CN112442523B - Method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution - Google Patents

Method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution Download PDF

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CN112442523B
CN112442523B CN201910793322.9A CN201910793322A CN112442523B CN 112442523 B CN112442523 B CN 112442523B CN 201910793322 A CN201910793322 A CN 201910793322A CN 112442523 B CN112442523 B CN 112442523B
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tetrahydroisoquinoline
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吴坚平
居述云
杨立荣
钱明心
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Tongli Biomedical Co ltd
Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The invention discloses a new method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution, which comprises the following steps: the (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid or (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid is prepared by selectively catalyzing 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 with 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 and using isolated L-pipecolic acid oxidase or cells expressing the L-pipecolic acid oxidase in cells as a catalyst, and retaining the catalyst in a reaction system. The method has the characteristics of high conversion rate, ee value up to more than 99%, mild reaction condition, strong stereoselectivity, high reaction efficiency, relatively simple process and the like.

Description

Method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution
Technical Field
The invention belongs to the technical field of biocatalysis, and particularly relates to a novel method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution.
Background
Optically pure 1,2,3, 4-tetrahydroisoquinoline compounds are an important chiral building block and are widely applied to synthesis of various medicaments. 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 levopraziquantel (chinese patent 201310487924.4).
In the prior art, the method for preparing the optical pure (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid comprises two methods of chemical chiral synthesis and biocatalytic chiral resolution. The chemical chiral synthesis method is characterized in that (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid is synthesized from chiral raw materials, such as Kurata and the like, optical pure olefin isoquinoline is taken as a starting material, and is subjected to ozonolysis, naBH 4 in-situ reduction, tetramethyl piperidine nitrogen oxide (TEMPO) oxidation and trifluoroacetic acid mediated N-tert-butoxycarbonyl deprotection, and the method has the advantages of low yield, complicated steps and incapacity of being applied to industrialization. Pa L and the like utilize lipase dynamic dynamics to split 1,2,3, 4-tetrahydroisoquinoline-1-ethyl formate to prepare (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid, 17.74g/L substrate, the enzyme adding amount is 20mg/mL lipase, the temperature is 3 ℃, the reaction is carried out for 24 hours, the yield is 85%, the ee value of the product is 98％(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)., the reaction condition of the method is mild, the stereoselectivity is strong, the process is relatively simple, but the reaction efficiency still needs to be further improved.
In the prior art, the method for preparing the optically pure (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid is mainly a biocatalytic chiral resolution method. Pa L and the like utilize lipase dynamic dynamics to split 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-ethyl formate to prepare (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid, 53.46g/L substrate, the enzyme adding amount is 100mg/mL lipase, the reaction is carried out for 7 hours under the condition of 25 ℃ and pH value of 8.5, the yield is 85 percent, the ee value of the product is 92％(Directed(R)-or(S)-Selective Dynamic Kinetic Enzymatic Hydrolysis of1,2,3,4-Tetrahydroisoquinoline-1-carboxylic Esters[J].European Journal of Organic Chemistry,2008,2008(31):5269-76)., the reaction condition is mild, the stereoselectivity is strong, the process is relatively simple, and the optical purity of the obtained product is still to be further improved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof. The method has the characteristics of mild reaction conditions, strong stereoselectivity, high reaction efficiency, relatively simple process and the like, and has industrial application prospect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
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, the method comprising:
(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;
(2) Separating the compound of the formula (I) from the reaction system to obtain the compound.
Further, in formula (I), R 1,R2 is independently selected from hydrogen, methyl, ethyl, isopropyl, methoxy, or ethoxy.
Preferably, the salt is a monovalent salt, particularly preferably an alkali metal salt or an ammonium salt, wherein the alkali metal salt may be, for example, a lithium salt, a sodium salt, a potassium salt.
According to some preferred and specific aspects of the present invention, 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.
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: an L-pipecolic acid oxidase derived from Pseudomonas putida (Pseudomonas putida) KT2440 or a mutant thereof or an L-pipecolic acid oxidase having an amino acid sequence homology of more than 80%, an L-pipecolic acid oxidase derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 or a mutant thereof or an L-pipecolic acid oxidase having an amino acid sequence homology of more than 80%, an L-pipecolic acid oxidase derived from Pseudomonas putida (Pseudomonas entomophila str.) L48 or a mutant thereof or an L-pipecolic acid oxidase having an amino acid sequence homology of more than 80%, an L-pipecolic acid oxidase derived from Aspergillus oryzae (Aspergillus oryzae) RIB40 or a mutant thereof or an L-pipecolic acid oxidase having an amino acid sequence homology of more than 80%, an L-pipecolic acid oxidase derived from Schizosaccharomyces pombe (Schizosaccharomyces pombe) or a mutant thereof or an L-pipecolic acid oxidase having an amino acid sequence homology of more than 80%.
Further preferably, the L-pipecolic 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, SEQ ID NO. 5.
According to some specific and preferred aspects of the invention, 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 cell or a pure enzyme of the L-pipecolic acid oxidase or an immobilized enzyme of the L-pipecolic acid oxidase.
As a preferred embodiment of the present invention: the cell is an engineering bacterium for expressing L-pipecolic acid oxidase, and the host cell of the engineering bacterium is E.coli BL21 (DE 3).
Specifically, the engineering bacteria contain an expression vector pET-28a (+), and the L-pipecolic acid oxidase gene is connected to the expression vector pET-28a (+).
According to some specific and preferred aspects of the invention, the catalyst is added in an amount of 1 to 5% by weight of the reaction system based on the wet weight of cells after centrifugation at 8000rpm for 10 min.
According to some specific and preferred aspects of the invention, the oxidative dehydrogenation reaction is carried out in an aerobic environment, the oxidative dehydrogenation reaction further producing hydrogen peroxide, the method further comprising adding catalase for catalytically decomposing the hydrogen peroxide to the reaction system at one or more of a point in time before, during and after the oxidative dehydrogenation reaction is carried out.
Further, the catalase is freeze-dried bovine liver catalase powder. According to a specific aspect of the invention, the enzymatic activity of the freeze-dried bovine liver catalase powder is 4000U/mg.
According to some preferred aspects of the invention, the enzyme activity ratio of the catalase to the L-pipecolic acid oxidase is from 100 to 400:1.
Further, in the step (1), a reaction system is first constructed, and then the oxidative dehydrogenation reaction is carried out in an aerobic environment at a set temperature, wherein the reaction system comprises the substrate, a solvent, the catalyst and optionally catalase for catalyzing the decomposition of hydrogen peroxide, and optionally a pH buffer and/or a pH regulator.
According to a preferred aspect of the present invention, 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. More preferably, the pH of the substrate solution is controlled to 7 to 8.
According to a specific and preferred aspect of the present invention, the pH buffer is phosphate, which can be formulated as a phosphate buffer solution by dissolving it in water.
According to some preferred aspects of the invention, the pH adjuster is preferably aqueous ammonia, an alkali metal hydroxide or an aqueous solution thereof.
According to a specific and preferred aspect of the present invention, the pH adjustor is 20wt% to 35wt% ammonia water.
According to yet another specific aspect of the 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 20g/L.
According to a preferred aspect of the present invention, the set temperature is 20 to 70 ℃. More preferably, the set temperature is 30 to 50 ℃.
In the step (2), the pH value of the reaction system is adjusted to 5.0-6.0, the protein is denatured and separated out by heating, the protein is filtered, the filtrate is concentrated, cooled and crystallized, and the compound shown in the formula (I) is obtained after drying.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following beneficial effects:
In the research process of the invention, the L-pipecolic acid oxidase can catalyze (S) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid or (S) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid with high efficiency and perform oxidative dehydrogenation reaction, and has 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 for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid or (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid has the advantages of high reaction efficiency, high conversion rate, mild reaction condition, strong stereoselectivity (ee value can reach more than 99 percent) and simple process.
Drawings
FIG. 1 is a high performance liquid chromatography detection chart of the reaction system in example 3 sampled at 0 hour, wherein the retention time 8.923min is (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid; the retention time 13.282min is (S) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid;
FIG. 2 is a high performance liquid chromatography detection chart of the reaction system in example 3, wherein the reaction is carried out for 24 hours and sampling is carried out.
Detailed Description
The invention provides a novel method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof, which takes 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 ammonia salt and the like), and uses isolated L-pipecolic acid oxidase or cells expressing L-pipecolic acid oxidase in cells as a catalyst to perform 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 as follows: the (S) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid or (S) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid is catalyzed stereoselectively by L-pipecolic acid oxidase with racemic 1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (1) or racemic 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid as a substrate to generate corresponding imidic acid, and the (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. Hydrogen peroxide produced in the catalytic process is decomposed into water and oxygen under the catalysis of catalase. The reaction process is schematically as follows:
Preferably, the L-pipecolic acid oxidase is derived from Pseudomonas putida, pseudomonas aeruginosa, pseudomonas arborescens, aspergillus oryzae, schizosaccharomyces pombe. Specifically, the L-pipecolic acid oxidase is derived from Pseudomonas putida (Pseudomonas putida) KT2440 or a mutant thereof or an L-pipecolic acid oxidase having homology of more than 80% with the amino acid sequence, an L-pipecolic acid oxidase derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 or a mutant thereof or an L-pipecolic acid oxidase having homology of more than 80% with the amino acid sequence, an L-pipecolic acid oxidase derived from Pseudomonas arborescens (Pseudomonas entomophila str.) L48 or a mutant thereof or an L-pipecolic acid oxidase having homology of more than 80% with the amino acid sequence, an L-pipecolic acid oxidase derived from Comamonas testosteroni (Comamonas testosterone) ATCC 11996 or a mutant thereof or an L-pipecolic acid oxidase having homology of more than 80% with the amino acid sequence, an L-pipecolic acid oxidase derived from Aspergillus oryzae (Aspergillus oryzae) RIB40 or a mutant thereof or a yeast having homology of more than 80% with the amino acid sequence, and a yeast Schizosaccharomyces pombe having homology of L-pipecolic acid sequence or a mutant thereof.
Preferably, the cell is an engineering bacterium for expressing L-pipecolic acid oxidase, and the host cell of the engineering bacterium is E.coli BL21 (DE 3). Specifically, the engineering bacteria contain an expression vector pET-28a (+), and the L-pipecolic 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 liquid, engineering bacteria resting cells expressing recombinant enzyme, pure enzyme or immobilized enzyme. The catalase is used in the form of a lyophilized 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 20g/L.
Preferably and specifically, in the catalyst system, the catalyst is added in an amount of 1 to 5% by weight of the reaction solution based on the wet weight of the cells after centrifugation at 8000rpm for 10 min.
Preferably, the ratio of the enzymatic activity of catalase to that of L-pipecolic acid oxidase in the reaction system is 100 to 400:1.
As a specific aspect, the catalase is freeze-dried powder of bovine liver catalase, and the enzyme activity is 4000U/mg.
Preferably, in the catalytic system, the reaction temperature is 20-70 ℃, the reaction time is 6-72 hours, and the pH value of the reaction solution is 6-9; more preferably, the temperature is 30 to 50℃and the time is 12 to 48 hours. The pH value of the phosphoric acid buffer solution is controlled to be 7-8.
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods in the examples of the present invention are conventional methods unless otherwise specified.
The genes used in the examples of the present invention were synthesized by the division of biological engineering (Shanghai). Coli BL21 (DE 3) strain was purchased from Novagen; DNA MARKER, PRIMESTAR DNA polymerase, low molecular weight standard proteins, and other biological assay reagents were purchased from TaKaRa. Specific procedures for gene cloning and expression can be found in the guidelines for molecular cloning experiments, which are compiled by J.Sam Brookfield et al.
The present invention analyzes the respective products and substrates of the catalytic reaction by High Performance Liquid Chromatography (HPLC). The HPLC analysis method of the racemization 1,2,3, 4-tetrahydroisoquinoline-1-formic acid comprises the following steps: chromatographic column-ZWIX (-), respectively; column temperature/25 ℃; flow rate/0.4 mL/min; detection wavelength/UV 220 nm; mobile phase: HPLC grade methanol (50 mM formic acid and 25mM dihexylamine added). The peak of each related substance is shown in figure 1.
EXAMPLE 1 construction of genetically engineered bacterial species
1.1 Screening of L-pipecolic acid oxidase and construction of genetically engineered bacterium expressing L-pipecolic acid oxidase
Pseudomonas putida (Pseudomonas putida) KT2440, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, pseudomonas arborescens (Pseudomonas entomophila str.) L48, aspergillus oryzae (Aspergillus oryzae) RIB40 and Schizosaccharomyces pombe (Schizosaccharomyces pombe) derived L-pipecolic acid oxidase were cloned, respectively (as shown in Table 1).
TABLE 1L-pipecolic acid oxidase from three different sources
PCR upstream and downstream primers were designed based on the corresponding gene DNA sequences.
Primer derived from Pseudomonas putida KT 2440L-pipecolic acid oxidase:
KT2440-F：5’-GGAATTCCATATGGAGCCGGCCATGTCCGAACTG-3’(Nde I)
KT2440-R：5’-CCCAAGCTTTTACAACACAACCCTCAGGCAC-3’(Hind III)
primer derived from Pseudomonas aeruginosa PAO L-pipecolic acid oxidase:
PAO1-F：5’-CGGGATCCATGGGCGGTTTGCAGCAACGGTG-3’(BamH I)
PAO1-R：5’-CCCAAGCTTCTAGAGAACCACGCGCAAGC-3’(Hind III)
Primer derived from L-pipecolic acid oxidase of Pseudomonas entomophila str.L48:
L48-F：5’-CGGGATCCATGTCCGAACTGCGTCAAGAATGTC-3’(BamH I)
L48-R：5’-CCCAAGCTTCTACAACACCACCCGCAAACACTG-3’(Hind III)
Restriction sites are added to the upstream and downstream primers, respectively, and specific restriction enzymes are shown in parentheses in the primer sequences as indicated by the underlines. The PCR amplification reaction is respectively carried out by taking Pseudomonas putida (Pseudomonas putida) KT2440, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 and Pseudomonas arboxylis (Pseudomonas entomophila str.) L48 genome DNA as templates and utilizing corresponding upstream and downstream primers, wherein 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 ℃ for 10s; annealing: 58 ℃ for 15s; extension: 72 ℃ for 10s; cycling for 35 times;
3) Extension: 72 ℃ for 10min;
4) Preserving heat at 4 ℃.
After the PCR amplification reaction is finished, the amplification result is detected by using 1.0% agarose gel electrophoresis, and the result shows that the amplification product is a single band with the size of about 1000bp. The target band is recovered by using the DNA recovery and purification kit, and specific steps are referred to the instruction of the purification kit.
The expression vector pET-28a (+) and the PCR amplified product were digested simultaneously with the corresponding restriction enzymes. And (5) recovering the target band by using a DNA recovery and purification kit after the enzyme digestion is completed. Subsequently, the double digested PCR amplified product was ligated to the expression vector pET-28a (+) with a corresponding cohesive end using T4 DNA ligase, the ligation system is shown in Table 2 below:
table 2 recombinant expression plasmid ligation System
The enzyme-linked product is transformed into E.coli DH5a competent cells, plated, picked up and cultured in LB liquid medium, bacterial liquid PCR identified positive transformant, and sent to sequencing company to verify the correctness of the inserted sequence. Plasmids were extracted from positive transformants verified to be error-free, and the related methods refer to plasmid extraction kits. And transferring the recombinant expression vector into an expression host E.coli BL21 (DE 3), and after bacterial liquid PCR and sequencing verification, adding 25% glycerol into the obtained engineering bacterial liquid and preserving at-80 ℃ for later use.
The gene sequences of the Aspergillus oryzae (Aspergillus oryzae) RIB40 and Schizosaccharomyces pombe (Schizosaccharomyces pombe) derived L-pipecolic acid oxidase were codon optimized and transferred to the division of biological engineering (Shanghai) for total gene synthesis, and cloned into plasmid pET-28a (+) respectively. Transferring the recombinant plasmid into an expression host E.coli BL21 (DE 3), and after sequencing verification, adding 25% glycerol into the obtained engineering bacteria liquid and preserving at-80 ℃ 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 sterilized at 121 deg.C for 20 min. In the case of solid LB medium, an additional 15g/L of agar is added.
The engineering bacteria containing L-pipecolic acid oxidase gene are inoculated into 5mL of liquid LB (containing 50 mug/mL kanamycin) culture medium, and cultured for about 8 hours at 37 ℃ under shaking at 200 rpm. The cells were inoculated into 50mL of liquid LB medium (containing 50. Mu.g/mL kanamycin) at an inoculum size of 1% (V/V), cultured, OD 600 was 0.6-0.8, and then the cells were induced at 18℃for 15 hours by adding the inducer IPTG (final concentration: 0.1 mM). After the completion of the culture, the culture solution was poured into a 100mL centrifuge tube, centrifuged at 4000rpm for 10min, the supernatant was discarded, the cells were collected, washed twice with 50mM phosphate buffer (pH 8.0), and then stored in an ultra-low temperature refrigerator at-80℃for use.
2.2 Preparation of crude enzyme solution
The bacterial suspension is subjected to ultrasonic disruption in 50mM phosphate buffer (pH 8.0) or deionized water, and the supernatant obtained after centrifugation is a crude enzyme solution containing L-pipecolic acid oxidase.
EXAMPLE 3 PpLPO resolution of (E 1) preparation of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid
Preparing a substrate solution: 5g/L of a solution of racemic 1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid was prepared with 50mM phosphate buffer solution (pH=8.0) and the pH of the solution was adjusted to 8.0 with 30% aqueous ammonia.
0.5ML of the substrate solution was added to a 10mL reaction tube, followed by adding 0.5mL PpLPO crude enzyme solution, 1mg of catalase lyophilized powder, and the volume of the reaction solution was made up to 2mL with phosphate buffer solution (50 mM, pH 8.0). After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for reaction for 24 hours. After the reaction is finished, the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method.
As shown in FIGS. 1 and 2, ppLPO (E 1) showed a strict (S) -configuration stereoselectivity, the conversion was 50% (conversion = [ (initial racemic substrate concentration (g/L) -residual substrate concentration (g/L))/initial racemic substrate concentration (g/L) ]. Times.100%), (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid had an ee value of 99% or more.
EXAMPLE 4 PaLPO resolution of (E 2) preparation of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid
The substrate solution was prepared as in example 3.
1ML of the substrate solution was added to a 10mL reaction tube, followed by addition of 0.5mL PaLPO crude enzyme solution, 1mg of catalase lyophilized powder, and the volume of the reaction solution was made up to 2mL with phosphate buffer solution (50 mM, pH 8.0). After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for reaction for 24 hours. After the reaction is finished, the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method. The detection result shows that the conversion rate is 49% (the ee value of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid reaches 98% in the same manner as in example 3).
EXAMPLE 5 PeLPO resolution of (E 3) preparation of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid
The substrate solution was prepared as in example 3.
1.5ML of the substrate solution was added to a 10mL reaction tube, followed by addition of 0.5mL PeLPO crude enzyme solution and 1mg of catalase lyophilized powder. After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for reaction for 12 hours. After the reaction is finished, the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method. The detection result shows that the conversion rate is 49% (the ee value of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid reaches 95% in the same manner as in example 3).
EXAMPLE 6 AoLPO resolution of (E 4) preparation of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid
The substrate solution was prepared as in example 3.
1.5ML of the substrate solution was added to a 10mL reaction tube, followed by addition of 0.5mL AoLPO crude enzyme solution and 1mg of catalase lyophilized powder. After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for reaction for 12 hours. After the reaction is finished, the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method. The detection result shows that the conversion rate is 50% (the calculation method is the same as that of example 3), and the ee value of the (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid reaches more than 99%.
EXAMPLE 7 SpLPO resolution of (E 5) preparation of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid
The substrate solution was prepared as in example 3.
1ML of the substrate solution was added to a 10mL reaction tube, followed by addition of 1mL SpLPO crude enzyme solution and 1mg of catalase lyophilized powder. After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for reaction for 12 hours. After the reaction is finished, the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method. The detection result shows that the conversion rate is 50% (the calculation method is the same as that of example 3), and the ee value of the (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid reaches more than 99%.
Example 8 PeLPO (E 3) preparation of (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid
Preparing a substrate solution: 5g/L of racemic 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid solution was prepared with 50mM phosphate buffer solution (pH=8.0), and the pH of the solution was adjusted to 8.0 with 30% aqueous ammonia.
0.5ML of the substrate solution was added to a 10mL reaction tube, followed by addition of 1mL PeLPO crude enzyme solution and 1mg of catalase lyophilized powder, and the volume of the reaction solution was made up to 2mL with phosphate buffer solution (50 mM, pH 8.0). After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for 16 hours. After the reaction, the content of two configurations of 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method. The conversion was 48% (calculated in the same manner as in example 3), and the ee value of (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid was 96%.
EXAMPLE 9 AoLPO resolution preparation of (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (E 4)
The substrate solution was prepared as in example 8.
1ML of the substrate solution was added to a 10mL reaction tube, followed by addition of 1mL AoLPO crude enzyme solution and 1mg of catalase lyophilized powder. After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for reaction for 12 hours. After the reaction, the content of two configurations of 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method. The detection result shows that the conversion rate is 50% (the calculation method is the same as that of example 3), and the ee value of the (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid reaches more than 99%.
EXAMPLE 10 SpLPO resolution preparation of (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (E 5)
The substrate solution was prepared as in example 8.
1ML of the substrate solution was added to a 10mL reaction tube, followed by addition of 1mL SpLPO crude enzyme solution and 1mg of catalase lyophilized powder. After mixing, 50. Mu.L was removed as "0 hours" and analyzed by HPLC. The reaction tube was placed in a constant temperature water bath at 30℃and magnetically stirred for reaction for 12 hours. After the reaction, the content of two configurations of 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid in the reaction system is detected by an HPLC method. The detection result shows that the conversion rate is 50% (the calculation method is the same as that of example 3), and the ee value of the (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid reaches more than 99%.
EXAMPLE 11 SpLPO (E 5) preparation of (R) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid with Large reaction System
Preparing a substrate solution: 8g/L of racemic 1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid solution was prepared with deionized water and the pH of the solution was adjusted to 8.0 with 30% aqueous ammonia.
200ML of a substrate solution, 200mL SpLPO of a crude enzyme solution, and 100mg of a catalase lyophilized powder were added to the reactor. After being evenly mixed, the mixture is placed in a constant temperature water bath at 30 ℃ for magnetic stirring and reaction for 12 hours. After the reaction is finished, the pH value of the reaction system is adjusted to 5.0-6.0. And (3) carrying out water bath at 99 ℃, and carrying out suction filtration after the protein is denatured and separated out. The filtrate was distilled off at 65℃and the reaction volume was concentrated 5 times. Placing on ice, cooling, and suction filtering. The white crystals which precipitated were carefully scraped off, placed in an oven, dried and weighed. 0.1g of white dried crystals were weighed and fixed to a volume of 50ml with 50mM phosphate buffer solution (pH 8.0). And detecting the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-1-formic acid in the taken sample by high performance liquid chromatography. The conversion 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 99% or more, and the isolated yield was 87% (isolated yield=the amount of the product actually isolated (mg)/the amount of the theoretical product (mg) ×100%).
EXAMPLE 12 SpLPO (E 5) preparation of (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid
Preparing a substrate solution: 5g/L of racemic 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid solution was prepared with deionized water and the pH of the solution was adjusted to 8.0 with 30% aqueous ammonia.
200ML of a substrate solution, 200mL SpLPO of a crude enzyme solution, and 100mg of a catalase lyophilized powder were added to the reactor. After being evenly mixed, the mixture is placed in a constant temperature water bath at 30 ℃ for magnetic stirring and reaction for 24 hours. After the reaction is finished, the pH value of the reaction system is adjusted to 5.0-6.0. And (3) carrying out water bath at 99 ℃, and carrying out suction filtration after the protein is denatured and separated out. The filtrate was distilled off at 65℃and the reaction volume was concentrated 5 times. Placing on ice, cooling, and suction filtering. The white crystals which precipitated were carefully scraped off, placed in an oven, dried and weighed. 0.1g of white dried crystals were weighed and fixed to a volume of 50ml with 50mM phosphate buffer solution (pH 8.0). And detecting the content of two configurations of 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-formic acid in the taken sample by high performance liquid chromatography. The conversion was 49.9% (the calculation was the same as in example 3), the ee value of (R) -6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline-1-carboxylic acid was 99% or more, and the isolated yield was 86%.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
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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 ℃.
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CN107384885A (en) * 2017-09-05 2017-11-24 武汉大学 The application of imine reduction enzyme and its mutant in the tetrahydroisoquinoline of (S) 1 aryl 1,2,3,4 is synthesized
CN108359651A (en) * 2018-02-26 2018-08-03 遵义医学院 A kind of monoamine oxidase and its gene and application

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