Chemical enzyme synthesis method of chiral aporphine alkaloid

Abstract

The invention discloses a chemical enzyme synthesis method of chiral aporphine alkaloids, and relates to the field of biochemistry, the method comprises the following steps: taking a phenylethylamine compound and a phenylacetic acid compound as initial raw materials, and carrying out amide condensation and dehydration cyclization to form a compound IV; then reducing through imine reductase or an imine reductase mutant to form a V-type compound; then, a compound VI is formed through a methylation reaction of methyltransferase or Eschweiler-Clarke or methyl iodide, and the compound VI is formed through a methylation reaction of methyltransferase or Eschweiler-Clarke or methyl iodide; and finally, carrying out photocatalytic coupling to obtain the aporphine alkaloid. According to the preparation method disclosed by the invention, purification is not needed in the preparation reaction process from the IV-type compound to the VI-type compound, and the compound can be directly used for the last step of photocatalytic coupling; the optically pure (S)-VII aporphine alkaloid can be obtained; the method is mild in reaction condition, green, environment-friendly, simple in process, low in cost, capable of reaching gram-level scale and suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, and particularly relates to a chemical enzyme synthesis method of chiral aporphine alkaloids. BACKGROUND

[0002] Apophine alkaloids are a kind of aromatic heterocyclic alkaloids containing biphenyl-type tetracyclic structure, belonging to benzyl isoquinoline alkaloids. At present, more than 500 aporphine alkaloids have been isolated from nature, and the core skeleton of all of them is a biphenyl-type tetracyclic structure, but they have different oxidation states, substituents and chiral centers, which endow them with a wide range of physiological activities, including anticancer, antiviral, antimalarial, anti-inflammatory, lipid-lowering, anti-platelet aggregation and vasodilation, and have good development and utilization value and medicinal prospects.

[0003]

[0004] Most of the known aporphine alkaloids have 1 and 2 positions in the A ring mainly substituted, and the substituents are mainly dimethoxy and methylenedioxy; the D ring is mainly substituted at the 9 and 10 positions, and the substituents are mainly hydrogen, dimethoxy, methylenedioxy or hydroxy. Studies on structure-activity relationships have shown that the diverse physiological activities of aporphine alkaloids are closely related to the absolute configuration of the core skeleton and the substituents on the nitrogen and benzene ring, such as ( R )-apomorphine is a dopamine receptor agonist, while ( S )-apomorphine is a dopamine receptor antagonist; pavatrine has anti-inflammatory and analgesic effects, and its antitussive effect is better than codeine, with a higher therapeutic index; nuciferine has lipid-lowering, antihypertensive and lipid-lowering effects, and is the main component of many weight loss health products. Therefore, it has great application value to invent a precise and efficient synthesis method of aporphine alkaloids.

[0005] At present, aporphine alkaloids can be prepared by extraction from plants, biosynthesis and chemical synthesis. Among them, extraction from plants is the main method for obtaining aporphine alkaloids in industry, but the plant culture period is long, the extraction efficiency is low, and a large amount of organic solvent is needed; biosynthesis is still mainly in the laboratory research stage and is difficult to be industrialized. Chemical synthesis often uses phenylacetic acid and phenethylamine compounds as raw materials, and the reported yield is about 76% through Bischer-Napieralski reaction or Pictet-Spengler reaction to obtain tetrahydroisoquinoline compounds, and then reduced by NaBH4 and other reducing agents and metal-catalyzed coupling reaction to obtain aporphine alkaloids, with a reported yield of about 50%. This method relies on toxic and expensive metal reagents, and it is difficult to obtain optically pure products for aporphine alkaloids containing chiral centers, and the cost of industrial application is high and the environmental pollution is large.

[0006] Existing methods for preparing apophene alkaloids suffer from problems such as complex processes, low yields, raw material waste, and environmental unfriendliness. Therefore, those skilled in the art are dedicated to developing a chemical enzymatic synthesis method for S-type apophene alkaloids that is environmentally friendly, simple, low-cost, and suitable for industrial production. Summary of the Invention

[0007] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to develop a chemical enzymatic synthesis method for S-type apophene alkaloids, and how this method is green and environmentally friendly, simple in process, low in cost, and suitable for industrial production.

[0008] To achieve the above objectives, this invention provides a chemical enzymatic synthesis method for S-type apophene alkaloids, the steps of which are as follows: Step (1) Using phenylethylamine compounds represented by chemical formula I and phenylacetic acid compounds represented by chemical formula II as raw materials, an amide compound or a compound whose benzene ring skeleton has been modified is obtained through an amide condensation reaction; Step (2) uses the amide compound obtained in step (1) or the compound whose benzene ring skeleton of the amide compound has been modified as the second raw material, and obtains dihydroisoquinoline compound or the compound whose benzene ring skeleton of the dihydroisoquinoline compound has been modified through cyclization dehydration reaction; the general formula of the dihydroisoquinoline compound is shown in chemical formula IV. Step (3) uses the dihydroisoquinoline compound obtained in step (2) or a compound whose benzene ring skeleton has been modified as a third raw material, and in the presence of imine reductase or its mutant in the first in vivo or in vitro environment, a reduction reaction is carried out to obtain S Tetrahydroisoquinoline compounds or S Compounds in the tetrahydroisoquinoline class whose benzene ring skeleton has been modified; S The general formula of tetrahydroisoquinoline compounds is as follows (chemical formula) S As shown in )-V; Step (4) is obtained from step (3). S Tetrahydroisoquinoline compounds or S A compound with a modified benzene ring skeleton, belonging to the tetrahydroisoquinoline class, serves as the fourth starting material. In the presence of a methyltransferase or methylating agent, and either in vivo or in vitro, a first methylation reaction is performed to obtain the desired product. S type N methyltetrahydroisoquinoline compounds or S type N -Methyltetrahydroisoquinoline compounds are compounds in which the benzene ring skeleton has been modified; S type N The general formula of methyltetrahydroisoquinoline compounds is as shown in the chemical formula (S ) as shown in formula (VII) ; the compound of formula (VII) is prepared by the first coupling reaction of the compound of formula (VI) and the compound of formula (VII) in the presence of the third solvent and light. S type N - methyl tetrahydroisoquinoline compound or S type N The compound of formula (VII) is prepared by the first coupling reaction of the compound of formula (VI) and the compound of formula (VII) in the presence of the third solvent and light. S type aporphine alkaloid or S The compound of formula (VII) is prepared by the first coupling reaction of the compound of formula (VI) and the compound of formula (VII) in the presence of the third solvent and light. S The general formula of the compound of formula (VII) is shown in formula (VII). S

[0009] wherein R1 and / or R2 include hydrogen, alkoxy, methylenedioxy, alkyl and / or halogen; The imine reductase includes an enzyme with an amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or an enzyme with a homologous sequence of more than 80% to the amino acid sequence of SEQ ID NO: 1; the mutant includes L1-L9, the coding gene sequence of which is shown in SEQ ID NO: 3-SEQ ID NO: 11 in the sequence listing, and the mutant further includes an enzyme with a homologous sequence of more than 80% to any one of the coding gene sequences of SEQ ID NO: 3-SEQ ID NO: 11; the methylase includes an enzyme with a coding gene sequence shown in SEQ ID NO: 2 in the sequence listing or an enzyme with a homologous sequence of more than 80% to the coding gene sequence of SEQ ID NO: 2.

[0010] Further, S type N - methyl tetrahydroisoquinoline compound or S type N The compound of formula (VII) is prepared by the first coupling reaction of the compound of formula (VI) and the compound of formula (VII) in the presence of the third solvent and light.

[0011] Further, the molar ratio of the phenethylamine compound or the compound with the modified benzene ring skeleton of the phenethylamine compound and the phenylacetic acid compound or the compound with the modified benzene ring skeleton of the phenylacetic acid compound in step (1) is 1:3 or 1:5; the molar ratio of the phenethylamine compound or the compound with the modified benzene ring skeleton of the phenethylamine compound and the condensing agent is 1:3 or 1:5; the molar ratio of the phenethylamine compound or the compound with the modified benzene ring skeleton of the phenethylamine compound and the organic base is 1:3 or 1:5; and the molar ratio of the amide compound obtained in step (1) or the compound with the modified benzene ring skeleton of the amide compound and the cyclizing agent in step (2) is 1:3 or 1:5. ​

[0012] Furthermore, the reaction conditions in the first in vitro step (3) also include: using a buffer solution with pH = 6 to 8 as the reaction solution in the presence of imine reductase or its mutant; the reaction conditions in the first in vivo step also include: using a recombinant strain containing the gene encoding imine reductase or its mutant as the biotransformation strain.

[0013] Furthermore, the reaction conditions in the second in vitro step (4) also include: using a buffer solution with a pH of 6 to 8 as the reaction solution in the presence of methyltransferase and S-adenosylmethionine (SAM); the reaction conditions in the second in vivo step also include: using a recombinant strain containing a methyltransferase encoding gene as the biotransformation strain; and using formaldehyde and / or iodomethane as the methylation reagent.

[0014] Furthermore, the third solvent is hydrochloric acid or an aqueous solution of sodium hydroxide; the illumination is high-pressure mercury lamp or LED lamp; the concentration of the hydrochloric acid or sodium hydroxide aqueous solution is 0.1 mM to 8 mM.

[0015] Furthermore, ( S )-VII apophene compounds or ( S Compounds of class VII whose benzene ring skeleton has been modified are optically pure compounds.

[0016] Furthermore, imine reductase and its mutants possess the following physicochemical properties (a) to (c): (a) Reduction of IV dihydroisoquinoline compounds in an NADPH-dependent manner ( S )-V tetrahydroisoquinoline compounds; (b) Coenzyme-dependent: Using NADPH as the coenzyme for the reduction reaction, and NADP... + As a coenzyme for oxidation reactions; (c) Molecular weight: Approximately 30,000 Daltons according to sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0017] Furthermore, methyltransferases possess the following physicochemical properties (d) and (e): (d) Catalyzing tetrahydroisoquinoline compounds of formula V in a SAM-dependent manner to generate tetrahydroisoquinoline compounds of formula VI; (e) Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, the result was approximately 40,000 Daltons.

[0018] Further, in step (3), the imine reductase original gene is obtained by NCBI database BLAST method (http: / / www.ncbi.nlm.nih.gov / blast / ), the DNA sequence is optimized against E. coli, sent to Kings River Biotech Co., Ltd. for gene synthesis, and constructed on pET28a plasmid; the mutant is subjected to enzyme engineering modification on the basis of the original imine reductase; the buffer pH is 6-8.

[0019] Further, in step (4), the methyltransferase gene is synthesized by Kings River Biotech Co., Ltd., and constructed on pET28a plasmid; the buffer pH is 6-8.

[0020] Further, S The compounds with modified benzene ring skeleton of the aporphine alkaloids include compounds Ia, Ib, Ic, Id, Ie, Ig, Ih, Ii, and Ik, the benzene ring skeleton C 1、 C2, C 9、 C 10 The groups are shown in Table 1.

[0021] Table 1 VII Compounds with modified benzene ring skeleton of the aporphine compounds

[0022] In a preferred embodiment 1 of the present application, the chemical enzymatic synthesis method of optically pure (R)-VII aporphine alkaloids is described in detail; S In another preferred embodiment 2 of the present application, the in vitro synthesis process of aporphine compound Ia is described in detail; In another preferred embodiment 3 of the present application, the in vivo synthesis process of aporphine compound Ia is described in detail; In another preferred embodiment 4 of the present application, the synthesis process of aporphine compound Ib is described in detail; In another preferred embodiment 5 of the present application, the synthesis process of aporphine compound Ic is described in detail; In another preferred embodiment 6 of the present application, the synthesis process of aporphine compound Id is described in detail; In another preferred embodiment 7 of the present application, the synthesis process of aporphine compound Ie is described in detail; In another preferred embodiment 8 of the present application, the synthesis process of aporphine compound Ig is described in detail; In another preferred embodiment 9 of the present application, the synthesis process of aporphine compound Ih is described in detail; In another preferred embodiment 9 of the present application, the synthesis process of aporphine compound Ih is described in detail; In another preferred embodiment 10 of the present application, the synthesis process of the aporphine compound Ii is illustrated in detail; In another preferred embodiment 11 of the present application, the synthesis process of the aporphine compound Ik is illustrated in detail; In another preferred embodiment 12 of the present application, the determination process of the imine reductase kinetic constant is illustrated in detail; In another preferred embodiment 13 of the present application, the process of identifying the optical purity of the photocatalytic product is illustrated in detail.

[0023] The positive beneficial effects of the present application are as follows: The present application synthesizes S-type aporphine alkaloids with different substituents on the mother nucleus through one-step simple chemical synthesis, two-step enzyme catalysis and one-step photocatalysis, which has the advantages of green and mild reaction conditions, cheap and easily available raw materials, convenient post-treatment and more concise synthesis route compared with the previous methods, and is suitable for various reaction substrates. The imine reductase and methyltransferase provided by the present application have a conversion number of substrate higher than 2000, and the highest can reach 30000, and the conversion rate can reach 100%, S The stereoselectivity of type S can reach 99%, which is much higher than the previously reported methods, and reaches the industrial application level. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a chemical synthesis route map of the aporphine compound of the prior art of the present application; Figure 2 is a synthesis route map of the aporphine compound of a preferred embodiment 1 of the present application; Figure 3 is a synthesis route map of the compound IV-1 in the first step of a preferred embodiment 2 of the present application; Figure 4 is a synthesis route map of the compound IV-1 in the second step of a preferred embodiment 2 of the present application; S Figure 5 is another synthesis route map of the compound IV-1 in the second step of a preferred embodiment 2 of the present application; S Figure 6 is a synthesis route map of the compound IV-1 in the third step of a preferred embodiment 2 of the present application; S Figure 7 is a kinetic parameter and fitting curve map of the wild-type imine reductase and three mutant proteins of a preferred embodiment 12 of the present application; Figure 8 is an electrophoresis map of the wild-type imine reductase and mutant proteins of a preferred embodiment 12 of the present application. ​​​Detailed Implementation

[0025] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0026] Example 1: Chemical Enzymatic Synthesis of Apophyte Alkaloids

[0027] like Figure 2 As shown, using phenylethylamine compounds represented by chemical formula I and phenylacetic acid compounds represented by chemical formula II as raw materials, the following steps were performed sequentially: (1) amide condensation, (2) cyclization and dehydration, (3) imine reductase-catalyzed reduction, (4) methyltransferase-catalyzed or Eschweiler-Clarke methylation or iodomethane methylation, and (5) photocatalytic coupling, to synthesize ( S The apophene-like compounds shown in VII-VII.

[0028] Example 2: In vitro synthesis of apophene-like compound Ia

[0029] Step 1: Synthesis of Compound IV-1

[0030] like Figure 3 As shown, compound I-1 and compound II-1 were used to synthesize compound IV-1. Specifically, compound I-1 (60 mmol, 10.86 g) was dissolved in 60 mL of dichloromethane, compound II-1 (60 mmol, 12.9 g) was added, followed by 1-hydroxybenzotriazole (66 mmol, 8.91 g). The mixture was stirred at 0 °C for 30 min, then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (66 mmol, 12.6 g) and triethylamine (180 mmol, 18.18 g) were added. The reaction was monitored by TLC and stirred at room temperature for 6 h until completion. The organic phase was extracted by washing with saturated sodium bicarbonate water and concentrated to obtain 22.5 g of white solid (compound III-1). This solid was dissolved directly in 60 mL of dichloromethane without further treatment and stirred at 0 °C for 30 min. Phosphorus pentachloride (120 mmol, 24.96 g) was slowly added in portions and stirred at room temperature for 18 h. h, the reaction was monitored by TLC to indicate completion, 100 ml of ice-cold saturated sodium bicarbonate aqueous solution was added, and the mixture was extracted with dichloromethane (50 mL × 3 times). The organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to remove the solvent, yielding 21.29 g of a pale yellow solid (compound IV-1), with a yield of 98.1%. 1 H NMR (400 MHz, Chloroform-d) delta 7.60 (d,J = 8.0 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 15.0 Hz, 1H), 7.14 (s, 1H), 7.09 (s, 1H), 6.75 (s, 1H), 4.90 (s, 2H), 4.01 (s, 2H), 3.96 (s, 3H), 3.78 (s, 3H), 3.06 (s, 1H).

[0031] Step 2: Compound ( S Synthesis of VI-1

[0032] Compounds ( S )-VI-1 can be obtained through a first reaction, which is either a first reduction reaction followed by a methylation reaction or a first methylation reaction followed by a reduction reaction.

[0033] The first step is a reduction reaction followed by a methylation reaction, as follows: Figure 4 As shown, compound IV-1 was synthesized into compound ( S The specific steps for compound IV-1 are as follows: The reaction system is 500 μL, and the buffer is 100 mM pH 7.0 potassium phosphate buffer containing 20 mM Md-glucose, 0.2 mg / mL glucose dehydrogenase, 5 mM nicotinamide adenine dinucleotide phosphate (NADP+), 2 mM compound IV-1, and 5% (v / v) DMSO as a solubilizer. Finally, 0.2 mg / mL purified imine reductase is added, and the mixture is placed at 30℃ and 200 rpm for 24 hours. After the reaction, 30 μL of 10M NaOH is added, and the mixture is extracted three times with ethyl acetate (1 mL × 3). The ethyl acetate phases are combined, washed with saturated saline, dried with anhydrous magnesium sulfate for 1 hour, filtered, and the filtrate is evaporated to obtain compound IV-1. S The transformation number of substrate IV-1 reached 14000; subsequently, 2 mM potassium phosphate buffer was added to a 500 μL reaction system containing 100 mM pH 7.0 potassium phosphate buffer. S -Sysyladenosylmethionine (SAM), 2 mM compound ( S The compound was prepared by adding 5% (v / v) of DMSO as a co-solvent, followed by 0.2 mg / mL of purified methyltransferase. The mixture was then incubated at 30°C and 200 rpm for 24 hours until the substrate transformation number reached 3000. After the reaction, 30 μL of 10M NaOH was added, and the mixture was extracted three times with ethyl acetate (1 mL × 3). The combined ethyl acetate phases were washed with saturated brine, dried over anhydrous magnesium sulfate for 1 hour, and the solvent was evaporated to obtain the compound (V-1).S )-VI-1. 1 H NMR (700 MHz, Chloroform-d) delta 7.55 (dd, J = 8.0, 1.3 Hz, 1H), 7.17(td, J = 7.5, 1.3 Hz, 1H), 7.07 (td, J = 7.6, 1.8 Hz, 1H), 6.99 (dd, J = 7.6,1.8 Hz, 1H), 6.58 (s, 1H), 5.92 (s, 1H), 3.91-3.88 (m, 1H), 3.84 (s, 3H),3.50 (s, 3H), 3.33-3.27 (m, 2H), 2.95-2.90 (m, 2H), 2.84 (ddd, J = 9.1, 6.2,3.0 Hz, 1H), 2.62 (ddd, J = 16.2, 5.3, 3.1 Hz, 1H), 2.54 (s, 3H)。

[0034] The first methylation reaction followed by reduction is shown in Figure 5 Scheme 1. The specific steps for the synthesis of compound S -VI-1 from compound IV-1 are as follows: the reaction system is 500 μL, the buffer is 100 mM pH 7.0 potassium phosphate buffer, which contains 2 mM SS-adenosylmethionine (SAM), 2 mM compound IV-1, and 5% (v / v) DMSO cosolvent were added, followed by 0.2 mg / mL purified methyltransferase. The mixture was incubated at 30°C and 200 rpm for 24 hours until the substrate transformation number reached 2000. After the reaction, 30 μL of 10M NaOH was added, and the mixture was extracted three times with ethyl acetate (1 mL × 3). The ethyl acetate phases were combined, washed with saturated brine, and dried over anhydrous magnesium sulfate for 1 hour to obtain compound V'-1. Subsequently, in a 500 μL reaction system with 100 mM pH 7.0 potassium phosphate buffer, 20 mM D-glucose, 0.2 mg / mL glucose dehydrogenase, 5 mM nicotinamide adenine dinucleotide phosphate (NADP+), 2 mM compound IV'-1, and 5% (v / v) DMSO cosolvent were added, followed by 0.2 mg / mL purified imine reductase. The mixture was incubated at 30°C and 200 rpm. After 24 hours of reaction, the substrate transformation number reached 5000. After the reaction was complete, 30 μL of 10M NaOH was added, and the mixture was extracted three times with ethyl acetate (50 mL × 3), 300 μL each time. The combined ethyl acetate phases were washed with saturated brine, dried over anhydrous magnesium sulfate for 1 hour, filtered, and the filtrate was evaporated to dryness to obtain the compound (…). S )-VI-1

[0035] Step 3: Synthesis of compound (S)-VII-1

[0036] like Figure 6 As shown, the compound ( S )-VI-1 synthetic compound ( S Formula VI-1, specifically: 50 ml of 3 mol / L hydrochloric acid solution was added to a quartz container, followed by the addition of compound VI-1 (0.5 mmol, 0.19 g) and 5 ml of methanol. The mixture was irradiated with a high-pressure mercury lamp for 7 h. After the reaction, 1 M sodium hydroxide solution was added to adjust the pH to weakly alkaline. The mixture was extracted three times with ethyl acetate (50 mL × 3). The ethyl acetate phases were combined and dried over anhydrous magnesium sulfate for 30 min. The mixture was filtered, and the filtrate was evaporated to dryness to remove the solvent, yielding a white solid. S 0.141 g of compound VII-1, yield 95%. 1 H NMR (700 MHz, Acetone-d6) delta 8.30 (dd, J =8.0, 1.2 Hz, 1H), 7.32-7.29 (m, 2H), 7.23 (td, J= 7.4, 1.3 Hz, 1H), 6.78 (s,1H), 3.88 (s, 3H), 3.65 (s, 3H), 3.17 (dd, J = 13.8, 4.0 Hz, 1H), 3.10-3.04(m, 1H), 3.00 (ddd, J = 11.4, 6.0, 1.3 Hz, 1H), 2.90-2.86 (m, 1H), 2.67 (dd, J = 15.9, 3.5 Hz, 1H), 2.49 (s, 3H), 2.47-2.32 (m, 2H)。

[0037] Example 3 In vivo synthesis of aporphine compound Ia

[0038] Compound IV-1 (14 mmol, 5.03 g) was dissolved in 20 ml of dimethyl sulfoxide, 200 g of overexpressed L9 mutant protein was added, 250 ml of M9 salt solution was added, and the mixture was incubated at 30°C for 72 h on a shaker. Then 250 ml of methyltransferase resuspended in M9 salt solution was added, and the mixture was incubated at 30°C for another 72 h on a shaker. After the reaction was completed, 30 ml of 10 M NaOH solution was added, and the mixture was extracted with ethyl acetate (200 mL x 3) three times. The organic phase was combined, dried, dissolved in 50 ml of methanol, and placed in a quartz container. 150 ml of 3 mol / L hydrochloric acid solution was added, and the mixture was irradiated with a 365 nm wavelength high pressure mercury lamp (100 W) for 7 h. After the reaction was completed, 1 M sodium hydroxide solution was added to adjust the solution to weak alkaline, and the mixture was extracted with ethyl acetate (100 mL x 3) three times. The ethyl acetate phase was combined and dried with anhydrous magnesium sulfate for 30 min. The filtrate was filtered and the solvent was removed by rotary evaporation to obtain white solid compound (compound VII-1) with a mass of 3.42 g and a yield of 96%. S

[0039] Example 4 Synthesis of aporphine compound Ib

[0040] Referring to the in vitro synthesis method of compound (S)-VII-1 in Example 2, compound II-1 was replaced with 2-bromo-4,5-methoxyphenylacetic acid, and the other conditions were unchanged. Yellow solid (compound Ib) 1.91 g was obtained with a yield of 93%. 1 H NMR (600MHz, MeOH) delta ​7.76 (s, 1H), 6.98 (s, 1H), 6.72 (s, 1H), 4.46 (m, 2H), 3.91 (s,3H), 3.88 (s, 3H), 3.73 (s,3H), 3.40 (s, 3H), 3.28 (m, 2H), 3.25 (td,1H),3.12 (dd, 1H), 3.02 (dd, 1H), 2.94 (s, 3H)。

[0041] Example 5 Synthesis of aporphine compound Ic

[0042] Referring to the in vitro synthesis method of compound (S)-VII-1 of Example 2, compound II-1 was replaced with 2-bromo-4-methoxyphenylacetic acid, and other conditions were unchanged. White solid (compound Ic) 1.41 g was obtained, with a yield of 95%. 1 H NMR (600 MHz, Chloroform-d) delta 7.17 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 2.7 Hz, 1H), 6.93 (s,1H), 6.75 (dd, J = 8.6, 2.7 Hz, 1H), 4.11 (m, 2H), 3.88 (s, 3H), 3.81 (s,3H), 3.75 (s, 3H), 3.73 – 3.70 (m, 2H), 2.98 (m, 2H), 2.64 (s, 3H)。

[0043] Example 6 Synthesis of aporphine compound Id

[0044] Referring to the in vitro synthesis method of compound (S)-VII-1 of Example 2, compound II-1 was replaced with 2-bromo-4,5-methylenedioxyphenylacetic acid, and other conditions were unchanged. Light yellow solid (compound Id) 1.33 g was obtained, with a yield of 93%. 1 H NMR (600 MHz, Chloroform-d) delta 7.93 (s, 1H), 6.75 (s, 1H), 6.59 (s, 1H), 5.97 (s,2H), 3.87 (s, 3H), 3.66 (s, 3H), 3.20– 2.43 (m, 7H), 2.52 (s, 3H)。

[0045] Example 7 Synthesis of aporphine compound Ie

[0046] The in vitro synthesis of compound (S)-VII-1 of Example 2 was followed, replacing compound I-1 with 3,4-methylenedioxyphenethylamine, and without further modification. A yellowish solid (compound Ie) 1.04 g was obtained in 93% yield. 1 H NMR (600MHz, Acetonitrile-d3) delta 8.05 (dd, J = 7.8, 1.4 Hz, 1H), 7.37-7.32 (m, 1H),7.31 (dt, J = 5.4, 1.4 Hz, 1H), 7.26 (td, J = 7.4, 1.3 Hz, 1H), 6.61 (s, 1H),6.01 (dt, J = 76.1, 1.3 Hz, 2H), 3.23 (dd, J = 14.3, 4.3 Hz, 1H), 3.06-3.02(m, 1H), 3.01-2.96 (m, 2H), 2.66-2.59 (m, 1H), 2.55-2.49 (m, 1H), 2.48 (d, J = 1.4 Hz, 3H), 2.45-2.38 (m, 1H)。

[0047] Example 8 Synthesis of aporphine compound Ig

[0048] The in vitro synthesis of compound (S)-VII-1 of Example 2 was followed, replacing compound I-1 with 3,4-methylenedioxyphenethylamine, and replacing compound II-1 with 2-bromo-5-methoxyphenylacetic acid, and without further modification. A brown solid (compound Ie) 1.22 g was obtained in 94% yield. 1 H NMR (400 MHz, Chloroform-d) delta 8.00 (d, J = 9.5, 1H),6.85 (dd, J = 9.5, 1H), 6.80 (d, 1H), 6.52 (s, 1H), 6.05 (s, 2H), 3.87 (s,3H), 3.10-3.00 (m, 2H), 2.89-2.83 (m, 1H), 2.57 (s, 3H), 2.47-2.32 (m, 2H).

[0049] Example 9 Synthesis of aporphine compound Ih

[0050] The in vitro synthesis method of compound (S)-VII-1 in Example 2 was referred to, compound I-1 was replaced with 3,4-methylenedioxyphenethylamine, compound II-1 was replaced with 2-bromo-4,5-dimethoxyphenylacetic acid, and other conditions were unchanged. White solid (compound Ih) 1.18 g was obtained with a yield of 93%. 1 H NMR (600 MHz, Chloroform-d) delta 7.67 (s, 1H),6.78 (s, 1H), 6.52 (s, 1H), 6.08 (d, J = 1.4 Hz, 1H), 5.93 (d, J = 1.4 Hz,1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.16-2.61 (m, 4H), 2.68-2.61 (m, 2H), 2.55(s, 3H), 2.52-2.48 (m, 1H)。

[0051] Example 10 Synthesis of aporphine compound Ii

[0052] The in vitro synthesis method of compound (S)-VII-1 in Example 2 was referred to, compound I-1 was replaced with 3,4-methylenedioxyphenethylamine, compound II-1 was replaced with 2-bromo-4-dimethoxyphenylacetic acid, and other conditions were unchanged. Light yellow solid (compound Ih) 1.23 g was obtained with a yield of 94%. 1 H NMR (600 MHz, Chloroform-d) δ 7.98 (d, J = 9.5,1H), 6.85 (dd, J = 9.5, 1H), 6.79 (d, 1H), 6.50 (s, 1H), 6.05 (s, 2H),3.57 (s, 3H), 3.04-3.00 (m, 2H), 2.89-2.86 (m, 1H), 2.57 (s, 3H), 2.27-2.12(m, 2H)。

[0053] Example 11 Synthesis of aporphine compound Ik

[0054] The in vitro synthesis method of compound (S)-VII-1 in Example 2 was referred to, compound I-1 was replaced with 3,4-methylenedioxyphenethylamine, compound II-1 was replaced with 2-bromo-4,5-methylenedioxyphenylacetic acid, and other conditions were unchanged. Light yellow solid (compound Ih) 1.35 g was obtained with a yield of 93%. 1H NMR (400 MHz, Chloroform-d) delta 7.58 (s, 1H), 6.71 (s, 1H) , 6.48 (s, 1H), 6.02 (d, J = 2.6 Hz, 1H) , 6.01 (dd, 2H) , 5.88(d, J = 2.6 Hz, 1H), 3.17 (m, 4H), 2.76 (s, 1H), 2.64 (m, 4H), 2.57 (s, 3H).

[0055] Example 12 Determination of the kinetic constant of imine reductase

[0056] The catalytic efficiency of the enzyme was determined by measuring the rate of NADPH consumption per unit time. The concentration of compound IV varied from 0.001 mM to 2 mM, while the NADPH concentration was maintained at 1 mM. The appropriate amount of enzyme was wild-type imine reductase or one of its three mutants (L4, L6, or L9). 100 mM (pH 7.0) KPB buffer was added to a total volume of 1 mL. The reaction was carried out at a constant temperature of 30 °C, with one point taken every second for 1 min. A curve was plotted, and the specific enzyme activity was calculated. The kinetic constant K was obtained by fitting the Michaelis constant equation using OriginPro 9.0 software. m and V max According to V max Calculate k cat value and k cat / K m Value. For example... Figure 7 The kinetic parameters and fitting curves of wild-type imine reductase and three mutant proteins are shown in the figure. Part A represents the wild-type imine reductase, Part B represents the L4 mutant, Part C represents the L6 mutant, and Part D represents the L9 mutant. The horizontal axis represents reaction time, and the vertical axis represents specific enzyme activity. m K is the characteristic constant of an enzyme, representing the concentration of the substrate when the reaction rate reaches half of its maximum rate. It reflects the affinity between the enzyme and the substrate. m The smaller the size, the greater the affinity; V max This refers to the maximum reaction rate; k cat It indicates the amount of substrate converted by one mole of enzyme per unit time. k cat The larger the value, the faster the enzyme converts the substrate; k cat / K mThe ratio is an important parameter for measuring the catalytic efficiency of the enzyme, and the larger the ratio, the better the catalytic efficiency of the enzyme.

[0057] The sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis was used to detect the electrophoretogram of the expressed wild-type imine reductase and mutant protein. As shown in Figure 8 Figure 2, M is a two-color pre-stained protein marker (Marker) with a size of 10 KD-250 KD. Lanes 2-11 are the 10 imine reductase proteins L1-L9 expressed and purified by the present application.

[0058] Example 13 Optical purity identification of photocatalytic product

[0059] The optical purity of the obtained compound VII was identified by the in vitro synthesis method of Reference Example 1, and the optical purity was analyzed by normal phase HPLC.

[0060] Instrument: Agilent 1260 Infinity Series HPLC System

[0061] Column: Daicel CHIRALPAK ® OD-H 250 mm × 4.6 mm, 5 μm.

[0062] Mobile phase: Mobile phase A n-hexane: Mobile phase B isopropyl alcohol

[0063] Flow rate: 0.5 mL / min

[0064] Detection wavelength: 280 nm or 320 nm

[0065] The liquid chromatography conditions used for optical purity analysis of the photocatalytic product are shown in Table 2: Table 2 Liquid chromatography conditions used for optical purity analysis of the photocatalytic product and R Retention time of the photocatalytic product of type S Retention time of the photocatalytic product of type

[0066] The above detailed the preferred embodiments of the present application. It should be understood that those skilled in the art can make many modifications and changes without creative labor based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment based on the prior art according to the concept of the present application shall be within the protection scope determined by the claims.

Claims

1. A method for the chemoenzymatic synthesis of S- aporphine alkaloids, characterized in that, The method steps are as follows: Step (1) taking phenethylamine compounds and phenylacetic acid compounds as raw materials, amide condensation reaction is carried out to obtain amide compounds or compounds with modified benzene ring skeleton of the amide compounds; Step (2) taking the amide compounds obtained in step (1) or the compounds with modified benzene ring skeleton of the amide compounds as the second raw material, cyclization dehydration reaction is carried out to obtain dihydroisoquinoline compounds or compounds with modified benzene ring skeleton of the dihydroisoquinoline compounds; the general formula of the dihydroisoquinoline compounds is shown in chemical formula IV; Step (3) is to use the dihydroisoquinoline compound obtained in step (2) or a compound in which the benzene ring skeleton of the dihydroisoquinoline compound is modified as a third raw material, and to obtain a compound of formula (I) or a compound in which the benzene ring skeleton of the compound of formula (I) is modified by a reduction reaction in the presence of an imine reductase or a mutant thereof in a first in vivo or a first in vitro S The compound of formula (I) or the compound in which the benzene ring skeleton of the compound of formula (I) is modified is a compound of formula (I) S The compound of formula (I) or the compound in which the benzene ring skeleton of the compound of formula (I) is modified is a compound of formula (I) S The general formula of the compound of formula (I) or the compound in which the benzene ring skeleton of the compound of formula (I) is modified is shown in the chemical formula (I) S )-V Step (4) is the compound obtained in step (3) S the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) N the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) N the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) N the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) N the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) N the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) S the compound of formula (I) or the compound of formula (II) wherein R1 and / or R2 include hydrogen, alkoxy, methylenedioxy, alkyl and / or halogen; The imine reductase includes an enzyme with an amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or an enzyme with a homologous sequence of more than 80% to the amino acid sequence of SEQ ID NO: 1; the mutant includes L1-L9, the coding gene sequence of which is shown in SEQ ID NO: 3-SEQ ID NO: 11 in the sequence listing, and the mutant also includes an enzyme with a homologous sequence of more than 80% to any one of the coding gene sequences of SEQ ID NO: 3-SEQ ID NO: 11; the methylase includes an enzyme with a coding gene sequence shown in SEQ ID NO: 2 in the sequence listing or an enzyme with a homologous sequence of more than 80% to the coding gene sequence of SEQ ID NO:

2.

2. The production method according to claim 1, wherein The S type N - methyltetrahydroisoquinoline compounds or the S type N - compounds in which the benzene ring skeleton of the methyltetrahydroisoquinoline compound is modified are subjected to the first coupling reaction as the fifth raw material without purification.

3. The production method according to claim 1, wherein In step (1), the molar ratio of the phenethylamine compounds or the compounds with modified benzene ring skeleton of the phenethylamine compounds to the phenylacetic acid compounds or the compounds with modified benzene ring skeleton of the phenylacetic acid compounds is 1:3 or 1:5; the molar ratio of the phenethylamine compounds or the compounds with modified benzene ring skeleton of the phenethylamine compounds to the condensing agent is 1:3 or 1:5; the molar ratio of the phenethylamine compounds or the compounds with modified benzene ring skeleton of the phenethylamine compounds to the organic base is 1:3 or 1:5; in step (2), the molar ratio of the amide compounds obtained in step (1) or the compounds with modified benzene ring skeleton of the amide compounds to the cyclizing agent is 1:3 or 1:

5.

4. The production method according to claim 1, wherein In step (3), the first in-vitro reaction condition further includes: taking a buffer solution with pH = 6-8 as the reaction solution in the presence of the imine reductase or its mutant; the first in-vivo reaction condition further includes: taking a recombinant strain containing the coding gene of the imine reductase or its mutant as the bioconversion strain.

5. The production method according to claim 1, wherein In step (4), the second in-vitro reaction condition further includes: taking a buffer solution with pH of 6-8 as the reaction solution in the presence of the methylase and S-adenosyl methionine (SAM); the second in-vivo reaction condition further includes: taking a recombinant strain containing the coding gene of the methylase as the bioconversion strain; and the methylating agent is formaldehyde and / or iodomethane.

6. The production method according to claim 1, wherein The third solvent is hydrochloric acid or sodium hydroxide aqueous solution; the light irradiation is high-pressure mercury lamp or LED lamp irradiation; the concentration of the hydrochloric acid or sodium hydroxide aqueous solution is 0.1 mM-8 mM. The third solvent is hydrochloric acid or sodium hydroxide aqueous solution; the light irradiation is high-pressure mercury lamp or LED lamp irradiation; the concentration of the hydrochloric acid or sodium hydroxide aqueous solution is 0.1 mM-8 mM.

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