Acetamidase mutant and use thereof in synthesis of (r)-2-trifluoromethyl-2-hydroxypropionic acid

Abstract

Provided are an acetamidase mutant and use thereof in the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid. The amino acid sequence of the acetamidase mutant in the present invention is set forth in SEQ ID NO. 1. (R)-2-trifluoromethyl-2-hydroxypropionic acid is synthesized by using the acetamidase mutant and optimizing reaction conditions. By means of the method, a product having an ee value of 99% or more can be obtained, the substrate concentration can reach 10%, and the maximum conversion can reach 45% or more.

Description

An acetamidinase mutant and its application in the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510475742.8, filed on April 16, 2025, entitled "An Acetamidinase Mutant and Its Application in the Synthesis of (R)-2-Trifluoromethyl-2-Hydroxypropionic Acid", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, and more specifically, to an acetamidinase mutant and its application in the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid. Background Technology

[0004] (R)-2-trifluoromethyl-2-hydroxypropionic acid, also known as (R)-2-trifluoromethyl-2-hydroxypropionic acid or (R)-(+)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, is a fluorinated chiral organic acid compound with the molecular formula CHFO. It contains one chiral carbon atom (R configuration) and is an optically active substance. As a pharmaceutical intermediate, (R)-2-trifluoromethyl-2-hydroxypropionic acid is commonly used in the synthesis of chiral drugs or fluorinated compounds. The introduction of the fluorinated group can improve the metabolic stability and bioavailability of drug molecules, thus possessing potential value in drug development.

[0005] Currently, there are chemical and biological methods for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid. The chemical method, as described in JP4529419B2, requires a nitrogen atmosphere and ultra-low temperatures, with harsh reaction conditions and a yield of only 36%.

[0006] Although the biological method has the advantages of being green and environmentally friendly, and having mild reaction conditions, as shown in Figure 1, the kinetic separation of 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (2) using Arthrobacterium S-2 and R-6 series in a two-step reaction requires two transformations using two enzymes sequentially, and the substrate concentration is only 2%, resulting in low efficiency and making it unsuitable for large-scale production.

[0007] Another technique for synthesizing (R)-2-trifluoromethyl-2-hydroxypropionic acid via whole-cell catalysis has been disclosed, such as in the literature DOI10.1007 / s00253-016-7921, whose synthetic route is shown in Figure 2. Although this method can achieve high substrate concentrations and relatively high conversion rates, it can only yield a product with an ee value of 95%. However, for chiral pharmaceutical intermediates intended for use in products, to avoid potential side effects from ineffective or toxic enantiomers, an ee value of at least 99% is generally required.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide an acetamidinase mutant and its application in the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid. The acetamidinase mutant and synthesis method of this invention can effectively improve the conversion rate, so that the ee value of the target product reaches more than 99%.

[0010] This invention is implemented as follows:

[0011] In a first aspect, the present invention provides an acetamidinase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0012] Secondly, the present invention provides the following biomaterials:

[0013] (A1) is the nucleic acid molecule encoding the above-mentioned acetamidinase mutant;

[0014] (A2) An expression cassette containing the nucleic acid molecule described in (A1);

[0015] (A3) A vector containing the nucleic acid molecule described in (A1) or a vector containing the expression cassette described in (A2);

[0016] (A4) A recombinant microorganism containing the recombinant microorganism described in (A1), or a recombinant microorganism containing the expression cassette described in (A2), or a recombinant microorganism containing the vector described in (A3).

[0017] Thirdly, the present invention provides a recombinant Escherichia coli containing the above-mentioned mutant or biological material.

[0018] Fourthly, the present invention provides applications of the above-mentioned acetamidinase mutants or biological materials in the following aspects:

[0019] (B1) Catalytic synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid;

[0020] (B2) Prepare the product of catalytic synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid.

[0021] Fifthly, the present invention provides a product for the catalytic synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid, comprising the above-mentioned acetamidinase mutant, biological material or recombinant Escherichia coli.

[0022] In a sixth aspect, the present invention provides a method for synthesizing (R)-2-trifluoromethyl-2-hydroxypropionic acid, comprising: adding the above-mentioned acetamidinase mutant to a solution containing 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide for a catalytic reaction, and then removing impurities and purifying the solution to obtain (R)-2-trifluoromethyl-2-hydroxypropionic acid. Attached Figure Description

[0023] Figure 1 shows the route for the two-step synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid using the S-2 and R-6 series of Arthrobacterium spp.;

[0024] Figure 2 shows the route for the synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid via whole-cell catalysis.

[0025] Figure 3 shows the synthetic route of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid in this invention;

[0026] Figure 4 shows the plasmid map in Example 1;

[0027] Figure 5 shows the central control result in Example 2;

[0028] Figure 6 shows the detection results after extraction in Example 2;

[0029] Figure 7 shows the chiral detection results of Example 2 after reaction and post-treatment. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] The synthetic route for (R)-2-trifluoromethyl-2-hydroxypropionic acid according to this invention is shown in Figure 3. It uses 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide as a substrate, which is converted to (R)-2-trifluoromethyl-2-hydroxypropionic acid by acetamidinase. To improve the synthetic efficiency of (R)-2-trifluoromethyl-2-hydroxypropionic acid, this invention involves site-directed mutagenesis of acetamidinase, thereby increasing the efficiency of converting 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide to (R)-2-trifluoromethyl-2-hydroxypropionic acid.

[0032] The acetamidinase mutant of the present invention is obtained by mutation based on the amino acid sequence shown in SEQ ID NO.3. Specifically, the mutation sites of the acetamidinase mutant include positions 135 and 188, and the amino acid sequence of the acetamidinase mutant is shown in SEQ ID NO.1.

[0033] The mutation at position 135 is a change from lysine to phenylalanine, i.e., L135F.

[0034] The mutation at position 188 is as follows: glycine is mutated to lysine, i.e., G188L.

[0035] Based on the above-mentioned acetamidinase mutant, related biological materials can be obtained, including: nucleic acid molecules encoding the above-mentioned acetamidinase mutant; expression cassettes containing the above-mentioned nucleic acid molecules; recombinant vectors containing the above-mentioned nucleic acid molecules or recombinant vectors containing the above-mentioned expression cassettes; recombinant bacteria containing the above-mentioned nucleic acid molecules, or recombinant bacteria containing the above-mentioned expression cassettes, or recombinant bacteria containing the above-mentioned recombinant vectors.

[0036] To obtain this acetamidinase mutant, this invention achieves this by constructing a recombinant *E. coli* strain containing the aforementioned mutant or biological material.

[0037] In some embodiments, the nucleotide sequence of the acetamidinase mutant suitable for host Escherichia coli is shown in SEQ ID NO.2.

[0038] The acetamidinase mutant of the present invention can be used to catalyze the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid, or to prepare products of the catalytic synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid. Products of the catalytic synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid include bacterial powder, bacterial liquid, and whole-cell catalysts.

[0039] Based on the above-mentioned acetamidinase mutant, the present invention can also provide a method for synthesizing (R)-2-trifluoromethyl-2-hydroxypropionic acid, which includes: adding the above-mentioned acetamidinase mutant to a solution containing 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide for catalytic reaction, and then removing impurities and purifying to obtain (R)-2-trifluoromethyl-2-hydroxypropionic acid.

[0040] To further improve conversion efficiency and product quality, this invention optimizes the conditions in the reaction process based on the above method.

[0041] In some embodiments, the concentration of 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide in the reaction system of the catalytic reaction is 1% to 10% g / mL, the acetamidinase mutant accounts for 1% to 5% of the substrate mass, and the cobalt chloride solution accounts for 0.1% to 0.5% of the substrate mass.

[0042] In some embodiments, the catalytic reaction conditions are: pH = 8.0, temperature 25°C, and the reaction process is monitored in advance by high-performance liquid chromatography (HPLC) under the following conditions:

[0043] Chromatographic column: C18 column, 250*4.6mm;

[0044] Mobile phase: 0.1% phosphoric acid: acetonitrile = 90:10;

[0045] Wavelength: 210nm / uv;

[0046] Flow rate: 0.8 mL / min;

[0047] Injection volume: 20 μl.

[0048] In some embodiments, impurity removal includes filtering the obtained reaction solution through diatomaceous earth and extracting it multiple times with ethyl acetate to control the impurity content to be below 0.5%.

[0049] In some embodiments, purification includes re-extraction and refining.

[0050] The second extraction involves adjusting the pH of the aqueous phase to 2-3, then performing multiple extractions with ethyl acetate, combining the ethyl acetate phases, and then dehydrating them with anhydrous sodium sulfate.

[0051] The refining process here involves concentrating and drying the dehydrated ethyl acetate phase, then dissolving it in ethyl acetate, filtering, concentrating it again, and drying it.

[0052] The relevant parameters of the reaction conditions in the above-mentioned impurity removal and purification process can be adjusted according to the scenario (such as scale-up reaction), and the present invention does not limit this.

[0053] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0054] Example 1

[0055] This example demonstrates the acquisition of an acetamidinase mutant, specifically as follows:

[0056] (1) First, the original amidase Bmi gene (as shown in SEQ ID NO.4) was fully synthesized and ligated into the pET28(+) vector to obtain the plasmid expressing the target gene: pET28-Bmi, and its plasmid map is shown in Figure 4.

[0057] (2) Soluble expression of the wild-type gene was performed in Escherichia coli BL21(DE3). The expressed protein has a 6-his-tag tag at the N-terminus, which facilitates the purification of the target protein using Ni-NTA.

[0058] (3) By using the three-dimensional crystal structure of the biological enzyme and the molecular docking model of the target reactant, and by using Rosetta energy calculation and identification software to identify the defects of key amino acids, potential mutation sites were determined. The PCR mutation primers used in Table 1 were synthesized according to the Quick Change Mutagenesis Kit instructions. PCR product amplification, Dpn1 digestion and subsequent nucleic acid recovery were all performed according to the kit instructions.

[0059] Table 1 Primers used for mutations

[0060] (4) Using pET28-Bmi plasmid as a template, primers 1 and 2 were used to introduce the L135F mutation into the plasmid and transform it into competent Escherichia coli DH5α cells. The plasmid was extracted and sequenced to confirm that the mutant plasmid pET28-Bmi L135F was obtained. The mutation site was confirmed to be correct by gene sequencing technology.

[0061] (5) Using the obtained single mutant plasmid as a template, primers 3 and 4 were used to introduce the G188L mutation into the mutant to obtain a double mutant plasmid. The obtained double mutant plasmid was transformed into E. coli BL21(DE3) competent cells and the plasmid was extracted. The mutation site was then verified by sequencing. The amino acid sequence of the mutant was verified as shown in SEQ ID NO.1.

[0062] (6) Escherichia coli BL21(DE3) containing the double mutant plasmid was cultured in 1L LB medium with shaking at 220 rpm at 37°C. When the OD600 of the cells reached about 0.6, IPTG (isopropyl-β-d-thiogalactopyranoside) was added to a final concentration of 1mM and the cells were induced at 16°C for 12-18h to induce protein expression.

[0063] (7) After induction, the induced cells were collected by centrifugation at 8,000 rpm for 15 min. The collected cells were then resuspended in PBS buffer solution of 20 mM imidazole and sonicated at low temperature. The enzyme solution was collected for later use.

[0064] Example 2

[0065] This embodiment describes a method for synthesizing (R)-2-trifluoromethyl-2-hydroxypropionic acid, and the reaction system is shown in Table 2:

[0066] Table 2 Reaction System

[0067] The steps of the catalytic reaction are as follows:

[0068] 1) Add 85 mL of water, 10 mL of 2% (g / mL) enzyme suspension, and 0.5 mL of 1% (g / mL) cobalt chloride solution to the reaction vessel, and stir thoroughly until homogeneous;

[0069] 2) Control the temperature at 25℃, weigh 5g of substrate and slowly add it to the reaction vessel, then adjust the pH to about 8.0 with a 0.5% (g / mL) sodium bicarbonate solution, and react at 25℃;

[0070] 3) Start central control after 3 hours of reaction.

[0071] Central control conditions for the reaction:

[0072] Chromatographic column: C18 column, 250*4.6mm;

[0073] Mobile phase: 0.1% phosphoric acid: acetonitrile = 90:10;

[0074] Wavelength: 210nm / uv;

[0075] Flow rate: 0.8 mL / min;

[0076] Injection volume: 20 μl.

[0077] The results from the central control are shown in Figure 5: the peak at 9 min is amide, and the peak at 15 min is trifluoromethylhydroxypropionic acid, indicating a reaction rate of 9.3%.

[0078] 4) Detect every hour to ensure maximum reaction and keep the target central reaction ≤50% to prevent over-reaction.

[0079] The post-reaction processing steps are as follows:

[0080] 1) Filter the reaction solution through diatomaceous earth.

[0081] 2) Extracted three times with 120 mL of ethyl acetate, using 50 mL, 40 mL, and 30 mL portions respectively. The extraction was performed under controlled conditions, and the amide residue was found to be below 0.5%. The product is shown in Figure 6.

[0082] 3) Adjust the pH of the aqueous phase to 2-3 using hydrochloric acid.

[0083] 4) Extract with 120 mL of ethyl acetate in three fractions of 50 mL, 40 mL, and 30 mL. Then combine the ethyl acetate phases and dehydrate with anhydrous sodium sulfate.

[0084] 5) Concentrate the ethyl acetate phase to dryness, add 40 mL of ethyl acetate to dissolve the product, and filter.

[0085] 6) The ethyl acetate phase was concentrated to dryness to obtain 1.6 g of the target product (R)-2-trifluoromethyl-2-hydroxypropionic acid, with a yield of 16% and an ee value of 99.75%. The product is shown in Figure 7.

[0086] The chiral detection conditions are as follows:

[0087] Chiral column: Chiral Pak IG-3 (250×4.6)mm;

[0088] Mobile phase: n-Heptane:Methanol:Isopropanol:Trifluoroacetic acid = 950mL:30mL:20mL:1mL;

[0089] Wavelength: 210nm / uv;

[0090] Flow rate: 0.7 mL / min;

[0091] Column oven: 30℃;

[0092] Injection volume: 10 μl.

[0093] Example 3

[0094] This example compares the effect of different enzyme dosages on the reaction. All other conditions are the same as in Example 2, only the enzyme dosage is changed. Specific conditions and results are as follows:

[0095] Table 3 Reaction conditions and results

[0096] As can be seen from the results in Table 3, high concentrations of enzyme can improve reaction efficiency, but they can also lead to rapid consumption of the substrate, which in turn prompts the enzyme to act on another enantiomer. A reaction exceeding 50% can affect the chirality of the product.

[0097] Example 4

[0098] This example compares the effect of different substrate concentrations on the reaction. All other conditions are the same as in Example 2, only the substrate concentration is changed. Specific conditions and results are as follows:

[0099] Table 4 Reaction conditions and results

[0100] As can be seen from the results in Table 4, the catalytic efficiency decreases significantly when the substrate concentration is higher than 10%, indicating that the conversion efficiency is better when the substrate concentration is ≤10%.

[0101] Example 5

[0102] This example compares the effects of different substrate and enzyme concentrations on the reaction. All other conditions are the same as in Example 2, except for the substrate concentration and enzyme amount. Specific conditions and results are as follows:

[0103] Table 5 Reaction conditions and results

[0104] As can be seen from Table 5, in order to balance reaction efficiency and quality, and to facilitate the control of the conversion process and cost, when the substrate concentration range is 1%-10% and the enzyme amount accounts for 1%-5% of the substrate, the conversion rate can reach more than 45%. When the enzyme amount is above 3%, the control interval after 8 hours of reaction is 0.5 hours to ensure that the conversion rate is ≤50% and the reaction is maximized, while not affecting the ee value of the target product.

[0105] Example 6

[0106] This embodiment optimizes the reaction conditions based on the results of Examples 2-5 and scales up the reaction. The specific steps are as follows:

[0107] (1) Prepare the reaction system according to the ratio of substrate mass concentration of 10% and enzyme mass of 5% of substrate mass: In a 1L reaction system, add 650mL water, 250mL enzyme suspension and 5mL cobalt chloride solution to the reaction vessel and stir thoroughly; control the temperature at 25℃, weigh 100g of substrate and slowly add it to the reaction vessel, adjust the pH to about 8.0 with sodium bicarbonate solution, and react at 25℃; after 3 hours of reaction, perform intermediate control every 1 hour. When the conversion progress reaches 40%, perform intermediate control every 0.5 hours. When the reaction progress reaches 48.2%, stop the reaction.

[0108] (2) The reaction solution was filtered through diatomaceous earth. The filtrate was extracted three times with 1200 mL of ethyl acetate, in portions of 500 mL, 400 mL, and 300 mL respectively. The aqueous phase was tested and confirmed to be free of amide.

[0109] (3) The pH of the aqueous phase was adjusted to 2-3 with hydrochloric acid; the product was extracted three times with 1200 mL of ethyl acetate (500 mL, 400 mL, and 300 mL), and the ethyl acetate phases were combined and dehydrated with anhydrous sodium sulfate; the ethyl acetate phase was concentrated to dryness, and the product was dissolved in 400 mL of ethyl acetate, filtered, and the filtrate was concentrated to dryness to obtain 45.4 g of the target product (R)-2-trifluoromethyl-2-hydroxypropionic acid, with a yield of 45.1% and an ee value of 99.85%.

[0110] Comparative Example 1

[0111] Using the original acetamidinase sequence, and following the production method in Example 2, the experiment was conducted with a 5% substrate concentration and different enzyme dosages. The results are shown in Table 6.

[0112] Table 6 Synthetic effects of wild-type acetamidoses

[0113] As can be seen from the results in Table 6, the target product has a low ee value and does not meet the quality requirements.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Industrial applicability

[0115] This invention provides a method for synthesizing (R)-2-trifluoromethyl-2-hydroxypropionic acid. The method uses 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide as a substrate, involves site mutation of acetylamidinase, and optimizes reaction conditions. This method can obtain a product with an ee value of over 99%, a substrate concentration of up to 10%, and a maximum conversion rate exceeding 45%. Therefore, the method of this invention for the production of (R)-2-trifluoromethyl-2-hydroxypropionic acid is highly efficient, environmentally friendly, and low-cost, showing promising prospects for industrial application.

Claims

1. An acetyl amidase mutant, characterized in that, The amino acid sequence of the acetamidase mutant is shown in SEQ ID NO.

1.

2. Biomaterial, characterized in that, The biological material includes any one of the following (A1)-(A4): (A1) a nucleic acid molecule encoding the acetamidase mutant of claim 1; (A2) an expression cassette containing the nucleic acid molecule of (A1); (A3) a vector containing the nucleic acid molecule of (A1) or the expression cassette of (A2); (A4) a recombinant microorganism containing the recombinant microorganism of (A1), or the expression cassette of (A2), or the vector of (A3).

3. A recombinant E. coli bacterium, characterized in that, The recombinant Escherichia coli contains the mutant of claim 1 or the biological material of claim 2; The nucleotide sequence of the acetamidase mutant is shown in SEQ ID NO.

2.

4. Use of the acetamidase mutant of claim 1 or the biological material of claim 2 in the following aspects: (B1) catalyzing the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid; (B2) preparing a product for catalyzing the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid.

5. A product of the catalytic synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid, characterized by The recombinant Escherichia coli of claim 3 contains the acetamidase mutant of claim 1, the biological material of claim 2, or the recombinant Escherichia coli of claim 3.

6. A process for the synthesis of (R)-2-trifluoromethyl-2-hydroxypropionic acid, characterized in that, It comprises: The acetamidase mutant of claim 1 is added to a solution containing 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide for catalytic reaction, followed by impurity removal and purification to obtain (R)-2-trifluoromethyl-2-hydroxypropionic acid.

7. The method of claim 6, wherein, The concentration of 3,3,3-trifluoro-2-hydroxy-2-methylpropionamide in the reaction system of the catalytic reaction is 1%-10% g / mL, the acetamidase mutant accounts for 1%-5% of the mass of the substrate, and the cobalt chloride solution accounts for 0.1%-0.5% of the mass of the substrate.

8. The method of claim 7, wherein, The conditions of the catalytic reaction are as follows: pH=7.0-8.0, temperature 20-30°C, and in-process control is performed during the reaction process, which is detected by high performance liquid chromatography with the following conditions: Column: C18 column 250*4.6mm; Mobile phase: 0.1% phosphoric acid: acetonitrile=90:10; Wavelength: 210nm / uv; Flow rate: 0.8mL / min; Injection volume: 20μl.

9. The method of claim 6, wherein, The impurity removal includes filtration and extraction, and the extraction includes multiple extractions of the filtrate with ethyl acetate to obtain an aqueous phase solution.

10. The method of claim 6, wherein, The purification includes re-extraction and refinement; The re-extraction includes adjusting the pH of the aqueous phase to 2-3, then multiple extractions with ethyl acetate, combining the ethyl acetate phase, and then dehydrating with anhydrous sodium sulfate; The refinement includes concentrating and drying the ethyl acetate phase after dehydration, then dissolving in ethyl acetate, filtering, re-concentrating and drying.

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