蔡永通创新答辩

软珊瑚Sarcophytontortuosum内生真菌SarcoF5代谢产物研究报告人：蔡永通指导老师：李厚金副教授图1扭曲肉芝软珊瑚的照片海底照片水上照片实验背景一、软珊瑚Sarcophytontortuosum•1986年本院天然室的苏镜娱等从软珊瑚Sarcophytontortuosum中得到2个罕见的四环四萜化合物；•2004年中山大学药学院蓝文健等又从该软珊瑚分离得到3个新的四萜化合物。发表的文章：•[1]J.Y.Su,T.S.Pengeta1.StudiesontheChineseSoftCoral,TheStructureofMethylsartortuoate.ActaChem.sinica,1986,1,101.•[2]JingyuSu,kanghouLong,TangshengPang.TheStructureofMethylIsosartortuoate,aNovelTetracyclicTetraterpenoidfromtheSoftCoralSarcophytontortuosum.J.Am.Chem.Soc.1986,108,177.•[3]JingyuSu,KanghouLong,TangshenPengetal.,DeterminationofMolecularandCrystalStuctureofaUniqueNewTetraterpenoid-MethylSartotuoate.ScienceSinica(SeriesB),1988,10,1172.•[4]ZengLong-Mei,LanWen-Jian,SuJing-Yu,ZhangGuang-Wen,FengXiao-Long,LiangYong-Ju,YangXiao-Ping.TwonewcytotoxictetracyclictetraterpenoidsfromthesoftcoralSarcophytontortuosum.JournalofNaturalProducts.2004,67(11),1915-8.•[5]LanW-J,LiH-J,YanS-J,SuJ-Y,ZengL-M.NewtetraterpenoidfromthesoftcoralSarcophytontortuosum.JournalofAsianNaturalProductsResearch.2007,9(3-5),267-271.OOHCOOCH3HOHOOOHmethylsartortuoateOOOHCOOCH3OmethylisosartortuoateOHOHOOHCOOCH3HOHOOmethyltortuoateAHOHOOOHCOOCH3OmethyltortuoateBOHOHOHOOHCOOCH3OmethyltortuoateCOHHOHOH图2四萜结构图(1)以上分离得到的四萜化合物对老鼠S180，CNE-2，P-388，HT-29，CNE-1显示了不同程度的抑制作用。化合物活性因为这些四萜化合物具有显著的抗癌活性，因此在医药方面有很好的应用价值，但是因为其结构复杂，合成比较困难，因此对寻找其生源途径的研究变得非常重要。•共附生或内生微生物（真菌、细菌、蓝菌等）是与海洋动植物处于共生、共栖、寄生或附生的关系的微生物。•从海洋附生微生物中分离得到许多具有生理活性的化合物。•海洋植物和无脊椎动物产生的活性物质实际上是由与其共附生的微生物产生的？二、海洋共附生微生物•分离得到的这些四萜化合物是来源于软珊瑚本身，还是由软珊瑚附生微生物产生而在珊瑚体内积累？•通过分离和纯化软珊瑚的共附生微生物，并且系统地研究附生微生物的代谢产物，将能揭示这些具抗肿瘤活性的四萜类化合物的真正来源。菌种的筛选我们课题已经开展了该珊瑚附生微生物的分离工作，已经分离、纯化了30多株真菌和细菌，发现一株真菌（SarcoF5）在GYP培养基中生长良好。(A)培养10天的生长情况(B)培养20天的生长情况(C)培养30天的生长情况(D)培养40天的生长情况图3SarcoF5生长情况图微生物的培养以葡萄糖(10g/L)、蛋白胨(3g/L)、酵母膏(1g/L)、100%人工海水(粗海盐23g/L)、pH7.5为培养基（GYP培养基）配制150L培养液分装到若干个1000ml的锥形瓶中，在124℃高温高压下灭菌1h。接入菌种扭曲肉芝软珊瑚内生真菌SarcoF5，28℃下静置培养40天。代谢产物的提取待内生真菌SarcoF5长到40天后，用纱布过滤，培养液用乙酸乙酯提取三次，菌体用甲醇浸泡。合并乙酸乙酯提取液，旋转蒸发至干，浓缩得到棕色提取物43.16g，菌体的甲醇提取物也得到45.2g。培养液提取物的分离培养液乙酸乙酯提取物(43.16g)硅胶柱层析以PE、EA和MO为洗脱剂进行梯度洗脱48个组分薄层层析合并化学成分相似组分，选择几个组分进行细分8-9合并液13-15合并液16-19合并液硅胶柱层析以PE、EA和MO为洗脱剂进行梯度洗脱PE:EA＝2:1/1:1硅胶柱层析以PE、EA和MO为洗脱剂进行梯度洗脱100%乙酸乙酯用乙酸乙酯和石油醚混合液进行多次重结晶化合物1（sarcoF5-1）化合物2（sarcoF5-2）化合物3（sarcoF5-3）图4分离流程图实验结果图5分离得到化合物结构图OOH3COHOHHOHOH3CCH3H3CHOHH3COH3CH3CCH3OHOOCH3HSarcoF5-1SarcoF5-2(new)SarcoF5-3(new)结构鉴定－SarcoF5-1实验分离出来的SarcoF5－1（青霉酸）是一种无色针状的晶体，分子式为C8H10O4,相对分子量为170.16。据文献报导它的熔点为83℃,极易溶于热水、乙醇、乙醚和氯仿,但不溶于戊烷、己烷。345O2OH3C129OHO10HH11167810a10b图6SarcoF5-1结构图ppm(f1)0.01.02.03.04.05.06.07.08.00100020003000400050006.0536.0496.0476.0446.0416.0396.0366.0326.0296.0255.4285.1445.1033.8791.7181.000.910.870.020.822.512.44图7SarcoF5-1-1HNMR345O2OH3C129OHO10HH11167810a10bppm(f1)050100150200-500005000100001500020000250003000035000179.694172.230139.408116.594103.54989.46077.83677.41276.98960.33117.723图8SarcoF5-1-13CNMR345O2OH3C129OHO10HH11167810a10bppm(f1)050100150200-1000100200300400116.58889.63360.71518.408图9SarcoF5-1-DEPT-135345O2OH3C129OHO10HH11167810a10bPositionδCDEPTδH234589101112172.0286.32171.17109.5857.88133.97114.36－17.43CCHCCCH3CCH2－CH3－5.23－－3.93－5.21(10a)5.5010b)5.931.96表1SarcoF5－1NMR数据(1HNMR300MHz，13CNMR75MHz，CDCl3，TMS)SarcoF5-1互变反应SarcoF5-1为青霉酸，是一个很强的多聚乙酰类霉菌毒素，首先由Alsberg等在1912年分离得到。OOOHOHHCOOHOHHO乙醇中重结晶熔融状态下结晶Tautomerismofpenicillicacid.Munday,C.W.Nature(London,UnitedKingdom)(1949),163443-4.CODEN:NATUASISSN:0028-0836.Journallanguageunav-ailable.CAN43:31631AN1949:31631CAPLUStheketoacidthelactone结构鉴定－SarcoF5-2图10SarcoF5-2结构图H3C11658942H3C12CH3OHOOCH3H13710131514SarcoF5-2为无色透明针状晶体，ESI-MS[m/z213.2(M＋1)+]，分子式为C12H20O3。ppm(f1)0.05.010.00500100013.17113.1697.2777.1922.6822.6582.4252.4002.2762.2542.2312.2092.1862.1342.1112.0892.0752.0682.0441.8271.0010.9791.001.211.991.881.061.280.9510.55图11SarcoF5-2-1HNMRH3C11658942H3C12CH3OHOOCH3H13710131514ppm(f1)05010015020001000020000300004000050000158.752156.871138.040123.38477.75677.33376.91142.09539.79328.59827.36723.07722.595图12SarcoF5-2-13CNMRH3C11658942H3C12CH3OHOOCH3H13710131514图13SarcoF5-2-DEPT-135ppm(f1)0501001502000500123.29542.61140.32229.21427.99323.736H3C11658942H3C12CH3OHOOCH3H13710131514表2SarcoF5－2NMR数据(1HNMR300MHz，13CNMR75MHz，CDCl3，TMS)PositionδCDEPTδH2345689101112131415172.93－124.87150.66203.9035.6327.2022.9443.6526.6222.8022.9422.80C－CHCCCH2CHCH3CH2CHCH3CH3CH3－13.1536.60(4a)－－2.451.890.902.472.090.950.900.95结构鉴定－SarcoF5-3图14SarcoF5-3结构图956OHO1023H3C1213CH3H3CHOHH3CO1478111416159aSarcoF5-3为无色透明树枝状晶体，EI-MS[m/z228.2(M)+]，分子式为C12H20O4，不饱和度为3。图15SarcoF5-3-1HNMRppm(f1)0.05.010.0010020030040050011.87211.86711.86311.85811.85611.85311.84911.84611.84411.83811.83211.83011.82511.82211.81711.81511.81311.8077.2687.2554.2814.2583.4922.7412.7172.6952.6702.6462.6242.6002.2712.2492.2262.2042.1812.1592.1362.1072.0842.0742.0622.0522.0392.0171.9951.6991.2621.0741.0520.9830.9610.9370.9151.000.560.560.910.291.474.370.530.131.091.05956OHO1023H3C1213CH3H3CHOHH3CO1478111416159appm(f1)05010015020005000138.481121.92377.71577.29276.87074.68442.02934.60327.28022.98319.29418.135图16SarcoF5-3-13CNMR956OHO1023H3C1213CH3H3CHOHH3CO14781114