Method for synthesizing 2-N-benzylamino-4-phenyl ethyl butyrate by enzyme method

Abstract

The invention discloses a method for synthesizing 2-N-benzylamino-4-phenyl ethyl butyrate by an enzymic method, which comprises the following steps: taking 2-oxo-4-phenyl ethyl butyrate and benzylamine as substrates, and carrying out asymmetric reductive amination reaction through two mutants of reductive aminase. The reaction is carried out under mild conditions, and a series of treatment is carried out after the reaction to obtain the product. According to the present invention, the synthesis route is shortened, the cost is reduced, the material loss is reduced, the conversion rate and the stereoselectivity are high, the green development idea is met, the effective method and the theoretical guidance are provided for the synthesis of potential ACEI drugs, and the gene sequence and the amino acid sequence of the related mutant are determined.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to the application of a reductive amination enzyme in asymmetric synthesis of chiral amines. Background Art

[0002] At present, the methods for synthesizing chiral amines mainly include physical and chemical synthesis methods, but these methods have problems such as harsh reaction conditions, long reaction time, environmental damage, and low optical purity of products. Biocatalysis technology has typical characteristics of green biomanufacturing such as environmental friendliness, high catalytic efficiency, and good selectivity, and has developed into the first choice for replacing or expanding traditional chemical synthesis in the field of pharmaceuticals and chemicals. The currently established biocatalytic synthesis of chiral amines mainly includes oxidative resolution using oxidase (theoretical yield 50%), transaminase-catalyzed transamination reaction (low atomic utilization, limited to the synthesis of primary amines), and ammonia dehydrogenase-catalyzed reductive amination (low catalytic efficiency).

[0003] In the prior art, 1 is usually used as a raw material, and a metal is used as a catalyst to react under high-pressure hydrogen, and recrystallization is performed to obtain 2. 2 is subjected to a three-step reaction under high pressure and catalyzed by palladium carbon to obtain 3. 3 is subjected to a two-step reaction to obtain 4, and 4 is used as a substrate to undergo substitution with benzylamine under high temperature and high pressure to obtain the final product 5.

[0004]

[0005]

[0006] The original synthesis route is long, the process route is cumbersome, and the yield is low; high temperature and high pressure reaction conditions are required during the reaction process, which is not suitable for industrial production; expensive metal catalysts (such as palladium, platinum and nickel, etc.) and reducing agents are used, which are easy to cause environmental pollution and difficult to remove. So far, there has been no report on the use of enzymes to synthesize 2-N-benzylamino-4-phenylbutyric acid ethyl ester. The present invention synthesizes two selective drug intermediates by catalyzing asymmetric reductive amination reactions with different mutants of reductive amination enzymes, which provides certain guidance methods and theoretical guidance for the synthesis of potential ACEI drugs. Summary of the invention

[0007] The present invention uses ethyl 2-oxo-4-phenylbutyrate and benzylamine as substrates to perform asymmetric reductive amination through two mutants of reductive amination enzymes to obtain two highly selective intermediates. Compared with traditional chemical synthesis, enzymatic synthesis not only shortens the synthesis route, reduces production costs, and reduces material loss, but also can meet the requirements of green, economical, efficient and sustainable development, which is in line with the green development of the current era.

[0008] In the present invention, the reductive amination enzyme can directly use equimolar ketones and amines (primary amines or secondary amines) as substrates, and use nicotinamide cofactor NADPH as a hydrogen donor to catalyze the intermolecular reductive amination reaction to synthesize the corresponding secondary or tertiary chiral amine products. The one-step reaction catalyzed by the reductive amination enzyme is the most direct and atom-economically efficient way to synthesize chiral amines.

[0009] In one embodiment, the present invention provides an enzymatic method for synthesizing ethyl 2-N-benzylamino-4-phenylbutyrate, using ethyl 2-oxo-4-phenylbutyrate and benzylamine as substrates, and performing an asymmetric reductive amination reaction by a reductive amination enzyme mutant.

[0010] In another embodiment, the reductaminase mutant is M5-L200F or F260G.

[0011] In another embodiment, the reaction system comprises nicotinamide cofactor NADPH as a hydrogen donor, and glucose dehydrogenase GDH and D-glucose for coenzyme cycle.

[0012] In another embodiment, the molar ratio of ketone to amine in the reaction is 1:2, the mass volume ratio of the wet weight of the reductive aminase mutant cells to the reaction mixture is 10 g / L, the molar ratio of D-glucose to ketone is 2:1, and NADP + The amount of is 1.0 mM / L, the amount of GDH enzyme powder is 1 mg / L, the amount of phosphate is 100 mM / L, and the volume of DMSO is 10% of the total volume of the reaction solution.

[0013] In another embodiment, the reaction solution has a pH of 7.0, a temperature of 30° C., and a reaction time of 24 hours.

[0014] In another embodiment, the reaction steps include: adding DMSO and sodium phosphate buffer in a volume ratio of 1:4 into a reactor; adding a mixture of benzylamine, D-glucose and nicotinamide adenine dinucleotide phosphate disodium salt into the reactor and reacting at 30° C.; adding ethyl 2-oxo-4-phenylbutyrate, with the molar ratio of amine to ketone being 2:1; adding cell lysate of the reductive amination enzyme mutant and GDH enzyme powder into the reaction solution for reaction.

[0015] In another embodiment, it further includes the step of obtaining the product after reacting for 24 hours: adding 50 μL of 1M dilute hydrochloric acid to adjust the pH of the mixed solution to 4-5, then adding 1 mL of ethyl acetate and vortexing for 30 s, centrifuging at 12000 rpm for 10 min, discarding the organic phase, repeating three times, then adding 50 μL of saturated sodium carbonate solution to adjust the pH of the mixed solution to 9-10, then adding 1 mL of ethyl acetate and vortexing for 30 s, centrifuging at 12000 rpm for 10 min, aspirating all the upper organic phase, and concentrating under reduced pressure to obtain 2-N-benzylamino-4-phenylbutyric acid ethyl ester.

[0016] In another embodiment, the amino acid sequence of the reductase mutant M5-L200F is shown in SEQ ID NO.4.

[0017] In another embodiment, the amino acid sequence of the reductase mutant M5-F260G is shown in SEQ ID NO.6.

[0018] In one embodiment, it also includes the use of 2-N-benzylamino-4-phenylbutyric acid ethyl ester synthesized by the method described in the present invention in the synthesis of potential ACEI drugs.

[0019] Beneficial effects of the present invention: In the present invention, the reaction of synthesizing chiral amines catalyzed by reductive aminase is the research object. By rationally designing the key active sites of the enzyme substrate binding pocket, the specific recognition of complex substrates is improved and the stereoselectivity is controlled to generate the target chiral amine product. At the same time, the crystal structure of the optimal mutant is analyzed, and the association mechanism between the reconstruction of the enzyme active center site and the recognition of the substrate chemical structure and chirality control is explained by using computational simulation technology, providing theoretical guidance for the design of new enzyme-catalyzed reactions of the same type and the synthesis of complex new molecules.

[0020] The present invention is based on the analysis of the structure of imine reductase, and the molecular structure of imine reductase is engineered in combination with rational design and semi-rational design, which reduces the manpower and time cost consumed in protein engineering transformation compared to traditional directed evolution; the enzyme has extremely high safety and stereo and regional selectivity, and its reaction conditions are mild, the reaction energy consumption is low, the cost is low, and pollutant emissions can be reduced. It is determined that the optimal mutants M5-L200F and F260G are screened out, and the efficient synthesis of ACEI drug molecular intermediate-2-N-benzylamino-4-phenylbutyric acid ethyl ester can be achieved. Compared with the industrial synthesis route, the enzymatic synthesis route has the advantages of high conversion rate / high stereoselectivity, which makes up for the raw materials lost by the industrial synthesis route due to chiral resolution, improves the yield, and can greatly shorten the synthesis steps. The final conversion rates of M5-L200F and F260G are 43% and 57%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is an enzymatic synthesis route.

[0022] Figure 2 HPLC spectra of the racemic standard, (R) chiral standard and M5-F260G catalytic product of compound 1a.

[0023] Figure 3 HPLC spectra of the racemic standard, (R) chiral standard and M5-L200F catalytic product of compound 1a. DETAILED DESCRIPTION

[0024] The following examples are only used to further illustrate the present invention, but do not constitute any limitation to the present invention.

[0025] Unless otherwise specified, the reagents, materials and instruments used in the following embodiments and test examples can be obtained from commercial sources; operations not otherwise specified are routine operations in the art.

[0026] In a specific embodiment of the present invention, the molar ratio of ketone to amine added in the reaction is 1:2.

[0027] In a specific embodiment of the present invention, a cell lysate of a reductive aminase mutant is added to the reaction, the mass volume ratio of the wet weight of the reductive aminase mutant cells to the reaction mixture is 10 g / L; the molar ratio of D-glucose to ketone is 2:1; the amount of NADP+ added to the reaction is 1.0 mM / L; the amount of glucose dehydrogenase GDH enzyme powder is 1 mg / L; the amount of phosphate is 100 mM / L; and the volume usage of DMSO is 10% of the total volume of the reaction solution.

[0028] In a specific embodiment of the present invention, the pH of the reaction solution is 7.0.

[0029] In a specific embodiment of the present invention, the reaction temperature is 30°C.

[0030] In a specific embodiment of the present invention, the reaction time is 24 hours.

[0031] In a specific embodiment of the present invention, the reaction steps include: adding DMSO and sodium phosphate buffer in a volume ratio of 1:4 into a reactor; adding a mixed solution of benzylamine, D-glucose and nicotinamide adenine dinucleotide phosphate disodium salt into the reactor and reacting at 30°C; the amount of 2-oxo-4-phenylbutyric acid ethyl ester added is 30mM / L; the molar ratio of amine to ketone is 2:1; adding cell lysate of the reductive amination enzyme mutant IR-G36-M5 mutant and GDH enzyme powder to the reaction solution for reaction; and obtaining a product after reacting for 24 hours.

[0032] In a specific embodiment of the present invention, after obtaining the product after reacting for 24 hours, the method further includes the following steps: adding 50 μL of 1 M dilute hydrochloric acid to adjust the pH of the mixed solution to 4-5, then adding 1 mL of ethyl acetate and vortexing for 30 s, centrifuging at 12000 rpm for 10 min, discarding the organic phase, repeating three times, then adding 50 μL of saturated sodium carbonate solution to adjust the pH of the mixed solution to 9-10, then adding 1 mL of ethyl acetate and vortexing for 30 s, centrifuging at 12000 rpm for 10 min, aspirating all the upper organic phase, and concentrating under reduced pressure to obtain two imine reductase catalytic products.

[0033] Example 1. Preparation of reductase IR-G36 mutant M5 The reductive aminase IR-G36 mutant M5 is referred to as M5 hereafter. Its co-crystallization results with the cofactor NADP(H) show that its structure is a classic homodimer. The two monomers are composed of the Rossman domain at the N-terminus and the helical bundle at the C-terminus. Two catalytic active centers are formed at the junction of the C-terminal and N-terminal domains of the two monomers, and the cofactor NADP(H) binds to the two active pockets.

[0034] Ethyl 2-oxo-4-phenylbutyrate and benzylamine were selected as substrates for the reductive amination reaction catalyzed by M5. Discovery studio 2019 software was used to perform molecular docking simulations on the imine intermediate 1a' and the wild-type M5 protein, in which amino acid residues within 5 Å of the imine intermediate 1a' were selected. From the docking results, it can be seen that 5 amino acid residues within 5 Å of the imine intermediate 1a' were selected, namely I149, L200, M203, F260 and Q265. By comparing with the molecular docking results of 1a', it was found that both had interactions between L200, M203 and F260 and the substrate, indicating that these amino acids may be key amino acids affecting the catalytic activity and stereoselectivity of imine reductase IR-G36-M5. At the I149 and Q265 sites, it was observed that the configuration of the product amine 1a was completely reversed, from the original S The configuration is reversed to R The configurations indicate that the I149 (Table 1) and Q265 (Table 2) sites are both involved in the chiral inversion of the imine reductase for the formation of bulky chiral imines from ethyl 2-oxo-4-phenylbutyrate 2 and benzylamine b.

[0035] Table 1. Results of ee value determination of I149 site saturation mutation

[0036] Table 2. Results of ee value determination of Q265 site saturation mutation

[0037] Referring to Table 3, at position M203, it can be found that except for the mutation of methionine to alanine, the chirality of the amine product is maintained. S In addition to the configuration, the remaining mutants all reversed the chirality of the amine product, indicating that the isoleucine at position 149, the methionine at position 203, and the glutamine at position 265 also played an important role in stabilizing the configuration of the selectivity of substrate 1a. Several mutants at two of the sites showed good reverse selectivity, such as M203E, M203G, and M203K at the M203 site, and Q265I, Q265L, and Q265M at the Q265 site. R The selectivity is as high as 95%-96%, see Table 2.

[0038] Table 3. Results of ee value determination of M203 site saturation mutation

[0039] At positions L200 (Table 4) and F260 (Table 5), it was observed that the products of the reactions catalyzed by most of the mutants at these sites were similar to the original S When the leucine at position 200 was mutated to threonine, the activity of the mutant was lost. When the leucine was mutated to the hydrophobic amino acid phenylalanine, the stereoselectivity of the mutant for substrate 1a' was greatly improved, from the original 93% to 99%. These phenomena collectively indicate that L200 is effective in catalyzing the formation of ethyl 2-oxo-4-phenylbutyrate 2 by imine reductase. S - plays a key role in selectivity. When the phenylalanine at the F260 position was mutated to glycine, which is also hydrophobic but smaller in size, the stereoselectivity of the mutant substrate 1a' was reversed to the maximum value of 97%. R .

[0040] Table 4. Results of ee value determination of L200 site saturation mutation

[0041] Table 5. Results of ee value determination of F260 site saturation mutation

[0042] Example 2. Expression and purification of mutants M5-L200F and M5-F260G in Escherichia coli The genes of mutants M5-L200F and M5-F260G were connected to the pET28a plasmid and transformed into E. coli BL21 (DE3) competent cells for protein expression. Single clones were picked and cultured in LB medium (10 mL) containing 50 µg / mL kanamycin at 37 ℃ and 220 rpm overnight. The above bacterial liquid was inoculated into LB medium containing 50 µg / mL kanamycin at an inoculum of 1:100 and cultured at 37 ℃ and 220 rpm until the OD600 value was 0.6-0.8. IPTG (0.15 mM / L) was added to the culture medium to induce gene expression, and the culture was continued at 18 ℃ and 180 rpm for 16 hours. The cells were then harvested by centrifugation at 5000 rpm for 10 minutes, resuspended in sodium phosphate buffer (100 mM / L, pH 7.0), and centrifuged at 5000 rpm for 10 minutes to collect the cells. The cells were washed twice with sodium phosphate buffer, resuspended in sodium phosphate buffer (100 mM / L, pH 7.0), and disrupted using a high-pressure disruptor. The disrupted solution was centrifuged at 1,2000 rpm, the supernatant was collected, and the protein was purified by Ni+2 affinity chromatography.

[0043] Example 3: Purified mutants M5-L200F and M5-F260G catalyze the reductive amination reaction of ethyl 2-oxo-4-phenylbutyrate and benzylamine The purified mutant proteins M5-L200F and M5-F260G were used to determine the reductive amination reaction between 2-oxo-4-phenylbutyric acid ethyl ester and benzylamine. 1 mM / L NADP+, 10 mg / mL purified enzyme, 30 m / L 2-oxo-4-phenylbutyric acid ethyl ester, the amount of benzylamine added to the ketone substance ratio of 2:1, the amount of D-glucose added to the ketone substance ratio of 2:1, 1 mg / mL GDH, 10% DMSO by volume, and the concentration of the sodium phosphate buffer was 100 mM / L (pH 7.0). All reactions were reacted at 30°C and 220 rpm for 24 hours, and 5 mL of acetonitrile containing 1M / L acetic acid was added to quench. Then, centrifuged at 12000 rpm for 10 minutes, and the supernatant was analyzed by HPLC. The experimental results are shown in Table 6.

[0044] Table 6 Results of the reductive amination reaction of ethyl 2-oxo-4-phenylbutyrate and benzylamine catalyzed by mutants M5-L200F and M5-F260G .

Claims

1. A method for enzymatically synthesizing ethyl 2-N-benzylamino-4-phenylbutyrate, characterized in that: Asymmetric reductive amination of ethyl 2-oxo-4-phenylbutyrate and benzylamine was carried out by reductive amination enzyme mutants.

2. The method according to claim 1, characterized in that: The reductase mutant is M5-L200F, and its amino acid sequence is shown in SEQ ID NO.

4.

3. The method according to claim 1, characterized in that: The reductase mutant is F260G, and its amino acid sequence is shown in SEQ ID NO.

6.

4. The method according to any one of claims 1 to 3, characterized in that: The reaction system contains nicotinamide cofactor NADPH as a hydrogen donor, and glucose dehydrogenase GDH and D-glucose for coenzyme circulation.

5. The method according to any one of claims 1 to 4, characterized in that: The molar ratio of ketone to amine in the reaction was 1:2, the mass volume ratio of the wet weight of the reductive aminase mutant cells to the reaction mixture was 10 g / L, the molar ratio of D-glucose to ketone was 2:1, and NADP + The amount of is 1.0 mM / L, the amount of GDH enzyme powder is 1 mg / L, the amount of phosphate is 100 mM / L, and the volume of DMSO is 10% of the total volume of the reaction solution.

6. The method according to any one of claims 1 to 5, characterized in that: The reaction solution pH was 7.0, the temperature was 30° C., and the reaction time was 24 hours.

7. The method according to any one of claims 1 to 6, characterized in that: The reaction steps include: adding DMSO and sodium phosphate buffer in a volume ratio of 1:4 into a reactor; adding a mixed solution of benzylamine, D-glucose and nicotinamide adenine dinucleotide phosphate disodium salt into the reactor and reacting at 30° C.; adding ethyl 2-oxo-4-phenylbutyrate, with the molar ratio of amine to ketone being 2:1; and adding cell lysate of a reductive amination enzyme mutant and GDH enzyme powder into the reaction solution for reaction.

8. The method according to any one of claims 1 to 7, characterized in that: The method further includes the following steps after obtaining the product after reacting for 24 hours: adding 50 μL of 1 M dilute hydrochloric acid to adjust the pH of the mixed solution to 4-5, then adding 1 mL of ethyl acetate and vortexing for 30 seconds, centrifuging at 12000 rpm for 10 minutes, discarding the organic phase, repeating three times, then adding 50 μL of saturated sodium carbonate solution to adjust the pH of the mixed solution to 9-10, then adding 1 mL of ethyl acetate and vortexing for 30 seconds, centrifuging at 12000 rpm for 10 minutes, absorbing all the upper organic phase, and concentrating under reduced pressure to obtain 2-N-benzylamino-4-phenylbutyric acid ethyl ester.

9. The method according to any one of claims 1 to 8, characterized in that: The reductive aminase mutant was prepared by the following steps: using Discovery studio 2019 software to perform molecular docking simulation on the imine intermediate 1a' and the wild-type M5 protein, selecting amino acid residues within 5 Å of the imine intermediate 1a', and performing saturation mutation and ee value determination on key amino acid residues to screen out mutants with desired stereoselectivity.

10. Use of ethyl 2-N-benzylamino-4-phenylbutyrate synthesized according to the method described in any one of claims 1 to 9 in the synthesis of potential ACEI drugs.

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