A method for synthesizing chiral 1,3-disubstituted tetrahydroisoquinolines by a chemical enzyme cascade system

Abstract

The application discloses a method for synthesizing chiral 1,3-disubstituted tetrahydroisoquinoline by using a chemical enzyme cascade system and belongs to the technical field of bioengineering. The application establishes a method for preparing chiral 1,3-disubstituted tetrahydroisoquinoline by using an enzyme cascade catalysis. Simple non-chiral ketone is used as a raw material, a transaminase obtained through screening is used to generate a chiral amine, a B-N cyclization reaction is used to obtain a chiral imine, and finally, an imine reductase is used to generate 1,3-disubstituted tetrahydroisoquinoline. The method has a high yield, can be close to 100%, has excellent diastereoselectivity and enantioselectivity, the de value and the ee value are close to 99%, and has good application value and prospect.

Description

Technical Field

[0001] This invention relates to a method for synthesizing chiral 1,3-disubstituted tetrahydroisoquinoline using a chemical enzyme cascade system, belonging to the field of bioengineering technology. Background Technology

[0002] Tetrahydroisoquinoline (THIQ) alkaloids are among the largest-scale natural products currently available and play an indispensable role in drug development. In recent decades, THIQ-type natural products have been extensively studied, exhibiting broad structural diversity and biological activity.

[0003] Among these different THIQs, 1,3-disubstituted THIQ, with at least two chiral carbon centers, is a unique THIQ alkaloid, distinct from tetrahydroisoquinolines generally believed to be formed via the Pictet-Spengler (PS) reaction between aldehydes and arylethylamines derived from aromatic amino acids. It is generated from further reactions of polyketides formed via the acetate-malonic acid pathway, which involves unidentified polyketide synthases and other enzymes. However, the unusual substitution pattern of 1,3-disubstituted THIQs, particularly the presence of methyl groups at C1 and C3 positions, presents significant obstacles to the conventional PS pathway for these important compounds due to the difficulty in pre-installing α-methyl groups on aromatic amino acids to construct C3 methyl groups via biocatalysis and the restriction of acetaldehyde to C1 methyl groups via NCS substrates.

[0004] Although their biosynthesis remains challenging, numerous 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 phenethylamine derivatives and aldehydes, Bischler-Napieralski (BN) / reduction cascades of chiral acylated phenethylamines, and palladium-catalyzed three-component Catellani / gold-catalyzed cyclization / reduction cascades. While these strategies are effective, they often rely on complex substrates with pre-constructed chiral centers, heavy metal catalysts, flammable and hazardous hydrogen, and harsh conditions of high temperature and high pressure. Therefore, developing a novel, efficient, and green method to construct chiral 1,3-disubstituted THIQs from simple achiral substrates is highly desirable. Summary of the Invention

[0005] To address the aforementioned problems, this invention presents a method for the diastereoselective and enantioselective modular synthesis of 1,3-disubstituted THIQ. Figure 1 ).

[0006] This invention provides a recombinant *Escherichia coli* strain expressing transaminases, using *Escherichia coli* BL21(DE3) as the host, expressing any of the transaminases represented by a1 to a9:

[0007] a1. ArR-TA, a transaminase derived from Arthrobacter sp., has the amino acid sequence shown in SEQ ID NO. 1;

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

[0009] a3. ArRmut-11, a transaminase derived from Arthrobacter sp., has the amino acid sequence shown in SEQ ID NO.3;

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

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

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

[0013] a7. The transaminase EX-TA derived from the foreign body bottle mold (Exophiala xenobiotica), the amino acid sequence of which is shown in SEQ ID NO.7;

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

[0015] a9. VF-TA, a transaminase derived from Vibrio fluvialis, has the amino acid sequence 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 the 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 from the recombinant Escherichia coli.

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

[0020] In one embodiment, the steps are as follows: the recombinant Escherichia coli is inoculated into LB medium and cultured at 20-40°C for 10-12 hours, then transferred to 2YT medium and cultured at 20-40°C until OD200. 600 When the pH reaches approximately 0.6-0.8, cool the temperature to 15-25℃, add IPTG at a final concentration of 0.05-0.5mM to induce induction for 10-20 hours, and then collect the bacterial cells.

[0021] This invention also provides the application of the transaminase in the synthesis of 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 involves mixing the transaminase protein with a substrate in a reaction system involving the lactate dehydrogenase (LDH) cycle.

[0024] In one implementation, the application includes the following steps:

[0025] (1) The transaminase protein and the substrate are mixed and reacted in the presence of the lactate dehydrogenase (LDH) cycle system to prepare amine 2; the substrate is ketone 1, D-alanine or L-alanine, pyridoxal phosphate (PLP); the LDH cycle system includes LDH, glucose dehydrogenase (GDH), NADH, and glucose.

[0026] (2) The amine 2 obtained in step (1) is reacted with acyl chloride, the reaction product is collected, and the product is then reacted with phosphorus oxychloride to obtain imine 3.

[0027] (3) Using the imine 3 obtained in step (2) as a substrate, a catalytic reaction is carried out with imine reductase protein and a GDH cycle system to obtain 1,3-disubstituted THIQ; the GDH cycle system includes GDH and NADP. + ,glucose.

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

[0029] In one embodiment, in step (3), the amount of imine 3 added is 1 mM, the amount of the imine reductase mutant added is 1.6-3 mol%, the amount of GDH added is 1 mol%, and the amount of NADP... + The amount of added glucose is 10 mol%, and the amount of added 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, and at least 1 equivalent of acyl chloride. 0.5M amide is dissolved in acetonitrile, and 5 equivalents of phosphorus oxychloride. The acetonitrile is preferably dried 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 includes ketone 1, D-alanine or L-alanine, or pyridoxal phosphate (PLP).

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

[0034] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction 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 amount of ketone 1 added is 5-10 mM; the amount of D-alanine or L-alanine added is 100 mM; the amount of PLP added is 1 mM; the amount of glucose added is 100 mM; and the amount of NADH added is 1 mM.

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

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

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

[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, and (R)-3l.

[0045]

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

[0047]

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

[0049] Beneficial effects:

[0050] (1) The present invention provides a transaminase (ArR-TA) that can efficiently convert substrate ketones 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 substrate ketones 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) that can convert racemic imine substrates into (1S,3R)-4a with a conversion rate of 11%, a de value of 44, and an ee value of 59%.

[0053] (4) This invention also provides a method for synthesizing 1,3-disubstituted THIQ from non-chiral ketones. 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%, 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 a raw material to synthesize 179.8 mg of (1S,3R)-4a in a modular chemical enzyme process with a yield of 81%, >99de, >99ee. Using ketone 1d as a raw material, 31.1 mg of (1S,3R)-4d is synthesized in a modular chemical enzyme process with a yield of 76%, 97de, >99ee. Attached Figure Description

[0055] Figure 1 The synthetic route for 1,3-disubstituted THIQ.

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

[0057] Figure 3 This is a liquid phase analysis diagram from the chemistry module.

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

[0059] Figure 5 To identify the chirality of the enzyme-catalyzed synthesis of (1S,3R)-4a.

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

[0061] Figure 7 To utilize imine reductase for substrate spectrum expansion.

[0062] Figure 8 To identify the chirality of modular chemoenzyme conjugates (1S,3S)-4d. Detailed Implementation

[0063] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, 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] Acyl 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] The structure of compound 1,3-disubstituted THIQ is any of the following:

[0072]

[0073] (II) Amino Acid and Nucleotide Sequence

[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 this invention, including ampicillin sodium, kanamycin sulfate, and streptomycin sulfate, are all from Shanghai Sangon Biotech. NADP + NADH was obtained from Shanghai Bid Pharmaceutical Technology Co., Ltd.; the PCR enzymes involved in the molecular experiments in this invention were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; the chemical reagents used in this invention, including methanol, dichloromethane, ethyl acetate (EtOAc), petroleum ether (PE), acetonitrile (MeCN), sodium sulfate, sodium carbonate, sodium bicarbonate, diethylamine (DEA), ammonia, sodium hydroxide, and hydrochloric acid, were all purchased from China National Pharmaceutical Group Corporation and Shanghai Titan Technology Co., Ltd.

[0089] (iv) Testing methods

[0090] Transaminase activity assay: Substrate 1a (10 mM), transaminase (0.2 mol%), GDH (0.15 mol%), LDH (0.03 mol%), D,L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM), and DMSO (5% v / v) were incubated in a 200 μL reaction system (pH = 8) containing 100 mM at 30 °C for 24 h. After the reaction, 24 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20000 g for 5 min. 20 μL of the sample was loaded onto a chromatographic column for conversion analysis. The remaining reaction solution was then adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with ethyl acetate. The organic layers were combined and concentrated under vacuum to remove the organic solvent. The ee value of the product was analyzed by forward HPLC.

[0091] Chemical module detection: Amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalent) were dissolved in dichloromethane (0.5 M), and acetyl chloride 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 obtain crude amide, which could be used directly for the next step without purification. A two-necked round-bottom flask (50 mL) equipped with a magnetic stir bar was thoroughly rinsed with argon for 3 cycles. The crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalent) was added. The mixture was added to the solution, and the mixture was refluxed at 90 °C for 2 hours. A 1 μL sample was diluted 1000 times with chromatographic grade methanol for liquid chromatography analysis.

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

[0093] High-performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature 25℃, detection wavelength 280nm. The detection method employed a gradient separation using 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), with a mobile phase flow rate of 1 mL / min. -1 The linear gradients are shown in Table 3.

[0094] Table 3. Detection conditions for high performance liquid chromatography (HPLC)

[0095]

[0096] Protein purification method: The collected bacterial cells were resuspended in lysis buffer (50 mmol / L Tris-HCl, 300 mmol / L NaCl, 20 mM imidazole, pH=8), and then the resuspended solution was homogenized using a high-pressure homogenizer. The homogenized resuspended solution was centrifuged at low temperature and high speed (4°C, 10,000 rpm for 30 min) to obtain crude enzyme solution. The solution was then desalted by nickel affinity chromatography and a Histrap™ 5 mL desalting column to obtain the purified protein.

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

[0098] 1. Construction of plasmids expressing transaminases

[0099] Taking ArR-TA as an example, the codons of the enzyme ArR-TA (SEQ ID NO.1) derived from Arthrobacter sp. were optimized, and the optimized gene sequence was synthesized and ligated into 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) derived from Chromobacterium violaceum was codon optimized, and the optimized gene sequence was synthesized and ligated into the pET-21b(+) vector to obtain the plasmid pET-21b-CV-TA.

[0101] Following the same method described 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 E. coli BL21 competent cells. The correctly verified strains were the recombinant E. coli expressing the corresponding transaminases. Single colonies of E. coli BL21 were picked and cultured in 5 mL LB medium containing antibiotics (ampicillin) at 37°C on a shaker at 220 rpm for 8 h. Then, they were transferred to 500 mL 2xYT medium at a 1% (v / v) inoculation rate, and 500 μL of 100 mg / mL ampicillin was added. The culture was then incubated at 37°C until the bacterial density at 600 nm (OD600) reached 0.5-0.7. The culture was then cooled to 18°C, and isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM to induce protein expression.

[0104] The culture was centrifuged (8000 rpm, 5 min, 4 °C) to collect cells, and the supernatant was discarded. Cell particles were then resuspended in 35 mL of pre-chilled (4 °C) Tris-HCl buffer (25 mM, pH 8.0) containing 0.1 mM pyridoxal-5-phosphate (PLP), 0.3 M sodium chloride, 20 mM imidazole, and 10% (v / v) glycerol. The resuspended cells were homogenized using an autoclave (800 bar) and centrifuged (18300 rpm, 30 min, 4 °C) to remove cell debris. Proteins were purified using metal affinity chromatography (IMAC), with the nickel column equilibrated with 10 column volumes of lysis buffer. Non-specifically bound proteins were removed by applying 10 column volumes of 20 mM imidazole. Specifically bound proteins were eluted from the nickel column with two column volumes of 250 mM imidazole. The purified transaminase fraction was collected and then concentrated using a 30 kDa ultrafiltration tube, and the stock solution (25 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol, 0.1 mM MPLP, pH 8.0) was replaced to obtain a purified enzyme solution with a final concentration of 10 mg / mL, which was then aliquoted and stored at -80°C for later use.

[0105] Example 2: SnIR protein expression and purification

[0106] 1. Obtaining SnIR plasmids

[0107] The codons of SnIR (SEQ ID NO. 10) encoding Stackebrandtia nassauensis were optimized, and the optimized gene sequence was synthesized and ligated into 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 E. coli BL21 competent cells. The correctly identified strains were the recombinant E. coli expressing imine reductase. Protein expression and purification were performed according to the method in Example 1 to obtain SnIR enzyme protein, which was aliquoted and adjusted to a final concentration of 10 mg / mL, and stored at -80°C for later use.

[0110] Example 3: Preparation of LDH and GDH

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

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

[0113] The LDH (SEQ ID NO. 12) encoding Lactobacillus fermentum JN248 was codon optimized, and the optimized gene sequence was synthesized and ligated into the pET-21b(+) vector to obtain plasmid pET-21b-LDH.

[0114] 2. Protein Expression and Purification

[0115] The protein was expressed and purified according to the method in Example 1, and LDH with a specific enzyme activity of 903 U / mg and GDH with a specific enzyme activity of 885 U / mg were obtained. The proteins were aliquoted and adjusted to a final concentration of 10 mg / mL and stored at -80°C for later use.

[0116] Example 4: Construction and Validation of the Modular Enzymatic Cascade Reaction Pathway for the Model Product

[0117] like Figure 1As shown, the first step (verification of the transaminase module): using (3,5-dimethoxyphenyl)ethyl ketone (1a) as a model substrate, the transaminase constructed in Example 1, along with cofactors PLP, D,L-alanine, glucose, lactose dehydrogenase (LDH) and glucose dehydrogenase (GDH) constructed in Example 3, were converted into the corresponding amines (R-(2a) or S-(2a)) under the action of cofactor NADH. The second step (verification of the chemical module): racemic (3,5-dimethoxyphenyl)ethylamine 2a reacted with acyl chloride to form an amide, which was then converted into 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): using rac-3a as a substrate, the imine reductase constructed in Example 2, along with glucose, GDH, and cofactor NADH, were converted into the corresponding 4a.

[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 incubated in a 200 μL reaction system containing 100 mM (pH = 8) at 30 °C for 24 hours. After the reaction was completed, 24 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20000 g for 5 min. 200 μL of the sample was loaded onto a chromatographic column for conversion analysis. The remaining reaction mixture was then alkalized to pH 10 with 5 M sodium hydroxide solution and extracted three times with dichloromethane. The solvent in the combined organic layer was removed by vacuum concentration, and the ee value was determined by forward HPLC.

[0120] Table 4. Screening results of transaminases using 1a as a substrate.

[0121]

[0122] (2) Second step reaction: Amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalent) were dissolved in dichloromethane (0.5 M), and acetyl chloride was added dropwise at 0 °C. After the reaction was complete, the solution was diluted with dichloromethane, washed twice with brine, dried with sodium sulfate, filtered, and concentrated to obtain crude amide, which was used directly for the next step without purification. A double-necked round-bottom flask (50 mL) equipped with a magnetic stir bar was 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 equivalent) was added. The mixture was added and refluxed at 90 °C for 2 hours. 1 μL of the sample was diluted 1000 times with chromatographic grade methanol for liquid chromatography analysis.

[0123] (3) Third step reaction: imine rac-3a (1 mM), imine reductase (1.6 mol%), GDH (1 mol%), NADP + The reaction mixture consisted of 10 mol% glucose (100 mM) and DMSO (5% v / v), where the molar percentages of each reactant were based on the substrate imine rac-3a. The reaction mixture was incubated at 30°C for 24 h in a 200 μL reaction system containing 100 mM Tris-HCl. After the reaction was complete, 30 μL of the reaction mixture was diluted to 300 μL with chromatographic grade methanol, and the conversion and de value were determined by HPLC and a standard curve. The remaining reaction solution was then adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with ethyl acetate. The organic layers were combined and concentrated under vacuum to remove the organic solvent, and the ee value of the product was analyzed by forward HPLC. The results showed that the conversion of imine reductase SnIR was 11%, the de value was 44, and the ee value was 59%.

[0124] Example 5: Substrate Spectrum Expansion

[0125] The experimental results indicate that the main rate-limiting step is the imine reductase module. Yield calculations in the substrate spectrum extension mainly focus on the imine reduction module. The de and ee values ​​represent the results of the modular cascade reaction of the chemical enzyme in preparing the final product from the substrate ketone. The structural formula of the corresponding product is shown below. Figure 7 As shown.

[0126] Transaminase ArR-TA was prepared according to the method of Example 1, imine reductase SnIR was prepared according to the method of Example 2, glucose dehydrogenase GDH and lactose dehydrogenase LDH were prepared according to the method of Example 3, and enantiomeric enrichment of 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 incubated in a 200 μL reaction system (pH = 8) containing 100 mM Tris-HCl at 30 °C for 24 h. After the reaction, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with dichloromethane. The combined organic layers were dried over sodium sulfate and concentrated under vacuum to remove the organic solvent. Crude amine 2 was used directly in the next reaction.

[0128] Amine 2 (1 equivalent) and triethylamine (TEA) (1.5 equivalent) were dissolved in dichloromethane (0.5 M), and acyl chloride (≥1 equivalent) 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 obtain crude amide, which was used directly for the next step without purification. A two-necked round-bottom flask (50 mL) with a magnetic stir bar was thoroughly rinsed with argon for 3 cycles. Crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalent) was added. The mixture was added, and the mixture was refluxed at 90 °C for 2 hours. The solvent was evaporated under vacuum, the residue was quenched with ammonia, extracted three times with ethyl acetate, washed three times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated under vacuum, and the residue was purified by column chromatography to obtain imine 3.

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

[0130] The results showed that all substrates ( Figure 7 All of them 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 table below.

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

[0132]

[0133]

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

[0135] Transaminase ArR-TA was prepared according to the method of Example 1, imine reductase SnIR was prepared according to the method of Example 2, glucose dehydrogenase GDH and lactose dehydrogenase LDH were prepared according to the method of Example 3, and the scale-up reaction of (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 incubated in a 200 mL reaction system (pH = 8) containing 100 mM Tris-HCl at 30 °C for 24 h. After the reaction, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with dichloromethane. The combined organic layers were dried over sodium sulfate and concentrated under vacuum to remove the organic solvent. The crude amine(R)-2a was used directly in the next reaction.

[0137] Crude amine (R)-2a (1 equivalent) and triethylamine (1.5 equivalent) were dissolved in dichloromethane (0.5 M), and acyl chloride (≥1 equivalent) 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 obtain crude amide, which was used directly for the next step without purification. A double-necked round-bottom flask (50 mL) with a magnetic stir bar was thoroughly rinsed with argon for 3 cycles. Crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalent) was added. The mixture was added and refluxed at 90 °C for 2 hours. The solvent was evaporated under vacuum, the residue was quenched with ammonia, extracted three times with ethyl acetate, washed three times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated under vacuum, and purified by column chromatography to obtain crude imine (R)-3a.

[0138] Crude imine(R)-3 (1 mM), SnIR (3 mol%, added in three equal portions, 16 h apart), GDH (1 mol%, added in three equal portions, 16 h apart), 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 layer was then washed with brine, dried over sodium sulfate, and concentrated to obtain a crude mixture. The crude mixture was purified by column chromatography to obtain 77.8 mg of (1S,3R)-4a, with a separation yield of 35%, 74% de, and >99% ee.

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

[0140] To scale up the preparation of (1S,3S)-4d, a system-wide reaction was performed. Transaminase CV-TA was prepared according to the method of Example 1, imine reductase SnIR was prepared according to the method of Example 2, and 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 incubated for 24 hours at 30°C in a 40 mL reaction system (pH = 8) containing 100 mM Tris-HCl. After the reaction, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with dichloromethane. The combined organic layers were dried over sodium sulfate and vacuum concentrated to remove the organic solvent. The crude amine (S)-2d was used directly in the next reaction.

[0142] Crude amine (S)-2d (1 equivalent) and triethylamine (1.5 equivalent) were dissolved in dichloromethane (0.5 M), and acyl chloride (≥1 equivalent) 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 obtain crude amide, which was used directly for the next step without purification. A double-necked round-bottom flask (50 mL) with a magnetic stir bar was thoroughly rinsed with argon for 3 cycles. Crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalent) was added. The mixture was added and refluxed at 90 °C for 2 hours. The solvent was evaporated under vacuum, the residue was quenched with ammonia, extracted three times with ethyl acetate, washed three times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated under vacuum, and the crude imine (S)-3d was purified by column chromatography.

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

[0144] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for synthesizing 1,3-disubstituted THIQ, characterized in that, Includes the following steps: (1) The transaminase and the substrate are mixed and reacted in the presence of the lactate dehydrogenase cycle system to obtain the product amine 2; the substrate is ketone 1, D-alanine or L-alanine, pyridoxal phosphate; the lactate dehydrogenase cycle system includes lactate dehydrogenase LDH, glucose dehydrogenase GDH, NADH, and glucose. The transaminase uses Escherichia coli BL21 (DE3) as the host and expresses the transaminase ArRTA with the amino acid sequence shown in SEQ ID NO.1; (2) The amine 2 obtained in step (1) is reacted with acyl chloride, the reaction product is collected, and then the product is reacted with phosphorus oxychloride to collect the reaction product imine 3; (3) Using the imine 3 obtained in step (2) as a substrate, a reaction is catalyzed by imine reductase protein and the GDH cycle system to obtain 1,3-disubstituted THIQ; the GDH cycle system includes glucose dehydrogenase GDH and NADP. + ,glucose; The imine reductase is an imine reductase expressed using Escherichia coli BL21 as the host, with the amino sequence shown in SEQ ID NO.

10. The glucose dehydrogenase GDH is expressed in Escherichia coli BL21 as shown in SEQ ID NO.

11. The lactate dehydrogenase LDH is expressed by Escherichia coli BL21 as shown in SEQ ID NO.

12. The ketone 1 is at least one of the following 1a-1f: 。 2. The method according to claim 1, characterized in that, The pH of the reaction in step (1) is 5 to 10, the temperature is 20 to 40°C, and the reaction time is 1 to 24 hours.

3. The method according to claim 1 or 2, characterized in that, The initial concentration of ketone 1 in step (1) is 1-50 mM, and the initial concentration of imine 3 in step (3) is 1-50 mM.

4. The method according to claim 1 or 2, characterized in that, The imine 3 is at least one of the following: 。 5. The method according to claim 1 or 2, characterized in that, The acyl chloride includes at least one of the following A and B: 。 6. The method according to claim 1 or 2, characterized in that, The 1,3-disubstituted THIQ structure is any of the following: 。

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