WO2023184791A1 - 一种酶法合成布瓦西坦手性中间体的方法 - Google Patents

一种酶法合成布瓦西坦手性中间体的方法 Download PDF

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WO2023184791A1
WO2023184791A1 PCT/CN2022/106271 CN2022106271W WO2023184791A1 WO 2023184791 A1 WO2023184791 A1 WO 2023184791A1 CN 2022106271 W CN2022106271 W CN 2022106271W WO 2023184791 A1 WO2023184791 A1 WO 2023184791A1
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enzyme
acid
nitrilase
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郑仁朝
郑裕国
詹侃
吴哲明
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浙江工业大学
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    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
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    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)
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Definitions

  • the present invention relates to a method for enzymatic synthesis of brivaracetam chiral intermediates, in particular to a method for synthesizing brivaracetam chiral intermediates using an enzyme with nitrile hydrolysis activity.
  • Brivaracetam is clinically used to treat generalized epilepsy. It is a high-affinity ligand for synaptic vesicle protein 2A (SV2A) and has certain inhibitory properties on sodium ion channels.
  • FDA U.S. Food and Drug Administration
  • Compound (II) and compound (III) are key chiral intermediates in many synthesis routes of brivaracetam, and they can be synthesized by ring closure of (R)-3-aminomethylhexanoic acid (IV). Therefore, constructing a green, efficient, intrinsically safe and highly atom-economic preparation method for (R)-3-aminomethylhexanoic acid (IV) is of great significance for the industrial production of brivaracetam.
  • Route 1 Use chiral phenylethylamine to resolve compound (VIII) to obtain compound (VII), and then undergo Hofmann degradation under the action of sodium hypochlorite or sodium hypobromite to obtain compound (IV) (CN113045468A). This method has complex chiral resolution steps, low resolution yield, and produces a small amount of isomerized impurities, which is not conducive to industrial production.
  • Biocatalytic hydrolysis of organic nitriles has significant advantages such as strict selectivity, mild reaction conditions, and sustainable development, and has become an important method for the preparation of chiral carboxylic acids.
  • the present invention efficiently synthesizes (R)-3-cyanocaproic acid (V) through regional and stereoselective hydrolysis of 3-cyanocapronitrile by nitrilase, and then hydrogenates and reduces it to synthesize (R)-3-aminomethylcaproic acid. (IV), and further prepare brivaracetam intermediate compound (II) and compound (III).
  • the object of the present invention is to provide a method for enzymatic synthesis of chiral intermediates of Brvaracetam, especially the use of an enzyme with nitrile hydrolysis activity to catalyze the hydrolysis of 3-cyanocapronitrile to synthesize (R)-3-cyanocaproic acid. method, and then synthesize (R)-3-aminomethylcaproic acid through hydrogenation and reduction of (R)-3-cyanocaproic acid to construct a new enzymatic synthesis process of brivaracetam, which is useful for realizing brivaracetam.
  • the industrial green synthesis is of great significance.
  • the present invention provides a method for enzymatic synthesis of chiral intermediates of Brvaracetam, which method utilizes an enzyme with nitrile hydrolysis activity to catalyze the hydrolysis of 3-cyanocapronitrile (VI) to synthesize (R)- 3-cyanocaproic acid, specifically: wet cells obtained by induced expression of recombinant genetically engineered bacteria containing enzyme encoding genes with nitrile hydrolyzing activity are used as catalysts, and 3-cyanocapronitrile (IV) is used as substrate , use pH 7-8 buffer or water as the medium to form a reaction system.
  • the reaction solution is separated and purified to obtain (R)-3- Cyanocaproic acid (V);
  • the enzyme with nitrile hydrolysis activity is a nitrilase, which is a single or double mutation of the 140th or 175th amino acid of the amino acid sequence shown in SEQ ID NO.2 Acquired by mutation.
  • the nitrilase is one of the following mutations of the amino acid sequence shown in SEQ ID NO.2: (1) The tryptophan at position 140 is mutated to glycine (W140, the nucleotide sequence is such as SEQ ID NO.3 As shown, the amino acid sequence is as shown in SEQ ID NO.4); (2) The tryptophan at position 140 is mutated to glycine, and the methionine at position 175 is mutated to threonine (W140G/M175T, the nucleotide sequence is as SEQ ID Shown in NO.5, the amino acid sequence is shown in SEQ ID NO.6).
  • the amount of catalyst used is 1-10g/L (preferably 3g/L) based on the dry weight of the bacteria, and the substrate concentration is 100-300g/L (preferably 150g/L).
  • the catalyst is a crude enzyme extracted from the wet bacterial cells after crushing, a pure enzyme after purification of the crude enzyme, immobilized wet bacterial cells or an immobilized enzyme; that is, the catalyst can be a recombinant genetically engineered bacterium expressing nitrilase.
  • Whole cells can also be used in the form of crude enzyme without purification, or in the form of partially purified or completely purified enzyme.
  • the nitrilase mutants of the present invention can also be made into biocatalysts in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.
  • the 3-cyanocapronitrile is prepared as follows: react a mixture of n-butyraldehyde, piperidine, ethyl cyanoacetate and n-hexane at 30-40°C for 10-15h (preferably 35°C, 12h) , distill the solvent from the obtained reactant under reduced pressure to obtain an oily substance; add isopropyl alcohol and an aqueous solution containing potassium cyanide to the oily substance, maintain the reaction at 30-40°C for 3-8h (preferably 35°C for 5h), and then react at 80 Reflux at -110°C for 3-8 hours (preferably 95°C for 5 hours); cool the reactant to room temperature, dilute with water, and extract with methyl tert-butyl ester.
  • the mass ratio of the piperidine to n-butyraldehyde, ethyl cyanoacetate and potassium cyanide is 1:80 -90: 100-150: 60-100 (preferably 1:85-86:122-123:70); the volume of n-hexane is 1- based on the total amount of n-butyraldehyde, piperidine and ethyl cyanoacetate.
  • volume ratio of the isopropyl alcohol to n-hexane is 5:3; the volume ratio of the aqueous solution containing potassium cyanide to n-hexane is 5:3; the dilution
  • the volume ratio of water to n-hexane is 2:3; the volume ratio of methyl tert-butyl ester to n-hexane is 5:3.
  • the method for preparing (R)-3-aminomethylcaproic acid (IV) from the (R)-3-cyanocaproic acid (V) is: taking (R)-3-cyanocaproic acid in a hydrogenation reaction kettle , add the catalyst Raney nickel, replace the air with nitrogen for 3 times, then add hydrogen to the final pressure of 2MPa, stop stirring after 9 hours of reaction at room temperature, and vent the hydrogen; after the reaction liquid is filtered, the catalyst in the filter cake is recovered, and the filtrate is purified.
  • the present invention provides a nitrilase mutant for synthesizing the chiral intermediate (R)-3-cyanocaproic acid of Brivaracetam, the mutant is shown in SEQ ID NO.2 It is obtained by performing single or double mutation on the 140th or 175th amino acid of the amino acid sequence.
  • the present application also relates to the coding gene of the nitrilase mutant, a recombinant vector containing the coding gene and a recombinant genetically engineered bacterium constructed by the recombinant vector; the recombinant vector uses pET-28a plasmid, and the recombinant genetically engineered bacterium is E. coli BL21(DE3) is the host bacteria.
  • 3-cyanocapronitrile (VI) is prepared through chemical methods; using 3-cyanocapronitrile (VI) as the substrate, it is prepared through biocatalysis (R)-3-Cyanohexanoic acid (V). Using (R)-3-cyanocaproic acid (V) as the substrate, (R)-3-aminomethylcaproic acid (IV) is obtained by catalytic hydrogenation. Finally, compounds (II) and (III) are obtained through esterification or amidation using (R)-3-aminomethylcaproic acid (IV) as the substrate.
  • the present invention provides a method for enzymatic synthesis of brivaracetam chiral intermediates, that is, using nitrilase to catalyze 3-cyanocapronitrile (VI ) method for hydrolyzing and synthesizing (R)-3-cyanocaproic acid.
  • the nitrilase is a mutant of nitrilase from Burkholderia paragramii. Compared with the wild type, the mutant activity increased 10 times, and the E value increased from 39 to more than 300.
  • the nitrilase mutant is used to catalyze the synthesis of (R)-3-cyanocaproic acid from 3-cyanocapronitrile, with a substrate conversion rate of 45% and a product ee of up to 98.5%; and then using (R)-3-cyanocaproic acid (R)-3-aminomethylcaproic acid is synthesized by catalytic hydrogenation of hexanoic acid, and the yield of (R)-3-aminomethylcaproic acid (IV) reaches more than 85%.
  • the invention has a short synthesis route, mild reaction conditions and high atom economy, and can be applied to the industrial synthesis of brivaracetam intermediates.
  • Figure 1 is a 1 H NMR chart of 3-cyanocapronitrile.
  • Figure 2 is a 13 C NMR chart of 3-cyanocapronitrile.
  • Figure 3 is a gas chromatogram showing the hydrolysis of 3-cyanocaproic acid catalyzed by nitrilase to synthesize (R)-3 cyanocaproic acid.
  • the room temperature described in this application is 25-30°C.
  • the nitrilase Pg-Nit gene derived from Paraburkholderia graminis (GenBank NO.WP006050412.1, the nucleotide sequence is shown in SEQ ID NO.1) and the vector pET- After 28a was digested with Nco I and Hind III respectively, T4 ligase was used to ligate to construct the pET-Pg-Nit plasmid; transformed into E.coli BL21 (DE3) competent cells by heat shock, and after recovery, spread on cells containing 50 ⁇ g/ Culture on LB plates with mL kanamycin at 37°C overnight.
  • Site-directed mutation was performed on Trp at position 140 in the amino acid sequence shown in SEQ ID NO.2 of wild-type nitrilase Pg-Nit, and corresponding primers were designed.
  • the primer sequences are shown in Table 1.
  • N A/G/C/T
  • K G/T
  • M A/C
  • the composition of the biocatalytic reaction system (10 mL) is: 50mM KH 2 PO 4 -K 2 HPO 4 buffer (pH 7.2), 5g/L 3-cyanocapronitrile (VI) prepared by the method of Example 1, wild-type wet bacteria 0.1g of wet bacteria or mutant cells.
  • the reaction solution was preheated at 30°C for 10 min, and reacted at 30°C and 600 rpm for 15 min. Take a 500 ⁇ L sample and add 20 ⁇ L 2M HCl to terminate the reaction and extract with an equal amount of ethyl acetate. Take the upper organic phase and dry it with anhydrous sodium sulfate.
  • Enzyme activity definition The amount of enzyme required to generate 1 ⁇ mol of product per minute at 30°C is one unit (U).
  • the gas chromatograph model is 7890N (Agilent), and the capillary column model is BGB-175 (BGB Analytik Switzerland).
  • the chromatographic conditions are: the injection volume is 1.0 ⁇ L, the injection port and detector temperatures are both 250°C, the column temperature is maintained at 120°C for 17 min, and then the temperature is raised to 170°C with a 10°C/min program and maintained for 8 min.
  • the carrier gas was high-purity helium, the flow rate was 1.0mL/min, and the split ratio was 50:1.
  • step 2 Based on the mutant Pg-Nit-W140G with improved activity and stereoselectivity obtained in step 2, extract the plasmid of the wet bacterial cell containing the mutant Pg-Nit-W140G, and use the Pg-Nit-W140G plasmid as a mutation template.
  • M175F/R is the primer.
  • step 2 method Use the step 2 method to perform PCR amplification of the whole plasmid to construct the mutant Pg-Nit-W140G/M175T (the nucleotide sequence is shown in SEQ ID NO.5 and the amino acid sequence is shown in SEQ ID NO. .6 shown).
  • the wild-type E.coli BL21(DE3)-Pg-Nit and the recombinant nitrilase mutant E.coli BL21(DE3)-Pg-Nit-W140G/M175T constructed in Example 2 were inoculated into cards containing 50 ⁇ g/mL respectively.
  • OD 600 0.6
  • add IPTG with a final concentration of 0.1mM induce culture at 28°C for 12 hours, centrifuge at 4°C, 12000rpm for 10 minutes, collect wet cells, and obtain wild-type Pg-Nit wet cells and mutant W140G/M175T wet cells respectively. Bacteria.
  • Example 3 In a 1L reaction system, use water as the reaction medium, and add the recombinant nitrilase mutant W140G/M175T and wild-type Pg-Nit wet cells obtained in Example 3 at an amount of 3g/L (dry weight) respectively. Then add the substrate 3-cyanocapronitrile prepared by the method of Example 1 with a final concentration of 100g/L, and react at 30°C and 600rpm for 24 hours. The reaction solution was sampled using gas chromatography as described in Example 2 to detect the concentrations of the substrate 3-cyanocapronitrile and the product (R)-3-cyanocaproic acid ( Figure 3). After sampling, the reaction solution was centrifuged at 8000 rpm for 10 minutes.
  • the supernatant (900 mL) was added to 300 mL of ethyl acetate to extract the remaining substrate.
  • 300 mL of ethyl acetate was added for extraction.
  • the substrate conversion rate of wild-type Pg-Nit is 35%, and the ee value of the product (R)-3-cyanocaproic acid is 92.0%; the substrate conversion rate of mutant W140G/M175T is 45 %, the ee value of the product (R)-3-cyanocaproic acid is 98.4%.
  • step 1 Take 100mL of the (R)-3-cyanocaproic acid aqueous solution obtained in step 1 (containing 20g of (R)-3-cyanocaproic acid) in a 500mL hydrogenation reaction kettle, add 30g of catalyst Raney nickel, and replace with nitrogen Air was added 3 times, hydrogen was introduced to the final pressure of 2MPa, and after 9 hours of reaction at room temperature, stirring was stopped and the hydrogen was vented. After the reaction liquid is filtered, the catalyst in the filter cake is recovered. The filtrate is sampled using the gas phase described in Example 2 to detect the residual amount of (R)-3-cyanocaproic acid, and the high-performance liquid chromatography is used to detect the (R)-3-aminomethyl. Content of caproic acid (CAS:1314557-10-2).
  • the wet bacterial cells in step 1 in Example 4 were changed to the recombinant nitrilase mutant W140G/M175T, the substrate addition concentration was changed to 150g/L, the reaction time was changed to 13h, and other operations were the same to obtain 200g/L (R )-3-cyanocaproic acid aqueous solution.
  • the results showed that after 13 hours of reaction, the substrate conversion rate of mutant W140G/M175T was 45.0%, and the ee value of the product (R)-3-cyanocaproic acid was 98.5%.
  • Example 4 The wet bacterial cells in Example 4 were changed to the recombinant nitrilase mutant W140G/M175T, and the substrate addition concentration was changed to 300g/L. The other operations were the same to obtain a 200g/L (R)-3-cyanocaproic acid aqueous solution. . The results showed that after 24 hours of reaction, the conversion rate of mutant W140G/M175T was 39.0%, and the ee value of the product (R)-3-cyanocaproic acid was 98.9%.
  • step 1 Take 100mL of the (R)-3-cyanocaproic acid aqueous solution obtained in step 1 (containing 20g of (R)-3-cyanocaproic acid) in a 500mL hydrogenation reaction kettle, add 30g of catalyst Raney nickel, and replace with nitrogen Air was added 3 times, hydrogen was introduced to the final pressure of 2MPa, and after 9 hours of reaction at room temperature, stirring was stopped and the hydrogen was vented.
  • the reaction solution was tested using the method of Example 4.
  • the immobilization method is: take the wet bacterial cells and resuspend the bacterial cells in 100mM baking soda (NaHCO 3 ) aqueous solution to prepare a 100g/L bacterial suspension, and stir for 20 minutes. After there are no obvious lumps, add diatomaceous earth in an amount of 6g/L and continue stirring for 20 minutes. Add a 5% polyethyleneimine aqueous solution in an amount of 30mL/L. After continuing to stir for 1 hour, add 10mL/L of polyethylenimine aqueous solution.

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Abstract

本发明公开了一种酶法合成布瓦西坦手性中间体的方法，即利用具有腈水解活性的酶催化3-氰基己腈水解合成布瓦西坦手性中间体(R)-3-氰基己酸的方法，具有腈水解活性的酶是将SEQ ID NO.2所示氨基酸序列第140位或第175位氨基酸进行单突变或双突变获得的。本发明腈水解酶突变体与野生型相比活力提高了10倍，ee值由39提升至300以上，底物转化率45％，产物ee可达98.5％；然后利用(R)-3-氰基己酸经催化加氢合成(R)-3-氨甲基己酸的收率达到85％以上。本发明合成路线短、反应条件温和、原子经济性高，可应用于工业化合成布瓦西坦中间体。

Description

一种酶法合成布瓦西坦手性中间体的方法 (一)技术领域
本发明涉及一种酶法合成布瓦西坦手性中间体的方法，特别涉及利用具有腈水解活性的酶合成布瓦西坦手性中间体的方法。
(二)背景技术
布瓦西坦，化学名为(2S)-2-[(4R)-2-氧代-4-丙基-1-吡咯烷基]丁酰胺，是比利时优时比制药公司(UCB)研发的新一代抗癫痫药物，其化学结构如式(I)所示。
Figure PCTCN2022106271-appb-000001
布瓦西坦在临床上用于治疗全身性癫痫，是突触囊泡蛋白2A(SV2A)的高亲合体配体，同时对钠离子通道具有一定的抑制性。2016年2月，美国食品药品管理局(FDA)批准布瓦西坦上市，成为治疗癫痫的新一代药物，市场前景广阔。化合物(II)和化合物(III)是众多布瓦西坦合成路线的关键手性中间体，它们可由(R)-3-氨甲基己酸(IV)关环合成。因此，构建一种绿色高效、本质安全和高原子经济性的(R)-3-氨甲基己酸(IV)制备方法对布瓦西坦工业化生产具有重要意义。
Figure PCTCN2022106271-appb-000002
目前，化合物(IV)的合成路线主要有以下两条：
路线一：以手性苯乙胺拆分化合物(VIII)，得到化合物(VII)，再在次氯酸钠或者次溴酸钠作用下，经霍夫曼降解得到化合物(IV)(CN113045468A)。该方法手性拆分步骤复杂，拆分收率较低，同时产生少量的异构化杂质，不利于工业化生产。
Figure PCTCN2022106271-appb-000003
路线二：以化合物(X)为起始原料，分别与二异丁胺和溴代乙酸酯反应合成化合物(IX)，再经转氨酶催化还原胺化制备手性化合物(IV)(WO2016075082)。该方法合成工艺复杂，产物ee值仅为90％左右，难以满足工业化生产需求。
Figure PCTCN2022106271-appb-000004
生物催化有机腈水解具有选择性严格、反应条件温和以及可持续发展等显著优势，已成为制备手性羧酸的重要方法。本发明通过腈水解酶区域、立体选择性水解3-氰基己腈高效合成(R)-3-氰基己酸(V)，再加氢还原合成(R)-3-氨甲基己酸(IV)，进而制备布瓦西坦中间体化合物(II)和化合物(III)。
(三)发明内容
本发明目的是提供一种酶法合成布瓦西坦手性中间体的方法，特别是利用具有腈水解活性的酶催化3-氰基己腈水解合成(R)-3-氰基己酸的方法，再通过(R)-3-氰基己酸加氢还原合成(R)-3-氨甲基己酸，构建一种全新的布瓦西坦酶法合成工艺，对于实现布瓦西坦的工业化绿色合成具有重要意义。
本发明采用的技术方案是：
第一方面，本发明提供一种酶法合成布瓦西坦手性中间体的方法，所述方法为利用具有腈水解活性的酶催化3-氰基己腈(VI)水解合成(R)-3-氰基己酸，具体为：以含具有腈水解活性的酶编码基因的重组基因工程菌经诱导表达后获得的湿菌体为催化剂，以3-氰基己腈(IV)为底物，以pH 7-8的缓冲液或水为介质构成反应体系，在20-40℃、200-600rpm(优选30℃、300rpm)条件下反应完全后，反应液分离纯化获得(R)-3-氰基己酸(V)；所述具有腈水解活性的酶为腈水解酶，所述腈水解酶是将SEQ ID NO.2 所示氨基酸序列第140位或第175位氨基酸进行单突变或双突变获得的。
优选，所述腈水解酶是将SEQ ID NO.2所示氨基酸序列进行下列之一的突变：(1)第140位色氨酸突变为甘氨酸(W140，核苷酸序列如SEQ ID NO.3所示、氨基酸序列如SEQ ID NO.4所示)；(2)第140位色氨酸突变为甘氨酸、同时第175位蛋氨酸突变为苏氨酸(W140G/M175T，核苷酸序列如SEQ ID NO.5所示、氨基酸序列如SEQ ID NO.6所示)。
优选，所述反应体系中，催化剂的用量以菌体干重计为1-10g/L(优选3g/L)，底物浓度为100-300g/L(优选150g/L)。
优选，所述催化剂按如下方法制备：将含具有腈水解活性的酶编码基因的重组基因工程菌接种至含50μg/mL卡那霉素的LB液体培养基中，37℃培养过夜，再以2％(v/v)接种量接种到含50μg/mL卡那霉素LB培养基中，37℃，150rpm培养至菌体浓度OD 600＝0.6，加入终浓度为0.1mM的IPTG，28℃诱导培养12h后，4℃、12000rpm离心10min，收集湿菌体。
优选，催化剂为所述湿菌体经破碎提取的粗酶、粗酶纯化后的纯酶、固定化湿菌体细胞或者固定化酶；即所述催化剂可以是表达腈水解酶的重组基因工程菌全细胞，也可以是未经纯化的粗酶形式使用，也可以是部分纯化的或完全纯化的酶的形式使用。还可以利用本领域已知的固定化技术将本发明的腈水解酶突变体制成固定化酶或固定化细胞形式的生物催化剂。
优选，所述3-氰基己腈按如下方法制备：将正丁醛、哌啶、氰基乙酸乙酯和正己烷的混合物在30-40℃反应10-15h后(优选35℃，12h)，将得到的反应物减压蒸馏除去溶剂得到油状物；在油状物中加入异丙醇和含氰化钾的水溶液，维持30-40℃反应3-8h(优选35℃反应5h)后，在80-110℃温度下回流3-8h(优选95℃反应5h)；将反应物冷却至室温后，用水稀释，并用甲基叔丁酯萃取，将萃取液用水洗涤三次后，用无水硫酸镁干燥，并在真空条件下浓缩至无液体流出，得到油状产物3-氰基己腈(VI)；所述哌啶与正丁醛、氰基乙酸乙酯和氰化钾的质量比为1：80-90：100-150：60-100(优选1:85-86:122-123:70)；所述正己烷体积用量以正丁醛、哌啶和氰基乙酸乙酯总量计为1-5mL/g(优选2-3mL/g)；所述异丙醇与正己烷体积比为5:3；所述含氰化钾的水溶液与正己烷体积比为5:3；所述稀释用的水与正己烷体积比为2:3；所述甲基叔丁酯与正己烷体积比为5:3。
所述(R)-3-氰基己酸(V)制备(R)-3-氨甲基己酸(IV)的方法为：取(R)-3-氰基己酸于 加氢反应釜中，加入催化剂Raney镍，通入氮气置换空气3次，再通入氢气至最终压力2MPa，室温反应9h后停止搅拌，放空氢气；反应液抽滤后，回收滤饼中的催化剂，滤液提纯，获得(R)-3-氨甲基己酸；所述(R)-3-氰基己酸与催化剂质量比为1:1-5(优选1：1.5)。所述滤液提纯是指：取样后的反应液8000rpm离心10min后，取上清按照体积比3:1加入乙酸乙酯萃取剩余底物，水相通过6M HCl调pH至1后，加入乙酸乙酯萃取，有机相加入pH＝12的NaOH水溶液进行反萃取，取水相即获得(R)-3-氨甲基己酸水溶液。
第二方面，本发明提供一种用于合成布瓦西坦手性中间体(R)-3-氰基己酸的腈水解酶突变体，所述突变体是将SEQ ID NO.2所示氨基酸序列第140位或第175位氨基酸进行单突变或双突变获得的。
本申请还涉及所述腈水解酶突变体的编码基因，包含所述编码基因的重组载体以及重组载体构建的重组基因工程菌；所述重组载体采用pET-28a质粒，重组基因工程菌以E.coli BL21(DE3)为宿主菌。
本发明以正丁醛(XI)和氰基乙酸乙酯为原料制备(R)-3-氨甲基己酸(IV)的合成路线及应用反应式如下：
Figure PCTCN2022106271-appb-000005
以正丁醛(XI)和氰基乙酸乙酯为原料，通过化学法制备得到3-氰基己腈(VI)；以3-氰基己腈(VI)为底物，通过生物催化制备得到(R)-3-氰基己酸(V)。以(R)-3-氰基己酸(V)为底物，通过催化加氢得到(R)-3-氨甲基己酸(IV)。最后以(R)-3-氨甲基己酸(IV)为底物，通过酯化或者酰胺化得到化合物(II)和(III)。
与现有技术相比，本发明的有益效果主要体现在：本发明提供了一种酶法合成布瓦西坦手性中间体的方法，即利用腈水解酶催化3-氰基己腈(VI)水解合成(R)-3-氰基己酸的方法。所述腈水解酶为来自格拉米氏副伯克霍尔德菌腈水解酶的突变体。与野生型相比，突变体活力提高了10倍，E值由39提升至300以上。利用所述腈水解酶突 变体催化3-氰基己腈合成(R)-3-氰基己酸，底物转化率45％，产物ee可达98.5％；然后利用(R)-3-氰基己酸经催化加氢合成(R)-3-氨甲基己酸，(R)-3-氨甲基己酸(IV)收率达到85％以上。本发明合成路线短、反应条件温和、原子经济性高，可应用于工业化合成布瓦西坦中间体。
(四)附图说明
图1为3-氰基己腈的 1H NMR图。
图2为3-氰基己腈的 13C NMR图。
图3为腈水解酶催化3-氰基己腈水解合成(R)-3氰基己酸气相色谱图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：本申请所述室温为25-30℃。
实施例1、化合物3-氰基己腈(VI)的合成
将正丁醛(51.4g)，哌啶(0.6g)，氰基乙酸乙酯(73.7g)和正己烷(300mL)的混合物在35℃连续反应12h后，将得到的反应物减压蒸馏除去溶剂得到油状物。在油状物中加入500mL异丙醇和500mL含42.0g氰化钾的水溶液，维持35℃反应5小时后，在95℃温度下回流5小时。将反应物冷却至室温后，用水(200mL)稀释，并用甲基叔丁酯(500mL)萃取，将萃取液用水洗涤三次后，用无水硫酸镁干燥，并在真空条件下浓缩至无液体流出，得到油状产物3-氰基己腈(VI)(CAS:1116-53-6)73g，收率92％。 1H NMR图见图1， 13C NMR图见图2所示。
1H NMR(400MHz，CDCl 3)：(δ，ppm)，3.0-2.9(m，1H)，2.8-2.7(d，2H)，1.9-1.7(m，2H)，1.7-1.4(m，2H)，1.1-0.9(t，3H)。
13C NMR(400MHz，CDCl 3)：(δ，ppm)，120，116，34，28，22，20，14。
实施例2、腈水解酶突变体的构建
1、重组腈水解酶Pg-Nit构建
将源自格拉米氏副伯克霍尔德菌(Paraburkholderia graminis)的腈水解酶Pg-Nit基因(GenBank NO.WP006050412.1，核苷酸序列如SEQ ID NO.1所示)及载体pET-28a分别用Nco I和Hind III酶切后，采用T4连接酶连接，构建pET-Pg-Nit质粒；热击转化到E.coli BL21(DE3)感受态细胞中，复苏后涂布于含50μg/mL卡那霉素的LB平板上培养，37℃过夜。
将平板上的菌落接种至含50μg/mL卡那霉素的LB液体培养基中，37℃培养过夜， 再以2％(v/v)接种量接种到含50μg/mL卡那霉素LB培养基中，37℃，150rpm培养至菌体浓度OD 600＝0.6，加入终浓度为0.1mM的IPTG，28℃诱导培养12h后，4℃、12000rpm离心10min，收集野生型湿菌体E.coli BL21(DE3)-Pg-Nit。
SEQ ID No.1
Figure PCTCN2022106271-appb-000006
SEQ ID No.2
Figure PCTCN2022106271-appb-000007
2、腈水解酶单突变体构建
将野生型腈水解酶Pg-Nit的SEQ ID NO.2所示氨基酸序列中的第140位点Trp 进行定点突变，设计相对应的引物，引物序列见表1。以含SEQ ID NO.1所示野生型腈水解酶基因Pg-Nit的pET-Pg-Nit质粒为模板，利用表1引物进行全质粒PCR扩增，对第140位的色氨酸进行饱和突变，即将140位对应的核苷酸利用NNK代替原有密码子。
表1、140位点定点饱和突变引物设计表
Figure PCTCN2022106271-appb-000008
注：N＝A/G/C/T，K＝G/T，M＝A/C。
PCR扩增及PCR产物转化构建重组腈水解酶大肠杆菌方法参照专利CN202011383565.4。
按照步骤1方法制备突变体湿菌体。生物催化反应体系(10mL)组成为：50mM KH 2PO 4-K 2HPO 4缓冲液(pH 7.2)，5g/L实施例1方法制备的3-氰基己腈(VI)，野生型湿菌体或突变体湿菌体0.1g。反应液于30℃预热10min后，30℃、600rpm反应15min。取样500μL加入20μL 2M HCl终止反应并用等量乙酸乙酯进行萃取，取上层有机相用无水硫酸钠干燥后，加30μL甲醇和15μL重氮甲烷混合后，采用气相色谱测定底物3-氰基己腈和产物(R)-3-氰基己酸(V，CAS:528566-19-6)的含量，计算底物转化率、产物对映体过量值(ee)和酶的对映体选择率(E值)。
酶活定义：在30℃下，每分钟生成1μmol产物所需的酶量为一个单位(U)。
气相色谱型号为7890N(安捷伦)，毛细管柱型号为BGB-175(BGB Analytik Switzerland)。色谱条件为：进样量1.0μL，进样口、检测器温度均为250℃，柱温120℃保持17min，然后以10℃/min程序升温到170℃，保持8min。载气为高纯氦气，流速为1.0mL/min，分流比为50:1。
表2、腈水解酶突变前后的活力和E值变化
Figure PCTCN2022106271-appb-000009
Figure PCTCN2022106271-appb-000010
结果表明，突变体Pg-Nit-W140G(核苷酸序列如SEQ ID NO.3所示、氨基酸序列如SEQ ID NO.4所示)活力达136U/g，为野生型5倍，同时立体选择性也大幅提高，E值由野生型的39提升至300以上。
3、腈水解酶双突变体构建
在步骤2获得活力和立体选择性均提高的突变体Pg-Nit-W140G基础上，提取含突变体Pg-Nit-W140G的湿菌体的质粒，以Pg-Nit-W140G质粒作为突变模板，以表1中M175F/R为引物，采用步骤2方法进行全质粒PCR扩增，构建突变体Pg-Nit-W140G/M175T(核苷酸序列如SEQ ID NO.5所示、氨基酸序列如SEQ ID NO.6所示)。
根据步骤2方法将突变体Pg-Nit-W140G/M175T转入E.coli BL21(DE3)，并制备得到相应的湿菌体，采用步骤2方法检测3-氰基己腈和产物(R)-3-氰基己酸的含量，计算酶活力和E值，结果见表3所示，突变体Pg-Nit-W140G/M175T水解活力达271U/g，为突变体Pg-Nit-W140G的2倍。
表3、腈水解酶活力和立体选择性对比
Figure PCTCN2022106271-appb-000011
实施例3、重组腈水解酶全细胞培养
将实施例2构建的野生型E.coli BL21(DE3)-Pg-Nit、重组腈水解酶突变体E.coli BL21(DE3)-Pg-Nit-W140G/M175T，分别接种至含50μg/mL卡那霉素的LB液体培养基中，37℃培养过夜，再以2％(v/v)接种量接种到含50μg/mL卡那霉素LB培养基中，37℃，150rpm培养至菌体浓度OD 600＝0.6，加入终浓度为0.1mM的IPTG，28℃诱导培养12h后，4℃、12000rpm离心10min，收集湿菌体，分别获得野生型Pg-Nit湿菌体和突变体W140G/M175T湿菌体。
实施例4、腈水解酶催化合成(R)-3-氨甲基己酸(一)
1、3-氰基己腈合成(R)-3-氰基己酸
在1L反应体系中，以水为反应介质，分别按3g/L(干重)的量加入实施例3中获得的重组腈水解酶突变体W140G/M175T及野生型Pg-Nit的湿菌体，再加入终浓度为100g/L的实施例1方法制备的底物3-氰基己腈，于30℃，600rpm反应24h。反应液取样采用实施例2所述气相色谱检测底物3-氰基己腈和产物(R)-3-氰基己酸的浓度(图3)。取样后的反应液8000rpm离心10min后，取上清(900mL)加入300mL乙酸乙酯萃取剩余底物，水相通过6M HCl调pH至1后，加入300mL乙酸乙酯萃取，有机相加入pH＝12的NaOH水溶液100mL进行反萃取，取水相即获得(R)-3-氰基己酸水溶液，加水调整至浓度为200g/L。
结果表明，反应24h后，野生型Pg-Nit的底物转化率为35％，产物(R)-3-氰基己酸ee值为92.0％；突变体W140G/M175T的底物转化率为45％，产物(R)-3-氰基己酸ee值为98.4％。
MS(m/z):C 7H 11NO 2(M+H +)理论计算值141.1，实测值141.1。
2、(R)-3-氰基己酸加氢催化合成(R)-3-氨甲基己酸
取步骤1得到的(R)-3-氰基己酸水溶液100mL(含(R)-3-氰基己酸20g)于500mL的加氢反应釜中，加入30g催化剂Raney镍，通入氮气置换空气3次，通入氢气至最终压力2MPa，室温反应9h后停止搅拌，放空氢气。反应液抽滤后，回收滤饼中的催化剂，滤液取样采用实施例2所述气相检测(R)-3-氰基己酸残留量，采用高效液相色谱检测(R)-3-氨甲基己酸(CAS:1314557-10-2)的含量。
液相检测条件为：色谱柱
Figure PCTCN2022106271-appb-000012
检测波长210nm；流动相为pH 6.3磷酸缓冲液(终浓度3.4g/L):甲醇＝850:150(v/v)。
结果表明，体系中(R)-3-氰基己酸无残留，转化率100％，(R)-3-氨甲基己酸的收率达到85.2％。
MS(m/z):C 7H 11NO 2(M+H +)理论计算值145.2，实测值145.2。
实施例5、腈水解酶催化合成(R)-3-氨甲基己酸(二)
1、3-氰基己腈合成(R)-3-氰基己酸
将实施例4中步骤1中湿菌体改为重组腈水解酶突变体W140G/M175T，底物加入浓度改为150g/L，反应时间改为13h，其他操作相同，得到200g/L的(R)-3-氰基 己酸水溶液。结果表明，反应13h，突变体W140G/M175T的底物转化率为45.0％，产物(R)-3-氰基己酸ee值为98.5％。MS(m/z):C 7H 11NO 2(M+H +)理论计算值141.1，实测值141.1。
2、(R)-3-氰基己酸加氢催化合成(R)-3-氨甲基己酸
取步骤1得到的100mL(R)-3-氰基己酸水溶液(含(R)-3-氰基己酸20g)于500mL的加氢反应釜中，加入30g催化剂Raney镍，通入氮气置换空气3次，通入氢气至最终压力2MPa，室温反应9h后停止搅拌，放空氢气。反应液采用实施例4方法检测。
结果表明，体系中(R)-3-氰基己酸无残留，转化率100％，(R)-3-氨甲基己酸的收率达到85.0％。
实施例6、腈水解酶催化合成(R)-3-氨甲基己酸(三)
1、3-氰基己腈合成(R)-3-氰基己酸
将实施例4中湿菌体改为重组腈水解酶突变体W140G/M175T，底物加入浓度改为300g/L，其他操作相同，得到200g/L的(R)-3-氰基己酸水溶液。结果表明，反应24h，突变体W140G/M175T的转化率为39.0％，产物(R)-3-氰基己酸ee值为98.9％。
GC-MS(m/z):C 7H 11NO 2(M+H +)理论计算值141.1，实测值141.1。
2、(R)-3-氰基己酸加氢催化合成(R)-3-氨甲基己酸
取步骤1得到的(R)-3-氰基己酸水溶液100mL(含(R)-3-氰基己酸20g)于500mL的加氢反应釜中，加入30g催化剂Raney镍，通入氮气置换空气3次，通入氢气至最终压力2MPa，室温反应9h后停止搅拌，放空氢气。反应液采用实施例4方法检测。
结果表明，体系中(R)-3-氰基己酸无残留，转化率100％，(R)-3-氨甲基己酸的收率达到86.3％。
实施例7、腈水解酶催化合成(R)-3-氨甲基己酸(四)
1、3-氰基己腈合成(R)-3-氰基己酸
将实施例5中步骤1中湿菌体改为固定化细胞，固定化方法为：取湿菌体先用100mM小苏打(NaHCO 3)水溶液重悬菌体配制100g/L菌悬液，搅拌20min无明显块状物后，以6g/L的量加入硅藻土继续搅拌20min，并以30mL/L的量加入质量浓度5％聚乙烯亚胺水溶液，继续搅拌1h后，以10mL/L的量加入体积浓度25％戊二醛水溶液，继续搅拌1h，随后进行抽滤，弃去液体，将得到的固定化细胞进行水洗， 最终得到腈水解酶固定化细胞。固定化细胞添加量为15g/L，其他操作相同，得到200g/L的(R)-3-氰基己酸水溶液。结果表明，反应13h，底物转化率为44.5％，产物(R)-3-氰基己酸ee值为98.7％；。MS(m/z):C 7H 11NO 2(M+H +)理论计算值141.1，实测值141.1。
2、(R)-3-氰基己酸加氢催化合成(R)-3-氨甲基己酸
取步骤1得到的100mL(R)-3-氰基己酸水溶液(含(R)-3-氰基己酸20g)于500mL的加氢反应釜中，加入30g催化剂Raney镍，通入氮气置换空气3次，通入氢气至最终压力2MPa，室温反应9h后停止搅拌，放空氢气。反应液采用实施例4方法检测。
结果表明，体系中(R)-3-氰基己酸无残留，转化率100％，(R)-3-氨甲基己酸的收率达到85.5％。

Claims (10)

  1. 一种酶法合成布瓦西坦手性中间体的方法，其特征在于，所述方法为利用具有腈水解活性的酶催化3-氰基己腈水解合成布瓦西坦手性中间体(R)-3-氰基己酸的方法。
  2. 如权利要求1所述的方法，其特征在于，所述的方法为：以含具有腈水解活性的酶编码基因的重组基因工程菌经诱导表达后获得的湿菌体为催化剂，以3-氰基己腈为底物，以pH7-8的缓冲液或水为介质构成反应体系，在20-40℃、200-600rpm条件下反应完全后，反应液分离纯化获得(R)-3-氰基己酸。
  3. 如权利要求1或2所述的方法，其特征在于所述的具有腈水解活性的酶为腈水解酶。
  4. 如权利要求3所述的方法，所述腈水解酶是将SEQ ID NO.2所示氨基酸序列第140位或第175位氨基酸进行单突变或双突变获得的。
  5. 如权利要求4所述的方法，其特征在于，所述腈水解酶的氨基酸序列为SEQ ID NO.4或SEQ ID NO.6所示。
  6. 如权利要求2所述的方法，其特征在于，所述反应体系中，催化剂的用量以菌体干重计为1-10g/L，底物浓度为100-300g/L。
  7. 如权利要求2所述的方法，其特征在于，所述催化剂按如下方法制备：将含具有腈水解活性的酶编码基因的重组基因工程菌接种至含50μg/mL卡那霉素的LB液体培养基中，37℃培养过夜，再以体积浓度2％接种量接种到含50μg/mL卡那霉素LB培养基中，37℃，150rpm培养至菌体浓度OD 600＝0.6，加入终浓度为0.1mM的IPTG，28℃诱导培养12h后，4℃、12000rpm离心10min，收集湿菌体。
  8. 如权利要求2所述的方法，所述催化剂为所述湿菌体经破碎提取的粗酶、粗酶纯化后的纯酶、固定化湿菌体细胞或者固定化酶。
  9. 一种用于权利要求1合成布瓦西坦手性中间体的腈水解酶突变体，其特征在于，所述突变体是将SEQ ID NO.2所示氨基酸序列第140位或第175位氨基酸进行单突变或双突变获得的。
  10. 一种含权利要求9所述腈水解酶突变体的编码基因的重组基因工程菌。
PCT/CN2022/106271 2022-04-02 2022-07-18 一种酶法合成布瓦西坦手性中间体的方法 WO2023184791A1 (zh)

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