Use of imine reductase or mutant thereof in synthesis of chiral piperidine amine intermediates

Abstract

The application provides application of imine reductase or a mutant thereof in synthesis of chiral piperidine amine intermediates, and belongs to the field of enzyme engineering. The application provides an imine reductase mutant, which is used for biocatalysis of 1-benzyl-5-methyl-1,6-dihydropyridin-3(2H)-one to synthesize a precursor compound of a chiral piperidine amine intermediate of nalofoxacin, so as to overcome the problems of complicated reaction steps, harsh conditions, great environmental pressure and other industrial application difficulties in the prior art for synthesis of chiral piperidine amine intermediate compounds, and has the advantages of mild reaction conditions, green environmental protection, short reaction steps, high selectivity and the like, and has more industrial application prospects.

Description

Technical Field

[0001] This invention relates to the application of imine reductase or its mutants in the synthesis of intermediate compounds of nemonoxacin chiral piperidineamine, and belongs to the field of enzyme engineering. Background Technology

[0002] Nemonoxacin, developed by Procter & Gamble, is the world's first fluoroquinolone antibiotic. It is often marketed as its malate salt, known as nemonoxacin malate. Its oral capsule formulation was first launched in March 2014, and subsequently received drug approval from the China National Medical Products Administration in 2016. Nemonoxacin's antibacterial mechanism primarily involves inhibiting bacterial DNA gyrase and topoisomerase IV, thus preventing bacterial DNA replication and exerting a bactericidal effect. It exhibits broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. It has also shown significant efficacy against many pathogens associated with respiratory and skin infections, including multidrug-resistant Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus. It is widely used in the treatment of skin infections such as acne and folliculitis, and has significant market application value.

[0003] The main method for synthesizing nemonoxacin active pharmaceutical ingredient is to react the chiral piperidineamine intermediate MAP (N-[(3S,5S)-5-methyl-3-piperidinyl]-aminocarbamate ethyl ester-1,1-dimethyl ethyl ester) with a fluoroquinolone building block through an aromatic nucleophilic substitution reaction, followed by the removal of Boc and hydroxyl protecting groups to obtain the target product.

[0004] Currently, the industrial synthesis routes for MAP, the key intermediate of nemonoxacin (e.g., TW200808729A, CN200610074122.0, CN200910160465.2), rely on the chiral source L-pyroglutamic acid. This requires multiple functional group transformations and protection / deprotection operations, involving carboxyl methyl esterification, Boc protection, palladium reduction on carbon for lactam ring opening, introduction of leaving groups (e.g., MsCl) into the dihydroxy compound intermediate, and benzylamine ring closure. This method has the following drawbacks: ① It uses large quantities of highly toxic reagents such as MsCl, putting pressure on production safety and the environment; ② It requires the use of expensive tert-butoxybis(dimethylamino)methane as a key reagent, resulting in high production costs; ③ The complex reaction steps and multiple reactions limit the overall yield, and the chiral source dependence reduces the flexibility of the process.

[0005] CN121108037A reports a key chiral piperidineamine intermediate for the industrial synthesis of the quinolone antibacterial drug nemonoxacin, which is prepared by condensation reactions of 5-hydroxynicotinic acid with different alcohols, followed by reactions with different aryl halides, asymmetric hydrogenation, methyl ester reduction, dehydroxylation, Mitsunobu reaction, and deprotection. However, this synthetic route is cumbersome and requires expensive metals and complex chiral ligands. The metal residue problem is environmentally unfriendly, and the reaction conditions are harsh, which is not conducive to large-scale industrial production.

[0006] (3S,5S)-1-benzyl-5-methylpiperidin-3-amine is a precursor for the synthesis of the chiral piperidinamine intermediate MAP. This precursor compound only requires a simple debenzylation and addition of a Boc group to obtain the nanofloxacin chiral piperidinamine intermediate, with the following structural formula: .

[0007] Reference: Hu Yuliang. Semi-rational design of imine reductase IR-G36-M5 and its application in the synthesis of chiral amines [D]. Jiangxi Agricultural University. 2024. This reference discloses that the imine reductase mutant IR-G36-M5 exhibits good stereoselectivity in the reductive amination reaction of two chiral building blocks. IR-G36-M5 is used to catalyze the asymmetric synthesis of the key intermediate (R)-N-demethylselegiline 1a of the chiral amine drug (R)-selegiline using phenylacetone 1 and propargylamine a, generating the corresponding chiral amine product (R)-1a. IR-G36-M5 is then used to catalyze the simultaneous synthesis of the potential drug intermediate chiral amine molecule 2-N-benzylamino-4-phenylbutyrate 2b from ethyl 2-oxo-4-phenylbutyrate and benzylamine b. This reference only generates secondary and tertiary amine compounds and only synthesizes chiral amines containing a single chiral center.

[0008] Currently, there are very limited methods for synthesizing (3S,5S)-1-benzyl-5-methylpiperidin-3-amine compounds, and no biosynthetic methods have been reported. Summary of the Invention

[0009] The present invention relates to the application of imine reductase or its mutant in the synthesis of chiral piperidineamine intermediates.

[0010] This invention provides an imine reductase mutant, which is a synergist obtained by mutating the imine reductase IR-B9 derived from Streptomyces aureocirculatus. The mutant has the following amino acid sequence: The mutant IR-B9-A219Y was obtained by mutating alanine at position 219 of the parental line to tyrosine.

[0011] Furthermore, it is a mutant obtained by mutating IR-B9-A219Y as the parent, and the mutant has the following amino acid sequence:

[0012] The parental serine at position 197 was mutated to cysteine ​​to obtain the mutant IR-B9-A219Y-S197C.

[0013] The parental serine at position 197 was mutated to valine to obtain the mutant IR-B9-A219Y-S197V.

[0014] The parental serine at position 197 was mutated to arginine to obtain the mutant IR-B9-A219Y-S197R.

[0015] The parental serine at position 197 was mutated to proline to obtain the mutant IR-B9-A219Y-S197P.

[0016] The parental line was mutated to phenylalanine at position 194 to obtain the mutant IR-B9-A219Y-V194F.

[0017] The valine at position 194 of the parental line was mutated to tyrosine, resulting in the mutant IR-B9-A219Y-V194Y.

[0018] Furthermore, it is a mutant obtained by mutating IR-B9-A219Y-S197R as the parent, and the mutant has the following amino acid sequence:

[0019] The parental valine was mutated to tyrosine at position 194 to obtain the mutant IR-B9-A219Y-S197R-V194Y; the amino acid sequence is shown in SEQ ID NO. 3.

[0020] The present invention provides a DNA molecule that encodes the imine reductase mutant.

[0021] The present invention provides an expression vector containing the aforementioned DNA molecule.

[0022] The present invention provides a host cell containing the expression vector described above.

[0023] This invention also provides the application of the aforementioned imine reductase mutant, the aforementioned DNA molecule, the aforementioned expression vector, or the aforementioned host cell in the synthesis of chiral piperidine amine intermediates; the aforementioned imine reductase IR-B9 is derived from Streptomyces aureocirculatus, and its amino acid sequence is shown in SEQ ID No. 1; the structural formula of the aforementioned chiral piperidine amine intermediate is shown in Formula I. .

[0024] This invention provides a method for synthesizing chiral piperidine amine intermediates. Using Formula II as a substrate, and the imine reductase mutant, the DNA molecule, the expression vector, or the host cell as a biocatalyst, the method synthesizes chiral piperidine amine intermediates. The synthetic route is shown below:

[0025] .

[0026] The beneficial effects of this invention are:

[0027] Imine reductase is an NADPH-dependent oxidoreductase that catalyzes the asymmetric hydrogenation of the proximal chiral C=N bond of imine to form corresponding chiral amine compounds. This invention provides an imine reductase and its mutant, which, without the need for a chiral amine donor, biocatalyzes a one-step synthesis of a precursor compound for the chiral piperidine amine intermediate of nemonoxacin. This overcomes the industrial application challenges of existing technologies for synthesizing chiral piperidine amine intermediates, such as cumbersome reaction steps, harsh conditions, and significant environmental impact. It offers advantages such as mild reaction conditions, environmental friendliness, short reaction steps, high selectivity, high conversion rate, and good stereoselectivity, making it more promising for industrial applications. Attached Figure Description

[0028] Figure 1 The pET28a-IR-B9 plasmid map;

[0029] Figure 2 The pET28a-GDH plasmid map;

[0030] Figure 3 The standard curve of the product of (3S,5S)-1-benzyl-5-methylpiperidin-3-amine compound;

[0031] Figure 4 The chiral separation LC-MS spectrum of the compound (3S,5S)-1-benzyl-5-methylpiperidin-3-amine is shown. Detailed Implementation

[0032] Example 1: Construction of imine reductase expression strain and investigation of initial reactivity

[0033] 1. Construction of an imine reductase IR-B9 expression strain

[0034] IR-B9 is a wild-type imine reductase derived from *Streptomyces aureocirculatus*, and its amino acid sequence is shown in SEQ ID No. 1. First, codon optimization of IR-B9 was performed using *Escherichia coli* as the host cell. Through whole-gene synthesis and vector construction, a recombinant expression vector containing the IR-B9 encoding gene was obtained, named pET28a-IR-B9. The vector map is shown below. Figure 1 The pET28a-IR-B9 recombinant expression vector was transformed into Escherichia coli TSR2566 competent cells to construct an IR-B9 expression strain.

[0035] 2. Protein expression and crude enzyme solution preparation of imine reductase

[0036] The successfully constructed expression strain IR-B9 was inoculated into 3 mL of LB liquid medium (containing Kan resistance) and activated overnight at 37°C and 250 rpm on a benchtop shaker. After activation, 1 mL of the activated bacterial culture was inoculated into an Erlenmeyer flask containing 50 mL of LB liquid medium (containing Kan resistance) and cultured at 37°C and 250 rpm for 2.5–3 h until the bacterial culture showed OD values. 600 When the OD200 reaches 0.6-0.8, add IPTG to a final concentration of 0.5 mM and induce expression for 18-20 h at 22℃ and 150 rpm. After induction, centrifuge the bacterial culture, discard the supernatant, collect the bacterial cells, resuspend the cell pellet in potassium phosphate (KPi, pH=7.0, 100 mM) buffer, vortex to mix, and then add the OD200. 600 Adjust the temperature to approximately 30 to obtain a crude enzyme solution for later use.

[0037] 3. Construction of glucose dehydrogenase (GDH) expression strain, protein expression, and preparation of crude enzyme solution.

[0038] Glucose dehydrogenase (GDH) derived from *Bacillus subtilis*, with its amino acid sequence shown in SEQ ID No. 2, was codon-optimized using *E. coli* as the host cell. The entire gene was synthesized and a vector was constructed to obtain the recombinant expression vector pET28a-GDH containing the glucose dehydrogenase gene. The vector map is shown below. Figure 2The protein was transformed into TSR2566 competent E. coli cells to obtain a GDH-expressing strain. The protein expression pattern was the same as that of imine reductase. OD was obtained. 600 After resuspending approximately 30 units of whole cells, high-pressure homogenization was performed using a high-pressure homogenizer under the following conditions: 4°C, 750 bar, 30 min, to obtain crude GDH enzyme solution.

[0039] 4. Initial reaction activity of imine reductase

[0040] The initial reactivity of the previously expressed wild-type imine reductase IR-B9 against the substrate 1-benzyl-5-methyl-1,6-dihydropyridin-3(2H)-one (hereinafter referred to as the unsaturated ketone substrate) was investigated. The specific procedures were as follows: 200 μL of crude IRED enzyme solution, 200 μL of crude GDH enzyme solution, and 120 μL of NADP were added sequentially to a 4 mL reaction flask. + A mixture of sodium salt (0.5 mM, KPi solution) and D-Glucose (50 mM, KPi solution) was prepared, followed by the addition of 40 μL of unsaturated ketone substrate (10 mM, DMSO solution) and 40 μL of ammonium bicarbonate (50 mM, KPi solution). The reaction was carried out at 30 °C and 800 rpm for 20 h with shaking. After the reaction, a sample was taken, and the reaction was quenched with twice the volume of anhydrous ethanol. After mixing, 1 mL was centrifuged, and 220 μL of the supernatant was transferred to a 96-well microplate for LC-MS analysis. The conversion rate was determined by detecting substrate standards at different concentration gradients using LC-MS. A standard curve was plotted using the peak area of ​​the standard and its corresponding concentration. The peak area of ​​the substrate was measured, and the corresponding conversion rate was calculated by substituting it into the standard curve. The standard curve is shown below. Figure 3 As shown in the figure. The ee and dr values ​​are calculated from the product peak area. The ee value represents the percentage by which the content of a particular enantiomer exceeds that of the other enantiomer in a pair of enantiomers. The dr value refers to the ratio of the relative contents of two isomers in a group of diastereomers. ee (%) = ((S,SR,R) / (S,S+R,R))*100%, dr = ((S,S)+(R,R) / (S,R)+(R,S)). The initial reaction activity results are shown in Table 1.

[0041] Table 1. Results of initial reactivity studies for unsaturated ketone substrates.

[0042]

[0043] Example 2: Directed evolution of IR-B9 using wild-type IR-B9 as the parent and unsaturated ketone substrate.

[0044] 1. Construction of the IR-B9 mutant library

[0045] First, the AlphaFold2 software was used to predict the three-dimensional structure of the candidate enzyme IR-B9. Then, the CB-dock online tool was used to combine IR-B9 with unsaturated ketone substrates and NADP. + Molecular docking was performed, and site-directed saturation mutagenesis was carried out on amino acids surrounding the active site. Using IR-B9 as the parent, site-directed saturation mutagenesis was performed on amino acid residues such as K43, I127, E161, S197, R25, Q235, T236, A219, S237, and E215. First, using pET28a-IR-B9 plasmid as a template, forward and reverse primers for the corresponding sites were designed using the 22-codon trick. Mutations were introduced by PCR, and the PCR reaction system is shown in Table 2.

[0046] Table 2 PCR reaction system

[0047]

[0048] The PCR reaction procedure is shown in Table 3:

[0049] Table 3 PCR reaction procedure

[0050]

[0051] The linear DNA products obtained by PCR were subjected to 1% agarose gel electrophoresis. After electrophoretic separation, the gel was excised and recovered. The nucleic acid concentration of the recovered products was determined using an ultra-micro nucleic acid analyzer, and ligation was performed according to the DNA seamless cloning kit (ClonExpress® II One Step Cloning Kit, Novizan). The ligation was incubated at 37°C for 30 min. The ligation system is shown in Table 4.

[0052] Table 4 PCR product ligation system

[0053]

[0054] 2. Reactivity screening

[0055] (1) The ligation product after incubation was transformed into TSR2566 competent cells and incubated upside down in a 37℃ incubator for 14-16 h. Single colonies were picked from the plates with toothpicks and inoculated into 96-well shallow plates containing 300 μL of LB-Kan liquid medium. The cells were activated at 37℃ and 250 rpm for 5-6 h. After activation, 50 μL of the bacterial culture was transferred to 96-well deep plates containing 900 μL of LB-Kan liquid medium and incubated at 37℃ and 250 rpm for 3 h. Subsequently, 50 μL of LPTG stock solution (10 mM) was added to induce expression, and the cells were incubated at 22℃ and 500 rpm for 20 h. The cells were collected by centrifugation at 4℃, and 100 μL of potassium phosphate buffer (pH 7.0, 100 mM) was added to a pipette and vortexed to resuspend the cells. Then, 80 μL of the prepared crude GDH enzyme solution and 60 μL of NADP were added. + A mixture of sodium salt (1 mM, KPi solution) and D-Glucose (100 mM, KPi solution), 40 μL of unsaturated ketone substrate (10 mM, DMSO solution), and 40 μL of ammonium bicarbonate (50 mM, KPi solution) were sealed and reacted at 30 °C with shaking at 800 rpm for 20 h. After the reaction was complete, 900 μL of anhydrous ethanol was added for treatment, and the reaction activity was determined by LC-MS.

[0056] The mutants with improved activity and dr value from the initial screening were subjected to shake-flask culture and reaction activity verification, following the procedures outlined in Examples 1-4. The relative activity, ee value, and dr value of the unsaturated ketone reaction were measured (relative activity: mutant activity / parental activity in this round). The results of the first round of directed evolution verification are shown in Table 5.

[0057] Table 5. Results of the first round of directed evolution validation of unsaturated ketones

[0058]

[0059] (2) As shown in Table 5, the mutant IR-B9-A219Y has the best relative activity and dr value. Based on this, a second round of site-directed saturation mutagenesis was performed on the S197, V194, and A218 sites. The mutant library construction, protein expression, reaction screening, and detection methods are the same as described above. The results of the second round of directed evolution are shown in Table 6.

[0060] Table 6. Results of the second round of directed evolution validation of unsaturated ketones

[0061]

[0062] (3) As shown in Table 6, IR-B9-A219Y-S197R has the best relative activity and dr value. Based on this, a third round of site-directed saturation mutagenesis was performed on sites V194, A218, V205, G245, and R233. The methods for constructing the mutant library, protein expression, reaction screening, and detection were the same as described above. The results of the third round of directed evolution are shown in Table 7.

[0063] Table 7 Results of the third round of directed evolution validation of unsaturated ketones

[0064]

[0065] Three rounds of directed evolution were performed on IR-B9, and the optimal mutant IR-B9-A219Y-S197R-V194Y (M3) was obtained. This mutant was created by mutating wild-type IR-B9 to tyrosine at position 219, serine at position 197 to arginine, and valine at position 194 to tyrosine. The amino acid sequence is shown in SEQ ID No. 3. Compared with wild-type IR-B9, M3 increased the conversion rate of unsaturated ketone substrates from 28% to 50%, the dr ratio from 40:1 to >400:1, and the ee value to 99%.

[0066] The activity assay was performed following the procedure described above. The relative reactivity was calculated as: mutant reactivity / original parent reactivity. The results are shown in Table 8.

[0067] Table 8. Relative reactivity results of IR-B9 mutants

[0068]

[0069] Example 3: Optimization of reaction conditions for mutant M3

[0070] To further enhance the reactivity, the reaction conditions for the unsaturated ketone were optimized, including reaction temperature, buffer type and pH, type and ratio of co-solvent, and substrate to ammonium bicarbonate equivalent ratio. The relative activity, dr value, and ee value were measured. Relative activity is defined as the product concentration under optimized conditions divided by the product concentration under initial conditions. The results of the optimized reaction conditions are shown in Table 9.

[0071] Table 9 Results of reaction condition optimization experiments

[0072]

[0073] Based on the results of the optimization study, the optimal reaction conditions were determined to be: reaction temperature 30℃, buffer solution (NaPi buffer) pH 8.0, co-solvent (DMSO) content 10%, and substrate to ammonium bicarbonate equivalent ratio 1:10. Under these optimal conditions, the conversion rate was 87%, dr value > 400:1, and ee value 99%. The results before and after optimization are shown in Table 10.

[0074] Table 10 Results of the investigation before and after optimization of the reaction conditions for the M3 mutant

[0075]

[0076] The results of the precursor chiral separation detection of the nemonoxacin chiral piperidine intermediate are as follows: Figure 4 As shown. The detection instruments were an Agilent 1260 Infinity II liquid chromatograph, an Agilent Ultivo G6465B mass spectrometer, a chiral detection column (Chiral IG column - Daicel CHIRALPAK (5μm packing, 4.6×250 mm)), a flow rate of 1.0 mL / min, a column oven temperature of 30℃, and a mobile phase of acetonitrile:water = 50:50 (containing 20 mM ammonium bicarbonate).

[0077] SEQ ID No.1

[0078] IR-B9 amino acid sequence

[0079] MNVTTPEPTTVAVIGLGNLGQVLARTLLDQGHKVTVWNRSQDKADDLVARGATRAATPADAIRASDLVIICVLDYTTVRDLLTPAADALAGRVVVNVTSGIPEPARELATRVNGSGAAYVDGAVYAIPQTIGTPEAFVLYSGDEEA FARHRALLETLGTAEFVGADAGLAAVHDVALLSGMYGMFAGFFQTVAVSGSAGIEAVRVTELLVRWLKEAIAALPAFAAEIDAGDYRTQTSNLDINAVGLANILAATRAQGVGVELLAPLHALFEQQVSAGHGAESLSRTIESLRLE

[0080] SEQ ID No.2

[0081] GDH amino acid sequence

[0082] MGSSHHHHHHSSGLVPRGSHMYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0083] SEQ ID No.3

[0084] Amino acid sequence of IR - B9 - A219Y - S197R - V194Y

[0085] MNVTTPEPTTVAVIGLGNLGQVLARTLLDQGHKVTVWNRSQDKADDLVARGATRAATPADAIRASDLVIICVLDYTTVRDLLTPAADALAGRVVVNVTSGIPEPARELATRVNGSGAAYVDGAVYAIPQTIGTPEAFVLYSGDEEAFARHRALLETLGTAEFVGADAGLAAVHDVALLSGMYGMFAGFFQTVAYSGRAGIEAVRVTELLVRWLKEAIAYLPAFAAEIDAGDYRTQTSNLDINAVGLANILAATRAQGVGVELLAPLHALFEQQVSAGHGAESLSRTIESLRLE

Claims

1. An imine reductase mutant, characterized in that: It is derived from Streptomyces aureocirculatus The imine reductase IR-B9 is the parent, and a synergist is obtained by mutation; the amino acid sequence of imine reductase IR-B9 is shown in SEQ ID No. 1; the mutant is obtained by mutating alanine at position 219 of the parent to tyrosine, resulting in mutant IR-B9-A219Y.

2. The imine reductase mutant according to claim 1, characterized in that: It is a mutant obtained by mutating IR-B9-A219Y as the parent: The parental serine at position 197 was mutated to cysteine ​​to obtain the mutant IR-B9-A219Y-S197C. The parental serine at position 197 was mutated to valine to obtain the mutant IR-B9-A219Y-S197V. The parental serine at position 197 was mutated to arginine to obtain the mutant IR-B9-A219Y-S197R. The parental serine at position 197 was mutated to proline to obtain the mutant IR-B9-A219Y-S197P. Mutating valine at position 194 of the parental line to phenylalanine yields the mutant IR-B9-A219Y-V194F; or, The valine at position 194 of the parental line was mutated to tyrosine, resulting in the mutant IR-B9-A219Y-V194Y.

3. The imine reductase mutant according to claim 2, characterized in that: It is a mutant obtained by mutating IR-B9-A219Y-S197R as the parent: The parental valine was mutated to tyrosine at position 194 to obtain the mutant IR-B9-A219Y-S197R-V194Y; the amino acid sequence is shown in SEQ ID NO.

3.

4. A DNA molecule, characterized in that: The DNA molecule encodes the imine reductase mutant according to any one of claims 1-3.

5. An expression vector, characterized in that: The expression vector contains the DNA molecule as described in claim 4.

6. A host cell, characterized in that: The host cell contains the expression vector as described in claim 5.

7. The use of the imine reductase mutant according to any one of claims 1-3, the DNA molecule according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6 in the synthesis of chiral piperidine amine intermediates; wherein the imine reductase IR-B9 is derived from... Streptomyces aureocirculatus The amino acid sequence is shown in SEQ ID No. 1, and the structural formula of the chiral piperidineamine intermediate is shown in Formula I: ； The structural formula of the synthesized substrate is shown in Formula II: .

8. A method for synthesizing a chiral piperidineamine intermediate compound, characterized in that: It uses Formula II as a substrate, the imine reductase mutant of any one of claims 1-3, the DNA molecule of claim 4, the expression vector of claim 5, or the host cell of claim 6 as a biocatalyst to synthesize chiral piperidineamine intermediate compounds. The synthetic route is shown below: 。

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