WO2020107783A1 - Procédé de préparation d'acide (s)-1,2,3, 4-tétrahydroisoquinoléine-3-carboxylique au moyen d'un couplage enzymatique multiple - Google Patents

Procédé de préparation d'acide (s)-1,2,3, 4-tétrahydroisoquinoléine-3-carboxylique au moyen d'un couplage enzymatique multiple Download PDF

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WO2020107783A1
WO2020107783A1 PCT/CN2019/083916 CN2019083916W WO2020107783A1 WO 2020107783 A1 WO2020107783 A1 WO 2020107783A1 CN 2019083916 W CN2019083916 W CN 2019083916W WO 2020107783 A1 WO2020107783 A1 WO 2020107783A1
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amino acid
tetrahydroisoquinoline
reductase
carboxylic acid
acid
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PCT/CN2019/083916
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Chinese (zh)
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吴坚平
居述云
施俊巍
杨立荣
钱明心
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苏州同力生物医药有限公司
浙江大学
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    • C—CHEMISTRY; METALLURGY
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    • 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

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  • the invention belongs to the technical field of biocatalysis, and in particular relates to a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by multi-enzyme coupling.
  • (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) is an important pharmaceutical intermediate and is widely used in Synthesis of various organic small molecule drugs and peptide-based drugs.
  • (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is an important component of the antihypertensive drug quinapril (Diversity-oriented synthesis of medically imported 1, 2, 3, 4 -tetrahydroisoquinoline-3-carboxylic(acic(Tic)derivatives and higher analogs[J].Org Biomol Chem, 2014, 12(45):9054-91.).
  • (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid can be used to synthesize small molecule antagonists containing tetrahydroisoquinoline nucleus, acting on the chemokine receptor CXCR4, thus It is expected to be used to treat HIV and other diseases (Discovery of tetrahydroisoquinoline-based CXCR4 antagonists [J]. ACS Med Chem Lett, 2013, 4(11): 1025-30.).
  • the method has low yield and many steps, which is not easy for industrial application.
  • Gong et al. prepared (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (racemic phenylalanine) as a raw material by chemical enzymatic method, and synthesized the racemic 1 by Pictet-Spengler reaction. 2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, followed by esterification and lipase kinetic resolution to prepare (S)-configuration product. 23.8g of racemic ester hydrochloride (0.1mol), the mass ratio of lipase to substrate is 0.2, the reaction is 48h, the product ee>99%, the yield is 49.1%.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a new method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
  • the method includes the following steps:
  • the imine acid represented by the formula (II) is converted into the (S)-1,2,3,4-tetrahydroisoquinoline in the presence of pipecolic acid reductase and a coenzyme capable of supplying hydride ions -3-carboxylic acid.
  • 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid salt may be an alkali metal salt or ammonium salt of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Etc., for example, sodium 1,2,3,4-tetrahydroisoquinoline-3-carboxylate, potassium 1,2,3,4-tetrahydroisoquinoline-3-carboxylate, 1,2,3,4-tetrakis Hydrogen isoquinoline-3-carboxylic acid ammonium.
  • the oxidative dehydrogenase is an enzyme capable of selectively catalyzing (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and the selectivity is greater than or equal to 80%, preferably greater than or equal to 90%.
  • the oxidative dehydrogenase is a D-amino acid oxidase.
  • the D-amino acid oxidase is a combination of one or more selected from the following D-amino acid oxidases: D-amino acid oxidase derived from Trigonopsis variabilis CBS 4095 or a mutant thereof Or other D-amino acid oxidases with amino acid sequence homology greater than 80%, D-amino acid oxidase from Fusarium Graminearum CS3005 or its mutants or other homology with amino acid sequence homology greater than 80% D-amino acid oxidase, D-amino acid oxidase from Fusarium poae 2516 or its mutants or other D-amino acid oxidases with amino acid sequence homology greater than 80%, from Fusarium solanacearum ( Fusarium (solani) M-0718 D-amino acid oxidase or its mutants or other D-amino acid oxidases with amino acid sequence homology greater than 80%
  • 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.
  • the added amount of the D-amino acid oxidase is based on the wet weight of the cells after centrifugation at 8000 rpm for 10 minutes, and the added amount of the cells is 1 to 5% of the weight of the reaction system.
  • the use form of the D-amino acid oxidase is isolated D-amino acid oxidase, or a crude enzyme solution or pure enzyme of the isolated D-amino acid oxidase or immobilization Enzymes, or cells that express D-amino acid oxidase intracellularly.
  • the cell is an engineered bacterium that expresses D-amino acid oxidase and contains an expression vector pET-28a(+), and the host cell of the engineered bacterium is E. coli BL21(DE3); wherein, the D-amino acid The oxidase gene is linked to the expression vector pET-28a(+).
  • the pipecolic acid reductase is a combination of one or more selected from the following pipecolic acid reductases: pipecolic acid reductase derived from Pseudomonas putida KT2440 or A mutant or a pipecolic acid reductase with an amino acid sequence homology greater than 80%, a pipecolic acid reductase derived from Pseudomonas aeruginosa PAO1 or a mutant thereof or a homology with an amino acid sequence greater than 80% Pipecolic acid reductase, pipecolic acid reductase derived from Pseudomonas fluorescens Pf0-1 or a mutant thereof, or pipecolic acid reductase with amino acid sequence homology greater than 80%, derived from insects Piperonic acid reductase of Pseudomonas entomophila str. L48 or its mutants or pipecolic acid reductase with
  • the pipecolic acid reductase has an amino acid sequence as shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8.
  • the added amount of pipecolic acid reductase is based on the wet weight of the cells after centrifugation at 4000 rpm for 10 minutes, and the added amount of the cells is 0.1 to 5% of the weight of the reaction system.
  • the use form of the pipecolic acid reductase is isolated pipecolic acid reductase, a crude enzyme liquid or pure enzyme containing the isolated pipecolic acid reductase or immobilized Enzymes, or cells that express pipecolic acid reductase intracellularly.
  • the cell is an engineered bacterium expressing pipecolic acid reductase and contains an expression vector pET-28a(+), and the host cell of the engineered bacterium is E. coli BL21(DE3); wherein, the pipecolic acid The reductase gene is linked to the expression vector pET-28a(+).
  • the coenzyme capable of supplying hydride ions is NADH and/or NADPH.
  • the reaction for producing imidic acid is also carried out in the presence of flavin adenine dinucleotide (FAD).
  • FAD flavin adenine dinucleotide
  • the reaction in the presence of FAD helps to further increase the conversion rate.
  • FAD is equivalent to or excessive to the substrate.
  • the prepared crude enzyme solution of D-amino acid oxidase already contains a sufficient amount of FAD. In the case of directly using the crude enzyme solution, it is not necessary to add FAD. In the case of using pure D-amino acid oxidase enzyme, an appropriate amount of FAD may be added as needed.
  • the reaction to produce imidic acid is also carried out in the presence of catalase.
  • the reaction to form the imidic acid is performed in a set temperature and an aerobic environment.
  • the set temperature is 20 to 70°C. More preferably, the set temperature is 20-50°C. Further preferably, the set temperature is 30-40°C.
  • the implementation process of the method includes: first constructing a reaction system, and then controlling the reaction system to perform the reaction in a set temperature and an aerobic environment, the reaction system includes the bottom Substances, the oxidative dehydrogenase, the pipecolic acid reductase, the coenzyme, the coenzyme regeneration system, the solvent, the reaction system also optionally includes a pH buffer and/or pH adjuster, and the coenzyme includes NAD+( Oxidized nicotinamide adenine dinucleotide) and/or NADH (reduced nicotinamide adenine dinucleotide), or, the coenzyme includes NADP+ (oxidized nicotinamide adenine dinucleotide phosphate) and// Or NADPH (reduced nicotinamide adenine dinucleotide phosphate).
  • the pH value of the reaction system is controlled to 6-9. More preferably, the pH of the reaction system is controlled to 7-8.5.
  • the concentration of the starting substrate in the reaction system is controlled to be 1-20 g/L.
  • the pH buffering agent is phosphate, which can be formulated into a phosphate buffer solution by dissolving it in water.
  • the pH adjusting agent is ammonia water, alkali metal hydroxide or its aqueous solution.
  • the pH adjusting agent is 20 wt% to 35 wt% ammonia.
  • the pH adjusting agent is an aqueous solution of sodium hydroxide or potassium hydroxide.
  • the amount of the coenzyme added is 1 ⁇ -1% of the substrate concentration.
  • the coenzyme regeneration system includes a coenzyme regeneration enzyme and a coenzyme regeneration substrate.
  • the coenzyme regenerating enzyme is glucose dehydrogenase and the coenzyme regenerating substrate is glucose; or, the coenzyme regenerating enzyme is alcohol dehydrogenase and the coenzyme regenerating substrate is isopropyl alcohol.
  • the glucose specifically uses D-glucose.
  • the glucose dehydrogenase is derived from Bacillus subtilis (Bacillus subtilis) 168; and/or the alcohol dehydrogenase is derived from Lactobscillus kefir DSM20587.
  • the glucose dehydrogenase has the amino acid sequence shown in SEQ ID NO.9.
  • the alcohol dehydrogenase has the amino acid sequence shown in SEQ ID NO. 10.
  • the reaction system further includes catalase.
  • the catalase is bovine liver catalase lyophilized powder.
  • the enzyme activity of the lyophilized powder of bovine liver catalase is 4000 U/mg.
  • the enzyme activity ratio of the catalase to the oxidative dehydrogenase is 1000-2000:1.
  • the reaction system further includes flavin adenine dinucleotide.
  • the method further includes a separation step.
  • the separation step includes: adjusting the pH value of the reaction system after the reaction to 5.0-6.0, heating to denature and precipitate the protein, suction filtration, after the filtrate is concentrated, cooling and crystallization, and drying to obtain the formula ( I) S-isomer of the compound shown.
  • the present invention has the following beneficial effects compared with the prior art:
  • the present invention finds that in the presence of pipecolic acid reductase and a coenzyme capable of supplying hydride ions, it can efficiently convert imidic acid to obtain (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid It has good selectivity, high yield and mild reaction conditions.
  • the ee value of the S-isomer relative to the R-isomer in the prepared product is >99%, and the process is relatively simple.
  • Example 1 is a high performance liquid phase detection spectrum of two optical isomers of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid sampled at 0 hours in the reaction system in Example 3;
  • the retention time 8.877min is (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
  • the retention time 11.308min is (S)-1,2,3,4-tetrahydroisoquinoline Porphyrin-3-carboxylic acid
  • FIG. 2 is a detection spectrum of high performance liquid chromatography for sampling of the reaction in Example 3 for 12 hours.
  • the invention provides a new method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
  • the method of the invention has mild reaction conditions, strong stereoselectivity, high reaction efficiency and yield High characteristics, with industrial application prospects.
  • the method uses racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid as a substrate, which is catalyzed by a multi-enzyme system to obtain (S)-1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid
  • the multi-enzyme system can be composed of oxidative dehydrogenase (preferably D-amino acid oxidase), catalase, pipecolic acid reductase and coenzyme (preferably NADP + and And/or NADPH), coenzyme regeneration system, etc.
  • Imine acid is asymmetrically reduced by pipecolic acid reductase to (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
  • reduced coenzyme II reduced nicotinamide adenine dinucleotide phosphate (NADPH)
  • NADP + oxidized nicotinamide adenine dinucleotide phosphate
  • the reaction process is as follows:
  • the reaction to generate imidic acid is also carried out in the presence of flavin adenine dinucleotide (FAD).
  • FAD flavin adenine dinucleotide
  • a molecule of oxygen is reduced to hydrogen peroxide (H 2 O 2 )
  • FADH 2 is oxidized to FAD.
  • Hydrogen peroxide is decomposed into water and oxygen under the catalysis of catalase.
  • the D-amino acid oxidase is derived from Triangle yeast, Fusarium graminearum, Fusarium oxysporum, and Fusarium solani.
  • the D-amino acid oxidase is derived from Trigonopsis variabilis CBS 4095, Fusarium graminearum CS3005, Fusarium poae 2516, or Fusarium solani M -0718.
  • the pipecolic acid reductase is derived from Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas arborea.
  • the pipecolic acid reductase is derived from Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens Pf0-1, or Pseudomonas arborea
  • the coenzyme regeneration system includes a coenzyme regeneration enzyme and a coenzyme regeneration substrate.
  • the coenzyme regeneration enzyme is derived from Bacillus subtilis and Lactobacillus.
  • the coenzyme regenerating enzyme is derived from the glucose dehydrogenase of Bacillus subtilis (Bacillus subtilis) 168 and the alcohol dehydrogenase of LSM (Lactobscillus kefir) DSM20587.
  • the use form of the enzyme in the multi-enzyme system may be an ex vivo enzyme, a crude enzyme solution, or a pure enzyme, or an immobilized enzyme, or a resting cell of an engineered bacteria expressing a recombinant enzyme.
  • the concentration of the starting substrate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction system is 1-20 g/L.
  • the added amount of D-amino acid oxidase is calculated as the wet weight of the cells after centrifugation at 4000 rpm for 10 min.
  • the added amount of the cells is 1 to 5% of the weight of the reaction solution.
  • the catalase is bovine liver catalase lyophilized powder
  • the enzyme activity is 4000 U/mg
  • the enzyme activity ratio of catalase to D-amino acid oxidase is 1000-2000:1.
  • the added amount of pipecolic acid reductase is calculated as the wet weight of the cells after centrifugation at 4000 rpm for 10 minutes, and the added amount of the cells is 0.1 to 5% of the weight of the reaction solution.
  • the added amount of coenzyme regenerating enzyme is based on the wet weight of the cells after centrifugation at 4000 rpm for 10 minutes, and the added amount of the cells is 0.1 to 5% of the weight of the reaction solution.
  • oxidized nicotinamide adenine dinucleotide phosphate may be added to the initial coenzyme in an amount of 1 ⁇ to 1%.
  • the reaction temperature is 20 to 70°C
  • the time is 6 to 72 hours
  • the pH of the reaction solution is 6 to 9; more preferably, the temperature is 30 to 40°C and the time is 12 to 48 hours.
  • Phosphate buffer solution controls the pH of the reaction from 7 to 8.5.
  • genes used in the examples of the present invention are synthesized by Biotechnology (Shanghai) Co., Ltd.
  • E.coli BL21 (DE3) strains were purchased from Novagen;
  • DNA marker, PrimeStar DNA polymerase, low molecular weight standard protein and other molecular biological reagents were purchased from TaKaRa.
  • DNA marker, PrimeStar DNA polymerase, low molecular weight standard protein and other molecular biological reagents were purchased from TaKaRa.
  • the present invention analyzes each product and substrate of the catalytic reaction by high-performance liquid chromatography (HPLC).
  • HPLC analysis method of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is: chromatography column/ ZWIX(-); column temperature/25°C; flow rate/0.5mL/min; detection wavelength/UV210nm; mobile phase: HPLC grade methanol/acetonitrile (50/50, v/v) (add 50mM formic acid and 25mM dihexylamine) .
  • HPLC grade methanol/acetonitrile 50/50, v/v
  • microbial-derived D-amino acid oxidases can be divided into two categories: 1) Preference for amino acids with smaller substrate side chain groups (such as D-alanine), such as Fusarium oxysporum (Fusarium oxysporum)-derived D-amino acid oxidase; 2) Preference for amino acids with larger substrate side chain groups (such as D-phenylalanine), such as D-amino acid oxidase from Trigonopsis variabilis ( POLLEGIONI L, MOLLA G, SACCHI S, et al. Properties, and applications, of microbial, D-amino acid, oxidationases: current state, and perspectives [J].
  • D-alanine such as Fusarium oxysporum (Fusarium oxysporum)-derived D-amino acid oxidase
  • D-phenylalanine such as D-amino acid oxidase from Trigonopsis variabilis
  • the above D-amino acid oxidase gene sequence was codon optimized and sent to Biotech (Shanghai) Co., Ltd. for full gene synthesis, and cloned into the recombinant expression plasmid pET-28a(+).
  • the recombinant plasmid was transferred into the expression host E. coli BL21 (DE3). After verification by sequencing, glycerol with an initial concentration of 25% was added to the obtained engineering bacterial solution and stored at -80°C for later use.
  • KT2440-F 5'-CG GGATCC ATGTCCGCACCTTCCACCAGCAC-3 '(BamH I)
  • KT2440-R 5'-CCC AAGCTT TCAGCCAAGCAGCTCTTTCAGG-3' (Hind III)
  • PAO1-F 5'-CG GGATCC GTGATCCGAATGACGCTGGAC-3 '(BamH I)
  • PAO1-R 5'-CCC AAGCTT TCACTCCAGCAACGCCAGC-3'(Hind III)
  • Pf0-1-F 5'-CG GGATCC ATGTCTGCGCCACACGATC-3 '(BamH I)
  • Pf0-1-R 5'-CCG CTCGAG TTACTCGCCGGCCAGTTCAC-3' (Xho I)
  • the amplification result was detected by 1.0% agarose gel electrophoresis. The result showed that the amplification product was a single band with a size of about 1000 bp.
  • Use DNA recovery and purification kit to recover the target band please refer to the instructions of the purification kit.
  • the expression vector pET-28a(+) and PCR amplification products were double-digested with the corresponding restriction enzymes. After the digestion is completed, use DNA recovery purification kit to recover the target band. Afterwards, the double-digested PCR amplification product was ligated to the expression vector pET-28a(+) with corresponding sticky ends using T4DNA ligase.
  • the ligation system is shown in Table 3 below:
  • the enzyme-linked product was transformed into E. coli DH5a competent cells, plated, single colonies were cultured in LB liquid base, bacterial solution PCR identified positive transformants, and sent to the sequencing company to verify the correctness of the inserted sequence. Extract the plasmids from the positive transformants that have been verified, and refer to the plasmid extraction kit for related methods. Then transfer the recombinant expression vector into the expression host E. coli BL21 (DE3), after verification by bacterial solution PCR and sequencing, add the initial concentration of 25% glycerol to the obtained engineering bacterial solution and place at -80°C Save for future use.
  • the glucose dehydrogenase (NCBI accession number: NP_388275.1, SEQ ID NO. 9) gene was cloned from the Bacillus subtilis 168 genome; the alcohol dehydrogenase was cloned from the genome of Lactobacillus kefiri DSM20587 ( NCBI accession number: AAP94029.1, SEQ ID NO.10) gene.
  • the specific method steps please refer to the construction method of the strain expressing pipecolic acid reductase in 1.2.
  • the relevant PCR upstream and downstream primers are as follows:
  • BGdh-F 5'-GA AGATCT GATGTATCCGGATTTAAAAGGAAAAGTC-3'(Bgl II)
  • LAdh-F 5'-CC GAATTC ATGACCGATCGTCTGAAGGGC-3'(EcoR I)
  • LAdh-R 5'-CCC AAGCTT TCACTGTGCGGTATACCCGCC-3' (Hind III).
  • liquid LB medium peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in deionized water, set the volume, sterilized at 121°C for 20min, and ready for use. If it is solid LB medium, add 15g/L agar.
  • the engineered bacteria containing the D-amino acid oxidase gene were inoculated in 5 mL liquid LB (containing 50 ⁇ g/ml kanamycin) medium, and cultured at 37° C. and shaking at 200 rpm for about 8 hours. Inoculated at 1% (V/V) inoculated in 100mL liquid LB (containing 50 ⁇ g/ml kanamycin) culture medium, OD600 reached 0.6-0.8, add the inducer isopropyl thiogalactoside ( The initial concentration was 0.1 mM) and induced at 18°C for 15h. After the cultivation, the culture solution was poured into a 100 mL centrifuge tube and centrifuged at 4000 rpm for 10 min. The supernatant was discarded, the bacterial cells were collected, the cells were washed twice with 50 mM phosphate buffer (pH 8.0), and stored in an ultra-low temperature refrigerator at -80°C spare.
  • V/V inducer isoprop
  • crude D-amino acid oxidase enzyme solution derived from Fusarium solani M-0718 and crude pipecolic acid reductase enzyme from Pseudomonas putida KT2440 were prepared respectively And Bacillus subtilis (Bacillus subtilis) 168 glucose dehydrogenase crude enzyme liquid.
  • Example 2 a crude D-amino acid oxidase enzyme solution derived from Fusarium poae 2516, a crude pipecolic acid reductase enzyme solution of Pseudomonas aeruginosa PAO1, and lactobacilli were prepared respectively (Lactobacillus Kefiri) DSM20587 alcohol dehydrogenase crude enzyme solution.
  • the concentration of 3,4-tetrahydroisoquinoline-3-carboxylic acid was 5 g/L, the concentration of NADP + was 0.03 mM, and the concentration of isopropyl alcohol was 20 mM.
  • the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution
  • the concentration of isoquinoline-3-carboxylic acid in two configurations (g/L).
  • the yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction solution was 82.1% (calculated in the same manner as in Example 3), and the ee value was 99.4%.
  • Example 2 a crude D-amino acid oxidase enzyme solution derived from Fusarium graminearum CS3005 and a crude pipecolic acid reductase enzyme from Pseudomonas fluorescens Pf0-1 were prepared respectively. And Bacillus subtilis (Bacillus subtilis) 168 glucose dehydrogenase crude enzyme liquid.
  • the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution
  • concentration of isoquinoline-3-carboxylic acid in two configurations g/L
  • the reaction yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction solution was 79.8% (calculated in the same manner as in Example 3), and the ee value was 99.1%.
  • the concentration of 3,4-tetrahydroisoquinoline-3-carboxylic acid was 7.5 g/L, the concentration of NADP + was 0.05 mM, and the concentration of isopropyl alcohol was 25 mM.
  • the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution
  • the concentration of isoquinoline-3-carboxylic acid in two configurations (g/L).
  • the reaction yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction solution was 78.6%, and the ee value was 99.2%.
  • crude D-amino acid oxidase enzyme solution derived from Fusarium solani M-0718 and crude pipecolic acid reductase enzyme from Pseudomonas putida KT2440 were prepared respectively And Bacillus subtilis (Bacillus subtilis) 168 glucose dehydrogenase crude enzyme liquid.
  • the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution
  • concentration of isoquinoline-3-carboxylic acid in two configurations g/L
  • the reaction yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was 84.9% (calculated in the same way as in Example 3), and the ee value was 99.2%.
  • the substrate solution and the reaction system are as in Example 3.
  • the content of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography is detected, and then the 1,2,3,4-tetra The content of hydrogen isoquinoline-3-carboxylic acid in two configurations.
  • Preparation of substrate solution prepare 50g/L racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid solution with 50mM phosphate buffer solution (pH8.0) and adjust the solution pH with 30% ammonia water To 8.0.

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Abstract

L'invention concerne un procédé de préparation d'acide (S)-1,2,3, 4-tétrahydroisoquinoléine-3-carboxylique au moyen d'un couplage enzymatique multiple, comprenant l'utilisation d'un racémate d'acide 1,2,3, 4-tétrahydroisoquinoline-3-carboxylique ou un racémate de 1,2,3, 4-tétrahydroisoquinoléine-3-carboxylate en tant que substrat, la réaction de l'acide (R)-1,2,3, 4-tétrahydroisoquinoléine-3-carboxylique dans le substrat sous l'action catalytique d'une déshydrogénase oxydante pour produire un acide imidique représenté par la formule (II); et la transformation de l'acide imidique représenté par la formule (II) dans l'acide (S)-1,2,3, 4-tétrahydroisoquinoléine-3-carboxylique en présence d'une réductase d'acide pipéridine et d'une coenzyme capable de donner un ion hydrure. L'invention présente des caractéristiques telles que des conditions de réaction douces, une forte stéréosélectivité, une efficacité de réaction élevée et un taux de conversion élevé.
PCT/CN2019/083916 2018-11-30 2019-04-23 Procédé de préparation d'acide (s)-1,2,3, 4-tétrahydroisoquinoléine-3-carboxylique au moyen d'un couplage enzymatique multiple WO2020107783A1 (fr)

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