Method for preparing (S)-1-phenethyl alcohol by enzyme method

Abstract

The invention discloses a method for preparing (S)-1-phenethyl alcohol by an enzyme method. According to the method, acetophenone is used as a substrate and is converted into (S)-1-phenethyl alcohol under the catalytic action of ketoreductase, when a hydrogen donor is isopropanol, the concentration of the substrate can reach 200 g / L, when the hydrogen donor is glucose, the concentration of the substrate can reach 600 g / L, and the conversion rate and ee value can reach 99%. The method disclosed by the invention is simple to operate and high in catalytic efficiency, and can be used for industrial production.

Description

Technical Field:

[0001] The present invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing (S)-1-phenylethanol by enzymatic method. Background Art:

[0002] (S)-1-Phenylethanol, with the structure shown in Formula I and CAS number 1445-91-6, is an important pharmaceutical intermediate. It is a precursor of drugs such as the JAK1 / TYK2 dual inhibitor Brepocitinib and esomeprazole sodium for inhibiting gastric acid secretion. In the future, this compound can be used to prepare more drugs.

[0003]

[0004] Currently, the preparation methods of (S)-1-phenylethanol are mainly divided into chemical methods and biological methods. Compared with traditional chemical synthesis techniques, the asymmetric bioreduction of carbonyl has the advantages of strong stereoselectivity, high catalytic efficiency, mild reaction conditions, and environmental friendliness in the production process. Therefore, in recent years, the use of reductase to asymmetrically reduce acetophenone has become the main method for producing (S)-1-phenylethanol, and the preparation route is shown in Scheme 1.

[0005]

[0006] Patent CN109652463B shows that using the reductase derived from Rauvolfia serpentina Benth. ex Kurz Perakine as a catalyst, 0.8 mM of the substrate acetophenone can be catalyzed to convert into (S)-1-phenylethanol, with a reaction yield of 75.6% and an ee value of 99.2%.

[0007] Patent CN102417889B shows that using Rhodococcus erythropolis WZ010 as a catalyst, up to 40 mM of the substrate can be catalyzed to convert into (S)-1-phenylethanol. The reaction time is 72 hours, the conversion rate is only 56.4%, and the ee value is 94.5%.

[0008] Patent CN107586763B shows that using the carbonyl reductase mutant derived from Bacillus subtilis as a catalyst, under the action of dimethyl sulfoxide and xylose, etc., 500 mM of the substrate acetophenone can be catalyzed to convert into (S)-1-phenylethanol, with a substrate conversion rate of 88.2% and an ee value of 99%.

[0009] Therefore, we need to develop a method for preparing (S)-1-phenylethanol by enzymatic method with high conversion rate, high catalytic efficiency, and good selectivity. Summary of the Invention:

[0010] The object of the present invention is to provide a method for preparing (S)-1-phenylethanol with simple operation and easy industrialization in view of the deficiencies of the prior art.

[0011] The technical solution adopted by the present invention is as shown in Scheme 2 and Scheme 3:

[0012]

[0013] The present invention provides a method for preparing (S)-1-phenylethanol, which specifically comprises the following steps: using acetophenone as a substrate and converting it into (S)-1-phenylethanol under the catalytic action of a ketoreductase mutant.

[0014] Furthermore, the ketoreductase mutant is obtained by site-directed mutagenesis with the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO.1 as a reference sequence.

[0015] Furthermore, the wild-type ketoreductase is derived from Candida parapsilosis, the NCBI accession number of the wild-type amino acid sequence is KAF6051446.1, and the amino acid sequence and nucleotide sequence are respectively shown in SEQ ID NO.1 and SEQ ID NO.2.

[0016] Furthermore, alanine (Ala) at the 72nd position of the wild-type ketoreductase is mutated to glutamic acid (Glu), isoleucine (Ile) at the 177th position is mutated to tyrosine (Tyr), lysine (Lys) at the 203rd position is mutated to glycine (Gly), tryptophan (Trp) at the 286th position is mutated to leucine (Leu), and valine (Val) at the 307th position is mutated to serine (Ser) to obtain the ketoreductase mutant Mut1 (A72E / I177Y / K203G / W286L / V307S).

[0017] Furthermore, the amino acid sequence and nucleotide sequence of the ketoreductase mutant Mut1 are respectively shown in SEQ ID NO.3 and SEQ ID NO.4.

[0018] Furthermore, the ketoreductase mutant participates in the catalytic reaction in the form of ketoreductase enzyme powder, ketoreductase enzyme solution, ketoreductase homogenate, ketoreductase lyophilized powder, cells containing ketoreductase, etc., and preferably ketoreductase enzyme powder and cell homogenate.

[0019] Furthermore, the expression receptor strain of the ketoreductase mutant is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis, and preferably Escherichia coli.

[0020] Furthermore, coenzymes can be added to the reaction system to promote the reaction. When using cells containing a ketoreductase mutant, there is a small amount of coenzyme inside the cells, and in this case, coenzymes may not be added; in some cases, a small amount of coenzyme is also contained in the prepared ketoreductase mutant enzyme powder, and coenzymes may not be added in this case either. 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 their combinations, preferably NAD + .

[0021] Furthermore, the concentration of the added coenzyme is 0.02 - 0.40 g / L, preferably 0.10 - 0.25 g / L.

[0022] Furthermore, the coenzymes used in this technical solution are all selected from the coenzyme products sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0023] Further, a hydrogen donor needs to be added to the reaction, and the hydrogen donor is selected from glucose and isopropanol.

[0024] Furthermore, when the hydrogen donor is selected from isopropanol, the substrate concentration in the reaction is preferably 20 - 200 g / L.

[0025] Furthermore, when the hydrogen donor is selected from isopropanol, the dosage of the isopropanol is preferably 5 - 20%.

[0026] Furthermore, when the hydrogen donor is selected from isopropanol, the reaction pH is preferably 6.5 - 7.5.

[0027] Furthermore, when the hydrogen donor is selected from isopropanol, the reaction temperature is preferably 30 - 40 °C.

[0028] Furthermore, when the hydrogen donor is selected from glucose, the substrate concentration in the reaction is preferably 200 - 600 g / L.

[0029] Furthermore, when the hydrogen donor is selected from glucose, the dosage of the glucose is preferably 1.0 - 1.5 equivalents.

[0030] Furthermore, when the hydrogen donor is selected from glucose, the reaction pH is preferably 6.0 - 7.0.

[0031] Furthermore, when the hydrogen donor is selected from glucose, the reaction temperature is preferably 25 - 35 °C.

[0032] The beneficial effects of the present invention are as follows. The present invention provides a method for preparing (S)-1-phenylethanol. In this method, a new ketoreductase is used, which can selectively convert acetophenone into (S)-1-phenylethanol. When the hydrogen donor is isopropanol, the substrate concentration can reach 200 g / L. When the hydrogen donor is glucose, the substrate concentration can reach 600 g / L, and both the conversion rate and the ee value can reach 99%. This method is simple to operate, has high catalytic efficiency, good selectivity, and mild reaction conditions, and can be used for industrial production. Description of the Drawings

[0033] Figure 1 HPLC analysis chromatogram of the purity of (S)-1-phenylethanol in Example 7

[0034] Figure 2 Chiral HPLC analysis chromatogram of (S)-1-phenylethanol in Example 7 Detailed Description of the Invention

[0035] The technical content of the present invention will be further elaborated below in combination with specific examples, with the aim of better understanding the content of the present invention, but the protection scope of the present invention is not limited thereto. Example 1 Preliminary screening of ketoreductase - isopropanol system

[0036] The enzymes in the ketoreductase enzyme library of Shangke Biopharmaceuticals (Shanghai) Co., Ltd. were used to screen the reaction of the substrate. 1.50 g of acetophenone and 15 mL of water were added to the system, and after stirring evenly, the pH was adjusted to 7.5 with an aqueous NaOH solution. Then, 3.75 g of isopropanol, 0.10 g of ketoreductase enzyme powder, and 0.005 g of NAD were successively added to the system, and the reaction was carried out at 30 °C for 12 hours. After the reaction was completed, the sample was extracted with ethyl acetate and analyzed by HPLC. The results showed that the ketoreductase derived from Candida parapsilosis detected the product (peak area ratio 10%), and the ee value was 92.5%.

[0037] Example 2 Preliminary screening of ketoreductase - glucose system

[0038] The enzyme in the ketoreductase enzyme library of Shangke Biopharmaceuticals (Shanghai) Co., Ltd. was used to screen the reaction of the substrate. 1.50 g of acetophenone and 15 mL of water were added to the system. After stirring evenly, the pH was adjusted to 7.5 with an aqueous NaOH solution. Then, 3.72 g of glucose monohydrate, 0.08 g of ketoreductase enzyme powder, 0.02 g of glucose dehydrogenase enzyme powder (ES-GDH-110), and 0.004 g of NAD were successively added to the system. The reaction was carried out at 30 °C for 12 hours. After the reaction, the sample was extracted with ethyl acetate and analyzed by HPLC. The results showed that the ketoreductase derived from Candida parapsilosis (the same enzyme as in Example 1) detected the product (peak area ratio 18%), and the ee value was 93.4%. Subsequently, directed evolution modification was carried out on this enzyme.

[0039] Example 3 Obtaining of ketoreductase mutants

[0040] Through substrate docking and protein structure analysis, it was confirmed that the amino acid sites A72, I177, K203, W286, and V307 in the ketoreductase were relatively crucial for catalytic activity. Semi-rational design mutations and screening were carried out for these 5 sites, and the optimal mutant Mut1 (A72E / I177Y / K203G / W286L / V307S) was obtained. Its amino acid sequence is shown in SEQ ID NO.3.

[0041] Example 4 Optimization of reaction conditions --- Substrate concentration

[0042] The substrate concentration in the reaction process was optimized. The substrate concentration was set to 100 - 800 g / L, and other reaction conditions were the same as in Examples 1 and 2. The reaction results were analyzed. The specific data are shown in Table 1. The reaction results showed that when the hydrogen donor was isopropanol, the highest conversion substrate concentration was 200 g / L, and when the hydrogen donor was glucose, the highest conversion substrate concentration was 600 g / L.

[0043] Table 1 Optimization results of substrate concentration

[0044]

[0045] Example 5 Optimization of reaction conditions --- Selection of pH

[0046] The pH in the reaction process was optimized. The pH was set to 6.0 - 8.0, and other reaction conditions were the same as in Examples 1 and 2. The reaction results were analyzed. The specific data are shown in Table 2. The reaction results showed that when the hydrogen donor was isopropanol, the optimal reaction pH was 6.5 - 7.5, and when the hydrogen donor was glucose, the optimal reaction pH was 6.0 - 7.0.

[0047] Table 2 Optimization results of different pH values

[0048]

[0049]

[0050] Example 6 Optimization of Reaction Conditions - Selection of Temperature

[0051] Optimize the temperature during the reaction. The temperature is set at 25 - 45 °C, and other reaction conditions are the same as in Examples 1 and 2. Analyze the reaction results. The specific data are shown in Table 3. The reaction results show that when the hydrogen donor is isopropanol, the optimal reaction temperature is 30 - 40 °C; when the hydrogen donor is glucose, the optimal reaction temperature is 25 - 35 °C.

[0052] Table 3 Optimization Results at Different Temperatures

[0053]

[0054] Example 7 Preparation of (S)-1-Phenylethanol - Isopropanol System

[0055] Add 150 g of acetophenone and 500 mL of water to a three-necked reaction flask in sequence. After mixing evenly, adjust the pH to 6.0 with an aqueous NaOH solution. Then, continue to add 80 g of isopropanol, 10 g of ketoreductase enzyme powder, and 0.075 g of NAD to the reaction system. Stir and react at 35 °C for 12 hours. After the reaction is completed, distill off the solvent and remove the enzyme by ultrafiltration. The obtained crude product is extracted 3 times with ethyl acetate. Combine the organic phases and distill under reduced pressure to obtain 145.6 g of (S)-1-phenylethanol, with a conversion rate of 99.73%, a product purity of 99.89%, and an ee value of 99.97%. The HPLC chromatogram of the product purity is shown in Figure 1 as shown, and the HPLC chromatogram of the product chiral purity is shown in Figure 2 as shown.

[0056] Example 8 Preparation of (S)-1-Phenylethanol - Glucose System

[0057] Add 150 g of acetophenone and 100 mL of water to a three-necked reaction flask in sequence. After mixing evenly, adjust the pH to 6.5 with an aqueous NaOH solution. Then, continue to add 248 g of glucose monohydrate, 8 g of ketoreductase enzyme powder, 2 g of glucose dehydrogenase enzyme powder (ES-GDH-110), and 0.0625 g of NAD to the reaction system. Stir and react at 30 °C for 12 hours. After the reaction is completed, distill off the solvent and remove the enzyme by ultrafiltration. The obtained crude product is extracted 3 times with ethyl acetate. Combine the organic phases and distill under reduced pressure to obtain 147.6 g of (S)-1-phenylethanol, with a conversion rate of 99.65%, a product purity of 99.9%, and an ee value of 99.97%.

Claims

1. A method for preparing (S)-1-phenylethanol by enzymatic method, characterized in that, This method uses acetophenone as a substrate and is converted into (S)-1-phenylethanol under the catalytic action of ketoreductase, and the amino acid sequence of the ketoreductase is as shown in SEQ ID NO.

3.

2. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the ketoreductase is as shown in SEQ ID NO.

4.

3. The preparation method according to claim 1, characterized in that, The ketoreductase participates in the catalytic reaction in the form of ketoreductase enzyme powder, ketoreductase enzyme solution, ketoreductase homogenate, ketoreductase lyophilized powder, cells containing ketoreductase, etc.

4. The preparation method according to claim 1, characterized in that, The ketoreductase expression receptor strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis.

5. The preparation method according to claim 1, characterized in that, The reaction requires the addition of a coenzyme, which is selected from NAD + , NADH, NADP + , NADPH or a combination thereof.

6. The preparation method according to claim 1, characterized in that, A hydrogen donor needs to be added in the reaction, and the hydrogen donor is selected from glucose and isopropanol.

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