【作者】 程敬丽；

【导师】 林福呈； 朱国念；

【作者基本信息】 浙江大学 ， 植物保护， 2010， 博士

【摘要】 近年来,基于天然产物的新颖杀菌剂创制研究层出不穷,像源自于微生物的代谢产物井冈霉素,作为农用杀菌剂多年来对水稻纹枯病的防治效果一直很好。Stroblurins类杀菌剂也是以天然产物为母体经过结构修饰,创制出的一系列高效、广谱、.安全的杀菌剂,成为当今杀菌剂市场最活跃、最有发展潜力的一类新品种。木霉菌是植物内生真菌系列中很重要的一类生防菌,木霉菌的广泛应用与其主要代谢产物木霉菌素广谱高效的抑菌活性是分不开的。为了更细致的研究木霉属系列代谢产物,更好的发挥木霉菌的用途,也为了寻求更优异的结构骨架或新颖先导结构,本文以南方红豆杉Taxus chinesis var. mairei内生真菌紫杉木霉Trichoderma taxi sp. nov.菌的代谢产物木霉菌素为先导化合物,根据活性亚结构连接、生物等排性原理等方法,对此类倍半萜结构进行详细的结构修饰,细致的生物活性筛选,具体研究内容及结果如下：1、系统总结了内生真菌中单端胞霉烯类化合物的研究现状,木霉菌生物防治应用、木霉菌素的发酵、结构、活性,以及细菌、真菌及高等生物都具有的P450酶-CYP51酶的研究进展及其作为靶标来创制筛选新型抑菌活性物的应用前景。2、首先对木霉菌素倍半萜结构进行“破坏性”的骨架改变,结合生物活性测定,筛选确定了其重要的抑菌活性基团：Δ-9,10(双键)、12,13-环氧结构、C4位的乙酰基,C4位的立体构型：R构型的比S构型的活性好很多。3、分别在C4位、C8位、C9位和环氧位置进行衍生设计与合成,合成了化合物102个,其中88个是新化合物。所有化合物结构都经过1H NMR、MS,部分经过13C NMR确证结构,每章代表性化合物2.5、2.8、32f、32d、42e和5.1通过X-ray衍射,获得其晶体结构,进一步确定了其化学结构,为后续的小分子化合物与大分子靶标对接打下良好的基础。4、以《农药生物活性测定标准操作规范(SOP)》为准则,通过生物活性测定,筛选出系列抑菌活性良好的新化合物：在50μg/mL的浓度下,木霉菌素C4位衍生物中,化合物31e、31f和35a对稻瘟病菌的抑制活性明显比木霉菌素原药2.1高,都达到100%抑制率；对黄瓜灰霉病菌的抑制活性：化合物31e、31f和2.1都是100%,35a也达到94.3%的抑制率；2.1对水稻纹枯病菌的活性最高为100%,31e、31f、35a分别为95.5%、92.5%、95.5%；化合物31j和31k对赤霉的抑制率都为100%；化合物31e、35a和木霉菌素2.1对油菜菌核病菌的抑制率为87.7%、86.6%、95.9%。C8位衍生物中,化合物42g对油菜菌核病菌和稻瘟病菌的抑制活性明显都比木霉菌素原药2.1高,抑制率都为100%。C9衍生物中化合物5.1对灰霉菌、稻曲病菌的抑制率为100%。进一步的初筛、复筛表明：化合物31e、35a、2.1对黄瓜灰霉病菌的EC50值分别4.23、2.99和0.45μg/mL。化合物31e、35a、2.1对油菜菌核病菌的EC50值分别为10.86、12.06、2.20μg/mL。木霉菌素2.1对小麦赤霉病菌的EC50值为5.60μg/mL。化合物31e、31f和35a对稻瘟病菌也表现出良好的生物活性,EC50分别为6.31、12.9和6.00μg/mL,均超过母体木霉菌素的活性。31e、35a、2.1和商品化药剂丙环唑对水稻纹枯病菌的EC50值分别为2.48、4.18、0.72和6.45μg/mL,与丙环唑的毒效比分别为2.60、1.54、8.96。化合物42g对油菜菌核病菌的EC50值分别1.77μg/mL,高于母体木霉菌素。木霉菌素原药2.1、新化合物42g对稻瘟病病菌的EC50值分别为39.47和0.74μg/mL。化合物42g对稻瘟病菌的抑制活性远超过木霉菌素原药,与现今普遍应用的防治稻瘟病菌的杀菌剂咪鲜胺(EC50=0.98μg/mL)相当,比常规的三环唑和甲基硫菌灵活性要高。另外42g对水稻纹枯病菌的EC50值分别为3.58μg/mL,虽然没有母体木霉菌素的抑制率高,但是也超过了商品化药剂丙环唑(EC50为6.45μg/mL)。42g对水稻稻曲病菌的抑制活性较好,EC50为1.48μg/mL,超过木霉菌素2.1的EC50(2.11μg/mL),比常用的防治稻曲病的常用杀菌剂咪鲜胺(EC50为0.76μg/mL)略低一些。合成C8位衍生物烯丙位氧化过程中得到了另外一个产物,经1HNMR、MS、X-ray结构确证为C9醛木霉(5.1),经生物测定新颖倍半萜醛化合物5.1对多种植物病原菌活性优异,对黄瓜灰霉的抑制中浓度(EC50)为0.48μg/mL,接近木霉菌素的抑制中浓度0.45μg/mL;对水稻稻瘟病的EC50为11.82μg/mL,高于甲基托布津(EC50为36.72μg/mL),对水稻纹枯病菌的活性EC50为7.40μg/mL,虽然低于木霉菌素母体的活性,但也与丙环唑(EC50=6.45μg/mL)、井冈霉素(EC50为1.26μg/mL)在同一个活性数量级。尤其是对于水稻稻曲病菌的抑制活性很高,EC50为0.80μg/mL,与现在市场上畅销的杀菌剂咪鲜胺(EC50为0.76μg/mL)非常接近,高于母体木霉菌素一个数量级,在稻曲病防治上有一定的开发价值。5、应用两种不同软件计算log p,比较相互结果发现：C4衍生物中活性好的化合物log P值基本上在2.2到3.2之间,C8位衍生物中对稻瘟病抑制活性好的化合物42g的log P值基本上在3.6-4.0之间。并且通过比较发现用ALOGPS 2.1预测计算一些同分异构体化合物的log P更为精确一些。6、因为活性化合物5.1在水稻重要病害-稻曲病、纹枯病和稻瘟病防治上显示出良好的活性,化合物42g对水稻稻瘟病防治具有优异的活性,所以进一步研究了其对水生生物-典型大型蚤和栅藻的毒性,通过DS软件预测与实际检测,结果显示42g对大型蚤的毒性都是低毒级别,化合物5.1对大型蚤的毒性预测值与实际测定值都为中等毒性级别,对栅藻的毒性测定值为高毒。所以新颖化合物42g和5.1都可以作为二级先导化合物进一步优化开发或者直接作为潜在活性物用于开发新型倍半萜类广谱高效杀菌剂,具有进一步研究开发的价值。7、初步研究了活性小分子与CYP51酶的互作机制。采用计算机模拟计算,重点选择细菌、真菌、高等动植物中都很重要的P450家族中CYP51为靶标对象,采用同源建模,选择基因序列最接近的模板建立了灰霉的CYP51蛋白结构-BCCYP51酶,利用Profiles-3D得分值186,应用Autodock 4和DS Libdock对活性化合物分别进行分子对接。结果发现,同样对灰霉模建蛋白CYP51进行对接,不同软件结果一样,母体2.1与活性物5.1在活性口袋中的作用方式与作用位点非常相似,主要结合于HEME所处的活性口袋中央,环氧乙烷指向狭长口袋。小分子的乙酰基和金属铁相互作用。HEME以及金属离子是抑制剂结合的识别作用点。但是42g与5.1的结合方式不同，苯环疏水作用于狭长口袋,倍半萜结构中主要是C8位-丙烯酸酯中的酰基与HEME中的金属铁直接作用。通过小分子化合物与大分子靶标之间的分子对接,初步探讨了活性化合物的作用机制,为进一步的衍生设计打下了良好的基础。更多还原

【Abstract】 Currently, many novel natural bactericides were developed, such as Validamycin which is a lead compound against Rhizoctonia solani causing many root rots of plants. Series of derivatives from Strobilurin were also developed as high effective, broad-spectrum and safe bactericides based on its original structure modification.Trichodernia sp. could be a very important endophytic fungus. Recently, we found that the natural product, Trichodermin from this fungus, was able to be against Botrytis cinerea, Rhizoctonia solani and Sclerotinia sclerotiorum with the broad-spectrum, high antifungal activities. Trichodermin would be potentially used as the lead compound for the development of a new generation of industrial fungicides for crop protection by modifying this sesquiterpenoids structure and bioactive assaying, due to its novelty, safety and powerful antifungal molecules. In this thesis, we report the following results:1) The researches of Trichothecene mycotoxins were systematically reviewed, and the bioactive application, the formation, structure and bioactivities of the secondary metabolite, Trichodermin in an endophytic fungus, Trichodema sp. were also detailly investigated. CYP51, an enzyme of P450 family, widely in bacterium, fungi and other advanced organisms, was used as a target to develop novel antifungal compounds in this study as well.2) After destroying some moieties of Trichodermin and assaying their bioactivities, the key pharmacophores of this compound was A-9,10,12,13-epoxide moiety and C4-ester.4R-Trichodermin is more active than 4S-Trichodermin.3) 102 new derivatives of Trichodermin were synthesized by modifying the C4, C8, C9 and epoxide moiety and 88 of them are new compounds. All of these compounds were confirmed by 1H NMR, MS, and compound 2.5,2.8,32f,32d,42e,5.1 were confirmed by X-ray, which will be helpful in studying the structural relationship of small molecular with the target enzyme.4) According to《Stand operation process of testing pesticidal bioactivity》, a series of novel compounds with good antifungal activities were gotten:at 50μg/mL, compound 31e,31f and 35a have higher activity than lead compound Trichodermin in inhibiting Magnaporthe oryzae. These three compounds also show high bioactivities against B. cinerea and R. solani. Compound 31e,35a and 2.1 inhibit S. sclerotiorum. In the derivatives of C8 of Trichodermin, The inhibition rate of 42g on S. sclerotiorum and M. oryzae is better than that of 2.1. Compound 5.1 has a 100% inhibition on B. cinerea and M. oryzae at 50μg/mL. Further assay indictaed that the EC50 of 31e,35a and 2.1 on B. cinerea is 4.23,2.99 and 0.45μg/mL, respectively, and 10.86,12.0 and 2.20μg/mL on S. sclerotiorum, respectively. The EC50 of 2.1 on Gibberella zeae is 5.60μg/mL. Meanwhile, compound 31e,31f and 3.5a also showed good inbition activity with EC50 6.31,12.9 and 6.00μg/mL on M. oryzae, respectively. The EC50 of 31e,35a,2.1 and Propiconazol on R. solani is up to 2.48,4.18,0.72 and 6.45μg/mL, respectively. Obviously, the bioactivity of 31e,35a and 2.1 on R. solani is several times than Propiconazol.Interestingly, compound 42g has good potential to develop as a broad-spectrum antifungal pesticide. The EC50 of it on S. sclerotiorum and on M. oyzae is 1.77μg/mL and 0.74μg/mL respectively, which is better than lead compound 2.1. Moreover, the bioactivity of 42g is almost the same as Prochloraz and better than Tricyclazole and Thiophanate methyl. Although the EC50 of 42g is only 3.58μg/mL on R. solani, a little less than 2.1, it is still more active that Propiconazol. And the bioactivity of 42g is also better than 2.1 and almost the same as Prochloraz.Compound 9-aldehyde Trichodermin (5.1) is another potential antifungal reagent. It was formed by oxidation ofΔ-9,10 with SeO2. This compound showed broad-spectrum antifungal activities and with an EC50 of 0.48μg/mL on B. cinerea. Its EC50 on M. oyzae and R. solani is also up to 11.82μg/mL and 7.40μg/mL respectively, which is a little more active than Thiophanate methyl and almost the same as Propiconazol and Validamycin. More interestingly, its EC50 on Ustilaginoidea virens reaches 0.80μg/mL, almost the same as Prochloraz (EC50,0.76μg/mL) and much better than 2.1. Therefore, 5.1 have the potential to develop as an antifungal reagent on B. cinerea and Ustilaginoidea virens.5) Two different softwares were used to calculate the log p value and its possible relationship with derivatives’ bioactivities. It was found that the log p value range from 2.2 to 3.2 of the derivatives of C4 of Trichodermin which have good antifungal activities. The log p value range from 3.6-4.0 of the C8 derivatives such as 42g.6) Based on the excellent antifungal activities of 5.1 and 42g against paddy fungi, we further evaluated their toxicities on typical Daphnia magna and Scenedesmus subspicatus. Through DS software calculation and practical evaluation,42g has low toxicity both on Daphnia magna and on Scenedesmus subspicatus; 5.1 has middle toxicity on Daphnia magna and high toxicity on Scenedesmus subspicatus. Thus,42g and 5.1 can be used as new lead compound or even as potential antifungal reagents on paddy fungi for further development.7) CYP51, which is an enzyme of P450 family and exists widely in bacterium, fungi and other advanced organisms, was used as a target to study the mechanism of small molecular. First, we construct BCCYP51 protein of Botrytis cinerea used by the same enzyme resource model and similar gene sequence. Then to gain a structural understanding and visualize the interaction of the CYP51 protein with compound 2.1, 5.1 and 42g, docking studies were performed using Autodock 4 and Libdock. Although different softwares were used to simulate and calculation 2.1,5.1 and 42g’s position in CYP51, the result is the same. Results indicated that lead compound 2.1 and 5.1 have the similar effective manner and action position in target enzyme, but the 42g is different in the position of C8 acrylate. This primary conclusion will help design and prepare novel antifungal pesticides.更多还原