A method for preparing chiral alpha-trifluoromethyl alcohols using an imine reductase AtRedAm

Abstract

The application relates to the technical field of biological chemical industry, in particular to a method for preparing chiral alpha-trifluoromethyl alcohol by using imine reductase AtRedAm. The imine reductase AtRedAm is used to reduce trifluoromethyl ketone into corresponding chiral trifluoromethyl alcohol with excellent conversion rate and high enantioselectivity. The reaction depends on the coenzyme cycle system of NADPH, 10% dimethyl sulfoxide is used as a reaction cosolvent, and the reaction is carried out in 100 mM (pH=7.5) phosphate buffer for 16 h; the highest molar conversion rate reaches 99%, and the highest optical purity reaches 95%.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to a method for preparing chiral α-trifluoromethyl alcohol using imine reductase AtRedAm. Background Technology

[0002] Trifluoromethyl-substituted chiral alcohols (CF3-substituted chiral alcohols) are key building blocks in the synthesis of numerous bioactive molecules, pharmaceutical components (such as the antidepressant befluridone, ethyl telotristat for treating carcinoid syndrome diarrhea, and bitopertin for treating erythropoietic protoporphyria), and natural products. As a crucial functional group in medicinal chemistry, the trifluoromethyl group can significantly alter the electron distribution of molecules, profoundly affecting their binding interactions with target proteins. Furthermore, these fluoroalcohols also have applications in organocatalysis and coordination chemistry. Currently, methods for synthesizing structurally diverse CF3-substituted chiral alcohols mainly include asymmetric transfer hydrogenation with metal catalysts, organocatalytic aldol reactions, and microbial biotransformation. However, chemical methods are often limited by harsh conditions (high temperature and pressure) and the use of expensive and complex chiral catalysts, while biotransformation often performs poorly in terms of product yield and stereoselectivity. Although there have been some reports on the biocatalytic reduction of CF3-substituted acetophenone, the choice of reducing agents remains limited. Therefore, there is an urgent need to develop new routes for the synthesis of CF3-substituted chiral alcohols that can circumvent problems such as expensive metal catalysts, harsh reaction conditions, and low stereoselectivity.

[0003] Enzyme-catalyzed synthesis is increasingly popular in industry due to its advantages such as mild reaction conditions, excellent stereoselectivity, and environmental friendliness. Among them, imine reductases, as a class of NAD(P)H-dependent oxidoreductases, have attracted widespread attention for their ability to catalyze the direct reduction of prochiral imines and the reductive amination of ketones (aldehydes) and amines, thus efficiently synthesizing chiral amine compounds with primary, secondary, and tertiary amine structures. It is noteworthy that although imine reductases have shown great potential in the synthesis of chiral amines, there are almost no reports on their application in the synthesis of chiral alcohols (especially CF3-substituted chiral alcohols). Summary of the Invention

[0004] The purpose of this invention is to solve the problem of how to apply imine reductase to the synthesis of chiral alcohols (especially CF3-substituted chiral alcohols), and to provide a method for preparing chiral α-trifluoromethyl alcohols using the imine reductase AtRedAm.

[0005] To achieve the above objectives, this invention discloses a method for preparing chiral α-trifluoromethyl alcohol using imine reductase AtRedAm, comprising the following steps:

[0006] S1, Dissolve the trifluoromethyl ketone substrate in dimethyl sulfoxide to obtain a mixed solution;

[0007] S2, the mixed solution obtained in step S1 is added to the reaction buffer containing imine reductase AtRedAm and NADPH coenzyme system, and chiral trifluoromethyl alcohol is obtained after the reaction.

[0008] In step S1, the trifluoromethyl ketone compound is compound 1a, 2a, 3a, 4a, or 5a, with the following structural formula:

[0009] .

[0010] In step S2, the concentration of trifluoromethyl ketone in the reaction system is 10 mM, and the concentration of imine reductase AtRedAm is 0.2 mM.

[0011] In step S2, the NADPH coenzyme system is a mixture of NADP+, glucose dehydrogenase GDH-105, and glucose.

[0012] The NADPH coenzyme system contains 0.5-1 μM NADP+, 10 U glucose dehydrogenase GDH-105, and 20-100 mM glucose.

[0013] In step S2, the imine reductase AtRedAm is obtained by expressing a recombinant bacterium containing the gene sequence shown in SEQ ID NO.1.

[0014] In step S2, the amino acid sequence of the imine reductase AtRedAm is shown in SEQ ID NO.2.

[0015] In step S2, the reaction conditions are: pH 4.5~10.0, 20~30℃, 150-200 rpm for 16~24 h.

[0016] In step S2, the trifluoromethyl alcohol compound is compound 1b, 2b, 3b, 4b, or 5b, and its structural formula is shown below:

[0017] .

[0018] The reductase involved in this invention is an imine reductase, which contains 319 amino acids and has the accession number 5OJL on PDB. Its amino acid sequence is shown in SEQ ID NO.2. The gene encoding this protein contains 957 bp, and its nucleotide sequence is shown in SEQ ID NO.1.

[0019] The catalyst used in this invention is an imine reductase with an amino acid sequence as shown in SEQ ID NO.2, which can participate in the reaction in any form. For example, the gene can be introduced into the recipient bacteria to express olefin reductase to participate in the reaction, or the fermented sludge can be directly used in the reaction.

[0020] After inducing the expression of olefin reductase with the gene sequence shown in SEQ ID NO.2, the enzyme was reacted with 10 mM trifluoromethyl ketone, 10% dimethyl sulfoxide, 0.5–1 μM NADP+, 10 U glucose dehydrogenase GDH-105, and 10–20 mM glucose at pH 4.5–10.0, 20–30°C, and 150–200 rpm for 16–24 h to obtain chiral trifluoromethyl alcohol. The added NADP+ reacts with glucose to generate NADPH under the action of glucose dehydrogenase GDH-105, allowing the reaction to proceed cyclically, thus reducing the amount added and lowering production costs.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention is the first to apply the imine reductase with an amino acid sequence as shown in SEQ ID NO.2 to the asymmetric reduction of trifluoromethyl ketone to prepare chiral trifluoromethyl alcohol, achieving very good results. It has a high conversion rate of the substrate trifluoromethyl ketone (up to 99%), high optical activity of the product chiral trifluoromethyl alcohol (ee% value up to 95%), and high yield, which greatly reduces the production cost.

[0022] The enzymatic asymmetric reduction method for preparing chiral trifluoromethyl alcohol of the present invention has the following advantages compared with other preparation methods:

[0023] 1. The reaction conditions are mild, the process is simple and rapid, and it efficiently produces the target product with high enantiomeric purity;

[0024] 2. The catalyst is tolerant of high substrate concentrations, and the reaction can be completed with a single feed, making it easy to operate and scale up.

[0025] 3. The coenzyme recycling function built within the system greatly reduces the cost of cofactor consumption, laying an economic foundation for industrial application. Attached Figure Description

[0026] Figure 1 One of the synthetic routes of this invention;

[0027] Figure 2 Chiral analysis spectrum of the racemic product of standard 1b;

[0028] Figure 3 Chiral analysis spectrum of product 1b obtained by sampling after 16 hours of reaction;

[0029] Figure 4Chiral analysis spectrum of the 2b standard racemic product;

[0030] Figure 5 Chiral analysis spectrum of product 2b obtained by sampling after 16 hours of reaction;

[0031] Figure 6 Chiral analysis spectrum of the racemic product of standard 3b;

[0032] Figure 7 Chiral analysis spectrum of product 3b obtained by sampling after 16 hours of reaction;

[0033] Figure 8 Chiral analysis spectrum of the 4b standard racemic product;

[0034] Figure 9 Chiral analysis spectrum of product 4b obtained by sampling after 16 hours of reaction;

[0035] Figure 10 Chiral analysis spectrum of the 5b standard racemic product;

[0036] Figure 11 Chiral analysis spectrum of product 5b obtained by sampling after 16 hours of reaction;

[0037] Figure 12 NMR of reduction product 1b of compound 1a 1 H-NMR spectrum;

[0038] Figure 13 NMR of the reduction product 2b of compound 2a 1 H-NMR spectrum;

[0039] Figure 14 NMR of the reduction product 3b of compound 3a 1 H-NMR spectrum;

[0040] Figure 15 NMR of the reduction product 4b of compound 4a 1 H-NMR spectrum;

[0041] Figure 16 NMR of the reduction product 5b of compound 5a 1 H-NMR spectrum, the green curve in the figure represents... 1 The integral curve of the H-NMR spectrum, the height of which represents the integral area of ​​each hydrogen signal. Detailed Implementation

[0042] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0043] Example 1

[0044] Using E. coli to express imine reductase:

[0045] 1. Preparation of recombinant vectors:

[0046] The preparation method of the AtRedAm (PDB No.: 5OJL) protein described in this embodiment is as follows:

[0047] The AtRedAm expression vector was constructed by Nanjing Genscript Biotech Co., Ltd. using the pET28a(+) vector (synthesized by Genscript). Specifically, the AtRedAm gene fragment was used as a template, and after polymerase chain reaction, the AtRedAm gene was cloned into the pET28a(+) vector. The corresponding promoter is the T7 promoter, and a 6X His tag was added to its N-terminus. The tag was then cloned after the ribosome binding site (RBS) of the pET-28a(+) vector. The resulting recombinant vector was denoted as AtRedAm_pET28a(+).

[0048] DNA sequence of the AtRedAm gene:

[0049] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTACCACCACCACCACCACCAAACTGACCATCTTCGGCCTCGGCGCCATGGGCACCGCCATGGCCACCCAATTCCTCAAACAAGGCCACACCCCGACAGTCTGGAACCGCACCGCCGCCAAAGCAAACCCCCTCGTCGAACAAGGCGCCCACCTGGCCGCAACAATCCCCGCCGCCATCGCCGCCAGCCCCCTCCTCATCTTCTGTCTCCTCGACAACGCCGCCGTAGAACAAACCCTGGCCGCGGGCCCGCCCTCCCTCGCGGGGAAAACCATCCTCAACCTCACAAACGGCACGCCCAGCCAGGCCCGCCGCCTCGCCACGCTCGCCTCCGCGCGCGGCGCGCGCTACTTCCACGGCGGCATCATGGCAACCCCCGATATGATCGGGGCGCCGCACGCGGTGATTCTCTACAGCGGCGGCGGGTCTGCTGAGACCTACGCGTCCGTCGAGGGCGTTCTCGCCGTGCTCGGGTCGGGCAAGTACCTAGGCGACGATGCGGGGTCGGCCTCGCTGCACGATCTGGCGCTGCTGTCGGGCATGTATGGCCTGTTCGCGGGGTTCTTGCATGCCACGGCGCTGGTGCGCTCTGAGGGAGAGGGTGTCTCGGCGACGGAGTTCCTGGGGCTGCTGGCGCCGTGGTTGCAGGCTATGACGGGGTATCTGGGGCTGCTGGCCAGGCAGATCGATGATGGGGTGTATACGGCGCAGACGTCCAATCTGGAGATGCAGCTGGTTGCGCTGGAGAATGCGTGCGCGGCGAGTAGGGAGCAGGGGGTTTCGGCGGAGGTGATGCTGCCGTTGAAGGGGTTGGTGGAACGCGCTGTGCGGGAGGGGAGGGGAGGGCATGATATTTCGAGTTTGATTGATTATTTCAGGAATGCCTCTGTATAA(SEQ ID NO.1,From 5’ to3’)。

[0050] The amino acid sequence of the 6X His is shown below: HHHHHH (SEQ ID NO.3).

[0051] 2. Expression of imine reductase AtRedAm:

[0052] A plasmid containing the AtRedAm enzyme gene was used to transform *E. coli* BL21 (DE3) competent cells using the heat shock method. The cells were cultured overnight on LB agar plates containing a final concentration of 50 μg / mL kanamycin. Colonies that grew overnight were considered positive colonies. A single positive colony containing the recombinant plasmid was inoculated into 5 mL of LB medium containing 30 μg / mL kanamycin and cultured at 37°C with shaking at 180 rpm for 18 hours. 5 mL of the pre-cultured bacterial solution was then inoculated into 1 L of LB medium and cultured at 37°C with shaking at 220 rpm until the absorbance at 600 nm reached 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was then added to a final concentration of 1 mM to induce gene expression, and the cells were cultured overnight at 18°C ​​with shaking at 180 rpm.

[0053] Centrifuge for 20 min, collect and resuspend the bacterial cells; resuspend the collected bacterial cells in 50 mM Tris-NaCl buffer (pH=7.5), add PMSF (phenylmethylsulfonyl fluoride) to the working concentration of 1 mM, and sonicate on ice. The sonication parameters are: power 20 W, sonication, working time 2 s, interval 3 s, for a total of 40 min; then centrifuge at 4℃, 10000 rpm for 40 min to obtain the supernatant containing protein.

[0054] 3. Purification of protein AtRedAm:

[0055] Purification was performed using a nickel column. The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a pre-equilibrated Ni-NTA column, followed by washing with loading buffer (20 mM imidazole, 50 mM Tris, 300 mM sodium chloride, pH 7.5). The bound protein was eluted using a linear gradient of buffer containing 300 mM imidazole. The eluent was collected and dialyzed overnight in dialysis buffer (50 mM Tris-NaCl buffer, pH 7.5). The eluent was then concentrated using a 30 kDa ultracentrifuge tube at 4000 rpm. Using this method, 100 mg of protein could be purified per liter of bacteria.

[0056] The amino acid sequence of imine reductase AtRedAm:

[0057] MGSSHHHHHHSSGLVPRGSHMATTTTTTKLTIFGLGAMGTAMATQFLKQGHTPTVWNRTAAKANPLVEQGAHLAATIPAAIAASPLLIFCLLDNAAVEQTLAAGPPSLAGKTILNLTNGTPSQARRLATLASARGARYFHGGIMATPDMIGAPHAVILYSG GGSAETYASVEGVLAVLGSGKYLGDDAGSASLHDLALLSGMYGLFAGFLHATALVRSEGEGVSATEFLGLLAPWLQAMTGYLGLLARQIDDGVYTAQTSNLEMQLVALENACAASREQGVSAEVMLPLKGLVERAVREGRGGHDISSLIDYFRNASV* (SEQ ID NO.2).

[0058] Example 2

[0059] Preparation of chiral α-trifluoromethyl alcohol 1b using imine reductase AtRedAm:

[0060] 10 μmol of trifluoromethyl ketone substrate 1a was dissolved in 100 μL of DMSO and added to a reaction buffer (100 mM pH 7.5) containing 2% (0.2 mM) AtRedAm, 0.1 mg GDH (~10 U), 80 μmol of D-glucose, and 0.5 μmol of NADP+. The reaction was carried out at 220 rpm for 16 h at 25 °C. Samples were then taken for chiral column analysis. The chromatogram is shown below. Figures 2-3 See NMR spectrum Figure 12 (Sample reacted for 16 h, retention time 25 min), the product yield was 86%, and the optical purity ee% value was greater than 90%.

[0061] The detection methods for the product are as follows:

[0062] After the reaction was complete, an equal volume of ethyl acetate was added, the mixture was shaken vigorously for 10 min, and then allowed to stand for two hours. The organic and aqueous layers were separated by centrifugation at 8000 rpm for 10 min. The upper ethyl acetate layer was carefully pipetted through an organic membrane and the sample was stored.

[0063] The determination of enantiomeric excess of chiral trifluoromethyl alcohol was performed using Shimadzu LC with a CHIRALPAK OD-H solvent-resistant bonded chiral column. The mobile phase used was 99% n-hexane and 1% isopropanol, and the program was as follows: column temperature 40℃, detector absorption wavelengths: 214 nm and 254 nm, flow rate 1 mL / min, and isogradient elution for 30 min.

[0064] The enantiomeric excess value (ee%) of the product chiral trifluoromethyl alcohol is calculated by the following formula:

[0065] Enantiomer excess (ee%) ,

[0066] In the formula, S is the peak area of ​​(S)-trifluoromethyl alcohol, and R is the peak area of ​​(R)-trifluoromethyl alcohol.

[0067] The retention time of (S)-trifluoromethyl alcohol was 25.7 min, and the retention time of (R)-trifluoromethyl alcohol was 27.8 min. The optical purity ee% was 95%.

[0068] Example 3

[0069] Preparation of chiral α-trifluoromethyl alcohol 2b using imine reductase AtRedAm:

[0070] 10 μmol of trifluoromethyl ketone substrate 2a was dissolved in 100 μL of DMSO and added to 1 mL of reaction buffer (100 mM pH 7.5 phosphate buffer) containing 2% mol (0.2 mM) AtRedAm, 0.1 mg GDH (~10 U), 80 μmol of D-glucose, and 0.5 μmol of NADP+. The reaction was carried out at 220 rpm for 16 h at 25 °C. Samples were then taken for chiral column analysis. The chromatogram is shown below. Figures 4-5 See NMR spectrum Figure 13 (Sample reacted for 16 h, retention time 21 min), the product yield was 86%, and the optical purity ee% value was greater than 23%.

[0071] The detection methods for the product are as follows:

[0072] After the reaction was complete, an equal volume of ethyl acetate was added, the mixture was shaken vigorously for 10 min, and then allowed to stand for two hours. The organic and aqueous layers were separated by centrifugation at 8000 rpm for 10 min. The upper ethyl acetate layer was carefully pipetted through an organic membrane and the sample was stored.

[0073] The determination of enantiomeric excess of chiral trifluoromethyl alcohol was performed using Shimadzu LC with a CHIRALPAK OJ-H solvent-resistant bonded chiral column. The mobile phase used was 90% n-hexane and 10% isopropanol. The program was as follows: column temperature 40℃, detector absorption wavelengths: 214 nm and 254 nm, flow rate 1 mL / min, and isogradient elution for 30 min.

[0074] The enantiomeric excess value (ee%) of the product chiral trifluoromethyl alcohol is calculated by the following formula:

[0075] Enantiomer excess (ee%) ,

[0076] In the formula, S is the peak area of ​​(S)-trifluoromethyl alcohol, and R is the peak area of ​​(R)-trifluoromethyl alcohol.

[0077] The retention time of (S)-trifluoromethyl alcohol was 20 min, and the retention time of (R)-trifluoromethyl alcohol was 21.6 min. The optical purity ee% was 23%.

[0078] Example 4

[0079] Preparation of chiral α-trifluoromethyl alcohol 3b using imine reductase AtRedAm:

[0080] 10 μmol of trifluoromethyl ketone substrate 3a was dissolved in 100 μL of DMSO and added to 1 mL of reaction buffer (100 mM pH 7.5 phosphate buffer) containing 2% mol (0.2 mM) AtRedAm, 0.1 mg GDH (~10 U), 80 μmol of D-glucose, and 0.5 μmol of NADP+. The reaction was carried out at 220 rpm for 16 h at 25 °C. Samples were then taken for chiral column analysis. The chromatogram is shown below. Figures 6-7 See NMR spectrum Figure 14 (Sample reacted for 16 h, retention time 7.5 min), the product yield was 72%, and the optical purity ee% value was greater than 90%.

[0081] The detection methods for the product are as follows:

[0082] After the reaction was complete, an equal volume of ethyl acetate was added, the mixture was shaken vigorously for 10 min, and then allowed to stand for two hours. The organic and aqueous layers were separated by centrifugation at 8000 rpm for 10 min. The upper ethyl acetate layer was carefully pipetted through an organic membrane and the sample was stored.

[0083] The determination of enantiomeric excess of chiral trifluoromethyl alcohol was performed using Shimadzu LC with a CHIRALPAK OJ-H solvent-resistant bonded chiral column. The mobile phase used was 90% n-hexane and 10% isopropanol. The program was as follows: column temperature 40℃, detector absorption wavelengths: 214 nm and 254 nm, flow rate 1 mL / min, and isogradient elution for 30 min.

[0084] The enantiomeric excess value (ee%) of the product chiral trifluoromethyl alcohol is calculated by the following formula:

[0085] Enantiomer excess (ee%) ,

[0086] In the formula, S is the peak area of ​​(S)-trifluoromethyl alcohol, and R is the peak area of ​​(R)-trifluoromethyl alcohol.

[0087] The retention time of (S)-trifluoromethyl alcohol was 7.2 min, and the retention time of (R)-trifluoromethyl alcohol was 8 min. The optical purity ee% was 90%.

[0088] Example 5

[0089] Preparation of chiral α-trifluoromethyl alcohol 4b using imine reductase AtRedAm:

[0090] 10 μmol of trifluoromethyl ketone substrate 4a was dissolved in 100 μL of DMSO and added to 1 mL of reaction buffer (100 mM pH 7.5 phosphate buffer) containing 2% mol (0.2 mM) AtRedAm, 0.1 mg GDH (~10 U), 80 μmol of D-glucose, and 0.5 μmol of NADP+. The reaction was carried out at 220 rpm for 16 h at 25 °C. Samples were then taken for chiral column analysis. The chromatogram is shown below. Figures 8-9 See NMR spectrum Figure 15 (Sample reacted for 16 h, retention time 7 min), the product yield was 67%, and the optical purity ee% value was greater than 62%.

[0091] The detection methods for the product are as follows:

[0092] After the reaction was complete, an equal volume of ethyl acetate was added, the mixture was shaken vigorously for 10 min, and then allowed to stand for two hours. The organic and aqueous layers were separated by centrifugation at 8000 rpm for 10 min. The upper ethyl acetate layer was carefully pipetted through an organic membrane and the sample was stored.

[0093] The determination of enantiomeric excess of chiral trifluoromethyl alcohol was performed using Shimadzu LC with a CHIRALPAK OJ-H solvent-resistant bonded chiral column. The mobile phase used was 90% n-hexane and 10% isopropanol, and the program was as follows: column temperature 40℃, detector absorption wavelengths: 214 nm and 254 nm, flow rate 1 mL / min, and isogradient elution for 30 min.

[0094] The enantiomeric excess value (ee%) of the product chiral trifluoromethyl alcohol is calculated by the following formula:

[0095] Enantiomer excess (ee%) ,

[0096] In the formula, S is the peak area of ​​(S)-trifluoromethyl alcohol, and R is the peak area of ​​(R)-trifluoromethyl alcohol.

[0097] The retention time for (S)-trifluoromethyl alcohol was 7 min, and the retention time for (R)-trifluoromethyl alcohol was 7.5 min. The optical purity ee% was 62%.

[0098] Example 6

[0099] Preparation of chiral α-trifluoromethyl alcohol 5b using imine reductase AtRedAm:

[0100] 10 μmol of trifluoromethyl ketone substrate 5a was dissolved in 100 μL of DMSO and added to 1 mL of reaction buffer (100 mM pH 7.5 phosphate buffer) containing 2% mol (0.2 mM) AtRedAm, 0.1 mg GDH (~10 U), 80 μmol of D-glucose, and 0.5 μmol of NADP+. The reaction was carried out at 220 rpm for 16 h at 25 °C. Samples were then taken for chiral column analysis. The chromatogram is shown below. Figures 10-11 See NMR spectrum Figure 16 (Sample reacted for 16 h, retention time 9 min), the product yield was 99%, and the optical purity ee% value was greater than 17%.

[0101] The detection methods for the product are as follows:

[0102] After the reaction was complete, an equal volume of ethyl acetate was added, the mixture was shaken vigorously for 10 min, and then allowed to stand for two hours. The organic and aqueous layers were separated by centrifugation at 8000 rpm for 10 min. The upper ethyl acetate layer was carefully pipetted through an organic membrane and the sample was stored.

[0103] The determination of enantiomeric excess of chiral trifluoromethyl alcohol was performed using Shimadzu LC with a CHIRALPAK OJ-H solvent-resistant bonded chiral column. The mobile phase used was 90% n-hexane and 10% isopropanol. The program was as follows: column temperature 40℃, detector absorption wavelengths: 214 nm and 254 nm, flow rate 1 mL / min, and isogradient elution for 30 min.

[0104] The enantiomeric excess value (ee%) of the product chiral trifluoromethyl alcohol is calculated by the following formula:

[0105] Enantiomer excess (ee%) ,

[0106] In the formula, S is the peak area of ​​(S)-trifluoromethyl alcohol, and R is the peak area of ​​(R)-trifluoromethyl alcohol.

[0107] The retention time of (S)-trifluoromethyl alcohol was 8.1 min, and the retention time of (R)-trifluoromethyl alcohol was 9.5 min. The optical purity ee% was 17%.

[0108] Example 7

[0109] Chiral α-trifluoromethyl alcohol 1b was prepared using imine reductase AtRedAm under different pH conditions:

[0110] A 100 mM phosphate buffer with a pH gradient of 0.5 (pH 4.5–10.0) was prepared. 10 μmol of trifluoromethyl ketone substrate 1a was dissolved in 100 μL of DMSO and added to 1 mL of reaction buffer containing 2% mol (0.2 mM) AtRedAm, 0.1 mg GDH (~10 U), 80 μmol of D-glucose, and 0.5 μmol of NADP+. The reaction was carried out at 220 rpm for 16 h at 25 °C. Samples were taken for HPGC yield detection. The results are shown in Table 1. The enantiomeric excess of the obtained chiral trifluoromethyl alcohol was determined, and the optical purity ee% values ​​were all greater than 90%.

[0111] The detection methods for the product are as follows:

[0112] After the reaction was complete, an equal volume of ethyl acetate and 10 μmol naphthalene (internal standard) were added, and the mixture was shaken vigorously for 10 min and then allowed to stand for two hours. The organic layer and the aqueous layer were separated by centrifugation at 8000 rpm for 10 min. The upper ethyl acetate layer was carefully pipetted through an organic membrane and the sample was stored.

[0113] The chromatographic column used was a Shimadzu GC-2030, and the column was an Agilent HP-FFAP. The program was: column temperature 100~220 ℃, and the detector was a flame ionization detector.

[0114] Table 1. Results of preparation of chiral α-trifluoromethyl alcohol 1b using imine reductase AtRedAm under different pH conditions.

[0115]

[0116] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for preparing chiral α-trifluoromethyl alcohol using imine reductase AtRedAm, characterized in that, Includes the following steps: S1, Dissolve the trifluoromethyl ketone substrate in dimethyl sulfoxide to obtain a mixed solution; S2, the mixed solution obtained in step S1 is added to the reaction buffer containing imine reductase AtRedAm and NADPH coenzyme system, and chiral trifluoromethyl alcohol is obtained after the reaction; In step S1, the trifluoromethyl ketone compound is compound 1a or 3a, with the following structural formula: 、 ； In step S2, the reaction conditions are: pH 7.5, 20-30℃, 150-200 rpm for 16-24 h; the amino acid sequence of imine reductase AtRedAm is shown in SEQ ID NO.2; the NADPH coenzyme system is a mixture of NADP+, glucose dehydrogenase GDH-105, and glucose; the trifluoromethyl alcohol compound is compound 1b or 3b, and its structural formula is shown below: 、 。 2. The method for preparing chiral α-trifluoromethyl alcohol using imine reductase AtRedAm as described in claim 1, characterized in that, In step S2, the concentration of trifluoromethyl ketone in the reaction system is 10 mM, and the concentration of imine reductase AtRedAm is 0.2 mM.

3. The method for preparing chiral α-trifluoromethyl alcohol using imine reductase AtRedAm as described in claim 1, characterized in that, The NADPH coenzyme system contains 0.5-1 μM NADP+, 10 U glucose dehydrogenase GDH-105, and 20-100 mM glucose.

4. The method for preparing chiral α-trifluoromethyl alcohol using imine reductase AtRedAm as described in claim 1, characterized in that, In step S2, the imine reductase AtRedAm is obtained by expressing a recombinant bacterium containing the gene sequence shown in SEQ ID NO.

1.

5. The method for preparing chiral α-trifluoromethyl alcohol using imine reductase AtRedAm as described in claim 1, characterized in that, In step S2, the reaction conditions are: pH 7.5, 25℃, 220 rpm for 16 h.

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