CN111254181B - Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by chemical enzyme method - Google Patents
Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by chemical enzyme method Download PDFInfo
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Abstract
The invention discloses a method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by using a chemical enzyme method, which comprises the following steps: racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid is taken as a substrate, D-amino acid oxidase is utilized to stereoselectively catalyze R-type isomer, corresponding imidic acid is generated through oxidative dehydrogenation, S-type isomer is not catalyzed and remains in a reaction system, imidic acid generates a racemic substrate through the action of imidic acid reducer, and R-type isomer in the substrate is stereoselectively catalyzed under the action of D-amino acid oxidase, so that S-type isomer is prepared. The method has the advantages of high reaction yield up to more than 80.6%, ee value higher than 99%, mild reaction condition, strong stereoselectivity, high reaction efficiency, high yield, relatively simple process and the like.
Description
Technical Field
The invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by using a chemical enzyme method.
Background
(S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (1, 2,3, 4-tetrahydroisoquinoline-3-carboxilic acid) is an important drug intermediate, and is widely applied to synthesis of various small organic molecule drugs and peptide-based drugs. For example, (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is an important component of the antihypertensive drug quinapril (Diversity-oriented synthesis of medicinally important1,2,3, 4-tetrahydroisoquinoline-3-carboxilic acid (Tic) derivatives and higher analogs [ J ]. Org Biomol Chem,2014,12 (45): 9054-91.). In addition, (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is useful for the synthesis of small molecule antagonists containing tetrahydroisoquinoline parent nuclei, acting on the chemokine receptor CXCR4, and thus is expected to be useful in the treatment of diseases such as HIV (Discovery of tetrahydroisoquinoline-based CXCR4 antagonts [ J ]. ACS Med Chem Lett,2013,4 (11): 1025-30.).
In the prior art, the method for preparing the optical pure (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid comprises two steps of chemical chiral synthesis and biocatalytic kinetic resolution. Researchers initially use Pictet-Spengler reaction to prepare optically pure (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, L-phenylalanine is taken as a raw material, and is condensed with formaldehyde under the conditions of concentrated acid and high temperature to generate a target product, and the process is relatively simple, but the generated product can be partially racemized. Subsequently, bischler-Nepieralski reaction, [2+2+2] cycloaddition method and the like are also used for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid and its derivatives, which are relatively complicated route and costly (Diversity-oriented synthesis of medicinally important1,2,3, 4-tetrahydroisoquinoline-3-carboxilic acid (Tic) derivatives and higher analogs [ J ]. Org Biomol Chem 2014,12 (45): 9054-91.). In recent years, kurata et al did not synthesize (S) -1,2,3, 4-Tetrahydroisoquinoline-3-Carboxylic acid (Synthesis of Optically Pure (R) -and (S) -Tetrahydroisoquinoline-1-and-3-carboxilic Acids [ J ]. Synthesis,2015,47 (09): 1238-44.) in three steps by ozonolysis, oxidation, and deprotection. The method has low yield and more steps, and is not easy for industrial application. Gong' S et al uses chemical enzyme method to prepare (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid, namely, uses racemic phenylalanine as raw material, synthesizes racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid by Pictet-Spengler reaction, then prepares (S) -configuration product by esterification and lipase kinetic resolution. 23.8g of racemic ester hydrochloride (0.1 mol), lipase and substrate in a mass ratio of 0.2, 48h, product ee >99% and yield of 49.1%. The product obtained by the method has high stereoselectivity and relatively simple process, but still has the problem that the maximum theoretical yield is only 50 percent (research on synthesizing optical pure (S) -1,2,3, 4-tetrahydroquinoline-3-carboxylic acid by a chemical enzyme method [ J ]. Modern chemical engineering, 2003,23 (12): 23-5.).
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a novel method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid. 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 (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid (I) by a chemical enzyme method,
the method comprises the following steps:
(1) Taking a racemate of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid or a racemate of 1,2,3, 4-tetrahydroisoquinoline-3-formate as a substrate, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to perform oxidative dehydrogenation reaction by using D-amino acid oxidase as a catalyst to generate iminoacid shown in a formula (II), wherein (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is not catalyzed and remains in a reaction system;
wherein an imidic acid reducing agent is added to the reaction system at one or more time points among before, during and after the oxidative dehydrogenation reaction, the imidic acid reducing agent being used for reducing the imidic acid generated by the oxidative dehydrogenation reaction to a racemate of the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or a racemate of a salt thereof;
(2) Separating the (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid from the reaction system.
Further, the 1,2,3, 4-tetrahydroisoquinoline-3-formate may be an alkali metal salt or ammonium salt of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or the like, specifically, for example, sodium 1,2,3, 4-tetrahydroisoquinoline-3-formate, potassium 1,2,3, 4-tetrahydroisoquinoline-3-formate, ammonium 1,2,3, 4-tetrahydroisoquinoline-3-formate.
According to the present invention, the D-amino acid oxidase is a combination of one or more selected from the group consisting of: a D-amino acid oxidase derived from trigonomycetes (Trigonopsis variabilis) CBS 4095 or a mutant thereof or other D-amino acid oxidase having greater than 80% amino acid sequence homology thereto, a D-amino acid oxidase derived from Fusarium graminearum (Fusarium graminearum) CS3005 or a mutant thereof or other D-amino acid oxidase having greater than 80% amino acid sequence homology thereto, a D-amino acid oxidase derived from Fusarium pyriform 2516 or a mutant thereof or other D-amino acid oxidase having greater than 80% amino acid sequence homology thereto, a D-amino acid oxidase derived from Fusarium solani (Fusarium solani) M-0718 or a mutant thereof or other D-amino acid oxidase having greater than 80% amino acid sequence homology thereto.
Preferably, the D-amino acid oxidase has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.
According to some specific and preferred aspects of the invention, the catalyst is a crude enzyme solution or a pure enzyme or an immobilized enzyme or a cell expressing the D-amino acid oxidase in cells containing the isolated D-amino acid oxidase.
Further, the cell is an engineering bacterium for expressing D-amino acid oxidase, and a host cell of the engineering bacterium is E.coli BL21 (DE 3).
According to a specific aspect of the invention, the engineering bacterium contains an expression vector pET-28a (+), and the D-amino 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 D-amino acid oxidase is 1000 to 2000:1.
according to some preferred aspects of the invention, in step (1), the reaction is carried out in the presence of coenzyme Flavin Adenine Dinucleotide (FAD). Allowing the reaction to proceed in the presence of FAD helps to further increase conversion. Further, FAD is either equivalent to the substrate or in excess. In general, the crude enzyme solution of the D-amino acid oxidase to be produced already contains a sufficient amount of FAD, and when the crude enzyme solution is directly used, it is not necessary to add FAD. In the case of using the D-amino acid oxidase pure enzyme, an appropriate amount of FAD may be added as needed.
According to some specific and preferred aspects of the present invention, in step (1), the reaction system is first constructed and then the reaction system is controlled to be in a set temperature and aerobic environment to perform a reaction, wherein the reaction system comprises the substrate, the catalyst, a solvent, an imidic acid reducing agent and optionally a catalase for catalytically decomposing 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 selectively added to prepare a substrate solution having a pH of 6 to 9, and the catalyst, the imidic acid reducing agent and/or 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 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 ℃.
According to the present invention, the imidic acid reducing agent may be a reducing agent well known in the art. According to some specific and preferred aspects of the present invention, the imidic acid reducing agent is a combination of one or more selected from sodium cyanoborohydride, borane amine and sodium borohydride, which have proved to be very reactive towards imidic acid.
According to some specific and preferred aspects of the invention, the imidic acid reducing agent is added in an amount of 3 to 20 equivalents of the molar amount of the substrate fed.
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 mixture is filtered by suction, freeze-dried, dissolved and filtered by hot ethanol, and the filtrate is concentrated, cooled, crystallized and dried to obtain the compound shown in the formula (I).
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the invention surprisingly discovers that the D-amino acid oxidase can efficiently and selectively catalyze (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to carry out oxidative dehydrogenation reaction, and has no catalytic effect on (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid basically, and meanwhile, the yield is further improved by combining with the use of an imidic acid reducing agent. The method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid has the advantages of mild reaction conditions, high reaction efficiency and yield, strong stereoselectivity (ee value is more than 99%) 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 is 8.77min (R) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid; the retention time 11.238min was (S) -1,2,3, 4-tetrahydroisoquinoline-3-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 (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid and derivatives thereof, which takes racemized 1,2,3, 4-tetrahydroisoquinoline-3-formic acid (or ammonia salt) as a substrate, uses isolated D-amino acid oxidase or cells expressing the D-amino acid oxidase in cells and the like as a catalyst, combines an imidic acid reducing agent, and performs oxidative dehydrogenation-chemical reduction reaction to obtain (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid.
The specific principle is as follows: racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid is taken as a substrate, D-amino acid oxidase is utilized to stereoselectively catalyze (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid, corresponding imidic acid is generated through oxidative dehydrogenation, and the (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is not catalyzed and remains in a reaction system. The imidic acid is reacted by an imidic acid reducing agent to generate a racemization substrate, and then (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is stereoselectively catalyzed by D-amino acid oxidase, so that the yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid can be improved, and the ee value is more than 99%. Wherein the hydrogen peroxide generated can be decomposed into water and oxygen by catalase catalysis. The reaction process is schematically as follows:
further, it is preferable that the reaction is carried out in the presence of coenzyme Flavin Adenine Dinucleotide (FAD), and in the catalytic process, coenzyme Flavin Adenine Dinucleotide (FAD) is reduced to FADH 2 Subsequently, one molecule of oxygen is reduced to hydrogen peroxide (H 2 O 2 ) While FADH 2 Oxidized to FAD. Hydrogen peroxide is catalyzed by catalase to decompose into water and oxygen. The reaction process is schematically as follows:
preferably, the D-amino acid oxidase is derived from Trigonella, fusarium graminearum, fusarium pyriform, and Fusarium solani. Specifically, the D-amino acid oxidase is derived from Trigonella (Trigonopsis variabilis) CBS 4095, fusarium graminearum (Fusarium graminearum) CS3005, fusarium pyriform (Fusarium poae) 2516 or Fusarium solani (Fusarium solani) M-0718. Preferably, the cell is an engineering bacterium for expressing D-amino 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 D-amino acid oxidase gene is connected to the expression vector pET-28a (+).
In the reaction system, the D-amino acid oxidase is used in the form of crude enzyme liquid, pure enzyme, immobilized enzyme or engineering bacteria resting cells for expressing recombinant enzyme. The catalase is used in the form of a lyophilized powder.
Preferably, the concentration of the substrate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction system is 1-20 g/L.
In a specific and preferred aspect, the catalyst D-amino acid oxidase is added in an amount of 1 to 5% by weight of the reaction solution based on the wet weight of cells after centrifugation at 8000rpm for 10 minutes.
Preferably, the imidic acid reducing agent in the reaction system may be sodium cyanoborohydride, borane amine, sodium borohydride or other chemical agent capable of reducing imines. The adding amount of the iminoacid reducing agent in the reaction system is 3-20 equivalent of the substrate feeding molar amount.
Preferably, in the reaction system, the enzyme activity ratio of the catalase to the D-amino acid oxidase is 1000 to 2000:1.
as a specific aspect, the catalase is freeze-dried powder of bovine liver catalase, and the enzyme activity is 4000U/mg.
Preferably, in the reaction 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 reaction temperature is 30 to 50℃and the time is 12 to 48 hours. As a specific and preferred aspect, the pH of the reaction is controlled to 7-8 by phosphate buffer solution.
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; the biological assay reagents such as DNAmaroker, primeStar DNA polymerase, low molecular weight standard protein 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-3-formic acid comprises the following steps: chromatographic column-
ZWIX (-); column temperature/25 ℃; flow rate/0.5 mL/min; detection wavelength/UV 210nm; mobile phase: HPLC grade methanol/acetonitrile (50/50, v/v) (50 mM formic acid and 25mM dihexylamine added). The peak of each related substance is shown in figure 1.
Example 1 screening of D-amino acid oxidase and construction of genetically engineered bacterium expressing D-amino acid oxidase
According to the substrate specificity, D-amino acid oxidases of microbial origin can be divided into two broad categories, 1) amino acids with smaller substrate side chain groups (e.g., D-alanine) are preferred, such as D-amino acid oxidases of Fusarium oxysporum (Fusarium oxysporum) origin; 2) Amino acids with larger substrate side chain groups (e.g., D-phenylalanine) are preferred, such as D-amino acid oxidase from Trigonella (Trigonopsis variabilis) (POLLEGIONI L, MOLLAG, SACCHI S, et al properties and applications of microbial D-amino acid oxidases: current state and perspectives [ J ]. Appl Microbiol Biotechnol,2008,78 (1): 1-16.). BLASTP analysis was performed in the National Center for Biotechnology Information (NCBI) database (https:// www.ncbi.nlm.nih.gov /) using the amino acid sequences of the two D-amino acid oxidases, respectively, and 4D-amino acid oxidases having different sequence identity were selected for further study (as shown in Table 1).
TABLE 1 four different sources of D-amino acid oxidase
The D-amino acid oxidase gene sequence is sent to a division company of biological engineering (Shanghai) to carry out total gene synthesis after codon optimization, and cloned to a recombinant expression plasmid pET-28a (+). Transferring the recombinant plasmid into an expression host E.coliBL21 (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 20min. In the case of solid LB medium, an additional 15g/L of agar is added.
Inoculating engineering bacteria containing D-amino acid oxidase gene into 5mL liquid LB (containing 50 mug/mL kanamycin) culture medium, and shaking culturing at 37deg.C and 200rpmAbout 8 hours. Inoculated in 100mL of liquid LB (containing 50. Mu.g/mL kanamycin) medium at an inoculum size of 1% (V/V), cultured, and OD 600 After reaching 0.6-0.8, the inducer isopropyl thiogalactoside (final concentration 0.1 mM) was added and induced at 18℃for 15h. 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 stored in an ultra-low temperature refrigerator at-80 ℃ for use.
2.2 preparation of crude enzyme solution
The bacterial suspension was resuspended in 25mL of phosphate buffer (50 mM, pH=8.0), the bacterial suspension was sonicated, and the supernatant obtained after centrifugation was a crude enzyme solution containing D-amino acid oxidase.
2.3 high Performance liquid chromatography detection of the content of each enantiomer in the reaction System
Reaction system (1 ml): 10g/L E 1 、E 2 、E 3 Or E is 4 Wet cells (sonicated), 2g/L substrate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, the reaction medium was phosphate buffer (50 mm, ph=8.0). The prepared reaction system is placed in a 30 ℃ metal bath oscillation reactor for reaction for 120min. The reaction system in which the phosphate buffer was used instead of the crude enzyme solution was used as a control. The samples were diluted 10-fold with mobile phase and analyzed qualitatively by high performance liquid chromatography.
The results show that: e compared with the control 1 、E 2 、E 3 E and E 4 Can stereoselectively catalyze the reaction of (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid, while the content of (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is kept basically unchanged.
EXAMPLE 3 FsDAAO-NH 3 ·BH 3 Preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
Preparing a substrate solution: a5 g/L solution of racemic 1,2,3,4-tetrahydroisoquinoline-3-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.
Into a 100mL reactor, 24mL of substrate solution and 6mL of FsDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, so that the crude enzyme solution reaction system does not need to additionally add FAD) are added, and 12mg of peroxidationFreeze-dried powder of hydrogenase and 0.4g NH 3 ·BH 3 . Immediately after mixing, samples were taken as "0 hours". The reaction system was placed in a constant temperature water bath at 30℃and magnetically stirred, reacted for 24 hours, and sampled. And detecting the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the taken sample by high performance liquid chromatography.
As shown in FIGS. 1 and 2, fsDAAO shows strict R-configuration stereoselectivity, the reaction yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is 81.2% (reaction yield=actual product concentration (g/L)/theoretical product concentration (g/L). Times.100%), and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is 99.2%.
EXAMPLE 4 FsDAAO-NH 3 ·BH 3 Preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
Preparing a substrate solution: 2.5g/L of racemic 1,2,3,4-tetrahydroisoquinoline-3-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.
Into a 100mL reactor, 24mL of substrate solution, 6mL of FsDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, therefore, the crude enzyme solution reaction system does not need to additionally add FAD), 12mg of catalase freeze-dried powder and 0.25g of NH 3 ·BH 3 . Immediately after mixing, samples were taken as "0 hours". The reaction system was placed in a constant temperature water bath at 30℃and magnetically stirred, reacted for 16 hours, and sampled. And detecting the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the taken sample by high performance liquid chromatography. The reaction yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 84.1%, and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99.7%.
EXAMPLE 5 FgDAAO-NaBH 4 Preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
Into a 100mL reactor, 20mL of substrate solution, 10mL of FgDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, so that the crude enzyme solution reaction system does not need to additionally add FAD), 15mg of catalase freeze-dried powder and 0.2g of NaBH are added 4 . After mixing evenly, standI.e. sampled as "0 hours". The reaction system was placed in a constant temperature water bath at 30℃and magnetically stirred, reacted for 24 hours, and sampled. And detecting the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the taken sample by high performance liquid chromatography. The reaction yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 78.9%, and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99.1%.
EXAMPLE 6 FpDAAO-NaCNBH 3 Preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
Into a 100mL reactor, 20mL of substrate solution, 10mL of FpDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, so that the crude enzyme solution reaction system does not need to additionally add FAD), 10mg of catalase lyophilized powder and 0.35g of NaCNBH were added 3 . Immediately after mixing, samples were taken as "0 hours". The reaction system was placed in a constant temperature water bath at 30℃and magnetically stirred, reacted for 30 hours, and sampled. And detecting the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the taken sample by high performance liquid chromatography. The reaction yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 78.2%, and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99.6%.
EXAMPLE 7 TvDAAO-NH 3 ·BH 3 Preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
10mL of substrate solution, 20mL of TvDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, so that the crude enzyme solution reaction system does not need to additionally add FAD), 10mg of catalase lyophilized powder and 0.17g of NH are added into a 100mL reactor 3 ·BH 3 . Immediately after mixing, samples were taken as "0 hours". The reaction system was placed in a constant temperature water bath at 30℃and magnetically stirred, reacted for 36 hours, and sampled. And detecting the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the taken sample by high performance liquid chromatography. The reaction yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is 80.6%, and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is 99.1%.
EXAMPLE 8 pure enzyme FsDAAO-NH 3 ·BH 3 Preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
Adding 1.6mL substrate solution into 5mL reaction tube, adding FsDAAO pure enzyme solution, flavin adenine dinucleotide sodium salt, catalase, NH 3 ·BH 3 And the total reaction volume was made up to 2ml with phosphate buffer (50 mM, pH=8.0), the final concentration of FsDAAO pure enzyme was 0.2mg/ml, the final concentration of FAD was 100. Mu.M, 28mg NH 3 ·BH 3 (20 equivalents) the final concentration of catalase was 1mg/ml. 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-3-formic acid in the reaction system is detected by high performance liquid chromatography. The reaction yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is 86.5%, and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is 99.4%.
Example 9 preparation and isolation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution and the reaction system were as in example 3.
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 taken, freeze-dried, dissolved with hot ethanol and filtered to remove excess ammonia borane. The filtrate was taken and distilled at 50℃and the reaction volume was concentrated 10 times. Placing on ice, cooling, and suction filtering. The white crystals which precipitated were carefully scraped off, placed in an oven, dried and weighed. 0.01g of white dried crystals were weighed and fixed to a volume of 10ml with 50mM phosphate buffer solution (pH=8.0). And detecting the content of two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the taken sample by high performance liquid chromatography. The isolated yield of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 74.2% (isolated yield = amount of product actually isolated (mg)/amount of theoretical product (mg). Times.100%), (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid had an ee value of 99.4%.
Comparative example FsDAAO preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
Preparing a substrate solution: a5 g/L solution of racemic 1,2,3,4-tetrahydroisoquinoline-3-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.
1.6mL of substrate solution is taken and added into a 5mL reaction tube, and then 0.4mL of FsDAAO crude enzyme solution (the crude enzyme solution already contains enough coenzyme FAD, so that the additional FAD is not needed to be added into the crude enzyme solution reaction system). After mixing, samples were taken 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-3-formic acid in the reaction system is detected by an HPLC method, and the concentration (g/L) of the two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the reaction system can be obtained. FsDAAO exhibits a strict R-configuration stereoselectivity, and the conversion of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is 49.9% (conversion = [ (initial racemic substrate concentration (g/L) -residual substrate concentration (g/L))/initial racemic substrate concentration (g/L) ]. Times.100%), (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid has an ee value of 99% or more.
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 (13)
1. A method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid (I) by a chemical enzyme method,
the method comprises the following steps:
(1) Taking a racemate of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid or a racemate of 1,2,3, 4-tetrahydroisoquinoline-3-formate as a substrate, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to perform oxidative dehydrogenation reaction by using D-amino acid oxidase as a catalyst to generate iminoacid shown in a formula (II), wherein (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is not catalyzed and remains in a reaction system; the D-amino acid oxidase has an amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4;
wherein an imidic acid reducing agent is added to the reaction system at one or more time points among before, during and after the oxidative dehydrogenation reaction, the imidic acid reducing agent being used for reducing the imidic acid generated by the oxidative dehydrogenation reaction into a racemate of the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or a racemate of the 1,2,3, 4-tetrahydroisoquinoline-3-formate;
(2) Separating the (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid from the reaction system.
2. The process of claim 1, wherein the 1,2,3, 4-tetrahydroisoquinoline-3-carboxylate salt is an alkali metal or ammonium salt of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
3. The method of claim 1, wherein the catalyst is a crude enzyme solution or a pure enzyme or an immobilized enzyme of the D-amino acid oxidase, or the catalyst is a cell that expresses the D-amino acid oxidase in a cell.
4. The method of claim 3, wherein the cell is an engineered bacterium that expresses D-amino acid oxidase and the host cell of the engineered bacterium is e.collbl21 (DE 3).
5. The method of claim 4, wherein the engineering bacterium comprises an expression vector pET-28a (+), and the D-amino acid oxidase gene is linked to the expression vector pET-28a (+).
6. 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.
7. The method of claim 6, wherein the catalase is bovine liver catalase lyophilized powder.
8. The method of claim 6, wherein the enzyme activity ratio of the catalase to the D-amino acid oxidase is 1000 to 2000:1.
9. the method of claim 1, wherein in step (1), the oxidative dehydrogenation reaction is performed in the presence of a coenzyme flavin adenine dinucleotide.
10. The method according to claim 1, wherein in the step (1), the reaction system is first constructed, and then the reaction system is controlled to be in a set temperature and an aerobic environment to perform a reaction, wherein the reaction system comprises the substrate, the catalyst, a solvent, an imidic acid reducing agent and optionally a catalase for catalytically decomposing hydrogen peroxide, and the reaction system further optionally comprises a pH buffer and/or a pH regulator.
11. The method of claim 10, wherein the solvent is water, the substrate is dissolved in the aqueous solution of the pH buffer, the pH regulator is selectively added to prepare a substrate solution having a pH of 6 to 9, and the catalyst, the imidic acid reducing agent and the selective catalase are added to obtain the reaction system.
12. 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, and the set temperature is set to be 20 to 70 ℃; the addition amount of the imidic acid reducing agent is 3-20 equivalents of the molar amount of the substrate.
13. The method of claim 1, wherein the imidic acid reducing agent is a combination of one or more selected from the group consisting of sodium cyanoborohydride, borane amine, and sodium borohydride.
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| PCT/CN2019/083840 WO2020107780A1 (en) | 2018-11-30 | 2019-04-23 | Method for preparing (s)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by means of chemoenzymatic method |
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| Peter Benz等.Amino acid oxidase-catalysed resolution and Pictet–Spengler reaction towards chiral and rigid unnatural amino acids.Journal of Chemical Technology and Biotechnology.2007,第82卷第1082-1086页. * |
| Wolf Dieter Fessner.现代生物催化——高离体选择及环境友好的反应.中国轻工业出版社,2016,第190-191页. * |
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