Method for preparing (S)-2-hydroxy-3, 3-dimethylbutyric acid by enzyme method

Abstract

The invention discloses a method for preparing (S)-2-hydroxy-3, 3-dimethyl butyric acid by an enzyme method. Trimethylpyruvic acid is used as a substrate and converted into (S)-2-hydroxy-3, 3-dimethylbutyric acid under the catalytic action of ketoreductase, the conversion rate is larger than or equal to 90%, and the chiral purity of the product is larger than or equal to 98%. The method disclosed by the invention is simple to operate, green, environment-friendly, high in atom economy and suitable for industrial production.

Description

Technical field:

[0001] The invention belongs to the technical field of biocatalysis, and specifically relates to a method for preparing (S)-2-hydroxy-3,3-dimethylbutyric acid by an enzymatic method. Background technology:

[0002] (S)-2-Hydroxy-3,3-dimethylbutyric acid, with the structural formula shown in I and CAS number 21641-92-9, is an important pharmaceutical intermediate that can be used to prepare protease inhibitors and Flaviviridae macrocyclic inhibitor precursor compounds to treat osteoporosis, hepatitis C, meningitis and other diseases. In the future, this compound can be used to prepare more new drugs.

[0003]

[0004] The preparation route of compound I reported in Bulletin of the Chemical Society of Japan (1968), 41(9), 2178-9. is shown in Scheme 1. This route uses pinacolone as a starting material, generates DL-2-hydroxy-3,3-dimethylbutyric acid through oxidation and reduction reactions, and then resolves it with cinchonidine to obtain the target compound I. In this route, the material loss in the resolution step is large, the yield is low, and the resolution agent is expensive.

[0005]

[0006] The preparation route of compound I disclosed in patent CN115677482A is shown in Scheme 2. In this route, L-tert-leucine is used as a substrate, and a chiral hydroxyl group is introduced into compound I by a diazotization reaction with sodium nitrite. The reaction requires strict control of low temperature, and sodium nitrite is a hazardous chemical with certain safety hazards, which is not suitable for industrial production.

[0007]

[0008] Therefore, we need to develop a preparation method that is simple to operate, high in yield, safe and environmentally friendly, and suitable for industrialization. Summary of the invention:

[0009] The purpose of the present invention is to provide a method for preparing (S)-2-hydroxy-3,3-dimethylbutyric acid by an enzymatic method which is simple to operate and easy to industrialize in view of the deficiencies of the prior art.

[0010] The technical solution adopted by the present invention is as follows:

[0011]

[0012] The present invention provides a method for preparing (S)-2-hydroxy-3,3-dimethylbutyric acid, which specifically comprises the following steps: using trimethylpyruvic acid as a substrate and converting it into (S)-2-hydroxy-3,3-dimethylbutyric acid under the catalytic action of ketoreductase.

[0013] Furthermore, the ketoreductase participates in the catalytic reaction in the form of ketoreductase enzyme powder, ketoreductase enzyme solution, ketoreductase homogenous solution, ketoreductase lyophilized powder, cells containing ketoreductase, etc., preferably ketoreductase homogenous solution and lyophilized powder.

[0014] Furthermore, the ketoreductase is selected from the ketoreductase ES-KRED-101 to ES-KRED-296 produced by Shangke Biopharmaceutical (Shanghai) Co., Ltd., preferably ES-KRED-266, ES-KRED-267, and ES-KRED-279.

[0015] Furthermore, the ketoreductase expression strain is selected from Escherichia coli.

[0016] Furthermore, coenzymes can be added to the reaction system to promote the reaction. When cells containing ketoreductase are used, a small amount of coenzymes are contained in the cells, and the coenzymes may not be added at this time. In some cases, the prepared ketoreductase enzyme powder also contains a small amount of coenzymes, and the coenzymes may not be added at this time. However, coenzymes can also be added to the reaction system to further promote the reaction. When coenzymes are added to the reaction system to promote the reaction, the coenzymes are selected from NAD + , NADH, NADP + , NADPH or a combination thereof, preferably NADPH and NADP + .

[0017] Furthermore, the concentration of the added coenzyme is 0.02 to 0.4 g / L, preferably 0.04 to 0.2 g / L.

[0018] Furthermore, the coenzymes used in this technical solution are all selected from the coenzyme products sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0019] Furthermore, a hydrogen donor needs to be added in the reaction, and the hydrogen donor is selected from methanol, ethanol, isopropanol, butanol, and glucose, preferably glucose.

[0020] Furthermore, the substrate concentration is 10 to 200 g / L, preferably 50 to 100 g / L.

[0021] Furthermore, the reaction time is 1 h to 48 h, preferably 12 h to 24 h.

[0022] Furthermore, the reaction needs to be controlled at a pH of 6.0 to 8.0, preferably a pH of 6.5 to 7.5.

[0023] Furthermore, the reaction temperature is 20°C to 50°C, preferably 30°C to 40°C.

[0024] The beneficial effect of the present invention is that the present invention provides an enzymatic method for preparing (S)-2-hydroxy-3,3-dimethylbutyric acid, wherein trimethylpyruvic acid is converted into a target product I by ketoreductase, the conversion rate is ≥90%, and the chiral purity of the product is ≥98%. The method is simple to operate, has high atom economy, and can be used for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Example 2 Product purity HPLC analysis chart.

[0026] Figure 2 Chiral HPLC analysis spectrum of the product in Example 2. DETAILED DESCRIPTION

[0027] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0028] Example 1 Screening of ketoreductase

[0029] The enzymes in the ketoreductase enzyme library of Shangke Biopharmaceuticals (Shanghai) Co., Ltd. were used to screen the substrates. The reaction system components are as follows: substrate 100g / L, glucose 160-240g / L, NADPH 0.2g / L, KRED enzyme powder 10g / L, glucose dehydrogenase enzyme powder 5g / L. The reaction was carried out at 30°C for 18h. After the reaction was completed, HPLC detection and analysis were performed. The results showed that the best enzymes screened were ketoreductase ES-KRED-266, ES-KRED-267, and ES-KRED-279, with a conversion rate of ≥85%, and chiral HPLC detection showed that the chiral purity of the product was ≥98.0%.

[0030] Example 2 Preparation of Compound I

[0031] 250 g of trimethylpyruvic acid was added to a three-necked flask, mixed with 2 L of water, and adjusted to pH 6.5 with an aqueous NaOH solution (10%), followed by the addition of 400 g of glucose, 10 g of ketoreductase freeze-dried powder (ES-KRED-266, Shangke Biopharmaceuticals (Shanghai) Co., Ltd.), 5 g of glucose dehydrogenase freeze-dried powder (ES-GDH-110, Shangke Biopharmaceuticals (Shanghai) Co., Ltd.) and 0.2 g of coenzyme NADP, heated to 30 ° C, stirred for 12 hours, and after GC monitoring of the reaction, the reaction solution was filtered to remove the enzyme, and the pH was adjusted to 1-2, then extracted three times with ethyl acetate, the organic phases were combined, and concentrated under reduced pressure to obtain 240 g of compound I, with a conversion rate of 99.1%, a product purity of 99.7%, and a purity HPLC spectrum as shown in FIG. Figure 1 As shown, the chiral purity of the product is 99.3%, and the chiral HPLC spectrum is shown in Figure 2 shown.

[0032] Example 3 Preparation of Compound I

[0033] Add 250 g of trimethylpyruvic acid to a three-necked flask, mix well with 2 L of water, and adjust the pH to 7.5 with an aqueous NaOH solution (10%), then add 400 g of glucose, 10 g of ketoreductase freeze-dried powder (ES-KRED-267 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.), 5 g of glucose dehydrogenase freeze-dried powder (ES-GDH-110 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.) and 0.2 g of coenzyme NADPH in sequence, heat to 40 ° C, stir and react for 18 hours, monitor the reaction by GC, filter the reaction solution to remove the enzyme, adjust the pH to 1-2, then extract three times with ethyl acetate, combine the organic phases, and concentrate under reduced pressure to obtain 226.7 g of compound I, with a conversion rate of 97.2%, a product purity of 99.9%, and a chiral purity of 99.1%.

[0034] Example 4 Preparation of Compound I

[0035] Add 250 g of trimethylpyruvic acid to a three-necked flask, mix well with 2 L of water, and adjust the pH to 6.5 with a NaOH aqueous solution (10%), then add 400 g of ethanol, 10 g of ketoreductase freeze-dried powder (ES-KRED-269, Shangke Biopharmaceutical (Shanghai) Co., Ltd.) and 0.2 g of coenzyme NADH in sequence, heat to 25 ° C, stir and react for 15 hours, and after GC monitoring of the reaction, filter the reaction solution to remove the enzyme, adjust the pH to 1-2, and then extract three times with ethyl acetate, combine the organic phases, and concentrate under reduced pressure to obtain 203.4 g of compound I, with a conversion rate of 92.3%, a product purity of 99.8%, and a chiral purity of 98.2%.

[0036] Example 5 Preparation of Compound I

[0037] 250 g of trimethylpyruvic acid was added to a three-necked flask, mixed evenly with 2 L of water, and adjusted to pH 7.5 with an aqueous NaOH solution (10%), followed by the addition of 400 g of glucose, 30 g of ketoreductase cell homogenate (ES-KRED-266 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.), 5 g of glucose dehydrogenase cell homogenate (ES-GDH-110 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.) and 0.25 g of coenzyme NADPH, the temperature was raised to 40 ° C, and the reaction was stirred for 24 hours. After the reaction was completed by GC monitoring, the reaction solution was filtered to remove the enzyme, and the pH was adjusted to 1-2, and then extracted three times with ethyl acetate, the organic phases were combined, and concentrated under reduced pressure to obtain 245 g of compound I, with a conversion rate of 99.5%, a product purity of 99.3%, and a chiral purity of 99.4%.

[0038] Example 6 Preparation of Compound I

[0039] Add 250 g of trimethylpyruvic acid to a three-necked flask, mix evenly with 2 L of water, and adjust the pH to 7.0 with an aqueous NaOH solution (10%), then add 600 g of glucose, 30 g of ketoreductase cell homogenate (ES-KRED-267 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.), 5 g of glucose dehydrogenase cell homogenate (ES-GDH-110 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.) and 0.25 g of coenzyme NADP in sequence, heat to 30 ° C, stir and react for 18 hours, monitor the reaction by GC, filter the reaction solution to remove the enzyme, adjust the pH to 1-2, then extract three times with ethyl acetate, combine the organic phases, and concentrate under reduced pressure to obtain 221.6 g of compound I, with a conversion rate of 94.1%, a product purity of 99.3%, and a chiral purity of 99.3%.

[0040] Example 7 Preparation of Compound I

[0041] Add 250 g of trimethylpyruvic acid to a three-necked flask, mix well with 2 L of water, and adjust the pH to 6.0 with a NaOH aqueous solution (10%), then add 300 g of butanol, 30 g of ketoreductase cell homogenate (ES-KRED-269, Shangke Biopharmaceutical (Shanghai) Co., Ltd.) and 0.25 g of coenzyme NADH in sequence, heat to 30 ° C, stir and react for 12 hours, monitor the reaction by GC, filter the reaction solution to remove the enzyme, adjust the pH to 1-2, then extract three times with ethyl acetate, combine the organic phases, and concentrate under reduced pressure to obtain 209.2 g of compound I, with a conversion rate of 90.1%, a product purity of 99.0%, and a chiral purity of 98.3%.

Claims

1. A method for preparing (S)-2-hydroxy-3,3-dimethylbutyric acid by enzymatic method, characterized in that: Under the catalysis of ketoreductase, trimethylpyruvate is converted into (S)-2-hydroxy-3,3-dimethylbutyrate. The reaction equation is as follows:

2. The preparation method according to claim 1, characterized in that The reaction temperature is 20°C to 50°C.

3. The preparation method according to claim 1, characterized in that: The reaction needs to control the pH to be 6.0-8.

0.

4. The preparation method according to claim 1, characterized in that: The ketoreductase participates in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenized solution, freeze-dried powder, cells containing the enzyme, etc.

5. The preparation method according to claim 1, characterized in that: The ketoreductase is selected from the ketoreductase ES-KRED-101 to ES-KRED-296 produced by Shangke Biopharmaceutical (Shanghai) Co., Ltd.

6. The method according to claim 1, characterized in that The ketoreductase is selected from genetically engineered Escherichia coli or yeast.

7. The preparation method according to claim 1, characterized in that: The coenzyme is selected from NAD + , NADH, NADP + , NADPH or a combination thereof.

8. The preparation method according to claim 1, characterized in that: The substrate concentration is 10-200 g / L.

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