WO2020156176A1 - 苯氧乙酸衍生物及利用其酶法制备青霉素v盐的方法 - Google Patents

苯氧乙酸衍生物及利用其酶法制备青霉素v盐的方法 Download PDF

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WO2020156176A1
WO2020156176A1 PCT/CN2020/072246 CN2020072246W WO2020156176A1 WO 2020156176 A1 WO2020156176 A1 WO 2020156176A1 CN 2020072246 W CN2020072246 W CN 2020072246W WO 2020156176 A1 WO2020156176 A1 WO 2020156176A1
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penicillin
phenoxyacetic acid
salt
derivative
acid derivative
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PCT/CN2020/072246
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English (en)
French (fr)
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许岗
张娇
曾红宇
黄斌
宋子博
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湖南福来格生物技术有限公司
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Priority to CN202080011415.2A priority Critical patent/CN113365973B/zh
Publication of WO2020156176A1 publication Critical patent/WO2020156176A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C12P37/04—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by acylation of the substituent in the 6 position

Definitions

  • This application belongs to the technical field of antibiotic medicines, and relates to a phenoxyacetic acid derivative and a method for preparing penicillin V salt by its enzymatic method.
  • Penicillin V salts such as penicillin V potassium and penicillin V sodium, are commonly used clinical antibiotics, and penicillin V potassium is more widely used.
  • Penicillin V potassium belongs to ⁇ -lactamase antibiotics. Its antibacterial spectrum is similar to penicillin G. It can destroy bacterial cell walls and has a bactericidal effect. It is the drug of choice for the treatment of Gram-positive and part-negative bacteria infections. It has been widely used in clinical treatment. Abscesses, purulent meningitis, pneumonia, gonorrhea and other diseases. Compared with penicillin G potassium/sodium, penicillin V potassium is more stable to acid, not easily destroyed by gastric acid, and better oral absorption. Its oral preparation is especially suitable for children, and it is destroyed by ⁇ -lactamase more slowly. It is more effective against infections caused by resistant Staphylococcus aureus.
  • Penicillin V potassium tablets were first used in clinical practice in 1952, and were approved by the US FDA in September 1957, and are included in the US Pharmacopoeia, British Pharmacopoeia, and Japanese Pharmacopoeia. At present, the United States, Canada, the United Kingdom, Germany, Japan and other countries have produced penicillin V potassium tablets, which is an oral antibiotic that is widely used clinically in the world.
  • Penicillin has been used so far to have a higher probability of allergic reactions.
  • Common allergic reactions include skin rash, urticaria, dermatitis, fever, angioedema, asthma, anaphylactic shock, etc.
  • anaphylactic shock is the most serious and can even lead to death.
  • penicillin injections need to be tested for skin sensitivity before use. Drugs with a negative skin test can be used by patients, and those with a positive skin test are forbidden to be used abroad; Oral penicillin drugs are exempt from skin testing.
  • Penicillin V potassium raw materials and preparations may contain its synthetic precursors, process by-products and various degradation products; in addition, penicillin V potassium may also polymerize by itself to produce high molecular polymers.
  • the largest source of process by-products, degradation products and polymer impurities is the production process. The longer the production process, the greater the probability of impurity generation. Allergic reactions caused by ⁇ -lactam antibiotics have been proved to be related to the high molecular polymers present in drugs after years of research.
  • the existing method for producing penicillin V potassium has a long fermentation cycle and a long production process, so various process by-products, degradation products and polymer impurities are high in content.
  • Penicillin V salt has not been prepared by biological enzymatic method, and has not been prepared by reversely catalyzing the reaction of 6-aminopenicillin acid (6-APA) and phenoxyacetic acid derivatives with penicillin acylase.
  • the primary purpose of this application is to provide phenoxyacetic acid derivatives so as to be able to use their better enzymatic method to prepare penicillin V salt.
  • this application provides phenoxyacetic acid derivatives, the derivatives are phenoxyacetic acid glycerides (including monophenoxyacetate, diphenoxyacetate, triphenyl Glyceryl oxyacetate).
  • the application provides a phenoxyacetic acid derivative, wherein the derivative is glycerol monophenoxyacetate.
  • the application provides a phenoxyacetic acid derivative, wherein the derivative has the structure shown in the following formula (I):
  • the second purpose of the application is to provide a method for preparing penicillin V salt by enzymatic method using the aforementioned phenoxyacetic acid derivative, so as to better prepare penicillin V salt by enzymatic method.
  • this application provides a method for preparing penicillin V salt by using the aforementioned phenoxyacetic acid derivative enzymatically, and the method includes the following steps:
  • Step (1) In an aqueous solution, under the catalysis of penicillin acylase, react 6-APA with the derivative;
  • Step (2) After the reaction is completed, the penicillin V obtained by the reaction is acidified, and the penicillin V salt is isolated after salting.
  • the present application provides a method for preparing penicillin V salt using the aforementioned phenoxyacetic acid derivative enzymatically, wherein in step (1), the penicillin acylase is immobilized penicillin Acylase.
  • this application provides a method for preparing penicillin V salt by enzymatic method using the aforementioned phenoxyacetic acid derivative, wherein in step (1), the penicillin acylase is a natural penicillin acylate
  • the mutant of the enzyme has the amino acid sequence shown in SEQ ID NO.1.
  • the present application provides a method for preparing penicillin V salt using the aforementioned phenoxyacetic acid derivative enzymatically, wherein in step (1),
  • the molar ratio of the 6-APA and the derivative is 1:1.1-1.5, and the weight ratio of the 6-APA and the penicillin acylase is 1:2-4,
  • the reaction temperature is 5-40°C
  • the reaction pH is 4.0-8.0
  • the reaction time is 1-2h.
  • the present application provides a method for preparing penicillin V salt using the aforementioned phenoxyacetic acid derivative enzymatically, wherein in step (1), the reaction temperature is 10-30° C., and the reaction pH is 5.0-6.0, the reaction time is 1-2h.
  • the application provides a method for preparing penicillin V salt by enzymatic method using the aforementioned phenoxyacetic acid derivative, wherein in step (2), the acidification is to adjust the pH of the solution to 2.5 with an acid. -3.5, grow crystals for 10-30 minutes.
  • this application provides a method for preparing penicillin V salt by using the aforementioned phenoxyacetic acid derivative enzymatically, wherein in step (2), the salinization is first adjusted with carbonate The pH of the solution is 5.5-7.5, and then a water-carrying agent (the water-carrying agent is one or more of ethanol, isopropanol, n-butanol, and isobutanol) is added to the solution, and concentrated by evaporation to penicillin V salt Crystals precipitated.
  • a water-carrying agent is one or more of ethanol, isopropanol, n-butanol, and isobutanol
  • this application provides a method for preparing penicillin V salt by using the aforementioned phenoxyacetic acid derivative enzymatically, wherein in step (2), before the acidification, the The penicillin acylase described is isolated from the solution.
  • penicillin V salt of the present application is short, and the whole process of penicillin V salt synthesis, purification and crystallization does not exceed 24 hours.
  • the synthesis process of the penicillin V salt of the present application is green and pollution-free, and does not need to use butyl acetate for extraction and purification.
  • the penicillin V salt synthesis of the present application uses 6-APA and phenoxyacetic acid derivatives as raw materials.
  • the phenoxyacetic acid derivatives have good water solubility and low synthesis cost. They can be combined with 6-APA in solution under the catalyst of penicillin acylase. The rapid reaction to produce penicillin V requires a small amount of penicillin acylase catalyst, and the penicillin acylase can be repeatedly used with low cost.
  • the penicillin V salt prepared in this application has few impurities, good product quality, high product purity and high yield, far exceeding the penicillin V salt produced by the existing fermentation method, and higher than the quality standard of similar raw materials abroad, 6-APA conversion
  • the rate can reach 99.5%
  • the liquid phase purity can reach more than 99.7%
  • 4-hydroxyphenoxymethyl penicillin (impurity D) is not detected
  • the polymer impurity is less than 0.01%, which can change the existing penicillin V drugs in China. Status of skin test.
  • Figure 1 is the NMR spectrum of glycerol monophenoxyacetate prepared in Example 1.
  • Step (1) Weigh 30.0g of phenoxyacetic acid, add 270.0g of glycerin, heat up to 60°C to fully dissolve the phenoxyacetic acid, pour it into a closed reactor, add 30.0g of immobilized lipase, mix evenly, and make the material Keep the flow, keep the vacuum degree of the reaction system lower than -0.1MPa, keep the reaction temperature at 50°C, react for 360 minutes, and separate the immobilized enzyme from the reaction liquid on a screen to obtain the reaction product.
  • Step (2) Add 200ml of water to the reaction product obtained in step (1), adjust the pH value to 7.0 with a 10% (m/v) sodium carbonate solution, and then adsorb through a macroporous adsorption resin, using 30% (v/v) ) Is eluted with ethanol solution, and the solution of compound I whose purity is higher than 98.0% is collected, and distilled to the minimum volume under reduced pressure at 50° C., and the concentrated solution is collected.
  • the reaction product obtained in step (1) and the concentrated solution obtained in step (2) were sampled separately for HPLC analysis.
  • the analysis conditions are as follows:
  • Preparation of the test solution Take 30-35mg of the sample to be tested in a 10ml volumetric flask, dissolve it with the mobile phase and dilute to the mark, and get it.
  • Step (2) Sampling the obtained concentrated solution for ESI mass spectrometry and NMR analysis (ESI conditions: Thermo Finnigan mass spectrometer, model LCQ advantage, solvent methanol; NMR conditions: Swiss Bruker nuclear magnetic resonance spectrometer, model AVANCE III HD 400MHz, solvent deuteration Chloroform), the obtained HPLC analysis results are shown in Table 1 below, the obtained mass spectrometry result is 474.69 [2M+Na], and the obtained NMR spectrum is shown in Figure 1 ( 1 H-NMR(CDCl 3 ,400MHz) ⁇ 7.293(1H, d,H-2); ⁇ 7.274(1H,d,H-6); ⁇ 6.908(1H,S,H-3); ⁇ 6.888(1H,S,H-5); ⁇ 7.002( 1H,t,H-4); ⁇ 4.699(2H,m,H-7); ⁇ 4.269(2H,m,H-9); ⁇ 3.918(1H,t,H-10); ⁇ 3. 6
  • Mobile phase A phase pH3.5 phosphate buffer (take 0.5moL/L potassium dihydrogen phosphate and adjust the pH to 3.50 with phosphoric acid)-methanol-water (10:30:60)
  • Mobile phase B phase pH3.5 phosphate buffer (take 0.5moL/L potassium dihydrogen phosphate and adjust the pH to 3.50 with phosphoric acid)-methanol-water (10:55:35)
  • the immobilized enzyme and the reaction solution are separated by a screen, and the pH of the reaction solution is adjusted to 2.8 with 6 mol/L hydrochloric acid, the crystals are cultivated for 20 minutes, and filtered to obtain a penicillin V acid filter cake.
  • the filter cake is transferred to 4 times the weight of pure water, the pH value of the mixed solution is adjusted to 6.3 with 10% (m/v) potassium carbonate solution, and then n-butanol is added to contain 63% (v/v) of n-butanol . It is concentrated by evaporation under reduced pressure until penicillin V salt crystals are precipitated, and the crystals are filtered, washed, and dried to obtain penicillin V potassium crystal powder.

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Abstract

本申请属于抗生素药物技术领域，涉及苯氧乙酸衍生物及利用其酶法制备青霉素V盐的方法。所述的衍生物为苯氧乙酸甘油酯。所述的方法包括如下步骤：(1)在水溶液中，在青霉素酰化酶的催化下，使6-APA和所述的衍生物反应；(2)反应完成后，酸化反应得到的青霉素V，并经盐化后分离得到青霉素V盐。利用本申请的苯氧乙酸衍生物及利用其酶法制备青霉素V盐的方法，能够更好的酶法制备青霉素V盐。

Description

苯氧乙酸衍生物及利用其酶法制备青霉素V盐的方法
相关申请的交叉引用
本申请要求于2019年1月30日提交的申请号为201910089112.1的中国专利申请的优先权，出于全部目的，该专利申请的全部内容通过引用的方式结合至本文。
技术领域
本申请属于抗生素药物技术领域，涉及苯氧乙酸衍生物及利用其酶法制备青霉素V盐的方法。
背景技术
青霉素V盐，如青霉素V钾和青霉素V钠，是临床上常用的抗生素药物，其中青霉素V钾应用更广泛。青霉素V钾属β-内酰胺酶类抗生素，抗菌谱与青霉素G相似，能够破坏细菌细胞壁，具有杀菌作用，是治疗革兰阳性菌及部分阴性菌感染的首选药物，临床上已广泛应用于治疗脓肿、化脓性脑膜炎，肺炎、淋病等疾病。青霉素V钾与青霉素G钾/钠相比，其对酸更稳定，不易被胃酸破坏，口服吸收更好，其口服制剂尤其适合儿童使用，且其被β-内酰胺酶的破坏更慢，故对耐药性金葡菌引起的感染更有效。
青霉素V钾片最早于1952年开始应用于临床，1957年9月获美国FDA批准，美国药典、英国药典、日本药局方等均有收载。目前，美国、加拿大、英国、德国、日本等国家均有生产青霉素V钾片，其是国际上临床使用较广泛的一种口服抗生素。
青霉素应用至今发生过敏反应的机率较高，常见的过敏反应包括皮疹、荨麻疹、皮炎、发热、血管神经性水肿、哮喘、过敏性休克等，其中以过敏性休克最为严重，甚至可导致死亡。为了防止过敏反应的发生，特别是严重过敏反应的发生，国内规定青霉素注射剂在使用前需要做皮肤敏感试验，皮试阴性的药物方可以给病人使用，皮试阳性的则禁止使用；而在国外口服的青霉素类药品均是免皮试的。
近年来口服青霉素V制剂的需求量大大增加，但是青霉素V盐原料药产品质量仍有较大问题。因此，生产得到高质量水平的青霉素V盐原料药(杂质含量低，尤其是聚合物杂质含量低)有重大意义。青霉素V钾原料及制剂中可能会含有其合成前体、工艺副产物以及各种降解产物；另外，青霉素V钾也可能自身聚合，产生高分子聚合物。工艺副产物、降解产物和聚合物杂质最大来源是生产过程，生产过程越长，杂质产生几率越大。β-内酰胺类抗生素所致的过敏反应，经多年来的研究已经证明和药物中存在的高分子聚合物有关。现有的生产青霉素V钾的方法因发酵周期长、生产工序长，故各种工艺副产物、降解产物和聚合物杂质含量高。
青霉素V盐目前未见用生物酶法制备，更未见用青霉素酰化酶逆向催化6-氨基青霉素酸(6-APA)和苯氧乙酸衍生物反应制备。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本申请的首要目的是提供苯氧乙酸衍生物，以能够利用其更好的酶法制备青霉素V盐。
为实现此目的，在基础的实施方案中，本申请提供苯氧乙酸衍生物，所述的衍生物为苯氧乙酸甘油酯(包括单苯氧乙酸甘油酯、二苯氧乙酸甘油酯、三苯氧乙酸甘油酯)。
在一种可选的实施方案中，本申请提供苯氧乙酸衍生物，其中所述的衍生物为单苯氧乙酸甘油酯。
在一种可选的实施方案中，本申请提供苯氧乙酸衍生物，其中所述的衍生物具有如下式(I)所示的结构：
Figure PCTCN2020072246-appb-000001
本申请的第二个目的是提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，以能够更好地酶法制备青霉素V盐。
为实现此目的，在基础的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，所述的方法包括如下步骤：
步骤(1)：在水溶液中，在青霉素酰化酶的催化下，使6-APA和所述的衍生物反应；
步骤(2)：反应完成后，酸化反应得到的青霉素V，并经盐化后分离得到青霉素V盐。
在一种可选的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，其中步骤(1)中，所述的青霉素酰化酶为固定化的青霉素酰化酶。
在一种可选的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，其中步骤(1)中，所述的青霉素酰化酶为天然青霉素酰化酶的突变体，具有SEQ ID NO.1所示的氨基酸序列。
在一种可选的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，其中步骤(1)中，
所述的6-APA和所述的衍生物的摩尔比为1：1.1-1.5，所述的6-APA和所述的青霉素酰化酶的重量比为1：2-4，
反应温度为5-40℃，反应pH为4.0-8.0，反应时间为1-2h。
在一种更加可选的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，其中步骤(1)中，反应温度为10-30℃，反应pH为5.0-6.0，反应时间为1-2h。
在一种可选的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，其中步骤(2)中，所述的酸化为用酸调节溶液pH至2.5-3.5，养晶10-30分钟。
在一种可选的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，其中步骤(2)中，所述的盐化为先用碳酸盐调节溶液pH至5.5-7.5，再向溶液中添加带水剂(所述带水剂为乙醇、异丙醇、正丁醇、异丁醇中的一种或几种)，蒸发浓缩至青霉素V盐晶体 析出。
在一种可选的实施方案中，本申请提供一种利用前述苯氧乙酸衍生物酶法制备青霉素V盐的方法，其中步骤(2)中，在所述的酸化前，还可以先将所述的青霉素酰化酶从溶液中分离。
本申请实施例的有益效果在于：
1)本申请首次发现最新报道的青霉素G酰化酶突变体和青霉素V酰化酶突变体对6-APA和苯氧乙酸衍生物之间的反应具有很好的催化活性，可以实现青霉素V的高效合成，以进一步获得高纯度青霉素V盐。
2)本申请的青霉素V盐的生产周期短，青霉素V盐合成、纯化、结晶全过程不超过24小时。
3)本申请的青霉素V盐的生产过程操作简单，可以只需一步合成和两步结晶步骤。
4)本申请的青霉素V盐的合成过程绿色无污染，不需使用醋酸丁酯等进行萃取纯化。
5)本申请的青霉素V盐合成以6-APA和苯氧乙酸衍生物为原材料，苯氧乙酸衍生物水溶性好、合成成本低，可与6-APA在青霉素酰化酶催化剂催化下在溶液中快速进行反应生成青霉素V，所需的青霉素酰化酶催化剂量少，且青霉素酰化酶可以反复利用，成本低。
6)本申请制备的青霉素V盐杂质少、产品质量好，产品纯度、得率高，远远超过现有的发酵法生产的青霉素V盐，高于国外同类原料药质量标准，6-APA转化率可达99.5％，液相纯度可达99.7％以上，4-羟基苯氧甲基青霉素(杂质D)未检出，聚合物杂质低于0.01％，可改变国内现有的青霉素V药物必须做皮试的现状。
7)本申请的青霉素V盐合成的整个过程中产生的副产物为甘油，对酶无毒害，对环境无污染。
附图说明
图1为实施例1制备的单苯氧乙酸甘油酯的NMR检测图谱。
具体实施方式
以下结合实施例和附图对本申请的具体实施方式作出进一步地说明。
实施例1：式(I)所示结构的单苯氧乙酸甘油酯的制备
步骤(1)：称取30.0g苯氧乙酸，加入270.0g甘油，升温至60℃使苯氧乙酸充分溶解后，倒入密闭反应器中，加入30.0g固定化脂肪酶，混合均匀，使物料保持流动，保持反应体系真空度低于-0.1MPa，保持反应温度50℃，反应360min，筛网分离固定化酶与反应液，得到反应产物。
步骤(2)：步骤(1)所得反应产物中加入200ml水，用10％(m/v)的碳酸钠溶液调节pH值至7.0，通过大孔吸附树脂进行吸附，用30％(v/v)的乙醇溶液洗脱，收集化合物I纯度高于98.0％的溶液，50℃减压蒸馏至最小体积，收集浓缩液。
步骤(1)所得反应产物和步骤(2)所得浓缩液分别取样进行HPLC分析，分析条件如下：
色谱柱：Diamosil C18(2)5μm 4.6×250mm
流动相：乙腈：0.1％(m/v)乙酸氨水溶液＝60:40
柱温：25℃
检测波长：220nm
总流速：1.0ml/min
进样量：5μl
供试品溶液的配制：取30-35mg的待测样品于10ml容量瓶中，用流动相溶解并定容至刻度，即得。
步骤(2)所得浓缩液取样进行ESI质谱分析和NMR分析(ESI条件：Thermo Finnigan质谱仪，型号LCQ advantage，溶剂甲醇；NMR条件：瑞士布鲁克核磁共振波谱仪，型号AVANCE Ⅲ HD 400MHz，溶剂氘代氯仿)，所得HPLC分析结果如下表1所示，所得质谱检测结果为474.69[2M+Na]，所得NMR图谱如图1所示( 1H-NMR(CDCl 3,400MHz)δ7.293(1H,d,H-2)；δ7.274(1H,d,H-6)；δ6.908(1H,S,H-3)；δ6.888(1H,S,H-5)；δ7.002(1H,t,H-4)；δ4.699(2H,m,H-7)；δ4.269(2H,m,H-9)；δ3.918(1H,t,H-10)；δ3.656(2H,m,H-11)；δ3.319(1H,S,OH-10)；δ2.927 (1H,S,OH-11))。
表1 HPLC分析结果
Figure PCTCN2020072246-appb-000002
其中，化合物I结构为：
Figure PCTCN2020072246-appb-000003
化合物II结构为：
Figure PCTCN2020072246-appb-000004
化合物III结构为：
Figure PCTCN2020072246-appb-000005
实施例2：酶法制备青霉素V盐
取25.0g 6-APA，加入400mL纯水，滴加3mol/L的氨水溶液使6-APA充分溶解，加入苯氧乙酸衍生物，混合均匀，再加入SEQ ID NO.1所示氨基酸序列的固定化的青霉素酰化酶(即中国专利申请CN201510736957.7中SEQ ID NO.3所示氨基酸序列的突变型青霉素G酰化酶SPGA-4，其制备方法参见中国专利申请CN201510736957.7的实施例1-3)，滴加3mol/L的氨水溶液调节反应pH。温度20℃下反应，定时取样做HPLC检测，检测条件如下：
色谱柱：Diamosil C18，5μm，4.6×250mm
流动相A相：pH3.5磷酸盐缓冲液(取0.5moL/L磷酸二氢钾用磷酸调pH至3.50)-甲醇-水(10:30:60)
流动相B相：pH3.5磷酸盐缓冲液(取0.5moL/L磷酸二氢钾用磷酸调pH至3.50)-甲醇-水(10:55:35)
柱温：25℃
检测波长：268nm
总流速：1.0mL/min
进样量：20μl。
梯度洗脱，其程序如下：
时间(min) 流动相A(％) 流动相B(％)
0 40 60
27 40 60
47 0 100
62 0 100
65 40 60
反应完成后，筛网分离固定化酶与反应液，将反应液用6mol/L的盐酸调节pH值至2.8，养晶20分钟，过滤，得到青霉素V酸滤饼。滤饼转 至4倍重量的纯水中，用10％(m/v)的碳酸钾溶液调节混合溶液pH值至6.3，再补加正丁醇至含正丁醇63％(v/v)。减压蒸发浓缩至有青霉素V盐晶体析出，晶体经过滤、洗涤、干燥后得到青霉素V钾结晶粉。
以上不同的操作条件及检测结果如下表2、表3所示。
表2不同的操作条件
Figure PCTCN2020072246-appb-000006
表3不同操作条件下对应的检测结果
Figure PCTCN2020072246-appb-000007
在阅读并理解了附图和详细描述后，可以明白其他方面。显然，本领域的 技术人员可以对本申请实施例进行各种改动和变型而不脱离本申请的精神和范围。这样，倘若对本申请的这些修改和变型属于本申请权利要求及其同等技术的范围之内，则本申请也意图包含这些改动和变型在内。上述实施例或实施方式只是对本申请实施例的举例说明，本申请也可以以其它的特定方式或其它的特定形式实施，而不偏离本申请的要旨或本质特征。因此，描述的实施方式从任何方面来看均应视为说明性而非限定性的。本申请的范围应由附加的权利要求说明，任何与权利要求的意图和范围等效的变化也应包含在本申请的范围内。

Claims (10)

  1. 苯氧乙酸衍生物，其中，所述的衍生物为苯氧乙酸甘油酯。
  2. 根据权利要求1所述的苯氧乙酸衍生物，其中，所述的衍生物为单苯氧乙酸甘油酯。
  3. 根据权利要求1或2所述的苯氧乙酸衍生物，其中，所述的衍生物具有如下式(I)所示的结构：
    Figure PCTCN2020072246-appb-100001
  4. 一种利用权利要求1-3之一所述的苯氧乙酸衍生物酶法制备青霉素V盐的方法，其包括如下步骤：
    步骤(1)：在水溶液中，在青霉素酰化酶的催化下，使6-APA和所述的衍生物反应；
    步骤(2)：反应完成后，酸化反应得到的青霉素V，并经盐化后分离得到青霉素V盐。
  5. 根据权利要求4所述的方法，其中，步骤(1)中所述的青霉素酰化酶为固定化的青霉素酰化酶。
  6. 根据权利要求4所述的方法，其中，步骤(1)中所述的青霉素酰化酶为天然青霉素酰化酶的突变体，具有SEQ ID NO.1所示的氨基酸序列。
  7. 根据权利要求4所述的方法，其中，步骤(1)中所述的6-APA和所述的衍生物的摩尔比为1：1.1-1.5，所述的6-APA和所述的青霉素酰化酶的重量比为1：2-4，
    反应温度为5-40℃，反应pH为4.0-8.0，反应时间为1-2h。
  8. 根据权利要求4所述的方法，其中，步骤(2)中所述的酸化为用酸调节溶液pH至2.5-3.5，养晶10-30分钟。
  9. 根据权利要求4所述的方法，其中，步骤(2)中所述的盐化为先用碳酸盐调节溶液pH至5.5-7.5，再向溶液中添加带水剂，蒸发浓缩至青霉素V盐晶体析出。
  10. 根据权利要求4所述的方法，其中，步骤(2)中，在所述的酸化前，还可以先将所述的青霉素酰化酶从溶液中分离。
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* Cited by examiner, † Cited by third party
Title
COURSE ON MEDICAL CHEMISTRY, VOCATIONAL SECONDARY SCHOOL, 31 January 2006 (2006-01-31), pages 237 - 239 *
MANOVICIU, IONEL ET AL.: "Esters of phenoxyacetic acids with trihydric and tetrahydric alcohols", MATERIALE PLASTICE, vol. 3, no. 31, 31 December 1994 (1994-12-31), Bucharest, pages 202 - 205 *

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