A mutant of r-type alpha fluoroimine reductase and preparation method and application thereof
By directing the evolution of imine reductase and mutating its active site amino acids, the problem of high-cost synthesis of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline in existing technologies has been solved, achieving efficient and low-cost biocatalytic synthesis suitable for the synthesis of key intermediates in the drug STX-478.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING UNIV
- Filing Date
- 2026-06-04
- Publication Date
- 2026-07-03
Smart Images
Figure CN122326554A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to enzyme mutants, and more particularly to an R-type α-fluoroimine reductase mutant, its preparation method, and its application. Background Technology
[0002] Fluorinated chiral amine drugs possess fluorine atoms, chiral centers, and amine groups. Fluorine enhances drug binding affinity, metabolic stability, and bioavailability, while the chiral center ensures target selectivity and reduces side effects. Synthesized through methods such as asymmetric catalysis, they form the core framework of drug development and are widely used in anti-tumor, anti-HIV, and antidepressant fields. For example, STX-478 (LY4064809) is a mutation-selective allosteric PI3Kα inhibitor. This drug is convenient to take orally and can cross the blood-brain barrier. It has strong affinity for PI3Kα mutants such as H1047R and E545K, weakly inhibits the wild-type, and has superior safety. In vitro IC50 results show... 50 With a concentration of 15-319 nmol / L, it exhibits dose-dependent tumor suppression. In its drug synthesis, the key fragment (R)-1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethylamine mainly relies on chiral resolution, which is costly and severely restricts the green enzymatic synthesis of intermediates for fluorinated chiral amine drugs. Therefore, there is an urgent need to develop a highly active, stereoselective, and substrate-adaptive imine reductase mutant to achieve efficient and low-cost biocatalytic synthesis of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline and the key intermediate STX-478. Summary of the Invention
[0003] Purpose of the invention: The purpose of this invention is to provide an R-type α-fluoroimine reductase mutant with high catalytic activity, high stereoselectivity and high production of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline, and to provide a method for preparing the enzyme mutant, the related nucleotide sequence, recombinant vector, and recombinant cells.
[0004] Technical solution: The R-type α-fluoroimine reductase mutant of the present invention is obtained by amino acid mutation of the sequence shown in SEQ ID NO.1, wherein the mutation is at least one of S237T, I241L or R248L.
[0005] The preferred R-type α-fluoroimine reductase mutants are S237T, S237T / I241L, and S237T / I241L / R248L, with amino acid sequences of SEQ ID NO.2~4, respectively.
[0006] This invention uses bacteria from Erythrobacterium tumefaciens Luteolibacter luteusThe imine reductase (UniProt:UPI00197BC338) is the original enzyme (wild type), with its amino acid sequence SEQ ID NO.1 and the gene sequence encoding this enzyme SEQ ID NO.5. This invention utilizes the sequence and structural information of publicly reported imine reductases, and through non-redundant searches in databases, screens potential enzyme genes based on principles such as protein structural similarity, conserved site analysis, and host origin diversity. After functional expression in an *E. coli* expression system, followed by purification, a pure original enzyme is obtained. Finally, imine reductase BcIRED is selected as the original enzyme, possessing broad substrate applicability and capable of catalyzing the formation of corresponding olefin compounds from various aromatic alkane substrates. This invention employs rational design to perform directed evolutionary modification of the original enzyme, mutating the amino acids in its active site, ultimately screening for an imine reductase mutant with high activity, stereoselectivity, and (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline yield.
[0007] This invention uses standard single-letter codes for amino acids and standard substitution notation. For example, S237T means that the serine (S) at position 237 of the N-terminus is mutated to threonine (T); S237T / I241L means that the serine (S) at position 237 of the N-terminus is mutated to threonine (T), and the isoleucine (I) at position 241 of the N-terminus is mutated to leucine (L).
[0008] The present invention also provides a nucleotide sequence encoding the R-type α-fluoroimine reductase mutant. The nucleotide sequence can be obtained by base mutation of the sequence shown in SEQ ID NO. 5, such as SEQ ID NO. 6-8.
[0009] The present invention also provides a recombinant vector comprising the aforementioned nucleotide sequence. The recombinant vector includes a cloning vector or an expression vector, optionally a plasmid or a virus, and is capable of maintaining replication in a host cell to amplify or express the nucleotide sequence. Preferably, the recombinant vector is a PET series expression vector.
[0010] The present invention also provides a recombinant cell comprising the aforementioned recombinant vector. The recombinant cell is preferably *Escherichia coli*.
[0011] The present invention also provides a method for preparing the R-type α-fluoroimine reductase mutant, comprising the following steps: (1) Design point mutation primers, use plasmids carrying wild-type imine reductase genes as templates, perform PCR reactions using point mutation primers, and obtain linearized plasmids after purification; (2) After the linearized plasmid is transferred into the host bacteria, the R-type α-fluoroimine reductase mutant is induced to express.
[0012] The present invention also provides a method for preparing the R-type α-fluoroimine reductase mutant, comprising the following steps: (1) Design point mutation primers, use plasmids carrying wild-type imine reductase genes as templates, perform PCR reactions using point mutation primers, and obtain mutant gene fragments and linearized plasmids after purification; (2) The mutant gene fragment was connected to the linearized plasmid to construct an expression vector. The expression vector was then transferred into the host bacteria and induced to express the R-type α-fluoroimine reductase mutant.
[0013] Preferably, in step (1), the point mutation primer is:
[0014] Preferably, in step (1), the PCR reaction system is as follows:
[0015] Preferably, in step (1), the PCR reaction conditions are as follows:
[0016] Preferably, in step (2), after expression is completed, cells are collected; or cells are broken and crude enzyme solution is collected; or cells are broken and purified R-type α-fluoroimine reductase mutant is collected.
[0017] The present invention also provides the application of the R-type α-fluoroimine reductase mutant, or the nucleotide sequence, or the recombinant vector, or the recombinant cell in the biocatalytic synthesis of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline.
[0018] Preferably, the biocatalysis uses (Z)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine as a substrate and reacts for 16-24 h at pH 4.2-9 and temperature 20-50°C.
[0019] Preferably, the present invention also provides the application of the R-type α-fluoroimine reductase mutant, or the nucleotide sequence, or the recombinant vector, or the recombinant cell in the key intermediate of the biocatalytic synthesis of the drug STX-478 (LY4064809).
[0020] The process by which the imine reductase-catalyzed reaction produces (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline is as follows:
[0021] This invention is based on the enzymatic imine reduction reaction, using (Z)-1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoro-N-(4-methoxyphenyl)ethane-1-imine (1) as a substrate and imine reductase as a biocatalyst, as a reaction pathway for the asymmetric reduction preparation of (R)-N-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)-4-methoxyaniline (2); constructing a candidate enzyme library, screening imine reductase using model reactions to obtain enzymes with initial reactivity; constructing a mutant library with different amino acid residue sites, screening the reactivity and selectivity of mutants using directed evolution; obtaining superior mutants with high reactivity and high selectivity, and obtaining (R)-1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethylamine (3) through deprotection.
[0022]
[0023] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The present invention obtains imine reductase mutants with high activity and stereoselectivity by performing single-point or combined mutations (S237T, I241L, R248L) on the active site of wild-type imine reductase through directed evolution. This enables the efficient synthesis of inexpensive (Z)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine to (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline, and can be applied to the synthesis of key intermediates in the drug STX-478 (LY4064809), showing good application prospects in the pharmaceutical field such as drug synthesis. Attached Figure Description
[0024] Figure 1 Schematic diagram of the yield and stereoselectivity of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline catalyzed by the original enzyme WT and the mutant; Figure 2 Schematic diagram of the yield and stereoselectivity of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline catalyzed by mutants S237T / I241L / R248L at different temperatures; Figure 3 Schematic diagram of the yield and stereoselectivity of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline catalyzed by mutants S237T / I241L / R248L at different pH levels; Figure 4 Application of R-type α-fluoroimine reductase mutant in the catalytic synthesis of key intermediates for the drug STX-478 (LY4064809); Figure 5 The chiral separation results of (R)-N-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)-4-methoxyaniline are shown in the figure. Figure 6 The 1H NMR spectrum of (R)-N-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)-4-methoxyaniline; Figure 7 The carbon NMR spectrum of (R)-N-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)-4-methoxyaniline; Figure 8 The NMR fluorine spectrum of (R)-N-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)-4-methoxyaniline. Detailed Implementation
[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0026] Example 1: An imine reductase mutant S237T This embodiment uses bacteria from *Acanthocephala*. Luteolibacter luteus The imine reductase (UniProt:UPI00197BC338) was used as the original enzyme, and the serine (S) at position 237 of its N-terminus was mutated to threonine (T).
[0027] The preparation method is as follows: (1) Design point mutation primers, perform PCR reaction, and obtain the mutant gene fragment; (2) Construct an expression vector and transform it into a host bacterium for induced expression. Details are as follows: (1) Design point mutation primers, perform PCR reaction, and obtain mutant gene fragments. A mutant plasmid for imine reductase was constructed. The wild-type imine reductase BcIRED gene was synthesized and constructed into the pET22b vector by Genewiz (Suzhou) Co., Ltd., and the vector was then transformed into... E. coli DH5α strain. Recombinant bacteria E. coli DH5α / pET22b-BcIRED cells were inoculated into 5 mL culture medium tubes and cultured at 37°C with shaking at 200 rpm for 12 h. After culture, the cells were centrifuged at 12,000 rpm for 1 min and collected. Using a high-purity plasmid miniprep kit, plasmids were extracted from the recombinant bacteria as templates for iterative mutagenesis to construct the pET22b-BcIRED mutant.
[0028] Expression and preparation of wild-type imine reductase. The expression vector was transformed into an expression strain to obtain a recombinant expression strain capable of expressing imine reductase. The successfully constructed recombinant expression strain... E. coli BL21(DE3) / pET22b-BcIRED was plated onto agar plates containing 100 μg / mL ampicillin. A single colony was picked and inoculated into 6 mL of LB medium containing antibiotic resistance. The culture was incubated overnight at 37 °C and 200 rpm. A 1% inoculum was then transferred to 500 mL of LB medium containing antibiotic resistance. When the OD600 reached approximately 0.6, 0.5 mM IPTG was added, and the culture was induced at 18 °C for approximately 14 h. Cells containing imine reductase were obtained for subsequent catalytic studies and enzyme activity assays.
[0029] Construction of recombinant Escherichia coli E. coli The BL21(DE3) / pET22b-BCIRED mutant S237T was obtained using whole-plasmid PCR. Primers for S237T are shown in Table 1, and single-point iterative mutagenesis was performed. The amino acid sequence of the mutant S237T is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.6.
[0030] Table 1 Primer sequences for mutant S237T
[0031] The PCR reaction system is shown in Table 2: Table 2 PCR reaction system
[0032] The PCR reaction conditions are shown in Table 3.
[0033] Table 3 PCR reaction conditions
[0034] After PCR amplification, the amplification product was detected by 0.9% agarose gel electrophoresis. The results showed that the amplification product was a single band, approximately 7000 bp in size. The amplification product was purified and recovered using a DNA recovery and purification kit.
[0035] The purified gene fragment was digested with DpnI to remove the template, and then recombined with recombinase. The recombinant product was transformed into... E. coliDH5α competent cells were plated on LB solid medium containing 100 mg / mL ampicillin and incubated at 37°C for 14 h. Single colonies were picked and transferred to LB liquid medium. Positive transformants were identified by PCR, and the correctness of the mutation sites was verified by sequencing. After verification, a portion of the cells were treated with 25% sterile glycerol, numbered, and stored at -80°C for later use. Plasmids were extracted from the remaining cells using a plasmid extraction kit, and the recombinant plasmids were stored at -20°C.
[0036] (2) After constructing the expression vector and transforming it into the host bacteria, the expression was induced. The successfully sequenced recombinant expression plasmid pET22b was transformed into... E. coli Using BL21(DE3) as the expression host, a recombinant mutant expression strain was constructed. E. coli BL21(DE3) / pET22b-BCIRED.
[0037] The successfully constructed recombinant mutant expression strain E. coli BL21(DE3) / pET22b-BcIRED was plated onto agar plates containing ampicillin at a final concentration of 100 μg / mL. A single colony was picked and inoculated into 6 mL of LB medium containing the resistant culture medium and incubated overnight at 37°C and 200 rpm. A 1% inoculum was then transferred to 500 mL of LB medium containing the resistant culture medium and cultured at OD... 600 When the concentration reaches approximately 0.6, add IPTG to a final concentration of 0.5 mM and induce at 18°C for approximately 14 h.
[0038] After centrifugation to obtain bacterial cells, the cells were resuspended in a buffer and sonicated in an ice bath (3 s intervals, 6 s intervals, total working time 30 min). The cells were then centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected and filtered through a 0.22 μm aqueous filter as a sample, which was then purified using a nickel column enzyme. Based on the amino acid sequence of BcIRED, the molar absorptivity e of the protein was calculated. The absorbance of the purified protein was measured using the A280 method to determine the protein concentration.
[0039] Example 2: An imine reductase mutant S237T / I241L This embodiment uses the same original enzyme as in Example 1, but mutates serine (S) at position 237 of its N-terminus to threonine (T), and isoleucine (I) at position 241 to leucine (L). The amino acid sequence of the mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.7. The preparation method is basically the same as in Example 1, except for the mutation primers, as shown in Table 4. Table 4 Primer sequences for mutant S237T / I241L
[0040] Example 3: An imine reductase mutant S237T / I241L / R248L This embodiment uses the same original enzyme as in Example 1, but mutates serine (S) at position 237 of its N-terminus to threonine (T), isoleucine (I) at position 241 to leucine (L), and arginine (R) at position 248 to leucine (L). The amino acid sequence of the mutant is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.8. The preparation method is basically the same as in Example 1, except for the mutation primers, as shown in Table 5. Table 5 Primer sequences for mutant S237T / I241L / R248L
[0041] Performance testing 1. Yield and stereoselectivity of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline catalyzed by imine reductase and its mutants. Wild-type enzymes and whole-cell bacterial cultures of mutants obtained in Examples 1-3 were used as catalysts.
[0042] The reaction system consisted of: whole-cell bacterial culture with OD=40, 10 mM (Z)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine (1), 5% DMSO, and 0.2 mM NADP. + 4 mg / mL GDH, 80 mM glucose. The reaction buffer was 200 mM pH 7.5 kpi. The reaction temperature was controlled at 30 °C using a water bath with magnetic stirring, and the reaction was carried out for 18 h. The concentration and stereoselectivity of the product (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline were determined by high-performance liquid chromatography (HPLC). Results are shown below. Figure 1 .
[0043] Depend on Figure 1 (The bar chart represents the yield, and the line graph represents the stereoselectivity.) It can be seen that, among the wild-type enzyme and the mutant, the mutant showed improvements in both yield and stereoselectivity compared to WT, with S237T / I241L / R248L showing the most significant improvement compared to WT.
[0044] 2. Optimal temperature for the synthesis of 4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline catalyzed by imine reductase mutant. Whole-cell bacterial culture of mutant S237T / I241L / R248L was used as a catalyst.
[0045] The reaction system consisted of: whole-cell bacterial culture with an OD of 40, 10 mM (Z)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine, 5% DMSO, and 0.2 mM NADP. + 4 mg / mL GDH, 80 mM glucose. The reaction buffer was 100 mM pH 8.5 Tris-HCl. The reaction temperature was controlled at 20℃, 25℃, 30℃, 37℃, and 45℃ using a water bath with magnetic stirring. The reaction was carried out for 18 h. The concentration and stereoselectivity of the product (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline were determined by high-performance liquid chromatography (HPLC). Results are shown below. Figure 2 .
[0046] Depend on Figure 2 (The bar chart represents yield, and the line graph represents selectivity.) It can be seen that different temperatures have a significant impact on the catalytic activity of the enzyme mutant, and its reactivity exhibits a pre-positive trend with temperature changes. The optimal catalytic effect was observed at 30℃, while enzyme activity was relatively low at temperatures below or above this level.
[0047] 3. The optimal pH for the synthesis of 4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline catalyzed by imine reductase mutants. Whole-cell bacterial culture of mutant S237T / I241L / R248L was used as a catalyst.
[0048] The reaction system consisted of: whole-cell bacterial culture with an OD of 40, 10 mM (Z)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine, 5% DMSO, and 0.2 mM NADP. + 4 mg / mL GDH and 80 mM glucose were used. The reaction buffers were different pH values: 50 mM disodium hydrogen phosphate-citrate buffer at pH 4.2, 50 mM disodium hydrogen phosphate-citrate buffer at pH 6, 50 mM sodium phosphate buffer at pH 7, 50 mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer at pH 7.5, 100 mM Tris-HCl buffer at pH 8, 100 mM Tris-HCl buffer at pH 8.5, and 100 mM Tris-HCl buffer at pH 9. The reaction temperature was controlled at 30 °C using a water bath with magnetic stirring, and the reaction was carried out for 18 h. The concentration and stereoselectivity of the product (R)-4-methoxy-N-(2,2,2-trifluoro-phenylethyl)aniline were determined by high-performance liquid chromatography (HPLC). Results are shown below. Figure 3 .
[0049] Depend on Figure 3(The bar chart represents yield, and the line graph represents stereoselectivity.) It can be seen that the optimal pH for enzyme activity in the enzyme mutant is 7.5. Under acidic conditions, the enzyme's cleavage activity is relatively limited, but it reaches its maximum value as the pH increases to 7.5. The activity gradually decreases with increasing buffer alkalinity.
[0050] 4. Application of imine reductase mutants in key intermediates of the catalytic synthesis of drug STX-478 (LY4064809) (see...) Figure 4 ) Whole-cell bacterial culture of mutant S237T / I241L / R248L was used as a catalyst.
[0051] The reaction system consisted of: whole-cell bacterial culture with OD=40, 2 mM (Z)-1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoro-N-(4-methoxyphenyl)ethane-1-imine (1), 5% DMSO, and 0.2 mM NADP. + 4 mg / mL GDH, 80 mM Glucose. The reaction buffer was 100 mM pH 8.5 Tris-HCl. The reaction temperature was controlled at 30℃ by water bath, and the mixture was magnetically stirred for 18 h. The concentration and stereoselectivity of the product (R)-N-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)-4-methoxyaniline (2) were determined by high performance liquid chromatography. The chiral separation results are shown in […]. Figure 5 .
[0052] Depend on Figure 5 It can be seen that the enzyme mutant can generate the target product (R)-1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethylamine (3) with single chirality, which has good application prospects.
[0053] (R)-N-(1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethyl)-4-methoxyaniline (2) was deprotected to give (R)-1-(5,7-difluoro-3-methylbenzofuran-2-yl)-2,2,2-trifluoroethylamine (3), NMR spectrum shown in [reference needed]. Figure 6 , 7 8.
Claims
1. A mutant of an R-type a-fluoroimine reductase, characterized in that, It is obtained by amino acid mutation of the sequence shown in SEQ ID NO.1, wherein the mutation is at least one of S237T, I241L or R248L.
2. The R-type a-fluoroimine reductase mutant of claim 1, wherein, The mutations are S237T, S237T / I241L, and S237T / I241L / R248L.
3. A nucleotide sequence, characterized in that, The R-type α-fluoroimine reductase mutant of claim 1 is encoded.
4. A recombinant vector, characterized in that, It comprises the nucleotide sequence of claim 3.
5. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 4.
6. A method for preparing the R-type α-fluoroimine reductase mutant according to claim 1, characterized in that, Includes the following steps: (1) Design point mutation primers, use plasmids carrying wild-type imine reductase genes as templates, perform PCR reactions using point mutation primers, and obtain linearized plasmids after purification; (2) After the linearized plasmid is transferred into the host bacteria, the R-type α-fluoroimine reductase mutant is induced to express.
7. The preparation method according to claim 6, characterized in that, In step (1), the point mutation primer is: 。 8. The use of the R-type α-fluoroimine reductase mutant of claim 1 or 2, the nucleotide sequence of claim 3, the recombinant vector of claim 4, or the recombinant cell of claim 5 in the biocatalytic synthesis of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline.
9. The application according to claim 8, wherein the biocatalysis uses (Z)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine as a substrate and reacts at pH 4.2-9 and temperature 20-50°C for 18-24 h.
10. The use of the R-type α-fluoroimine reductase mutant of claim 1 or 2, the nucleotide sequence of claim 3, the recombinant vector of claim 4, or the recombinant cell of claim 5 in the key intermediate of the biocatalytic synthesis of the drug STX-478 (LY4064809).