CN111254180A - 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
- Publication number
- CN111254180A CN111254180A CN201811454845.2A CN201811454845A CN111254180A CN 111254180 A CN111254180 A CN 111254180A CN 201811454845 A CN201811454845 A CN 201811454845A CN 111254180 A CN111254180 A CN 111254180A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- tetrahydroisoquinoline
- gly
- val
- acid oxidase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a new method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution, which comprises the following steps: using racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid or salt thereof as a substrate, and using an isolated D-amino acid oxidase or a cell expressing the D-amino acid oxidase intracellularly as a catalyst, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to perform oxidative dehydrogenation reaction, wherein (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is not catalyzed and is retained in a reaction system, thereby preparing and obtaining (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid. The (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is a key intermediate of a hypotensor quinapril. The invention has the advantages of reaction yield of over 49 percent, ee value of 99 percent, 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-tetrahydroquinoline-3-carboxylic 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 hypotensive drug quinapril (conversion-oriented synthesis of metabolic antigens 1,2,3, 4-tetrahydroquinoline-3-carboxylic acid (Tic) derivatives and highers analytes [ J ]. Org Biomol Chem,2014,12(45): 9054-91.). In addition, (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid can be used for synthesizing small molecule antagonists containing tetrahydroisoquinoline mother nucleus, and acts on CXCR4 of chemokine receptor, thereby being expected to be used for treating diseases such as HIV (Discovery of tetrahydroquinoline-based CXCR4antagonists [ J ]. ACS Med Chem Lett,2013,4(11): 1025-30.).
In the prior art, methods for preparing optically pure (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid include two methods, namely chemical chiral synthesis and biocatalytic kinetic resolution. Researchers originally utilized the Pictet-Spengler reaction to prepare optically pure (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, and condensed with formaldehyde under the conditions of concentrated acid and high temperature using L-phenylalanine as raw material to produce the target product. Subsequently, Bischler-Nepieralski reaction, [2+2+2] cycloaddition method, etc. have been used to prepare (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid and its derivatives, which are complicated in route and high in cost (decentralized-oriented synthesis of catalytic antigens 1,2,3, 4-tetrahydroquinoneine-3-carboxylic acid (Tic) derivatives and highers analytes [ J ] Org Biomol Chem,2014,12(45): 9054-91.). In recent years, Kurata et al have not synthesized (S) -1,2,3, 4-Tetrahydroisoquinoline-3-Carboxylic acid (Synthesis of optical Pure (R) -and (S) -tetrahydroquinoline-1-and-3-Carboxylic Acids [ J ] Synthesis,2015,47(09):1238-44.) by three steps of ozonolysis, oxidation and deprotection. The method has low yield and more steps, and is not easy to be applied industrially. Gong, etc. utilizes a chemoenzymatic method to prepare (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid, namely racemic phenylalanine as a raw material, synthesizes racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid through a Pictet-Spengler reaction, and then prepares an (S) -configuration product through esterification and lipase kinetic resolution. 23.8g racemic ester hydrochloride (0.1mol), lipase and substrate mass ratio of 0.2, reaction for 48h, ee of product > 99%, yield 49.1%. The product obtained by the method has high stereoselectivity and relatively simple process (research on synthesizing optically pure (S) -1,2,3, 4-tetrahydroquinoline-3-carboxylic acid by a chemical enzyme method [ J ]. modern chemical industry, 2003,23(12): 23-5.).
In the existing process, there is no method for preparing (S) -configuration product by directly utilizing enzymatic kinetic resolution of racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide 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 an industrial application prospect.
In order to achieve the 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) using 1,2,3, 4-tetrahydroisoquinoline-3-formic acid racemate or 1,2,3, 4-tetrahydroisoquinoline-3-formate racemate as substrate, using isolated D-amino acid oxidase or cell expressing D-amino acid oxidase intracellularly as catalyst, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to perform oxidative dehydrogenation reaction, while (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is not reacted and remains in the reaction system;
(2) separating the (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid from the reaction system.
The 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid salt may be an alkali metal salt or an ammonium salt of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and specifically, for example, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid sodium salt, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid potassium salt, and 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid ammonium salt.
According to the 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 Trigonopsis variabilis CBS 4095 or a mutant thereof or another D-amino acid oxidase having an amino acid sequence homology of more than 80%, a D-amino acid oxidase derived from Fusarium graminearum CS3005 or a mutant thereof or another 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 peaorum 2516 or a mutant thereof or another 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 solani M-0718 or a mutant thereof or another D-amino acid oxidase having an amino acid sequence homology of more than 80% (preferably more than 85%, more preferably more than 90%, even more preferably more than 95%) of other D-amino acid oxidases.
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, for example, engineering bacteria of which host cells are 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 (+).
Further, in the step (1), a reaction system is firstly constructed, and then the reaction system is controlled to be in a set temperature and aerobic environment to perform the oxidative dehydrogenation reaction, wherein the reaction system comprises the substrate, the pH buffer solution and/or the 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 in vitro D-amino acid oxidase or immobilized enzyme or cells for expressing the D-amino acid oxidase in cells; the set temperature is 20-70 ℃.
Preferably, the catalyst adopts crude enzyme liquid containing D-amino acid oxidase and flavin adenine dinucleotide in vitro.
According to one aspect of the invention, the addition amount of the catalyst is calculated by wet cell weight after centrifugation at 8000rpm for 10min, and the addition amount of the cells is 1-5% of the weight of the reaction system.
Preferably, in the step (1), the set temperature is 30-50 ℃, and the pH value of the reaction system is 7-8.
Preferably, in the 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 equivalent to the substrate or in excess. In general, a sufficient amount of FAD is already contained in a crude enzyme solution of a D-amino acid oxidase to be produced, and in the case where the crude enzyme solution is used as it is, it is not necessary to separately add FAD. When a D-amino acid oxidase pure enzyme is used, an appropriate amount of FAD may be added as needed.
According to a particular and preferred aspect of the invention, the pH buffer solution is a phosphate buffer solution.
According to a further specific and preferred aspect of the present invention, the pH adjusting agent is 20 wt% to 35 wt% aqueous ammonia.
Further, in the step (2), the pH value of the reaction system is adjusted to 5.0-6.0, the protein is denatured and precipitated by heating, the filtration is carried out, the filtrate is concentrated, and then the (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is obtained by cooling, crystallization and drying.
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 basically has no catalytic action on (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic 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 percent) and simple process.
Drawings
FIG. 1 is a HPLC analysis chart of 0 hour sampling in the reaction system in example 3, wherein the retention time 8.673min is (R) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid; retention time 10.969min is (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid;
FIG. 2 is a high performance liquid chromatography chromatogram obtained by sampling a reaction time of 24 hours in the reaction system of example 3.
Detailed Description
The invention provides a new method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid, which takes racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid (or ammonia salt) as a substrate and takes an isolated D-amino acid oxidase or a cell expressing the D-amino acid oxidase intracellularly as a catalyst to carry out oxidative dehydrogenation reaction to obtain (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid.
The specific principle is as follows: using racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid (shown as formula II) as a substrate, and using D-amino acid oxidase to stereoselectively catalyze (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to perform oxidative dehydrogenation reaction to generate corresponding imidic acid (shown as formula III), wherein (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid (shown as formula I) is not catalyzed and is remained in a reaction system. The reaction process is schematically shown as follows:
further, it is preferable that the reaction is carried out in the presence of coenzyme Flavin Adenine Dinucleotide (FAD)In the catalytic process, coenzyme Flavin Adenine Dinucleotide (FAD) is reduced to FADH2Subsequently, a molecule of oxygen is reduced to hydrogen peroxide (H)2O2) And FADH2Then oxidized to FAD, and the reaction process is schematically shown as follows:
according to the invention, the introduction of the D-amino acid oxidase can be effected by adding an enzyme ex vivo or cells expressing the D-amino acid oxidase intracellularly. The isolated enzyme may be in the form of crude enzyme liquid, pure enzyme, or immobilized enzyme, without limitation. The cell for intracellular expression of the D-amino acid oxidase can be specifically an engineering bacteria resting cell for expression of recombinase.
According to a preferred aspect of the present invention, the D-amino acid oxidase is derived from Trigonopsis variabilis, Fusarium graminearum, Fusarium peaorum, Fusarium solani, etc., and specifically, the D-amino acid oxidase may be a D-amino acid oxidase derived from Trigonopsis variabilis (Trigonopsis variabilis) CBS 4095, Fusarium graminearum (Fusarium graminearum) CS3005, Fusarium peaorum (Fusarium poae)2516, or Fusarium solani (Fusarium solani) M-0718.
According to still another preferred aspect of the present invention, the cell which intracellularly expresses the D-amino acid oxidase is an engineered bacterium which expresses 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, recombinant D-amino acid oxidase is obtained using an engineered bacterium that expresses the D-amino acid oxidase, and is used as a catalyst.
According to a preferred aspect of the present invention, the concentration of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the catalytic system is 1 to 20 g/L.
According to a preferable aspect of the invention, in the catalytic system, the addition amount of the catalyst is 1-5% of the weight of the reaction solution, based on the wet weight of the cells after centrifugation at 8000rpm for 10 min.
According to a preferable 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 30-50 ℃ and the reaction time is 12-48 hours.
Preferably, the pH value of the reaction is controlled to be 7-8 by adopting a phosphoric acid buffer solution.
The present invention is further illustrated by the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The experimental procedures in the examples of the present invention are conventional ones unless otherwise specified.
The gene used in the examples of the present invention was synthesized by Biotechnology engineering (Shanghai) Co., Ltd. Coli bl21(DE3) strain was purchased from Novagen. The specific operation of gene cloning and expression can be found in molecular cloning experimental guidelines compiled by J. SammBruk et al.
The present invention analyzes each product and substrate of the catalyzed reaction by High Performance Liquid Chromatography (HPLC). The HPLC analysis method of racemic 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 210 nm; mobile phase: HPLC grade methanol/acetonitrile (50/50, v/v) (50mM formic acid and 25mM dihexylamine added). The specific peak appearance 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 derived from microorganisms can be classified into two major classes, 1) amino acids with a smaller side chain group (e.g., D-alanine), such as DAAO derived from Fusarium oxysporum (Fusarium oxysporum); 2) amino acids with larger side chain groups (e.g., D-phenylalanine) are preferred, such as DAAO from Trigonopsis variabilis (POLEGIONI L, MOLLAG, SACCHI S, et al, properties and applications of microbial D-amino acid oxides: current state and fields [ J ]. appl Microbiol Biotechnol,2008,78(1): 1-16.). The amino acid sequences of these two D-amino acid oxidases were subjected to BLASTp analysis in the National Center for Biotechnology Information (NCBI) database (https:// www.ncbi.nlm.nih.gov /), respectively, and 4D-amino acid oxidases differing in sequence identity were selected for further study (as shown in Table 1).
TABLE 1 four D-amino acid oxidases of different origins
The D-amino acid oxidase gene sequence is sent to the company of biological engineering (Shanghai) to be synthesized into a whole gene after codon optimization, and is cloned to a recombinant expression plasmid pET-28a (+). Transferring the recombinant plasmid into an expression host E.coliBL21(DE3), and after sequencing verification, adding 25% of glycerol to the obtained engineering bacteria liquid and preserving at-80 ℃ for later use.
Example 2
2.1 cultivation of the microorganism
Liquid LB medium composition: 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl, dissolving with deionized water, fixing the volume, and sterilizing at 121 ℃ for 20min for later use. If the solid LB medium is used, 15g/L of agar is added.
The engineered bacterium containing the D-amino acid oxidase gene constructed in the manner described in example 1 was inoculated into 5mL of liquid LB medium (containing 50. mu.g/mL kanamycin) and cultured at 37 ℃ for about 8 hours with shaking at 200 rpm. Inoculating to 50mL liquid LB (containing 50. mu.g/mL kanamycin) medium at an inoculum size of 1% (V/V), and culturing at OD600After reaching 0.6-0.8, inducer IPTG (final concentration of 0.1mM) is added, and induction is carried out for 15h at 18 ℃. After the culture is finished, the culture solution is poured into a 100mL centrifuge tube and centrifuged at 4000rpm for 10min, the supernatant is discarded, the somatic cells are collected, the cells are washed twice by 50mM phosphate buffer solution (pH 8.0), and then the cells are stored in an ultra-low temperature refrigerator at minus 80 ℃ for standby.
2.2 preparation of crude enzyme solution
The cells were resuspended in 50mM phosphate buffer (pH 8.0), the cell suspension was disrupted by sonication, and the supernatant obtained after centrifugation was the crude enzyme solution containing D-amino acid oxidase.
2.3 HPLC method for detecting the content of each enantiomer in the reaction system
Reaction system (1 ml): 10g/L E1、E2、E3、E4Wet thalli (ultrasonic disruption), 2g/L substrate racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid, reaction medium for pH 8.0 phosphate buffer solution. And placing the prepared reaction system in a metal bath oscillation reactor at 30 ℃ for reaction for 120 min. As a control, a reaction system in which a phosphate buffer solution (pH 8.0) was used in place of the crude enzyme solution. Samples were diluted 10-fold with mobile phase and qualitatively analyzed by HPLC.
The results show that: number E compared to control1、E2、E3And E4The D-amino acid oxidase of (A) 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 basically kept unchanged.
EXAMPLE 3 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid by resolution of FsDAAO
Preparation of a substrate solution: using 50mM phosphate buffer solution (pH 8.0) to prepare 5g/L racemic 1,2,3, 4-four hydrogen isoquinoline-3-formic acid solution and using 30% ammonia water to adjust the pH value of the solution to 8.0.
1.6mL of substrate solution and 0.4mL of crude FsDAAO enzyme solution (the crude enzyme solution already contains enough coenzyme FAD, so that no FAD needs to be added in the reaction system of the crude enzyme solution) are added into a 5mL reaction tube. After mixing, 50. mu.L of the mixture was taken out as "0 hour" and analyzed by HPLC. The reaction tube is placed in a constant temperature water bath at 30 ℃, and is magnetically stirred to react for 24 hours. After the reaction is finished, the contents of the two configurations of the 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the reaction system are detected by an HPLC method.
As shown in FIG. 2, FsDAAO exhibited strict R-configuration stereoselectivity, the conversion rate was 49.9% (i.e., [ (initial racemic substrate concentration (g/L) — residual substrate concentration (g/L))/initial racemic substrate concentration (g/L) ]. times.100%), and the ee value of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid was 99% or more.
EXAMPLE 4 FgDAAO resolution preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
A substrate solution was prepared in the same manner as in example 3.
0.8mL of substrate solution and 1.2mL of FgDAAO crude enzyme solution (the crude enzyme solution already contains enough coenzyme FAD, so that no FAD needs to be added in the reaction system of the crude enzyme solution) are added into a 5mL reaction tube. After mixing, 50. mu.L of the mixture was taken out as "0 hour" and analyzed by HPLC. The reaction tube is placed in a constant temperature water bath at 30 ℃, and is magnetically stirred to react for 30 hours. After the reaction is finished, the contents of the two configurations of the 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the reaction system are detected by an HPLC method. The detection result is as follows: the conversion rate of 49.8% (calculation formula as shown in example 3), (S) -1,2,3, 4-four hydrogen isoquinoline-3-carboxylic acid ee value reaches more than 99%.
EXAMPLE 5 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid by FpDAAO resolution
A substrate solution was prepared in the same manner as in example 3.
0.4mL of substrate solution and 1.6mL of crude FpDAAO enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, so that no FAD needs to be added additionally in the reaction system of the crude enzyme solution) are added into a 5mL reaction tube. After mixing, 50. mu.L of the mixture was taken out as "0 hour" and analyzed by HPLC. The reaction tube is placed in a constant temperature water bath at 30 ℃, and is magnetically stirred to react for 24 hours. After the reaction is finished, the contents of the two configurations of the 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the reaction system are detected by an HPLC method. The detection result is as follows: the conversion rate of 49.9% (calculation formula as shown in example 3), (S) -1,2,3, 4-four hydrogen isoquinoline-3-carboxylic acid ee value up to 99%.
EXAMPLE 6 TvDAAO resolution preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
A substrate solution was prepared in the same manner as in example 3.
0.8mL of substrate solution and 1.2mL of TvDAAO crude enzyme solution (the crude enzyme solution already contains enough coenzyme FAD, so that no FAD needs to be added in the reaction system of the crude enzyme solution) are added into a 5mL reaction tube. After mixing, 50. mu.L of the mixture was taken out as "0 hour" and analyzed by HPLC. The reaction tube is placed in a constant temperature water bath at 30 ℃, and is magnetically stirred to react for 25 hours. After the reaction is finished, the contents of the two configurations of the 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the reaction system are detected by an HPLC method. The detection result is as follows: the conversion rate of 49.9% (calculation formula as shown in example 3), (S) -1,2,3, 4-four hydrogen isoquinoline-3-carboxylic acid ee value up to 99%.
EXAMPLE 7 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid by resolution of pure enzyme FsDAAO
A substrate solution was prepared in the same manner as in example 3.
Adding 1.6mL of substrate solution into a 5mL reaction tube, adding FsDAAO pure enzyme solution (obtained by nickel column affinity chromatography purification) and flavin adenine dinucleotide sodium salt (FAD), and supplementing the total reaction volume to 2mL by using phosphate buffer solution (50mM, pH 8.0), (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid final concentration of 4g/L, FsDAAO pure enzyme final concentration of 0.1mg/mL, FAD final concentration of 100 μ M. After mixing, 50. mu.L of the mixture was taken out as "0 hour" and analyzed by HPLC. The reaction tube is placed in a constant temperature water bath at 30 ℃, and is magnetically stirred to react for 24 hours. After the reaction, HPLC method is used to detect the content of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction system, 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-carboxylic acid reaches more than 99%.
EXAMPLE 8 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid by FsDAAO
Preparation of a substrate solution: 5g/L 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 substrate solution and 0.4mL of crude FsDAAO enzyme solution (the crude enzyme solution already contains enough coenzyme FAD, so that no FAD needs to be added in the reaction system of the crude enzyme solution) are added into a 5mL reaction tube. After mixing, 50. mu.L of the mixture was taken out as "0 hour" and analyzed by HPLC. The reaction tube is placed in a constant temperature water bath at 30 ℃, and is magnetically stirred to react for 24 hours. After the reaction is finished, the contents of the two configurations of the 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the reaction system are detected by an HPLC method. The detection result is as follows: the conversion rate of 49.9% (calculation formula as shown in example 3), (S) -1,2,3, 4-four hydrogen isoquinoline-3-carboxylic acid ee value up to 99%.
EXAMPLE 9 preparation of (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid using the FsDAAO Large reaction System
Preparation of a substrate solution: preparing 4g/L racemic 1,2,3, 4-tetrahydroisoquinoline-3-formic acid solution by deionized water and adjusting the pH value of the solution to 8.0 by 30% ammonia water.
200mL of substrate solution, 20mL of crude FsDAAO enzyme solution were added to the reactor. After mixing, placing in a constant temperature water bath at 30 ℃, stirring by magnetic force, and reacting for 30 hours. After the reaction is finished, the pH value of the reaction system is adjusted to 5.0-6.0. Water bath at 99 deg.c, and suction filtering after protein denaturation and separation. The filtrate was rotary evaporated at 65 ℃ and the reaction volume was concentrated 10 times. Placing on ice, cooling and filtering. The precipitated white crystals were carefully scraped off, placed in an oven, dried and weighed. 0.01g of white dried crystal is weighed and made up to 10ml with deionized water. Detecting the contents of two configurations of 1,2,3, 4-tetrahydroisoquinoline-3-formic acid in the sample by high performance liquid chromatography. The conversion was 49.9% (the calculation formula is shown in example 3), (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid ee value reached more than 99%, the isolation yield was 85% (the isolation yield ═ the amount of the product isolated actually (mg)/the theoretical amount of the product (mg). times.100%).
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Sequence listing
<110> Suzhou synergetics biomedical Co., Ltd
<120> method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>361
<212>PRT
<213> human (Homo)
<400>1
Met Ser Asn Thr Ile Val Val Val Gly Ala Ile Val Ser Gly Leu Thr
1 5 10 15
Ser Ala Leu Leu Leu Ser Lys Asn Lys Gly Asn Lys Ile Thr Val Val
20 25 30
Ala Lys His Met Pro Gly Asp Tyr Asp Val Glu Tyr Ala Ser Pro Phe
35 40 45
Ala Gly Ala Asn His Ser Pro Met Ala Thr Glu Glu Ser Ser Glu Trp
50 55 60
Glu Arg Arg Thr Trp Tyr Glu Phe Lys Arg Leu Val Glu Glu Val Pro
65 70 75 80
Glu Ala Gly Val His Phe Gln Lys Ser Arg Ile Gln Arg Arg Asn Val
85 90 95
Asp Thr Glu Lys Ala Gln Arg Ser Gly Phe Pro Asp Ala Leu Phe Ser
100 105 110
Lys Glu Pro Trp Phe Lys Asn Met Phe Glu Asp Phe Arg Glu Gln His
115 120 125
Pro Ser Glu Val Ile Pro Gly Tyr Asp Ser Gly Cys Glu Phe Thr Ser
130 135 140
Val Cys Ile Asn Thr Ala Ile Tyr Leu Pro Trp Leu Leu Gly Gln Cys
145 150155 160
Leu Lys Asn Gly Val Ile Val Lys Arg Ala Ile Leu Asn Asp Ile Ser
165 170 175
Glu Ala Lys Lys Leu Ser His Ala Gly Lys Thr Pro Asn Ile Ile Val
180 185 190
Asn Ala Thr Gly Leu Gly Ser Tyr Lys Leu Gly Gly Val Glu Asp Lys
195 200 205
Thr Met Ala Pro Ala Arg Gly Gln Ile Val Val Val Arg Asn Glu Ser
210 215 220
Ser Pro Met Leu Leu Thr Ser Gly Val Glu Asp Gly Gly Ala Asp Val
225 230 235 240
Met Tyr Leu Met Gln Arg Ala Ala Gly Gly Gly Thr Ile Leu Gly Gly
245 250 255
Thr Tyr Asp Val Gly Asn Trp Glu Ser Gln Pro Asp Pro Asn Ile Ala
260 265 270
Asn Arg Ile Met Gln Arg Ile Val Glu Val Arg Pro Glu Ile Ala Asn
275 280 285
Gly Lys Gly Val Lys Gly Leu Ser Val Ile Arg His Ala Val Gly Met
290 295 300
Arg Pro Trp Arg Lys Asp Gly Leu Arg Ile Glu Glu Glu Lys Leu Asp
305 310 315320
Asp Glu Thr Trp Ile Val His Asn Tyr Gly His Ser Gly Trp Gly Tyr
325 330 335
Gln Gly Ser Tyr Gly Cys Ala Glu Asn Val Val Gln Leu Val Asp Lys
340 345 350
Val Gly Lys Ala Ala Lys Ser Lys Leu
355 360
<210>2
<211>361
<212>PRT
<213> human (Homo)
<400>2
Met Ala Asn Thr Ile Ile Val Val Gly Ala Gly Val Ser Gly Leu Thr
1 5 10 15
Ser Ala Tyr Leu Leu Ser Lys Asn Lys Gly Asn Lys Ile Thr Val Val
20 25 30
Ala Lys His Met Pro Gly Asp Tyr Asp Ile Glu Tyr Ala Ser Pro Phe
35 40 45
Ala Gly Ala Asn Val Cys Pro Met Ala Thr Gln Glu Asn Ser Arg Trp
50 55 60
Glu Arg Arg Thr Trp Val Glu Phe Lys Arg Leu Cys Glu Gln Val Pro
65 70 75 80
Glu Ala Gly Ile His Phe Gln Lys Cys His Ile Ala Arg Arg Lys Lys
85 90 95
Asp Val Glu Glu Ala Lys Ser Ser Thr Phe Pro Asp Ala Leu Phe Gln
100 105 110
Glu Glu Pro Trp Tyr Lys Glu Leu Phe Glu Asp Phe Arg Glu Gln Asn
115 120 125
Pro Asn Glu Val Thr Arg Gly Tyr Asp Ser Gly Cys Glu Phe Thr Ser
130 135 140
Val Cys Ile Asn Thr Ala Ile Tyr Leu Pro Trp Leu Ala Gly Gln Cys
145 150 155 160
Leu Lys Asn Gly Val Val Leu Lys Arg Thr Ile Leu Thr Asp Ile Ser
165 170 175
Glu Ala Lys Lys Leu Ser His Thr Gly Lys Val Pro Asn Ile Ile Val
180 185 190
Asn Ala Thr Gly Leu Gly Ser Leu Lys Leu Gly Gly Val Lys Asp Glu
195 200 205
Thr Met Ala Pro Ala Arg Gly Gln Ile Val Val Val Arg Asn Glu Ser
210 215 220
Thr Pro Met Leu Ile Thr Ser Gly Val Glu Asp Gly Gly Ser Asp Val
225 230 235 240
Met Tyr Leu Met Gln Arg Ala Ala Gly Gly Gly Thr Ile Leu Gly Gly
245250 255
Thr Tyr Asp Val Gly Asn Trp Glu Ser Gln Pro Asp Pro Asn Ile Ala
260 265 270
Gln Arg Ile Met Gln Arg Ile Val Glu Ala Arg Pro Glu Val Ala Asp
275 280 285
Gly Lys Gly Val Lys Gly Leu Ser Ile Ile Arg His Ala Val Gly Leu
290 295 300
Arg Pro Trp Arg Lys Gly Gly Leu Arg Leu Glu Glu Glu Lys Leu Asp
305 310 315 320
Asp Glu Thr Trp Ile Val His Asn Tyr Gly His Ser Gly Trp Gly Tyr
325 330 335
Gln Gly Ser Tyr Gly Cys Ala Glu Gly Val Val Glu Leu Val Asp Lys
340 345 350
Val Gly Lys Gly Ala Lys Ala Lys Leu
355 360
<210>3
<211>361
<212>PRT
<213> human (Homo)
<400>3
Met Ala Asn Thr Ile Val Val Val Gly Ala Gly Val Ser Gly Leu Thr
1 5 10 15
Ser Ala Tyr Leu Leu Ser Lys Asn Lys Gly Asn Lys Ile Thr Val Val
20 25 30
Gly Lys His Met Pro Gly Asp Tyr Asp Ile Glu Tyr Ala Ser Pro Phe
35 40 45
Ala Gly Ala Asn Val Cys Pro Met Ala Thr Gln Glu Asn Ser Arg Trp
50 55 60
Glu Arg Arg Thr Trp Val Glu Phe Lys Arg Leu Cys Glu Gln Val Pro
65 70 75 80
Glu Ala Gly Ile His Phe Gln Lys Cys His Ile Ala Arg Arg Lys Lys
85 90 95
Asp Val Glu Glu Ala Lys Ser Asn Thr Phe Pro Asp Ala Leu Phe Gln
100 105 110
Glu Glu Pro Trp Tyr Lys Glu Leu Phe Glu Asp Phe Arg Glu Leu Asn
115 120 125
Pro Ser Glu Val Thr Arg Gly Tyr Asp Thr Gly Cys Glu Phe Thr Ser
130 135 140
Val Cys Ile Asn Thr Ala Ile Tyr Leu Pro Trp Leu Ala Gly Gln Cys
145 150 155 160
Leu Lys Lys Gly Val Val Ile Lys Arg Ala Ser Leu Thr Asp Ile Ser
165 170 175
Glu Ala Lys Lys Leu Ser His Thr Gly Asn Val Pro Asn Ile Ile Val
180 185 190
Asn Ala Thr Gly Leu Gly Ser Leu Lys Leu Gly Gly Val Lys Asp Glu
195 200 205
Thr Met Ala Pro Ala Arg Gly Gln Ile Val Val Val Arg Asn Glu Ser
210 215 220
Thr Pro Met Leu Ile Thr Ser Gly Val Glu Asp Gly Gly Ser Asp Val
225 230 235 240
Met Tyr Leu Met Gln Arg Ala Ala Gly Gly Gly Thr Ile Leu Gly Gly
245 250 255
Thr Tyr Asp Ile Gly Asn Trp Glu Ser Gln Pro Asp Pro Asn Val Ala
260 265 270
Gln Arg Ile Leu Gln Arg Ile Val Glu Ala Arg Pro Glu Val Ala Asp
275 280 285
Gly Lys Gly Val Lys Gly Leu Ser Ile Ile Arg His Ala Val Gly Leu
290 295 300
Arg Pro Trp Arg Lys Asp Gly Leu Arg Leu Glu Glu Glu Lys Leu Asp
305 310 315 320
Asp Glu Thr Trp Ile Val His Asn Tyr Gly His Ser Gly Trp Gly Tyr
325 330 335
Gln Gly Ser Tyr Gly Cys Ala Glu Gly Val Val Glu Leu Val Asp Lys
340 345 350
Val Gly Lys Gly Ala Lys Ala Lys Leu
355 360
<210>4
<211>356
<212>PRT
<213> human (Homo)
<400>4
Met Ala Lys Ile Val Val Ile Gly Ala Gly Val Ala Gly Leu Thr Thr
1 5 10 15
Ala Leu Gln Leu Leu Arg Lys Gly His Glu Val Thr Ile Val Ser Glu
20 25 30
Phe Thr Pro Gly Asp Leu Ser Ile Gly Tyr Thr Ser Pro Trp Ala Gly
35 40 45
Ala Asn Trp Leu Thr Phe Tyr Asp Gly Gly Lys Leu Ala Asp Tyr Asp
50 55 60
Ala Val Ser Tyr Pro Ile Leu Arg Glu Leu Ala Arg Ser Ser Pro Glu
65 70 75 80
Ala Gly Ile Arg Leu Ile Ser Gln Arg Ser His Val Leu Lys Arg Asp
85 90 95
Leu Pro Lys Leu Glu Val Ala Met Ser Ala Ile Cys Gln Arg Asn Pro
100 105 110
Trp Phe Lys Asn Thr Val Asp Ser Phe Glu Ile Ile Glu Asp Arg Ser
115 120 125
Arg Ile Val His Asp Asp Val Ala Tyr Leu Val Glu Phe Arg Ser Val
130 135 140
Cys Ile His Thr Gly Val Tyr Leu Asn Trp Leu Met Ser Gln Cys Leu
145 150 155 160
Ser Leu Gly Ala Thr Val Val Lys Arg Arg Val Asn His Ile Lys Asp
165 170 175
Ala Asn Leu Leu His Ser Ser Gly Ser Arg Pro Asp Val Ile Val Asn
180 185 190
Cys Ser Gly Leu Phe Ala Arg Phe Leu Gly Gly Val Glu Asp Lys Lys
195 200 205
Met Tyr Pro Ile Arg Gly Gln Val Val Leu Val Arg Asn Ser Leu Pro
210 215 220
Phe Met Ala Ser Phe Ser Ser Thr Pro Glu Lys Glu Asn Glu Asp Glu
225 230 235 240
Ala Leu Tyr Ile Met Thr Arg Phe Asp Gly Thr Ser Ile Ile Gly Gly
245 250 255
Cys Phe Gln Pro Asn Asn Trp Ser Ser Glu Pro Asp Pro Ser Leu Thr
260 265 270
His Arg Ile Leu Ser Arg Ala Leu Asp Arg Phe Pro Glu Leu Thr Lys
275 280 285
Asp Gly Pro Leu Asp Ile Val Arg Glu Cys Val Gly His Arg Pro Gly
290 295 300
Arg Glu Gly Gly Pro Arg Val Glu Leu Glu Lys Ile Pro Gly Val Gly
305 310 315 320
Phe Val Val His Asn Tyr Gly Ala Ala Gly Ala Gly Tyr Gln Ser Ser
325 330 335
Tyr Gly Met Ala Asp Glu Ala Val Ser Tyr Val Glu Arg Ala Leu Thr
340 345 350
Arg Pro Asn Leu
355
Claims (10)
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) using 1,2,3, 4-tetrahydroisoquinoline-3-formic acid racemate or 1,2,3, 4-tetrahydroisoquinoline-3-formate racemate as substrate, using isolated D-amino acid oxidase or cell expressing D-amino acid oxidase intracellularly as catalyst, selectively catalyzing (R) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid to perform oxidative dehydrogenation reaction, while (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid is not reacted and remains in the reaction system;
(2) separating the (S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid from the reaction system.
2. The process according to claim 1, wherein the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid salt is an alkali metal salt or an ammonium salt of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
3. The method of claim 1, wherein the D-amino acid oxidase is a combination of one or more selected from the group consisting of: d-amino acid oxidase derived from triangular yeast (Trigonopsis variabilis) CBS 4095 or a mutant thereof or other D-amino acid oxidase having an amino acid sequence homology of more than 80%, 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%, D-amino acid oxidase derived from Fusarium peaorum (Fusarium poae)2516 or a mutant thereof or other D-amino acid oxidase having an amino acid sequence homology of more than 80%, D-amino acid oxidase derived from Fusarium solani (Fusarium solani) M-0718 or a mutant thereof or other D-amino acid oxidase having an amino acid sequence homology of more than 80%.
4. The method of claim 1 or 3, wherein the cell is an engineered bacterium expressing a D-amino acid oxidase, and the host cell of the engineered bacterium is E.coli BL21(DE 3).
5. The method of claim 4, wherein the engineered bacterium comprises an expression vector pET-28a (+), and the D-amino acid oxidase gene is ligated to the expression vector pET-28a (+).
6. 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 an aerobic environment at a set temperature, wherein the reaction system comprises the substrate, the pH buffer solution and/or the pH regulator, and the catalyst.
7. The method according to claim 6, wherein 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 in vitro D-amino acid oxidase or immobilized enzyme or cells for expressing the D-amino acid oxidase in cells; the set temperature is 20-70 ℃.
8. The method according to claim 6, wherein in the step (1), the set temperature is 30 to 50 ℃ and the pH of the reaction system is 7 to 8.
9. The method of claim 7, wherein the catalyst is a crude enzyme solution containing D-amino acid oxidase and flavin adenine dinucleotide ex vivo.
10. The method according to claim 1 or 6, wherein in step (1), the oxidative dehydrogenation reaction is carried out in the presence of coenzyme flavin adenine dinucleotide.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811454845.2A CN111254180B (en) | 2018-11-30 | 2018-11-30 | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution |
| 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811454845.2A CN111254180B (en) | 2018-11-30 | 2018-11-30 | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111254180A true CN111254180A (en) | 2020-06-09 |
| CN111254180B CN111254180B (en) | 2023-04-28 |
Family
ID=70852635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811454845.2A Active CN111254180B (en) | 2018-11-30 | 2018-11-30 | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN111254180B (en) |
| WO (1) | WO2020107781A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117003880A (en) * | 2022-07-06 | 2023-11-07 | 珠海重链生物科技有限公司 | Anti-thiocyanate fluorescein monoclonal antibody and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5252738A (en) * | 1991-01-24 | 1993-10-12 | Hoechst Aktiengesellschaft | Process for the preparation of racemic and optically active 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and its precursors |
| US20100016309A1 (en) * | 2008-07-16 | 2010-01-21 | National Defense Medical Center | 1,2,3,4-Tetrahydroisoquinoline Derivatives, Preparation Process therefor and Pharmaceutical Composition Containing the Same |
| CN102174632A (en) * | 2009-05-19 | 2011-09-07 | 重庆邮电大学 | Bio-catalytic deracemization preparation method of non-natural L-amino acid |
| CN104327077A (en) * | 2013-10-17 | 2015-02-04 | 苏州同力生物医药有限公司 | Levopraziquantel crystal form and preparation method and application thereof |
| CN104557911A (en) * | 2013-10-17 | 2015-04-29 | 苏州同力生物医药有限公司 | Preparation method of (R)-praziquantel |
-
2018
- 2018-11-30 CN CN201811454845.2A patent/CN111254180B/en active Active
-
2019
- 2019-04-23 WO PCT/CN2019/083877 patent/WO2020107781A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5252738A (en) * | 1991-01-24 | 1993-10-12 | Hoechst Aktiengesellschaft | Process for the preparation of racemic and optically active 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and its precursors |
| US20100016309A1 (en) * | 2008-07-16 | 2010-01-21 | National Defense Medical Center | 1,2,3,4-Tetrahydroisoquinoline Derivatives, Preparation Process therefor and Pharmaceutical Composition Containing the Same |
| CN102174632A (en) * | 2009-05-19 | 2011-09-07 | 重庆邮电大学 | Bio-catalytic deracemization preparation method of non-natural L-amino acid |
| CN104327077A (en) * | 2013-10-17 | 2015-02-04 | 苏州同力生物医药有限公司 | Levopraziquantel crystal form and preparation method and application thereof |
| CN104557911A (en) * | 2013-10-17 | 2015-04-29 | 苏州同力生物医药有限公司 | Preparation method of (R)-praziquantel |
Non-Patent Citations (7)
| Title |
|---|
| HISASHI MURAMATSU等: "The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of D-Lysine and D-Proline", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| SHUYUN JU等: "Chemoenzymatic approach to (S)-1,2,3,4-tetrahydroisoquinoline carboxylic acids employing D-amino acid oxidase", 《ADVANCED SYNTHESIS AND CATALYSIS》 * |
| 夏仕文等: "氨基酸氧化酶催化合成非天然手性氨基酸研究进展", 《分子催化》 * |
| 居述云: "生物催化氧化还原不对称合成手性羧基取代四氢异喹啉类化合物", 《中国博士学位论文全文数据库工程科技I辑》 * |
| 居述云等: "D-氨基酸氧化酶的分子改造及应用研究进展", 《化工进展》 * |
| 朱瑞恒: "基于各种拆分方法的四氢异喹啉生物碱、手性亚磺酰胺和顺式哌虫啶的合成研究", 《中国博士学位论文全文数据库工程科技I辑》 * |
| 程峰等: "NAD(P)H依赖型氧化还原酶不对称还原胺化制备手性胺的研究进展", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117003880A (en) * | 2022-07-06 | 2023-11-07 | 珠海重链生物科技有限公司 | Anti-thiocyanate fluorescein monoclonal antibody and application thereof |
| CN117003880B (en) * | 2022-07-06 | 2024-01-16 | 珠海重链生物科技有限公司 | Anti-thiocyanate fluorescein monoclonal antibody and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020107781A1 (en) | 2020-06-04 |
| CN111254180B (en) | 2023-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2017119731A1 (en) | Co hydratase and method for preparing formic acid by using same | |
| CN111996176B (en) | Carbonyl reductase mutant and application thereof | |
| CN110628841B (en) | Novel method for synthesizing key intermediate of dextromethorphan through enzyme catalysis asymmetry | |
| CN109971802B (en) | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution | |
| CN113151201B (en) | High-thermal-stability and high-activity isoeugenol monooxygenase mutant and application thereof | |
| CN111254180B (en) | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by enzymatic resolution | |
| CN110628736B (en) | Tryptophan 2,3 dioxygenase mutant and preparation method and application thereof | |
| CN111254181B (en) | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by chemical enzyme method | |
| CN111254170B (en) | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-3-formic acid by multienzyme coupling | |
| CN109943542B (en) | Alcohol dehydrogenase for producing atazanavir intermediate | |
| CN111394289A (en) | Genetically engineered bacterium and application thereof, and method for producing prostaglandin E2 | |
| CN110835639B (en) | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof | |
| CN110317849B (en) | Method for preparing (S) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof | |
| CN113652408A (en) | Carbonyl reductase mutant and application thereof in synthesis of (R) -4-chloro-3-hydroxybutyric acid ethyl ester | |
| AU2020103435A4 (en) | Method for preparing (s)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof | |
| CN111518851A (en) | Continuous preparation of immobilized enzyme14/15N]Method for producing L-citrulline | |
| CN108690836B (en) | Cyclohexanone monooxygenase and application thereof in synthesis of prazole | |
| CN112852768B (en) | Carbonyl reductase mutant and application thereof | |
| CN112442523A (en) | Method for preparing (R) -1,2,3, 4-tetrahydroisoquinoline-1-formic acid and derivatives thereof by enzymatic resolution | |
| CN114480315B (en) | Baeyer-Villiger monooxygenase and application thereof in brivaracetam synthesis | |
| CN116536279B (en) | Genetically engineered bacterium and application thereof in preparation of dehydroepiandrosterone | |
| CN114574454B (en) | Short-chain dehydrogenase, mutant and application thereof | |
| CN113388627B (en) | Reductase LX05 gene, genetic engineering bacterium containing gene and application of gene | |
| CN114317631B (en) | Application of monoamine oxidase in preparation of topiroxone | |
| CN109722456B (en) | Biosynthesis method and application of acrylic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |