temperature of the fixed bed reactor is 25-50° C., and the reaction liquid flow rate is 10-500 mL / h.

4. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 1, characterized in that: The preparation process of hydrophobic SiO2 nanoparticles is as follows: ultrasonically disperse SiO2 nanoparticles in an organic solvent, add hydrophobic silane and triethylamine, and stir and reflux at 100-120°C for 3-6 hours under nitrogen protection; centrifuge after cooling, wash with toluene 3-5 times, and dry in a vacuum drying oven at 50-100°C to obtain hydrophobic SiO2 nanoparticles with interface activity.

5. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 4, characterized in that: The SiO2 nanoparticles are The hydrophobic silane is prepared by a synthetic method and has a size of 60 to 80 nm; the hydrophobic silane is selected from one of methyltrimethoxysilane, dichlorodimethylsilane and octyltrimethoxysilane.

6. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 4, characterized in that: The molar ratio of SiO2 nanoparticles to hydrophobic silane is 1:0.06-0.6, and the molar ratio of hydrophobic silane to triethylamine is 1:0.4-3.

7. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 1, characterized in that: The organic solvent is one of toluene, n-heptane, hexane, ethyl acetate, methyl tert-butyl ether or octane; the ketoreductase is selected from one or more of ES-KRED-101 to ES-KRED-287 and mutants thereof; the cofactor is selected from one of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; the linker is selected from one of polyacrylic acid, polyethyleneimine, tetraethylenepentamine and pentaethylenehexamine; and the buffer solution is a PBS buffer solution with a concentration of 80 to 200 mM.

8. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 1, characterized in that: The ketoreductase content in the buffer solution is 1-500 μL / mL, the dosage of the cofactor is 0.01-0.03 mg / mL; the amount of the hydrophobic SiO2 nanoparticles added to each milliliter of the buffer solution is 0.04-0.08 g; and the volume ratio of the buffer solution to the organic solvent is 1:0.5-10.

9. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 1, characterized in that: The water-oil phases of the Pickering emulsion are mixed by pouring method, and then stirred at high speed to obtain a water-in-oil Pickering emulsion with uniform particle size for immobilizing ketoreductase and cofactor.

10. The method for continuously preparing an ezetimibe intermediate by enzyme immobilization according to claim 9, characterized in that: The high-speed stirring speed is 5000-10000 rpm, and the particle size of the Pickering emulsion droplets is 5-300 μm.

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