A process for the enzymatic preparation of (r)-2-trifluoromethyl-2-hydroxypropionic acid
The preparation of (R)-2-trifluoromethyl-2-hydroxypropionic acid by direct hydrolysis of 3,3,3-trifluoro-2-hydroxy-2-methylpropionitrile with nitrile hydrolase solves the problems of complex operation and low yield of existing enzymatic methods, and realizes efficient and simple industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SYNCOZYMES SHANGHAI
- Filing Date
- 2024-12-27
- Publication Date
- 2026-06-30
AI Technical Summary
Existing enzymatic methods for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid are complex to operate and have low yields, making them difficult to apply to industrial production.
The target compound was prepared by direct hydrolysis of 3,3,3-trifluoro-2-hydroxy-2-methylpropionitrile with nitrile hydrolase. The nitrile hydrolase used was selected from ES-NIT-101 to ES-NIT-140 of Shangke Biopharmaceutical Co., Ltd. The reaction conditions included pH 6.5 to 8.0, temperature 20 to 35℃, time 8 to 40 h, substrate concentration 1 to 75 g/L, and enzyme to substrate weight ratio 0.5:1 to 3:1.
A preparation method with short route, high efficiency and simple operation has been realized, which is suitable for industrial production. The yield and chiral purity reach 42.2% to 83.0%, and the chiral purity is 99.1% to 99.6%.
Smart Images
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Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biocatalysis technology, specifically relating to an enzymatic method for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid. Background Technology
[0002] (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one (CAS: 2875067-31-3), with the structure shown in Formula I, is primarily used in the synthesis of VX-548, a novel Nav1.8 inhibitor developed by Vertex Pharmaceuticals Inc. VX-548 is intended for the treatment of pain, neuropathic pain, and diabetic peripheral neuropathy. Compound M1 is a key intermediate in the synthesis of (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one.
[0003]
[0004] Patent DE19725802 A1 discloses a method for preparing compound M1 using an ester hydrolase, as shown in Scheme 1. This method uses compound SM1 as a starting material, undergoes alcoholysis to obtain compound M1-1, and then proceeds to esterase-catalyzed hydrolysis to obtain compound M1, with an overall yield of only 12% and an ee value of only 71.7%.
[0005]
[0006] Patent WO1998001568 A2 discloses a method for preparing compound M1 using an amide hydrolase, as shown in Scheme 2. This method uses compound SM1 as a raw material, hydrolyzes it to obtain compound M1-2, and then hydrolyzes it using an amide hydrolase to obtain compound M1, with an overall yield of 38% and an ee value of 100%.
[0007]
[0008] Existing enzymatic methods require first converting compound SM1 into an amide or ester, followed by selective hydrolysis to obtain compound M1 with a specific configuration. This process is complex and yields low results. Therefore, we need to develop a method for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid that is short, efficient, easy to operate, and suitable for industrial production. Summary of the Invention
[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing a novel enzymatic method for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid.
[0010] The technical solution adopted in this invention is shown in Scheme 3:
[0011]
[0012] On one hand, the present invention provides a method for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid, specifically including the following steps: using 3,3,3-trifluoro-2-hydroxy-2-methylpropionitrile as raw material, the target compound (R)-2-trifluoromethyl-2-hydroxypropionic acid is obtained by direct hydrolysis under the action of nitrile hydrolase.
[0013] Furthermore, the nitrile hydrolase is selected from ES-NIT-101 to ES-NIT-140 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.
[0014] Furthermore, the nitrile hydrolase participates in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenate, cell slurry containing nitrile hydrolase, immobilized cells, immobilized enzyme, etc., preferably nitrile hydrolase solution.
[0015] Furthermore, the nitrile hydrolase expression receptor strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis.
[0016] Furthermore, the weight ratio of the nitrile hydrolase to the substrate is selected from 0.5:1 to 3:1, preferably 1:1.
[0017] Furthermore, the concentration of the substrate 3,3,3-trifluoro-2-hydroxy-2-methylpropionitrile (SM1) is selected from 1 to 75 g / L, preferably 50 g / L.
[0018] Furthermore, the pH value of the enzymatic reaction is selected from 6.5 to 8.0, preferably 7.5 ± 0.2.
[0019] Furthermore, the temperature of the enzymatic reaction is selected from 20 to 35°C, preferably 25 ± 2°C.
[0020] Furthermore, the reaction time of the enzymatic method is selected from 8 to 40 hours, preferably 20 hours.
[0021] On the other hand, the (R)-2-trifluoromethyl-2-hydroxypropionic acid can be used to prepare the intermediate (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one of VX-548, as shown in Scheme 4.
[0022]
[0023] The beneficial effect of the present invention is that it discloses a method for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid that is short in route, highly efficient, easy to operate and suitable for industrial production. Attached Figure Description
[0024] none. Detailed Implementation
[0025] The technical content of the present invention will be further described below with reference to specific embodiments, in order to better understand the content of the present invention, but the scope of protection of the present invention is not limited thereto.
[0026] Example 1: Preparation of compound M1
[0027] In a 500 mL three-necked flask equipped with a mechanical stirrer, add phosphate buffer solution (40.0 mL, 0.1 N, pH = 7.0), deionized water (50.0 mL), and compound SM1 (5.0 g, 1.0 eq). Stir until dissolved at 20–30 °C. Adjust the pH to 7.5 with 10% sodium carbonate aqueous solution. Add nitrile hydrolase (5.0 g, ES-NIT-109 enzyme solution). Stir the reaction at 25 ± 2 °C. During the reaction, maintain the pH at 7.5 ± 0.2 using automatic potentiometric titration with 10% sodium carbonate aqueous solution. React for 20 hours. After the reaction is complete, add 10.0 g of diatomaceous earth, stir for 30 minutes, and filter. Wash the filter cake once with 20.0 mL of water, then extract twice with 25.0 mL of ethyl acetate, stirring for 30 minutes each time, followed by standing for 30 minutes. Separate the liquid and aqueous phases. Adjust the pH to 2.0–3.0 by adding concentrated hydrochloric acid dropwise. Extract three times with 50.0 mL of ethyl acetate, stirring for 30 minutes each time, and allowing to stand for 30 minutes. Separate the liquid and aqueous phases. Combine the organic phases and wash twice with 5.0 mL of water, stirring for 30 minutes each time, and allowing to stand for 30 minutes. Separate the liquid and aqueous phases. Concentrate the organic phase to dryness under reduced pressure at 40±2℃ to give compound M1 (2.4 g, yield 42.2%, chiral purity 99.1%).
[0028] Example 2 Preparation of Compound I
[0029] Compound M1 (2.0 g, 1.0 eq) and diethoxymethane (10.0 mL, 5.0 v / w) were added to a 50 mL three-necked flask equipped with a mechanical stirrer and nitrogen protection. The mixture was purged with nitrogen three times and cooled to 5 °C. A 2.7 M lithium methyl ether solution (14.0 mL, 3.0 eq) was slowly added dropwise at 5–15 °C. After the addition was complete, the mixture was stirred at 20–30 °C. After the reaction was complete, the mixture was cooled to 0 °C, and the reaction was quenched dropwise with 2 N HCl at 0–10 °C. The pH was adjusted to 8.0–9.0, and the mixture was stirred for 30 minutes and allowed to stand for 30 minutes. The aqueous phase was separated, and extracted twice with 10.0 mL of diethoxymethane, stirring for 30 minutes each time, followed by standing for 30 minutes. The organic phases were then separated and combined, washed twice with 5.0 mL of water, stirring for 30 minutes each time, followed by standing for 30 minutes. The liquid phase was separated and concentrated to dryness under reduced pressure at 30±2℃ to give compound I (1.6g, yield 81.0%, chiral purity 99.5%).
[0030] Example 3 Preparation of compound M1
[0031] In a 500 mL three-necked flask equipped with a mechanical stirrer, add phosphate buffer solution (40.0 mL, 0.1 N, pH = 7.0), deionized water (50.0 mL), and compound SM1 (8.0 g, 1.0 eq). Stir until dissolved at 20–30 °C. Adjust the pH to 7.5 with 10% sodium carbonate aqueous solution. Add nitrile hydrolase (8.0 g, ES-NIT-101 homogenate). Stir the reaction at 25 ± 2 °C. During the reaction, maintain the pH at 7.5 ± 0.2 using automatic potentiometric titration with 10% sodium carbonate aqueous solution. React for 20 hours. After the reaction is complete, add 10.0 g of diatomaceous earth, stir for 30 minutes, and filter. Wash the filter cake once with 32.0 mL of water, then extract twice with 40.0 mL of ethyl acetate, stirring for 30 minutes each time, followed by standing for 30 minutes. Separate the liquid and aqueous phases. Adjust the pH to 2.0–3.0 by adding concentrated hydrochloric acid dropwise. Extract three times with 80.0 mL of ethyl acetate, stirring for 30 minutes each time, and allowing to stand for 30 minutes. Separate the liquid and aqueous phases. Combine the organic phases and wash twice with 8.0 mL of water, stirring for 30 minutes each time, and allowing to stand for 30 minutes. Separate the liquid and aqueous phases. Concentrate the organic phase to dryness under reduced pressure at 40±2℃ to give compound M1 (3.2 g, yield 35.8%, chiral purity 99.3%).
[0032] Example 4 Preparation of Compound I
[0033] Compound M1 (3.0 g, 1.0 eq) and diethoxymethane (15.0 mL, 5.0 v / w) were added to a 100 mL three-necked flask equipped with a mechanical stirrer and nitrogen protection. The mixture was purged with nitrogen three times and cooled to 5 °C. A 2.7 M methyl lithium diethyl ether solution (21.0 mL, 3.0 eq) was slowly added dropwise at 5–15 °C. After the addition was complete, the mixture was stirred at 20–30 °C. After the reaction was complete, the mixture was cooled to 0 °C, and the reaction was quenched dropwise with 2 N HCl at 0–10 °C. The pH was adjusted to 8.0–9.0, and the mixture was stirred for 30 minutes and allowed to stand for 30 minutes. The aqueous phase was separated, and extracted twice with 15.0 mL of diethoxymethane, stirring for 30 minutes each time, followed by standing for 30 minutes. The organic phases were then separated and combined, washed twice with 7.5 mL of water, stirring for 30 minutes each time, followed by standing for 30 minutes. The liquid phase was separated and concentrated to dryness under reduced pressure at 30±2℃ to give compound I (2.5g, yield 83.0%, chiral purity 99.6%).
Claims
1. A method for the enzymatic preparation of (R)-2-trifluoromethyl-2-hydroxypropionic acid, characterized in that, The method uses 3,3,3-trifluoro-2-hydroxy-2-methylpropionitrile as a raw material, and hydrolyzes it under the action of nitrile hydrolase to obtain the target compound (R)-2-trifluoromethyl-2-hydroxypropionic acid. The method is shown below.
2. The preparation method according to claim 1, characterized in that, The nitrile hydrolase is selected from ES-NIT-101 to ES-NIT-140 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.
3. The preparation method according to claim 1, characterized in that, The weight ratio of the nitrile hydrolase to the substrate is selected from 0.5:1 to 3:
1.
4. The preparation method according to claim 1, characterized in that, The concentration of the 3,3,3-trifluoro-2-hydroxy-2-methylpropionitrile is selected from 1 to 75 g / L.
5. The preparation method according to claim 1, characterized in that, The pH value of the enzymatic reaction is selected from 6.5 to 8.
0.
6. The preparation method according to claim 1, characterized in that, The temperature of the enzymatic reaction is selected from 20 to 35°C.
7. The preparation method according to claim 1, characterized in that, The reaction time for the enzymatic method is selected from 8 to 40 hours.
8. The preparation method according to claim 1, characterized in that, The (R)-2-trifluoromethyl-2-hydroxypropionic acid can be used to prepare the VX548 intermediate (R)-4,4,4-trifluoro-3-hydroxy-3-methylbut-2-one.