WO2020034660A1 - 一种制备(s)-1，2，3，4-四氢异喹啉-1-甲酸及其衍生物的方法 - Google Patents

一种制备(s)-1，2，3，4-四氢异喹啉-1-甲酸及其衍生物的方法 Download PDF

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WO2020034660A1
WO2020034660A1 PCT/CN2019/083813 CN2019083813W WO2020034660A1 WO 2020034660 A1 WO2020034660 A1 WO 2020034660A1 CN 2019083813 W CN2019083813 W CN 2019083813W WO 2020034660 A1 WO2020034660 A1 WO 2020034660A1
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amino acid
reductase
piperidinate
tetrahydroisoquinoline
reaction
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French (fr)
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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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Priority to US17/268,455 priority Critical patent/US20230107679A1/en
Publication of WO2020034660A1 publication Critical patent/WO2020034660A1/zh
Priority to AU2020103435A priority patent/AU2020103435A4/en

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    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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Definitions

  • the invention belongs to the field of biocatalysis technology, and particularly relates to a method for preparing (S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof.
  • 1,2,3,4-tetrahydroisoquinoline compounds are a very important class of pharmaceutical intermediates and are widely used in the synthesis of many drugs.
  • Hu et al. Discovery of small-molecule, inhibitor, and cellular probe of Keap1-Nrf2 protein-protein interactions [J]. Bioorg Med Chem Lett, 2013, 23 (10): 3039-43.) Take (S)- 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid was used as a starting compound to synthesize a Kelch-like ECH-associated protein 1 (Keap1) inhibitor, which is expected to be used in cancer and diabetes. Treatment and prevention of Alzheimer's disease, Parkinson's and other diseases.
  • isoquinoline with medicinal value contain 6,7-dimethoxy (such as papaverine, turanine, etc.), which is conducive to reducing the hydrophobicity of the drug molecule and improving the drugability, such as 6, 7 -Dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid.
  • the method has low yield and complicated steps, and is not suitable for industrial application.
  • Bulyszko et al Used the Petasis reaction and the Pomeranz-Fritsch-Bobbitt reaction to diastereoselectively fully synthesize (S) -6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid.
  • Ee value is 90% (Synthesis of (+)-6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, a Diastereoselective Approach [J] .European Journal of Organic Chemistry, 2015, 2015 (2): 383-8.).
  • the biocatalytic method has the advantages of high stereoselectivity and mild reaction conditions, and is a potential advantage for the preparation of (S) -1, 2, 3, 4-tetrahydroisoquinoline-1-carboxylic acid or its derivatives. method. Paál et al.
  • 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-1-carboxylic acid and its derivatives.
  • R 1 and R 2 are independently selected from hydrogen, C1-C6 alkyl, and C1-C6 alkoxy.
  • the method includes the following steps:
  • a racemic form of the compound represented by the formula (I) or a racemic form of a salt of the compound represented by the formula (I) is used as a substrate, and the R form of the compound represented by the formula (I) in the substrate is used.
  • the isomers react under the catalysis of oxidative dehydrogenase to form imine acids represented by formula (II);
  • the imine represented by the formula (II) is converted into an S-type isomer of the compound represented by the formula (I) in the presence of a piperidinate reductase and a coenzyme capable of supplying a hydrogen anion.
  • R 1 and R 2 are independently selected from hydrogen, methyl, ethyl, isopropyl, methoxy or ethoxy.
  • the salt is a monovalent salt, particularly preferably an alkali metal salt or an ammonium salt, wherein the alkali metal salt may be, for example, a lithium salt, a sodium salt, or a potassium salt.
  • the S-isomer of the compound represented by formula (I) is (S) -1, 2, 3, 4-tetrahydroisoquinoline-1-carboxylic acid or (S) -6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid.
  • the oxidative dehydrogenase is an enzyme capable of selectively catalyzing the R-isomer of the compound represented by formula (I), and has a selectivity of 80% or more, preferably 90% or more.
  • 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 oxidase or a mutant thereof derived from Trigonopsis variabilis CBS 4095 Or other D-amino acid oxidases with a homology of more than 80% of its amino acid sequence, D-amino acid oxidases from Fusarium graminearum CS3005 or its mutants, or others with a homology of more than 80% D-amino acid oxidase, D-amino acid oxidase from Fusarium poae 2516 or a mutant thereof, or other D-amino acid oxidase with homology greater than 80% of its amino acid sequence, from Fusarium solanacearum ( Fusarium (solarani) D-07 amino acid oxidase of M-0718 or a mutant thereof or other D-amino acid oxidase having homology of more 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% by weight of the reaction system.
  • the use form of the D-amino acid oxidase is an isolated D-amino acid oxidase, a crude enzyme solution containing an isolated D-amino acid oxidase, and a D-amino acid oxidase Pure enzymes, immobilized enzymes of D-amino acid oxidase, or cells expressing 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 ligated to the expression vector pET-28a (+).
  • the piperidinate reductase is a combination of one or more selected from the group consisting of a piperidinate reductase derived from Pseudomonas putida KT2440 or a combination thereof A mutant or a piperidinate reductase with homology greater than 80% of its amino acid sequence, a piperidinate reductase derived from Pseudomonas aeruginosa PAO1 or a mutant thereof, or a mutant having a homology of more than 80% in its amino acid sequence Piperidinate reductase, piperidinate reductase derived from Pseudomonas fluorescens Pf0-1 or a mutant thereof, or piperidinate reductase with homology greater than 80% in its amino acid sequence, derived from insects Piperidinate reductase or mutant thereof of Pseudomonas entomophila L48 or piperidinate reductase having homology of
  • the piperidinate 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 piperidinate 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 1 to 5% by weight of the reaction system.
  • the piperidinate reductase is used in the form of piperidinate reductase in vitro, a crude enzyme solution containing piperidinate reductase in vitro, and piperidinate reductase Pure enzyme, piperidinate reductase immobilized enzyme or intracellular expression of piperidinate reductase.
  • the cell is an engineered bacterium expressing piperidinate reductase and containing an expression vector pET-28a (+), and the host cell of the engineered bacterium is E. coli BL21 (DE3); wherein the piperidinate The reductase gene is ligated to the expression vector pET-28a (+).
  • the coenzyme capable of supplying a hydrogen anion is NADH and / or NADPH.
  • the reaction for generating imine is also performed in the presence of flavin adenine dinucleotide (FAD). Allowing the reaction to proceed in the presence of FAD helps to further increase the conversion. Further, the FAD is equivalent to the substrate or is in excess.
  • FAD flavin adenine dinucleotide
  • 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, there is no need to add additional FAD. In the case of using a pure D-amino acid oxidase, an appropriate amount of FAD may be further added as required.
  • the reaction for generating imine is also performed in the presence of catalase.
  • the reaction for generating the imine acid is performed at 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 to 40 ° C.
  • the implementation process of the method includes: first constructing a reaction system, and then controlling the reaction system to perform the reaction at a set temperature and an aerobic environment, the reaction system including the substrate Substance, the oxidative dehydrogenase, the piperidinate reductase, coenzyme, a coenzyme regeneration system, a solvent, the reaction system optionally further includes a pH buffer and / or a 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).
  • NAD + Oxidized nicotinamide adenine dinucleotide
  • the pH of the reaction system is controlled to be 6-9. More preferably, the pH of the reaction system is controlled from 7 to 8.5.
  • the concentration of the starting substrate in the reaction system is controlled to be 1 to 20 g / L. More preferably, the concentration of the starting substrate in the reaction system is controlled to be 1 to 20 g / L. According to a specific aspect of the present invention, the concentration of the starting substrate in the reaction system is controlled to be 5 g / L.
  • the pH buffer is a phosphate, which can be formulated into a phosphate buffer solution by dissolving it in water.
  • the pH adjusting agent is ammonia water, an alkali metal hydroxide or an aqueous solution thereof.
  • the pH adjusting agent is 20 wt% to 35 wt% ammonia water.
  • the pH adjusting agent is an aqueous solution of sodium hydroxide or potassium hydroxide.
  • the coenzyme is added in an amount of 1 ⁇ to 1% of the substrate concentration.
  • the coenzyme regeneration system includes a coenzyme regeneration enzyme and a coenzyme regeneration substrate.
  • the coenzyme regeneration enzyme is glucose dehydrogenase, and the coenzyme regeneration substrate is glucose; or, the coenzyme regeneration enzyme is alcohol dehydrogenase, and the coenzyme regeneration substrate is isopropyl alcohol.
  • the glucose specifically uses D-glucose.
  • the glucose dehydrogenase is derived from Bacillus subtilis 168; and / or the alcohol dehydrogenase is derived from Lactobacillus kefir DSM20587.
  • the glucose dehydrogenase has an amino acid sequence as shown in SEQ ID NO.9.
  • the alcohol dehydrogenase has an amino acid sequence as shown in SEQ ID NO.10.
  • the reaction system further comprises a catalase.
  • the catalase is a bovine liver catalase lyophilized powder.
  • the enzyme activity of the bovine liver catalase lyophilized powder is 4000 U / mg.
  • the enzyme activity ratio of the catalase to the oxidative dehydrogenase is 100-400: 1.
  • the reaction system further includes flavin adenine dinucleotide.
  • the method further comprises 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, concentrating the filtrate, cooling and crystallization, and drying to obtain the formula ( I) The S-isomer of the compound shown.
  • the present invention has the following beneficial effects compared with the prior art:
  • the imine in the coexistence of a piperidinate reductase and a coenzyme capable of supplying a hydrogen anion, the imine can be efficiently converted to obtain the S-isomer of the compound represented by the formula (I), which has good selectivity and yield.
  • High, 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.
  • FIG. 1 is a HPLC detection spectrum (1g / L) of two optical isomers of the racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid;
  • the retention time of 8.810min is (R) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid; the retention time of 12.685min is (S) -1,2,3,4-tetrahydroisoquinoline Chloro-1-carboxylic acid
  • Example 2 is a high-performance liquid chromatography detection spectrum of a sample taken at 0 hours in the reaction system in Example 3;
  • FIG. 3 is a high performance liquid chromatography detection spectrum of a 16-hour sample taken in the reaction in Example 3.
  • FIG. 3 is a high performance liquid chromatography detection spectrum of a 16-hour sample taken in the reaction in Example 3.
  • the invention provides a new method for preparing (S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof.
  • the method of the invention has mild reaction conditions, strong stereoselectivity and reaction efficiency. It has the characteristics of high productivity and high yield, and has industrial application prospects.
  • the method is racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or racemic 6,7-dimethoxy-1,2,3,4 -Tetrahydroisoquinoline-1-carboxylic acid as a substrate, and (S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (S) -6,7- Dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid, the multi-enzyme system can be oxidative dehydrogenase (preferably D-amino acid oxidase), catalase, piperidine An acid reductase and a coenzyme (preferably NADP + and / or NADPH), a coenzyme regeneration system, and the like.
  • oxidative dehydrogenase preferably D-amino acid oxidase
  • catalase preferably D-amino acid oxidase
  • piperidine An
  • racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline 1-formic acid is used as a substrate, and D-amino acid oxidase is used to stereoselectively catalyze (R) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (R) -6,7-dimethylformate
  • the hydrogen peroxide produced during the reaction is decomposed into water and oxygen by catalase.
  • Amino acid is asymmetrically reduced by piperidinate reductase to (S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (S) -6,7-dimethoxy-1, 2,3,4-tetrahydroisoquinoline-1-carboxylic acid.
  • reduced coenzyme II reduced nicotinamide adenine dinucleotide phosphate (NADPH)
  • NADP + oxidized nicotinamide adenine dinucleotide phosphate
  • NADP + is reduced by the coenzyme regeneration system For NADPH.
  • the reaction for generating imine is also performed 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 triangular yeast, Fusarium graminearum, Fusarium pear spore, and Fusarium solani.
  • the D-amino acid oxidase is derived from Trigonopsis variabilis CBS 4095, Fusarium graminearum CS3005, Fusarium poae 2516, or Fusarium solsolani M -0718.
  • the piperidinate reductase is derived from Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas arborea.
  • the piperidinate reductase is derived from Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens Pf0-1, or Pseudomonas fluorescens Pseudomonas entomophila str.
  • the coenzyme regeneration system includes a coenzyme regeneration enzyme and a coenzyme regeneration substrate, and the coenzyme regeneration enzyme is derived from Bacillus subtilis and Lactobacillus.
  • the coenzyme regenerating enzyme is derived from a glucose dehydrogenase of Bacillus subtilis 168 and an alcohol dehydrogenase of Lactobacillus kefir DSM20587.
  • the use form of the enzyme in the multi-enzyme system may be an ex vivo enzyme, a crude enzyme solution, or an engineered bacteria resting cell expressing a recombinant enzyme, or a pure enzyme, or an immobilized enzyme.
  • the starting substrate in the reaction system is racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or racemic 6,7-dimethoxy-1,2,3,4 -
  • concentration of tetrahydroisoquinoline-1-carboxylic acid is 1 to 20 g / L. More preferably, the concentration of the starting substrate in the reaction system is controlled to be 1 to 20 g / L. According to a specific aspect of the present invention, the concentration of the starting substrate in the reaction system is controlled to be 5 g / L.
  • the added amount of D-amino acid oxidase 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 1 to 5% by weight of the reaction solution.
  • the catalase is lyophilized powder of bovine liver catalase
  • the enzyme activity is 4000 U / mg
  • the enzyme activity ratio of catalase to D-amino acid oxidase is 100-400: 1.
  • the added amount of piperidinate 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 1 to 5% by weight of the reaction solution.
  • the added amount of the 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 1 to 5% by weight of the reaction solution.
  • the starting coenzyme may be added with oxidized nicotinamide adenine dinucleotide phosphate (NADP + ), and the added amount is 1 ⁇ to 1%.
  • NADP + oxidized nicotinamide adenine dinucleotide phosphate
  • 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.
  • the pH value of the reaction is controlled by a phosphate buffer solution from 7 to 8.5.
  • the genes used in the examples of the present invention were synthesized by Biotech Bioengineering (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 experimental reagents were purchased from TaKaRa.
  • DNA marker, PrimeStar DNA polymerase, low molecular weight standard protein and other molecular biological experimental reagents were purchased from TaKaRa.
  • the 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-1-carboxylic acid is: ZWIX (-); column temperature / 25 ° C; flow rate / 0.4mL / min; detection wavelength / UV220nm; mobile phase: HPLC-grade methanol (add 50mM formic acid and 25mM dihexylamine). See Figure 1 for specific peaks of related substances.
  • HPLC analysis method of racemic 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is: Column / Chirobiotic TAG; column temperature / 25 ° C; flow rate / 0.8 mL / min; detection wavelength / UV220nm and 232nm; mobile phase: HPLC-grade methanol / water (1: 1) (Directed (R) -or (S) -Selective Dynamic Kinetic Enzymatic Hydrolysis of 1,2,3,4-Tetrahydroisoquinoline -1-carboxylic Esters [J]. European Journal of Organic Chemistry, 2008, 2008 (31): 5269-76).
  • D-amino acid oxidases derived from microorganisms can be divided into two categories: 1) Amino acids with a smaller side chain group in the substrate (such as D-alanine), such as Fusarium oxysporum (Fusarium oxysporum) -derived D-amino acid oxidase; 2) Prefers amino acids with larger substrate side chain groups (such as D-phenylalanine), such as D-amino acid oxidase derived from Trigonopsis variabilis ( POLLEGIONI, MOLLA, G, SACCHI, et al. Properties and applications of D-amino acididoxidases: current state and perspectives [J]. Appl.
  • D-alanine such as Fusarium oxysporum (Fusarium oxysporum) -derived D-amino acid oxidase
  • Prefers amino acids with larger substrate side chain groups such as D-phenylalanine
  • the D-amino acid oxidase gene sequence was codon optimized and sent to Shenggong Bioengineering (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 future use.
  • the upstream and downstream primers of PCR were designed according to the DNA sequences of the corresponding genes.
  • 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)
  • Restriction enzyme restriction sites are added to the upstream and downstream primers, respectively, as shown by the underline. For specific restriction enzymes, see the primer sequence brackets. Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens Pf0-1, Pseudomonas entomomia L48 genomic DNA, respectively As a template, the corresponding upstream and downstream primers were used to perform PCR amplification reactions. The PCR reaction system and reaction conditions are as follows:
  • the amplification results were detected by 1.0% agarose gel electrophoresis. The results showed that the amplified product was a single band with a size of about 1000 bp.
  • Use the DNA recovery purification kit to recover the target band For specific steps, refer to the purification kit instructions.
  • the expression vector pET-28a (+) and the PCR amplified products were double-digested with the corresponding restriction enzymes, respectively. After the digestion was completed, the target band was recovered using a DNA recovery and purification kit. Subsequently, T4DNA ligase was used to ligate the double-digested PCR amplification product to the expression vector pET-28a (+) with a corresponding sticky end.
  • the ligation system is shown in Table 3 below:
  • the enzyme-linked product was transformed into E. coli DH5a competent cells, plated, and single colonies were cultured in LB liquid base.
  • the positive transformants were identified by PCR of the bacterial liquid, and sent to a sequencing company to verify the correctness of the inserted sequence. Plasmids were extracted from the positive transformants that were verified to be correct, and the relevant methods were referred to the plasmid extraction kit.
  • the recombinant expression vector was transferred into the expression host E. coli BL21 (DE3). After verification by PCR and sequencing of the bacterial solution, glycerol with an initial concentration of 25% was added to the obtained engineering bacterial solution and placed at -80 ° C. Keep it 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, and the alcohol dehydrogenase (Lactobacillus kefiri) DSM20587 genome was cloned from the Bacillus subtilis 168 genome, respectively.
  • NCBI accession number: AAP94029.1, SEQ ID NO. 10 gene.
  • 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 culture medium composition peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water to make up the volume, sterilized at 121 ° C for 20min, and set aside. For solid LB medium, add 15g / L agar.
  • the engineered bacteria containing the D-amino acid oxidase gene was inoculated into 5 mL of liquid LB (containing 50 ⁇ g / ml kanamycin) medium, and cultured at 37 ° C. with shaking at 200 rpm for about 8 hours.
  • 1% (V / V) inoculation amount was inoculated in 100 mL liquid LB (containing 50 ⁇ g / ml kanamycin) culture medium, after the OD 600 reached 0.6-0.8, the inducer isopropyl thiogalactoside was added (Initial concentration is 0.1 mM), induced at 18 ° C for 15 h.
  • the bacterial weight was suspended in 25 mL of phosphate buffer solution (50 mM, pH 8.0), the bacterial suspension was sonicated, and the supernatant obtained after centrifugation was a crude enzyme solution containing D-amino acid oxidase or piperidinate reductase or coenzyme regenerating enzyme. .
  • Example 2 a crude D-amino acid oxidase solution derived from Fusarium solani M-0718, and a crude piperidinate reductase from Pseudomonas putida KT2440 were prepared, respectively. Solution and crude glucose dehydrogenase solution of Bacillus subtilis 168.
  • the reaction temperature was controlled to 30 ° C using a water bath, magnetic stirring was performed, the pH of the reaction was adjusted within the range of 8 to 8.5 with ammonia, and the reaction was sampled for 16 hours.
  • the concentration of 3,4-tetrahydroisoquinoline-1-carboxylic acid was 10 g / L, the concentration of NADP + was 0.1 mM, and the concentration of isopropanol was 25 mM.
  • the reaction temperature was controlled to 30 ° C with a water bath, magnetically stirred, and the reaction was sampled for 30 hours.
  • HPLC detection of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid content in the samples taken we can know the 1,2,3,4-tetrahydro in the reaction solution Concentration (g / L) of isoquinoline-1-carboxylic acid in two configurations.
  • the yield of (S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction solution was 95.2% (the calculation method is the same as in Example 3), and the ee value was 99.4%.
  • Example 2 a crude D-amino acid oxidase solution derived from Fusarium graminearum CS3005 and a crude piperidinate reductase from Pseudomonas fluorescens Pf0-1 were prepared. Solution and crude glucose dehydrogenase solution of Bacillus subtilis 168.
  • the reaction temperature was controlled to 30 ° C using a water bath, magnetic stirring, and the pH of the reaction was adjusted to be in the range of 8 to 8.5 with ammonia water, and the reaction was sampled for 6 hours.
  • HPLC detection of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid content in the samples taken we can know the 1,2,3,4-tetrahydro in the reaction solution Concentration (g / L) of isoquinoline-1-carboxylic acid in two configurations.
  • the reaction yield of (S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the reaction solution was 99.4% (the calculation method is the same as in Example 3), and the ee value was 99.1%.
  • Example 2 a crude D-amino acid oxidase solution derived from Trigonopsis variabilis CBS 4095, and a crude piperidinate reductase from Pseudomonas entomophila L. 48 were prepared. Solution and crude alcohol dehydrogenase solution of Lactobacillus kefiri DSM20587.
  • the concentration of 3,4-tetrahydroisoquinoline-1-carboxylic acid was 20 g / L, the concentration of NADP + was 0.1 mM, and the concentration of isopropanol was 50 mM.
  • the reaction temperature was controlled to 30 ° C with a water bath, magnetically stirred, and the reaction was sampled for 50 hours.
  • HPLC detection of 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid content in the samples taken we can know the 1,2,3,4-tetrahydro in the reaction solution Concentration (g / L) of isoquinoline-1-carboxylic acid in two configurations.
  • Example 2 a crude D-amino acid oxidase solution derived from Fusarium solani M-0718, and a crude piperidinate reductase from Pseudomonas putida KT2440 were prepared, respectively. Solution and crude glucose dehydrogenase solution of Bacillus subtilis 168.
  • the reaction temperature was controlled to 30 ° C using a water bath, magnetic stirring was performed, the pH of the reaction was adjusted within the range of 8 to 8.5 with ammonia, and the reaction was sampled for 19 hours.
  • Example 2 a crude D-amino acid oxidase solution derived from Fusarium poae 2516 and a crude piperidinate reductase from Pseudomonas fluorescens Pf0-1 were prepared. Solution and crude alcohol dehydrogenase solution of Lactobacillus kefiri DSM20587.
  • D-amino acid oxidase crude enzyme solution derived from Fusarium graminearum CS3005, crude piperidinate reductase solution of Pseudomonas aeruginosa PAO1, and subtilis spore Crude glucose dehydrogenase solution from Bacillus subtilis 168.
  • Example 2 a crude D-amino acid oxidase solution derived from Trigonopsis variabilis CBS 4095, and a crude piperidinate reductase from Pseudomonas entomophila L. 48 were prepared. Solution and crude alcohol dehydrogenase solution of Lactobacillus kefiri DSM20587.
  • the initial concentration of pure glucose dehydrogenase is 0.1 mg / ml
  • the initial concentration of NADP + is 0.01 mM
  • the initial concentration of catalase is 0.01 mg / ml
  • the initial concentration of D-glucose is 8mM.
  • Example 2 a crude D-amino acid oxidase solution derived from Fusarium solani M-0718, and a crude piperidinate reductase from Pseudomonas putida KT2440 were prepared, respectively. Solution and crude glucose dehydrogenase solution of Bacillus subtilis 168.
  • Example 2 a crude D-amino acid oxidase solution derived from Fusarium solani M-0718, and a crude piperidinate reductase from Pseudomonas putida KT2440 were prepared, respectively. Solution and crude glucose dehydrogenase solution of Bacillus subtilis 168.
  • the water temperature was used to control the reaction temperature to 30 ° C, magnetic stirring was performed, and the reaction pH was adjusted to be in the range of 8 to 8.5 with a 0.5M potassium hydroxide solution, and the reaction was sampled for 18 hours.
  • the substrate solution and reaction system are as in Example 3.
  • the substrate solution and reaction system are as in Example 7.
  • the content of the two configurations of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in the sample was determined by high performance liquid chromatography. Contents of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid in two configurations. The yield of (S) -6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid was 78.5%, and the ee value was 99.8%.
  • FsDAAO shows strict stereoselectivity in the R-configuration, with a yield of 49.9%, and the ee value of (S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid is above 99%.

Abstract

本发明公开了一种制备(S)-1，2，3，4-四氢异喹啉-1-甲酸及其衍生物的方法，包括：以所述式(I)所示的化合物的外消旋体或式(I)所示的化合物的盐的外消旋体为底物，使该底物中式(I)所示化合物的R型异构体在氧化脱氢酶的催化作用下反应生成式(II)所示的亚胺酸；使所述式(II)所示的亚胺酸在哌啶酸还原酶与能够供给氢负离子的辅酶的存在下转化为所述式(I)所示化合物的S型异构体；本发明具有反应条件温和、立体选择性强、反应效率高、转化率高等特点。

Description

一种制备(S)-1，2，3，4-四氢异喹啉-1-甲酸及其衍生物的方法 技术领域
本发明属于生物催化技术领域，具体涉及一种制备(S)-1，2，3，4-四氢异喹啉-1-甲酸及其衍生物的方法。
背景技术
1，2，3，4-四氢异喹啉类化合物是一类非常重要的药物中间体，被广泛应用于多种药物的合成。近年来，Hu等(Discovery of a small-molecule inhibitor and cellular probe of Keap1-Nrf2 protein-protein interaction[J].Bioorg Med Chem Lett，2013，23(10)：3039-43.)以(S)-1，2，3，4-四氢异喹啉-1-甲酸为起始化合物，合成一种靶向Kelch-like ECH-associated protein 1(Keap1)的抑制剂，从而有望用于癌症，糖尿病，阿尔兹海默症，帕金森等疾病的治疗和预防。而大部分具有药用价值的异喹啉碱都含有6，7-二甲氧基(如罂粟碱、吐根碱等)，有利于降低药物分子的疏水性，提高成药性，如6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸。
现有技术中，制备光学纯(S)-1，2，3，4-四氢异喹啉-1-甲酸或(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的方法主要有两大类：化学手性合成和生物催化手性拆分。
化学手性合成法从手性原料出发合成(S)-1，2，3，4-四氢异喹啉-1-甲酸，如Kurata等光学纯烯烃异喹啉为起始原料经臭氧分解和NaBH 4原位还原、四甲基哌啶氮氧化物(TEMPO)氧化以及三氟乙酸介导的N-叔丁氧羰基去保护作用三步不对成合成(S)-1，2，3，4-四氢异喹啉-1-甲酸(Synthesis of Optically Pure(R)-and(S)-Tetrahydroisoquinoline-1-and-3-Carboxylic Acids[J].Synthesis，2015，47(09)：1238-44.)。该法产率低，步骤繁琐，不适于工业化应用。Bulyszko等采用Petasis反应和Pomeranz-Fritsch-Bobbitt反应非对映体选择性全合成(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸，ee值为90％(Synthesis of(+)-6，7-Dimethoxy-1，2，3，4-tetrahydroisoquinoline-1-carboxylic Acid，a Diastereoselective Approach[J].European Journal of Organic Chemistry，2015，2015(2)：383-8.)。
相比之下，生物催化法具有立体选择性高、反应条件温和等优点，是制备(S)-1，2，3，4-四氢异喹啉-1-甲酸或其衍生物的潜在优势方法。Paál等利用枯草杆菌蛋白酶动态动力学拆分6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸乙酯制备(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸，53.46g/L底物，加酶量为80mg/mL固定化酶，3℃，pH＝8.5条件下，反应3天，产率92％，产物ee值为93％(Directed(R)-or(S)-Selective Dynamic Kinetic Enzymatic Hydrolysis of 1，2，3，4-Tetrahydroisoquinoline-1-carboxylic Esters[J].European Journal of Organic Chemistry，2008，2008(31)：5269-76)。该法反应条件温和，立体选择性强，工艺相对简单，但所得产物的光学纯度还有待进一步提高。
发明内容
本发明的目的在于克服现有技术的不足，提供一种新的制备(S)-1，2，3，4-四氢异喹啉-1-甲酸及其衍生物的方法。
为实现上述目的，本发明采取的技术方案如下：
一种制备如式(I)所示化合物的S型异构体的方法，
Figure PCTCN2019083813-appb-000001
式(I)中，R 1，R 2独立地选自氢、C1-C6烷基、C1-C6烷氧基，所述方法包括如下步骤：
以所述式(I)所示的化合物的外消旋体或式(I)所示的化合物的盐的外消旋体为底物，使该底物中式(I)所示化合物的R型异构体在氧化脱氢酶的催化作用下反应生成式(II)所示的亚胺酸；
Figure PCTCN2019083813-appb-000002
使所述式(II)所示的亚胺酸在哌啶酸还原酶与能够供给氢负离子的辅酶的存在下转化为所述式(I)所示化合物的S型异构体。
根据本发明的一些优选方面，式(I)中，R 1，R 2独立地选自氢、甲基、乙基、异丙基、甲氧基或乙氧基。
根据本发明的一些优选方面，所述的盐为一价盐，具体优选碱金属盐或铵盐，其中碱金属盐可以为例如锂盐、钠盐、钾盐。
根据本发明的一些优选方面，所述的式(I)所示化合物的S型异构体为(S)-1，2，3，4-四氢异喹啉-1-甲酸或(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸。
根据本发明，所述氧化脱氢酶是能够选择性催化式(I)所示化合物的R型异构体的酶，且选择性大于等于80％，优选大于等于90％。
根据本发明的一些优选方面，所述氧化脱氢酶为D-氨基酸氧化酶。
根据本发明，所述D-氨基酸氧化酶为选自如下D-氨基酸氧化酶中的一种或多种的组合：来源于三角酵母(Trigonopsis variabilis)CBS 4095的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自禾谷镰刀菌(Fusarium graminearum)CS3005的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自梨孢镰刀菌(Fusarium poae)2516的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶，来自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶。
优选地，所述D-氨基酸氧化酶具有如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的氨基酸序列。
根据本发明的一些具体且优选的方面，所述D-氨基酸氧化酶的添加量以8000rpm离心 10min后的细胞湿重计，所述细胞的添加量为反应体系重量的1～5％。
根据本发明的一些具体且优选的方面，所述D-氨基酸氧化酶的使用形式为离体的D-氨基酸氧化酶、含有离体的D-氨基酸氧化酶的粗酶液、D-氨基酸氧化酶的纯酶、D-氨基酸氧化酶的固定化酶或胞内表达D-氨基酸氧化酶的细胞。
进一步地，所述细胞为表达D-氨基酸氧化酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述D-氨基酸氧化酶基因连接在所述表达载体pET-28a(+)上。
根据本发明，所述哌啶酸还原酶为选自如下哌啶酸还原酶中的一中或多种的组合：来源于恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于绿脓杆菌(Pseudomonas aeruginosa)PAO1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于荧光假单胞菌(Pseudomonas fluorescens)Pf0-1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶。
优选地，所述哌啶酸还原酶具有如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的氨基酸序列。
根据本发明的一些具体且优选的方面，所述哌啶酸还原酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应体系重量的1～5％。
根据本发明的一些具体且优选的方面，所述哌啶酸还原酶的使用形式为离体的哌啶酸还原酶、含有离体的哌啶酸还原酶的粗酶液、哌啶酸还原酶的纯酶、哌啶酸还原酶的固定化酶或胞内表达哌啶酸还原酶的细胞。
进一步地，所述细胞为表达哌啶酸还原酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述哌啶酸还原酶基因连接在所述表达载体pET-28a(+)上。
根据本发明的一些优选方面，所述能够供给氢负离子的辅酶为NADH和/或NADPH。
根据本发明的一些优选方面，使所述生成亚胺酸的反应还在黄素腺嘌呤二核苷酸(FAD)的存在下进行。使反应在FAD存在下进行，有助于进一步提高转化率。进一步地，FAD与所述的底物等当量或者是过量的。一般情况下，所制备的D-氨基酸氧化酶的粗酶液中已经含有足够量的FAD，在直接采用粗酶液的情况下，无需再另外添加FAD。在使用D-氨基酸氧化酶纯酶的情况下，可以根据需要再外加适量的FAD。
根据本发明的一些优选方面，使所述生成亚胺酸的反应还在过氧化氢酶的存在下进行。
根据本发明的一些具体且优选的方面，使生成所述亚胺酸的反应在设定温度和有氧环境中进行。
根据本发明的优选方面，所述设定温度为20～70℃。更优选地，所述设定温度为20～50℃。进一步优选地，所述设定温度为30～40℃。
根据本发明的一些具体且优选的方面，所述方法的实施过程包括：首先构建反应体系， 然后控制所述反应体系处于设定温度和有氧环境中进行反应，所述反应体系包括所述底物、所述氧化脱氢酶、所述哌啶酸还原酶、辅酶、辅酶再生系统、溶剂，所述反应体系还选择性地包括pH缓冲剂和/或pH调节剂，所述辅酶包括NAD+(氧化型烟酰胺腺嘌呤二核苷酸)和/或NADH(还原型烟酰胺腺嘌呤二核苷酸)，或者，所述辅酶包括NADP+(氧化型烟酰胺腺嘌呤二核苷酸磷酸)和/或NADPH(还原型烟酰胺腺嘌呤二核苷酸磷酸)。
根据本发明的一些优选方面，控制所述反应体系的pH值为6～9。更优选地，控制所述反应体系的pH值为7～8.5。
根据本发明的一些优选方面，控制所述反应体系中起始底物的浓度为1～20g/L。更优选地，控制所述反应体系中起始底物的浓度为1～20g/L。根据本发明的一个具体方面，控制所述反应体系中起始底物的浓度为5g/L。
根据本发明的一个具体且优选方面，所述pH缓冲剂为磷酸盐，将其溶于水可以配制成磷酸盐缓冲溶液。
根据本发明的一些优选方面，所述的pH调节剂为氨水、碱金属氢氧化物或其水溶液。
根据本发明的一个具体且优选方面，所述的pH调节剂为20wt％～35wt％氨水。
根据本发明的又一具体方面，所述的pH调节剂为氢氧化钠或氢氧化钾的水溶液。
根据本发明的一些具体且优选的方面，所述辅酶的添加量为底物浓度的1‰～1％。
根据本发明，所述辅酶再生系统包括辅酶再生酶和辅酶再生底物。
根据本发明的一些优选方面，所述辅酶再生酶为葡萄糖脱氢酶，所述辅酶再生底物为葡萄糖；或者，所述辅酶再生酶为醇脱氢酶，所述辅酶再生底物为异丙醇。根据本发明的一个具体方面，所述葡萄糖具体使用D-葡萄糖。
根据本发明的一个具体方面，所述葡萄糖脱氢酶来源于枯草芽胞杆菌(Bacillus subtilis)168；和/或，所述醇脱氢酶来源于乳酸杆菌(Lactobscillus kefir)DSM20587。
优选地，所述葡萄糖脱氢酶具有如SEQ ID NO.9所示的氨基酸序列。
优选地，所述醇脱氢酶具有如SEQ ID NO.10所示的氨基酸序列。
根据本发明的一些优选方面，所述反应体系还包括过氧化氢酶。
根据本发明的一些优选方面，所述过氧化氢酶为牛肝过氧化氢酶冻干粉。根据本发明的一个具体方面，所述牛肝过氧化氢酶冻干粉的酶活为4000U/mg。
根据本发明的一些优选方面，所述过氧化氢酶与所述氧化脱氢酶的酶活比为100～400∶1。
根据本发明的一些优选方面，所述反应体系还包括黄素腺嘌呤二核苷酸。
根据本发明的一些具体且优选的方面，所述方法还包括分离步骤。
根据本发明，所述分离步骤包括：将反应后的反应体系的pH值调至5.0-6.0，加热使蛋白变性析出，抽滤，滤液浓缩后，冷却析晶，干燥，即得所述式(I)所示的化合物的S型异构体。
由于以上技术方案的实施，本发明与现有技术相比具有如下有益效果：
本发明发现在哌啶酸还原酶与能够供给氢负离子的辅酶的共同存在下可高效地使亚胺 酸转化得到式(I)所示化合物的S型异构体，其选择性好，收率高，反应条件温和，制备得到的产物中S型异构体相对R型异构体的ee值＞99％，且工艺相对简单。
附图说明
图1为底物外消旋1，2，3，4-四氢异喹啉-1-甲酸的两个光学异构体高效液相检测图谱(1g/L)；
其中，保留时间8.810min为(R)-1，2，3，4-四氢异喹啉-1-甲酸；保留时间12.685min为(S)-1，2，3，4-四氢异喹啉-1-甲酸；
图2为实施例3中反应体系中0小时取样的高效液相色谱检测图谱；
图3为实施例3中反应16小时取样的高效液相色谱检测图谱。
具体实施方式
本发明提供一种制备(S)-1，2，3，4-四氢异喹啉-1-甲酸及其衍生物的新方法，本发明方法具有反应条件温和、立体选择性强、反应效率高、产率高等特点，具有工业化应用前景。
根据本发明的一个具体方面，该方法以外消旋1，2，3，4-四氢异喹啉-1-甲酸或外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸为底物，经多酶体系催化获得(S)-1，2，3，4-四氢异喹啉-1-甲酸或(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸，所述多酶体系可由氧化脱氢酶(优选为D-氨基酸氧化酶)、过氧化氢酶、哌啶酸还原酶和辅酶(优选为NADP +和/或NADPH)、辅酶再生系统等组成。
具体原理为：以外消旋1，2，3，4-四氢异喹啉-1-甲酸或外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸为底物，利用D-氨基酸氧化酶立体选择性催化(R)-1，2，3，4-四氢异喹啉-1-甲酸或(R)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸进行氧化脱氢生成亚胺酸，(S)-1，2，3，4-四氢异喹啉-1-甲酸或(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸基本未被催化而保留在反应体系中。反应过程中产生的过氧化氢经过氧化氢酶催化分解成水和氧气。亚胺酸被哌啶酸还原酶不对称还原为(S)-1，2，3，4-四氢异喹啉-1-甲酸或(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸。在此过程中，还原型辅酶II即还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)被氧化为NADP +(氧化型烟酰胺腺嘌呤二核苷酸磷酸)，NADP +经辅酶再生系统还原为NADPH。
反应过程示意如下：
Figure PCTCN2019083813-appb-000003
进一步地，优选使生成亚胺酸的反应还在黄素腺嘌呤二核苷酸(FAD)存在下进行，在反应过程中，黄素腺嘌呤二核苷酸(FAD)被还原为FADH 2，随后，一分子氧被还原为过氧化氢(H 2O 2)，而FADH 2则被氧化为FAD。过氧化氢在过氧化氢酶的催化下分解成水和氧气。反应过程示意如下：
Figure PCTCN2019083813-appb-000004
作为优选，所述D-氨基酸氧化酶来源于三角酵母、禾谷镰刀菌、梨孢镰刀菌、茄病镰刀菌。具体地，所述D-氨基酸氧化酶来源于三角酵母(Trigonopsis variabilis)CBS 4095、禾谷镰刀菌(Fusarium graminearum)CS3005、梨孢镰刀菌(Fusarium poae)2516或茄病镰刀菌(Fusarium solani)M-0718。
作为优选，所述哌啶酸还原酶来源于恶臭假单胞菌，绿脓杆菌，荧光假单胞菌，虫媒假单胞菌。具体的，所述哌啶酸还原酶源于恶臭假单胞菌(Pseudomonas putida)KT2440、绿脓杆菌(Pseudomonas aeruginosa)PAO1、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1或虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶。
作为优选，所述辅酶再生系统包括辅酶再生酶和辅酶再生底物，所述辅酶再生酶来源于枯草芽胞杆菌，乳酸杆菌。具体地，所述辅酶再生酶来源于枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶，来源于乳酸杆菌(Lactobscillus kefir)DSM20587的醇脱氢酶。
具体地，反应体系中，多酶体系中的酶的使用形式可以为离体酶、粗酶液，或者表达重组酶的工程菌静息细胞，或者是纯酶，或者固定化酶。
作为优选，反应体系中起始底物外消旋1，2，3，4-四氢异喹啉-1-甲酸或外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的浓度为1～20g/L。更优选地，控制所述反应体系中起始底物的浓度为1～20g/L。根据本发明的一个具体方面，控制所述反应体系中起始底物的浓度为5g/L。
作为优选，反应体系中，D-氨基酸氧化酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应液重量的1～5％。
作为优选，反应体系中，过氧化氢酶为牛肝过氧化氢酶冻干粉末，酶活为4000U/mg，过氧化氢酶与D-氨基酸氧化酶的酶活比为100～400∶1。
作为优选，反应体系中，哌啶酸还原酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应液重量的1～5％。
作为优选，反应体系中，辅酶再生酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应液重量的1～5％。
作为优选，反应体系中，起始的辅酶可以添加氧化型烟酰胺腺嘌呤二核苷酸磷酸(NADP +)，其添加量为1‰～1％。
作为优选，反应体系中，反应的温度为20～70℃，时间为6～72小时，反应液的pH值为6～9；更优选，温度为30～40℃，时间为12～48小时。磷酸缓冲溶液控制反应的pH值为7～8.5。
以下结合具体实施例，对本发明做进一步说明。应理解，以下实施例仅用于说明本发明而非用于限制本发明的范围。
本发明实施例中的实验方法如无特别说明均为常规方法。
本发明实施例中所用基因由生工生物工程(上海)股份有限公司合成。E.coli BL21(DE3)菌种购自Novagen公司；DNA marker、PrimeStar DNA聚合酶、低分子量标准蛋白等分子生物学实验试剂购自TaKaRa。基因克隆及表达具体操作可参见J.萨姆布鲁克等编的《分子克隆实验指南》。
本发明通过高效液相色谱(HPLC)分析催化反应的各个产物和底物。外消旋1，2，3，4-四氢异喹啉-1-甲酸的HPLC分析方法为：色谱柱/
Figure PCTCN2019083813-appb-000005
ZWIX(-)；柱温/25℃；流速/0.4mL/min；检测波长/UV220nm；流动相：HPLC级甲醇(加入50mM甲酸和25mM二已胺)。具体各相关物质出峰情况见附图1。外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的HPLC分析方法为：色谱柱/Chirobiotic TAG；柱温/25℃；流速/0.8mL/min；检测波长/UV220nm和232nm；流动相：HPLC级甲醇/水(1∶1)(Directed(R)-or(S)-Selective Dynamic Kinetic Enzymatic Hydrolysis of 1，2，3，4-Tetrahydroisoquinoline-1-carboxylic Esters[J].European Journal of Organic Chemistry，2008，2008(31)：5269-76)。
实施例1基因工程菌菌种构建
1.1 D-氨基酸氧化酶的筛选及表达D-氨基酸氧化酶的基因工程菌的构建
根据底物特异性的不同，微生物来源的D-氨基酸氧化酶可分为两大类：1)偏好底物侧链基团较小的氨基酸(如D-丙氨酸)，如尖孢镰刀菌(Fusarium oxysporum)来源的D-氨基酸氧化酶；2)偏好底物侧链基团较大的氨基酸(如D-苯丙氨酸)，如三角酵母(Trigonopsis variabilis)来源的D-氨基酸氧化酶(POLLEGIONI L，MOLLA G，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.)。分别用这两种D-氨基酸氧化酶的氨基酸序列在美国国立生物技术信息中心(NCBI)数据库(https：//www.ncbi.nlm.nih.gov/)中进行BLASTp分析，选取序列一致性不同的4种D-氨基酸氧化酶作进一步研究(如表1所示)。
表1四种不同来源的D-氨基酸氧化酶
Figure PCTCN2019083813-appb-000006
将上述D-氨基酸氧化酶基因序列经密码子优化后送生工生物工程(上海)股份有限公司进行全基因合成，并克隆到重组表达质粒pET-28a(+)上。重组质粒转入表达宿主E.coli BL21(DE3)中，经测序验证无误后，向所得工程菌菌液中加入起始浓度为25％的甘油并置于-80℃保藏备用。
1.2表达哌啶酸还原酶的基因工程菌的构建
分别从恶臭假单胞菌(Pseudomonas putida)KT2440、绿脓杆菌(Pseudomonas aeruginosa)PAO1、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1、虫媒假单胞菌(Pseudomonas entomophila str.)L48基因组中克隆哌啶酸还原酶基因(如表2所示)。
表2四种不同来源的哌啶酸还原酶
Figure PCTCN2019083813-appb-000007
根据相应基因DNA序列设计PCR上游引物和下游引物。
来源于Pseudomonas putida KT2440的哌啶酸还原酶的引物：
KT2440-F：5’-CG GGATCCATGTCCGCACCTTCCACCAGCAC-3’(BamH I)
KT2440-R：5’-CCC AAGCTTTCAGCCAAGCAGCTCTTTCAGG-3’(Hind III)
来源于Pseudomonas aeruginosa PAO1的哌啶酸还原酶的引物：
PAO1-F：5’-CG GGATCCGTGATCCGAATGACGCTGGAC-3’(BamH I)
PAO1-R：5’-CCC AAGCTTTCACTCCAGCAACGCCAGC-3’(Hind III)
来源于Pseudomonas fluorescens Pf0-1的哌啶酸还原酶的引物：
Pf0-1-F：5’-CG GGATCCATGTCTGCGCCACACGATC-3’(BamH I)
Pf0-1-R：5’-CCG CTCGAGTTACTCGCCGGCCAGTTCAC-3’(Xho I)
来源于Pseudomonas entomophila str.L48的哌啶酸还原酶的引物：
L48-F：5’-CG GGATCCGTGCGCGTAGCCTTCAAC-3’(BamH I)
L48-R：5’-CCC AAGCTTTCACCTCGCCAGCGCCTTC-3’(Hind III)
在上、下游引物中分别加入限制性内切酶切位点，如下划线所示，具体限制性内切酶见引物序列括号内。分别以恶臭假单胞菌(Pseudomonas putida)KT2440、绿脓杆菌(Pseudomonas aeruginosa)PAO1、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1、虫媒假单胞菌(Pseudomonas entomophila str.)L48基因组DNA为模板，利用相应的上下游引物分别进行PCR扩增反应，PCR反应体系和反应条件如下：
PCR扩增体系：
Figure PCTCN2019083813-appb-000008
PCR扩增条件：
1)预变性：95℃5min；
2)变性：95℃10s；退火：58℃15s；延伸：72℃10s；共循环30次；
3)延伸：72℃10min；
4)4℃保温。
PCR扩增反应结束后，利用1.0％琼脂糖凝胶电泳检测扩增结果，结果显示扩增产物为单一条带，大小约为1000bp。用DNA回收纯化试剂盒回收目的条带，具体步骤参照纯化试剂盒说明书。
表达载体pET-28a(+)和PCR扩增产物分别用相应的限制性内切酶进行双酶切。酶切完成后用DNA回收纯化试剂盒回收目的条带。随后，利用T4DNA连接酶将双酶切后的PCR扩增产物连接到具有相对应黏性末端的表达载体pET-28a(+)上，连接体系如下表3所示：
表3重组表达质粒构建体系
Figure PCTCN2019083813-appb-000009
将酶连产物转化至E.coli DH5a感受态细胞中，涂平板、挑单菌落到LB液体基中培养，菌液PCR鉴定阳性转化子，并送测序公司来验证插入序列的正确性。从验证无误的阳性转化子中提取质粒，相关方法参照质粒提取试剂盒。再将重组表达载体转入表达宿主E.coli BL21(DE3)中，经菌液PCR和测序验证无误后，向所得工程菌菌液中加入起始浓度为25％的甘油并置于-80℃保藏备用。
1.3表达辅酶再生酶的基因工程菌的构建
分别从枯草芽胞杆菌(Bacillus subtilis)168基因组中克隆葡萄糖脱氢酶(NCBI登录号：NP_388275.1，SEQ ID NO.9)基因；从乳酸杆菌(Lactobacillus kefiri)DSM20587基因组中克 隆醇脱氢酶(NCBI登录号：AAP94029.1，SEQ ID NO.10)基因。具体方法步骤参考1.2中哌啶酸还原酶表达菌种的构建方法。相关PCR上游引物和下游引物如下：
来源于Bacillus subtilis 168的葡萄糖脱氢酶的引物：
BGdh-F：5’-GA AGATCTGATGTATCCGGATTTAAAAGGAAAAGTC-3’(Bgl II)
BGdh-R：5’-CATG CCATGGTTAACCGCGGC-3’(Nco I)
来源于Lactobacillus kefiri DSM20587的醇脱氢酶的引物：
LAdh-F：5’-CC GAATTCATGACCGATCGTCTGAAGGGC-3’(EcoR I)
LAdh-R：5’-CCC AAGCTTTCACTGTGCGGTATACCCGCC-3’(Hind III)。
实施例2
2.1微生物的培养
液体LB培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，用去离子水溶解后定容，121℃灭菌20min，待用。若为固体LB培养基，则另加15g/L琼脂。
将含有D-氨基酸氧化酶基因的工程菌接种于5mL液体LB(含50μg/ml卡那霉素)培养基中，37℃，200rpm振荡培养8小时左右。按1％(V/V)的接种量接种于100mL液体LB(含50μg/ml卡那霉素)培养基中培养，OD 600达到0.6-0.8后，加入诱导剂异丙基硫代半乳糖苷(起始浓度为0.1mM)，18℃诱导15h。培养结束后，将培养液倒入100mL离心管中4000rpm离心10min，弃上清，收集菌体细胞，用50mM磷酸缓冲液(pH 8.0)洗涤细胞两次，放于-80℃超低温冰箱中保存，备用。
2.2粗酶液的制备
将菌体重悬于25mL磷酸缓冲液(50mM，pH 8.0)中，超声破碎菌悬液，离心后得到的上清为含D-氨基酸氧化酶或哌啶酸还原酶或辅酶再生酶的粗酶液。
实施例3 FsDAAO-PpdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶粗酶液、恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。
称取0.2g外消旋1，2，3，4-四氢异喹啉-1-甲酸盐酸盐至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FsDAAO粗酶液(粗酶液中已含有足量辅酶FAD，因此，粗酶液反应体系中不需额外添加FAD)，7.6ml PpdpkA粗酶液，2.4ml葡萄糖脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及D-葡萄糖，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-1-甲酸的浓度为5g/L，NADP +的浓度为0.05mM，D-葡萄糖浓度为15mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，氨水调节反应pH在8～8.5范围内，反应16小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。
检测结果如图2和图3所示，FsDAAO表现出严格的R-构型立体选择性，反应液中(S)-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为98.4％(反应收率＝实际产物浓度(g/L)/理论产 物浓度(g/L)×100％)，ee值为99.2％。
实施例4 FpDAAO-PadpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自梨孢镰刀菌(Fusarium poae)2516的D-氨基酸氧化酶粗酶液、绿脓杆菌(Pseudomonas aeruginosa)PAO1的哌啶酸还原酶粗酶液以及乳酸杆菌(Lactobacillus kefiri)DSM20587的醇脱氢酶粗酶液。
称取0.4g外消旋1，2，3，4-四氢异喹啉-1-甲酸盐酸盐至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FpDAAO粗酶液，8ml PadpkA粗酶液，2ml醇脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及异丙醇，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-1-甲酸的浓度为10g/L，NADP +的浓度为0.1mM，异丙醇浓度为25mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，反应30小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。反应液中(S)-1，2，3，4-四氢异喹啉-1-甲酸的收率为95.2％(计算方式同实施例3)，ee值为99.4％。
实施例5 FgDAAO-PfdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自禾谷镰刀菌(Fusarium graminearum)CS3005的D-氨基酸氧化酶粗酶液、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。
称取0.04g外消旋1，2，3，4-四氢异喹啉-1-甲酸盐酸盐至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FgDAAO粗酶液，7.5ml PfdpkA粗酶液，2.5ml葡萄糖脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及D-葡萄糖，使起始反应体系中的底物外消旋1，2，3，4-四氢异喹啉-1-甲酸的浓度为1g/L，NADP +的浓度为0.01mM，D-葡萄糖的浓度为3mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，氨水调节反应pH在8～8.5范围内，反应6小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。反应液中(S)-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为99.4％(计算方式同实施例3)，ee值为99.1％。
实施例6 TvDAAO-PedpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自三角酵母(Trigonopsis variabilis)CBS 4095的D-氨基酸氧化酶粗酶液、虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶粗酶液以及乳酸杆菌(Lactobacillus kefiri)DSM20587的醇脱氢酶粗酶液。
称取0.8g外消旋1，2，3，4-四氢异喹啉-1-甲酸盐酸盐至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml TvDAAO粗酶液，8ml PedpkA粗酶液，2ml醇脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及异丙醇，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-1-甲酸的浓度为20g/L，NADP +的浓度为0.1mM，异丙醇浓度为50mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温 度为30℃，磁力搅拌，反应50小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。反应液中(S)-1，2，3，4-四氢异喹啉-1-甲酸的反应收率(计算方式同实施例3)为92.5％，ee值为99.2％。
实施例7 FsDAAO-PpdpkA多酶耦合制备(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶粗酶液、恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。
称取0.2g外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FsDAAO粗酶液，7.6ml PpdpkA粗酶液，2.4ml葡萄糖脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及D-葡萄糖，使起始反应体系中底物6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的浓度为5g/L，NADP +的浓度为0.05mM，D-葡萄糖浓度为15mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，氨水调节反应pH在8～8.5范围内，反应19小时取样。高效液相色谱检测所取样品中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。反应液中(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸反应收率为97.4％(计算方式同实施例3)，ee值为99.1％。
实施例8 FpDAAO-PfdpkA多酶耦合制备(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自梨孢镰刀菌(Fusarium poae)2516的D-氨基酸氧化酶粗酶液、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1的哌啶酸还原酶粗酶液以及乳酸杆菌(Lactobacillus kefiri)DSM20587的醇脱氢酶粗酶液。
称取0.16g外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FpDAAO粗酶液，8ml PfdpkA粗酶液，2ml醇脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及异丙醇，使起始反应体系中底物外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的浓度为4g/L，NADP +的浓度为0.01mM，异丙醇浓度为10mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，反应20小时取样。高效液相色谱检测所取样品中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为94.8％(计算方式同实施例3)，ee值为99.2％。
实施例9 FgDAAO-PadpkA多酶耦合制备(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自禾谷镰刀菌(Fusarium graminearum)CS3005的D-氨基酸氧化酶粗酶液、绿脓杆菌(Pseudomonas aeruginosa)PAO1的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。
称取0.1g外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸至100ml反应瓶中，加10ml 磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FgDAAO粗酶液，8ml PadpkA粗酶液，2ml葡萄糖脱氢酶液，2mg过氧化氢酶，NADP +以及D-葡萄糖，使起始反应体系中底物外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的浓度为2.5g/L，NADP +的浓度为0.01mM，D-葡萄糖浓度为5mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，氨水调节反应pH在8～8.5范围内，反应20小时取样。高效液相色谱检测所取样品中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为96.5％(计算方式同实施例3)，ee值为99.2％。
实施例10 TvDAAO-PedpkA多酶耦合制备(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自三角酵母(Trigonopsis variabilis)CBS 4095的D-氨基酸氧化酶粗酶液、虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶粗酶液以及乳酸杆菌(Lactobacillus kefiri)DSM20587的醇脱氢酶粗酶液。
称取0.5g外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH 8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml TvDAAO粗酶液，8ml PedpkA粗酶液，2ml醇脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及异丙醇，使起始反应体系中底物外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的浓度为12.5g/L，NADP +的浓度为0.02mM，异丙醇浓度为25mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，反应24小时取样。高效液相色谱检测所取样品中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为92.4％(计算方式同实施例3)，ee值为99.5％。
实施例11纯酶FsDAAO-PpdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
底物溶液的配制：用50mM的磷酸盐缓冲溶液(pH＝8.0)配制10g/L外消旋1，2，3，4-四氢异喹啉-1-甲酸溶液并用30％氨水调节溶液pH至8.0。
取1ml底物溶液加入到5mL反应管中，再加入FsDAAO纯酶液，黄素腺嘌呤二核苷酸钠盐，过氧化氢酶，PpdpkA纯酶液，NADP +，葡萄糖脱氢酶纯酶液，以及D-葡萄糖，并用磷酸盐缓冲溶液(50mM，pH＝8.0)将反应总体积补到2ml，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-1-甲酸的浓度为5g/L，FsDAAO纯酶的起始浓度为0.74mg/ml，FAD起始浓度为100μM，纯酶PpdpkA的起始浓度为2.4mg/ml，葡萄糖脱氢酶纯酶的起始浓度为0.1mg/ml，NADP +的起始浓度为0.01mM，过氧化氢酶起始浓度为0.01mg/ml，D-葡萄糖的起始浓度为15mM。混匀后，立即取样，作为“0小时”。将反应管置于30℃恒温水浴中，磁力搅拌，反应2小时。反应结束后用高效液相色谱检测反应体系中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。(S)-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为99.2％(计算方式同实施例3)，ee值达99.5％。
实施例12纯酶FsDAAO-PpdpkA多酶耦合制备(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸
底物溶液的配制：用50mM的磷酸盐缓冲溶液(pH＝8.0)配制10g/L外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸溶液并用30％氨水调节溶液pH至8.0。
取1ml底物溶液加入到5mL反应管中，再加入FsDAAO纯酶液，黄素腺嘌呤二核苷酸钠盐，过氧化氢酶，PpdpkA纯酶液，NADP +，葡萄糖脱氢酶纯酶液，以及D-葡萄糖，并用磷酸盐缓冲溶液(50mM，pH＝8.0)将反应总体积补到2ml，使起始反应体系中底物外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的浓度为4g/L，FsDAAO纯酶的起始浓度为0.74mg/ml，FAD起始浓度为100μM，纯酶PpdpkA的起始浓度为2.4mg/ml，葡萄糖脱氢酶纯酶的起始浓度为0.1mg/ml，NADP +的起始浓度为0.01mM，过氧化氢酶起始浓度为0.01mg/ml，D-葡萄糖的起始浓度为8mM。混匀后，立即取样，作为“0小时”。将反应管置于30℃恒温水浴中，磁力搅拌，反应3小时。反应结束后用高效液相色谱检测反应体系中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为99.1％(计算方式同实施例3)，ee值达99.4％。
实施例13 FsDAAO-PpdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶粗酶液、恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。
称取0.2g外消旋1，2，3，4-四氢异喹啉-1-甲酸盐酸盐至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用5M氢氧化钠溶液调节溶液pH值到8.0。加入20ml FsDAAO粗酶液，7.6ml PpdpkA粗酶液，2.4ml葡萄糖脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及D-葡萄糖，使底物外消旋1，2，3，4-四氢异喹啉-1-甲酸的起始浓度为5g/L，NADP +的起始浓度为0.01mM，D-葡萄糖起始浓度为15mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，0.5M氢氧化钠溶液调节反应pH在8～8.5范围内，反应16小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。(S)-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为99.1％(计算方式同实施例3)，ee值为99.4％。
实施例14 FsDAAO-PpdpkA多酶耦合制备(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸
按照实施例2的方法，分别制备源自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶粗酶液、恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。
称取0.2g外消旋6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用5M氢氧化钾溶液pH值到8.0。加入20ml FsDAAO粗酶液，7.6ml PpdpkA粗酶液，2.4ml葡萄糖脱氢酶粗酶液，2mg过氧化氢酶，NADP +以及D-葡萄糖，使底物6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的起始浓度为5 g/L，NADP +的起始浓度为0.01mM，D-葡萄糖起始浓度为15mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，用0.5M氢氧化钾溶液调节反应pH在8～8.5范围内，反应18小时取样。高效液相色谱检测所取样品中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的反应收率为98.6％(计算方式同实施例3)，ee值为99.1％。
实施例15(S)-1，2，3，4-四氢异喹啉-1-甲酸的制备与分离
底物溶液及反应体系如实施例3。
反应结束后，将反应体系的pH值调至5.0-6.0。99℃水浴，待蛋白变性析出后，抽滤。取滤液于65℃条件下旋蒸，将反应体积浓缩10倍。置于冰上，冷却后，抽滤。将析出的白色晶体，小心刮下，置于烘箱中，烘干并称重。称取0.2g白色烘干晶体，用50mM磷酸盐缓冲溶液(pH＝8.0)定容至50ml，取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知分离后产品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量。(S)-1，2，3，4-四氢异喹啉-1-甲酸的产率为80.2％，ee值达99.8％。
实施例16(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的制备与分离
底物溶液及反应体系如实施例7。
反应结束后，将反应体系的pH值调至5.0-6.0。99℃水浴，待蛋白变性析出后，抽滤。取滤液于65℃条件下旋蒸，将反应体积浓缩10倍。置于冰上，冷却后，抽滤。将析出的白色晶体，小心刮下，置于烘箱中，烘干并称重。称取0.25g白色烘干晶体，用50mM磷酸盐缓冲溶液(pH＝8.0)定容至50ml，取样。高效液相色谱检测所取样品中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知分离后产品中6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量。(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸的产率为78.5％，ee值达99.8％。
对比例1 FsDAAO制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
底物溶液的配制：用50mM的磷酸盐缓冲溶液(pH＝8.0)配制10g/L的外消旋1，2，3，4-四氢异喹啉-1-甲酸溶液并用30％氨水调节溶液pH至8.0。
取1ml底物溶液加入到5mL反应管中，再加入1mL FsDAAO粗酶液(粗酶液中已含有足量辅酶FAD，因此，粗酶液反应体系中不需额外添加FAD)。混匀后，取样，作为“0小时”并进行HPLC分析。将反应管置于30℃恒温水浴中，磁力搅拌，反应30小时。反应结束后用HPLC法检测反应体系中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应体系中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。
FsDAAO表现出严格的R-构型立体选择性，产率为49.9％，(S)-1，2，3，4-四氢异喹啉-1-甲酸的ee值达99％以上。
对比例2 FsDAAO-NaCNBH 3制备(S)-1，2，3，4-四氢异喹啉-1-甲酸
底物溶液的配制：用50mM的磷酸盐缓冲溶液(pH＝8.0)配制10g/L外消旋1，2，3，4-四氢异喹啉-1-甲酸溶液并用30％氨水调节溶液pH至8.0。
向100mL反应器中加入20mL底物溶液，20mL FsDAAO粗酶液(粗酶液中已含有足量辅酶FAD，因此，粗酶液反应体系中不需额外添加FAD)，8mg过氧化氢酶冻干粉末和0.3g NaCNBH 3。混匀后，立即取样，作为“0小时”。将反应体系置于30℃恒温水浴中，磁力搅拌，反应30小时，取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-1-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-1-甲酸两种构型的浓度(g/L)。FsDAAO表现出严格的R-构型立体选择性，(S)-1，2，3，4-四氢异喹啉-1-甲酸的收率为78.8％，ee值为99.2％。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (24)

  1. 一种制备如式(I)所示化合物的S型异构体的方法，
    Figure PCTCN2019083813-appb-100001
    式(I)中，R 1，R 2独立地选自氢、C1-C6烷基、C1-C6烷氧基，其特征在于，所述方法包括如下步骤：
    以所述式(I)所示的化合物的外消旋体或式(I)所示的化合物的盐的外消旋体为底物，使该底物中式(I)所示化合物的R型异构体在氧化脱氢酶的催化作用下反应生成式(II)所示的亚胺酸；
    Figure PCTCN2019083813-appb-100002
    使所述式(II)所示的亚胺酸在哌啶酸还原酶与能够供给氢负离子的辅酶的存在下转化为所述式(I)所示化合物的S型异构体。
  2. 如权利要求1所述的方法，其特征在于，式(I)中，R 1，R 2独立地选自氢、甲基、乙基、异丙基、甲氧基或乙氧基，所述的盐为碱金属盐或铵盐。
  3. 如权利要求1所述的方法，其特征在于，所述的式(I)所示化合物的S型异构体为(S)-1，2，3，4-四氢异喹啉-1-甲酸或(S)-6，7-二甲氧基-1，2，3，4-四氢异喹啉-1-甲酸。
  4. 如权利要求1所述的方法，其特征在于，所述氧化脱氢酶是能够选择性催化式(I)所示化合物的R型异构体的酶，且选择性大于等于80％，优选大于等于90％。
  5. 如权利要求1或4所述的方法，其特征在于，所述氧化脱氢酶为D-氨基酸氧化酶。
  6. 如权利要求5所述的方法，其特征在于，所述D-氨基酸氧化酶为选自如下D-氨基酸氧化酶中的一种或多种的组合：来源于三角酵母(Trigonopsis variabilis)CBS 4095的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自禾谷镰刀菌(Fusarium graminearum)CS3005的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自梨孢镰刀菌(Fusarium poae)2516的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶，来自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶。
  7. 如权利要求5所述的方法，其特征在于，所述D-氨基酸氧化酶的使用形式为离体的D-氨基酸氧化酶、含有离体的D-氨基酸氧化酶的粗酶液、D-氨基酸氧化酶的纯酶、D-氨基酸 氧化酶的固定化酶或胞内表达D-氨基酸氧化酶的细胞。
  8. 如权利要求7所述的方法，其特征在于，所述细胞为表达D-氨基酸氧化酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述D-氨基酸氧化酶基因连接在所述表达载体pET-28a(+)上。
  9. 如权利要求1所述的方法，其特征在于，所述哌啶酸还原酶为选自如下哌啶酸还原酶中的一中或多种的组合：来源于恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于绿脓杆菌(Pseudomonas aeruginosa)PAO1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于荧光假单胞菌(Pseudomonas fluorescens)Pf0-1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶。
  10. 如权利要求1所述的方法，其特征在于，所述哌啶酸还原酶的使用形式为离体的哌啶酸还原酶、含有离体的哌啶酸还原酶的粗酶液、哌啶酸还原酶的纯酶、哌啶酸还原酶的固定化酶或胞内表达哌啶酸还原酶的细胞。
  11. 如权利要求10所述的方法，其特征在于，所述细胞为表达哌啶酸还原酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述哌啶酸还原酶基因连接在所述表达载体pET-28a(+)上。
  12. 如权利要求1所述的方法，其特征在于，所述能够供给氢负离子的辅酶为NADH和/或NADPH。
  13. 如权利要求1所述的方法，其特征在于，使所述生成亚胺酸的反应还在黄素腺嘌呤二核苷酸(FAD)的存在下进行。
  14. 如权利要求1所述的方法，其特征在于，使所述生成亚胺酸的反应还在过氧化氢酶的存在下进行。
  15. 如权利要求1所述的方法，其特征在于，使生成所述亚胺酸的反应在设定温度和有氧环境中进行。
  16. 如权利要求15所述的方法，其特征在于，所述设定温度为20～70℃。
  17. 如权利要求1或15所述的方法，其特征在于，所述方法的实施过程包括：首先构建反应体系，然后控制所述反应体系处于设定温度和有氧环境中进行反应，所述反应体系包括所述底物、所述氧化脱氢酶、所述哌啶酸还原酶、辅酶、辅酶再生系统、溶剂，所述反应体系还选择性地包括pH缓冲剂和/或pH调节剂，所述辅酶包括NAD+和/或NADH，或者，所述辅酶包括NADP+和/或NADPH。
  18. 如权利要求17所述的方法，其特征在于，所述辅酶再生系统包括辅酶再生酶和辅酶再生底物。
  19. 如权利要求18所述的方法，其特征在于，所述辅酶再生酶为葡萄糖脱氢酶，所述辅酶再生底物为葡萄糖；或者，所述辅酶再生酶为醇脱氢酶，所述辅酶再生底物为异丙醇。
  20. 如权利要求19所述的方法，其特征在于，所述葡萄糖脱氢酶来源于枯草芽胞杆菌Bacillus subtilis 168；和/或，所述醇脱氢酶来源于乳酸杆菌Lactobscillus kefir DSM20587。
  21. 如权利要求17所述的方法，其特征在于，所述反应体系还包括过氧化氢酶。
  22. 如权利要求21所述的方法，其特征在于，所述过氧化氢酶为牛肝过氧化氢酶冻干粉。
  23. 如权利要求21所述的方法，其特征在于，所述过氧化氢酶与所述氧化脱氢酶的酶活比为100～400∶1。
  24. 如权利要求17所述的方法，其特征在于，所述反应体系还包括黄素腺嘌呤二核苷酸。
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