WO2019140687A1 - 一种酶法合成氯霉素中间体的方法 - Google Patents

一种酶法合成氯霉素中间体的方法 Download PDF

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WO2019140687A1
WO2019140687A1 PCT/CN2018/073631 CN2018073631W WO2019140687A1 WO 2019140687 A1 WO2019140687 A1 WO 2019140687A1 CN 2018073631 W CN2018073631 W CN 2018073631W WO 2019140687 A1 WO2019140687 A1 WO 2019140687A1
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chloramphenicol
aldolase
nitrobenzaldehyde
glycine
reaction
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PCT/CN2018/073631
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傅荣昭
刘立辉
张贵慰
江名
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邦泰生物工程(深圳)有限公司
深圳市邦泰绿色生物合成研究院
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Priority to PCT/CN2018/073631 priority Critical patent/WO2019140687A1/zh
Priority to CN201880000133.5A priority patent/CN108323173B/zh
Publication of WO2019140687A1 publication Critical patent/WO2019140687A1/zh

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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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  • the invention belongs to the technical field of biopharmaceuticals and biochemicals, and relates to a preparation method of a chloramphenicol intermediate.
  • Chloramphenicol is a broad-spectrum antibiotic isolated from Streptomyces venezuela. Very resistant to most Gram-negative and positive bacteria. Its antibacterial mechanism is to bind to the nucleoprotein 50S subunit and inhibit peptide acyltransferase, thereby inhibiting protein synthesis.
  • chloramphenicol has the advantages of small side effects and high efficacy. At present, the total domestic output has reached more than 3,000 tons, of which about 2,000 tons are exported, and domestic consumption is about 1,000 tons. In recent years, the use of the world is constantly expanding. Therefore, the development of a green and efficient chloramphenicol industrialization method has attracted the attention of researchers.
  • Chloramphenicol synthesis generally uses chemical synthesis pathways, and there are few reports on the synthesis of key intermediates by enzymatic methods. Among them, CN106566851A invents a method for preparing a chloramphenicol compound by using a ketoreductase. Its structure is significantly different from our structure.
  • the synthetic route is as follows:
  • a method for enzymatic synthesis of chloramphenicol intermediates characterized in that the method uses nitrobenzaldehyde and glycine as substrates, and the chloramphenicol intermediate is obtained by the reaction of aldolase, and the structural formula is:
  • the aldolase is a threonine aldolase.
  • amino acid sequence of the threonine aldolase is selected from one of SEQ ID NOs: 1-4.
  • the concentrations of nitrobenzaldehyde and glycine are 0.04 to 0.1 M and 0.5 to 1 M, respectively.
  • the above reaction system further contains a solvent.
  • the solvent is a buffer solution of a co-solvent, including but not limited to at least one of dichloromethane, ethyl acetate, ethanol, dimethyl sulfoxide, and dimethylformamide.
  • the buffer solution includes, but is not limited to, at least one of a phosphate buffer and an acetate buffer.
  • the cosolvent is preferably dimethyl sulfoxide; when the aldolase is the aldolase of SEQ ID NO: 4, the cosolvent is preferably dimethylformamide.
  • the volume concentration of the cosolvent in the solvent is from 20 to 50%.
  • the buffer has a pH of 5 to 6 and a concentration of 0.08 to 0.12 M.
  • the above reaction system further contains pyridoxal phosphate.
  • the pyridoxal phosphate has a concentration in the reaction system of 0.1 to 0.7 mM.
  • the temperature of the above reaction is 0 to 50 ° C, more preferably the reaction temperature is 5 to 40 ° C; further preferably, the reaction temperature is 5 to 10 ° C.
  • the above reaction time is 2 to 38 h, and more preferably the reaction time is 2 to 6 h.
  • the pH of the above reaction system is from 1 to 10, and more preferably, the pH is from 6 to 7.
  • the obtained product is isolated and purified by HPLC to obtain a chloramphenicol intermediate.
  • the method for producing a chloramphenicol intermediate of the present invention is obtained by reacting a substrate with nitrobenzaldehyde and glycine using an aldolase starting from an inexpensive and readily available starting material.
  • the reaction reduces the cost of the raw materials, and solves the defects of the prior art, such as complicated operation, large pollution, and low yield.
  • the method of the invention has high conversion rate and high yield of target product, so that the process of chloramphenicol intermediate is simple, and the aldolase used is easily obtained by Escherichia coli fermentation, and the production cost and product quality are superior to chemical methods, and is suitable for industrial production. .
  • Figure 1 is a HPLC detection diagram of the product chloramphenicol intermediate (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl)propionic acid;
  • Figure 2 is the HPLC detection result of the 2-amino-3-hydroxy-3-(4-nitrophenyl)propionic acid cis-conversion standard
  • Figure 3 is a graph showing the NMR of chloramphenicol intermediates.
  • the threonine aldolase (the amino acid sequence thereof is shown in SEQ ID NO: 1) is encoded by the LTA gene, and the gene sequence is optimized and the artificial gene fragment is inserted into the expression plasmid pET-22b to obtain the recombinant expression plasmid pET-22b. -LTA01.
  • the pET-22b-LTA01 plasmid was transformed into E. coli Rosetta (DE3) to express threonine aldolase.
  • a single clone containing the recombinant plasmid was picked and inoculated into 50 mL of LB liquid medium (50 ⁇ g/mL ampicillin), and cultured at 37 ° C, 200 rpm, overnight.
  • the trans form represents (2S,3S)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid.
  • Table 2 is a 2-amino-3-hydroxy-3-(4-nitrophenyl)propionic acid cis-conversion standard
  • the trans form represents (2S,3S)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid.
  • chloramphenicol intermediate obtained by the present invention is further characterized by nuclear magnetic resonance (NMR), and the obtained characterization chart is shown in FIG. 3, which shows that the chloramphenicol intermediate obtained by the present invention is (2S, 3R)-2-amino- 3-hydroxy-3-(4-nitrophenyl)propionic acid.
  • NMR nuclear magnetic resonance
  • the conversion rate of the present example was tested, the conversion rate of the substrate p-nitrobenzaldehyde was 91.79%, and the target product (2S, 3R)-2-amino-3-hydroxy-3-(4-nitrophenyl)propionic acid was obtained. The rate is 45.9%.
  • the threonine aldolase (the amino acid sequence thereof is shown in SEQ ID NO: 2) is encoded by the LTA gene, and the gene sequence is optimized and the artificial gene fragment is inserted into the expression plasmid pET-22b to obtain the recombinant expression plasmid pET-22b. -LTA02.
  • the pET-22b-LTA02 plasmid was transformed into E. coli Rosetta (DE3) to express threonine aldolase.
  • a single clone containing the recombinant plasmid was picked and inoculated into 50 mL of LB liquid medium (50 ⁇ g/mL ampicillin), and cultured at 37 ° C, 200 rpm, overnight.
  • the threonine aldolase (the amino acid sequence thereof is shown in SEQ ID NO: 3) is encoded by the LTA gene, and the gene sequence is optimized and the artificial gene fragment is inserted into the expression plasmid pET-22b to obtain the recombinant expression plasmid pET-22b. -LTA03.
  • the pET-22b-LTA03 plasmid was transformed into E. coli Rosetta (DE3) to express threonine aldolase.
  • a single clone containing the recombinant plasmid was picked and inoculated into 50 mL of LB liquid medium (50 ⁇ g/mL ampicillin), and cultured at 37 ° C, 200 rpm, overnight.
  • the threonine aldolase (the amino acid sequence thereof is shown in SEQ ID NO: 4) is encoded by the LTA gene, and the gene sequence is optimized and the artificial gene fragment is inserted into the expression plasmid pET-22b to obtain the recombinant expression plasmid pET-22b. -LTA04.
  • the pET-22b-LTA04 plasmid was transformed into E. coli Rosetta (DE3) to express threonine aldolase.
  • a single clone containing the recombinant plasmid was picked and inoculated into 50 mL of LB liquid medium (50 ⁇ g/mL ampicillin), and cultured at 37 ° C, 200 rpm, overnight.

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Abstract

本发明公开了一种酶法合成氯霉素中间体的方法，该方法采用硝基苯甲醛和甘氨酸为底物，在醛缩酶催化作用下，反应获得氯霉素中间体。本发明制备氯霉素中间体的方法，从廉价且易获得的原料开始，利用醛缩酶对底物硝基苯甲醛和甘氨酸进行反应获得。该反应降低原料成本，解决现有技术操作复杂、污染大、产率低等缺点。本发明方法转化率高，目标产物得率高，使得氯霉素中间体的工艺简单，所用醛缩酶经大肠杆菌发酵易得，生产成本和产品质量优于化学法，适合工业化生产。

Description

一种酶法合成氯霉素中间体的方法 技术领域
本发明属于生物制药和生物化工的技术领域，涉及一种氯霉素中间体的制备方法。
背景技术
氯霉素是由委内瑞拉链霉菌(Streptomyces venezuela)中分离提取的广谱抗生素。对大多数革兰氏阴性和阳性菌都具有很强抗性。其抗菌机制是与核蛋白体50S亚基结合，抑制肽酰基转移酶，从而抑制蛋白质合成。氯霉素作为一种临床应用广泛的抗生素药物，具副作用小、药效高等优点。目前，国内总产量达到3000吨以上，其中出口2000吨左右，国内消耗1000吨左右。近年来，全世界的用量正在不断地扩大。因此，开发一种绿色高效的氯霉素工业化方法受到广大研究者的关注。
氯霉素合成一般采用化学合成途径，几乎没有酶法合成关键中间体的报道。其中，CN106566851A发明一种利用酮还原酶制备一种氯霉素类化合物的方法。其结构与我们的结构有明显差异。
其合成路线如下：
Figure PCTCN2018073631-appb-000001
化学合成存在的缺点：制备工艺复杂，反应步骤长尤其是拆分的步骤非常繁琐，反应条件苛刻，整体收率偏低，消耗大量有机试剂，产生大量有害成分，对环境造成严重污染。CN 106566851 A给出的方法存在的缺点是原料价格较贵，获取困难。
发明内容
本发明的目的在于提供一种酶法合成氯霉素中间体的方法。
本发明所采取的技术方案是：
一种酶法合成氯霉素中间体的方法，其特征在于，该方法采用硝基苯甲醛和甘氨酸为底物，在醛缩酶催化作用下，反应获得氯霉素中间体，其结构式为：
Figure PCTCN2018073631-appb-000002
优选的，所述醛缩酶为苏氨酸醛缩酶。
更优选的，所述苏氨酸醛缩酶的氨基酸序列选自SEQ ID NO：1～4中的一种。
优选的，上述反应体系中，硝基苯甲醛和甘氨酸的浓度分别为0.04～0.1M、0.5～1M。
优选的，上述反应体系中还含有溶剂。
优选的，所述溶剂为助溶剂的缓冲溶液，所述助溶剂包括但不限于二氯甲烷、乙酸乙酯、乙醇、二甲基亚砜、二甲基甲酰胺中的至少一种，所述缓冲溶液包括但不限于磷酸缓冲液、乙酸缓冲液中的至少一种。
当醛缩酶为SEQ ID NO：2所示的醛缩酶时，助溶剂优选为二甲基亚砜；当醛缩酶为SEQ ID NO：4所示的醛缩酶时，助溶剂优选为二甲基甲酰胺。
优选的，所述溶剂中助溶剂的体积浓度为20～50％。
优选的，所述缓冲液的pH值为5～6，浓度为0.08～0.12M。
优选的，上述反应体系中还含有磷酸吡哆醛。
优选的，所述磷酸吡哆醛在反应体系中的浓度为0.1～0.7mM。
优选的，上述反应的温度为0～50℃，更优选反应温度为5～40℃；进一步优选的，反应温度为5～10℃。
优选的，上述反应时间为2～38h，更优选的反应时间为2～6h。
优选的，上述反应体系的pH值为1～10，更优选的，pH值为6～7。
优选的，上述反应结束后，所得产物经HPLC分离纯化得氯霉素中间体。
本发明的有益效果是：
(1)本发明制备氯霉素中间体的方法，从廉价且易获得的原料开始，利用醛缩酶对底物硝基苯甲醛和甘氨酸进行反应获得。该反应降低原料成本，解决现有技术操作复杂、污染大、产率低等缺点。
(2)本发明方法转化率高，目标产物得率高，使得氯霉素中间体的工艺简单，所用醛缩酶经大肠杆菌发酵易得，生产成本和产品质量优于化学法，适合工业化生产。
附图说明
图1产物氯霉素中间体(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸的HPLC检测图；
图2为2-氨基-3-羟基-3-(4-硝基苯)丙酸顺反构型标准品的HPLC检测结果；
图3为氯霉素中间体核磁共振的表征图。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例1一种氯霉素中间体的制备方法
(1)苏氨酸醛缩酶的制备
苏氨酸醛缩酶(其氨基酸序列如SEQ ID NO：1所示)由LTA基因编码，将基因序列优化后进行人工合成的基因片段，插入到表达质粒pET-22b得到重组表达质粒pET-22b-LTA01。pET-22b-LTA01质粒转化大肠杆菌Rosetta(DE3)用来表达苏氨酸醛缩酶。挑取含有重组质粒的单克隆接种到50mL LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm，过夜培养。吸取20mL培养液转接到1L LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm振荡培养。当OD 600为0.6时，加入IPTG终浓度为0.5mM，30℃诱导16-20h后，停止培养，5000rpm离心10min，收获菌体。菌体用适量PBS稀释后，超声波破碎，即得苏氨酸醛缩酶的粗酶液。
(2)氯霉素中间体的制备方法
取1.8g对硝基苯甲醛和9g甘氨酸，投入500ml反应瓶中，瓶中事先加入含40％v/v乙醇-乙酸乙酯助溶剂的乙酸缓冲液(pH5.5 0.1M)200mL。搅拌桨转速300rpm，使底物充分混匀。然后加入PLP(磷酸吡哆醛)使体系终浓度达0.6mM，20ml粗酶液，用10M NaOH的调节pH到6.0左右，30℃反应26h。反应结束后对产物进行HPLC分离纯化(如图1和表1)，获得氯霉素中间体，其为顺式构型，具体命名为(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸，其结构式为：
Figure PCTCN2018073631-appb-000003
将本实施例产物氯霉素中间体与2-氨基-3-羟基-3-(4-硝基苯)丙酸顺反构型标准品的HPLC检测结果(图2和表2)进行对比，进一步证实了本发明产物氯霉素中间体确实为顺式构型，即(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸。
表1产物氯霉素中间体(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸的HPLC检测结果
Figure PCTCN2018073631-appb-000004
注：N.A表示未知，
顺式表示(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸；
反式表示(2S，3S)-2-氨基-3-羟基-3-(4-硝基苯)丙酸。
表2为2-氨基-3-羟基-3-(4-硝基苯)丙酸顺反构型标准品的
Figure PCTCN2018073631-appb-000005
注：顺式表示(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸；
反式表示(2S，3S)-2-氨基-3-羟基-3-(4-硝基苯)丙酸。
另外，进一步对本发明所得产物氯霉素中间体进行核磁共振(NMR)表征，所得表征图如图3所示，说明本发明所得产物氯霉素中间体为(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸。
对本实施例的转化率进行检测，底物对硝基苯甲醛转化率达91.79％，目标产物(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸得率为45.9％。
实施例2一种氯霉素中间体的制备方法
(1)苏氨酸醛缩酶的制备
苏氨酸醛缩酶(其氨基酸序列如SEQ ID NO：2所示)由LTA基因编码，将基因序列优化后进行人工合成的基因片段，插入到表达质粒pET-22b得到重组表达质粒pET-22b-LTA02。pET-22b-LTA02质粒转化大肠杆菌Rosetta(DE3)用来表达苏氨酸醛缩酶。挑取含有重组质 粒的单克隆接种到50mL LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm，过夜培养。吸取20mL培养液转接到1L LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm振荡培养。当OD 600为0.6时，加入IPTG终浓度为0.5mM，30℃诱导16-20h后，停止培养，5000rpm离心10min，收获菌体。菌体用适量PBS稀释后，超声波破碎，即得苏氨酸醛缩酶的粗酶液。
(2)氯霉素中间体的制备方法
取3.02g对硝基苯甲醛和15g甘氨酸，投入500ml反应瓶中，瓶中事先加入含30％(V/V)二甲基亚砜溶剂的磷酸缓冲液(pH5.5 0.1M)200mL。搅拌桨转速300rpm，使底物充分混匀。然后加入PLP(磷酸吡哆醛)使体系终浓度达0.5mM，20ml粗酶液，用10M NaOH的调节pH到7.0，5℃反应4h，反应体系中，对硝基苯甲醛和甘氨酸的浓度分别为0.1M、1M。对硝基苯甲醛转化率达99％，反应结束后对产物进行HPLC分离纯化，获得氯霉素中间体，其为顺式构型，具体命名为(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸，得率48％。
实施例3一种氯霉素中间体的制备方法
(1)苏氨酸醛缩酶的制备
苏氨酸醛缩酶(其氨基酸序列如SEQ ID NO：3所示)由LTA基因编码，将基因序列优化后进行人工合成的基因片段，插入到表达质粒pET-22b得到重组表达质粒pET-22b-LTA03。pET-22b-LTA03质粒转化大肠杆菌Rosetta(DE3)用来表达苏氨酸醛缩酶。挑取含有重组质粒的单克隆接种到50mL LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm，过夜培养。吸取20mL培养液转接到1L LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm振荡培养。当OD 600为0.6时，加入IPTG终浓度为0.5mM，30℃诱导16-20h后，停止培养，5000rpm离心10min，收获菌体。菌体用适量PBS稀释后，超声波破碎，即得苏氨酸醛缩酶的粗酶液。
(2)氯霉素中间体的制备方法
取3.02g对硝基苯甲醛和15g甘氨酸，投入500ml反应瓶中，瓶中事先加入含20％(V/V)乙醇的磷酸缓冲液(pH5.5 0.1M)200mL。搅拌桨转速300rpm，使底物充分混匀。然后加入PLP(磷酸吡哆醛)使体系终浓度达0.6mM，20ml粗酶液，用5M NaOH的调节pH到7.0，20℃反应2h，反应体系中，对硝基苯甲醛和甘氨酸的浓度分别为0.1M、1M。对硝基苯甲醛转化率达92.1％，反应结束后对产物进行HPLC分离纯化，获得氯霉素中间体，其为顺式构型，具体命名为(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸，得率44.2％。
实施例4一种氯霉素中间体的制备方法
(1)苏氨酸醛缩酶的制备
苏氨酸醛缩酶(其氨基酸序列如SEQ ID NO：4所示)由LTA基因编码，将基因序列优化后进行人工合成的基因片段，插入到表达质粒pET-22b得到重组表达质粒pET-22b-LTA04。pET-22b-LTA04质粒转化大肠杆菌Rosetta(DE3)用来表达苏氨酸醛缩酶。挑取含有重组质粒的单克隆接种到50mL LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm，过夜培养。吸取20mL培养液转接到1L LB液体培养基(50微克/mL氨苄青霉素)，37℃，200rpm振荡培养。当OD 600为0.6时，加入IPTG终浓度为0.5mM，30℃诱导16-20h后，停止培养，5000rpm离心10min，收获菌体。菌体用适量PBS稀释后，超声波破碎，即得苏氨酸醛缩酶的粗酶液。
(2)氯霉素中间体的制备方法
取3.02g对硝基苯甲醛和15g甘氨酸，投入500ml反应瓶中，瓶中事先加入含20％(V/V)二甲基甲酰胺的磷酸缓冲液(pH5.5 0.1M)200mL。搅拌桨转速300rpm，使底物充分混匀。然后加入PLP(磷酸吡哆醛)使体系终浓度达0.7mM，20ml粗酶液，用5M NaOH的调节pH到6.0，10℃反应6h，反应体系中，对硝基苯甲醛和甘氨酸的浓度分别为0.1M、1M。对硝基苯甲醛转化率达94.1％，反应结束后对产物进行HPLC分离纯化，获得氯霉素中间体，其为顺式构型，具体命名为(2S，3R)-2-氨基-3-羟基-3-(4-硝基苯)丙酸，得率46.2％。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种酶法合成氯霉素中间体的方法，其特征在于，该方法采用硝基苯甲醛和甘氨酸为底物，在醛缩酶催化作用下，反应获得氯霉素中间体，其结构式为：
    Figure PCTCN2018073631-appb-100001
  2. 根据权利要求1所述的方法，其特征在于，所述醛缩酶为苏氨酸醛缩酶。
  3. 根据权利要求2所述的方法，其特征在于，所述苏氨酸醛缩酶的氨基酸序列选自SEQ ID NO：1～4中的一种。
  4. 根据权利要求1所述的方法，其特征在于，反应体系中，硝基苯甲醛和甘氨酸的浓度分别为0.04～0.1M、0.5～1M。
  5. 根据权利要求1所述的方法，其特征在于，反应体系中还含有溶剂。
  6. 根据权利要求5所述的方法，其特征在于，所述溶剂为助溶剂的缓冲溶液，所述助溶剂包括但不限于二氯甲烷、乙酸乙酯、乙醇、二甲基亚砜、二甲基甲酰胺中的至少一种，所述缓冲溶液包括但不限于磷酸缓冲液、乙酸缓冲液中的至少一种。
  7. 根据权利要求1所述的方法，其特征在于，反应体系中还含有磷酸吡哆醛。
  8. 根据权利要求7所述的方法，其特征在于，所述磷酸吡哆醛在反应体系中的浓度为0.1～0.7mM。
  9. 根据权利要求1所述的方法，其特征在于，反应温度为0～50℃。
  10. 根据权利要求1所述的方法，其特征在于，反应体系的pH值为1～10。
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