CN112442523B - A method for preparing (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and its derivatives by enzymatic separation - Google Patents

Abstract

The present invention discloses a novel method for preparing (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and its derivatives by enzymatic separation. The method comprises the following steps: using racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (1) or racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (2) as a substrate, using an isolated L-piperidinic acid oxidase or a cell expressing L-piperidinic acid oxidase in a cell as a catalyst, selectively catalyzing the reaction of (S)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid with the oxidase. The method is an oxidative dehydrogenation reaction of (R)-1,2,3,4-tetrahydroisoquinoline-1-formic acid or (S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-formic acid, wherein (R)-1,2,3,4-tetrahydroisoquinoline-1-formic acid or (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-formic acid is not catalyzed and remains in the reaction system, thereby preparing (R)-1,2,3,4-tetrahydroisoquinoline-1-formic acid or (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-formic acid. The method has high conversion rate, ee value can reach more than 99%, and has the characteristics of mild reaction conditions, strong stereoselectivity, high reaction efficiency, relatively simple process, etc.

Description

A method for preparing (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and its derivatives by enzymatic separation

Technical Field

The invention belongs to the technical field of biocatalysis, and specifically relates to a new method for preparing (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and its derivatives by enzymatic separation.

Background technique

Optically pure 1,2,3,4-tetrahydroisoquinoline compounds are an important class of chiral building blocks that are widely used in the synthesis of a variety of drugs. For example, (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is an important chiral intermediate for the synthesis of the broad-spectrum antiparasitic drug 1-praziquantel (Chinese Patent 201310487924.4).

In the prior art, there are two methods for preparing optically pure (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid: chemical chiral synthesis and biocatalytic chiral resolution. The chemical chiral synthesis method synthesizes (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid from chiral raw materials. For example, Kurata et al. used optically pure olefin isoquinoline as the starting material and synthesized (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in three steps by ozone decomposition and _NaBH4 in-situ reduction, tetramethylpiperidine nitrogen oxide (TEMPO) oxidation, and trifluoroacetic acid-mediated N-tert-butyloxycarbonyl deprotection (Synthesis of Optically Pure (R)-and (S)-Tetrahydroisoquinoline-1-and-3-Carboxylic Acids [J]. Synthesis, 2015, 47 (09): 1238-44.). This method has low yield and cumbersome steps and is not suitable for industrial application. Paál et al. used lipase to dynamically kinetically hydrolyze 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid ethyl ester to prepare (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, with 17.74 g/L substrate, 20 mg/mL lipase, 3°C, 24 hours of reaction, 85% yield, and 98% product ee value (Directed (R)-or (S)-Selective Dynamic Kinetic Enzymatic Hydrolysis of 1,2,3,4-Tetrahydroisoquinoline-1-carboxylic Esters [J]. European Journal of Organic Chemistry, 2008, 2008 (31): 5269-76). This method has mild reaction conditions, strong stereoselectivity, and relatively simple process, but the reaction efficiency still needs to be further improved.

In the prior art, the method for preparing optically pure (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is mainly a biocatalytic chiral resolution method. Paál et al. used lipase to dynamically kinetically hydrolyze 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid ethyl ester to prepare (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid. The substrate was 53.46 g/L, the enzyme dosage was 100 mg/mL lipase, the reaction time was 7 hours at 25°C and pH 8.5, the yield was 85%, and the product ee value was 92% (Directed (R)-or (S)-Selective Dynamic Kinetic Enzymatic Hydrolysis of 1,2,3,4-Tetrahydroisoquinoline-1-carboxylic Esters [J]. European Journal of Organic Chemistry, 2008, 2008 (31): 5269-76). This method has mild reaction conditions, strong stereoselectivity, and a relatively simple process, but the optical purity of the obtained product needs to be further improved.

Summary of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a new method for preparing (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and its derivatives. The method has the characteristics of mild reaction conditions, strong stereoselectivity, high reaction efficiency, relatively simple process, etc., and has industrial application prospects.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

A method for preparing a compound as shown in formula (I) by enzymatic resolution,

In formula (I), R ¹ and R ² are independently selected from hydrogen, C ₁ -C ₆ alkyl, and C ₁ -C ₆ alkoxy. The method comprises:

(1) using the racemate of the compound of formula (I) or the racemate of the salt of the compound of formula (I) as a substrate, using in vitro L-piperidinic acid oxidase or cells expressing L-piperidinic acid oxidase intracellularly as a catalyst, selectively catalyzing the oxidative dehydrogenation reaction of the (S) isomer of the compound of formula (I), while the compound of formula (I) is not catalyzed and remains in the reaction system;

(2) Separating the compound of formula (I) from the reaction system to obtain.

Furthermore, in formula (I), R ¹ and R ² are independently selected from hydrogen, methyl, ethyl, isopropyl, methoxy or ethoxy.

Preferably, the salt is a monovalent salt, and more preferably an alkali metal salt or an ammonium salt, wherein the alkali metal salt may be, for example, a lithium salt, a sodium salt, or a potassium salt.

According to some preferred and specific aspects of the present invention, the compound described by formula (I) is (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid.

According to the present invention, the L-pipecolic acid oxidase is preferably a combination of one or more of the following L-pipecolic acid oxidases: L-pipecolic acid oxidase derived from Pseudomonas putida KT2440 or its mutant or an L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, L-pipecolic acid oxidase derived from Pseudomonas aeruginosa PAO1 or its mutant or an L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, L-pipecolic acid oxidase derived from Pseudomonas entomophila str. L48 or its mutant or an L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, L-pipecolic acid oxidase derived from Aspergillus oryzae oryzae) RIB40 or its mutants or an L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, L-pipecolic acid oxidase derived from Schizosaccharomyces pombe or its mutants or an L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%.

Further preferably, the L-piperidinic acid oxidase has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

According to some specific and preferred aspects of the present invention, the catalyst is a crude enzyme solution containing the isolated L-piperidinic acid oxidase, or cells expressing the L-piperidinic acid oxidase intracellularly, or a pure enzyme of the L-piperidinic acid oxidase, or an immobilized enzyme of the L-piperidinic acid oxidase.

As a preferred embodiment of the present invention: the cell is an engineered bacterium expressing L-piperidinic acid oxidase, and the host cell of the engineered bacterium is E. coli BL21 (DE3).

Specifically, the engineered bacteria contains an expression vector pET-28a(+), and the L-piperidinic acid oxidase gene is connected to the expression vector pET-28a(+).

According to some specific and preferred aspects of the present invention, the amount of the catalyst added is based on the wet weight of cells after centrifugation at 8000 rpm for 10 min, and the amount of cells added is 1-5% of the weight of the reaction system.

According to some specific and preferred aspects of the present invention, the oxidative dehydrogenation reaction is carried out in an aerobic environment, the oxidative dehydrogenation reaction also generates hydrogen peroxide, and the method further comprises adding catalase for catalytically decomposing the hydrogen peroxide to the reaction system at one or more time points before, during and after the oxidative dehydrogenation reaction.

Furthermore, the catalase is bovine liver catalase lyophilized powder. According to a specific aspect of the present invention, the enzyme activity of the bovine liver catalase lyophilized powder is 4000 U/mg.

According to some preferred aspects of the present invention, the enzyme activity ratio of the catalase to the L-pipecolic acid oxidase is 100-400:1.

Furthermore, in step (1), a reaction system is first constructed, and then the reaction system is controlled to be in a set temperature and an aerobic environment to carry out the oxidative dehydrogenation reaction, wherein the reaction system comprises the substrate, the solvent, the catalyst and selectively a catalase for catalytically decomposing hydrogen peroxide, and also selectively comprises a pH buffer and/or a pH regulator.

According to a preferred aspect of the present invention, the solvent is water, the substrate is first dissolved in the aqueous solution of the pH buffer, the pH adjuster is selectively added, a substrate solution with a pH value of 6 to 9 is prepared, and then the catalyst and the catalase are added to obtain the reaction system. More preferably, the pH value of the substrate solution is controlled to be 7 to 8.

According to a specific and preferred aspect of the present invention, the pH buffer is a phosphate, which can be dissolved in water to prepare a phosphate buffer solution.

According to some preferred aspects of the present invention, the pH adjuster is preferably ammonia water, alkali metal hydroxide or an aqueous solution thereof.

According to a specific and preferred aspect of the present invention, the pH regulator is 20 wt% to 35 wt% ammonia water.

According to another specific aspect of the present invention, the pH adjuster is an aqueous solution of sodium hydroxide or potassium hydroxide.

According to some specific and preferred aspects of the present invention, in step (1), the concentration of the starting substrate in the reaction system is controlled to be 1 to 20 g/L.

According to a preferred aspect of the present invention, the set temperature is 20-70°C. More preferably, the set temperature is 30-50°C.

Furthermore, in step (2), the pH value of the reaction system is adjusted to 5.0-6.0, and the protein is heated to denature and precipitate, and then filtered. After the filtrate is concentrated, it is cooled to crystallize and dried to obtain the compound represented by formula (I).

Due to the implementation of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

During the research process of the present invention, it was unexpectedly found that L-piperidinic acid oxidase can efficiently and selectively catalyze (S)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid for oxidative dehydrogenation reaction, while it has basically no catalytic effect on (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid. The method of the present invention is used to prepare (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, with high reaction efficiency, high conversion rate, mild reaction conditions, strong stereoselectivity (ee value can reach more than 99%), and simple process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a high performance liquid chromatography detection spectrum of a sample taken at 0 hours in the reaction system in Example 3, wherein the retention time 8.923 min is (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid; the retention time 13.282 min is (S)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid;

FIG. 2 is a high performance liquid chromatography detection spectrum of samples taken after 24 hours of reaction in the reaction system in Example 3.

Detailed ways

The invention provides a new method for preparing (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof. The method comprises the following steps: using racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid as a substrate (or an ammonium salt, etc.) and utilizing an isolated L-piperidinic acid oxidase or a cell expressing L-piperidinic acid oxidase in the cell as a catalyst to carry out an oxidative dehydrogenation reaction to obtain (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid.

The specific principle is: using racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (1) or racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (2) as substrate, L-piperidinic acid oxidase is used to stereoselectively catalyze the oxidative dehydrogenation of (S)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid to generate the corresponding imidic acid, while (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is not catalyzed and remains in the reaction system. The hydrogen peroxide generated during the catalytic process is decomposed into water and oxygen under the catalysis of catalase. The reaction process is schematically shown as follows:

Preferably, the L-pipecolic acid oxidase is derived from Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas spp., Aspergillus oryzae, or Schizosaccharomyces pombe. Specifically, the L-pipecolic acid oxidase is derived from the L-pipecolic acid oxidase of Pseudomonas putida KT2440 or its mutant or the L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, the L-pipecolic acid oxidase derived from Pseudomonas aeruginosa PAO1 or its mutant or the L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, the L-pipecolic acid oxidase derived from Pseudomonas sentomophila str. L48 or its mutant or the L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, the L-pipecolic acid oxidase derived from Comamonas testosterone ATCC 11996 or its mutant or the L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, the L-pipecolic acid oxidase derived from Aspergillus oryzae oryzae) RIB40 or its mutants or an L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%, L-pipecolic acid oxidase derived from Schizosaccharomyces pombe or its mutants or an L-pipecolic acid oxidase with an amino acid sequence homology greater than 80%.

Preferably, the cell is an engineered bacterium expressing L-piperidinic acid oxidase, and the host cell of the engineered bacterium is E. coli BL21 (DE3). Specifically, the engineered bacterium contains an expression vector pET-28a (+), and the L-piperidinic acid oxidase gene is connected to the expression vector pET-28a (+).

In the reaction system, the catalyst can be used in the form of crude enzyme solution, or resting cells of engineered bacteria expressing recombinant enzyme, or pure enzyme, or immobilized enzyme. Catalase can be used in the form of freeze-dried powder.

Preferably, the concentration of the substrate racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the catalytic system is 1 to 20 g/L.

As a preference and specifically, in the catalytic system, the amount of catalyst added is based on the wet weight of cells after centrifugation at 8000 rpm for 10 min, and the amount of cells added is 1-5% of the weight of the reaction solution.

Preferably, in the reaction system, the enzyme activity ratio of catalase to L-piperidinic acid oxidase is 100-400:1.

As a specific aspect, the catalase is bovine liver catalase freeze-dried powder with an enzyme activity of 4000 U/mg.

Preferably, in the catalytic system, the reaction temperature is 20-70°C, the reaction time is 6-72 hours, and the pH value of the reaction solution is 6-9; more preferably, the temperature is 30-50°C, the reaction time is 12-48 hours. The phosphate buffer solution controls the reaction pH to 7-8.

The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

The experimental methods in the embodiments of the present invention are all conventional methods unless otherwise specified.

The genes used in the examples of the present invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. E. coli BL21 (DE3) strains were purchased from Novagen; DNA marker, PrimeStar DNA polymerase, low molecular weight standard protein and other molecular biology experimental reagents were purchased from TaKaRa. For specific operations of gene cloning and expression, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

The present invention analyzes the various products and substrates of the catalytic reaction by high performance liquid chromatography (HPLC). The HPLC analysis method of racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is: chromatographic column/ ZWIX(-); column temperature/25°C; flow rate/0.4 mL/min; detection wavelength/UV220 nm; mobile phase: HPLC grade methanol (with 50 mM formic acid and 25 mM diethylamine added). The specific peaks of the relevant substances are shown in Figure 1.

Example 1 Construction of genetically engineered bacteria

1.1 Screening of L-piperidinic acid oxidase and construction of genetically engineered bacteria expressing L-piperidinic acid oxidase

L-piperidinic acid oxidases from Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas entomophila str. L48, Aspergillus oryzae RIB40 and Schizosaccharomyces pombe were cloned respectively (as shown in Table 1).

Table 1 L-piperidinic acid oxidase from three different sources

PCR upstream primers and downstream primers were designed according to the corresponding gene DNA sequence.

Primers for L-pipecolic acid oxidase from Pseudomonas putida KT2440:

KT2440-F: 5'-GGAATTC CATATG GAGCCGGCCATGTCCGAACTG-3' (Nde I)

KT2440-R: 5'-CCC AAGCTT TTACAACACAACCCTCAGGCAC-3' (Hind III)

Primers for L-pipecolic acid oxidase from Pseudomonas aeruginosa PAO1:

PAO1-F: 5'-CG GGATCC ATGGGCGGTTTGCAGCAACGGTG-3' (BamH I)

PAO1-R: 5'-CCC AAGCTT CTAGAGAACCACGCGCAAGC-3' (Hind III)

Primers for L-pipecolic acid oxidase from Pseudomonas entomophila str.L48:

L48-F: 5'-CG GGATCC ATGTCCGAACTGCGTCAAGAATGTC-3' (BamH I)

L48-R: 5'-CCC AAGCTT CTACAACACCACCCGCAAACACTG-3' (Hind III)

Restriction endonuclease sites were added to the upstream and downstream primers, as shown by the underline. The specific restriction endonucleases are shown in the brackets of the primer sequences. The genomic DNA of Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, and Pseudomonas entomophila str. L48 were used as templates, and PCR amplification reactions were performed using the corresponding upstream and downstream primers. The PCR reaction system and reaction conditions are as follows:

PCR amplification system:

PCR amplification conditions:

1) Pre-denaturation: 95℃ for 5min;

2) Denaturation: 95°C for 10 s; annealing: 58°C for 15 s; extension: 72°C for 10 s; 35 cycles in total;

3) Extension: 72°C for 10 min;

4) Keep warm at 4℃.

After the PCR amplification reaction was completed, the amplification results were detected by 1.0% agarose gel electrophoresis, and the results showed that the amplified product was a single band with a size of about 1000 bp. The target band was recovered using a DNA recovery and purification kit, and the specific steps were referred to the purification kit instructions.

The expression vector pET-28a(+) and the PCR amplification product were double-digested with the corresponding restriction endonucleases. After the digestion was completed, the target band was recovered using a DNA recovery and purification kit. Subsequently, the PCR amplification product after double digestion was connected to the expression vector pET-28a(+) with the corresponding sticky ends using T4 DNA ligase. The connection system is shown in Table 2 below:

Table 2 Recombinant expression plasmid connection system

The enzyme-linked product was transformed into E.coli DH5a competent cells, plated, and single colonies were picked and cultured in LB liquid medium. The positive transformants were identified by bacterial liquid PCR, and sent to a sequencing company to verify the correctness of the inserted sequence. The plasmid was extracted from the verified positive transformants. The relevant method refers to the plasmid extraction kit. The recombinant expression vector was then transferred into the expression host E.coli BL21 (DE3). After verification by bacterial liquid PCR and sequencing, glycerol with a final concentration of 25% was added to the obtained engineering bacterial liquid and stored at -80°C for later use.

For L-piperidinic acid oxidase from Aspergillus oryzae RIB40 and Schizosaccharomyces pombe, their gene sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis after codon optimization, and cloned into plasmid pET-28a(+) respectively. The recombinant plasmid was transferred into the expression host E. coli BL21 (DE3), and after sequencing verification, glycerol with a final concentration of 25% was added to the obtained engineering bacterial solution and stored at -80°C for later use.

Example 2

2.1 Cultivation of microorganisms

Liquid LB medium composition: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in deionized water and fixed to volume, sterilized at 121℃ for 20min, and set aside. If it is solid LB medium, add 15g/L agar.

The engineered bacteria containing the L-piperidinic acid oxidase gene were inoculated into 5 mL of liquid LB (containing 50 μg/ml kanamycin) medium, and cultured at 37°C and 200 rpm for about 8 hours. The inoculation amount was inoculated into 50 mL of liquid LB (containing 50 μg/ml kanamycin) medium at 1% (V/V) and cultured. After OD ₆₀₀ reached 0.6-0.8, the inducer IPTG (final concentration of 0.1 mM) was added and induced at 18°C for 15 hours. After the culture was completed, the culture solution was poured into a 100 mL centrifuge tube and centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, the bacterial cells were collected, and the cells were washed twice with 50 mM phosphate buffer (pH 8.0), and then stored in a -80°C ultra-low temperature refrigerator for use.

2.2 Preparation of crude enzyme solution

The bacterial suspension was resuspended in 50 mM phosphate buffer (pH 8.0) or deionized water, and the bacterial suspension was disrupted by ultrasonication. The supernatant obtained after centrifugation was a crude enzyme solution containing L-piperidinic acid oxidase.

Example 3 Preparation of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by resolution of PpLPO (E ₁ )

Preparation of substrate solution: Prepare 5 g/L racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid solution with 50 mM phosphate buffer solution (pH=8.0) and adjust the solution pH to 8.0 with 30% ammonia water.

Take 0.5ml of substrate solution and add it to a 10mL reaction tube, then add 0.5mL of PpLPO crude enzyme solution, 1mg of catalase freeze-dried powder, and use phosphate buffer solution (50mM, pH 8.0) to make up the reaction volume to 2mL. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 24 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system.

The detection results are shown in Figures 1 and 2. PpLPO (E ₁ ) exhibits strict (S)-configuration stereoselectivity, with a conversion rate of 50% (conversion rate = [(initial racemic substrate concentration (g/L) - residual substrate concentration (g/L)) / initial racemic substrate concentration (g/L)] × 100%), and the ee value of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is over 99%.

Example 4 Preparation of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by resolution of PaLPO(E ₂ )

The substrate solution was prepared according to the method of Example 3.

Take 1ml of substrate solution and add it to a 10mL reaction tube, then add 0.5mL of PaLPO crude enzyme solution, 1mg of catalase freeze-dried powder, and use phosphate buffer solution (50mM, pH 8.0) to make the reaction solution volume up to 2mL. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 24 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system. The test results are a conversion rate of 49% (calculated in the same way as in Example 3), and the ee value of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is 98%.

Example 5 Preparation of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by resolution of PeLPO(E ₃ )

The substrate solution was prepared according to the method of Example 3.

Take 1.5ml substrate solution and add it to a 10mL reaction tube, then add 0.5mL PeLPO crude enzyme solution and 1mg catalase freeze-dried powder. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 12 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system. The test results are a conversion rate of 49% (calculated in the same way as in Example 3), and the ee value of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is 95%.

Example 6 Preparation of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by resolution of AoLPO (E ₄ )

The substrate solution was prepared according to the method of Example 3.

Take 1.5ml substrate solution and add it to a 10mL reaction tube, then add 0.5mL AoLPO crude enzyme solution and 1mg catalase freeze-dried powder. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 12 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system. The test results show that the conversion rate is 50% (calculation method is the same as Example 3), and the ee value of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is more than 99%.

Example 7 Preparation of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by resolution of SpLPO(E ₅ )

The substrate solution was prepared according to the method of Example 3.

Take 1ml of substrate solution and add it to a 10mL reaction tube, then add 1mL SpLPO crude enzyme solution and 1mg of catalase freeze-dried powder. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 12 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system. The test results are a conversion rate of 50% (calculation method is the same as Example 3), and the ee value of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is more than 99%.

Example 8 Preparation of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid from PeLPO (E ₃ )

Preparation of substrate solution: Prepare 5 g/L racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid solution with 50 mM phosphate buffer solution (pH=8.0) and adjust the pH of the solution to 8.0 with 30% ammonia water.

Take 0.5ml substrate solution and add it to a 10mL reaction tube, then add 1mL PeLPO crude enzyme solution, 1mg catalase freeze-dried powder, and use phosphate buffer solution (50mM, pH 8.0) to make the reaction solution volume up to 2mL. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 16 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system. The conversion rate is 48% (calculated in the same way as in Example 3), and the ee value of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is 96%.

Example 9 Preparation of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by resolution of AoLPO (E ₄ )

The substrate solution was prepared according to the method of Example 8.

Take 1ml of substrate solution and add it to a 10mL reaction tube, then add 1mL of AoLPO crude enzyme solution and 1mg of catalase freeze-dried powder. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 12 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system. The test results are a conversion rate of 50% (calculation method is the same as Example 3), and the ee value of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is more than 99%.

Example 10 Preparation of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by resolution of SpLPO (E ₅ )

The substrate solution was prepared according to the method of Example 8.

Take 1ml of substrate solution and add it to a 10mL reaction tube, then add 1mL SpLPO crude enzyme solution and 1mg of catalase freeze-dried powder. After mixing, take out 50μL as "0 hour" and perform HPLC analysis. Place the reaction tube in a 30℃ constant temperature water bath, stir magnetically, and react for 12 hours. After the reaction is completed, use HPLC to detect the content of the two configurations of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction system. The test results are a conversion rate of 50% (calculation method is the same as Example 3), and the ee value of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is more than 99%.

Example 11 Preparation of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid by SpLPO (E ₅ ) large reaction system

Preparation of substrate solution: Prepare 8 g/L racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid solution with deionized water and adjust the pH of the solution to 8.0 with 30% ammonia water.

Add 200mL substrate solution, 200mL SpLPO crude enzyme solution, and 100mg catalase freeze-dried powder to the reactor. After mixing, place in a 30℃ constant temperature water bath, stir magnetically, and react for 12 hours. After the reaction is completed, adjust the pH value of the reaction system to 5.0-6.0. Place in a 99℃ water bath, wait for the protein to denature and precipitate, and then filter. Take the filtrate and evaporate it at 65℃ to concentrate the reaction volume 5 times. Place on ice, cool, and filter. Carefully scrape off the precipitated white crystals, place in an oven, dry and weigh. Weigh 0.1g of white dried crystals and dilute to 50ml with 50mM phosphate buffer solution (pH 8.0). High performance liquid chromatography is used to detect the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the sample. The conversion rate was 49.9% (calculated in the same manner as in Example 3), the ee value of (R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid was over 99%, and the isolated yield was 87% (isolated yield = the amount of product actually isolated (mg)/theoretical amount of product (mg) × 100%).

Example 12 Preparation of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid using SpLPO (E ₅ ) large reaction system

Preparation of substrate solution: Prepare 5 g/L racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid solution with deionized water and adjust the pH of the solution to 8.0 with 30% ammonia water.

Add 200mL substrate solution, 200mL SpLPO crude enzyme solution, and 100mg catalase freeze-dried powder to the reactor. After mixing, place in a 30℃ constant temperature water bath, stir magnetically, and react for 24 hours. After the reaction is completed, adjust the pH value of the reaction system to 5.0-6.0. Place in a 99℃ water bath, wait for the protein to denature and precipitate, and then filter. Take the filtrate and evaporate it at 65℃ to concentrate the reaction volume 5 times. Place on ice, cool, and filter. Carefully scrape off the precipitated white crystals, place in an oven, dry and weigh. Weigh 0.1g of white dried crystals and dilute to 50ml with 50mM phosphate buffer solution (pH 8.0). High performance liquid chromatography is used to detect the content of two configurations of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the sample. The conversion rate was 49.9% (calculated in the same manner as in Example 3), the ee value of (R)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid was over 99%, and the isolated yield was 86%.

The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Sequence Listing

<110> Zhejiang University; Suzhou Tongli Biopharmaceutical Co., Ltd.

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Claims (16)

1. A method for preparing a compound shown in a formula (I) by enzymatic resolution,

In formula (I), R ¹,R² is independently selected from hydrogen, C ₁-C₆ alkyl, C ₁-C₆ alkoxy, characterized in that the method comprises:

(1) Selectively catalyzing the (S) isomer of the compound of the formula (I) to perform oxidative dehydrogenation reaction by using the racemate of the compound of the formula (I) or the racemate of the salt of the compound of the formula (I) as a substrate and using an isolated L-pipecolic acid oxidase or a cell which expresses the L-pipecolic acid oxidase in a cell as a catalyst, wherein the compound of the formula (I) is not catalyzed and remains in a reaction system; the amino acid sequence of the L-pipecolic acid oxidase is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5;

(2) Separating the compound of the formula (I) from the reaction system to obtain the compound.

2. The method of claim 1, wherein in formula (I), R ¹,R² is independently selected from hydrogen, methyl, ethyl, isopropyl, methoxy, or ethoxy, and the salt is an alkali metal salt or an ammonium salt.

3. The process of claim 1, wherein the compound of formula (I) is (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid or (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid.

4. The method of claim 1, wherein the catalyst is a crude enzyme solution containing the L-pipecolic acid oxidase ex vivo or a cell expressing the L-pipecolic acid oxidase in a cell or a pure enzyme of the L-pipecolic acid oxidase or an immobilized enzyme of the L-pipecolic acid oxidase.

5. The method of claim 4, wherein the cell is an engineered bacterium that expresses L-pipecolic acid oxidase and the host cell of the engineered bacterium is e.collbl21 (DE 3).

6. The method of claim 5, wherein the engineering bacterium comprises expression vector pET-28a (+), and the L-pipecolic acid oxidase gene is linked to expression vector pET-28a (+).

7. The method of claim 1, wherein the oxidative dehydrogenation reaction is conducted in an aerobic environment, the oxidative dehydrogenation reaction further producing hydrogen peroxide, the method further comprising adding catalase to the reaction system for catalytically decomposing the hydrogen peroxide at one or more of a point in time before, during, and after the oxidative dehydrogenation reaction.

8. The method of claim 7, wherein the catalase is bovine liver catalase lyophilized powder.

9. The method of claim 7, wherein the enzyme activity ratio of the catalase to the L-pipecolic acid oxidase is from 100 to 400:1.

10. The method according to claim 1, wherein in the step (1), a reaction system is first constructed, and then the oxidative dehydrogenation reaction is carried out by controlling the reaction system to be in a set temperature and an aerobic environment, wherein the reaction system comprises the substrate, a solvent, the catalyst and optionally catalase for catalytically decomposing hydrogen peroxide, and optionally a pH buffer and/or a pH adjuster.

11. The method according to claim 10, wherein the solvent is water, the substrate is dissolved in the aqueous solution of the pH buffer, the pH regulator is optionally added to prepare a substrate solution having a pH of 6 to 9, and the catalyst and the catalase are added to obtain the reaction system.

12. The method of claim 11, wherein the pH of the substrate solution is controlled to be 7 to 8.

13. The method of claim 11, wherein the pH buffer is phosphate, which is dissolved in water to prepare a phosphate buffer solution; or, the pH regulator is ammonia water, or alkali metal hydroxide or aqueous solution thereof.

14. The method according to claim 10, wherein in the step (1), the concentration of the starting substrate in the reaction system is controlled to be 1 to 20g/L.

15. The method of claim 10, wherein the set temperature is 20 to 70 ℃.

16. The method of claim 15, wherein the set temperature is between 30 and 50 ℃.