WO2020182203A1 - 一种万古霉素类似物的分离纯化方法 - Google Patents

一种万古霉素类似物的分离纯化方法 Download PDF

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WO2020182203A1
WO2020182203A1 PCT/CN2020/079200 CN2020079200W WO2020182203A1 WO 2020182203 A1 WO2020182203 A1 WO 2020182203A1 CN 2020079200 W CN2020079200 W CN 2020079200W WO 2020182203 A1 WO2020182203 A1 WO 2020182203A1
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vancomycin
chromatographic
solution
volume ratio
ratio
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PCT/CN2020/079200
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French (fr)
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张磊
夏兴
孙新强
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浙江医药股份有限公司新昌制药厂
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Priority to CN202080019140.7A priority Critical patent/CN113544137A/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof

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  • the invention belongs to the field of biopharmaceuticals, and specifically relates to a method for separating and preparing a vancomycin structural analog with a purity of more than 80% by using crude vancomycin as a raw material.
  • Vancomycin is a natural amphoteric glycopeptide antibiotic extracted from the fermentation broth of actinomycetes, such as Nocardia orientalis. Its function is to interact with the D-C of the bacterial cell wall precursor.
  • the combination of the dipeptide residues of aminoacyl-D-alanine (D-Ala-D-Ala) inhibits the synthesis of peptidoglycan, thereby inhibiting the synthesis of bacterial cell walls; in addition, it can also change the permeability of cell membranes and RNA Synthesized to play the expected pharmacological effect.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Vancomycin hydrochloride is mainly used for the initial treatment of severe or severe infections caused by staphylococcus against ⁇ -lactamine antibiotics, and also for the treatment of patients who are allergic to penicillin or who have no effect on penicillin and cephalosporins; vancomycin hydrochloride alone Use or in combination with other glucosamine to treat endocarditis caused by Staphylococcus, Streptococcus, Enterococcus or Diphtheria bacillus.
  • Vancomycin molecule is composed of two basic structures, namely the glycosyl part ( ⁇ -o-vancosamine- ⁇ -o-glucosy) and the peptidyl part of the central hepeptide core, whose structure determines its instability.
  • the glycosyl part ⁇ -o-vancosamine- ⁇ -o-glucosy
  • the peptidyl part of the central hepeptide core whose structure determines its instability.
  • Some modified compounds such as Oritavancin, Dalbavancin, Telavancin, have initially shown resistance to vancomycin-sensitive and resistant bacteria. (MRSA, VRSA, VRA) has a good antibacterial effect.
  • the patent CN 101724645A discloses a method for genetically engineered vancomycin producing strains to obtain vancomycin analogs-chlorine-free vancomycin.
  • Patent CN 101724644A discloses a method for genetically engineering a vancomycin producing strain to obtain a vancomycin analogue-norvancomycin.
  • Patent CN 101397333A discloses a method for obtaining vancomycin analog-deshydroxyvancomycin through a vancomycin production strain.
  • Vancomycin Structure and Transformation to CDP-I, Constance M. Harris., J. Am. Chem. Soc., Vol. 105, No. 23, 6915-6922, introduced the production of several analogues of vancomycin
  • the reaction path includes the compounds involved in this patent.
  • the inventor of the present application found a method for isolating and preparing a vancomycin analog with a purity of >80% using crude vancomycin as a raw material. After the compound was prepared, its structure was identified and it was confirmed that the compound was a vancomycin analog.
  • the present invention discloses a preparation method of vancomycin analogs represented by the following formula (I):
  • the present invention discloses a method for separating and purifying vancomycin analogs with a purity of >80% as shown in the above formula (I) using crude vancomycin as a raw material.
  • the separation and purification method includes the following steps: a) Vancomycin
  • the first chromatographic enrichment of element analogues the crude product containing vancomycin analogues is used as raw material, and polymer matrix chromatography packing is used for the first chromatographic enrichment, isocratic elution or gradient elution, segmented Collect the first chromatographic enrichment solution with a purity of> 35%; wherein the polymer matrix chromatographic packing is polystyrene/divinylbenzene copolymer UniPS, UniPSA (UniPS and UniPSA are both polystyrene/diethylene Based on phenyl, the synthesis parameters are different when bonding, resulting in differences in the hydrophilicity and polarity of the two), or UniPSN, a mixed copo
  • the polymer matrix chromatographic filler is Polystyrene/divinylbenzene UniPS, or UniPSA, or polyacrylic acid UniPSN from Nanomicro,
  • the particle size of the filler is 5 ⁇ m, or 10 ⁇ m, or 30 ⁇ m, preferably the particle size is 10 ⁇ m; the pore size of the filler is 100A, or 300A, or 500A, and the preferred pore size is 300A.
  • step a) when the chromatographic mobile phase is methanol aqueous solution, the methanol ratio is 25%-45% (volume ratio), preferably 30%-40% (volume Ratio), more preferably 33%-38% (volume ratio); when the chromatography mobile phase is ethanol aqueous solution, the ethanol ratio is 40%-60% (volume ratio), preferably 45%-55% (volume ratio), more preferably 48%-53% (volume ratio); when the chromatography mobile phase is acetonitrile aqueous solution, the acetonitrile ratio is 15%-35% (volume ratio), preferably 20%-30% (volume ratio), more preferably 23%-28% (Volume ratio).
  • the chromatography packing is a reversed-phase silica gel matrix chromatography packing, such as C8 or C18 from Nanomicro Corporation.
  • the particle size of the filler is 5 ⁇ m, or 10 ⁇ m, or 30 ⁇ m, preferably 10 ⁇ m; the pore size of the filler is 60A, or 100A, or 120A, preferably the pore size is 120A.
  • step b) when the chromatographic mobile phase is methanol aqueous solution, the methanol ratio is 5%-25% (volume ratio), preferably 10%-20% (volume Ratio), more preferably 12%-17% (volume ratio); when the chromatographic mobile phase is ethanol aqueous solution, the ethanol ratio is 10%-30% (volume ratio), preferably 15%-25% (volume ratio), more preferably 18%-23% (volume ratio); when the chromatographic mobile phase is acetonitrile aqueous solution, the acetonitrile ratio is 5%-25% (volume ratio), preferably 10%-20% (volume ratio), more preferably 12%-18% (Volume ratio).
  • the ratio of triethylamine in the triethylamine-water solution is 0.1%-0.3% (volume ratio), preferably 0.15%-0.25% (volume ratio) Ratio), more preferably 0.18%-0.23% (volume ratio).
  • step b) phosphoric acid is used to adjust the pH to 2.0-4.0, preferably 2.5-3.5, more preferably 2.8-3.3.
  • the chromatography packing is a reversed-phase silica gel matrix chromatography packing, such as C8 or C18 of Nanomicro Corporation.
  • the particle size of the filler is 5 ⁇ m, or 10 ⁇ m, or 30 ⁇ m, preferably 10 ⁇ m; the pore size of the filler is 60A, or 100A, or 120A, preferably the pore size is 120A.
  • step c) when the chromatographic mobile phase is methanol aqueous solution, the methanol ratio is 5%-25% (volume ratio), preferably 10%-20% (volume Ratio), more preferably 12%-17% (volume ratio); when the chromatographic mobile phase is ethanol aqueous solution, the ethanol ratio is 10%-30% (volume ratio), preferably 15%-25% (volume ratio), more preferably 18%-23% (volume ratio); when the chromatographic mobile phase is acetonitrile aqueous solution, the acetonitrile ratio is 5%-25% (volume ratio), preferably 10%-20% (volume ratio), more preferably 12%-18% (Volume ratio).
  • the buffer solution in step c) is formic acid-aqueous solution, acetic acid-aqueous solution, or trifluoroacetic acid-aqueous solution, wherein the ratio of added acid is 0.1-0.3% ( Volume ratio), preferably 0.15% to 0.25% (volume ratio), more preferably, 0.18% to 0.23% (volume ratio).
  • step d) during concentration under reduced pressure, the temperature is controlled at 10-50°C, preferably 20-40°C, more preferably 25-35°C.
  • step d) the pressure is controlled at -0.09mPa to -0.1mPa during concentration under reduced pressure.
  • the present invention uses crude vancomycin as a raw material to obtain vancomycin analogs with a purity of >80% through separation and purification, and has undergone structural identification, for further screening of biological activity, or structural modification before biological activity Screening laid the foundation. Through follow-up work, it is possible to obtain new compounds with better efficacy and fewer adverse reactions.
  • Figure 1 shows the elemental composition diagram of the vancomycin analog obtained in the present invention
  • Figure 2 shows the MS chart of the vancomycin analog obtained in the present invention
  • FIG. 3 shows the C spectrum of the vancomycin analog obtained in the present invention
  • Figure 4 shows the H diagram of the vancomycin analog obtained in the present invention
  • the crude vancomycin containing vancomycin analogues (the crude vancomycin is prepared according to Chinese patent CN101456903B), using polymer matrix chromatography packing, such as UniPS, with a particle size of 10 ⁇ m and a pore size of 300A, and the first layer Analyze the enrichment, the mobile phase is methanol aqueous solution, the methanol ratio is 33% (volume ratio), the isocratic elution, the fractional collection, the purity>35% part.
  • the part of the collected liquid with a purity of >35% is concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C8, with a particle size of 10 ⁇ m and a pore size of 100A, is used for the second chromatography
  • the chromatographic mobile phase is ethanol-buffer solution
  • the ethanol ratio is 18% (volume ratio)
  • the buffer is 0.18% (volume ratio) triethylamine solution
  • phosphoric acid adjusts the pH to 2.8
  • isocratic elution segmented collection
  • the purity>65% part enters the next step.
  • the fraction of the collected liquid with a purity> 65% was concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C18, with a particle size of 10 ⁇ m and a pore size of 120A, was used for the third chromatographic purification.
  • Chromatographic mobile phase is acetonitrile-buffer solution, the ratio of acetonitrile is 12% (volume ratio), the buffer solution is 0.18% (volume ratio) formic acid aqueous solution, isocratic elution, fractional collection, purity>80% part enters the next step .
  • the crude vancomycin containing vancomycin analogues (the crude vancomycin is prepared in accordance with Chinese patent CN101456903B), using polymer matrix chromatography packing, such as UniPSA, with a particle size of 30 ⁇ m and a pore size of 100A, for the first layer
  • polymer matrix chromatography packing such as UniPSA, with a particle size of 30 ⁇ m and a pore size of 100A
  • the mobile phase is ethanol aqueous solution
  • the ethanol ratio is 48% (volume ratio)
  • the isocratic elution the fractional collection, the purity>35% part.
  • the part of the collected liquid with a purity of >35% is concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C18, with a particle size of 10 ⁇ m and a pore size of 60A, is used for the second chromatography
  • the chromatographic mobile phase is methanol-buffer
  • the methanol ratio is 12% (volume ratio)
  • the buffer is 0.20% (volume ratio) triethylamine solution
  • phosphoric acid adjusts the pH to 3.0
  • isocratic elution isocratic elution
  • segmented collection The purity>65% part enters the next step.
  • Chromatographic mobile phase is ethanol-buffer solution
  • ethanol ratio is 18% (volume ratio)
  • buffer is 0.20% (volume ratio)
  • acetic acid solution isocratic elution, segmented collection, purity> 80% part enters the next step.
  • the crude vancomycin containing vancomycin analogues (the crude vancomycin is prepared according to Chinese patent CN101456903B), using polymer matrix chromatography packing, such as UniPSN, the particle size is 30 ⁇ m, the pore size is 300A, and the first layer Analyze enrichment, the mobile phase is acetonitrile aqueous solution, the proportion of acetonitrile is 23% (volume ratio), the isocratic elution is performed, and the fraction is collected, and the purity is greater than 35%.
  • polymer matrix chromatography packing such as UniPSN
  • the part of the collected liquid with a purity of more than 35% is concentrated under reduced pressure to remove the solvent, and then reverse-phase silica gel matrix chromatography packing, such as C18, with a particle size of 30 ⁇ m and a pore size of 120A, is used for the second chromatography Purification, chromatographic mobile phase is acetonitrile-buffer solution, acetonitrile ratio is 12% (volume ratio), buffer is 0.23% (volume ratio) triethylamine solution, phosphoric acid adjusts PH to 3.3, isocratic elution, segmented Collect, the purity>65% part goes to the next step.
  • chromatographic mobile phase is acetonitrile-buffer solution
  • acetonitrile ratio is 12% (volume ratio)
  • buffer is 0.23% (volume ratio) triethylamine solution
  • phosphoric acid adjusts PH to 3.3
  • isocratic elution segmented Collect
  • the crude vancomycin containing vancomycin analogues (the crude vancomycin is prepared according to Chinese patent CN101456903B), using polymer matrix chromatography packing, such as UniPS, the particle size is 30 ⁇ m, the pore size is 100A, and the first layer Analyze the enrichment, the mobile phase is methanol aqueous solution, the methanol ratio is 34% (volume ratio), the isocratic elution is performed, and the fractions are collected in sections, and the purity is greater than 35%.
  • polymer matrix chromatography packing such as UniPS
  • the part of the collected liquid with a purity of >35% is concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C8, with a particle size of 10 ⁇ m and a pore size of 100A, is used for the second chromatography
  • the chromatographic mobile phase is methanol-buffer solution
  • the methanol ratio is 14% (volume ratio)
  • the buffer is 0.19% (volume ratio) triethylamine solution
  • phosphoric acid adjusts the pH to 2.9
  • isocratic elution segmented collection
  • the purity>65% part enters the next step.
  • the fraction of the collected liquid with a purity> 65% was concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C18, with a particle size of 10 ⁇ m and a pore size of 120A, was used for the third chromatographic purification.
  • Chromatographic mobile phase is acetonitrile-buffer solution
  • the ratio of acetonitrile is 14% (volume ratio)
  • the buffer is 0.19% (volume ratio) formic acid aqueous solution
  • isocratic elution fractional collection, purity> 80% part enters the next step .
  • the crude vancomycin containing vancomycin analogues (the crude vancomycin is prepared according to Chinese patent CN101456903B), using polymer matrix chromatography packing, such as UniPSA, with a particle size of 10 ⁇ m and a pore size of 300A, and the first layer Analyze enrichment, the mobile phase is acetonitrile aqueous solution, the proportion of acetonitrile is 26% (volume ratio), isocratic elution, fractional collection, purity>35% part.
  • polymer matrix chromatography packing such as UniPSA
  • the part of the collected liquid with a purity of >35% is concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C18, with a particle size of 10 ⁇ m and a pore size of 120A, is used for the second chromatography
  • the chromatographic mobile phase is ethanol-buffer solution, ethanol ratio is 51% (volume ratio), buffer is 0.21% (volume ratio) triethylamine solution, phosphoric acid adjusts the pH to 3.1, isocratic elution, segmented collection , The purity>65% part enters the next step.
  • the fraction of the collected liquid with a purity of >65% was concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C18, with a particle size of 5 ⁇ m and a pore size of 120A, was used for the third chromatographic purification.
  • Chromatographic mobile phase is acetonitrile-buffer solution, the ratio of acetonitrile is 16% (volume ratio), the buffer is 0.21% (volume ratio) acetic acid solution, isocratic elution, fractional collection, purity>80% part enters the next step .
  • the crude vancomycin containing vancomycin analogues (the crude vancomycin is prepared in accordance with Chinese patent CN101456903B), using polymer matrix chromatography packing, such as UniPSN, with a particle size of 10 ⁇ m and a pore size of 100A, for the first layer Analyze the enrichment, the mobile phase is ethanol aqueous solution, the ethanol ratio is 51% (volume ratio), the isocratic elution, the fractional collection, the purity>35% part.
  • the part of the collected liquid with a purity of more than 35% is concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C8, with a particle size of 30 ⁇ m and a pore size of 120A, is used for the second chromatography
  • the chromatographic mobile phase is methanol-buffer solution
  • the methanol ratio is 16% (volume ratio)
  • the buffer is 0.23% (volume ratio) triethylamine solution
  • phosphoric acid adjusts the pH to 3.2
  • isocratic elution segmented collection
  • the purity>65% part enters the next step.
  • the fraction of the collected liquid with a purity of >65% was concentrated under reduced pressure to remove the solvent, and then reversed-phase silica gel matrix chromatography packing, such as C8, with a particle size of 10 ⁇ m and a pore size of 120A, was used for the third chromatographic purification.
  • Chromatographic mobile phase is methanol aqueous solution, methanol ratio is 15% (volume ratio), buffer is 0.2% (volume ratio) trifluoroacetic acid solution, isocratic elution, fractional collection, purity>80% part enters the next step .
  • UPLC-MS and nuclear magnetic resonance are used to detect the sample to determine the structure of the sample.
  • Analytical table 1 Mass spectrometry electrospray ionization source (ESI) positive ion detection, the [M+H] + peak of the compound is 1431.5046.
  • the elemental composition is determined to be C 66 H 73 N 8 O 24 Cl 2 , which is consistent with the elemental composition of vancomycin for removing NH 3 and adding H.
  • Test instrument Bruker 600MHz; test solvent: DMSO-d6.

Abstract

一种万古霉素类似物(I)的分离纯化方法，以万古霉素粗品为原料，先用聚合物基质色谱填料层析富集，然后再反相硅胶基质色谱填料层析，得到纯度＞80%的万古霉素类似物。

Description

一种万古霉素类似物的分离纯化方法 技术领域
本发明属于生物制药领域，具体涉及一种以万古霉素粗品为原料，分离制备一个纯度＞80％的万古霉素结构类似物的方法。
背景技术
自20世纪40年代青霉素应用于临床以来，抗感染药物的种类已达到几千种，在临床上常用的已有几百种。2006年全球畅销药500强中的抗感染药物数量已达77个，是19大类药物中居于首位的药物。
万古霉素(Vancomycin)是从放线菌，如东方诺卡氏菌等发酵液中提取获得的一种天然的两性糖肽类抗生素，其起作用的方式是与细菌细胞壁前体的D-丙氨酰-D-丙氨酸(D-Ala-D-Ala)的二肽残基结合，抑制肽聚糖的合成，从而抑制细菌细胞壁的合成；此外，还可以改变细胞膜的渗透性和RNA的合成，从而起到预期的药理作用。
随着抗生素的大规模使用，细菌逐渐进化出抗药性；特别是β-内酰胺类抗生素的大量使用，导致由甲氧西林耐药金葡球菌(MRSA)引起的感染逐渐流行。在这种情况下，盐酸万古霉素成为临床上用于治疗由MRSA引起的严重感染疾病的首选药物，在过去相当长的一段时间内，被国际抗生素专家誉为“人类对付顽固性耐药菌株的最后一道防线”和“王牌抗生素”。盐酸万古霉素主要适用于抗β-乳胺抗生素的葡萄球菌引起的严重或重症感染的初步治疗，也用于治疗对青霉素过敏或使用青霉素和头孢菌素没有效果的患者；盐酸万古霉素单独使用或与其它氨基葡糖苷结合使用，可以治疗由葡萄球菌、链球菌、肠球 菌或白喉杆菌引起的心内膜炎。
自临床大规模应用盐酸万古霉素开始，它在过去的几十年终一直作为抗革兰氏阳性菌的最后手段，在细菌感染治疗中广泛使用；但是，自1986年临床上第一次检测到耐万古霉素的肠球菌(VRE)以来，肠球菌对万古霉素的耐药性变得越来越普遍(＞20％)，并且开始扩散到人类以外的其他生物体中。最近多起报道显示，国外在对血液透析病人的抗感染治疗中，检测到了对万古霉素高度耐药的新的耐甲氧西林青霉素的金葡菌(VRSA)，这些报道引起了医学界的恐慌，医药行业中则把发现和改造抗生素的工作提到一个非常紧迫的日程上来，急需开发第二代对耐药致病菌株有特效的糖肽类抗生素。
万古霉素分子由两个基本结构组成，即糖基部分(α-o-vancosamine-β-o-glucosy)和肽基部分中心七肽核，其结构决定了它的不稳定性。在过去的数十年中，运用结构修饰的策略制备合成活性万古霉素类似物取得了一些重要进步。据知，目前已有数个经不同天然万古霉素类似物化学修饰得到的化合物进入了临床试验阶段，一些修饰化合物，如：Oritavancin、Dalbavancin、Telavancin初步显示出对万古霉素敏感菌和耐药菌(MRSA、VRSA、VRA)具有良好的抑菌作用。结合国际上具有生物活性的化合物的开发经验，利用现代生物技术和化学合成技术，对万古霉素进行结构修饰是其中一个研究方向，另一个研究方式是从源头进行筛选，针对万古霉素类似物，筛选具有更好的药理作用的新的化合物。
专利CN 101724645A，公开一种基因工程改造万古霉素生产菌株，获取万古霉素类似物-无氯万古霉素的方法。
专利CN 101724644A，公开一种基因工程改造万古霉素生产菌株，获取万古霉素类似物-去甲万古霉素的方法。
专利CN 101397333A，公开通过万古霉素生产菌株，获取万古霉素 类似物-去羟基万古霉素的方法。
文献Vancomycin:Structure and Transformation to CDP-Ⅰ,Constance M.Harris.，J.Am.Chem.Soc.,Vol.105,No.23,6915-6922中，介绍了万古霉素几个类似物的产生的反应路径，包括本专利涉及的化合物。
本申请的发明人在对Zalerion arboricola菌体发酵产生的万古霉素进行纯化时，找到了以万古霉素粗品为原料，分离制备一个纯度＞80％的万古霉素类似物的方法。制备得到该化合物后，对其进行了结构鉴定，确认该化合物为万古霉素类似物。
发明内容
本发明公开了一种如下式(I)所示的万古霉素类似物的制备方法：
Figure PCTCN2020079200-appb-000001
而万古霉素结构式(II)如下，两者仅在红框处存在差异：
Figure PCTCN2020079200-appb-000002
本发明公开了一种以万古霉素粗品为原料，分离纯化如上式(I)所示的纯度＞80％的万古霉素类似物的方法，所述分离纯化方法包括以下步骤：a)万古霉素类似物的第一次层析富集：以含有万古霉素类似物的粗品为原料，用聚合物基质色谱填料进行第一次层析富集，等度洗脱或梯度洗脱，分段收集纯度＞35％的第一次层析富集液；其中，所述聚合物基质色谱填料为聚苯乙烯/二乙烯基苯共聚物UniPS、UniPSA(UniPS和UniPSA都是聚苯乙烯/二乙烯基苯基质，键合时合成参数不同，导致两个亲水性和极性有差异)、或聚丙烯酸酯和二乙烯基苯混合体共聚物UniPSN，流动相为甲醇水溶液、乙醇水溶液、或乙腈水溶液；b)万古霉素类似物的第二次层析纯化：将步骤a)得到的纯度＞35％的第一次层析富集液进行减压浓缩以除去溶剂，用反相硅胶基质色谱填料进行第二次层析纯化，用磷酸调节pH至酸性，等度洗脱或梯度洗脱，分段收集纯度＞65％的第二次层析富集液；其中，所述反相硅胶基质色谱填料为辛烷基硅胶(C8)或者十八烷基硅胶(C18)，流动相为甲醇-缓冲溶液、乙醇-缓冲溶液、或乙腈-缓冲溶液，所述缓冲溶液为三乙胺-水溶液；c)万古霉素类似物的第三次层析纯化：将步骤b)得到的纯度＞65％的第二次层析富集液进行减压浓缩以除去溶剂，再用 反相硅胶基质色谱填料进行第三次层析纯化，等度洗脱或梯度洗脱，分段收集纯度＞80％的第三次层析富集液；其中，所述反相硅胶基质色谱填料为辛烷基硅胶(C8)或者十八烷基硅胶(C18)，流动相为甲醇-缓冲溶液、乙醇-缓冲溶液、或乙腈-缓冲溶液，所述缓冲溶液为甲酸-水溶液、乙酸-水溶液、或三氟乙酸-水溶液；以及d)将纯度＞80％的第三次层析富集液进行减压浓缩以除去溶剂，冻干，得到万古霉素类似物干粉。
在本发明所述分离纯化方法的优选技术方案中，在步骤a)中，所述聚合物基质色谱填料为纳微公司的聚苯乙烯/二乙烯基苯UniPS，或UniPSA，或聚丙烯酸UniPSN，填料粒径为5μm，或10μm，或30μm，优选粒径为10μm；填料孔径为100A，或300A,或500A，优选孔径为300A。
在本发明所述分离纯化方法的优选技术方案中，在步骤a)中，层析流动相为甲醇水溶液时，甲醇比例为25％-45％(体积比)，优选30％-40％(体积比)，更优选33％-38％(体积比)；层析流动相为乙醇水溶液时，乙醇比例为40％-60％(体积比)，优选45％-55％(体积比)，更优选48％-53％(体积比)；层析流动相为乙腈水溶液时，乙腈比例为15％-35％(体积比)，优选20％-30％(体积比)，更优选23％-28％(体积比)。
在本发明所述分离纯化方法的优选技术方案中，在步骤b)中，所述色谱填料为反相硅胶基质色谱填料，如纳微公司的C8或者C18。填料粒径为5μm，或10μm，或30μm，优选10μm；填料孔径为60A，或100A，或120A，优选孔径为120A。
在本发明所述分离纯化方法的优选技术方案中，在步骤b)中，层析流动相为甲醇水溶液时，甲醇比例为5％-25％(体积比)，优选10％-20％(体积比)，更优选12％-17％(体积比)；层析流动相为乙醇水溶液时，乙醇比例为10％-30％(体积比)，优选15％-25％(体积比)，更优选 18％-23％(体积比)；层析流动相为乙腈水溶液时，乙腈比例为5％-25％(体积比)，优选10％-20％(体积比)，更优选12％-18％(体积比)。
在本发明所述分离纯化方法的优选技术方案中，在步骤b)中，三乙胺-水溶液中，三乙胺比例为0.1％-0.3％(体积比)，优选0.15％-0.25％(体积比)，更优选0.18％-0.23％(体积比)。
在本发明所述分离纯化方法的优选技术方案中，在步骤b)中，用磷酸调节ph至2.0～4.0，优选2.5～3.5，更优选2.8～3.3。
在本发明所述分离纯化方法的优选技术方案中，在步骤c)中，所述色谱填料为反相硅胶基质色谱填料，如纳微公司的C8或者C18。填料粒径为5μm，或10μm，或30μm，优选10μm；填料孔径为60A，或100A，或120A，优选孔径为120A。
在本发明所述分离纯化方法的优选技术方案中，在步骤c)中，层析流动相为甲醇水溶液时，甲醇比例为5％-25％(体积比)，优选10％-20％(体积比)，更优选12％-17％(体积比)；层析流动相为乙醇水溶液时，乙醇比例为10％-30％(体积比)，优选15％-25％(体积比)，更优选18％-23％(体积比)；层析流动相为乙腈水溶液时，乙腈比例为5％-25％(体积比)，优选10％-20％(体积比)，更优选12％-18％(体积比)。
在本发明所述分离纯化方法的优选技术方案中，在步骤c)中缓冲溶液为甲酸-水溶液，乙酸-水溶液，或三氟乙酸-水溶液，其中，所加酸的比例为0.1～0.3％(体积比)，优选0.15％～0.25％(体积比)，更优选，0.18％-0.23％(体积比)。
在本发明所述分离纯化方法的优选技术方案中，在步骤d)中，减压浓缩时，温度控制在10-50℃，优选20-40℃，更优选25-35℃。
在本发明所述分离纯化方法的优选技术方案中，在步骤d)中，减压浓缩时，压力控制在-0.09mPa～-0.1mPa。
本发明以万古霉素粗品为原料，经过分离纯化得到纯度＞80％的万 古霉素类似物，并进行了结构鉴定，为进一步进行生物活性的筛选，或者对其进行结构修饰后再进行生物活性筛选，奠定了基础。通过后续工作，有可能得到药效更好、不良反应更少的新化合物。
附图说明
图1表示本发明得到的万古霉素类似物的元素组成图；
图2表示本发明得到的万古霉素类似物的MS图；
图3表示本发明得到的万古霉素类似物的C谱；
图4表示本发明得到的万古霉素类似物的H图；
具体实施方式
下面通过实施例来进一步说明本发明，本发明的实施例仅仅是用于说明本发明而给出，而不是对本发明的限制。
实施例1：
a)万古霉素类似物的第一次层析富集
含有万古霉素类似物的万古霉素粗品(该万古霉素粗品是按照中国专利CN101456903B进行制备)，用聚合物基质色谱填料，如UniPS，粒径为10μm，孔径为300A，进行第一次层析富集，流动相为甲醇水溶液，甲醇比例为33％(体积比)，等度洗脱，分段收集，纯度＞35％部分。
b)万古霉素类似物的第二次层析纯化
第一次层析富集后，纯度＞35％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C8，粒径为10μm，孔径为100A，进行第二次层析纯化，层析流动相为乙醇-缓冲溶液，乙醇比例为18％(体积比)，缓冲液为0.18％(体积比)三乙胺溶液，磷酸调教PH至2.8，等 度洗脱，分段收集，纯度＞65％部分进入下一步。
c)万古霉素类似物的第三次层析纯化
第二次层析纯化后，纯度＞65％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C18，粒径为10μm，孔径为120A，进行第三次层析纯化，层析流动相为乙腈-缓冲溶液，乙腈比例为12％(体积比)，缓冲溶液为0.18％(体积比)甲酸水溶液，等度洗脱，分段收集，纯度＞80％部分进入下一步。
d)万古霉素类似物的第三次层析纯化后，纯度＞80％部分收集液，减压浓缩，压力控制在-0.09mPa～-0.1mPa，控制温度为10℃，除去溶剂后冻干，得到万古霉素类似物干粉，液相检测纯度为85.1％，产品收率47.7％。
实施例2：
a)万古霉素类似物的第一次层析富集
含有万古霉素类似物的万古霉素粗品(该万古霉素粗品是按照中国专利CN101456903B进行制备)，用聚合物基质色谱填料，如UniPSA，粒径为30μm，孔径为100A，进行第一次层析富集，流动相为乙醇水溶液，乙醇比例为48％(体积比)，等度洗脱，分段收集，纯度＞35％部分。
b)万古霉素类似物的第二次层析纯化
第一次层析富集后，纯度＞35％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C18，粒径为10μm，孔径为60A，进行第二次层析纯化，层析流动相为甲醇-缓冲液，甲醇比例为12％(体积比)，缓冲液为0.20％(体积比)三乙胺溶液，磷酸调教PH至3.0，等度洗脱，分段收集，纯度＞65％部分进入下一步。
c)万古霉素类似物的第三次层析纯化
第二次层析纯化后，纯度＞65％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C8，粒径为30μm，孔径为120A，进行第三次层析纯化，层析流动相为乙醇-缓冲溶液溶液，乙醇比例为18％(体积比)，缓冲液为0.20％(体积比)乙酸溶液，等度洗脱，分段收集，纯度＞80％部分进入下一步。
d)万古霉素类似物的第三次层析纯化后，纯度＞80％部分收集液，减压浓缩，压力控制在-0.09mPa～-0.1mPa，控制温度为50℃，除去溶剂后冻干，得到万古霉素类似物干粉，液相检测纯度为91.5％，产品收率40.5％。
实施例3：
a)万古霉素类似物的第一次层析富集
含有万古霉素类似物的万古霉素粗品(该万古霉素粗品是按照中国专利CN101456903B进行制备)，用聚合物基质色谱填料，如UniPSN，粒径为30μm，孔径为300A，进行第一次层析富集，流动相为乙腈水溶液，乙腈比例为23％(体积比)，等度洗脱，分段收集，纯度＞35％部分。
b)万古霉素类似物的第二次层析纯化
第一次层析富集后，纯度＞35％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C18，粒径为30μm，孔径为120A，进行第二次层析纯化，层析流动相为乙腈-缓冲溶液溶液，乙腈比例为12％(体积比)，缓冲液为0.23％(体积比)三乙胺溶液，磷酸调教PH至3.3，等度洗脱，分段收集，纯度＞65％部分进入下一步。
c)万古霉素类似物的第三次层析纯化
第二次层析纯化后，纯度＞65％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C8，粒径为10μm，孔径为60A，进 行第三次层析纯化，层析流动相为甲醇-缓冲溶液，甲醇比例为12％(体积比)，缓冲液为0.23％(体积比)三氟乙酸溶液，等度洗脱，分段收集，纯度＞80％部分进入下一步。
d)万古霉素类似物的第三次层析纯化后，纯度＞80％部分收集液，减压浓缩，压力控制在-0.09mPa～-0.1mPa，控制温度为35℃，除去溶剂后冻干，得到万古霉素类似物干粉，液相检测纯度为83.2％，产品收率51.4％。
实施例4：
a)万古霉素类似物的第一次层析富集
含有万古霉素类似物的万古霉素粗品(该万古霉素粗品是按照中国专利CN101456903B进行制备)，用聚合物基质色谱填料，如UniPS，粒径为30μm，孔径为100A，进行第一次层析富集，流动相为甲醇水溶液，甲醇比例为34％(体积比)，等度洗脱，分段收集，纯度＞35％部分。
b)万古霉素类似物的第二次层析纯化
第一次层析富集后，纯度＞35％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C8，粒径为10μm，孔径为100A，进行第二次层析纯化，层析流动相为甲醇-缓冲溶液，甲醇比例为14％(体积比)，缓冲液为0.19％(体积比)三乙胺溶液，磷酸调教PH至2.9，等度洗脱，分段收集，纯度＞65％部分进入下一步。
c)万古霉素类似物的第三次层析纯化
第二次层析纯化后，纯度＞65％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C18，粒径为10μm，孔径为120A，进行第三次层析纯化，层析流动相为乙腈-缓冲溶液，乙腈比例为14％(体积比)，缓冲液为0.19％(体积比)甲酸水溶液，等度洗脱，分段 收集，纯度＞80％部分进入下一步。
d)万古霉素类似物的第三次层析纯化后，纯度＞80％部分收集液，减压浓缩，压力控制在-0.09mPa～-0.1mPa，控制温度为31℃，除去溶剂后冻干，得到万古霉素类似物干粉，液相检测纯度为87.9％，产品收率42.8％。
实施例5：
a)万古霉素类似物的第一次层析富集
含有万古霉素类似物的万古霉素粗品(该万古霉素粗品是按照中国专利CN101456903B进行制备)，用聚合物基质色谱填料，如UniPSA，粒径为10μm，孔径为300A，进行第一次层析富集，流动相为乙腈水溶液，乙腈比例为26％(体积比)，等度洗脱，分段收集，纯度＞35％部分。
b)万古霉素类似物的第二次层析纯化
第一次层析富集后，纯度＞35％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C18，粒径为10μm，孔径为120A，进行第二次层析纯化，层析流动相为乙醇-缓冲溶液，乙醇比例为51％(体积比)，缓冲液为0.21％(体积比)三乙胺溶液，磷酸调教PH至3.1，等度洗脱，分段收集，纯度＞65％部分进入下一步。
c)万古霉素类似物的第三次层析纯化
第二次层析纯化后，纯度＞65％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C18，粒径为5μm，孔径为120A，进行第三次层析纯化，层析流动相为乙腈-缓冲溶液，乙腈比例为16％(体积比)，缓冲液为0.21％(体积比)乙酸溶液，等度洗脱，分段收集，纯度＞80％部分进入下一步。
d)万古霉素类似物的第三次层析纯化后，纯度＞80％部分收集液， 减压浓缩，压力控制在-0.09mPa～-0.1mPa，控制温度为30℃，除去溶剂后冻干，得到万古霉素类似物干粉，液相检测纯度为86.1％，产品收率44.8％。
实施例6：
a)万古霉素类似物的第一次层析富集
含有万古霉素类似物的万古霉素粗品(该万古霉素粗品是按照中国专利CN101456903B进行制备)，用聚合物基质色谱填料，如UniPSN，粒径为10μm，孔径为100A，进行第一次层析富集，流动相为乙醇水溶液，乙醇比例为51％(体积比)，等度洗脱，分段收集，纯度＞35％部分。
b)万古霉素类似物的第二次层析纯化
第一次层析富集后，纯度＞35％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C8，粒径为30μm，孔径为120A，进行第二次层析纯化，层析流动相为甲醇-缓冲溶液，甲醇比例为16％(体积比)，缓冲液为0.23％(体积比)三乙胺溶液，磷酸调教PH至3.2，等度洗脱，分段收集，纯度＞65％部分进入下一步。
c)万古霉素类似物的第三次层析纯化
第二次层析纯化后，纯度＞65％部分收集液，减压浓缩除去溶剂，再用反相硅胶基质色谱填料，如C8，粒径为10μm，孔径为120A，进行第三次层析纯化，层析流动相为甲醇水溶液，甲醇比例为15％(体积比)，缓冲液为0.2％(体积比)三氟乙酸溶液，等度洗脱，分段收集，纯度＞80％部分进入下一步。
d)万古霉素类似物的第三次层析纯化后，纯度＞80％部分收集液，减压浓缩，压力控制在-0.09mPa～-0.1mPa，控制温度为40℃，除去溶剂后冻干，得到万古霉素类似物干粉，液相检测纯度为80.3％，产品收 率52.9％。
实施例7：
利用UPLC-MS和核磁共振对样品进行检测，确定样品的结构。
1、UPLC-MS分析：仪器：Q-TOF micro；测试条件：ES+源。
测试结果：
Figure PCTCN2020079200-appb-000003
解析表1：质谱电喷雾电离源(ESI)正离子检测,测得化合物的[M+H] +峰为1431.5046。确定元素组成为C 66H 73N 8O 24Cl 2，与万古霉素脱NH 3加H的元素组成一致。
Figure PCTCN2020079200-appb-000004
2、核磁图谱分析：
测试仪器：Bruker 600MHz；测试溶剂：DMSO-d6。
Figure PCTCN2020079200-appb-000005
根据 13C-NMR谱(图3)、 1H-NMR谱(图4)表明样品为如下式(I)化学结构如下。
Figure PCTCN2020079200-appb-000006
需要声明的是，上述发明内容及具体实施方式意在证明本发明所提供技术方案的实际应用，不应解释为对本发明保护范围的限定。本领域技术人员在本发明的精神和原理内，当可作各种修改、等同替换、或改进。

Claims (10)

  1. 一种万古霉素类似物(I)的分离纯化方法，其特征在于，所述分离纯化方法包括以下步骤：
    a)万古霉素类似物的第一次层析富集：
    以含有万古霉素类似物的粗品为原料，用聚合物基质色谱填料进行第一次层析富集，等度洗脱或梯度洗脱，分段收集纯度＞35％的第一次层析富集液；其中，所述聚合物基质色谱填料为聚苯乙烯/二乙烯基苯共聚物UniPS、UniPSA、或聚丙烯酸酯和二乙烯基苯混合体共聚物UniPSN，流动相为甲醇水溶液、乙醇水溶液、或乙腈水溶液；
    b)万古霉素类似物的第二次层析纯化：
    将步骤a)得到的纯度＞35％的第一次层析富集液进行减压浓缩以除去溶剂，用反相硅胶基质色谱填料进行第二次层析纯化，用磷酸调节pH至酸性，等度洗脱或梯度洗脱，分段收集纯度＞65％的第二次层析富集液；其中，所述反相硅胶基质色谱填料为辛烷基硅胶C8或者十八烷基硅胶C18，流动相为甲醇-缓冲溶液、乙醇-缓冲溶液、或乙腈-缓冲溶液，所述缓冲溶液为三乙胺-水溶液；
    c)万古霉素类似物的第三次层析纯化：
    将步骤b)得到的纯度＞65％的第二次层析富集液进行减压浓缩以除去溶剂，再用反相硅胶基质色谱填料进行第三次层析纯化，等度洗脱或梯度洗脱，分段收集纯度＞80％的第三次层析富集液；其中，所述反相硅胶基质色谱填料为辛烷基硅胶或者十八烷基硅胶，流动相为甲醇-缓冲溶液、乙醇-缓冲溶液、或乙腈-缓冲溶液，所述缓冲溶液为甲酸-水溶液、乙酸-水溶液、或三氟乙酸-水溶液；以及
    d)将纯度＞80％的第三次层析富集液进行减压浓缩以除去溶剂，冻干，得到万古霉素类似物(I)的干粉。
  2. 根据权利要求1所述的方法，其特征在于，在步骤a)中，所述聚合物基质色谱填料的粒径为5μm、10μm或30μm；所述聚合物基质色谱填料的孔径为100A、300A或500A。
  3. 根据权利要求1所述的方法，其特征在于，在步骤a)中，所述流动相为甲醇水溶液时，甲醇比例为25％-45％(体积比)；所述流动相为乙醇水溶液时，乙醇比例为40％-60％(体积比)；所述流动相为乙腈水溶液时，乙腈比例为15％-35％(体积比)。
  4. 根据权利要求1所述的方法，其特征在于，在步骤b)和步骤c)中，所述反相硅胶基质色谱填料的粒径为5μm，或10μm，或30μm，优选10μm；所述反相硅胶基质色谱填料的孔径为60A、100A、120A。
  5. 根据权利要求1所述的方法，其特征在于，在步骤b)和步骤c)中，所述流动相为甲醇水溶液时，甲醇比例为5％-25％(体积比)；所述流动相为乙醇水溶液时，乙醇比例为10％-30％(体积比)；所述流动相为乙腈水溶液时，乙腈比例为5％-25％(体积比)。
  6. 根据权利要求1所述的方法，其特征在于，在步骤b)中，所述三乙胺-水溶液中的三乙胺比例为0.1％-0.3％(体积比)。
  7. 根据权利要求1所述的方法，其特征在于，在步骤b)中，用磷酸调节pH至2.0～4.0。
  8. 根据权利要求1所述的方法，其特征在于，在步骤c)中，所述缓冲溶液中所加酸的比例为0.1～0.3％(体积比)。
  9. 根据权利要求1所述的方法，其特征在于，在步骤d)中，减压浓缩的温度控制在10-50℃。
  10. 根据权利要求1所述的方法，其特征在于，在步骤d)中，减压浓缩的压力控制在-0.09mPa～-0.1mPa。
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