A method for synthesizing ethyl 2-N-benzylamino-4-phenylbutyrate by enzymatic method

Abstract

The present invention discloses a method for enzymatically synthesizing ethyl 2-N-benzylamino-4-phenylbutyrate, wherein ethyl 2-oxo-4-phenylbutyrate and benzylamine are used as substrates, and an asymmetric reductive amination reaction is performed by two mutants of a reductive aminase. The reaction is carried out under mild conditions, and a product is obtained by a series of treatments after the reaction. The present invention shortens the synthetic route, reduces costs, reduces material loss, has high conversion rate and high stereoselectivity, conforms to the concept of green development, provides an effective method and theoretical guidance for the synthesis of potential ACEI drugs, and determines the gene sequence and amino acid sequence of the relevant mutants.

Description

Technical Field

[0001] The present 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] Currently, the main methods for synthesizing chiral amines are physical and chemical synthesis. However, these methods suffer from harsh reaction conditions, long reaction times, environmental hazards, and low product optical purity. Biocatalysis, with its environmentally friendly, highly efficient, and highly selective characteristics typical of green biomanufacturing, has become a leading alternative or expansion option for traditional chemical synthesis in the pharmaceutical and chemical industries. Currently established biocatalytic processes for synthesizing chiral amines include oxidative resolution using oxidases (theoretical yield 50%), transamination catalyzed by transaminases (low atom utilization, limited to the synthesis of primary amines), and reductive amination catalyzed by ammonia dehydrogenases (low catalytic efficiency).

[0003] In the prior art, 1 is usually used as a raw material, and a metal catalyst is used for reaction under high-pressure hydrogen. Recrystallization is performed to obtain 2. 2 is then subjected to a three-step reaction catalyzed by palladium on carbon under high pressure to obtain 3. 3 is subjected to a two-step reaction to obtain 4. 4 is then 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 existing synthetic route is long, complex, and yields are low. The reaction requires high temperatures and high pressures, making it unsuitable for industrial production. The use of expensive metal catalysts (such as palladium, platinum, and nickel) and reducing agents can easily cause environmental pollution and be difficult to remove. To date, there have been no reports of enzymatic synthesis of ethyl 2-N-benzylamino-4-phenylbutyrate. This invention synthesizes two selective drug intermediates through asymmetric reductive amination reactions catalyzed by different reductive amination enzyme mutants, providing methodological and theoretical guidance for the synthesis of potential ACEI drugs. Summary of the Invention

[0007] This invention utilizes ethyl 2-oxo-4-phenylbutyrate and benzylamine as substrates, undergoing asymmetric reductive amination via two mutant reductive amination enzymes to obtain two highly selective intermediates. Compared to traditional chemical synthesis, enzymatic synthesis not only shortens the synthesis route, reduces production costs, and minimizes material loss, but also achieves environmental, economical, efficient, and sustainable development, in line with the current era of green development.

[0008] The reductive amination enzyme described herein can directly utilize equimolar amounts of ketones and amines (primary or secondary amines) as substrates, using the nicotinamide cofactor NADPH as a hydrogen donor to catalyze an intermolecular reductive amination reaction to synthesize the corresponding secondary or tertiary chiral amine products. This one-step reaction catalyzed by reductive amination enzyme is the most direct and atom-efficient method for synthesizing 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 using 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 recycling.

[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 dTT was 1.0 mM / L, the amount of GDH enzyme powder was 1 mg / L, the amount of phosphate was 100 mM / L, and the volume of DMSO was 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 an amine to ketone molar ratio of 2:1; and adding cell lysate of the reductive amination enzyme mutant and GDH enzyme powder to the reaction solution for reaction.

[0015] In another embodiment, the method further comprises 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 mixture to 4-5, then adding 1 mL of ethyl acetate and vortexing for 30 seconds, centrifuging at 12,000 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 mixture to 9-10, then adding 1 mL of ethyl acetate and vortexing for 30 seconds, centrifuging at 12,000 rpm for 10 minutes, aspirating the entire upper organic phase, and concentrating under reduced pressure to obtain ethyl 2-N-benzylamino-4-phenylbutyrate.

[0016] In another embodiment, the amino acid sequence of the reductaminase 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:

[0020] This study examines the synthesis of chiral amines catalyzed by reductive amination enzymes. By rationally designing the key active site of the enzyme's substrate-binding pocket, the enzyme enhances specific recognition of complex substrates and controls its stereoselectivity, yielding the target chiral amine product. Furthermore, the crystal structure of the optimal mutant was determined, and computational simulations were used to elucidate the mechanism linking the remodeling of the enzyme's active center site with substrate chemical structure recognition and chirality control. This approach provides theoretical guidance for the design of novel enzymatic reactions of the same type and the synthesis of complex new molecules.

[0021] 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. Compared with traditional directed evolution, it reduces the manpower and time cost consumed in protein engineering transformation; the enzyme has extremely high safety and stereo and regioselectivity, 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 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 due to chiral resolution in the industrial synthesis route, 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

[0022] Figure 1 It is an enzymatic synthesis route.

[0023] Figure 2 HPLC spectra of the racemic standard, (R) chiral standard and M5-F260G catalytic product of compound 1a.

[0024] Figure 3 HPLC spectra of the racemic standard, (R) chiral standard and M5-L200F catalytic product of compound 1a. DETAILED DESCRIPTION

[0025] The following examples are only used to further illustrate the present invention, but are not intended to limit the present invention in any way.

[0026] Unless otherwise specified, the reagents, materials, and instruments used in the following examples and test examples can be obtained from commercial sources; operations not otherwise specified are routine operations in the art.

[0027] In a specific embodiment of the present invention, the molar ratio of ketone to amine added in the reaction is 1:2.

[0028] In a specific embodiment of the present invention, a cell lysate of a reductive amination enzyme mutant is added to the reaction, the mass volume ratio of the wet weight of the reductive amination enzyme 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 amount of DMSO used is 10% of the total volume of the reaction solution.

[0029] In a specific embodiment of the present invention, the pH of the reaction solution is 7.0.

[0030] In a specific embodiment of the present invention, the reaction temperature is 30°C.

[0031] In a specific embodiment of the present invention, the reaction time is 24 hours.

[0032] 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 to a reactor; adding a mixture of benzylamine, D-glucose and nicotinamide adenine dinucleotide phosphate disodium salt to the reactor and reacting at 30° C.; the amount of 2-oxo-4-phenylbutyric acid ethyl ester added is 30 mM / L; the molar ratio of the amine to the 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.

[0033] 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 mixture 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, and then adding 50 μL of saturated sodium carbonate solution to adjust the pH of the mixture to 9-10, then adding 1 mL of ethyl acetate and vortexing for 30 seconds, centrifuging at 12000 rpm for 10 minutes, aspirating all the upper organic phase, and concentrating under reduced pressure to obtain two imine reductase catalytic products.

[0034] Example 1. Preparation of Reductive Amination Enzyme IR-G36 Mutant M5

[0035] Cocrystallization of the reductive aminase IR-G36 mutant M5 (hereafter referred to as M5) with the cofactor NADP(H) revealed a classic homodimer structure. The two monomers are composed of an N-terminal Rossman domain and a C-terminal helical bundle. Two catalytically active sites are formed at the junction of the C-terminal and N-terminal domains of the two monomers, with the cofactor NADP(H) binding in both active pockets.

[0036] 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 from the imine intermediate 1a' were selected. The docking results show that 5 amino acid residues within 5 Å from 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 into R The configurations indicate that both I149 (Table 1) and Q265 (Table 2) sites participate in the chiral inversion of imine reductase for the formation of bulky chiral imines from ethyl 2-oxo-4-phenylbutyrate 2 and benzylamine b.

[0037] Table 1. Results of ee value determination of I149 site saturation mutation

[0038]

[0039] Table 2. Results of ee value determination of Q265 site saturation mutation

[0040]

[0041] 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 Except for the configuration, the other mutants all experienced the inversion of the chirality of the amine product, indicating that the isoleucine at position 149, the methionine at position 203, and the glutamine at position 265, these three amino acids also played an important role in stabilizing the configuration in the selectivity of substrate 1a. Among them, several mutants at two positions showed good reverse selectivity, such as M203E, M203G, and M203K at the M203 position, and Q265I, Q265L, and Q265M at the Q265 position. R The selectivity is as high as 95%-96%, see Table 2.

[0042] Table 3. Results of ee value determination of M203 site saturation mutation

[0043]

[0044] 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, rising from the original 93% to 99%. These phenomena collectively indicate that L200 is a promising imine reductase for the formation of ethyl 2-oxo-4-phenylbutyrate 2 S - plays a key role in selectivity. When the phenylalanine at the F260 position is mutated to glycine, which is also hydrophobic but smaller in size, the stereoselectivity of the mutant substrate 1a' is reversed to the maximum value of 97%. R .

[0045] Table 4. Results of ee value determination of L200 site saturation mutation

[0046]

[0047] Table 5. Results of ee value determination of F260 site saturation mutation

[0048]

[0049] Example 2. Expression and purification of mutants M5-L200F and M5-F260G in Escherichia coli

[0050] The genes encoding the mutants M5-L200F and M5-F260G were ligated into the pET28a plasmid and transformed into competent E. coli BL21 (DE3) cells for protein expression. A single colony was isolated and cultured overnight in 10 mL of LB medium supplemented with 50 µg / mL kanamycin at 37°C and 220 rpm. The culture was inoculated at a ratio of 1:100 into LB medium supplemented with 50 µg / mL kanamycin and grown at 37°C and 220 rpm to an OD600 of 0.6-0.8. IPTG (0.15 mM / L) was added to the culture to induce gene expression, and the culture was continued at 18°C ​​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 again 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 cell disruptor. The disrupted solution was centrifuged at 12000 rpm, and the supernatant was collected and the protein purified by Ni+2 affinity chromatography.

[0051] Example 3: Purified mutants M5-L200F and M5-F260G catalyze the reductive amination reaction of ethyl 2-oxo-4-phenylbutyrate and benzylamine

[0052] Purified mutant proteins M5-L200F and M5-F260G were used to measure the reductive amination reaction between ethyl 2-oxo-4-phenylbutyrate and benzylamine. To a 1 mL reaction mixture, 1 mM / L NADP+, 10 mg / mL purified enzyme, 30 mL / L ethyl 2-oxo-4-phenylbutyrate, a 2:1 molar ratio of benzylamine to ketone, a 2:1 molar ratio of D-glucose to ketone, 1 mg / mL GDH, and 10% DMSO were added. The sodium phosphate buffer concentration was 100 mM / L (pH 7.0). All reactions were incubated at 30°C and 220 rpm for 24 hours. The reaction was quenched by adding 5 mL of acetonitrile containing 1 M / L acetic acid. The reaction mixture was then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was analyzed by HPLC. The results are shown in Table 6.

[0053] Table 6 Results of the reductive amination reaction of ethyl 2-oxo-4-phenylbutyrate and benzylamine catalyzed by mutants M5-L200F and M5-F260G

[0054] .

Claims

1. A method for enzymatically synthesizing ethyl 2-N-benzylamino-4-phenylbutyrate, characterized in that: An asymmetric reductive amination reaction is carried out using ethyl 2-oxo-4-phenylbutyrate and benzylamine as substrates by a reductive amination enzyme mutant; wherein the reductive amination enzyme mutant is M5-L200F, whose amino acid sequence is shown in SEQ ID NO.4; or the reductive amination enzyme mutant is F260G, whose amino acid sequence is shown in SEQ ID NO.

6.

2. The method according to claim 1, characterized in that The reaction system contains nicotinamide cofactor NADPH as a hydrogen donor, and glucose dehydrogenase GDH and D-glucose for coenzyme circulation.

3. The method according to claim 1 or 2, characterized in that In the reaction, the molar ratio of ethyl 2-oxo-4-phenylbutyrate and benzylamine was 1:2, the wet weight of cells expressing the reductive aminase mutant to the mass volume ratio of the reaction mixture was 10 g / L, the molar ratio of D-glucose to ethyl 2-oxo-4-phenylbutyrate was 2:1, and NADP + The amount of dTT was 1.0 mM / L, the amount of GDH enzyme powder was 1 mg / L, the amount of phosphate was 100 mM / L, and the volume of DMSO was 10% of the total volume of the reaction solution.

4. The method according to claim 1 or 2, characterized in that The reaction solution pH was 7.0, the temperature was 30° C., and the reaction time was 24 hours.

5. The method according to claim 1 or 2, 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 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 ethyl 2-oxo-4-phenylbutyrate and benzylamine being 1:2; and adding cell lysate expressing a reductive amination enzyme mutant and GDH enzyme powder into the reaction solution for reaction.

6. The method according to claim 5, 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 mixture to 4-5, then adding 1 mL of ethyl acetate and vortexing for 30 seconds, centrifuging at 12,000 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 mixture to 9-10, then adding 1 mL of ethyl acetate and vortexing for 30 seconds, centrifuging at 12,000 rpm for 10 minutes, aspirating the entire upper organic phase, and concentrating under reduced pressure to obtain ethyl 2-N-benzylamino-4-phenylbutyrate.

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