An imine reductase PmIR mutant and a preparation method and use of (R)-5-fluoro-2-methoxy-3-(pyrrolidin-2-yl)pyridine

Abstract

The application discloses an imine reductase PmIR mutant and a preparation method and application of (R)-5-fluoro-2-methoxy-3-(pyrrolidin-2-yl)pyridine ((R)-FMPP) in the mutant, wherein the mutant is any one of single-point or combined mutation of glutamine at position 138, proline at position 140, glutamine at position 190, tryptophan at position 195, serine at position 228 and arginine at position 251 of imine reductase PmIR, and the amino acid sequence of the parent is shown in SEQ ID NO. 2.

Description

Technical Field

[0001] This invention belongs to the field of enzyme-catalyzed chiral synthesis, specifically relating to the preparation method and uses of an imine reductase PmIR mutant and (R)-5-fluoro-2-methoxy-3-(pyrrolidine-2-yl)pyridine ((R)-FMPP). Background Technology

[0002] (R)-FMPP is a key chiral building block for many TRK kinase inhibitors. LOXO-195 (seritrectinib) is a targeted therapy primarily used to treat NTRK fusion-positive tumors, such as non-small cell lung cancer, breast cancer, and soft tissue sarcoma. As a selective TRK inhibitor, LOXO-195 inhibits tumor cell growth by precisely suppressing TRK receptor activation induced by NTRK gene fusions. Compared to traditional chemotherapy, it offers higher selectivity, fewer side effects, and effectively overcomes resistance to some TRK inhibitors. LOXO-195 provides a new treatment option for NTRK fusion-positive patients, particularly those with advanced tumors resistant to other therapies, and has significant clinical implications.

[0003] In the original drug synthesis route published in 2018, (R)-FMPP is responsible for forming the R stereoconfiguration of the pyridine ring (US9902741(B2)). Currently, the construction of this chiral center usually requires the use of an additional chiral auxiliary (-)sparteine, such as (R)-2-methylpropane-2-sulfinamide disclosed in US Patent US9902741B2 and WIPO Patent WO2018081417, or asymmetric reduction using transition metals under high-pressure hydrogen. Summary of the Invention

[0004] To address the aforementioned technical problems, this application provides an imine reductase PmIR mutant and its synthetic method and application in (R)-FMPP. Compared to existing technologies, in a preferred embodiment of this application, the use of imine reductase for asymmetric reduction not only reduces dependence on toxic solvents and high-energy-consuming reaction conditions but also significantly improves the optical purity and reaction selectivity of the product, while possessing advantages such as mild reaction conditions and environmental friendliness. This application discloses for the first time an imine reductase mutant and its application in the synthesis of (R)-FMPP.

[0005] According to one aspect of this application, an imine reductase PmIR mutant is provided, said mutant being a single-point or combined mutation of any one of the hollow positions of glutamine at position 138, proline at position 140, glutamine at position 190, tryptophan at position 195, serine at position 228, and arginine at position 251 of the imine reductase PmIR, said parent having an amino acid sequence as shown in SEQ ID NO. 1.

[0006] In a preferred embodiment, the mutant is a single-point mutation of the parent as shown below.

[0007] Or combined mutations:

[0008] (1) Mutate the 138th amino acid residue from glutamine Q to methionine M;

[0009] (2) Mutate the 140th amino acid residue from proline P to alanine A;

[0010] (3) Mutate the 190th amino acid residue from glutamine Q to serine S;

[0011] (4) Mutate the 195th amino acid residue from tryptophan W to methionine M;

[0012] (5) Mutate the 228th amino acid residue from serine (S) to asparagine (N);

[0013] (6) The 251st amino acid residue was mutated from arginine R to asparagine N.

[0014] According to another aspect of this application, this application provides a gene or recombinant vector encoding a mutant of imine reductase PmIR, such as an imine reductase.

[0015] In a preferred embodiment, pET-28a(+) is used as the expression vector.

[0016] According to another aspect of this application, this application provides a recombinant microbial cell carrying the aforementioned genes or a recombinant vector.

[0017] In a preferred embodiment, the microorganism is Escherichia coli.

[0018] In a preferred embodiment, the recombinant microbial cells use Escherichia coli BL21(DE3) as the host cell.

[0019] According to another aspect of this application, this application also provides a method for preparing (R)-FMPP, wherein the method for preparing (R)-FMPP includes the following steps:

[0020] A catalytic system was constructed using 3-(3,4-dihydro-2H-pyrrolo-5-yl)-5-fluoro-2-methoxypyridine (DPFM) as a substrate and the imine reductase PmIR mutant as a catalyst to catalyze the generation of (R)-FMPP.

[0021] According to another aspect of this application, this application provides the use of (R)-FMPP prepared by the method described above, which is used as a TRK kinase inhibitor.

[0022] It is worth noting that amino acids within substrate channels not only affect substrate binding but may also directly participate in the catalytic process. Based on the wild-type structure model of PmIR, the applicant selected a series of key sites in the substrate channel for saturation mutations and found that multiple mutation sites significantly promoted the enzyme's catalytic activity. Through further combined mutation screening, PmIR mutants with optimized catalytic activity were obtained. The engineered imine reductase PmIR mutant exhibited higher space-time yields and a wider substrate catalytic range.

[0023] The imine reductase PmIR mutant involved in this invention can reduce the substrate DPFM to (R)-FMPP through its catalysis. In a reaction system with a substrate concentration of 19.8 g / L, the conversion rate can reach 100% within 2.5 h, and the optical purity ee value of the product is greater than 99%. Attached Figure Description

[0024] Figure 1 A schematic diagram of the enzyme-catalyzed reaction of the imine reductase in DPFM is shown.

[0025] Figure 2 This illustration shows a schematic diagram of the mutagenesis accumulation process of imine reductase in DPFM catalyzed in one embodiment of this application.

[0026] Figures 3A-3D Schematic diagrams of DPFM catalysis under conditions of PmIR and different contents of PmIR-M5 are shown respectively.

[0027] Figure 4 An example is shown of the enzymatic gram-level synthesis of (R)-FMPP 50mM and 100mM. Detailed Implementation

[0028] The following description is intended to disclose the present invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.

[0029] The following examples will help those skilled in the art to further understand the present invention, but do not limit the invention in any way.

[0030] The recombinant Escherichia coli carrying the imine reductase gene involved in this invention uses pET-28a(+) as the vector and BL21(DE3) as the host. Unless otherwise specified, the cofactor NADP, restriction endonucleases, and plasmid extraction kits used in the enzyme catalysis are all commercially available.

[0031] The specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the product instructions for the kit.

[0032] Example 1: Molecular docking and channel analysis using PmIR

[0033] Using the PmIR wild-type crystal structure as a template, the PmIR wild-type crystal model and the substrate DPFM were molecularly docked using AutoDock Tools. A docking box was set with the NADPH binding site as the center, and semi-flexible docking was performed using AutoDock 100 times, with other parameters left as default. After docking, reasonable conformations were selected for subsequent analysis.

[0034] Substrate transport channels of PmIR were predicted using CAVER Analyst 2.0β. First, the cavity probe radius was set to 2.20 Å. In the channel calculation settings, the region containing the cofactor NADPH was selected as the starting point for the channel search, with a minimum probe radius of 0.9 Å. The residue contribution analysis plot output by the software further revealed the influence of residues surrounding the channel on channel morphology, identifying 25 key residues that may affect channel properties, including M32, E59, S112, V137, Q138, V139, P140, P141, Q190, M191, F194, W195, S228, F232, Y236, P246, V249, D250, R251, G255, M254, S258, V282, M287F, and F296.

[0035] Example 2: Preparation of PmIR Expression

[0036] Primers were designed using NNK codons for the sites selected in Example 1. Base mutations were introduced into the imine reductase PmIR gene sequence (the nucleotide sequence of PmIR is shown in SEQ ID No. 2) using PCR amplification technology. The template used was the recombinant plasmid of imine reductase PmIR. After transformation, single colonies were picked from the plate and sequenced to obtain a saturated mutant library.

[0037] The recombinant pET28a(+)-PmIR plasmid obtained in Example 2 was transformed into *Escherichia coli* BL21(DE3). After preliminary screening, the host strain BL21(DE3) / pET28a(+)-PmIR containing the target plasmid was successfully obtained and preserved as a 20% glycerol bacterium. Subsequently, *E. coli* containing the target gene was inoculated at a 1% inoculum into 50 mL LB liquid medium containing 50 mg / mL kanamycin (the medium was purchased from Sangon Biotech Co., Ltd.). The culture was carried out in a shaker at 37 °C and 220 rpm until OD (dose expiratory volume) was reached. 600nm The expression level was increased to 0.6 to 0.8. At this point, 50 μL of 100 mM isopropyl β-D-thiogalactoside (IPTG) was added to induce protein expression, and induction was continued for 20 hours at 20 °C and 220 rpm. After induction, the bacterial cells were collected by centrifugation and washed once with physiological saline. After washing, excess water was removed, and the bacterial cells were prepared into a resting cell suspension of 50 mg / ml using 100 mM pH 6.0 phosphate buffer.

[0038] Example 3: Detection of transformation rate of PmIR saturated mutant library

[0039] Using DPFM as the substrate, the schematic diagram of the enzyme-catalyzed reaction is shown below. Figure 1 As shown in Table 1. 275 μL of mutant resting cells (50 mg / ml), 100 μL of glucose dehydrogenase resting cells (50 mg / ml), 50 μL of 500 mM glucose solution, and 50 μL of 2.5 mM NADP+ solution were reacted at 30 ℃ and 220 rpm for 10 min, followed by the addition of 25 μL of 200 mM DPFM in methanol. The reaction was continued at 30 ℃ and 220 rpm for 50 min, then 1 ml of IPA was added for inactivation. After centrifugation, the supernatant was collected, and the ee value and conversion rate were detected by HPLC. After multiple rounds of screening and comparison, as shown in Table 1... Figure 2 As shown, the dominant mutant PmIR-M5 (Q138M / P140A / Q190S / W195M / S228N / R251N) was obtained, and its nucleotide sequence is shown in SEQ ID No. 4.

[0040] Table 1 500 μL reaction system

[0041]

[0042] Chiral analytical column = AD-H; mobile phase = n-hexane:ethanol:isopropanol (95:2.5:2.5) + 0.1% diethylamine; flow rate = 1 ml / min; temperature = 30 ℃; detection wavelength = 254 nm. The retention time of the substrate DPFM was 4.958 (min), the R enantiomer retention time was 5.884 (min), and the S enantiomer retention time was 6.991 (min).

[0043] Example 4: Comparison of purification and catalytic performance of PmIR and PmIR-M5

[0044] PmIR and PmIR-M5 were purified using a nickel column. Impurities were eluted with 20 mM and 50 mM imidazole solutions, while the target protein was eluted with 200 mM imidazole solution. The purified enzyme solutions of PmIR and PmIR-M5 were concentrated using 10 kD ultrafiltration centrifuge tubes.

[0045] Take 275 μL of purified enzyme with a similar protein concentration, 100 μL of crude GDH enzyme solution, 50 μL of 2.5 mM NADP solution, and 50 μL of 500 mM glucose solution. After incubating for 10 min, add 400 mM, 200 mM, 100 mM, and 50 mM substrate DPFM to initiate the reaction. Figures 3A-3D As shown.

[0046] Figures 3A-3D For PmIR and PmIR-M5 at 2.5mM ( Figure 3A ), 5mM ( Figure 3B ), 10mM ( Figure 3C ), 20mM ( Figure 3D Catalytic DPFM under the following conditions.

[0047] Example 5 Enzymatic gram-level synthesis of (R)-FMPP

[0048] As shown in Tables 2 and 3, IRED dominant mutant cells, GDH cells, 2.5 mM NADP+ solution, glucose, and 0.1 M phosphate buffer were added sequentially. After reacting at 30 °C for 10 min, substrate solutions of 500 mM and 1000 mM concentrations were added. The substrate concentrations in the enzymatic reactions were 9.7 g / L and 19.4 g / L, respectively. The reactions were completed within 1.5 h and 2.5 h, respectively, with an optical purity greater than 99%. The reaction progress curves are shown below. Figure 2 As shown.

[0049] Table 2. Enzymatic synthesis in a 1 ml reaction system at a 50 mM concentration.

[0050]

[0051] Table 3. Enzymatic synthesis in 1 ml reaction system at 100 mM concentration.

[0052]

[0053] Compared to the wild type, the imine reductase PmIR mutant provided by this invention exhibits significantly improved conversion rate and optical purity in the production of (R)-FMPP. Catalysis can be completed at a concentration of 19.8 g / L, with both ee and conversion rate >99%.

[0054] It is worth mentioning that the sequence information involved in this invention is as follows:

[0055] SEQ ID No. 1 (Amino acid sequence of imine reductase PmIRED):

[0056] MKSSNRSENIRVGTENTVGKSKSVTVIGLGPMGKAMAAAFLEHGYKVTVWNRTSNKADELITKGAVRASTVHEALAANELVILSLTDYDAMYTILEPASENLSGKVLVNLSSDTPDKAREAAKWLANRGAGHITGGVQVPPSGIGKPESSTYYSG PKEVFEANKETLEVLTGTDYRGEDPGLAALYYQIQMDMFWTAMLSYLHATAVAQANGITAEQFLPYAAETMSSLPKFIEFYTPRINAGEYPGDVDRLAMGMASVEHVVHTTQDAGIDITLPTAVLEVFRRGMENGHAGNSFTSLIEIFKKSDIRP

[0057] SEQ ID No. 2 (nucleotide sequence of imine reductase PmIRED):

[0058] ATGAAAAGTAGCAATCGAAGTGAGAACATTCGTGTTGGAACCGAGAATACAGTTGGGAAGAGCAAATCGGTGACGGTCATCGGGCTTGGTCCGATGGGTAAGGCGATGGCGGCCGCTTTCCTTGAGCATGGCTATAAGGTGACCGTGTGGAACAGGACTTCAAACAAAGCAGACGAACTCATAACAAAAGGAGCCGTTAGGGCGTCCACGGTCCACGAAGCGTTGGCGGCCAATGAGCTAGTCATCCTCAGCCTAACGGACTACGATGCGATGTACACCATTCTTGAACCGGCATCGGAGAATCTGTCCGGCAAGGTTCTCGTCAACCTGAGCTCGGACACTCCGGATAAAGCGCGCGAGGCGGCGAAGTGGCTGGCCAACCGTGGAGCCGGGCACATCACAGGCGGCGTTCAGGTCCCTCCTTCTGGCATCGGTAAACCAGAGTCCTCCACTTACTACAGCGGCCCGAAAGAGGTCTTTGAGGCCAACAAGGAGACACTCGAAGTCCTGACCGGAACTGACTATCGGGGAGAAGATCCCGGACTTGCGGCACTGTACTACCAGATCCAGATGGACATGTTCTGGACAGCCATGCTCAGCTACCTCCATGCCACTGCGGTGGCCCAAGCGAACGGTATTACGGCTGAGCAGTTTCTGCCATACGCCGCCGAGACAATGTCGTCGCTGCCGAAGTTCATCGAGTTCTACACACCTCGGATTAACGCGGGTGAATATCCCGGTGACGTAGACAGACTTGCCATGGGAATGGCGAGCGTCGAGCACGTCGTTCATACGACCCAAGATGCCGGCATCGATATCACCCTGCCAACCGCTGTTTTGGAAGTTTTCAGACGCGGCATGGAGAACGGTCATGCCGGCAATAGCTTCACTAGCCTCATTGAAATCTTCAAGAAGTCCGATATTCGTCCATAA

[0059] SEQ ID No.3 (Amino acid sequence of imine reductase PmIRED-M5):

[0060] MKSSNRSENIRVGTENTVGKSKSVTVIGLGPMGKAMAAAFLEHGYKVTVWNRTSNKADELITKGAVRASTVHEALAANELVILSLTDYDAMYTILEPASENLSGKVLVNLSSDTPDKAREAAKWLANRGAGHITGGVMVAPSGIGKPESSTYYSGPKEVFEANKETLEVLTGTDYRGEDPGLAALYYQISMDMFMTAMLSYLHATAVAQANGITAEQFLPYAAETMSNLPKFIEFYTPRINAGEYPGDVDNLAMGMASVEHVVHTTQDAGIDITLPTAVLEVFRRGMENGHAGNSFTSLIEIFKKSDIRP

[0061] SEQ ID No. 4 (Nucleotide sequence of imine reductase PmIRED-M5):

[0062] ATGAAAAGTAGCAATCGAAGTGAGAACATTCGTGTTGGAACCGAGAATACAGTTGGGAAGAGCAAATCGGTGACGGTCATCGGGCTTGGTCCGATGGGTAAGGCGATGGCGGCCGCTTTCCTTGAGCATGGCTATAAGGTGACCGTGTGGAACAGGACTTCAAACAAAGCAGACGAACTCATAACAAAAGGAGCCGTTAGGGCGTCCACGGTCCACGAAGCGTTGGCGGCCAATGAGCTAGTCATCCTCAGCCTAACGGACTACGATGCGATGTACACCATTCTTGAACCGGCATCGGAGAATCTGTCCGGCAAGGTTCTCGTCAACCTGAGCTCGGACACTCCGGATAAAGCGCGCGAGGCGGCGAAGTGGCTGGCCAACCGTGGAGCCGGGCACATCACAGGCGGCGTTATGGTCGCACCTTCTGGCATCGGTAAACCAGAGTCCTCCACTTACTACAGCGGCCCGAAAGAGGTCTTTGAGGCCAACAAGGAGACACTCGAAGTCCTGACCGGAACTGACTATCGGGGAGAAGATCCCGGACTTGCGGCACTGTACTACCAGATCAGTATGGACATGTTCATGACAGCCATGCTCAGCTACCTCCATGCCACTGCGGTGGCCCAAGCGAACGGTATTACGGCTGAGCAGTTTCTGCCATACGCCGCCGAGACAATGTCGAATCTGCCGAAGTTCATCGAGTTCTACACACCTCGGATTAACGCGGGTGAATATCCCGGTGACGTAGACAACCTTGCCATGGGAATGGCGAGCGTCGAGCACGTCGTTCATACGACCCAAGATGCCGGCATCGATATCACCCTGCCAACCGCTGTTTTGGAAGTTTTCAGACGCGGCATGGAGAACGGTCATGCCGGCAATAGCTTTACTAGCCTCATTGAAATCTTCAAGAAGTCCGATATTCGTCCATAA。

[0063] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the accompanying drawings are merely examples and do not limit the invention. The advantages of the present invention have been fully and effectively realized. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments; any variations or modifications can be made to the implementation of the present invention without departing from these principles.

Claims

1. An imine reductase PmIR mutant, characterized in that, The amino acid sequence of the mutant is shown as SEQ ID NO.

3.

2. A gene, characterized in that, The gene encoding the imine reductase PmIR mutant of claim 1.

3. A recombinant vector, characterized in that, The gene of claim 2.

4. The recombinant vector of claim 3, wherein, The expression vector is pET-28a (+).

5. A recombinant microorganism, characterized in that, The recombinant vector of claim 3 or 4.

6. The recombinant microorganism of claim 5, wherein, The recombinant microbial cell takes Escherichia coli BL21 (DE3) as the host cell.

7. A kind of ( R The preparation method of )-FMPP, wherein ( R The preparation method of FMPP includes the following steps: A catalytic system is composed of 3-(3,4-dihydro-2H-pyrrol-5-yl)-5-fluoro-2- methoxypyridine (DPFM) as substrate and imine reductase PmIR mutant as catalyst, which catalyzes the generation of said (3-(3,4-dihydro-2H-pyrrol-5-yl)-5-fluoro-2- methoxypyridin-3-yl)phenyl phosphate (FPPP). R )-FMPP.

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