A mutant R-aminotransferase and a method for preparing an ubagepam intermediate thereof

Abstract

The invention provides a mutant R-transaminase and a method for preparing an intermediate of ubugepam from it. The mutant R-transaminase is obtained by site-directed mutagenesis, and is immobilized. In a fully organic phase reaction system without DMSO, 4-phenyl-2-(tert-butoxycarbonyl)amino-5-oxohexanoic acid isopropyl ester is used as a substrate, an amino donor and an immobilized R-transaminase are added, and a catalytic reaction is performed to obtain a key intermediate of ubugepam, (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-l-piperidin-2-one. The immobilized transaminase prepared by the method has an excellent yield and DR value, a high number of reusable times, and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the field of bioenzyme catalysis, and in particular to a mutant R-aminotransferase and a method for preparing an ubagepan intermediate thereof. Background Art

[0002] Ubrogepant is a small molecule CGRP receptor antagonist that has been approved by the FDA for acute migraine treatment in 2019. Calcitonin gene-related peptide (CGRP) is an important mediator of migraine attacks. Ubrogepant shows extremely high affinity for human CGRP receptors, preventing CGRP from binding to its receptors and counteracting the neurovascular effects of CGRP, thereby terminating migraine attacks.

[0003] (5S,6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-l-piperidin-2-one (Compound II) is an important lactam fragment in the preparation of ubugepam, which can be synthesized through the transamination reaction of 4-phenyl-2-(tert-butoxycarbonyl)amino-5-oxohexanoic acid isopropyl ester (Compound I). Compound I contains two chiral centers, and its corresponding four isomers D1, D2, S1, and S2. The third R-configuration chiral center is introduced at the 5th position using a specific R-transaminase, so that the enzyme only acts on the D1 and D2 configurations in Compound I, but is useless for S1 and S2. The corresponding products of enzyme catalysis are DW1 and DW2, and its ester bond can spontaneously break and cyclize to Compound II with (5S,6R) configuration. Due to phase isomerization, the remaining S1 and S2 configuration compounds in the reaction system spontaneously transform to D1 and D2 configuration as D1 and D2 decrease, thereby preparing high chiral purity compound II. However, if the aforementioned R-transaminase can transform to S1 and S2 configuration, it will lead to the production of (5R, 6R) configuration compound III, thereby affecting the chiral purity of compound II.

[0004]

[0005] US patent application US20150038707A1 (published on February 5, 2015) discloses a method for preparing compound II using Arthrobacter transaminase with compound I as substrate. The substrate concentration is 50 g / L, 50% dimethyl sulfoxide (DMSO) is added, and the reaction is carried out for 18-24 hours. The conversion rate is greater than 99%, and the product dr value is greater than 60:1. The conversion capacity of the enzyme is limited, and DMSO needs to be added to help dissolve when the substrate concentration exceeds 50 g / L. The addition of DMSO introduces a new organic solvent, which brings difficulties to subsequent processing and is often difficult to remove. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a mutant R-aminotransferase suitable for high substrate concentration and with high organic solvent tolerance, which can efficiently convert 4-phenyl-2-(tert-butoxycarbonyl)amino-5-oxohexanoic acid isopropyl ester into (5S,6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one with a high DR value; another purpose of the present invention is to provide a gene sequence encoding the aforementioned mutant R-aminotransferase; another purpose of the present invention is to provide a recombinant vector comprising the aforementioned gene sequence; another purpose of the present invention is to provide a host cell comprising the aforementioned recombinant vector; another purpose of the present invention is to provide a method for preparing the high DR value ubagepam intermediate (5S,6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one by using immobilized R-aminotransferase in a fully organic phase with a high concentration of substrate.

[0007] It should be noted here that the dr value described in the present invention is the "diastereoisomer ratio", specifically, it refers to the ratio of (5S, 6R) configuration compound II to (5R, 6R) configuration compound III in the product. The larger the ratio, the higher the chiral purity of compound II (target product).

[0008] The mutant R-aminotransferase provided by the present invention has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The enzyme is derived from a wild-type transaminase of Aspergillus terreus, the wild-type of which has an amino acid sequence as shown in SEQ ID NO: 9, and is obtained by mutation at any one or more positions at position 60, position 115, position 117, position 240, and position 276.

[0009] As a preferred embodiment of the present invention, the R-transaminase is as shown in SEQ ID NO: 2, and its mutation sites are Y60F, F115T, E117P and R240A, and the enzyme can achieve a conversion rate of more than 65% and a dr value of more than 100: 1. As the most optimal embodiment of the present invention, the R-transaminase is as shown in SEQ ID NO: 1, and its mutation sites are Y60F, F115T, E117P, A276G and R240A, and the enzyme can achieve a conversion rate of more than 85% and a dr value of more than 100: 1. Y (tyrosine) at position 60, F (phenylalanine) at position 115, E (glutamate) at position 117 and A (alanine) at position 276 in the wild-type transaminase are important amino acid residues that constitute the catalytic pocket for binding of the substrate to the enzyme active site. This pocket structure affects the attack of the active site K (lysine) at position 180 on compound I. The closer the distance between compound I and K at position 180, the easier it is to produce the R configuration, thereby affecting the catalytic activity and the configuration of compound II.

[0010] Furthermore, the gene sequence provided by the present invention is used to encode the mutant R-transaminase, which has a nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6, which encodes the amino acid sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0011] The present invention constructs a recombinant vector and adds it to a host cell in the sensory zone to obtain an engineered strain expressing the R-aminotransferase. The recombinant vector comprises the gene sequence described above, and the expression vector selected includes but is not limited to any one of pET, pGEX, and pMAL, preferably pET-28 plasmid. The host cell is selected from any one of Escherichia coli, Bacillus subtilis, and Pichia pastoris, wherein Escherichia coli is used as the host cell as a preferred embodiment.

[0012] The present invention provides a method for preparing (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one, comprising the following steps:

[0013] (1) inoculating and culturing the host cells, crushing and separating the supernatant to obtain a crude R-aminotransferase enzyme solution;

[0014] (2) mixing the crude R-aminotransferase solution with a macroporous resin carrier to prepare immobilized R-aminotransferase;

[0015] (3) In an organic phase reaction system free of DMSO, 4-phenyl-2-(tert-butoxycarbonyl)amino-5-oxohexanoic acid isopropyl ester is used as a substrate, the substrate is fully dissolved, an amino donor is added, and then the immobilized R-aminotransferase is added to obtain (5S,6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-l-piperidin-2-one.

[0016] The method provided by the present invention adopts a mutant R-aminotransferase with high tolerance to organic solvents, so that a high concentration of organic phase can be used to dissolve the substrate without adding DMSO, thereby avoiding the subsequent process of separating DMSO. Preferably, the organic phase is selected from any one or more combinations of saturated or unsaturated ethyl acetate, toluene, ethanol, acetonitrile, methyl tert-butyl ether or isopropyl acetate. In particular, a saturated organic phase solvent is used for dissolution. More preferably, the saturated organic solvent is selected from C1-C6 ester or ether solvents, such as saturated methyl tert-butyl ether or saturated isopropyl acetate. Compared with traditional solvents such as ethanol and acetonitrile, methyl tert-butyl ether and isopropyl acetate have smaller polarity and the protein structure is not easily destroyed.

[0017] Furthermore, due to the improvement of the solvent system, a higher concentration of substrate can be added to improve the yield. The initial concentration of the substrate in the reaction system is 80-150 g / L, and further, the substrate concentration can be selected from 100-150 g / L, or even 120-150 g / L.

[0018] Furthermore, the mass ratio of the immobilized R-aminotransferase to the substrate is 0.5-3:1.

[0019] Furthermore, the reaction system is reacted at a temperature of 40-65°C, more preferably at a temperature of 50-60°C.

[0020] Furthermore, the amino donor is selected from any one or more combinations of isopropylamine, 1,4-butanediamine, L-glutamic acid or L-alanine, wherein isopropylamine is preferably used as the amino donor.

[0021] Furthermore, the molar ratio of the amino group in the amino donor to the substrate is 2-5:1, and an excess of the amino donor is beneficial to improving the substrate conversion rate.

[0022] Furthermore, the immobilized R-aminotransferase also includes pyridoxal phosphate (PLP), which is added to the crude R-aminotransferase solution and mixed, and then mixed with the macroporous resin carrier, shaken overnight, filtered, washed, and refrigerated for use. The mass ratio of pyridoxal phosphate to immobilized enzyme is 1:200-500.

[0023]

[0024] Further, the macroporous resin carrier selected by the present invention is selected from any one or more combinations of LX-1000HA, LX-1000EP, LX-1000NH, SEPABEADSEC-HFA, SEPABEADSEC-HA, SEPABEADSEC-HFA / S, SEPABEADSEXE119, SEPABEADSEXE120, and DIAIONHP2MG. Among them, DIAIONHP2MG resin is preferably used as a carrier, which is a macroporous adsorption resin prepared by cross-linking methacrylic acid monomers, can effectively adsorb and separate polar substances, and has a high reuse rate. In particular, the resin can prevent enzyme protein from falling off the column to the greatest extent in a relatively low polarity reaction system, affecting the catalytic activity of the enzyme. Therefore, it is particularly suitable for industrial continuous production.

[0025] As a further optimization of the present invention, the immobilized enzyme of the present invention can be filled into an immobilized bed for repeated use in industrial production. After the immobilized R-aminotransferase is installed, the feed liquid (including substrate and organic phase) is prepared according to the above method and poured into the column, and the flow rate is adjusted by a peristaltic pump for cyclic catalysis. After the batch reaction is completed, the feed liquid is drained and the newly configured feed liquid is added for the next batch of production. Using the immobilized enzyme and the cyclic reaction system of the present invention, the immobilized enzyme can be produced repeatedly and can achieve a production effect of 15 times with a conversion rate of not less than 90%.

[0026] The (5S,6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-l-piperidin-2-one (Compound II) prepared by the above method has a conversion rate of not less than 99% and a dr value greater than 100:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison chart of the effects of different organic solvents on the activity of the immobilized enzyme prepared from SEQ ID NO: 1 in Example 4;

[0028] Figure 2 This is a schematic diagram of loading immobilized R-aminotransferase into a fixed bed reaction device in Example 6;

[0029] Figure 3 It is the number of times the immobilized enzyme is repeatedly operated and used in the fixed bed reaction device of Example 6. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0031] Example 1 Preparation of Crude Enzyme Solution of Mutant R-Transaminase

[0032] According to the soil mold ( Aspergillus terreus ) The gene sequence of the wild-type aminotransferase was obtained, and Anhui General Biotechnology Co., Ltd. was commissioned to perform full gene synthesis of the protein coding sequence and cloned into the pet28a(+) vector to obtain a vector that can express SEQ ID NO: 9. After sequencing verification, it was introduced into Escherichia coli BL21(DE3) to construct an expression strain.

[0033] Homology modeling of the sequence of SEQ ID NO: 9 was performed using SWISS-MODEL, and its three-dimensional structural features and the interactions between amino acid residues were analyzed using PyMOL software. Compound I was molecularly docked with the structure of SEQID NO: 9 using Discovery Studio software, and Y at position 60, F at position 115, E at position 117, and A at position 276 were screened as important amino acid residues constituting the catalytic pocket for binding the substrate to the enzyme active site. This pocket structure affects the attack of active site K at position 180 on compound I. The closer the distance between compound I and K at position 180, the easier it is to produce R configuration, thereby affecting the catalytic activity and configuration of compound II.

[0034] Site-directed mutagenesis was performed on SEQ ID NO: 9 to obtain the following four groups of mutants:

[0035] Y60F, F115T, the amino acid sequence of which is shown in SEQ ID NO: 4;

[0036] Y60F, F115T, E117P, whose amino acid sequence is shown in SEQ ID NO: 3;

[0037] Y60F, F115T, E117P, R240A, the amino acid sequence of which is shown in SEQ ID NO: 2;

[0038] Y60F, F115T, E117P, R240A, A276G, whose amino acid sequence is shown in SEQ ID NO: 1.

[0039] The above sequences were commissioned to Anhui General Biotechnology Co., Ltd. to perform full gene synthesis of the protein sequences encoded by each, and connected with pET-28a(+). After sequencing verification, they were introduced into Escherichia coli BL21 (DE3) to obtain the corresponding expression strains.

[0040] The above strains were inoculated into TB liquid medium containing 50 mg / L kanamycin, cultured at 37°C and 180 rpm until OD600 was about 1, and IPTG with a final concentration of 0.3 mM was added for induction, and induced at 25°C for 16 hours. Then, the cells were collected by centrifugation at 10,000 rpm for 10 minutes, and 20 g of the cells were resuspended in 80 g of phosphate buffer (0.1 mol / L, pH 7.0). After ultrasonic disruption, 0.2% (m / V) polyethyleneimine was added and stirred for flocculation for 20 minutes, and then centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken to obtain the crude enzyme solution of R-transaminase. The wild-type strain was cultured using the same steps as above to obtain the crude enzyme solution of wild-type transaminase.

[0041] Example 2 Preparation of immobilized R-aminotransferase

[0042] PLP was added to each crude enzyme solution of each mutant R-aminotransferase obtained in Example 1 to prepare a mixed solution containing 1 g / L PLP. 1 g of DIAIONHP2MG macroporous adsorption resin was mixed with 5 mL of the mixed solution, shaken overnight at 25°C and 180 rpm, filtered, and washed three times with 0.02 mol / L, pH 7.0 phosphate buffer to obtain immobilized R-aminotransferase, which was refrigerated for later use.

[0043] In the same manner as above, the crude enzyme solution of wild-type transaminase was used to prepare the immobilized enzyme, which was refrigerated and stored for later use.

[0044] Example 3 Production of Compound II in a Pure Organic Phase Reaction System Using Immobilized R-Transaminase

[0045] The immobilized R-aminotransferase and immobilized enzyme obtained in Example 2 were selected and fully dried in a vacuum oven at 40° C. for later use.

[0046] A 50 mL reaction system was prepared as follows: 1 g of compound I was added, 50 mL of water-saturated isopropyl acetate was added, stirred at 55°C to fully dissolve compound I, 0.4 g of isopropylamine was added, and 1 g of the above-mentioned immobilized R-aminotransferase and immobilized enzyme were added respectively. The reaction was carried out at 55°C for 12 h, and the conversion rate and product chirality were detected by HPLC. The results are shown in Table 1.

[0047]

[0048] It can be seen from the above that the immobilized R-aminotransferase prepared with SEQ ID NO: 1 and SEQ ID NO: 2 can already achieve a dr value greater than 100:1, but the immobilized R-aminotransferase prepared with SEQ ID NO: 1 has a higher conversion rate and is more suitable for industrial production.

[0049] Example 4

[0050] This example studies the effect of different organic solvents on the activity of the immobilized R-aminotransferase prepared by SEQ ID NO: 1. Prepare a 50 mL reaction system as follows: add 1 g of compound I, add 50 mL of ethyl acetate, toluene, ethanol, acetonitrile, methyl tert-butyl ether, and isopropyl acetate organic solvents, stir at 55°C to fully dissolve compound I, add 0.4 g of isopropylamine, and add the immobilized R-aminotransferase prepared by SEQ ID NO: 1 obtained in Example 2. React at 55°C for 12 h, and detect the conversion rate by high performance liquid chromatography HPLC. The highest conversion rate is defined as 100%, and the relative activity of different solvents is measured. The results Figure 1 shown.

[0051] Depend on Figure 1 It can be seen that the catalytic activity of immobilized enzyme in ethanol and acetonitrile solvents is poor. Since ethanol and acetonitrile have high polarity, the protein structure is easily destroyed, and the DIAIONHP2MG resin reacts with the enzyme by adsorption. The enzyme protein is easy to fall off in highly polar solutions, which in turn affects the catalytic activity; the catalytic activity is higher in methyl tert-butyl ether and isopropyl acetate, and these two solvents are more appropriate; while the polarity in toluene solution is smaller, which also affects the enzyme protein structure and thus the catalytic activity.

[0052] Example 5

[0053] This example provides a specific process for producing compound II in a 1L reaction system. Add 100g of compound I, add 1L of water-saturated isopropyl acetate, stir at 55°C to fully dissolve compound I, add 40g of isopropylamine, and then add 50g of immobilized enzyme prepared by SEQ ID NO: 1. React at 55°C for 40 h, and the conversion rate detected by HPLC is 99.2%, and the dr value of compound II is >100:1.

[0054] Example 6

[0055] In a fixed bed reactor, 15 g of immobilized R-aminotransferase prepared from SEQ ID NO: 1 was loaded into Figure 2 In the device column shown, 5g of compound I was mixed with 50mL of saturated aqueous isopropyl acetate. After the substrate was dissolved, 2g of isopropylamine was added to prepare a feed solution containing compound I. The feed solution was poured into the column, and two peristaltic pumps were used to adjust the appropriate flow rate to circulate the circulating water and the feed solution respectively. The circulating water temperature was controlled at 55°C for insulation. The reaction was carried out for 40 hours. The conversion rate was 99.5% as detected by HPLC, and the dr value of compound II was >100:1. After the reaction was completed, the immobilized enzyme was still retained in the column, the feed solution was discharged, and the newly prepared feed solution was added to repeat the reaction. The results are shown in Figure 3 As shown, the conversion rate of the immobilized enzyme was still greater than 90% after 15 repeated reactions.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant R-aminotransferase, wherein the amino acid sequence of the mutant R-aminotransferase is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A gene fragment encoding a mutant R-aminotransferase according to claim 1, wherein the nucleotide sequence of the gene fragment is shown in SEQ ID NO: 5 or SEQ ID NO:

6.

3. A recombinant vector, characterized in that: Contains the gene fragment according to claim 2.

4. A host cell, characterized in that: Comprising the recombinant vector according to claim 3.

5. A method for preparing (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one, characterized in that The steps include: (1) inoculating and culturing the host cell described in claim 4, crushing and separating the supernatant to obtain a crude R-aminotransferase enzyme solution; (2) mixing the crude R-aminotransferase solution with a macroporous resin carrier to prepare immobilized R-aminotransferase; (3) In an organic phase reaction system free of DMSO, 4-phenyl-2-(tert-butoxycarbonyl)amino-5-oxohexanoic acid isopropyl ester is used as a substrate, the substrate is fully dissolved, an amino donor is added, and then the immobilized R-aminotransferase is added to obtain (5S,6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-l-piperidin-2-one.

6. The method for preparing (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one according to claim 5, characterized in that: The initial concentration of the substrate in the reaction system is 80-150 g / L.

7. The method for preparing (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one according to claim 5, characterized in that: The mass ratio of the immobilized R-aminotransferase to the substrate is 0.5-3:

1.

8. The method for preparing (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one according to claim 5, characterized in that: The organic phase is selected from any one or more combinations of saturated or unsaturated ethyl acetate, toluene, ethanol, acetonitrile, methyl tert-butyl ether or isopropyl acetate.

9. The method for preparing (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one according to claim 5, characterized in that: The amino donor is selected from any one or more combinations of isopropylamine, 1,4-butanediamine, L-glutamic acid or L-alanine.

10. The method for preparing (5S, 6R)-3-(tert-butoxycarbonyl)amino-6-methyl-5-phenyl-1-piperidin-2-one according to claim 9, characterized in that: The molar ratio of the amino group in the amino donor to the substrate is 2-5:1.

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