Carbonyl reductase immobilization process and application of carbonyl reductase immobilization process in ezetimibe intermediate synthesis

Abstract

The invention discloses a carbonyl reductase immobilization process and application of the carbonyl reductase immobilization process in synthesis of an ezetimibe intermediate, immobilized enzyme particles are prepared through activation of epoxy resin and loading of carbonyl reductase and glucose dehydrogenase, and then ZT-4 is catalyzed by the immobilized enzyme particles to chirally synthesize a ZT-5 product. The method disclosed by the invention has the beneficial effects that the enzyme can be repeatedly utilized through an enzyme immobilization process, so that the use cost of the enzyme is effectively reduced, and the use amount of expensive coenzyme is reduced through a glucose regeneration method, so that the production cost is effectively saved; according to the method, the treatment step of free enzyme is omitted by separating the immobilized enzyme, so that the reaction short batch interval can be effectively shortened, the generation of waste gas in the reaction post-treatment is reduced, the environmental protection property of the reaction is ensured, the production requirement of green industrialization is met, and the method is also suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of enzyme-catalyzed synthesis of pharmaceutical intermediates, specifically a carbonyl reduction method. Background Technology

[0002] Ezetimibe is a novel cholesterol absorption inhibitor jointly developed by Schering-Plough and Merck. It is the first selective cholesterol absorption inhibitor to be approved by the U.S. FDA. Its brand name is "EZETROL", and its chemical name is (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-2-azacyclobutanecarbonyl.

[0003] Its mechanism of action is to inhibit the absorption of cholesterol from food in the intestines, reduce the transport of cholesterol from the small intestine to the liver, and lower plasma cholesterol levels. It can be used for various types of hyperlipidemia, especially for patients who do not respond well to or cannot tolerate statins, as well as some patients with hereditary and drug-induced hyperlipidemia. Merck's market sales reached $2.52 billion in 2015, and it has become a blockbuster drug in the lipid-lowering drug market with a broad market prospect. With its patent expiring in 2016, the development and production of this drug will have significant social and economic benefits.

[0004] (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazacyclopentane-2-carbonyl is a key chiral intermediate in the preparation of the drug ezetimibe. Currently, the reduction methods for producing (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazacyclopentane-2-carbonyl can be divided into chemical and biological methods. Chemical methods produce small amounts of isomers, affecting the quality of the final product; biological methods, with their mild reaction conditions, high stereospecificity, and high conversion rate, are widely studied and applied. However, most biological methods for producing this intermediate currently utilize liquid enzymes for the reaction. Compared with immobilized enzymes, each batch of reaction requires a new enzyme solution, which makes separation difficult, the operation cumbersome, and reduces the utilization efficiency of the enzyme. The waste liquid is not recyclable, generating a large amount of waste pollutants. At the same time, the product may contain microbial impurities such as proteins, which leads to the need for more complex post-processing and purification processes.

[0005] However, to date, there is no mature immobilized enzyme-catalyzed synthesis process for the preparation of ezetimibe intermediates, and further development of the production process conditions for the immobilized enzymes specifically required for this reaction is also lacking. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a carbonyl reductase immobilization process and its application in the synthesis of ezetimibe intermediates. The immobilized enzyme is obtained through a carbonyl reductase immobilization process and then applied to the synthesis of ezetimibe intermediates, enabling chiral catalysis and further optimizing the synthesis process of ezetimibe intermediates. The technical solution is as follows: A carbonyl reductase immobilization process and its application in the synthesis of ezetimibe intermediates include the following steps: The first step is the enzyme immobilization process. The epoxy resin carrier was washed three times with deionized water, vacuum filtered until semi-dry, then soaked in PBS buffer for 2 hours to swell, washed with deionized water, dried, and then soaked in 1M arginine solution to obtain the activated epoxy resin carrier. Carbonyl reductase and glucose dehydrogenase were dissolved sequentially in PBS buffer, and NADP⁺ was added to adjust the concentration to 5 mM. The enzyme solution was mixed with the activation carrier and cross-linked by microwave irradiation. After irradiation, Tris-HCl was added and the mixture was shaken at 25°C for 2 hours to obtain immobilized enzyme particles. Finally, the immobilized enzyme particles were washed three times with 0.1 M phosphate buffer and then stored in a buffer containing 5 mM glucose at 4°C. The second step is the catalytic synthesis process. In a reaction flask, 0.1M phosphate buffer (pH 6.5), ZT-4, immobilized enzyme particles, and glucose were added sequentially. The temperature was slowly raised to 35°C, and the mixture was stirred at 150 rpm for 6–8 hours. The reaction was considered complete when the residual raw material was less than 7%. After the reaction, the immobilized enzyme was recovered by filtration through a 100-mesh sieve, washed with PBS buffer, and directly added to the next batch. The reaction solution was directly filtered to obtain a clear filtrate. The aqueous phase was extracted three times with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and then desolventized under reduced pressure to obtain crude ZT-5.

[0007] Furthermore, the carbonyl reductase used in the first step has an activity of ≥500 U / mg, the glucose dehydrogenase used has an activity of ≥300 U / mg, and the mass ratio of the carbonyl reductase to the glucose dehydrogenase is 1:1.

[0008] Furthermore, the epoxy resin used in the first step is LX-1000HA resin.

[0009] Furthermore, the pH of the PBS buffer used in the first step is 7.5, the pH of the PBS buffer used for washing in the first step is 6.5, and the pH of the Tris-HCl reagent used is 8.0.

[0010] Furthermore, the microwave reactor irradiation crosslinking is achieved by placing the enzyme solution and the activated carrier mixture at 4°C in a 40W microwave reactor and irradiating for 3 minutes.

[0011] Furthermore, in the second step, the immobilized enzyme particles are added by adding the storage solution obtained in the first step, and the amount added is 20% v / v of the reaction system.

[0012] Furthermore, the concentration of the substrate ZT-4 in the second step reaction is 0.1–0.3 g / ml.

[0013] Furthermore, the pH of the PBS buffer used in the second step is 6.5.

[0014] Furthermore, the concentration of glucose in the reaction solution in the second step is 100 mM.

[0015] Furthermore, the carbonyl reductase is derived from frozen stem cells of *S. chizosaccharomycetes so ctosporus* ATCC 2 4 7 9, as disclosed in Chinese Patent CN112458143A.

[0016] The beneficial effects of this invention are: 1. Enzyme immobilization allows enzymes to be reused multiple times, effectively reducing enzyme usage costs. Glucose regeneration reduces the amount of expensive coenzymes used, effectively saving production costs; 2. By separating and immobilizing enzymes, the treatment steps of free enzymes are omitted, effectively shortening batch intervals and reducing the generation of waste gases in post-reaction processing, ensuring the environmental friendliness of the reaction, meeting the production requirements of green industrialization, and suitable for large-scale production. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the reaction route of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 The first step is the enzyme immobilization process. Weigh 100g of epoxy resin carrier and wash it three times with deionized water to ensure the removal of surface impurities. After each wash, use vacuum filtration to remove water until it is semi-dry. Immerse the carrier in 0.1M PBS buffer (pH 7.5) for 2 hours to allow it to fully swell and increase its surface activity. Remove the carrier and rinse it with deionized water to remove excess PBS buffer. Then allow the carrier to air dry. The dried carrier was immersed in 1M arginine solution (deionized water) for 1 hour to increase the hydrophilicity of the carrier and increase the enzyme loading. After immersion, it was rinsed with deionized water to obtain the activated epoxy resin carrier. 10,000 U of ZT8001 carbonyl reductase (≥500 U / mg) and 6,000 U of GDH (≥300 U / mg) were dissolved in 200 mL of PBS buffer (pH 7.5). NADP⁺ was added to adjust the concentration to 5 mM. The enzyme solution was mixed with the activation carrier and placed in a microwave reactor (40 W, 4 °C) for 3 minutes to promote rapid cross-linking. After irradiation, 50 mL of 1 M Tris-HCl (pH 8.0) was added, and the mixture was shaken at 25 °C for 2 hours to block unreacted epoxy groups. The immobilized enzyme particles were filtered and washed three times with 0.1 M phosphate buffer. The immobilized enzyme particles were then stored in a buffer containing 5 mM glucose at 4 °C. The second step is the catalytic synthesis process. In the reaction flask, 160 ml of 0.1 M phosphate buffer (pH 6.5), 21 g of ZT-4, and immobilized enzyme particles (40 ml of glucose buffer for storing the immobilized enzyme particles) were added sequentially. Glucose was added to adjust the concentration to 100 mM. The temperature was slowly raised to 35°C, and the mixture was stirred at 150 rpm for 6–8 hours until the residual raw material was less than 7%, at which point the reaction was considered complete. After the reaction, the immobilized enzyme particles were recovered by filtration through a 100-mesh sieve. After rinsing with PBS buffer, the mixture was directly added to the next batch. The reaction solution was directly filtered to obtain a clear filtrate. The aqueous phase was extracted three times with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and then subjected to desolventization under reduced pressure to obtain crude ZT-5 with a yield of 92.5% and a purity of 93.2%.

[0020] Example 2 In the reaction flask, 160 ml of 0.1 M phosphate buffer (pH 6.5) was added, followed by 63 g of ZT-4, immobilized enzyme particles (40 ml of glucose buffer for storing the immobilized enzyme particles), and glucose was added to adjust the concentration to 100 mM. The temperature was slowly raised to 35°C, and the mixture was stirred at 150 rpm for 6–8 hours until the residual raw material was less than 7%, at which point the reaction was considered complete. After the reaction, the immobilized enzyme particles were recovered by filtration through a 100-mesh sieve, washed with PBS buffer, and directly added to the next batch. The reaction solution was directly filtered to obtain a clear filtrate. The aqueous phase was extracted three times with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and then desolventized under reduced pressure to obtain crude ZT-5 with a yield of 93.2% and a purity of 94.0%.

[0021] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0022] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A carbonyl reductase immobilization process and its application in the synthesis of ezetimibe intermediates, characterized in that... Includes the following steps: The first step is the enzyme immobilization process. The epoxy resin carrier was washed three times with deionized water, vacuum filtered until semi-dry, then soaked in PBS buffer for 2 hours to swell, washed with deionized water, dried, and then soaked in 1M arginine solution to obtain the activated epoxy resin carrier. Carbonyl reductase and glucose dehydrogenase were dissolved sequentially in PBS buffer, and NADP⁺ was added to adjust the concentration to 5 mM. The enzyme solution was mixed with the activation carrier and cross-linked by microwave irradiation. After irradiation, Tris-HCl was added and the mixture was shaken at 25°C for 2 hours to obtain immobilized enzyme particles. Finally, the immobilized enzyme particles were washed three times with 0.1 M phosphate buffer and then stored in a buffer containing 5 mM glucose at 4°C. The second step is the catalytic synthesis process. In the reaction flask, 0.1M phosphate buffer (pH 6.5), ZT-4, immobilized enzyme particles, and glucose were added sequentially. The temperature was slowly raised to 35°C, and the mixture was stirred at 150 rpm for 6–8 hours. The reaction was considered complete when the residual raw material was less than 7%. After the reaction, the immobilized enzyme was recovered by filtration through a 100-mesh sieve. After rinsing with PBS buffer, the enzyme was directly added to the next batch. The reaction solution was directly filtered to obtain a clear filtrate. 30% sulfuric acid was added to the filtrate to adjust the pH to 3–4. The aqueous phase was extracted three times with dichloromethane. The organic phases were combined, washed with saturated brine, dried with anhydrous Na2SO4, filtered, and then desolventized under reduced pressure to obtain crude ZT-5.

2. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The carbonyl reductase used in the first step has an activity of ≥500 U / mg, and the glucose dehydrogenase used has an activity of ≥300 U / mg. The mass ratio of the carbonyl reductase to the glucose dehydrogenase is 1:

1.

3. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The epoxy resin used in the first step is LX-1000HA resin.

4. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The pH of the PBS buffer used in the first step is 7.5, the pH of the PBS buffer used for washing in the first step is 6.5, and the pH of the Tris-HCl reagent used is 8.

0.

5. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The microwave reactor crosslinking process involves placing the enzyme solution and the activated carrier mixture at 4°C in a 40W microwave reactor and irradiating for 3 minutes.

6. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: In the second step, the immobilized enzyme particles are added by adding the storage solution obtained in the first step, and the amount added is 20% v / v of the reaction system.

7. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The concentration of the substrate ZT-4 in the second step reaction is 0.1–0.3 g / ml.

8. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The pH of the PBS buffer used in the second step is 6.

5.

9. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The concentration of glucose in the reaction solution in the second step is 100 mM.

10. The carbonyl reductase immobilization process according to claim 1 and its application in the synthesis of ezetimibe intermediates, characterized in that: The carbonyl reductase was derived from frozen stem cells of *S. chizosaccharomyce so ctosporus* ATCC 2 4 7 9.

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