CN111254180B - Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution - Google Patents
Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution Download PDFInfo
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Abstract
The invention discloses a novel method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution, which comprises the following steps: the (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is prepared by taking racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid or salt thereof as a substrate, using isolated D-amino acid oxidase or cells expressing the D-amino acid oxidase in cells as a catalyst, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to perform oxidative dehydrogenation reaction, and keeping the (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid in a reaction system without being catalyzed. The (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is a key intermediate of quinapril which is a antihypertensive drug. The invention has the advantages of 49% of reaction yield, 99% of ee value, mild reaction condition, strong stereoselectivity, high reaction efficiency, relatively simple process and the like.
Description
Technical Field
The invention relates to a novel method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution.
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 (study [ J ]. Modern chemical engineering, 2003,23 (12): 23-5 ] of synthesizing optically pure (S) -1,2,3, 4-tetrahydroquinoline-3-carboxylic acid by a chemical enzyme method).
In the existing technology, there is no method for preparing (S) -configuration products by directly utilizing enzymatic kinetics to separate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
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 enzymatic resolution,
the method comprises the following steps:
(1) Taking a racemate of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or a racemate of 1,2,3, 4-tetrahydroisoquinoline-3-formate as a substrate, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid to carry out oxidative dehydrogenation reaction by taking isolated D-amino acid oxidase or cells expressing the D-amino acid oxidase in cells as a catalyst, wherein (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is unreacted and remains in a reaction system;
(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 preferably a combination of one or more selected from the group consisting of the following D-amino acid oxidases: a D-amino acid oxidase derived from trigonomycetes (Trigonopsis variabilis) CBS 4095 or a mutant thereof or other D-amino acid oxidase having an amino acid sequence homology of more than 80%, a D-amino acid oxidase derived from Fusarium graminearum (Fusarium graminearum) CS3005 or a mutant thereof or other D-amino acid oxidase having an amino acid sequence homology of more than 80% (preferably more than 85%, more preferably more than 90%, further preferably more than 95%), a D-amino acid oxidase derived from Fusarium pyriformis (Fusarium poae) 2516 or a mutant thereof or other D-amino acid oxidase having an amino acid sequence homology of more than 80% (preferably more than 85%, more preferably more than 90%, further preferably more than 95%) or a mutant thereof or other D-amino acid oxidase derived from Fusarium solani (Fusarium solani) M-0718.
Further 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.
As a preferred embodiment of the present invention: the cell is an engineering bacterium for expressing D-amino acid oxidase. The engineering bacteria are engineering bacteria with E.coli BL21 (DE 3) as host cells.
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 (+).
Further, in the step (1), a reaction system is firstly 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 pH buffer solution and/or a pH regulator and the catalyst.
Preferably, in the step (1), the concentration of the initial substrate in the reaction system is 1-20 g/L, and the pH value of the reaction system is 6-9; the catalyst is crude enzyme liquid or pure enzyme containing the isolated D-amino acid oxidase or immobilized enzyme or cell expressing the D-amino acid oxidase in cells; the set temperature is 20-70 ℃.
Preferably, the catalyst employs a crude enzyme solution containing an isolated D-amino acid oxidase and flavin adenine dinucleotide.
According to one aspect of the invention, the catalyst is added in an amount of generally 1 to 5% by weight of the reaction system based on the wet cell weight after centrifugation at 8000rpm for 10 min.
Preferably, in the step (1), the set temperature is 30-50 ℃, and the pH value of the reaction system is 7-8.
Preferably, in step (1), the reaction system contains coenzyme Flavin Adenine Dinucleotide (FAD). The oxidative dehydrogenation reaction is carried out in the presence of FAD, which contributes to further improvement of the reaction yield. 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 a specific and preferred aspect of the present invention, the pH buffer solution is a phosphate buffer solution.
According to yet another specific and preferred aspect of the present invention, the pH adjustor is 20wt% to 35wt% ammonia water.
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 by suction, the filtrate is concentrated, cooled and crystallized, and dried, thus obtaining the (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the invention surprisingly found that D-amino acid oxidase can efficiently and selectively catalyze (R) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid to perform oxidative dehydrogenation reaction, and has no catalytic effect on (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid. 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 of example 3 sampled at 0 hour, wherein the retention time 8.673min is (R) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid; the retention time was 10.969min to be (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, which takes racemized 1,2,3, 4-tetrahydroisoquinoline-3-formic acid (or ammonia salt) as a substrate, and uses isolated D-amino acid oxidase or cells expressing the D-amino acid oxidase in cells as a catalyst to perform oxidative dehydrogenation 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 (shown as a formula II) is taken as a substrate, D-amino acid oxidase is utilized to stereoselectively catalyze (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to generate corresponding imidic acid (shown as a formula III), and (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid (shown as a formula I) is not catalyzed and remains in a reaction system. 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, the reaction proceeds as follows:
according to the present invention, the introduction of the D-amino acid oxidase can be achieved by adding an ex vivo enzyme or a cell expressing the D-amino acid oxidase in a cell. The isolated enzyme may be in the form of a crude enzyme solution, a pure enzyme, or an immobilized enzyme, without limitation. The cell expressing the D-amino acid oxidase in the cell can be specifically an engineering bacterium resting cell expressing the recombinase.
According to a preferred aspect of the present invention, the D-amino acid oxidase is derived from Trigonella, fusarium graminearum, fusarium pyriform, fusarium solani, etc., and specifically, the D-amino acid oxidase may be a D-amino acid oxidase derived from Trigonella (Trigonopsis variabilis) CBS 4095, fusarium graminearum (Fusarium graminearum) CS3005, fusarium pyriform 2516 or Fusarium solani (Fusarium solani) M-0718.
According to still another preferred aspect of the present invention, the cell expressing the D-amino acid oxidase in the cell is an engineering bacterium expressing the D-amino acid oxidase. In a preferred embodiment, the host cell of the engineered 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 (+).
According to a specific aspect of the present invention, a recombinant D-amino acid oxidase is obtained using an engineering bacterium expressing the D-amino acid oxidase, and is used as a catalyst.
According to a preferred aspect of the invention, the concentration of the substrate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the catalytic system is from 1 to 20g/L.
According to a preferred aspect of the present invention, 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 minutes.
According to a preferred aspect of the invention, 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 reaction temperature is 30 to 50℃and the time is 12 to 48 hours.
Preferably, the pH of the reaction is controlled to 7-8 using a phosphate buffer solution.
The invention is further illustrated below 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. 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:
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 preferred side chain groups (e.g., D-alanine), such as DAAO from Fusarium oxysporum (Fusarium oxysporum); 2) Amino acids with larger side chain groups (e.g., D-phenylalanine) are preferred, such as DAAO (PolEGIONI 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) from Trigonella foenum-graecum (Trigonopsis variabilis). The amino acid sequences of these two D-amino acid oxidases were each subjected to BLASTP analysis in the National Center for Biotechnology Information (NCBI) database (https:// www.ncbi.nlm.nih.gov /), and 4D-amino acid oxidases differing in 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.
The engineering bacteria containing the D-amino acid oxidase gene constructed in the method of example 1 were inoculated into 5mL of liquid LB (containing 50. Mu.g/mL kanamycin) medium, and cultured with shaking at 37℃and 200rpm for about 8 hours. Inoculated in 50mL of liquid LB (containing 50. Mu.g/mL kanamycin) medium at 1% (V/V) inoculum size for cultivation, OD 600 After reaching 0.6-0.8, the inducer IPTG (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 then stored in an ultra-low temperature refrigerator at-80℃for use.
2.2 preparation of crude enzyme solution
The bacterial suspension was sonicated in 50mM phosphate buffer (pH 8.0), and the supernatant obtained after centrifugation was a crude enzyme solution containing D-amino acid oxidase.
2.3 HPLC method for detecting content of each enantiomer in reaction system
Reaction system (1 ml): 10g/L E 1 、E 2 、E 3 、E 4 Wet cells (sonicated), 2g/L substrate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, reaction medium pH 8.0 phosphate buffer. The prepared reaction system is placed in a 30 ℃ metal bath oscillation reactor for reaction for 120min. The reaction system in which the crude enzyme solution was replaced with phosphate buffer (pH 8.0) was used as a control. Samples were diluted 10-fold with mobile phase and analyzed qualitatively by HPLC.
The results show that: number E compared to control 1 、E 2 、E 3 E and E 4 The D-amino acid oxidase of (C) is capable of stereoselectively catalyzing the reaction of (R) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid while the content of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid remains substantially unchanged.
EXAMPLE 3 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid by FsDAAO resolution
Preparing a substrate solution: 5g/L of a 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 the substrate solution and 0.4mL 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 into a 5mL reaction tube. 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 an HPLC method.
As shown in FIG. 2, fsDAAO exhibits a strict R-configuration stereoselectivity, and the conversion 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.
EXAMPLE 4 FgDAAO resolution preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
To a 5mL reaction tube, 0.8mL of a substrate solution and 1.2mL of FgDAAO crude enzyme solution (the crude enzyme solution already contains a sufficient amount of coenzyme FAD, so that the crude enzyme solution reaction system does not need to additionally add FAD) were added. 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 30 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. The detection result is as follows: the conversion rate is 49.8% (the calculation formula is shown in example 3), and the ee value of the (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is more than 99%.
EXAMPLE 5 FpDAAO resolution preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
To a 5mL reaction tube, 0.4mL of a substrate solution and 1.6mL of a crude FpDAAO enzyme solution (the crude enzyme solution already contains a sufficient amount of coenzyme FAD, so that the crude enzyme solution reaction system does not need to additionally contain FAD) were added. 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 an HPLC method. The detection result is as follows: the conversion was 49.9% (the formula is shown in example 3), and the ee value of the (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99% or more.
EXAMPLE 6 resolution of TvDAAO preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
To a 5mL reaction tube, 0.8mL of a substrate solution and 1.2mL of TvDAAO crude enzyme solution (the crude enzyme solution already contains a sufficient amount of coenzyme FAD, so that the crude enzyme solution reaction system does not need to additionally add FAD) were added. 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 25 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. The detection result is as follows: the conversion was 49.9% (the formula is shown in example 3), and the ee value of the (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99% or more.
EXAMPLE 7 resolution of pure enzyme FsDAAO preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
The substrate solution was prepared as in example 3.
1.6mL of the substrate solution was added to a 5mL reaction tube, fsDAAO pure enzyme solution (obtained by nickel column affinity chromatography purification) and flavin adenine dinucleotide sodium salt (FAD) were further added, and the total reaction volume was made up to 2mL with phosphate buffer solution (50 mM, pH=8.0), the final concentration of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 4g/L, the final concentration of FsDAAO pure enzyme was 0.1mg/mL, and the final concentration of FAD was 100. Mu.M. 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 an HPLC method, the conversion rate is 50% (the calculation formula is shown in example 3), and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid reaches more than 99%.
EXAMPLE 8 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid by FsDAAO
Preparing a substrate solution: 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 substrate solution was adjusted to 8.0 with 5M sodium hydroxide solution.
1.6mL of the substrate solution and 0.4mL 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 into a 5mL reaction tube. 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 an HPLC method. The detection result is as follows: the conversion was 49.9% (the formula is shown in example 3), and the ee value of the (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99% or more.
EXAMPLE 9 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid by FsDAAO large reaction System
Preparing a substrate solution: a4 g/L solution of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was prepared with deionized water and the pH of the solution was adjusted to 8.0 with 30% aqueous ammonia.
200mL of substrate solution and 20mL of FsDAAO crude enzyme solution 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 30 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 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. Weigh 0.01g white dried crystal and volume to 10ml with deionized water. 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 conversion was 49.9% (the calculation formula is shown in example 3), the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99% or more, and the isolation yield was 85% (isolation yield=the amount of the product actually isolated (mg)/the amount of the theoretical product (mg) ×100%).
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 (9)
1. A method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid (I) by enzymatic resolution,
the method comprises the following steps:
(1) Taking a racemate of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or a racemate of 1,2,3, 4-tetrahydroisoquinoline-3-formate as a substrate, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid to carry out oxidative dehydrogenation reaction by taking isolated D-amino acid oxidase or cells expressing the D-amino acid oxidase in cells as a catalyst, wherein (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid is unreacted 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;
(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 cell is an engineered bacterium that expresses D-amino acid oxidase and the host cell of the engineered bacterium is e.collbl21 (DE 3).
4. The method of claim 3, 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 (+).
5. The method according to claim 1, wherein in the step (1), a reaction system is first constructed, and then the oxidative dehydrogenation reaction is performed by controlling the reaction system to be in a set temperature and an aerobic environment, wherein the reaction system comprises the substrate, a pH buffer solution and/or a pH adjuster, and the catalyst.
6. The method according to claim 5, wherein in the step (1), the concentration of the starting substrate in the reaction system is 1 to 20g/L and the pH value of the reaction system is 6 to 9; the catalyst is crude enzyme liquid or pure enzyme containing the isolated D-amino acid oxidase or immobilized enzyme or cell expressing the D-amino acid oxidase in cells; the set temperature is 20-70 ℃.
7. The method according to claim 5, wherein in the step (1), the set temperature is 30 to 50℃and the pH of the reaction system is 7 to 8.
8. The method of claim 6, wherein the catalyst is a crude enzyme solution comprising an isolated D-amino acid oxidase and flavin adenine dinucleotide.
9. The method of claim 1 or 5, wherein in step (1), the oxidative dehydrogenation reaction is performed in the presence of a coenzyme flavin adenine dinucleotide.
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| PCT/CN2019/083877 WO2020107781A1 (en) | 2018-11-30 | 2019-04-23 | Method for preparing (s)-1,2,3,4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution |
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| US8173648B2 (en) * | 2008-07-16 | 2012-05-08 | National Defense Medical Center | 1,2,3,4-tetrahydroisoquinoline derivatives, preparation process therefor and pharmaceutical composition containing the same |
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