Mutant m203a, mutant m203a / s241l, and uses thereof

Abstract

Mutants M203A and M203A / S241L of the reductive amination enzyme IR-G36-M5 are provided, as well as the use of said mutants in the catalytic asymmetric reductive amination synthesis of (R)-N-Boc-3-cyclopropylpiperidine or (R)-N-Boc-3-cyclopropylazheptanane.

Description

Mutant M203A, mutant M203A / S241L and their applications Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and particularly relates to the application of a reductive amination enzyme mutant in catalyzing asymmetric reductive amination synthesis of (R)-N-Boc-3-cyclopropylpiperidine and (R)-N-Boc-3-cyclopropylazepane. Background Art

[0002] (R)-3-Cyclopropyl nitrogen heterocycles are important building blocks and pharmacophores for pharmaceutical synthesis, widely used in drug molecule synthesis. Traditional chemical synthesis methods primarily involve expensive transition metal-catalyzed asymmetric reductive amination reactions to synthesize (R)-3-cyclopropyl nitrogen heterocycles, or chiral resolution of racemic 3-cyclopropyl nitrogen heterocycles using chiral resolving agents. Alternatively, (R)-3-amino nitrogen heterocycles can be synthesized enzymatically or chemically, followed by reaction with 1-ethoxy-1-trimethylsilyloxycyclopropane to synthesize (R)-3-cyclopropyl nitrogen heterocycles, but yields are only approximately 15%.

[0003] The efficient and green synthesis of chiral amines using enzymatic methods has always been a hot topic in scientific research. In 2017, Nicholas J. Turner discovered that reductive amination enzymes can catalyze the reductive amination of equal amounts of prochiral ketones or aldehydes and amines to produce secondary or tertiary amines, and the substrate spectrum is very broad (Nat Chem, 2017, 9(10):961-969.). The reductive amination reaction catalyzed by this enzyme has high atom economy and is the most direct strategy for synthesizing alkyl-substituted chiral amines. In 2019, GlaxoSmithKline used "directed evolution" technology to modify reductive amination enzymes and achieved industrial production of the anti-tumor drug GSK2879552 (LSD inhibitor) (Nature Catalysis, 2019, 2(10):909-915.). After three rounds of evolution, the final mutant achieved an ee value of 99.7% and a yield of up to 84% in a kilogram-scale preparation reaction. In 2021, Pfizer used an engineered imine reductase to achieve industrial production of abrocitinib, a JAK1 inhibitor, which was launched in 2022 (Nature Catalysis, 2021, 4(9):775-782.). Based on computer-aided design, the mutant was successfully applied to ton-scale industrial production, with an isolated yield of 73% and a stereoselectivity of >99:1.

[0004] In previous studies, the inventors engineered the reductive amination enzyme IR-G36 to obtain a highly efficient mutant, M5 (Chinese Patent ZL 202210294265.1.), which was used to catalyze the reductive amination synthesis of a series of R-alkylated piperidinamines and azepineamines. However, in the synthesis of (R)-N-Boc-3-cyclopropylpiperidine and (R)-N-Boc-3-cyclopropylazepane, M5 exhibited poor stereoselectivity, with an ee value of 87% (R), which fell short of the requirements for industrial application. Therefore, designing and modifying the highly active reductive amination enzyme to develop mutants with high catalytic efficiency and stereoselectivity is of great significance for the synthesis of optically pure (R)-N-Boc-3-cyclopropylpiperidine and (R)-N-Boc-3-cyclopropylazepane. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention designs and transforms the existing reductive amination enzyme mutant M5 to obtain a new mutant with significantly improved activity and stereoselectivity, thereby providing a green, efficient and highly selective enzymatic synthesis method for optically pure (R)-N-Boc-3-cyclopropylpiperidine and (R)-N-Boc-3-cyclopropylazepane.

[0006] The present invention mutates reductive aminase M5 having the amino acid sequence shown in SEQ ID No. 1. Methionine at position 203 is replaced with alanine to obtain mutant M203A. Simultaneously, methionine at position 203 is replaced with alanine, and serine at position 241 is replaced with leucine to obtain mutant M203A / S241L. Both mutants M203A and M203A / S241L exhibit significantly improved catalytic activity and stereoselectivity.

[0007] Imine reductase is used to catalyze the reductive amination reaction of N-Boc-3-piperidone or N-Boc-3-azepanone with cyclopropylamine to asymmetric synthesis of (R)-N-Boc-3-cyclopropylpiperidine and (R)-N-Boc-3-cyclopropylazepane. The reaction formula is as follows:

[0008] The imine reductase biocatalyst of the present invention can be recombinant engineered bacteria, engineered bacteria broken liquid, crude enzyme powder, pure enzyme or immobilized enzyme product.

[0009] In the biocatalytic system of the present invention, the solvent is an aqueous solution containing one or more buffer salts or a buffer salt solution containing a partial solubilizing organic solvent.

[0010] The (R)-N-Boc-3-cyclopropylpiperidine and (R)-N-Boc-3-cyclopropylazepane substrate concentrations described herein are typically 10 to 300 mmol / L, and are preferably 90 mmol / L or higher for production efficiency. The cyclopropylamine hydrochloride concentration is 1 to 4 times the ketone substrate concentration, and is preferably 1.1 to 2.0 times the ketone substrate concentration for cost savings.

[0011] The pH value of the reaction system of the present invention is 6.0 to 9.0, and preferably 7.0 to 7.5 to ensure the catalytic efficiency of the reductive amination enzyme.

[0012] The incubation temperature of the present invention is 20-40° C., preferably 28-32° C., to ensure the stability and catalytic efficiency of the reductive amination enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a chiral HPLC spectrum of (R)-N-Boc-3-cyclopropylpiperidine (1a) synthesized by M203A catalysis.

[0014] Figure 2 is the chiral HPLC spectrum of (R)-N-Boc-3-cyclopropylazepane (1b) synthesized by M203A.

[0015] Figure 3 is a chiral HPLC spectrum of (R)-N-Boc-3-cyclopropylpiperidine (1a) synthesized by M203A / S241L.

[0016] Figure 4 is a chiral HPLC spectrum of (R)-N-Boc-3-cyclopropylazepane (1b) synthesized by M203A / S241L catalysis.

[0017] Figure 5 is a diagram of (R)-N-Boc-3-cyclopropylpiperidine (1a) 1 H NMR spectrum (CDCl3, 500 MHz).

[0018] Figure 6 is a diagram of (R)-N-Boc-3-cyclopropylpiperidine (1a) 13 C NMR spectrum (CDCl3, 125 MHz).

[0019] Figure 7 is a diagram of (R)-N-Boc-3-cyclopropylazepane (1b) 1 H NMR spectrum (CDCl3, 500 MHz).

[0020] Figure 8 is a diagram of (R)-N-Boc-3-cyclopropylazepane (1b) 13 C NMR spectrum (CDCl3, 125 MHz).

[0021] Figure 9 is a graph showing the structure of 2-fluoro-4-(trifluoromethoxy)benzonitrile. 1H NMR spectrum (CDCl3, 400 MHz).

[0022] FIG10 is a low-resolution mass spectrum of (2-fluoro-4-(trifluoromethoxy)phenyl)methanamine.

[0023] Figure 11 is a graph showing the structure of tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate. 1 H NMR spectrum (CDCl3, 400 MHz).

[0024] Figure 12 is a graph showing the relationship between tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate 13 C NMR spectrum (CDCl3, 100 MHz).

[0025] FIG13 is a low-resolution mass spectrum of (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea.

[0026] Figure 14 is a diagram of (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxamide 1 H NMR spectrum (CDCl3, 400 MHz).

[0027] Figure 15 is a diagram of (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxamide 13 C NMR spectrum (CDCl3, 100 MHz). DETAILED DESCRIPTION

[0028] Example 1

[0029] The reductive aminase mutant M5 is designed and modified based on its structure. The reductive aminase mutant M5 has a DNA sequence as described in SEQ ID NO.1 and an amino acid sequence as described in SEQ ID NO.2 in the sequence list.

[0030] The pET28a plasmid was double-digested with restriction endonucleases XhoI and NdeI, and the gene sequence encoding M5 was ligated into the pET28a vector to construct a recombinant expression plasmid.

[0031] Site-saturation mutagenesis primers were designed using the degenerate codon NNK at sites M203 and S241. Plasmid-wide PCR was performed using the Fast Mutagenesis System (Full Gold Site-Directed Mutagenesis Kit). The PCR product was digested with DnpI and transformed into competent E. coli BL21(DE3) cells. The cells were plated onto kanamycin-resistant LB medium plates and cultured overnight at 37°C. Ninety-six single colonies from each plate were selected and cultured in a primary 96-well deep-well plate at 37°C, 800 rpm, overnight.

[0032] The base sequence of the mutant M203A is shown in the sequence listing SEQ ID NO.3, and the amino acid sequence thereof is shown in the sequence listing SEQ ID NO.4.

[0033] The base sequence of the mutant M203A / S241L is shown in the sequence listing as SEQ ID NO.5, and the amino acid sequence thereof is shown in the sequence listing as SEQ ID NO.6.

[0034] Example 2

[0035] Preparation of crude reductaminase

[0036] The recombinant E. coli clones containing mutants M203A and M203A / S241L described in Example 1 were transferred into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. The culture was inoculated into LB liquid medium containing 50 μg / mL kanamycin at a ratio of 1:100 and cultured at 37°C and 220 rpm until the OD 600 The value was 0.6 to 0.8. Protein expression was induced by adding 0.2 mM IPTG to the culture medium, and the culture was induced at 20°C and 180 rpm for 16 hours. The bacterial liquid was centrifuged at 4000 rpm for 20 minutes, the cells were harvested, and resuspended in sodium phosphate buffer (100 mM, pH 7.0). The cells were collected by centrifugation again, washed twice, and resuspended in phosphate buffer (100 mM, pH 7.0). The cells were broken using a high-pressure homogenizer. The broken liquid was centrifuged at 1,2000 rpm, the supernatant was collected, and placed in a vacuum freeze dryer for lyophilization. The collected powder was the crude enzyme powder of the reductive amination enzyme mutant.

[0037] Example 3

[0038] Preparation of immobilized enzyme M203A / S241L@NKA-9 and determination of its activity and circulation

[0039] Step 1: Pretreatment of NKA-9 macroporous resin: Soak 10g of macroporous resin in 50mL of anhydrous ethanol and gently stir for 12 hours. Then, add 50mL of distilled water to the suspension, continue stirring for 20 minutes, and remove the ethanol by filtration. Repeatedly wash with distilled water until the ethanol odor is gone, and remove the water by filtration. Finally, place the pretreated NKA-9 macroporous resin in a vacuum drying oven and dry at room temperature before use.

[0040] Step 2: Immobilization by Adsorption: The imine reductase mutant M203A / S241L prepared in Example 2 was purified by nickel affinity chromatography and diluted to a concentration of 3 mg / mL with pH 7.0 phosphate buffer. Next, 1 g of the NKA-9 macroporous resin pretreated in Step 1 was weighed and added to 10 mL of the enzyme solution. The mixture was then placed in a shaker at 25°C, 150 rpm, and adsorbed for 4 hours. The adsorbed macroporous resin was allowed to stand overnight at 4°C and centrifuged at 4000 rpm for 5 minutes. The supernatant was assayed for enzyme concentration using a BCA protein assay kit, and the adsorption efficiency of the macroporous resin was calculated. The immobilized macroporous resin was rinsed 3-5 times with deionized water, filtered to remove water, and dried in a vacuum drying oven at room temperature. Finally, the pretreated and immobilized NKA-9 macroporous resin was weighed and the adsorption capacity was calculated. The adsorption capacity of the NKA-9 macroporous resin was 26.7 mg / g, with an adsorption efficiency of 89%.

[0041] Step 3: Activity determination of immobilized enzyme M203A / S241L@NKA-9: The catalytic activity and recyclability of the immobilized enzyme were determined by the reaction of converting cyclopropylamine 1 and N-Boc-3-piperidone a into 1a. GDH (1 mg / mL), glucose (200 mM), N-Boc-3-piperidone (100 mM), cyclopropylamine (110 mM) and NADP were added to 50 mL of phosphate buffer solution (100 mM, pH 7.0). + The reaction system was prepared using a 2mM ion exchanger. 50mg of M203A / S241L free enzyme and 1.81g of M203A / S241L@NKA-9 were then added to each reaction system. Reactions were carried out at 30°C and 180rpm. After 24 hours of reaction, the reaction system was filtered, and the filtrate was analyzed for substrate conversion by HPLC. The results showed that the free enzyme and M203A / S241L@NKA-9 had comparable activities, with conversion rates of >99% and 98%, respectively.

[0042] Step 4: Cyclic Activity of the Immobilized Enzyme: After the reaction in Step 2, the M203A / S241L@NKA-9 was thoroughly rinsed with phosphate buffer. The reaction system from Step 2 was added again, and the same reaction method was repeated four times. The relative activity of M203A / S241L@NKA-9 was calculated. The results showed that the substrate conversion rates of M203A / S241L@NKA-9 over the five cycles were 97%, 95%, 82%, 66%, and 50%, respectively.

[0043] Example 4

[0044] Enzymatic Synthesis of (R)-N-Boc-3-Cyclopropyl Nitrogen Heterocycle

[0045] The present invention prepared crude enzyme powders of reductive amination enzymes M203A / S241L and M203A according to Example 2, and then used a 500 mL reaction system to synthesize (R)-N-Boc-3-cyclopropylpiperidine and (R)-N-Boc-3-cyclopropylazepane.

[0046] The reaction system includes N-Boc-3-pyrrolidone (180mM, 1 equivalent) or N-Boc-3-azepanone (90mM, 1 equivalent), cyclopropylamine hydrochloride (198 or 99mM, 1.1 equivalent), D-glucose (1.5 times the ketone equivalent), NADP + 2 mM), glucose dehydrogenase powder (50 mg / L), 100 mM sodium phosphate buffer (pH 7.0) and 20% (total volume of the reaction solution) DMSO, mutant M203A / S241L or M203A crude enzyme powder (1.0 g / L), the solution was adjusted to pH 7.0 with 1 M hydrochloric acid, and the mixed solution was placed in a bioreactor for biocatalytic reaction at 30°C and 220 rpm.

[0047] After 18 hours of reaction, acetic acid was added to the reaction system to quench the reaction, the pH was adjusted to 3.0, diatomaceous earth was added, and the mixture was filtered using a Buchner funnel, rinsed with water, and the filtrate was collected. The filtrate was extracted 3 times with 500 mL of dichloromethane to remove water-insoluble components. Saturated sodium carbonate was added to the remaining aqueous phase to adjust the pH to 10.0, and then extracted 3 times with 500 mL of dichloromethane. The dichloromethane phase was collected and the solvent was removed by distillation under reduced pressure to obtain the corresponding product.

[0048] Chiral HPLC analysis: N-Boc-3-cyclopropylpiperidine was analyzed on a CHIRALPAK AY-H column using n-hexane / ethanol (96:4, v / v, 0.2‰ diethylamine) at 1.0 mL / min and 210 nm. N-Boc-3-cyclopropylazepane was analyzed on a CHIRALPAK IG-H column using n-hexane / ethanol (80:20, v / v, 0.2‰ diethylamine) at 1.0 mL / min and 210 nm.

[0049] Example 5

[0050] Gram-scale enzymatic synthesis of (R)-N-Boc-3-cyclopropylpiperidine (1a):

[0051] According to the method of Example 3, recombinant Escherichia coli containing mutants M203A / S241L and mutant M203A were used to catalyze the reductive amination reactions of N-Boc-3-piperidone (180 mM and 140 mM) with cyclopropylamine hydrochloride, respectively, to obtain 18.1 g and 13.9 g of colorless oily (R)-N-Boc-3-cyclopropylpiperidine product, respectively. The conversion rates were both >99%, and the isolated yields were 84% and 83%.

[0052] The chiral HPLC spectrum of N-Boc-3-cyclopropylpiperidine (1a) catalyzed by recombinant E. coli containing mutant M203A is shown in Figure 1, with an ee(R) value of >99%. The chiral HPLC spectrum of (R)-N-Boc-3-cyclopropylpiperidine (1a) catalyzed by recombinant E. coli containing mutant M203A / S241L is shown in Figure 3, with an ee(R) value of 97%.

[0053] The NMR spectrum is shown in Figure 5, and the data are: 1 H NMR δ H (500MHz, CD3OD)4.08(1H,m),3.71(1H,m),3.20(2H,m),3.10(1H,m),2.66(1H,m),2 .13(1H,m),1.76(1H,m),1.64(1H,m),1.57(1H,m),1.47(9H,s),0.79-0.86(4H,m).

[0054] The carbon spectrum is shown in Figure 6. 13 C NMR δ C (125MHz, CD3OD)156.3,81.8,55.8,29.1,28.8,28.6,23.7,4.8,4.4.

[0055] Example 6

[0056] Enzymatic synthesis of (R)-N-Boc-3-cyclopropylazepane (1b):

[0057] According to the method of Example 3, recombinant E. coli containing mutants M203A and M203A / S241L were used to catalyze the reductive amination reaction of N-Boc-3-azepanone (90 mM) with cyclopropylamine hydrochloride, respectively, to obtain 9.2 g and 9.4 g of colorless oily (R)-N-Boc-3-cyclopropylazepane product, with conversion rates of >99% and isolated yields of 81% and 84%. The chiral HPLC spectrum of the synthesis of (R)-N-Boc-3-cyclopropylazepane (1b) catalyzed by recombinant E. coli containing mutant M203A is shown in Figure 2, with an ee(R) value of >99%. The chiral HPLC spectrum of the synthesis of (R)-N-Boc-3-cyclopropylazepane (1b) catalyzed by recombinant E. coli containing mutant M203A / S241L is shown in Figure 4, with an ee(R) value of 98%.

[0058] The NMR spectrum is shown in Figure 7, and the data are: 1 H NMR δ H (500MHz, CDCl3)3.97(0.5H,d,J=12.4Hz),3.71(1H,m),3.49(1H,m),3.32( 0.5H,m),3.24(0.5H,m),3.13(0.5H,m),2.99(1H,m),2.46(0.5H,m),2.29( 0.5H,m),2.18(0.5H,m),1.98(0.5H,m),1.86(1H,m),1.78(1H,m),1.57(1H ,m),1.47(4.5H,s),1.46(4.5H,s),1.38(2H,m),0.84(1H,m),0.65(3H,m).

[0059] The carbon spectrum is shown in Figure 8. 13 C NMR δ C (125MHz, CDCl3)156.9,155.5,80.5,79.8,59.1,58.9,49.7,49.6,49.3,47.0,3 4.0,31.5,28.8,28.75,28.7,28.6,28.5,27.6,22.5,22.45,6.3,5.3,5.2,4.8.

[0060] The invention obtains mutants M203A and M203A / S241L by designing and modifying reductive amination enzymes. The reductive amination reaction of N-Boc-pyrrolidone and N-Boc-3-azepanone with cyclopropylamine hydrochloride proves that the catalytic activity and stereoselectivity are improved, and the conversion rate and ee value both reach above 99%.

[0061] Example 7

[0062] Synthesis of 2-Fluoro-1-(isocyanomethyl)-4-(trifluoromethoxy)benzene

[0063] Step 1:

[0064] Zn(CN)2 (3.8 g, 14.67 mmol, 1.0 equiv), Pd2(dba)3 (671 mg, 0.733 mmol, 0.05 equiv), and 1,1'-bis(diphenylphosphino)ferrocene (813 mg, 1.47 mmol, 0.1 equiv) were added to a dry round-bottom flask. The atmosphere in the round-bottom flask was replaced with argon three times. 1-Bromo-2-fluoro-4-(trifluoromethoxy)benzene (3.8 g, 14.67 mmol, 1 equiv) was diluted with DMF (57 mL) and added to the mixture at room temperature. The reaction mixture was replaced with argon three times and then stirred at 100°C for 16 hours. The mixture was quenched with ice water (10 mL). The aqueous layer was extracted twice with ethyl acetate (EA, 10 mL x 2). The combined organic layers were washed with saturated aqueous sodium chloride (10 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The product was purified by silica gel column chromatography with gradient elution (PE:EA=100:1→10:1) to obtain a yellow oil, namely 2-fluoro-4-(trifluoromethoxy)benzonitrile (2.3 g, 11.21 mmol, 76.43%).

[0065] As shown in Figure 9, the NMR data is: 1 H NMR (400 MHz, CDCl3) δ H 7.72–7.66(1H,m),7.18-7.08(2H,m).

[0066] Step 2:

[0067] 2-Fluoro-4-(trifluoromethoxy)benzonitrile (2.3 g, 11.21 mmol, 1 equivalent) and ammonia / methanol solution (7 mol / L, 80 mL) were added to a dry round-bottom flask. The round-bottom flask was replaced with argon three times. Raney nickel (1.32 g) was added to the mixture at room temperature. The mixture was replaced with hydrogen three times at -60°C. The reaction was stirred at -60°C to room temperature for 16 hours. After the mixture was filtered, it was washed twice with methanol (20 mL×2). The filtrate was concentrated under reduced pressure to obtain a yellow oil, namely (2-fluoro-4-(trifluoromethoxy)phenyl)methylamine (2.1 g, crude product), which was used directly in the next reaction.

[0068] As shown in FIG10 , the low-resolution mass spectrometry result is: ESI-MS m / z = 210.17 [M+H] + ;Calculated MW:209.14.

[0069] Step 3:

[0070] (2-Fluoro-4-(trifluoromethoxy)phenyl)methanamine (2.1 g, 10.04 mmol, 1 eq) and toluene (210 mL) were added to a dry round-bottom flask. The round-bottom flask was purged with argon three times. Triphosgene (1.49 g, 5.02 mmol, 0.5 eq) was dissolved in toluene (21 mL) and then added dropwise to the mixture at -30°C. The reaction was stirred at 120°C for 3 hours. The mixture was cooled to room temperature and then concentrated under reduced pressure to give a yellow oil, 2-fluoro-1-(isocyanomethyl)-4-(trifluoromethoxy)benzene (2.36 g, crude product).

[0071] Example 8

[0072] Synthesis of tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate:

[0073] Tert-butyl (R)-3-(cyclopropylamino)piperidine-1-carboxylate 1a (2.0 g, 8.32 mmol, 1.0 equiv) and dichloromethane (DCM, 100 mL) were added to a dry round-bottom flask. The round-bottom flask was replaced with argon three times. Triethylamine (2.36 g, 23.30 mmol, 2.8 equiv) was added to the mixture at room temperature. A solution of 2-fluoro-1-(isocyanatomethyl)-4-(trifluoromethoxy)benzene 5 (2.35 g, 9.99 mmol, 1.2 equiv) in DCM (60 mL) was added dropwise to the mixture at 0 ° C. The reaction mixture was stirred at 0 ° C to room temperature for 16 hours and then quenched with ice water (100 mL). The aqueous layer was extracted twice with DCM (100 mL × 2). The organic phases were combined, washed with H2O (100 mL × 2), dried over MgSO4, filtered, and concentrated under reduced pressure. The product was purified by silica gel column chromatography with gradient elution (PE:EA=100:1→3:1) to give a yellow oil, namely tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate 6 (2.8 g, 5.89 mmol, 70.77%).

[0074] As shown in Figures 11 and 12, the NMR data are: 1 H NMR (400 MHz, CDCl3) δ H 7.38(1H,t,J=8.4Hz),7.01–6.88(2H,m),5.72(1H,t,J=6.0Hz),4.43(2H,d,J=6.0Hz),4.15–3.85(2H,br),3.65–3.45(1H,m),3.18(1H,t,J =11.9Hz),2.67–2.52(1H,br),2.50–2.42(1H,m),2.15–2.02(1H,m),1.85–1.77(1H,m),1.73–1.63(2H,m),1.43(9H,s),0.97–0.64(4H,m).

[0075] 13 C NMR (100 MHz, CDCl3) δ C =162.1,159.6,158.6,154.9,148.9(d,J C-F =10.0Hz),131.0(t,J C-F =29.0Hz),125.7,125.5,121.7,119.1,117.0,108.7,108.4,79.6,57.5,57.4,38.3,28.6,28.4,27.8,9.6,9.0.

[0076] The low-resolution mass spectrometry results are: ESI-MS m / z = 476.56 [M+H] + ;Calculated MW:475.48.

[0077] Example 9

[0078] Synthesis of (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea:

[0079] Tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate 6 (2.5 g, 5.26 mmol, 1 equiv) and dichloromethane (DCM, 50 mL) were added to a flame-dried round-bottom flask. The round-bottom flask was replaced with argon three times. Trifluoroacetic acid (TFA, 25 mL) was added dropwise to the mixture at 0°C. The reaction was stirred at 0°C to room temperature for 3 hours. The mixture was concentrated under reduced pressure to give the desired product (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea 7 (1.9 g, crude) as a yellow oil, which was used directly in the next reaction.

[0080] As shown in FIG13 , the low-resolution mass spectrometry result is: ESI-MS m / z = 376.46 [M+H] + ;Calculated MW:375.37.

[0081] Example 10

[0082] Synthesis of (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxamide:

[0083] (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea 7 (1.9 g, 5.06 mmol, 1.0 equivalent) and tetrahydrofuran (THF, 38 mL) were added to a dry round-bottom flask to obtain a furfuryl urea mixture. The round-bottom flask was replaced with argon three times. Triethylamine (3.07 g, 30.37 mmol, 6 equivalents) was added to the furfuryl urea mixture at 0°C to obtain a furfuryl urea amine mixture. Trimethylsilyl isocyanate (1.22 g, 10.63 mmol, 2.1 equivalents) was added dropwise to the furfuryl urea amine mixture at 0°C. The reaction was stirred at 0°C to room temperature for 16 hours. The reaction was quenched with ice water (100 mL). The aqueous layer was extracted twice with ethyl acetate (EtOAc, 100 mL×2). The combined organic phases were washed with saturated sodium chloride solution (100 mL x 2), dried over MgSO₄, filtered, and concentrated under reduced pressure. The product was triturated with 50 mL of solvent (PE:DCM = 100:1) to give a white solid, (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxamide JNT-517 (1.8 g, 4.3 mmol, 84.99%).

[0084] As shown in Figures 14 and 15, the NMR data are: 1 H NMR (400 MHz, CDCl3) δ H 7.36(1H,t,J=8.4Hz),7.10–6.84(2H,m),5.76(1H,t,J=6.1Hz),4.75(2H,s) ,4.43(2H,d,J=6.0Hz),4.00(1H,d,J=13.4Hz),3.80–3.63(2H,m),3.17–2.9 0(m,1H),2.65(1H,td,J=13.1,2.9Hz),2.44–2.37(1H,m),2.15–1.92(2H,m) ,1.77–1.69(1H,m),1.61–1.48(1H,m),1.04–0.80(2H,m),0.80–0.57(2H,m).

[0085] 13 C NMR (100 MHz, CDCl3) δ C =162.1,159.6,159.0,158.5,149.0(d,J C-F =12.0Hz),130.9(d,J C-F =7.0Hz),125.4(d,J C-F =15.0Hz),121.7,119.1,116.8(d,J C-F=4.0Hz),109.0(d,J C-F =25.0Hz),56.3,49.8,44.7,38.4(d,J C-F =3.0Hz),28.7,26.4,25.5,9.7,9.1.

[0086] The low-resolution mass spectrometry results are: ESI-MS m / z = 419.05 [M+H] + ;Calculated MW:418.16.

Claims

1. Mutant M203A, the amino acid sequence of which is shown in SEQ ID NO.

4.

2. Mutant M203A / S241L, characterized in that The serine at position 241 of the mutant M203A according to claim 1 is replaced by leucine.

3. A plasmid comprising the mutant M203A according to claim 1 or the mutant M203A / S241L according to claim 2.

4. A recombinant Escherichia coli characterized by: The plasmid according to claim 2 is transformed into Escherichia coli BL21 (DE3) competent cells to obtain the plasmid.

5. Use of the mutant M203A according to claim 1 in the catalytic synthesis of (R)-N-Boc-3-cyclopropylpiperidine or (R)-N-Boc-3-cyclopropylazepane.

6. Use of the mutant M203A / S241L according to claim 2 in the catalytic synthesis of (R)-N-Boc-3-cyclopropylpiperidine or (R)-N-Boc-3-cyclopropylazepane.

7. Use of the plasmid according to claim 3 in the catalytic synthesis of (R)-N-Boc-3-cyclopropylpiperidine or (R)-N-Boc-3-cyclopropylazepane.

8. Use of the recombinant Escherichia coli according to claim 4 in catalyzing the synthesis of (R)-N-Boc-3-cyclopropylpiperidine or (R)-N-Boc-3-cyclopropylazepane.

9. A method for synthesizing a compound, characterized in that: Tert-butyl (R)-3-(cyclopropylamino)piperidine-1-carboxylate reacts with 2-fluoro-1-(isocyanomethyl)-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate.

10. A method for synthesizing a compound, characterized in that: The tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate according to claim 9 is reacted with trifluoroacetic acid to produce (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea.

11. A method for synthesizing a compound, characterized in that: The (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea according to claim 10 is reacted with trimethylsilyl isocyanate to produce (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxamide.

12. The method for synthesizing the compound according to claim 9, characterized in that: The synthesis method of the 2-fluoro-1-(isocyanatomethyl)-4-(trifluoromethoxy)benzene comprises: 1-Bromo-2-fluoro-4-(trifluoromethoxy)benzene reacts with Zn(CN)2 to produce 2-fluoro-4-(trifluoromethoxy)benzonitrile; 2-Fluoro-4-(trifluoromethoxy)benzonitrile reacts with hydrogen to produce (2-fluoro-4-(trifluoromethoxy)phenyl)methylamine; (2-Fluoro-4-(trifluoromethoxy)phenyl)methylamine reacts with triphosgene to produce 2-fluoro-1-(isocyanomethyl)-4-(trifluoromethoxy)benzene.

13. The method for synthesizing the compound according to claim 9, characterized in that: The reaction of tert-butyl (R)-3-(cyclopropylamino)piperidine-1-carboxylate with 2-fluoro-1-(isocyanomethyl)-4-(trifluoromethoxy)benzene to generate tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate comprises: A DCM solution of 2-fluoro-1-(isocyanomethyl)-4-(trifluoromethoxy)benzene was added dropwise to the mixture at 0°C for reaction; The mixture was prepared by adding triethylamine to a solution of tert-butyl (R)-3-(cyclopropylamino)piperidine-1-carboxylate and dichloromethane.

14. The method for synthesizing the compound according to claim 10, characterized in that: The reaction of tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate with trifluoroacetic acid to generate (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea comprises: adding trifluoroacetic acid dropwise to a mixture of tert-butyl (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)ureido)piperidine-1-carboxylate and dichloromethane at 0°C for reaction.

15. The method for synthesizing the compound according to claim 11, characterized in that: The reaction of (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea with trimethylsilyl isocyanate to generate (R)-3-(1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)urea-1-piperidine-1-carboxamide includes: adding trimethylsilyl isocyanate dropwise to a furfurylamide mixture for reaction at 0°C; the preparation of the furfurylamide mixture is by adding triethylamine to the furfurylamide mixture at 0°C; the preparation of the furfurylamide mixture is by adding (R)-1-cyclopropyl-3-(2-fluoro-4-(trifluoromethoxy)benzyl)-1-(piperidin-3-yl)urea 7 (1.9 g, 5.06 mmol, 1.0 equivalent) to tetrahydrofuran (THF, 38 mL).

16. Immobilized enzyme M203A / S241L@NKA-9, characterized in that The invention comprises the mutant M203A / S241L according to claim 2 and NKA-9 macroporous resin.

17. Immobilized enzyme M203A / S241L@NKA-9, characterized in that The adsorption capacity of the mutant M203A / S241L in the NKA-9 macroporous resin was 26.7 mg / g.

18. Use of the immobilized enzyme M203A / S241L@NKA-9 according to any one of claims 16 or 17 in catalyzing the synthesis of (R)-N-Boc-3-cyclopropylpiperidine or (R)-N-Boc-3-cyclopropylazepane.

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