Method for synthesizing chiral 1, 3-disubstituted tetrahydroisoquinoline through chemoenzyme cascade system

Abstract

The invention discloses a method for synthesizing chiral 1, 3-disubstituted tetrahydroisoquinoline through a chemoenzyme cascade system, and belongs to the technical field of biological engineering. The invention establishes a method for preparing chiral 1, 3-disubstituted tetrahydroisoquinoline through enzyme cascade catalysis, simple achiral ketone is taken as a raw material, chiral amine is generated through screened transaminase, then chiral imine is obtained through B-N cyclization reaction, and finally the 1, 3-disubstituted tetrahydroisoquinoline is generated through imine reductase. The method is high in yield which can be up to 100%, has excellent diastereoselectivity and enantioselectivity, has a de value and an ee value which are close to 99%, and has good application value and prospect.

Description

Technical Field

[0001] The invention relates to a method for synthesizing chiral 1,3-disubstituted tetrahydroisoquinoline by a chemical enzyme cascade system, belonging to the technical field of bioengineering. Background Art

[0002] Tetrahydroisoquinoline (THIQ) alkaloids are one of the largest natural products and play an indispensable role in drug development. THIQ natural products have been intensively studied in recent decades and have a wide range of structural diversity and biological activities.

[0003] Among these different THIQs, 1,3-disubstituted THIQs with at least two chiral carbon centers are a special class of THIQ alkaloids that are different from tetrahydroisoquinolines, which are generally believed to be formed through the Pictet-Spengler (PS) reaction between aldehydes and aromatic amino acid-derived arylethylamines, and are further generated by polyketides formed by the acetate-malonate pathway, which involves an as-yet-unidentified polyketide synthase and other enzymes. However, the unusual substitution pattern of 1,3-disubstituted THIQs, especially the presence of methyl groups at the C1 and C3 positions, poses a huge obstacle to the formation of these important compounds through the traditional PS route due to the difficulty in biocatalytically pre-installing α-methyl groups on aromatic amino acids to construct C3 methyl groups and the NCS substrate-limited acetaldehyde to construct C1 methyl groups.

[0004] Although their biosynthesis remains challenging, many pioneering chemical synthetic methods have been developed to construct these valuable chiral structures, including transition metal-catalyzed asymmetric reduction of 1,3-disubstituted isoquinolines, asymmetric nucleophilic addition of chiral 3,4-dihydroisoquinolines, alkylation of chiral tetrahydroisoquinolines, PS reactions between chiral phenylethylamine derivatives and aldehydes, Bischler-Napieralski (BN) / reduction cascade reactions of chiral acylated phenylethylamines, and palladium-catalyzed three-component Catellani / gold-catalyzed cyclization / reduction cascade reactions. Although these strategies are effective, they often rely on complex substrates with pre-constructed chiral centers, heavy metal catalysts, flammable and hazardous hydrogen gas, and harsh conditions of high temperature and pressure. Therefore, it is highly desirable to develop a novel, efficient, and green method to construct chiral 1,3-disubstituted THIQs from simple achiral substrates. Summary of the invention

[0005] In order to solve the above problems, the present invention designs a method for modular synthesis of 1,3-disubstituted THIQ by diastereoselectivity and enantioselectivity ( Figure 1 ).

[0006] The present invention provides a recombinant Escherichia coli expressing a transaminase, using Escherichia coli BL21 (DE3) as a host to express any transaminase shown in a1 to a9:

[0007] a1, transaminase ArR-TA from Arthrobacter sp., whose amino acid sequence is shown in SEQ ID NO.1;

[0008] a2, transaminase CV-TA from Chromobacterium violaceum, the amino acid sequence of which is shown in SEQ ID NO.2;

[0009] a3, transaminase ArRmut-11 from Arthrobacter sp., whose amino acid sequence is shown in SEQ ID NO.3;

[0010] a4, transaminase AT-TA from Aspergillus terreus, the amino acid sequence of which is shown in SEQ ID NO.4;

[0011] a5, transaminase OA-TA from Ochrobactrum anthropic, the amino acid sequence of which is shown in SEQ ID NO.5;

[0012] a6, transaminase RS-TA from Ruegeria sp. TM1040, the amino acid sequence of which is shown in SEQ ID NO.6;

[0013] a7, transaminase EX-TA from Exophiala xenobiotica, whose amino acid sequence is shown in SEQ ID NO.7;

[0014] a8, transaminase PP-TA from Paraburkholderia phymatum, the amino acid sequence of which is shown in SEQ ID NO.8;

[0015] a9. Transaminase VF-TA from Vibrio fluvialis, whose amino acid sequence is shown in SEQ ID NO.9.

[0016] In one embodiment, pET series plasmids are used as expression vectors; the pET series plasmids include but are not limited to pET-21b(+).

[0017] In one embodiment, the recombinant Escherichia coli uses pET-21b(+) as an expression vector to express a gene encoding the transaminase shown in SEQ ID NO.1 or SEQ ID NO.2.

[0018] The present invention also provides a transaminase protein prepared by the recombinant Escherichia coli.

[0019] In one embodiment, the method for preparing the transaminase protein comprises: inoculating the recombinant Escherichia coli expressing the transaminase into a culture medium, culturing the strain to a logarithmic growth phase, inducing, collecting the bacteria, resuspending, breaking, centrifuging to collect the lysate, and purifying the protein.

[0020] In one embodiment, the steps are: inoculating the recombinant E. coli into LB medium, culturing at 20-40°C for 10-12h, transferring to 2YT medium, culturing at 20-40°C until OD 600 When the value reaches about 0.6-0.8, cool down to 15-25℃, add IPTG with a final concentration of 0.05-0.5mM to induce for 10-20h, and collect the bacteria.

[0021] The present invention also provides application of the transaminase in synthesizing 1,3-disubstituted THIQ.

[0022] In one embodiment, the transaminase is selected from any one of a1 to a13.

[0023] In one embodiment, the application is to mix the transaminase protein with a substrate in a reaction system involving a lactate dehydrogenase (LDH) circulation system.

[0024] In one embodiment, the application comprises the following steps:

[0025] (1) mixing the transaminase protein with a substrate in the presence of a lactate dehydrogenase (LDH) circulation system to produce an amine 2; the substrate is a ketone 1, D-alanine or L-alanine, pyridoxal phosphate (PLP); the LDH circulation system includes LDH, glucose dehydrogenase (GDH), NADH, and glucose;

[0026] (2) reacting the amine 2 obtained in step (1) with an acyl chloride, collecting the reaction product, and then reacting the product with phosphorus oxychloride to obtain an imine 3;

[0027] (3) using the imine 3 obtained in step (2) as a substrate, and catalyzing the reaction with an imine reductase protein and a GDH circulation system to obtain 1,3-disubstituted THIQ; the GDH circulation system includes GDH, NADP + ,glucose.

[0028] In one embodiment, the imine reductase protein is the imine reductase SnIR from Stackebrandtia nassauensis, and its amino acid sequence is shown in SEQ ID NO.10.

[0029] In one embodiment, the amount of imine 3 added in step (3) is 1 mM, the amount of imine reductase mutant added is 1.6-3 mol%, the amount of GDH added is 1 mol%, and the amount of NADP + The added amount of is 10 mol %, and the added amount of glucose is 100 mM.

[0030] In one embodiment, the reaction system of step (2) contains 0.5M amine 2 dissolved in dichloromethane, 1.5 equivalents of triethylamine, at least 1 equivalent of acyl chloride, 0.5M amide dissolved in acetonitrile, and 5 equivalents of phosphorus oxychloride. The acetonitrile is preferably dry acetonitrile. The phosphorus oxychloride is preferably freshly distilled phosphorus oxychloride.

[0031] In one embodiment, the reaction temperature in step (2) is 90°C.

[0032] In one embodiment, the substrate comprises ketone 1, D-alanine or L-alanine, pyridoxal phosphate (PLP).

[0033] In one embodiment, the LDH circulation system includes LDH, glucose dehydrogenase (GDH), NADH, and glucose.

[0034] In one embodiment, the reaction temperature is 20-40°C, and the reaction pH is 7.0-8.0.

[0035] In one embodiment, the reaction time is 12-24 hours, or the reaction time is not less than 12 hours.

[0036] In one embodiment, the added amount of the ketone 1 is 5-10 mM; the added amount of the D-alanine or L-alanine is 100 mM, the added amount of the PLP is 1 mM, the added amount of the glucose is 100 mM, and the added amount of the NADH is 1 mM.

[0037] In one embodiment, the amount of the transaminase added is 0.2-0.4 mol%, the amount of the GDH added is 0.15-0.3 mol%, and the amount of the LDH added is 0.03-0.06 mol%, wherein all molar percentages are calculated based on the substrate ketone 1.

[0038] In one embodiment, the added amount of the ketone 1 is 5-10 mM; the added amount of the D-alanine or L-alanine is 100 mM, the added amount of the PLP is 1 mM, the added amount of the glucose is 100 mM, and the added amount of the NADH is 1 mM.

[0039] In one embodiment, the amount of the transaminase added is 0.2-0.4 mol%, the amount of the GDH added is 0.15-0.3 mol%, and the amount of the LDH added is 0.03-0.06 mol%; the above molar percentages are calculated based on the corresponding substrates in the step.

[0040] In one embodiment, the ketone 1 includes at least one of the following 1a-1d, 1f:

[0041]

[0042] In one embodiment, the acyl chloride comprises at least one of the following AG:

[0043]

[0044] In one embodiment, the imine includes at least one of the following (R)-3a, (R)-3b, (R)-3c, (R or S)-3d, (R)-3e, (R)-3f, (R)-3g, (R)-3h, (R)-3i, (R)-3j, (R)-3k, (R)-3l:

[0045]

[0046] In one embodiment, the 1,3-disubstituted THIQ structure is as follows:

[0047]

[0048] The present invention also provides application of the mutant, or the recombinant cell, or the method in preparing 1,3-disubstituted THIQ.

[0049] Beneficial effects:

[0050] (1) The present invention provides a transaminase (ArR-TA) that can efficiently convert a substrate ketone into (R)-2a with a conversion rate of >90% and an ee value of >99%;

[0051] (2) The present invention provides a transaminase (CV-TA) that can efficiently convert a substrate ketone into (S)-2a with a conversion rate of >99% and an ee value of >99%;

[0052] (3) The present invention provides an imine reductase (SnIR), which can convert the substrate racemic imine into (1S, 3R)-4a with a conversion rate of 11%, a de value of 44, and an ee value of 59%;

[0053] (4) The present invention also provides a method for synthesizing 1,3-disubstituted THIQ using achiral ketone as raw material. The HPLC yields of (1S,3R)-4a to (1S,3R)-4l are 47%, 83%, 92%, 86%, 98%, >99%, 92%, 62%, 28%, 16%, 15%, and 16%, respectively. The de values ​​are 14%, 99%, >99%, >99%, >99%, >99%, >99%, 77%, 53%, >99%, >99%, 64%, and 47%, respectively. The ee values ​​are >99%, >99%, 98%, >99%, >99%, >99%, 97%, >99%, >99%, >99%, 99%, and 99%, respectively.

[0054] (5) The method of the present invention uses ketone 1a as raw material to synthesize 179.8 mg of (1S, 3R)-4a by chemical enzymatic modular cascade with a yield of 81%, >99de, >99ee. Use ketone 1d as raw material to synthesize 31.1 mg of (1S, 3R)-4d by chemical enzymatic modular cascade with a yield of 76%, 97de, >99ee. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The present invention is a synthetic route for 1,3-disubstituted THIQ.

[0056] Figure 2 This is the liquid phase analysis diagram of the transaminase module.

[0057] Figure 3 This is the liquid phase analysis diagram of the chemical module.

[0058] Figure 4 This is the liquid phase analysis diagram of the imine reductase module.

[0059] Figure 5 Chiral identification for the enzyme-catalyzed synthesis of (1S,3R)-4a.

[0060] Figure 6 NMR spectroscopy analysis of enzyme-catalyzed synthesis of (1S,3R)-4a 1 H NMR and 13 C NMR.

[0061] Figure 7 For substrate spectrum expansion using imine reductases.

[0062] Figure 8 Modular cascade synthesis of (1S,3S)-4d chiral identification for chemoenzymes. DETAILED DESCRIPTION

[0063] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0064] (I) Compounds

[0065] In some embodiments of the present invention, ketone 1 includes the following 1a-1d, 1f:

[0066]

[0067] Acid chlorides include the following AG:

[0068]

[0069] Imines include the following (R)-3a, (R)-3b, (R)-3c, (R or S)-3d, (R)-3e, (R)-3f, (R)-3g, (R)-3h, (R)-3i, (R)-3j, (R)-3k, (R)-3l:

[0070]

[0071] Compound 1,3-disubstituted THIQ has any of the following structures:

[0072]

[0073] (ii) Amino acid and nucleotide sequences

[0074] Table 1 Amino acid sequences of related proteins

[0075]

[0076]

[0077]

[0078]

[0079] Table 2 Nucleotide sequences encoding related proteins

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] (III) Reagents and Materials

[0088] The antibiotics used in the present invention, such as ampicillin sodium, kanamycin sulfate, and streptomycin sulfate, are all from Shanghai Biotechnology; NADP + , NADH comes from Shanghai BiDe Pharmaceutical Technology Co., Ltd.; the PCR enzyme involved in the molecular experiment in the present invention is purchased from Bao Biotechnology (Dalian) Co., Ltd.; the chemical reagents used in the present invention, such as methanol, dichloromethane, ethyl acetate (EtOAc), petroleum ether (PE), acetonitrile (MeCN), sodium sulfate, sodium carbonate, sodium bicarbonate, diethylamine (DEA), ammonia, sodium hydroxide, hydrochloric acid, etc., are all purchased from China Pharmaceutical Group Co., Ltd. and Shanghai Titan Technology Co., Ltd.

[0089] (IV) Detection methods

[0090] Transaminase module enzyme activity detection: Substrate 1a (10mM), transaminase (0.2mol%), GDH (0.15mol%), LDH (0.03mol%), D,L-alanine (100mM), PLP (1mM), glucose (100mM), NADH (1mM) and DMSO (5% v / v) were cultured in a 200μL reaction system (pH=8) containing 100mM at 30℃ for 24h. After the reaction, 24 times the volume of methanol was added to quench the reaction, shake, centrifuge at low temperature and high speed, centrifuge at 20000g for 5min, and take 20μL sample to load the chromatographic column to analyze the conversion rate. Then, the remaining reaction solution was adjusted to pH 10 with 5M sodium hydroxide solution and extracted with ethyl acetate three times. The organic layers were combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product was analyzed by normal HPLC.

[0091] Chemical module detection: Amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalents) were dissolved in dichloromethane (0.5M), and acetyl chloride was added dropwise at 0°C. After the reaction was completed, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to obtain the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 equivalent) was dissolved in dry MeCN (0.5M), and then phosphorus oxychloride (5 equivalents) was added. Add to the mixture and reflux at 90°C for 2 hours. Take 1μL sample and dilute 1000 times with chromatography-grade methanol for liquid phase analysis of the reaction.

[0092] Imine reductase activity detection: imine rac-3a (1mM), imine reductase (1.6mol%), GDH (1mol%), NADP + (10mol%), glucose (100mM) and DMSO (5% v / v) were added and incubated at 30°C for 24 hours in a 200μL reaction system containing 100mM Tris-HCl. After the reaction was completed, 30μL of the reaction mixture was diluted to 300μL with chromatography-grade methanol, and the conversion rate and de value were determined by HPLC and a standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5M sodium hydroxide solution and extracted 3 times with ethyl acetate. The organic layers were combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product was analyzed by positive HPLC.

[0093] High performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature of 25°C, detection wavelength of 280nm. The detection method uses a three-phase solvent system of methanol (containing 0.1% (v / v) trifluoroacetic acid), acetonitrile (containing 0.1% (v / v) trifluoroacetic acid) and double distilled water (containing 0.1% (v / v) trifluoroacetic acid) for gradient separation, and the mobile phase flow rate is 1mL min -1 The linear gradient is shown in Table 3.

[0094] Table 3 High performance liquid chromatography (HPLC) detection conditions

[0095]

[0096] Protein purification method: Resuspend the collected bacteria in lysis buffer (50mmol / L Tris-HCl, 300mmol / L NaCl, 20mM imidazole, pH=8), and then crush the resuspension by high-pressure homogenizer. The crushed resuspension is centrifuged at low temperature and high speed, 4°C, 10000rpm for 30min to obtain a crude enzyme solution. Desalt by nickel column affinity chromatography and desalting column (HistrapTM 5mL Desalting) to finally obtain the purified protein.

[0097] Example 1: Expression and purification of transaminase protein

[0098] 1. Construction of plasmid expressing transaminase

[0099] Taking ArR-TA as an example, the enzyme ArR-TA (shown in SEQ ID NO.1) derived from Arthrobacter sp. was codon optimized, and the optimized gene sequence was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-ArR-TA.

[0100] Taking CV-TA as an example, the enzyme CV-TA (shown in SEQ ID NO.2) from Chromobacterium violaceum was codon optimized, and the optimized gene sequence was synthesized and connected to the pET-21b (+) vector to obtain the plasmid pET-21b-CV-TA.

[0101] According to the same method as above, recombinant plasmids carrying AT-TA, CV-TA, EX-TA, LS-TA, ML-TA, MM-TA, OA-TA, PP-TA, PJ-TA, RS-TA, and VF-TA were constructed respectively.

[0102] 2. Protein expression and purification

[0103] The recombinant plasmids constructed in step 1 were transformed into competent E. coli BL21 cells, and the correct strain was verified to be the recombinant E. coli expressing the corresponding transaminase. Each E. coli BL21 single colony was picked in 5mL LB medium containing antibiotics (ampicillin), cultured on a shaker at 37°C and 220rpm for 8h, and then transferred to 500mL 2xYT medium with an inoculum volume of 1% (v / v), and 500ul 100mg / mL ampicillin was added to the medium and cultured at 37°C until the optical density of the bacteria at 600nm (OD600) was 0.5-0.7, then the culture was cooled to 18°C, and isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 0.2mM for protein induction expression.

[0104] The above culture was centrifuged (8000rpm, 5min, 4°C) to collect cells and discard the supernatant. The cell pellet was then resuspended in 35mL pre-cooled (4°C) Tris-HCl buffer (25mM, pH 8.0), which contained 0.1mM pyridoxal-5-phosphate (PLP), 0.3M sodium chloride, 20mM imidazole and 10% (v / v) glycerol. The resuspended cells were broken by a high-pressure homogenizer (800bar) and centrifuged (18300rpm, 30min, 4°C) to remove cell debris. The protein was purified by metal affinity chromatography (IMAC), and the nickel column was balanced with 10 column volumes of lysate. Non-specifically bound proteins were removed by applying ten column volumes of 20mM imidazole. The specifically bound proteins were eluted from the nickel column with two column volumes of 250mM imidazole. The purified transaminase fractions were pooled, then concentrated using a 30 KDa ultrafiltration tube and replaced with a storage solution (25 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol, 0.1 mM PLP, pH 8.0) to obtain a purified enzyme solution with a final concentration of 10 mg / mL, which was then stored in aliquots at -80°C until use.

[0105] Example 2: SnIR protein expression and purification

[0106] 1. Obtaining SnIR plasmid

[0107] The SnIR (shown in SEQ ID NO.10) encoding Stackebrandtia nassauensis was codon optimized, and the optimized gene sequence was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-SnIR.

[0108] 2. Protein expression and purification

[0109] The recombinant plasmids constructed in step 1 were transformed into competent E. coli BL21 cells, and the correct strain was verified to be the recombinant E. coli expressing imine reductase. Protein expression and purification were performed according to the method of Example 1 to obtain SnIR enzyme protein, which was packaged and adjusted to a final concentration of 10 mg / mL and stored at -80°C for use.

[0110] Example 3: Preparation of LDH and GDH

[0111] 1. Construction of plasmids expressing LDH and GDH

[0112] The codons of the GDH (shown in SEQ ID NO.11) from Bacillus subtilis were optimized, and the optimized gene sequence was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-GDH.

[0113] The codons encoding LDH derived from Lactobacillus fermentum JN248 (shown in SEQ ID NO.12) were optimized, and the optimized gene sequence was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-LDH.

[0114] 2. Protein expression and purification

[0115] The proteins were expressed and purified according to the method of Example 1 to obtain LDH with a specific enzyme activity of 903 U / mg and GDH with a specific enzyme activity of 885 U / mg, respectively. The proteins were packaged and adjusted to a final concentration of 10 mg / mL and stored at -80°C until use.

[0116] Example 4: Construction and verification of chemoenzymatic modular cascade reaction pathways for model products

[0117] like Figure 1As shown, the first step (verification of the transaminase module): (3,5-dimethoxyphenyl)ethanone (1a) is used as a model substrate, and the transaminase constructed in Example 1, the cofactor PLP, D,L-alanine, glucose, the lactose dehydrogenase (LDH) and glucose dehydrogenase (GDH) constructed in Example 3 are converted into the corresponding amine (R-(2a) or S-(2a)) under the action of the cofactor NADH. The first step (verification of the chemical module): racemic (3,5-dimethoxyphenyl)ethanamine 2a reacts with acyl chloride to form an amide, and then generates racemic 6,8-dimethoxy-1,3-dimethyl-3,4-dihydroisoquinoline (rac-3a) under the action of phosphorus oxychloride. The third step (verification of the imine reductase module): rac-3a is used as a substrate and converted into the corresponding 4a under the action of the imine reductase constructed in Example 2, glucose, GDH, and the cofactor NADH.

[0118] The specific steps are as follows:

[0119] (1) First step reaction: Substrate 1a (10 mM), transaminase (0.2 mol%), GDH (0.15 mol%, 885 U / mg), LDH (0.03 mol%, 903 U / mg), D,L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM) and DMSO (5% v / v) were cultured in a 200 μL reaction system (pH = 8) containing 100 mM at 30°C for 24 hours. After the reaction, 24 times the volume of methanol was added to quench the reaction, shaken, centrifuged at low temperature and high speed, centrifuged at 20000g for 5 min, and 200 μL of sample was loaded on the chromatographic column to analyze the conversion rate. The remaining reaction mixture was then alkalized to pH 10 with 5 M sodium hydroxide solution and extracted 3 times with dichloromethane. The solvent of the combined organic layer was removed by vacuum concentration, and the ee value was determined by normal HPLC.

[0120] Table 4 Results of transaminase screening using 1a as substrate

[0121]

[0122] (2) Second step reaction: Amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalents) were dissolved in dichloromethane (0.5 M), and acetyl chloride was added dropwise at 0°C. After the reaction was completed, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to obtain the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50 mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalents) was added. Add to the mixture and reflux at 90°C for 2 hours. Take 1 μL sample and dilute 1000 times with chromatography-grade methanol for liquid phase analysis of the reaction.

[0123] (3) Step 3: Imine rac-3a (1 mM), imine reductase (1.6 mol%), GDH (1 mol%), NADP + (10mol%), glucose (100mM) and DMSO (5% v / v), wherein the molar percentage of each reactant is based on the substrate imine rac-3a. Incubate at 30°C for 24 hours in a 200μL reaction system containing 100mM Tris-HCl. After the reaction is completed, 30μL of the reaction mixture is diluted to 300μL with chromatography-grade methanol, and the conversion rate and de value are determined by HPLC and a standard curve. Then, the remaining reaction solution is adjusted to pH 10 with 5M sodium hydroxide solution and extracted three times with ethyl acetate. The organic layers are combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product is analyzed by forward HPLC. The results show that the conversion rate of imine reductase SnIR is 11%, the de value is 44, and the ee value is 59%.

[0124] Example 5: Substrate spectrum expansion

[0125] According to the experimental results, the main rate-limiting step is the imine reductase module. The yield calculation in the expansion of the substrate spectrum is mainly focused on the imine reduction module. The de value and ee value can represent the results of the chemical enzyme modular cascade reaction to prepare the final product with the substrate ketone. The structural formula of the corresponding product is as follows Figure 7 shown.

[0126] According to the examples, the transaminase ArR-TA was prepared according to the method of Example 1, the imine reductase SnIR was prepared according to the method of Example 2, the glucose dehydrogenase GDH and the lactose dehydrogenase LDH were prepared according to the method of Example 3, and the enantiomer-enriched imine 3 was prepared according to the following steps:

[0127] Substrate 1 (5 mM), ArR-TA (0.4-1 mol%), GDH (0.15-0.3 mol%), LDH (0.03-0.06 mol%), D, L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM) and DMSO (5% v / v) were cultured in a 200 μL reaction system (pH = 8) containing 100 mM Tris-HCl at 30°C for 24 hours. After the reaction, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted with dichloromethane three times. The organic layers were combined, dried over sodium sulfate, and concentrated in vacuo to remove the organic solvent. The crude amine 2 was directly used in the next reaction.

[0128] Amine 2 (1 eq.) and triethylamine (TEA) (1.5 eq.) were dissolved in dichloromethane (0.5 M) and acid chloride (≥1 eq.) was added dropwise at 0 °C. After the reaction was complete, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to give the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50 mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 eq.) was dissolved in dry MeCN (0.5 M) and phosphorus oxychloride (5 eq.) was added. Add to the mixture and reflux at 90 °C for 2 h. The solvent was evaporated in vacuo, the residue was quenched with aqueous ammonia, extracted 3 times with ethyl acetate, washed 3 times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated in vacuo, and column chromatography was used to obtain imine 3.

[0129] Imine 3 (1 mM), SnIR (1.6 mol%), GDH (1 mol%), NADP + (10mol%), glucose (100mM) and DMSO (5% v / v) were added and incubated at 30°C for 24 hours in a 200μL reaction system containing 100mM Tris-HCl. After the reaction was completed, 30μL of the reaction mixture was diluted to 300μL with chromatography-grade methanol, and the conversion rate and de value were determined by HPLC and a standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5M sodium hydroxide solution and extracted 3 times with ethyl acetate. The organic layers were combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product was analyzed by positive HPLC.

[0130] The results showed that all substrates ( Figure 7 All of the above compounds (a1) and (b1) can be converted into the corresponding products (1S,3R)-4a to (1S,3R)-4l. The specific yields, de values ​​and ee values ​​are shown in the following table.

[0131] Table 5 Yield, de and ee of 1,3-disubstituted THIQ

[0132]

[0133]

[0134] Example 6: Scale-up preparation of (1S,3R)-4a

[0135] According to the examples, the aminotransferase ArR-TA was prepared according to the method of Example 1, the imine reductase SnIR was prepared according to the method of Example 2, the glucose dehydrogenase GDH and the lactose dehydrogenase LDH were prepared according to the method of Example 3, and the amplification system reaction for preparing (1S, 3R)-4a was carried out according to the following steps.

[0136] Substrate 1a (5 mM), ArR-TA (0.4 mol%), GDH (0.15 mol%), LDH (0.03 mol%), D-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM) and DMSO (5% v / v) were cultured in a 200 mL reaction system (pH = 8) containing 100 mM Tris-HCl at 30°C for 24 hours. After the reaction, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted with dichloromethane three times. The organic layers were combined, dried over sodium sulfate, and concentrated in vacuo to remove the organic solvent. The crude amine (R)-2a was directly used in the next reaction.

[0137] Crude amine (R)-2a (1 eq.) and triethylamine (1.5 eq.) were dissolved in dichloromethane (0.5 M) and acid chloride (≥1 eq.) was added dropwise at 0 °C. After the reaction was complete, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to give the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50 mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 eq.) was dissolved in dry MeCN (0.5 M) and phosphorus oxychloride (5 eq.) was added. Add to the mixture and reflux at 90 °C for 2 h. The solvent was evaporated in vacuo, the residue was quenched with ammonia, extracted 3 times with ethyl acetate, washed 3 times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated in vacuo, and purified by column chromatography to obtain crude imine (R)-3a.

[0138] Crude imine (R)-3 (1 mM), SnIR (3 mol%, added in 3 equal portions with an interval of 16 h), GDH (1 mol%, added in 3 equal portions with an interval of 16 h), NADP + (10 mol%), glucose (100 mM) and DMSO (5% v / v) were incubated at 30°C for 48 hours in a 1 L reaction system containing 100 mM Tris-HCl. The combined organic layers were then washed with brine, dried over sodium sulfate, and concentrated to give a crude mixture, which was purified by column chromatography to obtain 77.8 mg of (1S, 3R)-4a with an isolated yield of 35%, 74% de, and >99% ee.

[0139] Example 7: Scale-up preparation of (1S,3S)-4d

[0140] In order to prepare (1S, 3S)-4d on a large scale, a system amplification reaction was performed. The aminotransferase CV-TA was prepared according to the method of Example 1, the imine reductase SnIR was prepared according to the method of Example 2, and the glucose dehydrogenase GDH and lactose dehydrogenase LDH were prepared according to the method of Example 3.

[0141] Substrate 1d (5 mM), CV-TA (1 mol%), GDH (0.15 mol%), LDH (0.03 mol%), L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM) and DMSO (5% v / v) were cultured in a 40 mL reaction system (pH = 8) containing 100 mM Tris-HCl at 30°C for 24 hours. After the reaction, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted with dichloromethane three times. The organic layers were combined, dried over sodium sulfate, and concentrated in vacuo to remove the organic solvent. The crude amine (S)-2d was directly used in the next reaction.

[0142] The crude amine (S)-2d (1 eq.) and triethylamine (1.5 eq.) were dissolved in dichloromethane (0.5 M) and the acid chloride (≥1 eq.) was added dropwise at 0 °C. After the reaction was complete, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to give the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50 mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 eq.) was dissolved in dry MeCN (0.5 M) and phosphorus oxychloride (5 eq.) was added. Add to the mixture and reflux at 90 °C for 2 h. The solvent was evaporated in vacuo, the residue was quenched with ammonia, extracted 3 times with ethyl acetate, washed 3 times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated in vacuo, and purified by column chromatography to obtain the crude imine (S)-3d.

[0143] Crude imine (S)-3d (1 mM), SnIR (2 mol%, added in 3 equal portions with an interval of 16 h), GDH (1 mol%, added in 3 equal portions with an interval of 16 h), NADP + (10 mol%), glucose (100 mM) and DMSO (5% v / v) were incubated in a 200 mL reaction system containing 100 mM Tris-HCl at 30°C for 48 hours. The combined organic layers were then washed with brine, dried over sodium sulfate, and concentrated to give a crude mixture, which was purified by column chromatography to obtain 31.1 mg of (1S,3S)-4d with an isolated yield of 76%, 97% de, and >99% ee.

[0144] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli expressing a transaminase, characterized in that: Using Escherichia coli BL21 (DE3) as the host, express any of the aminotransferases shown in a1 to a9: a1, transaminase ArR-TA from Arthrobacter sp., whose amino acid sequence is shown in SEQ ID NO.1; a2, transaminase CV-TA from Chromobacterium violaceum, the amino acid sequence of which is shown in SEQ ID NO.2; a3, transaminase ArRmut-11 from Arthrobacter sp., whose amino acid sequence is shown in SEQ ID NO.3; a4, transaminase AT-TA from Aspergillus terreus, the amino acid sequence of which is shown in SEQ ID NO.4; a5, transaminase OA-TA from Ochrobactrum anthropic, the amino acid sequence of which is shown in SEQ ID NO.5; a6, transaminase RS-TA from Ruegeria sp. TM1040, the amino acid sequence of which is shown in SEQ ID NO.6; a7, transaminase EX-TA from Exophiala xenobiotica, whose amino acid sequence is shown in SEQ ID NO.7; a8, transaminase PP-TA from Paraburkholderia phymatum, the amino acid sequence of which is shown in SEQ ID NO.8; a9. Transaminase VF-TA from Vibrio fluvialis, whose amino acid sequence is shown in SEQ ID NO.

9.

2. A method for preparing a transaminase, characterized in that: The recombinant Escherichia coli according to claim 1 is cultured in a culture medium to induce enzyme production, and the enzyme protein is collected.

3. A method for synthesizing 1,3-disubstituted THIQ, characterized in that: The following steps are involved: (1) mixing a transaminase and a substrate for reaction under the participation of a lactate dehydrogenase circulation system; the substrate is ketone 1, D-alanine or L-alanine, pyridoxal phosphate; the LDH circulation system includes LDH, glucose dehydrogenase, NADH, and glucose; the transaminase contains an amino acid sequence shown in any one of SEQ ID NOs. 1 to 9; (2) reacting the amine 2 obtained in step (1) with an acyl chloride, collecting the reaction product, and then reacting the obtained product with phosphorus oxychloride, and collecting the reaction product imine 3; (3) using the imine 3 obtained in step (2) as a substrate, and catalyzing the reaction with an imine reductase protein and a GDH circulation system to obtain 1,3-disubstituted THIQ; the GDH circulation system includes GDH, NADP + ,glucose.

4. The method according to claim 3, characterized in that The pH of the biocatalytic reaction is 5-10, the reaction temperature is 20-40°C, and the reaction time is 1h-24h.

5. The method according to claim 3 or 4, characterized in that: In the biological reaction system, the initial concentration of the ketone 1 is 1-50 mM, and the initial concentration of the imine 3 is 1-50 mM.

6. The method according to any one of claims 3 to 5, characterized in that: The glucose dehydrogenase is derived from Bacillus subtilis, and its amino acid sequence is shown in SEQ ID NO.11; the lactate dehydrogenase is derived from Lactobacillus fermentum JN248, and its amino acid sequence is shown in SEQ ID NO.

12.

7. The method according to any one of claims 3 to 6, characterized in that: The ketone 1 includes at least one of the following 1a-1e: The imine 3 comprises at least one of the following:

8. The method according to any one of claims 3 to 7, characterized in that: The acyl chloride comprises at least one of the following AG:

9. The method according to any one of claims 3 to 8, characterized in that: The 1,3-disubstituted THIQ structure includes any of the following:

10. Use of the recombinant Escherichia coli according to claim 1 or the method according to any one of claims 2 to 9 in the preparation of chiral 1,3-disubstituted tetrahydroisoquinoline.

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