技术领域：Technical field:
本发明属于天然产物技术领域，具体涉及4’-N-demethyl-vicenistatin及其制法和用途。The invention belongs to the technical field of natural products, and specifically relates to 4'-N-demethyl-vicenistatin and its preparation method and application.
背景技术：Background technique:
广谱、高效、低毒性的抗生素一直被认为是20世纪人类最伟大的发明之一。作为抗生素的主要生产菌，放线菌生产的许多次级代谢产物被开发利用为医疗卫生上有临床应用价值的抗菌药物，例如应用于临床抗感染的抗生素红霉素(erythromycin)、万古霉素(vancomycin)、达托霉素(daptomycin)等。但是由于全球广泛存在的抗生素滥用、误用现象，抗生素耐药性的出现愈发频繁，病源微生物能轻而易举地从环境中获得耐药性基因，并导致临床上使用的抗生素对致病菌的有效性大大降低。因此，抗生素耐药性问题已成为临床抗感染药物治疗的严峻挑战，临床上现行有效的抗生素将不足以应对具有广谱耐药性的致病性“超级细菌”，研发具有新颖抗菌机制的抗生素药物成为解决目前细菌耐药性困境的重要手段。Broad-spectrum, high-efficiency, and low-toxicity antibiotics have always been considered one of the greatest inventions of mankind in the 20th century. As the main antibiotic-producing bacteria, many secondary metabolites produced by actinomycetes have been developed and utilized as antibacterial drugs with clinical application value in medical and health, such as erythromycin and vancomycin, which are used in clinical anti-infection antibiotics. (vancomycin), daptomycin (daptomycin), etc. However, due to the widespread abuse and misuse of antibiotics in the world, the emergence of antibiotic resistance is becoming more and more frequent, and pathogenic microorganisms can easily acquire drug resistance genes from the environment, which leads to the effectiveness of clinically used antibiotics against pathogenic bacteria. Sex is greatly reduced. Therefore, the problem of antibiotic resistance has become a serious challenge for clinical anti-infective drug treatment. The current clinically effective antibiotics will not be enough to deal with pathogenic "super bacteria" with broad-spectrum drug resistance. Antibiotics with novel antibacterial mechanisms should be developed. Drugs have become an important means to solve the current plight of bacterial drug resistance.
肿瘤是严重威胁人类生命、影响人类健康的重大疾病，根据世界卫生组织的报告，全球肿瘤的发病率和死亡率呈快速增长的趋势。化疗是肿瘤治疗的重要手段，各种化疗药物在肿瘤治疗过程中被广泛使用，然而肿瘤细胞对化疗药物产生多药耐药性、化疗药物的毒副作用以及部分患者对化疗药物的不敏感性是导致化疗失败的主要原因。因此，在肿瘤治疗领域，对于新机理、新靶点的创新药物有着迫切而巨大的需求。Tumor is a major disease that seriously threatens human life and affects human health. According to the report of the World Health Organization, the morbidity and mortality of tumors in the world are increasing rapidly. Chemotherapy is an important means of tumor treatment. Various chemotherapeutic drugs are widely used in the process of tumor treatment. However, tumor cells have multidrug resistance to chemotherapeutic drugs, toxic side effects of chemotherapeutic drugs, and insensitivity of some patients to chemotherapeutic drugs. leading cause of chemotherapy failure. Therefore, in the field of tumor treatment, there is an urgent and huge demand for innovative drugs with new mechanisms and new targets.
Vicenistatin是一种大环内酰胺类抗生素，其结构如式a所示，最早由Shindo等人在1993年从放线菌Streptomyces halstedii HC34中分离获得。从结构上分析，vicenistatin由一个特殊的氨基糖vicenisamine及大环内酰胺母核vicenilactam组成。对vicenistatin的生物活性研究发现，该化合物在体外条件下对人血癌细胞株HL-60(IC50＝0.12μg/mL)和人结肠癌细胞株COLO205(IC50＝0.19μg/mL)具有显著的细胞毒活性，并在植入人结肠癌细胞株Co-3的裸鼠模型中测得vicenistatin具有显著的体内抗肿瘤活性。因此，大环内酰胺类天然产物vicenistatin是一类具有成药潜力的先导化合物，该化合物的成药性值得深入研究。Vicenistatin is a macrolactam antibiotic whose structure is shown in formula a. It was first isolated from Actinomyces Streptomyces halstedii HC34 by Shindo et al. in 1993. Structurally, vicenistatin is composed of a special amino sugar vicenisamine and a macrolactam nucleus vicenilactam. The study on the biological activity of vicenistatin found that the compound has significant cytotoxicity to human blood cancer cell line HL-60 (IC50=0.12μg/mL) and human colon cancer cell line COLO205 (IC50=0.19μg/mL) in vitro Activity, and vicenistatin has significant anti-tumor activity in vivo measured in nude mice implanted with human colon cancer cell line Co-3. Therefore, vicenistatin, a natural product of macrocyclic lactams, is a kind of lead compound with druggability, and the druggability of this compound is worthy of further study.
近年来微生物来源药物的研发逐渐变得困难，以土壤放线菌为主的陆生微生物资源经历了近80年的长时期、高强度、大范围的筛选，导致菌株和次级代谢产物重复发现几率大大增加。因此，以“老药新用”策略为指导，扩宽现行有效的临床药物的治疗用途并充分挖掘已知先导化合物的成药潜力，可满足抗菌和抗肿瘤新药研发中对新机理和新靶点药物的迫切需求。另一方面，通过对原研药的结构进行理性改造和修饰，往往可以获得疗效更好的派生药，而对已知先导化合物的结构进行改造获得结构衍生物，是开发具有良好药理作用的候选药物的重要手段。因此，先导化合物vicenistatin生物活性评价及结构衍生化研究对抗感染、抗肿瘤新药开发具有重要的意义。In recent years, the development of microbial-derived drugs has gradually become difficult. The terrestrial microbial resources dominated by soil actinomycetes have undergone long-term, high-intensity, and large-scale screening for nearly 80 years, resulting in repeated discoveries of strains and secondary metabolites. The odds are greatly increased. Therefore, under the guidance of the "old drug new use" strategy, expanding the therapeutic use of currently effective clinical drugs and fully tapping the drug potential of known lead compounds can meet the needs of new mechanisms and new targets in the development of new antibacterial and anti-tumor drugs. urgent need for medicines. On the other hand, by rationally modifying and modifying the structure of the original drug, derivative drugs with better curative effect can often be obtained, while modifying the structure of known lead compounds to obtain structural derivatives is a candidate drug with good pharmacological effects. important means. Therefore, the biological activity evaluation and structural derivation research of the lead compound vicenistatin are of great significance for the development of new anti-infection and anti-tumor drugs.
发明内容：Invention content:
本发明的第一个目的是提供一种具有抗感染、抗肿瘤活性的N-脱甲基vicenistatin衍生物4’-N-demethyl-vicenistatin，所述的4’-N-demethyl-vicenistatin，其结构如式(Ⅰ)：The first object of the present invention is to provide a kind of N-demethyl vicenistatin derivative 4'-N-demethyl-vicenistatin with anti-infection and anti-tumor activity, said 4'-N-demethyl-vicenistatin, its structure Such as formula (Ⅰ):
本发明的第二个目的是提供如式(Ⅰ)所示的4’-N-demethyl-vicenistatin的制备方法，所述的4’-N-demethyl-vicenistatin是从Streptomycesparvus SCSIO Mla-L010的N-甲基转移酶失活突变株SCSIO Mla-L010/ΔvicG中分离得到，所述SCSIO Mla-L010/ΔvicG是利用同源重组技术将抗性标记基因替换S.parvus SCSIO Mla-L010基因组的vicenistatin基因簇中N-甲基转移酶基因vicG从而获得的，所述的vicG序列如SEQ IDNO.1所示。The second object of the present invention is to provide the preparation method of 4'-N-demethyl-vicenistatin as shown in formula (I), and described 4'-N-demethyl-vicenistatin is N- Methyltransferase inactivated mutant strain SCSIO Mla-L010/ΔvicG was isolated from the vicenistatin gene cluster of S.parvus SCSIO Mla-L010 genome by replacing the resistance marker gene with homologous recombination technology The N-methyltransferase gene vicG in N-methyltransferase gene vicG is thus obtained, and the vicG sequence is shown in SEQ ID NO.1.
本发明所涉及的海洋来源的vicenistatin生产菌株S.parvus SCSIO Mla-L010分离自海洋无脊椎动物单齿螺(Monodonata labio)样品，样品采集地点为中国南海大亚湾高潮间带。利用同源重组技术将抗性标记基因替换S.parvus SCSIO Mla-L010基因组的vicenistatin基因簇中N-甲基转移酶基因vicG，获得N-甲基转移酶失活突变株SCSIO Mla-L010/ΔvicG。The vicenistatin producing strain S.parvus SCSIO Mla-L010 of marine origin involved in the present invention is isolated from a marine invertebrate monodentate snail (Monodonata labio) sample, and the sampling location is the high intertidal zone of Daya Bay, South China Sea. Using homologous recombination technology to replace the N-methyltransferase gene vicG in the vicenistatin gene cluster of S.parvus SCSIO Mla-L010 genome with the resistance marker gene, and obtain the N-methyltransferase inactive mutant strain SCSIO Mla-L010/ΔvicG .
在一优选例中，所述的4’-N-demethyl-vicenistatin的制备方法包括如下步骤：In a preferred example, the preparation method of said 4'-N-demethyl-vicenistatin comprises the following steps:
(1)制备N-甲基转移酶失活突变株SCSIO Mla-L010/ΔvicG的发酵培养物，将发酵上清液和菌丝体离心分离，菌丝体用丙酮萃取，上清液用丁酮萃取，获得丙酮萃取液和丁酮萃取液，然后通过减压浓缩萃取液后得到菌丝体浸膏和上清液浸膏，将两个浸膏混合得到粗浸膏；(1) Prepare the fermentation culture of the N-methyltransferase inactivated mutant strain SCSIO Mla-L010/ΔvicG, centrifuge the fermentation supernatant and mycelium, extract the mycelium with acetone, and use butanone for the supernatant Extracting, obtaining acetone extract and butanone extract, then concentrating the extract under reduced pressure to obtain mycelium extract and supernatant extract, mixing the two extracts to obtain a crude extract;
(2)将步骤(1)得到的粗浸膏用硅胶拌样并进行正相硅胶柱层析，采用氯仿/甲醇体系按以下体积比进行梯度洗脱：100:0；98:2；96:4；94:6；92:8；9:1；8:2；6:4；5:5；0:100，顺序得到Fr.A1～10共10个组分；将组分氯仿/甲醇92:8；9:1；8:2；6:4洗脱的Fr.A5～8合并，通过HPLC纯化，得到所述的4’-N-demethyl-vicenistatin。(2) Mix the crude extract obtained in step (1) with silica gel and carry out normal phase silica gel column chromatography, and use chloroform/methanol system to carry out gradient elution according to the following volume ratio: 100:0; 98:2; 96: 4; 94:6; 92:8; 9:1; 8:2; 6:4; 5:5; 0:100, sequentially obtained 10 components of Fr.A1～10; :8; 9:1; 8:2; 6:4 The eluted Fr.A5-8 were combined and purified by HPLC to obtain the 4'-N-demethyl-vicenistatin.
进一步地，所述的对菌丝体和上清液进行萃取是向菌丝体沉淀加入3倍体积丙酮进行萃取，重复萃取3次后，将萃取液减压浓缩成浸膏，向发酵上清液中加入等体积丁酮萃取3次，将萃取液减压浓缩成浓缩浸膏。Further, the extraction of the mycelium and supernatant is to add 3 times the volume of acetone to the mycelium precipitation for extraction. After repeated extraction for 3 times, the extract is concentrated under reduced pressure into an extract, and then added to the fermentation supernatant An equal volume of butanone was added to the liquid for extraction three times, and the extract was concentrated under reduced pressure to form a concentrated extract.
进一步地，步骤(2)中所述的通过HPLC纯化，是以甲醇为流动相，采用SephadexLH-20羟丙基葡聚糖凝胶色谱柱进行层析并收集目标组分，利用半制备高效液相色谱和ODS色谱柱(250×10mm,5μm)，按以下条件制备目标峰：CH3CN/H2O(32:68，V/V，含0.1％醋酸)体系等度洗脱25min，流速2.5mL/min，得到所述的4’-N-demethyl-vicenistatin(tR＝14.1min)。Further, the HPLC purification described in step (2) uses methanol as the mobile phase, uses SephadexLH-20 hydroxypropyl dextran gel chromatography column for chromatography and collects target components, and uses semi-preparative high-efficiency liquid Phase chromatography and ODS chromatographic column (250×10mm, 5μm), prepare the target peak according to the following conditions: CH3 CN/H2 O (32:68, V/V, containing 0.1% acetic acid) system isocratic elution 25min, flow rate 2.5 mL/min to obtain the 4'-N-demethyl-vicenistatin (tR =14.1 min).
本发明的第三个目的是提供如式(Ⅰ)所示的4’-N-demethyl-vicenistatin在制备抗感染药物或抗肿瘤药物中的应用。The third object of the present invention is to provide the application of 4'-N-demethyl-vicenistatin represented by formula (I) in the preparation of anti-infective drugs or anti-tumor drugs.
在一优选例中，所述的抗感染药物为抗细菌感染药物或抗真菌感染药物；进一步地，所述的抗感染药物为抗金黄色葡萄球菌(Staphylococcus aureus)、耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus)、藤黄微球菌(Micrococcusluteus)、枯草芽孢杆菌(Bacillus subtilis)、新生隐球菌(Cryptoccus neoformans)，幽门螺杆菌(Helicobacter pylori)或白色念珠菌(Candida albicans)药物。In a preferred example, the anti-infective drug is an anti-bacterial infection drug or an anti-fungal infection drug; further, the anti-infective drug is anti-Staphylococcus aureus, methicillin-resistant grape Methicillin-resistant Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Cryptoccus neoformans, Helicobacter pylori, or Candida albicans drug.
在另一优选例中，所述的抗肿瘤药物为抗乳腺癌、肝癌、宫颈癌、非小细胞肺癌、结肠腺癌或白血病药物；进一步地，所述的抗肿瘤药物为抗人乳腺癌细胞MCF-7、人肝癌细胞HepG2、人宫颈癌细胞HeLa、人非小细胞肺癌细胞A549、人结肠腺癌细胞RKO或人急性早幼粒白血病细胞HL-60药物。In another preferred example, the anti-tumor drug is an anti-breast cancer, liver cancer, cervical cancer, non-small cell lung cancer, colon adenocarcinoma or leukemia drug; further, the anti-tumor drug is an anti-human breast cancer cell MCF-7, human liver cancer cell HepG2, human cervical cancer cell HeLa, human non-small cell lung cancer cell A549, human colon adenocarcinoma cell RKO or human acute promyelocytic leukemia cell HL-60 drug.
本发明的第四个目的是提供一种抗感染药物，其含有如式(Ⅰ)所示的4’-N-demethyl-vicenistatin作为活性成分。The fourth object of the present invention is to provide an anti-infective drug containing 4'-N-demethyl-vicenistatin represented by formula (I) as an active ingredient.
优选地，所述的抗感染药物为抗细菌感染药物或抗真菌感染药物；更优选地，所述的抗感染药物为抗金黄色葡萄球菌(Staphylococcus aureus)、耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus)、藤黄微球菌(Micrococcusluteus)、枯草芽孢杆菌(Bacillus subtilis)、新生隐球菌(Cryptoccus neoformans)，幽门螺杆菌(Helicobacter pylori)或白色念珠菌(Candida albicans)药物。Preferably, the anti-infective drug is an anti-bacterial infection drug or an anti-fungal infection drug; more preferably, the anti-infective drug is an anti-Staphylococcus aureus (Staphylococcus aureus), methicillin-resistant Staphylococcus aureus ( Methicillin-resistant Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Cryptoccus neoformans, Helicobacter pylori, or Candida albicans drug.
本发明的第五个目的是提供一种抗肿瘤药物，其含有如式(Ⅰ)所示的4’-N-demethyl-vicenistatin作为活性成分。The fifth object of the present invention is to provide an antineoplastic drug containing 4'-N-demethyl-vicenistatin represented by formula (I) as an active ingredient.
优选地，所述的抗肿瘤药物为抗乳腺癌、肝癌、宫颈癌、非小细胞肺癌、结肠腺癌或白血病药物；更优选地，所述的抗肿瘤药物为抗人乳腺癌细胞MCF-7、人肝癌细胞HepG2、人宫颈癌细胞HeLa、人非小细胞肺癌细胞A549、人结肠腺癌细胞RKO或人急性早幼粒白血病细胞HL-60药物。Preferably, the anti-tumor drug is an anti-breast cancer, liver cancer, cervical cancer, non-small cell lung cancer, colon adenocarcinoma or leukemia drug; more preferably, the anti-tumor drug is an anti-human breast cancer cell MCF-7 , human liver cancer cell HepG2, human cervical cancer cell HeLa, human non-small cell lung cancer cell A549, human colon adenocarcinoma cell RKO or human acute promyelocytic leukemia cell HL-60.
本发明的第六个目的是提供N-甲基转移酶失活突变株SCSIO Mla-L010/ΔvicG在制备4’-N-demethyl-vicenistatin中的应用。The sixth object of the present invention is to provide the application of the N-methyltransferase inactivated mutant strain SCSIO Mla-L010/ΔvicG in the preparation of 4'-N-demethyl-vicenistatin.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)试验结果表明，4’-N-demethyl-vicenistatin对细菌和真菌具有显著的抑制活性，对金黄色葡萄球菌Staphylococcus aureus ATCC 29213，6株耐甲氧西林金黄色葡萄球菌Methicillin-resistant Staphylococcus aureus(MRSA)shhs-E1、MRSA 16339、MRSA745524、MRSA 16162、MRSA718306和MRSA 6917，藤黄微球菌Micrococcus luteus，枯草芽孢杆菌Bacillus subtilis，新生隐球菌Cryptoccus neoformans，幽门螺杆菌Helicobacter pylori和白色念珠菌Candida albicans的最小抑菌浓度(minimuminhibitory concentration，MIC)在0.125～2μg/mL，显示出其在抗感染药物开发中的重要价值；(1) The test results show that 4'-N-demethyl-vicenistatin has significant inhibitory activity against bacteria and fungi, against Staphylococcus aureus ATCC 29213, 6 strains of methicillin-resistant Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) shhs-E1, MRSA 16339, MRSA745524, MRSA 16162, MRSA718306 and MRSA 6917, Micrococcus luteus, Bacillus subtilis, Cryptoccus neoformans, Helicobacter pylori and Candida albican the s The minimum inhibitory concentration (minimuminhibitory concentration, MIC) of 0.125 ~ 2μg/mL, showing its important value in the development of anti-infective drugs;
(2)N-脱甲基vicenistatin衍生物4’-N-demethyl-vicenistatin对真菌白色念珠菌Candida albicans的抗菌活性比vicenistatin强2-4倍，显示出该结构衍生物具有更好的成药潜力。(2) The antibacterial activity of N-demethyl vicenistatin derivative 4'-N-demethyl-vicenistatin against the fungus Candida albicans is 2-4 times stronger than that of vicenistatin, showing that the derivative of this structure has better drug-making potential.
(3)4’-N-demethyl-vicenistatin还对多种癌细胞显示出较强的细胞毒活性，具有开发成为抗肿瘤药物的潜力。(3) 4'-N-demethyl-vicenistatin also shows strong cytotoxic activity against various cancer cells, and has the potential to be developed as an antitumor drug.
本发明的放线菌菌株Streptomyces parvus SCSIO Mla-L010公开于http://hyyw.journalsystem.net/ch/reader/view_abstract.aspx？file_no＝202104020000001，该菌株本申请人也持有，保证自申请日起20年内向公众发放。Actinomyces strain Streptomyces parvus SCSIO Mla-L010 of the present invention is disclosed in http://hyyw.journalsystem.net/ch/reader/view_abstract.aspx? file_no=202104020000001, this strain is also held by the applicant, and it is guaranteed to be released to the public within 20 years from the date of application.
附图说明：Description of drawings:
图1是SCSIO Mla-L010/ΔvicG基因双交换突变株的构建；Figure 1 is the construction of the SCSIO Mla-L010/ΔvicG gene double exchange mutant;
图2是野生型菌株S.parvus SCSIO Mla-L010和vicG基因突变株SCSIO Mla-L010/ΔvicG的发酵提取液的HPLC分析；Fig. 2 is the HPLC analysis of the fermentation extract of wild-type bacterial strain S.parvus SCSIO Mla-L010 and vicG gene mutant strain SCSIO Mla-L010/ΔvicG;
图3为4’-N-demethyl-vicenistatin的高分辨质谱相关信息；Figure 3 is the high-resolution mass spectrum related information of 4'-N-demethyl-vicenistatin;
图4为4’-N-demethyl-vicenistatin的1H-NMR相关信息；Figure 4 is the1 H-NMR related information of 4'-N-demethyl-vicenistatin;
图5为4’-N-demethyl-vicenistatin的13C-NMR相关信息。Figure 5 shows the13 C-NMR related information of 4'-N-demethyl-vicenistatin.
具体实施方式：Detailed ways:
以下实施例是对本发明的进一步说明，而不是对本发明的限制。如无特殊说明，所使用的实验方法是本领域技术人员所熟知的，所使用的实验材料可以通过商业渠道获得的。The following examples are to further illustrate the present invention, rather than limit the present invention. Unless otherwise specified, the experimental methods used are well known to those skilled in the art, and the experimental materials used can be obtained through commercial channels.
一般性说明General Notes
本发明所涉及的海洋来源的菌株Streptomyces parvus SCSIO Mla-L010分离自海洋无脊椎动物单齿螺(Monodonata labio)样品，样品采集地点为中国南海大亚湾高潮间带。利用同源重组技术将抗性标记基因替换S.parvus SCSIO Mla-L010基因组的vicenistatin基因簇中N-甲基转移酶基因vicG，其核苷酸序列如SEQ ID NO.1所示，长度为711bp，获得N-甲基转移酶失活突变株SCSIO Mla-L010/ΔvicG。The marine-derived bacterial strain Streptomyces parvus SCSIO Mla-L010 involved in the present invention is isolated from a marine invertebrate monodentate snail (Monodonata labio) sample, and the sampling location is the high intertidal zone of Daya Bay, South China Sea. Use homologous recombination technology to replace the N-methyltransferase gene vicG in the vicenistatin gene cluster of the S.parvus SCSIO Mla-L010 genome with the resistance marker gene, its nucleotide sequence is shown in SEQ ID NO.1, and its length is 711bp , to obtain N-methyltransferase inactivation mutant SCSIO Mla-L010/ΔvicG.
(1)产孢培养基和发酵培养基的配制：(1) Preparation of sporulation medium and fermentation medium:
以MS固体培养基作为以上两株菌株的产孢平板，其组成如下：黄豆片10g/L，黄豆粉10g/L，D-甘露醇20g/L，调节pH值至7.3，分装后加入质量分数2.5％技术琼脂粉，121℃灭菌30min后备用。Use MS solid medium as the sporulation plate of the above two strains, and its composition is as follows: 10g/L of soybean flakes, 10g/L of soybean powder, 20g/L of D-mannitol, adjust the pH value to 7.3, add mass Fraction 2.5% technical agar powder, sterilized at 121°C for 30 minutes for later use.
以改良AM3液体培养基作为以上两株菌株的小瓶发酵及大规模发酵培养基培养基，其组成如下：黄豆粉2.5g/L，淀粉15g/L，甘油15g/L，细菌学蛋白胨7.5g/L，硫酸铵1g/L，海盐30g/L，调节pH值至7.3后加入碳酸钙5g/L，121℃灭菌30min后备用。Using the improved AM3 liquid medium as the vial fermentation and large-scale fermentation medium medium of the above two bacterial strains, its composition is as follows: soybean powder 2.5g/L, starch 15g/L, glycerol 15g/L, bacteriological peptone 7.5g/L L, ammonium sulfate 1g/L, sea salt 30g/L, adjust the pH value to 7.3, add calcium carbonate 5g/L, and sterilize at 121°C for 30min before use.
(2)菌株培养及发酵条件：(2) Strain cultivation and fermentation conditions:
4’-N-demethyl-vicenistatin生产菌株SCSIO Mla-L010/ΔvicG菌株使用MS固体培养基(含有50μg/mL奥普拉霉素)作为产孢平板，置于28℃孵箱培养7天至孢子成熟。The 4'-N-demethyl-vicenistatin producing strain SCSIO Mla-L010/ΔvicG strain uses MS solid medium (containing 50 μg/mL oppramycin) as a sporulation plate, and cultures it in a 28°C incubator for 7 days until the spores mature .
小瓶发酵：将MS固体培养基上成熟的孢子接种至改良AM3液体培养基(50mL)中，摇床振荡培养7d，摇床条件设置为28℃、200r/min。加入2倍体积的丁酮萃取发酵液，减压浓缩成浸膏后加入甲醇溶解并定容至1mL作为菌株发酵浓缩液，并取20μL浓缩液进样至HPLC分析，上述两株菌株发酵情况的HPLC分析结果如图2所示。Vial fermentation: Inoculate the mature spores on the MS solid medium into the modified AM3 liquid medium (50 mL), shake and cultivate for 7 days on a shaker, and set the shaker conditions at 28°C and 200r/min. Add 2 times the volume of methyl ethyl ketone to extract the fermentation liquid, concentrate it under reduced pressure to form an extract, add methanol to dissolve it and make it volume up to 1mL as a strain fermentation concentrate, and take 20 μL of the concentrate and inject it into HPLC for analysis. The fermentation status of the above two strains The results of HPLC analysis are shown in FIG. 2 .
规模发酵：将MS固体培养基上成熟的孢子接种至改良AM3液体培养基(50mL)中作为种子液，摇床振荡培养2d，将种子液转接至200mL的改良AM3液体培养基，发酵7d，摇床条件设置为28℃、200r/min。Scale fermentation: Inoculate the mature spores on the MS solid medium into the improved AM3 liquid medium (50mL) as the seed liquid, shake the shaker for 2d, transfer the seed liquid to 200mL of the improved AM3 liquid medium, and ferment for 7d. Shaking table conditions were set at 28°C, 200r/min.
实施例1甲基转移酶基因突变株SCSIO Mla-L010/ΔvicG的构建Construction of embodiment 1 methyltransferase gene mutant strain SCSIO Mla-L010/ΔvicG
1.S.parvus SCSIO Mla-L010基因组文库的构建及含有甲基转移酶基因的粘粒的筛选1. Construction of S.parvus SCSIO Mla-L010 Genomic Library and Screening of Cosmids Containing Methyltransferase Genes
1.1 S.parvus SCSIO Mla-L010基因组文库的构建1.1 Construction of S.parvus SCSIO Mla-L010 Genomic Library
参照SuperCos1 Cosmid Vector Kit(Agilent)和Gigapck III XL packingExtract(Epicentre)操作手册进行S.parvus SCSIO Mla-L010基因组文库的构建。液体深层培养S.parvus SCSIO Mla-L010菌株2天后，离心收集菌体，采用酚-氯仿抽提法提取基因组DNA。取适宜浓度的高纯度DNA加入一定浓度的Sau3AI酶，反应一定时间将基因组DNA酶切为适宜的片段大小，可跑核酸胶检测，酶切产物用酚-氯仿抽提纯化并进行去磷酸化处理。与此同时，用XbaI限制性内切酶处理SuperCos1载体，酚-氯仿抽提一次后同样进行去磷酸化处理，去磷酸化后再用BamHI限制性内切酶处理并抽提纯化。经上述方法处理后的基因组DNA和SuperCos1载体在T4连接酶的作用下随机连接，并跑胶验证连接效率。之后，将连接产物用包装蛋白进行包装并侵染文库菌E.coli LE392，把被侵染后的E.coli LE392涂布在含有卡那霉素的抗性平板上。待长出单克隆后，随机挑取约2000个克隆接种于96孔板中，37℃培养至一定浓度后置于-80℃冰箱保存，完成S.parvus SCSIO Mla-L010的基因组文库的构建。The S.parvus SCSIO Mla-L010 genomic library was constructed with reference to the SuperCos1 Cosmid Vector Kit (Agilent) and Gigapck III XL packingExtract (Epicentre) operating manuals. After submerged culture of S.parvus SCSIO Mla-L010 strain for 2 days, the cells were collected by centrifugation, and genomic DNA was extracted by phenol-chloroform extraction. Take a suitable concentration of high-purity DNA and add a certain concentration of Sau3AI enzyme, and react for a certain period of time to digest the genomic DNA into a suitable fragment size, which can be tested on a nucleic acid gel, and the digested product is extracted and purified with phenol-chloroform and dephosphorylated. . At the same time, the SuperCos1 carrier was treated with XbaI restriction endonuclease, dephosphorylated after phenol-chloroform extraction once, and then treated with BamHI restriction endonuclease and extracted and purified after dephosphorylation. The genomic DNA and SuperCos1 vector treated by the above method were randomly ligated under the action of T4 ligase, and the ligation efficiency was verified by gel running. Afterwards, the ligation product was packaged with packaging protein and infected with library bacteria E.coli LE392, and the infected E.coli LE392 was spread on the resistance plate containing kanamycin. After monoclonal growth, about 2000 clones were randomly picked and inoculated in 96-well plates, cultured at 37°C to a certain concentration and stored in a -80°C refrigerator to complete the construction of the genome library of S.parvus SCSIO Mla-L010.
1.2含有甲基转移酶基因vicG的cosmid质粒1-F11的筛选1.2 Screening of cosmid plasmid 1-F11 containing methyltransferase gene vicG
采用二代测序技术Oxford Nanopore对S.parvus SCSIO Mla-L010进行全基因组测序并拼接得到全基因组图谱和序列。将该序列上传至在线生物信息学分析软件antiSMASH(https://antismash.secondarymetabolites.org/#！/start)，搜索可能编码聚醚类化合物的次级代谢产物基因簇，通过基因敲除实验加以验证。进一步分析基因簇中各个基因的功能并筛选出编码甲基转移酶的基因为vicG(其核苷酸序列入SEQ ID NO.1所示)，并确定了其分布的位置。因为cosmid能承载的DNA片段大小为30-40kb，为了筛选出包含甲基转移酶基因vicG的cosmid质粒，在基因簇上合适的位置设计了三对筛库引物，序列如下：The whole genome of S.parvus SCSIO Mla-L010 was sequenced and spliced using the next-generation sequencing technology Oxford Nanopore to obtain the whole genome map and sequence. Upload the sequence to the online bioinformatics analysis software antiSMASH (https://antismash.secondarymetabolites.org/#!/start), search for the secondary metabolite gene clusters that may encode polyether compounds, and identify them through gene knockout experiments. verify. The function of each gene in the gene cluster was further analyzed and the gene encoding methyltransferase was screened out as vicG (its nucleotide sequence is shown in SEQ ID NO.1), and its distribution position was determined. Because the size of the DNA fragment that cosmid can carry is 30-40kb, in order to screen out the cosmid plasmid containing the methyltransferase gene vicG, three pairs of screening library primers were designed at appropriate positions on the gene cluster, and the sequences are as follows:
vic-upstream-F：5’-CGCTTCGTCCACATCTCCAC-3’，vic-upstream-F: 5'-CGCTTCGTCCACAATCTCCAC-3',
vic-upstream-R：5’-ACTTCGTCGGTTCCCACCAC-3’；vic-upstream-R:5'-ACTTCGTCGGTTCCCACCAC-3';
vic-midstream-F：5’-GACACCTACCGTCTGCACATTG-3’，vic-midstream-F:5'-GACACCTACCGTCTGCACATTG-3',
vic-midstream-R：5’-GTCACTCAGCCAGGTCTGCAC-3’；vic-midstream-R:5'-GTCACTCAGCCAGGTCTGCAC-3';
vic-downstream-F：5’-CGGAAGAGGCCACCGACCGG-3’，vic-downstream-F: 5'-CGGAAGAGGCCACCGACCGG-3',
vic-downstream-R：5’-GGTGTCGACGAGCAGAAAGC-3’。vic-downstream-R: 5'-GGTGTCGACGAGCAGAAAGC-3'.
利用这三对引物，通过PCR筛选得可包含甲基转移酶基因vicG的cosmid质粒1-F11，采用的PCR程序为：95℃5min；95℃45s，58℃45s，72℃1min，30个循环；72℃10min。Using these three pairs of primers, the cosmid plasmid 1-F11 containing the methyltransferase gene vicG was screened by PCR. The PCR program used was: 95°C for 5min; 95°C for 45s, 58°C for 45s, 72°C for 1min, 30 cycles ; 72°C for 10 min.
2.利用PCR-targeting技术构建甲基转移酶基因突变株基因敲除突变株的构建主要采用PCR-targeting技术，具体操作步骤为：首先将携带有甲基转移酶基因vicG的cosmid质粒1-F11导入到E.coli BW25113/pIJ790中，并将其制备成感受态。同时，设计各甲基转移酶基因的敲除引物用(正向引物5’-GCGGGGGAGGCCGACCGCATCCGCGAACTGGCCCTCGAAATTCCGGGGATCCGTCGACC-3’和反向引物5’-CACCGCGCACCCGGCCGACCGGAACGCCGCCTCGTACTCTGTAGGCTGGAGCTGCTTC-3’)以扩增两端具有同源臂的阿普拉霉素(Apr)抗性基因片段，采用的PCR程序为：95℃2min；95℃20s，60℃20s，72℃90s，30个循环；72℃10min。将该片段导入携带cosmid 1-F11的E.coli BW 25113/pIJ790感受态菌株中，利用E.coli BW 25113/pIJ790的同源重组系统，使阿普拉霉素(Apr)抗性基因片段与cosmid上目标基因vicG的关键功能区域发生同源重组，从而导致目标基因突变失活，无法正常表达。然后，提取突变的cosmid质粒1-F11并将其导入E.coli ET12567/pUZ8002中。最后，通过接合转移利用E.coli ET中非穿梭型pUZ8002质粒诱导携带突变基因的cosmid 1-F11转移到野生型链霉菌S.parvusSCSIO Mla-L010中，再经同源重组使基因组中的目标基因突变而得到甲基转移酶基因突变菌株Mla-L010/ΔvicG，经抗性验证和PCR验证(正向引物5’-CGACATCTACCGACGGCACAAG-3’和反向引物5’-AGAACAGCGACAGCTCCTGCTC-3’)以鉴定构建的突变株是否正确。获得突变cosmid 1-F11 E.coli ET 12567/pUZ8002菌，最后，保存验证正确的突变株于-80℃冰箱。具体构建过程如图1所示。2. Using PCR-targeting technology to construct methyltransferase gene mutants The construction of gene knockout mutants mainly adopts PCR-targeting technology. Introduce it into E.coli BW25113/pIJ790 and make it competent. At the same time, the knockout primers of each methyltransferase gene were designed (forward primer 5'-GCGGGGGAGGCCGACCGCATCCGCGAACTGGCCCTCGAAATTCCGGGGATCCGTCGACC-3' and reverse primer 5'-CACCGCGCACCCGGCCGACCGGAACGCCGCCTCGTACTCTGTAGGCTGGAGCTGCTTC-3') to amplify Apra with homology arms at both ends Mycin (Apr) resistance gene fragment, the PCR program adopted is: 95°C 2min; 95°C 20s, 60°C 20s, 72°C 90s, 30 cycles; 72°C 10min. This fragment was introduced into the E.coli BW 25113/pIJ790 competent strain carrying cosmid 1-F11, and the homologous recombination system of E.coli BW 25113/pIJ790 was used to make the apramycin (Apr) resistance gene fragment and Homologous recombination occurs in the key functional region of the target gene vicG on the cosmid, which leads to the inactivation of the target gene mutation and the failure to express normally. Then, the mutated cosmid plasmid 1-F11 was extracted and introduced into E. coli ET12567/pUZ8002. Finally, the non-shuttle pUZ8002 plasmid in E.coli ET was used to induce the cosmid 1-F11 carrying the mutant gene to be transferred to the wild-type Streptomyces S.parvusSCSIO Mla-L010 by conjugative transfer, and then the target gene in the genome was made by homologous recombination. Mutated to obtain the methyltransferase gene mutant strain Mla-L010/ΔvicG, through resistance verification and PCR verification (forward primer 5'-CGACATCTACCGACGGCACAAG-3' and reverse primer 5'-AGAACAGCGACAGCTCCTGCTC-3') to identify the constructed Whether the mutant strain is correct. Obtain mutant cosmid 1-F11 E.coli ET 12567/pUZ8002 bacteria, and finally, save the verified mutant strain in a -80°C refrigerator. The specific construction process is shown in Figure 1.
其中，接合转移的实验操作流程如下：Among them, the experimental operation process of conjugation transfer is as follows:
接种构建好的携带突变cosmid 1-F11 E.coli ET 12567/pUZ8002菌于3-5ml加有50μg/mL氯霉素(Cm)、50μg/mL阿普拉霉素(Apr)和50μg/mL卡那霉素(Kan)的LB液体培养基中，37℃，200rpm摇床中过夜培养。取过夜培养菌液500-1000ul(接种量根据菌液浓度而定)转接装有50ml LB的250ml三角瓶(加有50μg/mL氯霉素、50μg/mL阿普霉素和50μg/mL卡那霉素)，于37℃，200rpm摇床中培养2-3h，使其OD600值达到0.6～0.8范围即可。取45ml菌液至50ml离心管中，常温，4000rpm离心10min，弃上清并吸除残余液体。用40ml无抗LB洗涤菌体，洗去抗生素，常温，4000rpm离心10min，弃上清并吸除残余液体，重复洗一次。根据菌体浓度加入适量LB使菌体充分重悬并分装至1.5ml无菌EP管中。Inoculate the constructed mutant cosmid 1-F11 E.coli ET 12567/pUZ8002 bacteria into 3-5ml with 50μg/mL chloramphenicol (Cm), 50μg/mL apramycin (Apr) and 50μg/mL card Namycin (Kan) was cultured overnight in LB liquid medium at 37° C. in a shaker at 200 rpm. Take 500-1000ul of the overnight culture solution (the inoculation amount depends on the concentration of the bacteria solution) and transfer it to a 250ml Erlenmeyer flask filled with 50ml LB (added with 50μg/mL chloramphenicol, 50μg/mL apramycin and 50μg/mL card Namycin), cultured at 37° C., 200 rpm in a shaker for 2-3 hours, so that the OD600 value reaches the range of 0.6-0.8. Take 45ml of the bacterial solution into a 50ml centrifuge tube, centrifuge at 4000rpm for 10min at room temperature, discard the supernatant and suck off the residual liquid. Wash the cells with 40ml of anti-antibiotic-free LB to remove the antibiotics, centrifuge at 4000rpm for 10min at room temperature, discard the supernatant and suck off the residual liquid, and repeat the washing once. Add an appropriate amount of LB according to the cell concentration to fully resuspend the cells and distribute them into 1.5ml sterile EP tubes.
刮取链霉菌S.parvus SCSIO Mla-L010孢子至加有适量LB的50ml离心管，涡旋振荡使孢子充分分散。分装至1.5ml EP管中，用封口膜封口并置于50℃恒温水浴锅中热激10min，再用自来水冷却2-3min，外界温度的变化可刺激孢子预萌发。将EP管置于28℃，200rpm的摇床上复苏约5h。Scrape the Streptomyces S.parvus SCSIO Mla-L010 spores to a 50ml centrifuge tube with an appropriate amount of LB, and vortex to fully disperse the spores. Dispense into 1.5ml EP tubes, seal with parafilm and place in a constant temperature water bath at 50°C for heat shock for 10 minutes, then cool with tap water for 2-3 minutes. Changes in external temperature can stimulate spore pre-germination. Place the EP tube on a shaking table at 28°C and 200rpm to recover for about 5h.
将上述分装的E.coli ET加到分装的孢子中，混合均匀后涂布到加有10mM Mg2+或20mM Mg2+的MS固体培养基上，使菌液在平板上均匀铺开，晾干后置于28℃恒温培养16-20h后进行覆盖，覆盖步骤为：每个板加终浓度均为50μg/mL的阿普拉霉素(Apr)和50μg/mL甲氧苄啶(Tmp)，为了方便覆盖，可将抗生素加到1ml双蒸水中混匀后再加到平板上，并用玻璃涂布棒均匀涂布，晾干平板后置于28℃恒温培养，3-4天后可观察到长出接合子。即获得N-甲基转移酶失活突变株SCSIO Mla-L010/ΔvicG。Add the above-mentioned subpackaged E.coli ET to the subpackaged spores, mix evenly, and spread it on the MS solid medium with 10mM Mg2+ or 20mM Mg2+ , so that the bacterial solution is evenly spread on the plate , dried, placed at 28°C for 16-20h at a constant temperature, and then covered. The covering steps were as follows: each plate was added with apramycin (Apr) and 50 μg/mL trimethoprim ( Tmp), in order to facilitate coverage, add antibiotics to 1ml of double-distilled water and mix evenly before adding to the plate, and evenly spread it with a glass coating rod, dry the plate and place it at 28°C for constant temperature cultivation, after 3-4 days, it can be Outgrowth of zygotes was observed. That is, the N-methyltransferase inactivated mutant strain SCSIO Mla-L010/ΔvicG was obtained.
实施例2 4’-N-demethyl-vicenistatin的分离及结构鉴定Example 2 Separation and structure identification of 4'-N-demethyl-vicenistatin
采用实施例1所述的规模发酵条件制备4’-N-demethyl-vicenistatin生产菌株SCSIO Mla-L010/ΔvicG的发酵培养物，通过离心(4000r/min，15min)分离获得菌丝体沉淀和发酵上清液。向菌丝体沉淀加入3倍体积丙酮进行萃取，重复萃取3次后，将萃取液减压浓缩成浸膏。向发酵上清液中加入等体积丁酮萃取3次，将萃取液减压浓缩成浓缩浸膏。将以上两部分浓缩浸膏合并，加入硅胶拌样后进行正相硅胶柱层析，采用氯仿/甲醇体系按以下体积比进行梯度洗脱：100:0；98:2；96:4；94:6；92:8；9:1；8:2；6:4；5:5；0:100，顺序得到Fr.A1～10共10个组分。将组分Fr.A5～8(即92:8；9:1；8:2；6:4洗脱的馏分)合并，以甲醇为流动相，采用Sephadex LH-20羟丙基葡聚糖凝胶色谱柱进行层析并收集目标组分，利用半制备高效液相色谱和ODS色谱柱(250×10mm,5μm)，按以下条件制备目标峰：CH3CN/H2O(32:68，V/V，含0.1％醋酸)体系等度洗脱25min，流速2.5mL/min，得到4’-N-demethyl-vicenistatin(tR＝14.1min)The fermentation culture of the 4'-N-demethyl-vicenistatin production bacterial strain SCSIO Mla-L010/ΔvicG was prepared using the scale fermentation conditions described in Example 1, and the mycelium precipitate and fermentation surface were separated by centrifugation (4000r/min, 15min). Serum. Add 3 times the volume of acetone to the mycelium precipitate for extraction, repeat the extraction 3 times, and concentrate the extract under reduced pressure to form an extract. An equal volume of butanone was added to the fermentation supernatant for extraction three times, and the extract was concentrated under reduced pressure to form a concentrated extract. Combine the above two parts of concentrated extracts, add silica gel to mix the sample, and then perform normal phase silica gel column chromatography, and use chloroform/methanol system to carry out gradient elution according to the following volume ratio: 100:0; 98:2; 96:4; 94: 6; 92:8; 9:1; 8:2; 6:4; 5:5; 0:100, a total of 10 components of Fr.A1-10 were obtained in sequence. Combine components Fr.A5～8 (i.e. 92:8; 9:1; 8:2; 6:4 eluted fractions), use methanol as the mobile phase, and use Sephadex LH-20 hydroxypropyl dextran to coagulate Gel chromatographic column for chromatography and collect target components, use semi-preparative high performance liquid chromatography and ODS chromatographic column (250×10mm, 5μm), prepare the target peak according to the following conditions: CH3 CN/H2 O (32:68, V/V, containing 0.1% acetic acid) system isocratic elution for 25min, flow rate 2.5mL/min, to obtain 4'-N-demethyl-vicenistatin (tR =14.1min)
4’-N-demethyl-vicenistatin的鉴定：为白色无定型粉末；(+)HRESI-MS(图3)呈现[M+H]+(m/z 487.3544)峰(Cal.487.3536[M+H]+)，结合1H NMR(图4)和13C NMR(图5)确定其分子式为C29H46N2O4。其核磁数据如下：1H NMR(700MHz,Pyr)δ8.52(d,J＝7.7Hz,NH-19),7.63(dd,J＝14.7,11.2Hz,H-3),6.82(dd,J＝14.7,11.2Hz,H-14),6.29(d,J＝14.7Hz,H-2),6.22(dd,J＝15.4,11.2Hz,H-4),5.98(d,J＝11.2Hz,H-13),5.88(dd,J＝15.4,9.8Hz,H-5),5.71(m,H-15),5.36(d,J＝11.2Hz,H-1’),5.22(t,J＝7.0Hz,H-9),4.35(d,J＝1.4,H-3’),4.10(m,H-5’),4.05(dt,J＝13.3,9.8Hz,H-19a),3.40(t,J＝8.4Hz,H-7),3.10(dd,J＝15.4,8.4Hz,H-8a),3.05(ddd,J＝13.3,4.2,2.8Hz,H-19b),2.76(d,J＝15.4Hz,H-11a),2.68(dd,J＝9.8,1.4Hz,H-4’)2.65(d,J＝15.4Hz,H-11b),2.42(overlapped,H-6,H-16a,H-2’a),2.29(m,H-8b),2.09(overlapped,H-16b),2.00(overlapped,H-2’b),1.97(s,H-22),1.88(overlapped,H-18),1.72(s,H-21),1.58(overlapped,H-17a),1.52(d,J＝5.6Hz,H-6’),1.48(m,H-17b),1.10(d,J＝6.3Hz,H-20),0.86(d,J＝7.0Hz,H-23)。13C NMR(176MHz,Pyr)δ166.79(C-1),143.82(C-5),140.78(C-3),135.53(C-10),134.50(C-12),133.05(C-15),128.89(C-4,C-14),128.57(C-13),125.02(C-2),122.45(C-9),100.40(C-1’),86.50(C-7),72.12(C-5’),69.12(C-3’),57.88(C-4’),49.71(C-11),46.87(C-6),43.50(C-19),40.89(C-2’),37.08(C-8),33.98(C-18),33.15(C-17),28.06(C-16),20.00(C-6’),19.70(C-20),18.44(C-21),18.44(C-23),17.86(C-22)。Identification of 4'-N-demethyl-vicenistatin: white amorphous powder; (+) HRESI-MS (Figure 3) presents [M+H]+ (m/z 487.3544) peak (Cal.487.3536[M+H]+ ), combined with1 H NMR (Fig. 4) and13 C NMR (Fig. 5), it was determined that its molecular formula was C29 H46 N2 O4 . Its NMR data are as follows:1 H NMR (700MHz, Pyr) δ8.52 (d, J = 7.7Hz, NH-19), 7.63 (dd, J = 14.7, 11.2Hz, H-3), 6.82 (dd, J =14.7,11.2Hz,H-14),6.29(d,J=14.7Hz,H-2),6.22(dd,J=15.4,11.2Hz,H-4),5.98(d,J=11.2Hz, H-13), 5.88(dd, J=15.4, 9.8Hz, H-5), 5.71(m, H-15), 5.36(d, J=11.2Hz, H-1'), 5.22(t, J =7.0Hz, H-9), 4.35(d, J=1.4, H-3'), 4.10(m, H-5'), 4.05(dt, J=13.3, 9.8Hz, H-19a), 3.40 (t, J=8.4Hz, H-7), 3.10(dd, J=15.4, 8.4Hz, H-8a), 3.05(ddd, J=13.3, 4.2, 2.8Hz, H-19b), 2.76(d , J=15.4Hz, H-11a), 2.68 (dd, J=9.8, 1.4Hz, H-4'), 2.65 (d, J=15.4Hz, H-11b), 2.42 (overlapped, H-6, H -16a, H-2'a), 2.29(m, H-8b), 2.09(overlapped, H-16b), 2.00(overlapped, H-2'b), 1.97(s, H-22), 1.88( overlapped, H-18), 1.72 (s, H-21), 1.58 (overlapped, H-17a), 1.52 (d, J=5.6Hz, H-6'), 1.48 (m, H-17b), 1.10 (d, J=6.3Hz, H-20), 0.86 (d, J=7.0Hz, H-23). 13C NMR (176MHz, Pyr) δ166.79(C-1), 143.82(C-5), 140.78(C-3), 135.53(C-10), 134.50(C-12), 133.05(C-15) ,128.89(C-4,C-14),128.57(C-13),125.02(C-2),122.45(C-9),100.40(C-1'),86.50(C-7),72.12( C-5'),69.12(C-3'),57.88(C-4'),49.71(C-11),46.87(C-6),43.50(C-19),40.89(C-2') ,37.08(C-8),33.98(C-18),33.15(C-17),28.06(C-16),20.00(C-6'),19.70(C-20),18.44(C-21) , 18.44 (C-23), 17.86 (C-22).
综上鉴定，4’-N-demethyl-vicenistatin的结构如式(I)所示，In summary, the structure of 4'-N-demethyl-vicenistatin is as shown in formula (I),
实施例3 4’-N-demethyl-vicenistatin对系列活性指示菌株的最小抑菌浓度测定Example 3 4'-N-demethyl-vicenistatin measures the minimum inhibitory concentration of a series of active indicator strains
采用微量肉汤稀释法使对vicenisatin及4’-N-demethyl-vicenistatin进行抗菌活性评价。用二甲基亚砜(DMSO)溶解vicenisatin、4’-N-demethyl-vicenistatin和阳性对照品万古霉素(Vancomycin)、氨苄西林(Ampicillin)、两性霉素B(Amphotericin B)，均配制成终浓度为1.6mg/mL的药液，-20℃冰箱保藏备用。The antibacterial activity of vicenisatin and 4'-N-demethyl-vicenistatin was evaluated by broth microdilution method. Dissolve vicenisatin, 4'-N-demethyl-vicenistatin, and positive controls vancomycin (Vancomycin), ampicillin (Ampicillin), and amphotericin B (Amphotericin B) in dimethyl sulfoxide (DMSO) to prepare the final The drug solution with a concentration of 1.6mg/mL should be stored in a -20°C refrigerator for later use.
选用Mueller-Hinton(MH)肉汤液体培养基培养活性测试菌株，其配方如下牛肉浸膏粉300g/L，可溶性淀粉15g/L，酪蛋白酸水解物17.5g/L，调节pH值至7.3，121℃灭菌30min备用。Select Mueller-Hinton (MH) broth liquid medium to cultivate the active test bacterial strain, its formula is as follows beef extract powder 300g/L, soluble starch 15g/L, casein acid hydrolyzate 17.5g/L, adjust pH value to 7.3, Sterilize at 121°C for 30 minutes for later use.
将过夜培养的测试菌液利用MH培养基稀释至在600nm波长处的吸光值约等于1，再使用MH培养基稀释至原来的1‰待用。在96孔板上第1行第1列加入100μL无菌MH培养基，第一行其余微孔均加入50μL无菌MH培养基，作为空白对照。从第2行起，每行的第1列加入92μL无菌MH培养基，其余各列加入50μL无菌MH培养基，标记好后备用。在第1列(除第1行外)加入8μL待测化合物药液和阳性对照品，使其起始浓度为128μg/mL，每个样品做3个平行对照。将排枪体积设置为50μL，将第1列的测试药液小心上下吸取4～5次，混合均匀。从中吸取50μL加入第2列，重复之前步骤以混匀，以此类推，直至稀释到最后一列，取出50μL弃之，使得第1孔至第12孔的测试药液终浓度为128、64、32、16、8、4、2、1、0.5、0.25、0.125、0.0625μg/mL。取50μL稀释好的实验菌液分别加入每个微孔并混匀，置于37℃培养箱中培养16h后观察测试菌生长情况，能有效抑制菌株生长的最小样品浓度即为最小抑菌浓度(minimuminhibitory concentration,MIC)值。Dilute the overnight cultured test bacteria solution with MH medium until the absorbance value at 600nm wavelength is approximately equal to 1, and then use MH medium to dilute to the original 1‰ before use. Add 100 μL of sterile MH medium to the first row and first column of the 96-well plate, and add 50 μL of sterile MH medium to the remaining microwells in the first row as a blank control. From row 2 onwards, add 92 μL of sterile MH medium to the first column of each row, and add 50 μL of sterile MH medium to the remaining columns, and mark them for later use. Add 8 μL of test compound solution and positive control substance to the first column (except row 1) so that the initial concentration is 128 μg/mL, and make 3 parallel controls for each sample. Set the volume of the discharge gun to 50 μL, carefully pipette the test liquid in column 1 up and down 4 to 5 times, and mix well. Draw 50 μL from it and add it to the second column, repeat the previous steps to mix, and so on, until it is diluted to the last column, take out 50 μL and discard it, so that the final concentration of the test solution from the first to the 12th well is 128, 64, 32 , 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg/mL. Take 50 μL of the diluted experimental bacteria solution and add them to each microwell and mix them evenly. After culturing in a 37°C incubator for 16 hours, observe the growth of the test bacteria. The minimum sample concentration that can effectively inhibit the growth of the strain is the minimum inhibitory concentration ( minimumhibitory concentration, MIC) value.
试验结果表明，4’-N-demethyl-vicenistatin对金黄色葡萄球菌Staphylococcusaureus ATCC 29213，6株耐甲氧西林金黄色葡萄球菌Methicillin-resistantStaphylococcus aureus(MRSA)shhs-E1、MRSA 16339、MRSA745524、MRSA 16162、MRSA718306和MRSA 6917，藤黄微球菌Micrococcus luteus，枯草芽孢杆菌Bacillussubtilis，新生隐球菌Cryptoccus neoformans，幽门螺杆菌Helicobacter pylori和白色念珠菌Candida albicans具有显著的抑制活性，最小抑菌浓度(minimum inhibitoryconcentration，MIC)在0.125～2μg/mL，显示出其在抗感染药物开发中的重要价值。值得注意的是，对真菌白色念珠菌Candida albicans的抑制活性，N-脱甲基vicenistatin衍生物4’-N-demethyl-vicenistatin的抗菌活性比vicenistatin强2-4倍，显示出该结构衍生物具有更好的成药潜力。实验结果见表1。The test results showed that 4'-N-demethyl-vicenistatin was effective against Staphylococcus aureus ATCC 29213, 6 strains of methicillin-resistant Staphylococcus aureus (MRSA) shhs-E1, MRSA 16339, MRSA745524, MRSA 16162, MRSA718306 and MRSA 6917, Micrococcus luteus, Bacillus subtilis, Cryptoccus neoformans, Helicobacter pylori and Candida albicans have significant inhibitory activity, minimum inhibitory concentration (MIC) At 0.125～2μg/mL, it shows its important value in the development of anti-infective drugs. It is worth noting that the antibacterial activity of the N-demethyl vicenistatin derivative 4'-N-demethyl-vicenistatin is 2-4 times stronger than that of vicenistatin for the inhibitory activity of the fungus Candida albicans, showing that the structural derivative has Better druggability potential. The experimental results are shown in Table 1.
表1 Vincenistain和4’-N-demethyl-vicenistatin的MIC(μg/mL)值Table 1 MIC (μg/mL) values of Vincenistain and 4’-N-demethyl-vicenistatin
实施例4 4’-N-demethyl-vicenistatin对系列细胞株的半抑制浓度(IC50，μM)测定Example 4 Determination of the half-inhibitory concentration (IC50 , μM) of 4'-N-demethyl-vicenistatin on a series of cell lines
采用国际通用的肿瘤细胞株，即：人乳腺癌细胞MCF-7、人肝癌细胞HepG2、人宫颈癌细胞HeLa、人非小细胞肺癌细胞A549、人结肠腺癌细胞RKO、人急性早幼粒白血病细胞HL-60。试验方法为MTT法：International tumor cell lines are used, namely: human breast cancer cell MCF-7, human liver cancer cell HepG2, human cervical cancer cell HeLa, human non-small cell lung cancer cell A549, human colon adenocarcinoma cell RKO, human acute promyelocytic leukemia Cell HL-60. The test method is MTT method:
1)细胞培养：根据细胞生长速度，将处于对数生长期的肿瘤细胞以100μL/孔接种于96孔板，贴壁生长24小时。1) Cell culture: According to the cell growth rate, tumor cells in the logarithmic growth phase were inoculated in a 96-well plate at 100 μL/well, and grown adherently for 24 hours.
2)加样品(药品)：每孔加10μL不同浓度的4’-N-demethyl-vicenistatin的培养基稀释溶液，并设相应浓度的培养基溶媒对照及无细胞凋零孔。2) Add samples (drugs): add 10 μL of 4’-N-demethyl-vicenistatin medium dilution solution of different concentrations to each well, and set corresponding concentrations of medium vehicle control and no cell death wells.
3)加药细胞培养：肿瘤细胞在37℃、5％的CO2条件下培养68小时。3) Drug-added cell culture: the tumor cells were cultured for 68 hours at 37° C. and 5% CO2 .
4)活性测试：经培养后的细胞，每孔加入20μL(5mg/mL)的MTT溶液，继续培养4小时，洗掉上清培养基，加入150μL二甲基亚砜(DMSO)溶解MTT转变成的甲臜结晶。4) Activity test: Add 20 μL (5 mg/mL) of MTT solution to each well of the cultured cells, continue to cultivate for 4 hours, wash off the supernatant medium, and add 150 μL dimethyl sulfoxide (DMSO) to dissolve MTT into formazan crystals.
5)酶标仪540nm波长测定OD值。5) Measure the OD value with a microplate reader at a wavelength of 540 nm.
6)活性报告。每株细胞系做3个平行实验，实验结果见表2：6) Activity report. Three parallel experiments were performed for each cell line, and the experimental results are shown in Table 2:
表2 4’-N-demethyl-vicenistatin的对系列肿瘤细胞的抑制活性(IC50，μM)Table 2 Inhibitory activity of 4'-N-demethyl-vicenistatin on a series of tumor cells (IC50 , μM)
序列表sequence listing
<110> 中国科学院南海海洋研究所<110> South China Sea Institute of Oceanology, Chinese Academy of Sciences
南方海洋科学与工程广东省实验室（广州）Southern Ocean Science and Engineering Guangdong Laboratory (Guangzhou)
<120> 4’-N-demethyl-vicenistatin及其制备方法和应用<120> 4'-N-demethyl-vicenistatin and its preparation method and application
<160> 1<160> 1
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 711<211> 711
<212> DNA<212>DNA
<213> 海洋放线菌(Streptomyces parvus SCSIO Mla-L010)<213> Marine actinomycetes (Streptomyces parvus SCSIO Mla-L010)
<400> 1<400> 1
gtgtacgaag aggatttcgc ccgcgtctac gacgacatct accgacggca caagaactat 60gtgtacgaag aggatttcgc ccgcgtctac gacgacatct accgacggca caagaactat 60
gcgggggagg ccgaccgcat ccgcgaactg gccctcgaat accgacccga cgcgtcgagt 120gcgggggagg ccgaccgcat ccgcgaactg gccctcgaat accgacccga cgcgtcgagt 120
ctgctggacg tcggctgcgg cacgggcgag catctggccc gtctgcggca gcatttcgac 180ctgctggacg tcggctgcgg cacgggcgag catctggccc gtctgcggca gcatttcgac 180
gtggccggtg tggatctcgc cccgcccatg atccgtattg ccacagccaa gcttcccggg 240gtggccggtg tggatctcgc cccgcccatg atccgtattg ccacagccaa gcttcccggg 240
gtgcccctgc tccaggacga catgcgcacc ttctcgctgg accggacctt cgacgtcgtc 300gtgcccctgc tccaggacga catgcgcacc ttctcgctgg accggacctt cgacgtcgtc 300
tgttcgatgt acagctcggt gggctatctg gcgacggccg acgacctgtc caccgccgtc 360tgttcgatgt acagctcggt gggctatctg gcgacggccg acgacctgtc caccgccgtc 360
aagaacatga ccggtcatct gcgtccgggc ggtgtgctga tcgtcgagcc gtggatcctc 420aagaacatga ccggtcatct gcgtccgggc ggtgtgctga tcgtcgagcc gtggatcctc 420
cgggaggact ggaacggcgg cgacctcgtg caggccgact tcgagaacga ggagggcaag 480cgggaggact ggaacggcgg cgacctcgtg caggccgact tcgagaacga ggagggcaag 480
gtcgtccgga tgggccgctg gaccaccagg aacgggcgca gccgcgtcga gatgcactat 540gtcgtccgga tgggccgctg gaccaccagg aacgggcgca gccgcgtcga gatgcactat 540
ctggtcgcca cggactcggg accggtgggt cacttcgtgg acgagcagga gctgtcgctg 600ctggtcgcca cggactcggg accggtgggt cacttcgtgg acgagcagga gctgtcgctg 600
ttctcccgcg aggagtacga ggcggcgttc cggtcggccg ggtgcgcggt ggagtaccgc 660ttctcccgcg aggagtacga ggcggcgttc cggtcggccg ggtgcgcggt ggagtaccgc 660
ccggacggct acgcggaccg cggaatcttc gtgggcgtac ggcaggactg a 711ccggacggct acgcggaccg cggaatcttc gtgggcgtac ggcaggactg a 711
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| JPH05186493A (en)* | 1992-01-08 | 1993-07-27 | Kirin Brewery Co Ltd | New compound hc34, its use and production |
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