Biosynthesis method of brivaracetam and diastereoisomer thereof

Abstract

The invention discloses an imine reductase which is derived from Jianggellauris, Streptomyces aureococcus, Streptomyces alboflavus, Sandaracinus amylolyticus, Phyllobacterium SYSU D60010, Mesorhizobium, Labitorixococcus or a metagenome, the imine reductase is used as a biocatalyst for preparing the anti-epileptic drug brivaracetam and a diastereoisomer of the brivaracetam, the problems that an existing industrial synthesis route is poor in environmental protection property, low in atom utilization rate, long in route and the like are solved, and the imine reductase has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and relates to a method for the dynamic kinetic resolution reductive amination synthesis of brivaracetam and its diastereomers by using imine reductase to catalyze ethyl 3-formylhexanoate and (S)-2-aminobutyramide. Background Art

[0002] Epilepsy is one of the most common neurological diseases, with a total global epilepsy patient population of over 50 million. Brivaracetam is the third-generation anti-epileptic drug launched by Belgian company UCB after levetiracetam, and was approved for marketing by the European Medicines Agency and the US Food and Drug Administration in 2016. Brivaracetam belongs to chiral pyrrolidone derivatives. Compared with levetiracetam, it has an (R)-configured n-propyl group attached to the 4-position of the pyrrolidine hydrocarbon. The molecular formula of brivaracetam is C 11 H 20 N2O2, and its structural formula is as follows:

[0003]

[0004] Currently, the chiral center at the (2S) position in the brivaracetam molecule is mainly introduced through chiral raw materials, such as (R)-2-bromobutyric acid and (S)-2-aminobutyramide. Relatively speaking, the chiral center at the C4 position in the pyrrolidine hydrocarbon has become the key and difficult point in the synthesis. The methods that can achieve kilogram-scale synthesis include chiral column chromatography separation (WO2017076738A1) and lipase-catalyzed resolution (A. Schule, A. Merschaert, C. Szczepaniak, C. Marechal, N. Carly, J. O'Rourke, C. Ates, Org. Process Res. Dev. 2016, 20, 1566 - 1575.), but they have problems of poor environmental friendliness and complex routes with low yields (9%) respectively. The biocatalytic asymmetric amination method of α-substituted carbonyl compounds is one of the most important and direct methods for synthesizing β-chiral amines. Kroutil et al. used transaminase to catalyze the dynamic kinetic resolution amination of α-substituted aldehyde esters to synthesize the brivaracetam intermediate (C.S. Fuchs, J.E. Farnberger, G. Steinkellner, J.H. Sattler, M. Pickl, R.C. Simon, F. Zepeck, K. Gruber, W. Kroutil, Adv. Synth. Catal. 2018, 360, 768 - 778.), but it needs to further react with (R)-2-bromobutyric acid to obtain the final product, which will cause partial racemization at the C4 position.

[0005] Therefore, it is very necessary to develop a green, efficient, and highly stereoselective method for synthesizing brivaracetam. Summary of the Invention

[0006] The present invention provides a method for the catalytic synthesis of brivaracetam and its diastereoisomers by imine reductase, that is, by using the catalytic action of imine reductase, ethyl 3-formylhexanoate (Formula I) and (S)-2-aminobutanamide (Formula II) are subjected to asymmetric reductive amination to obtain ethyl (R)-3-(((S)-1-amino-1-oxobutan-2-yl)amino)methyl)hexanoate (Formula III) or ethyl (S)-3-(((S)-1-amino-1-oxobutan-2-yl)amino)methyl)hexanoate (Formula IV). Subsequently, cyclization can obtain (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide (i.e., brivaracetam, Formula V) or (S)-2-((S)-2-oxo-4-propylpyrrolidin-1-yl)butanamide (Formula VI). Some of Compounds III and IV can spontaneously cyclize, or 2-hydroxypyridine can be used to promote cyclization.

[0007]

[0008] The imine reductases described above include imine reductase IR61 (WP_053204479.1) derived from Jiangella muralis, or imine reductase IR104 (WP_078965966.1) derived from Streptomyces aureocirculatus, or imine reductase IR183 (>WP_087885906.1) derived from Streptomyces alboflavus, or imine reductase IR40 (WP_075097693.1) derived from Sandaracinus amylolyticus, or imine reductase IR55 (WP_119269405.1) derived from Phyllobacteriaceae bacterium SYSU D60010, or imine reductase IR58 (WP_036254014.1) derived from Mesorhizobium, or imine reductase IR70 (AKU97888.1) derived from Labilithrix luteola, or imine reductase IR100 derived from metagenome. The amino acid sequences of the imine reductases are shown in SEQ ID NO: 1-8 respectively, or amino acid sequences having at least 80% identity therewith, and the imine reductases have the activity of reductive amination.

[0009] The vector series used in the genetically engineered bacteria producing imine reductase in the present invention include: pET series plasmids, pTXB1 series, pGEX series, pETduet series, pTYB series, and preferably pET series plasmids.

[0010] The genetically engineered bacterium producing imine reductase in the present invention is characterized in that the host bacterium capable of highly expressing exogenous genes is one of the following: BL21 series, Rosetta series, Origami series, Tuner series.

[0011] The imine reductase for preparing brivaracetam can be the culture of the above-mentioned imine reductase genetically engineered bacterium, or the bacterial cells obtained by centrifuging the culture medium or their processed products. The processed products refer to the extracts, lysates obtained from the bacterial cells, or the separation products obtained by separating and / or purifying the benzaldehyde lyase mutant and carbonyl reductase of the extracts. In the present invention, the most used is the bacterial cells obtained by centrifuging the fermentation broth.

[0012] The present invention relates to a method for catalytically converting ethyl 3-formylhexanoate and (S)-2-aminobutyramide into brivaracetam and its diastereomers by whole cells. The configuration of the product is analyzed by liquid phase to ensure the synthesis of brivaracetam (αS,4R-configuration) and the diastereoisomer, i.e., (αS,4S-configuration) product.

[0013] Preferably, the method uses the wet bacterial cells obtained by fermentation culture of the engineering bacterium expressing the coding gene of the imine reductase as the catalyst; preferably, the catalyst is whole cells, and its dosage is 5-100 g / L, preferably 50 g / L.

[0014] In the specific implementation manner, a buffer solution with a pH of 5.0-10.0 is used as the reaction medium, and the reaction is carried out at 20°C-40°C. Preferably, the buffer solution used in the reaction is an aqueous solution with a pH of 7.0-8.5, and the temperature is 25-40°C. More preferably, a sodium phosphate buffer solution with a pH of 7.5 is used as the reaction medium, and the reaction is carried out at 25°C.

[0015] Optionally, the reaction system further includes a coenzyme regeneration system, which contains the coenzyme of glucose dehydrogenase or formate dehydrogenase to promote the stability of the coenzyme regeneration system reaction.

[0016] In the specific implementation manner, the concentration of the substrate ethyl 3-formylhexanoate is 1 mM-20 mM, preferably 5-15 mM. Considering the substrate conversion rate and product yield, the optimal choice is 10 mM. The substrate concentration of (S)-2-aminobutyramide is 2 times the molar equivalent of the former.

[0017] Furthermore, it also includes the step of purifying brivaracetam. Specifically, after the reaction is completed, the pH is adjusted with saturated sodium carbonate, and after extraction with ethyl acetate, the product is purified by silica gel column chromatography. In a specific example, the method is as follows: The genetically engineered bacteria of the imine reductase are cultured in a seed medium, inoculated into a fermentation medium at a certain ratio, cultured for a certain period of time, then an inducer IPTG is added and cultured for a period of time, and the cells are collected by centrifugation. 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + and 3 U / mL GDH are added to the wet cells of benzaldehyde lyase, and the reaction is carried out at 25 °C for 24 h with 100 mM sodium phosphate as the buffer solution. After the reaction is completed, the pH value is adjusted to above 10 with saturated sodium carbonate solution, the reaction solution is extracted with ethyl acetate, dried and then evaporated to dryness to collect the product. The enzyme product is cyclized with 2-hydroxypyridine, and the final product is purified by silica gel column chromatography.

[0018] The innovation or advantage of the present invention lies in that for the first time, the asymmetric reductive amination reaction of imine reductase catalyzing amino amide amino donors and aldehydes is realized. The successful implementation of this reaction provides a new enzymatic synthesis route for the antiepileptic drug brivaracetam and its diastereoisomers, which has the advantages of mild reaction conditions, short reaction steps, and high stereoselectivity compared with other routes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 1H NMR spectrum of the product (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyramide.

[0020] Figure 2 13C NMR spectrum of the product (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyramide. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The following is further illustrated by specific examples, the purpose of which is to better understand the content of the invention, but these examples do not constitute a limitation to the present invention.

[0022] Example 1: Construction and culture of high-expression genetically engineered bacteria

[0023] The whole gene synthesis was completed by Anhui General Biotech Co., Ltd. According to the imine reductase sequences from Jiangella muralis, Streptomyces aureocirculatus, Streptomyces alboflavus, Sandaracinus amylolyticus, Phyllobacteriaceae bacterium SYSU D60010, Mesorhizobium, Labilithrix luteola and Megagenome, codon optimization was carried out to enable the gene to be expressed in Escherichia coli expression hosts. Nde I and EcoRI restriction sites were added to both ends of the gene and constructed into the pET-28a(+) vector. The recombinant vector was transformed into Escherichia coli BL21, Rosetta or Origami by conventional methods to construct recombinant engineering bacteria, among which the recombinant bacteria with Escherichia coli BL21 as the host bacterium had relatively good expression of the target protein. The engineering bacteria with the target protein expression level not less than 20% were used as the engineering bacteria strains for production and stored in the form of glycerol bacteria at -80°C.

[0024] Thaw the bacteria strains stored at -80°C, streak them on the plate and incubate overnight in a 37°C constant temperature incubator. Pick a single colony on the plate and inoculate it into 20 mL of LB medium containing kanamycin antibiotic, and culture it for about 12 h as the seed solution. Inoculate it into 700 mL of LB medium containing the corresponding antibiotic at an inoculation amount of 1%, and culture it on a shaker at 37°C and 200 rpm until the OD 600 reaches about 0.6 - 0.8. Add IPTG with a final concentration of 0.1 mmol / L and induce it at 25°C for 12 h, and collect the bacteria by centrifugation at 6000 rpm.

[0025] Among them, the imine reductase sequences are as follows:

[0026] Imine reductase IR61 [Jiangella muralis] (SEQ ID NO: 1):

[0027]

[0028] Imine reductase IR104 [Streptomyces aureocirculatus] (SEQ ID NO: 2):

[0029]

[0030]

[0031] Imine reductase IR183 [Streptomyces alboflavus] (SEQ ID NO: 3):

[0032]

[0033] Imine reductase IR40 [Sandaracinus amylolyticus] (SEQ ID NO: 4):

[0034]

[0035] Imine reductase IR55 [Phyllobacteriaceae bacterium SYSU D60010] (SEQ ID NO: 5):

[0036]

[0037] Imine reductase IR58 [Mesorhizobium] (SEQ ID NO: 6):

[0038]

[0039] Imine reductase IR70 [Labilithrix luteola] (SEQ ID NO: 7):

[0040]

[0041]

[0042] Imine reductase IR100 [Metagenome] (SEQ ID NO: 8):

[0043]

[0044] Example 2: Purification of imine reductase

[0045] Since the carbonyl reductase or alcohol dehydrogenase in the background of Escherichia coli will reduce the substrate ethyl 3-formylhexanoate to the by-product ethyl 3-(hydroxymethyl)hexanoate, in order to prevent the generation of by-products, we purified the imine reductase and then established the reaction.

[0046] The imine reductase involved in the present invention was constructed onto the pET-28a vector using the Nde I and EcoRI restriction sites, and the native histidine tag could be fusion-expressed. Therefore, nickel column affinity chromatography was used for purification. The AKTA protein purification system and chromatography column (HisTrap TMHP-5 mL). The purification steps are as follows: (1) Preparation of equilibration buffer A: 20 mM sodium phosphate buffer containing 500 mM sodium chloride, 5% glycerol and 20 mM imidazole, pH 7.5; Preparation of elution buffer B: 20 mM sodium phosphate buffer containing 500 mM sodium chloride, 5% glycerol and 500 mM imidazole, pH 7.5. (2) The chromatography column was first equilibrated with buffer A. The bacterial cells were resuspended in 20 mL of sodium phosphate buffer and then sonicated and disrupted. Cell debris was removed by centrifugation (10,000 g, 20 min, 4 °C). The supernatant was filtered through a 0.45 μm filter membrane and then loaded onto the column. The column was then flushed with approximately 5 column volumes of buffer A to remove unbound miscellaneous proteins. Subsequently, the proportion of elution buffer B was linearly increased from 0% to 100% within 30 min, corresponding to an increase in the imidazole concentration from 20 mM to 500 mM. The elution peaks were collected in fractions, and the pure protein was collected after SDS-PAGE analysis. A desalting column was used to remove high concentrations of NaCl and imidazole. The pure enzyme was ultrafiltered and concentrated and then stored at -80 °C for later use.

[0047] Example 3 Synthesis of brivaracetam using purified IR104

[0048] A 50 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + , 3 U / mL GDH, 5 mg / mL pure enzyme and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. After the reaction was completed, the pH value was adjusted to above 10 with saturated sodium carbonate solution, and the product was collected by rotary evaporation after drying. The enzymatic product was cyclized in toluene, 2-hydroxypyridine was added, and the reaction was carried out at 80 °C. The final product was purified by silica gel column chromatography. The results were as follows: IR104 catalyzed the synthesis of brivaracetam, and the d.r. value (αS,4R:αS,4S) = 95:5, the conversion rate was 80%, and the total separation yield was 70%. The NMR results of the product are shown in Figure 1 and Figure 2 .

[0049] Example 4 Synthesis of brivaracetam using purified IR61

[0050] A 20 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + , 3 U / mL GDH, 5 mg / mL pure enzyme and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. The results were as follows: IR61 catalyzed the synthesis of brivaracetam, and the d.r. value (αS,4R:αS,4S) = 96:4, and the conversion rate was 45%.

[0051] Example 4 Synthesis of brivaracetam catalyzed by purified IR183

[0052] A 20 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + , 3 U / mL GDH, 5 mg / mL pure enzyme and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. The results were as follows: Brivaracetam was synthesized by IR183 catalysis, and the d.r. value (αS,4R:αS,4S) = 82:18, and the conversion rate was 84%.

[0053] Example 6 Synthesis of diastereomers of brivaracetam catalyzed by purified IR40

[0054] A 20 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + , 3 U / mL GDH, 1 mg / mL pure enzyme and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. The results were as follows: Diastereomers of brivaracetam were synthesized by IR40 catalysis, and the d.r. value (αS,4R:αS,4S) = 5:95, and the conversion rate was 79%.

[0055] Example 7 Synthesis of diastereomers of brivaracetam catalyzed by purified IR55

[0056] A 20 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + , 3 U / mL GDH, 1 mg / mL pure enzyme and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. The results were as follows: Diastereomers of brivaracetam were synthesized by IR55 catalysis, and the d.r. value (αS,4R:αS,4S) = 20:80, and the conversion rate was 60%.

[0057] Example 8 Synthesis of diastereomers of brivaracetam catalyzed by purified IR58

[0058] A 20 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP +, 3 U / mL GDH, 5 mg / mL pure enzyme, and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. The results were as follows: IR58 catalyzed the synthesis of the diastereoisomers of brivaracetam, with a d.r. value (αS,4R:αS,4S) = 6:94 and a conversion rate of 80%.

[0059] Example 9 The diastereoisomers of brivaracetam were synthesized using purified IR70

[0060] A 20 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + , 3 U / mL GDH, 5 mg / mL pure enzyme, and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. The results were as follows: IR70 catalyzed the synthesis of the diastereoisomers of brivaracetam, with a d.r. value (αS,4R:αS,4S) = 7:93 and a conversion rate of 90%.

[0061] Example 10 The diastereoisomers of brivaracetam were synthesized using purified IR100

[0062] A 20 mL conversion reaction was established, which contained 10 mM ethyl 3-formylhexanoate, 20 mM (S)-2-aminobutyramide, 20 mM glucose, 0.5 g / L NADP + , 3 U / mL GDH, 5 mg / mL pure enzyme, and sodium phosphate buffer (pH 7.5, 100 mM). The substrate conversion rate was detected by gas chromatography. The results were as follows: IR100 catalyzed the synthesis of the diastereoisomers of brivaracetam, with a d.r. value (αS,4R:αS,4S) = 18:82 and a conversion rate of 76%.

Claims

1. A method for the catalytic synthesis of bucindolol and / or diastereoisomers by imine reductase, characterized in that, Using the catalytic reaction of imine reductase, ethyl 3-formylhexanoate and (S)-2-aminobutyramide are used as substrates, and ethyl (R)-3-(((S)-1-amino-1-oxobutan-2-yl)amino)methyl)hexanoate or ethyl (S)-3-(((S)-1-amino-1-oxobutan-2-yl)amino)methyl)hexanoate is obtained by asymmetric reductive amination. Subsequently, cyclization can obtain (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyramide, i.e., bucillamine, or its diastereoisomer (S)-2-((S)-2-oxo-4-propylpyrrolidin-1-yl)butyramide; Among them, the imine reductase includes imine reductase IR61 (WP_053204479.1) derived from Jiangellamuralis or imine reductase IR104 (WP_078965966.1) derived from Streptomyces aureocirculatus or imine reductase IR183 (>WP_087885906.1) derived from Streptomyces alboflavus or imine reductase IR40 (WP_075097693.1) derived from Sandaracinus amylolyticus or imine reductase IR55 (WP_119269405.1) derived from the same as Phyllobacteriaceae bacterium SYSU D60010 or imine reductase IR58 (WP_036254014.1) derived from Mesorhizobium or imine reductase IR70 (AKU97888.1) derived from Labilithrix luteola or imine reductase IR100 from metagenome; Preferably, the amino acid sequence of the imine reductase is as shown in SEQ ID NO: 1-8, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identity thereto, and is derived from a related substance, and the imine reductase has the activity of reductive amination.

2. The method according to claim 1, characterized in that The imine reductase is expressed by a genetically engineered bacterium containing the gene encoding the imine reductase to obtain a catalyst, specifically, the culture of the genetically engineered bacterium of the imine reductase, or its whole cells, or the bacterial cells obtained after centrifuging the culture or its processed products are used as the catalyst; Among them, the whole cells use the wet bacterial cells obtained by fermenting and culturing the genetically engineered bacterium expressing the coding gene of the imine reductase as the catalyst; preferably, the catalyst is whole cells, and its dosage is 5-100 g / L, preferably 50 g / L; The processed products refer to the extracts, lysates obtained from the bacterial cells, or those obtained by subjecting the extracts to benzaldehyde lyase mutants and carbonyl reductase.

3. The method according to claim 2, wherein The starting bacterium of the genetically engineered bacterium is Escherichia coli, preferably Escherichia coli of the BL21 series, Rosetta series, Origami series, Tuner series.

4. The method according to claim 2, wherein The vector used for expressing the imine reductase is selected from pET series plasmids, pTXB1 series, pGEX series, pETduet series, pTYB series, preferably pET series plasmids.

5. The method according to any one of claims 1 to 4, characterized in that, The catalytic reaction uses a buffer solution with a pH of 5.0-10.0 as the reaction medium and is carried out at 20°C-40°C; preferably, the buffer solution used in the catalytic reaction is an aqueous solution with a pH of 7.0-8.5, and the temperature is 25-40°C; more preferably, a sodium phosphate buffer solution with a pH of 7.5 is used as the reaction medium and the reaction is carried out at 25°C.

6. The method according to claim 5, characterized in that, The reaction system further includes a coenzyme regeneration system, which also contains the coenzyme of glucose dehydrogenase or formate dehydrogenase.

7. The method according to any one of claims 1 to 4, characterized in that The concentration of the substrate ethyl 3-formylhexanoate is 1 mM - 20 mM, preferably 5 - 15 mM,( S ) and the concentration of the substrate ( )-2-aminobutanamide is 2 molar equivalents of the former.

8. The method according to claim 7, wherein Add 10 mM ethyl 3-formylhexanoate, 20 mM ( S )-2-aminobutanamide, 20 mM glucose, 0.5 g / L NADP + and 3 U / mL GDH to the wet cells of benzaldehyde lyase, and react at 25 °C for 24 h with 100 mM sodium phosphate as the buffer solution.

9. The method according to any one of claims 1 to 4, characterized in that It also includes the step of purifying brivaracetam, specifically, after the reaction is completed, the pH is adjusted with saturated sodium carbonate, and after extraction with ethyl acetate, the product is purified by silica gel column chromatography.

10. The method according to claim 9, characterized in that, The step of purifying brivaracetam is specifically as follows: after the reaction is completed, the pH value is adjusted to above 10 with saturated sodium carbonate solution, the reaction solution is extracted with ethyl acetate, dried and then rotary evaporated to collect the product, the enzyme product is cyclized with 2-hydroxypyridine, and the final product is purified by silica gel column chromatography.

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