| 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.
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