WO2020107781A1 - 一种酶法拆分制备(s)-1,2,3,4-四氢异喹啉-3-甲酸的方法 - Google Patents
一种酶法拆分制备(s)-1,2,3,4-四氢异喹啉-3-甲酸的方法 Download PDFInfo
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- WO2020107781A1 WO2020107781A1 PCT/CN2019/083877 CN2019083877W WO2020107781A1 WO 2020107781 A1 WO2020107781 A1 WO 2020107781A1 CN 2019083877 W CN2019083877 W CN 2019083877W WO 2020107781 A1 WO2020107781 A1 WO 2020107781A1
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- BWKMGYQJPOAASG-VIFPVBQESA-N OC([C@H]1NCc2ccccc2C1)=O Chemical compound OC([C@H]1NCc2ccccc2C1)=O BWKMGYQJPOAASG-VIFPVBQESA-N 0.000 description 1
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- 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
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- 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
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- the invention relates to a new method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by enzymatic resolution.
- (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 product obtained by this method has high stereoselectivity and relatively simple process (Study on synthesis of optically pure (S)-1,2,3,4-tetrahydroquinoline-3-carboxylic acid by chemical enzymatic method[J]. Modern Engineering, 2003 , 23(12): 23-5.).
- 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 has the characteristics of mild reaction conditions, strong stereoselectivity, high reaction efficiency, relatively simple process, etc., and has industrial application prospects.
- the method includes:
- the racemic form of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or the racemization of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid salt The body is used as a substrate, using isolated D-amino acid oxidase or cells expressing D-amino acid oxidase as a catalyst to selectively catalyze (R)-1,2,3,4-tetrahydroisoquinoline-3 -Formic acid undergoes oxidative dehydrogenation, while (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid remains unreacted and remains in the reaction system;
- 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 D-amino acid oxidase is preferably 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 mutation thereof Or D-amino acid oxidase with amino acid sequence homology greater than 80%, D-amino acid oxidase from Fusarium Graminearum CS3005 or its mutant or homology with amino acid sequence greater than 80% ( (Preferably greater than 85%, more preferably greater than 90%, further preferably greater than 95%) other D-amino acid oxidase, D-amino acid oxidase from Fusarium poae 2516 or mutants thereof or the same amino acid sequence Other D-amino acid oxidases with a source of greater than 80% (preferably greater than 85%, more preferably greater than 90%, further preferably greater than 95%), D-amino acid oxidase from
- 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 cell is an engineered bacterium expressing D-amino acid oxidase.
- the engineered bacteria are, for example, engineered bacteria whose host cell is E. coli BL21 (DE3).
- the engineered bacterium contains the expression vector pET-28a(+), and the D-amino acid oxidase gene is connected to the expression vector pET-28a(+).
- a reaction system is first constructed, and then the reaction system is controlled to perform the oxidative dehydrogenation reaction in a set temperature and an aerobic environment, wherein the reaction system includes the substrate, pH buffer solution and /Or pH adjuster and the catalyst.
- the concentration of the starting substrate in the reaction system is 1-20 g/L, and the pH of the reaction system is 6-9;
- the catalyst is D containing the ex vivo -A crude enzyme solution of amino acid oxidase or a pure enzyme or an immobilized enzyme or a cell expressing D-amino acid oxidase intracellularly;
- the set temperature is 20 to 70°C.
- the catalyst uses a crude enzyme solution containing ex vivo D-amino acid oxidase and flavin adenine dinucleotide.
- the added amount of the catalyst is the weight of wet cells after centrifugation at 8000 rpm for 10 minutes, and the added amount of the cells is generally 1 to 5% of the weight of the reaction system.
- the set temperature is 30-50°C, and the pH of the reaction system is 7-8.
- the reaction system contains coenzyme flavin adenine dinucleotide (FAD).
- FAD coenzyme 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, 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 pH buffer solution is a phosphate buffer solution.
- the pH adjusting agent is 20% to 35% by weight of ammonia.
- step (2) the pH value of the reaction system is adjusted to 5.0-6.0, the protein is denatured and precipitated by heating, suction filtration, the filtrate is concentrated, cooled and crystallized, and dried to obtain (S)-1,2 3,4-tetrahydroisoquinoline-3-carboxylic acid.
- the present invention has the following beneficial effects compared with the prior art:
- the present invention unexpectedly found that D-amino acid oxidase can efficiently catalyze the oxidative dehydrogenation of (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, while for (S)-1,2 , 3,4-tetrahydroisoquinoline-3-carboxylic acid has basically no catalytic effect.
- the method of the present invention is used to prepare (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid with mild reaction conditions, high reaction efficiency and yield, and strong stereoselectivity (ee value>99%) ,Simple process.
- Figure 1 is a high-performance liquid detection spectrum 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; Time 10.969min is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
- FIG. 2 is a high-performance liquid detection pattern of the reaction system sampled in the reaction system in Example 3 for 24 hours.
- the invention provides a new method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, racemic 1,2,3,4-tetrahydroisoquinoline-3- Formic acid (or ammonium salt) is used as a substrate, using the isolated D-amino acid oxidase or cells expressing D-amino acid oxidase in the cell as a catalyst to perform oxidative dehydrogenation reaction to obtain (S)-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid.
- racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (as shown in formula II) as a substrate, using D-amino acid oxidase stereoselective catalysis (R)-1 , 2,3,4-tetrahydroisoquinoline-3-carboxylic acid undergoes oxidative dehydrogenation to form the corresponding imidic acid (as shown in formula III), (S)-1,2,3,4-tetrahydroiso Quinoline-3-carboxylic acid (shown in Formula I) is not catalyzed but remains in the reaction system.
- the reaction process is as follows:
- the reaction is carried out in the presence of coenzyme flavin adenine dinucleotide (FAD).
- FAD coenzyme flavin adenine dinucleotide
- the coenzyme flavin adenine dinucleotide (FAD) is reduced to FADH 2 .
- Oxygen is reduced to hydrogen peroxide (H 2 O 2 ), and FADH 2 is oxidized to FAD.
- the reaction process is as follows:
- the introduction of D-amino acid oxidase can be achieved by adding an enzyme in vitro or a cell expressing D-amino acid oxidase in a cell.
- the enzyme in vitro can be in the form of crude enzyme solution, pure enzyme, or immobilized enzyme, without limitation.
- the cell that expresses D-amino acid oxidase intracellularly may specifically be a resting cell of an engineered bacteria expressing a recombinant enzyme.
- the D-amino acid oxidase is derived from Triangle yeast, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, etc.
- the D-amino acid oxidase may be derived from Triangle yeast (Trigonopsis variabilis) CBS 4095, Fusarium graminearum CS3005, Fusarium poae 2516 or Fusarium solani M-0718 D-amino acid oxidase.
- the cell that expresses D-amino acid oxidase intracellularly is an engineered bacterium that expresses D-amino acid oxidase.
- the host cell of the engineered bacterium is E. coli BL21(DE3).
- the engineered bacterium contains the expression vector pET-28a(+), and the D-amino acid oxidase gene is connected to the expression vector pET-28a(+).
- an engineered bacterium expressing D-amino acid oxidase is used to obtain a recombinant D-amino acid oxidase for use as a catalyst.
- the concentration of the substrate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the catalytic system is 1-20 g/L.
- the addition amount of the catalyst is based on the wet weight of the cells after centrifugation at 8000 rpm for 10 min, and the addition amount of the cells is 1 to 5% by weight of the reaction solution.
- 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 reaction temperature is 30 to 50°C , The time is 12 to 48 hours.
- a phosphate buffer solution is used to control the pH of the reaction to 7-8.
- 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: chromatographic 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
- Example 1 Screening of D-amino acid oxidase and construction of genetically engineered bacteria expressing D-amino acid oxidase
- microbial-derived D-amino acid oxidases can be divided into two categories: 1) Prefer amino acids with smaller side chain groups (such as D-alanine), such as Fusarium oxysporum) derived DAAO; 2) preference for amino acids with larger side chain groups (such as D-phenylalanine), such as DAAO (POLLEGIONI L, MOLLA G, SACCHI S, et al. from Trigonopsis variabilis).
- D-alanine such as Fusarium oxysporum
- DAAO Prefer amino acids with smaller side chain groups (such as D-alanine), such as Fusarium oxysporum) derived DAAO
- preference for amino acids with larger side chain groups such as D-phenylalanine
- DAAO POLLEGIONI L, MOLLA G, SACCHI S, et al. 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, the final concentration of 25% glycerol was added to the obtained engineering bacterial solution and placed at -80°C for preservation.
- 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 constructed according to the method of Example 1 were inoculated in 5 mL liquid LB (containing 50 ⁇ g/ml kanamycin) medium, and cultured with shaking at 200 rpm for about 8 hours at 37°C. Press 1% (V / V) was inoculated in 50mL of an LB liquid (containing kanamycin 50 ⁇ g / ml card) in the culture medium, OD 600 reached 0.6-0.8, addition of inducer IPTG (0.1 mM final concentration) , 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 then placed in a -80°C ultra-low temperature refrigerator Save and use.
- the bacterial weight was suspended in 50 mM phosphate buffer (pH8.0), the bacterial suspension was sonicated, and the supernatant obtained after centrifugation was the crude enzyme solution containing D-amino acid oxidase.
- the reaction system (1ml): 10g / L E 1, E 2, E 3, E 4 wet cells (sonication), an outer 2g / L substrate racemic 1,2,3,4-tetrahydroisoquinoline -3 -Formic acid, the reaction medium is pH 8.0 phosphate buffer.
- the prepared reaction system was placed in a 30°C metal bath shaking reactor for 120 min.
- the reaction system in which phosphate buffer (pH 8.0) replaced the crude enzyme solution was used as a control.
- the sample was diluted 10-fold by mobile phase and then qualitatively analyzed by HPLC.
- the substrate solution was prepared as in Example 3.
- the substrate solution was prepared as in Example 3.
- the substrate solution was prepared as in Example 3.
- the substrate solution was prepared as in Example 3.
- the reaction tube was placed in a constant temperature water bath at 30°C, magnetically stirred, and reacted for 24 hours. After the reaction, the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction system was detected by HPLC, and the conversion rate was 50% (the calculation formula is shown in Example 3).
- (S) 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid has an ee value of 99% or more.
- substrate solution 4g/L of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid solution was prepared with deionized water and the pH value of the solution was adjusted to 8.0 with 30% ammonia water.
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Abstract
本发明公开了一种酶法拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸的新方法,所述方法为:以外消旋1,2,3,4-四氢异喹啉-3-甲酸或其盐为底物,利用离体的D-氨基酸氧化酶或胞内表达D-氨基酸氧化酶的细胞作为催化剂,选择性催化(R)-1,2,3,4-四氢异喹啉-3-甲酸进行氧化脱氢反应,(S)-1,2,3,4-四氢异喹啉-3-甲酸未被催化,保留在反应体系中,由此制备获得(S)-1,2,3,4-四氢异喹啉-3-甲酸。所述(S)-1,2,3,4-四氢异喹啉-3-甲酸是降压药喹那普利的关键中间体。本发明反应收率>49%,ee值达99%,具有反应条件温和、立体选择性强、反应效率高、工艺相对简单等特点。
Description
本发明涉及一种酶法拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸的新方法。
(S)-1,2,3,4-四氢异喹啉-3-甲酸(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)是一种重要的药物中间体,被广泛应用于多种有机小分子药物以及肽基药物的合成。例如,(S)-1,2,3,4-四氢异喹啉-3-甲酸是降压药喹那普利的重要组成部分(Diversity-oriented synthesis of medicinally important 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic)derivatives and higher analogs[J].Org Biomol Chem,2014,12(45):9054-91.)。此外,(S)-1,2,3,4-四氢异喹啉-3-甲酸可用于合成含有四氢异喹啉母核的小分子拮抗剂,作用于趋化因子受体CXCR4,从而有望用于治疗HIV等疾病(Discovery of tetrahydroisoquinoline-based CXCR4 antagonists[J].ACS Med Chem Lett,2013,4(11):1025-30.)。
现有技术中,制备光学纯(S)-1,2,3,4-四氢异喹啉-3-甲酸的方法有化学手性合成和生物催化动力学拆分两种。研究人员最初利用Pictet-Spengler反应制备光学纯(S)-1,2,3,4-四氢异喹啉-3-甲酸,以L-苯丙氨酸为原料,在浓酸与高温条件下,与甲醛缩合生成目标产物,该法工艺相对简单,但生成的产物会发生部分消旋化。随后,Bischler-Nepieralski反应,[2+2+2]环加成方法等也被用于制备(S)-1,2,3,4-四氢异喹啉-3-甲酸及其衍生物,该类方法路线较复杂,成本高(Diversity-oriented synthesis of medicinally important 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic)derivatives and higher analogs[J].Org Biomol Chem,2014,12(45):9054-91.)。近年来,Kurata等通过臭氧分解、氧化及去保护作用三步不对成合成(S)-1,2,3,4-四氢异喹啉-3-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,步骤较多,不易于工业化应用。龚等利用化学酶法制备(S)-1,2,3,4-四氢异喹啉-3-甲酸即以外消旋苯丙氨酸为原料,经Pictet-Spengler反应合成外消旋1,2,3,4-四氢异喹啉-3-甲酸,然后经酯化作用、脂肪酶动力学拆分制备(S)-构型产物。23.8g外消旋酯盐酸盐(0.1mol),脂肪酶和底物质量比为0.2,反应48h,产物ee>99%,收率为49.1%。该法所得产物立体选择性高,工艺相对简单(化学酶法合成光学纯(S)-1,2,3,4-四氢喹啉-3-羧酸的研究[J].现代化工,2003,23(12):23-5.)。
目前存在的工艺中,尚没有直接利用酶法动力学拆分外消旋1,2,3,4-四氢异喹啉-3-甲酸制备(S)-构型产物的方法。
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-3-甲酸的方法。该方法具有反应条件温和、立体选择性强、反应效率高、工艺相对简单等特点,具有工业化应用前景。
为实现上述目的,本发明采取的技术方案如下:
一种酶法拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸(I)的方法,
所述方法包括:
(1)以1,2,3,4-四氢异喹啉-3-甲酸的外消旋体或1,2,3,4-四氢异喹啉-3-甲酸盐的外消旋体为底物,利用离体的D-氨基酸氧化酶或胞内表达D-氨基酸氧化酶的细胞作为催化剂,选择性催化(R)-1,2,3,4-四氢异喹啉-3-甲酸进行氧化脱氢反应,而(S)-1,2,3,4-四氢异喹啉-3-甲酸未反应,保留在反应体系中;
(2)将所述(S)-1,2,3,4-四氢异喹啉-3-甲酸与反应体系分离。
进一步地,所述1,2,3,4-四氢异喹啉-3-甲酸盐可以为1,2,3,4-四氢异喹啉-3-甲酸的碱金属盐或铵盐等,具体例如1,2,3,4-四氢异喹啉-3-甲酸钠、1,2,3,4-四氢异喹啉-3-甲酸钾、1,2,3,4-四氢异喹啉-3-甲酸铵。
根据本发明,所述D-氨基酸氧化酶优选为选自如下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%(优选大于85%,更优选大于90%,进一步优选大于95%)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%(优选大于85%,更优选大于90%,进一步优选大于95%)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%(优选大于85%,更优选大于90%,进一步优选大于95%)的其它D-氨基酸氧化酶。
进一步优选地,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。
作为本发明的一种优选实施方案:所述细胞为表达D-氨基酸氧化酶的工程菌。所述工程菌例如是宿主细胞为E.coli BL21(DE3)的工程菌。
具体地,所述工程菌含有表达载体pET-28a(+),所述D-氨基酸氧化酶基因连接在表达载体pET-28a(+)上。
进一步地,步骤(1)中,首先构建反应体系,然后控制反应体系处于设定温度和有氧环境中进行所述氧化脱氢反应,其中所述反应体系包含所述底物、pH缓冲溶液和/或pH调节剂以及所述催化剂。
优选地,步骤(1)中,所述反应体系中,起始底物的浓度为1~20g/L,反应体系的pH值为6~9;所述的催化剂为含有所述离体的D-氨基酸氧化酶的粗酶液或者纯酶或者固定化酶或胞内表达D-氨基酸氧化酶的细胞;所述设定温度为20~70℃。
优选地,所述催化剂采用含有离体的D-氨基酸氧化酶和黄素腺嘌呤二核苷酸的粗酶液。
根据本发明的一个方面,所述催化剂的添加量以8000rpm离心10min后的湿细胞重计,所述细胞的添加量一般为反应体系重量的1~5%。
优选地,步骤(1)中,所述设定温度为30~50℃,所述反应体系的pH值为7~8。
优选地,步骤(1)中,所述反应体系中含有辅酶黄素腺嘌呤二核苷酸(FAD)。使氧化脱氢反应在FAD存在下进行,有助于进一步提高反应收率。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。
根据本发明的一个具体且优选方面,所述pH缓冲溶液为磷酸盐缓冲溶液。
根据本发明的又一个具体且优选方面,所述的pH调节剂为20wt%~35wt%氨水。
进一步地,步骤(2)中,将反应体系的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(S)-1,2,3,4-四氢异喹啉-3-甲酸。
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:
本发明意外发现D-氨基酸氧化酶可高效选择性催化(R)-1,2,3,4-四氢异喹啉-3-甲酸进行氧化脱氢反应,而对于(S)-1,2,3,4-四氢异喹啉-3-甲酸基本无催化作用。采用本发明方法来制备(S)-1,2,3,4-四氢异喹啉-3-甲酸,反应条件温和,反应效率及收率高,立体选择性强(ee值>99%),工艺简单。
图1为实施例3中反应体系中0小时取样的高效液相检测图谱,其中,保留时间8.673min为(R)-1,2,3,4-四氢异喹啉-3-甲酸;保留时间10.969min为(S)-1,2,3,4-四氢异喹啉-3-甲酸;
图2为实施例3中反应体系中反应24小时取样的高效液相检测图谱。
本发明提供一种制备(S)-1,2,3,4-四氢异喹啉-3-甲酸的新方法,以外消旋1,2,3,4-四氢异喹啉-3-甲酸(或氨盐)为底物,利用离体的D-氨基酸氧化酶或胞内表达D-氨基酸氧化酶的细胞作为催化剂,进行氧化脱氢反应,获得(S)-1,2,3,4-四氢异喹啉-3-甲酸。
具体原理为:以外消旋1,2,3,4-四氢异喹啉-3-甲酸(如式II所示)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-3-甲酸发生氧化脱氢反应生成相应的亚胺酸(如式III所示),(S)-1,2,3,4-四氢异喹啉-3-甲酸(如式I所示)未被催化而保留在反应体系中。反应过程示意如下:
进一步地,优选使反应在辅酶黄素腺嘌呤二核苷酸(FAD)存在下进行,在催化过程中, 辅酶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,反应过程示意如下:
根据本发明,D-氨基酸氧化酶的引入可以通过加入离体的酶或胞内表达D-氨基酸氧化酶的细胞来实现。离体的酶可以是粗酶液形式、纯酶,或者固定化酶,没有限制。胞内表达D-氨基酸氧化酶的细胞,具体可以是表达重组酶的工程菌静息细胞。
根据本发明的一个优选方面,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、茄病镰刀菌等,具体地,D-氨基酸氧化酶可以是来源于三角酵母(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的D-氨基酸氧化酶。
根据本发明的又一优选方面,胞内表达D-氨基酸氧化酶的细胞为表达D-氨基酸氧化酶的工程菌。在一个优选实施方式中,该工程菌的宿主细胞为E.coli BL21(DE3)。具体地,所述工程菌含有表达载体pET-28a(+),所述D-氨基酸氧化酶基因连接在表达载体pET-28a(+)上。
根据本发明的一个具体方面,利用表达D-氨基酸氧化酶的工程菌获得重组D-氨基酸氧化酶,用作催化剂。
根据本发明的一个优选方面,催化体系中底物外消旋1,2,3,4-四氢异喹啉-3-甲酸的浓度为1~20g/L。
根据本发明的一个优选方面,催化体系中,催化剂的添加量以8000rpm离心10min后的细胞湿重计,所述细胞的添加量为反应液重量的1~5%。
根据本发明的一个优选方面,催化体系中,反应的温度为20~70℃,时间为6~72小时,反应液的pH值为6~9;更优选地,反应的温度为30~50℃,时间为12~48小时。
优选地,采用磷酸缓冲溶液控制反应的pH值为7~8。
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。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-3-甲酸的HPLC分析方法为:色谱柱
ZWIX(-);柱温/25℃;流速 /0.5mL/min;检测波长/UV210nm;流动相:HPLC级甲醇/乙腈(50/50,v/v)(加入50mM甲酸和25mM二已胺)。具体各相关物质出峰情况见图1。
实施例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DAAO;2)偏好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DAAO(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.)。分别将这两种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
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终浓度为25%的甘油并置于-80℃保藏备用。
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。
将按照实施例1方法构建的含有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8h左右。按1%(V/V)的接种量接种于50mL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IPTG(终浓度为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,之后,放于-80℃超低温冰箱中保存,待用。
2.2粗酶液的制备
将菌体重悬于50mM磷酸缓冲液(pH 8.0)中,超声破碎菌悬液,离心后得到的上清为含D-氨基酸氧化酶的粗酶液。
2.3 HPLC法检测反应体系中各对映体含量
反应体系(1ml):10g/L E
1、E
2、E
3、E
4湿菌体(超声波破碎),2g/L底物外消旋1,2,3,4-四氢异喹啉-3-甲酸,反应介质为pH 8.0磷酸盐缓冲液。将配好的反应体系置于30℃金属浴振荡反应器内反应120min。磷酸盐缓冲液(pH 8.0)代替粗酶液的反应体系作为对照。样品经流动相稀释10倍后用HPLC进行定性分析。
结果表明:与对照相比,编号为E
1、E
2、E
3以及E
4的D-氨基酸氧化酶能够立体选择性催化(R)-1,2,3,4-四氢异喹啉-3-甲酸反应,而(S)-1,2,3,4-四氢异喹啉-3-甲酸含量基本保持不变。
实施例3 FsDAAO拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸
底物溶液的配制:用50mM的磷酸盐缓冲溶液(pH=8.0)配置5g/L的外消旋1,2,3,4-四氢异喹啉-3-甲酸溶液并用30%氨水调节溶液pH值至8.0。
在5mL反应管中加入1.6ml底物溶液,0.4ml 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,取出50μL,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24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-3-甲酸两种构型的含量。
检测结果如图2所示,FsDAAO表现出严格的R-构型立体选择性,转化率为49.9%(转化率=[(初始外消旋底物的浓度(g/L)-残余的底物浓度(g/L))/初始外消旋底物的浓度(g/L)]×100%),(S)-1,2,3,4-四氢异喹啉-3-甲酸的ee值达99%以上。
实施例4 FgDAAO拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸
按实施例3的方法配制底物溶液。
在5mL反应管中加入0.8ml底物溶液,1.2ml FgDAAO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,取出50μL,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-3-甲酸两种构型的含量。检测结果为:转化率49.8%(计算公式如实施例3所示),(S)-1,2,3,4-四氢异喹啉-3-甲酸的ee值达99%以上。
实施例5 FpDAAO拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸
按实施例3的方法配制底物溶液。
在5mL反应管中加入0.4ml底物溶液,1.6ml FpDAAO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,取出50μL,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24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-3-甲酸两种构型的含量。检测结果为:转化率为49.9%(计算公式如实施例3所示),(S)-1,2,3,4-四氢异喹啉-3-甲酸的ee值达99%以上。
实施例6 TvDAAO拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸
按实施例3的方法配制底物溶液。
在5mL反应管中加入0.8ml底物溶液,1.2ml TvDAAO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,取出50μL,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25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-3-甲酸两种构型的含量。检测结果为:转化率为49.9%(计算公式如实施例3所示),(S)-1,2,3,4-四氢异喹啉-3-甲酸的ee值达99%以上。
实施例7 纯酶FsDAAO拆分制备(S)-1,2,3,4-四氢异喹啉-3-甲酸
按实施例3的方法配制底物溶液。
取1.6ml底物溶液加入到5mL反应管中,再加入FsDAAO纯酶液(经镍柱亲和层析纯化获得)以及黄素腺嘌呤二核苷酸钠盐(FAD),并用磷酸盐缓冲溶液(50mM,pH=8.0)将反应总体积补到2ml,(S)-1,2,3,4-四氢异喹啉-3-甲酸的终浓度为4g/L,FsDAAO纯酶的终浓度为0.1mg/ml,FAD终浓度为100μM。混匀后,取出50μL,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24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-3-甲酸两种构型的含量,转化率为50%(计算公式如实施例3所示),(S)-1,2,3,4-四氢异喹啉-3-甲酸的ee值达99%以上。
实施例8 FsDAAO制备(S)-1,2,3,4-四氢异喹啉-3-甲酸
底物溶液的配制:用50mM的磷酸盐缓冲溶液(pH=8.0)配置5g/L的外消旋1,2,3,4-四氢异喹啉-3-甲酸溶液并用5M氢氧化钠溶液调节底物溶液pH值至8.0。
在5mL反应管中加入1.6ml底物溶液,0.4ml 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,取出50μL,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24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-3-甲酸两种构型的含量。检测结果为:转化率为49.9%(计算公式如实施例3所示),(S)-1,2,3,4-四氢异喹啉-3-甲酸的ee值达99%以上。
实施例9 FsDAAO大反应体系制备(S)-1,2,3,4-四氢异喹啉-3-甲酸
底物溶液的配制:用去离子水配置4g/L的外消旋1,2,3,4-四氢异喹啉-3-甲酸溶液并用30%氨水调节溶液pH值至8.0。
向反应器中加入200mL底物溶液,20mL FsDAAO粗酶液。混匀后,置于30℃恒温水浴中,磁力搅拌,反应30小时。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0.01g白色烘干晶体,用去离子水定容至10ml。高效液相色谱检测所取样品中1,2,3,4-四氢异喹啉-3-甲酸两种构型的含量。转化率为49.9%(计算公式如实施例3所示),(S)-1,2,3,4-四氢异喹啉-3-甲酸的ee值达99%以上,分离产率为85%(分离产率=实际分离得到的产物的量(mg)/理论的产物的量(mg)×100%)。
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 (10)
- 如权利要求1所述的方法,其特征在于,所述1,2,3,4-四氢异喹啉-3-甲酸盐为1,2,3,4-四氢异喹啉-3-甲酸的碱金属盐或铵盐。
- 如权利要求1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。
- 如权利要求1或3所述的方法,其特征在于,所述细胞为表达D-氨基酸氧化酶的工程菌,所述工程菌的宿主细胞为E.coli BL21(DE3)。
- 如权利要求4所述的方法,其特征在于,所述工程菌含有表达载体pET-28a(+),所述D-氨基酸氧化酶基因连接在表达载体pET-28a(+)上。
- 如权利要求1所述的方法,其特征在于,步骤(1)中,首先构建反应体系,然后控制反应体系处于设定温度和有氧环境中进行所述氧化脱氢反应,其中所述反应体系包含所述底物、pH缓冲溶液和/或pH调节剂以及所述催化剂。
- 如权利要求6所述的方法,其特征在于,步骤(1)中,所述反应体系中,起始底物的浓度为1~20g/L,反应体系的pH值为6~9;所述的催化剂为含有所述离体的D-氨基酸氧化酶的粗酶液或者纯酶或者固定化酶或胞内表达D-氨基酸氧化酶的细胞;所述设定温度为20~70℃。
- 如权利要求6所述的方法,其特征在于,步骤(1)中,所述设定温度为30~50℃,所述反应体系的pH值为7~8。
- 如权利要求7所述的方法,其特征在于,所述催化剂采用含有离体的D-氨基酸氧化 酶和黄素腺嘌呤二核苷酸的粗酶液。
- 如权利要求1或6所述的方法,其特征在于,步骤(1)中,使所述氧化脱氢反应在辅酶黄素腺嘌呤二核苷酸存在下进行。
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