新型四元氮桥环内酯类化合物农抗N2拮抗植物病原真菌的抑菌机制及构效关系研究

In our previous work, a novel Streptomyces sp. N2 with a broad and effective antifungal activity on various soilboren phytopathogenic fungi was isolated and screened out, which could produce a new structure of four-membered azalids compound (1-methyl-3-oxa-6-azabicyclo[3.1.1]hept-5(7)-en-2-one, temporarily named “Agricultural antibiotics N2”). However, compared to the common agricultural fungicides with five-membered and six-membered nitrogen heterocycles, the inhibition mechanism and structure-activity relationship of the four-membered N-heterocyclic compound is still unknown. Based on this fact, this project aims to illuminate the inhibition mechanism and structure-activity relationship of Agricultural antibiotics N2 on the common rice diseases inducing strains R. solani and Pyricularia grisea, so as to provide a theoretic foundation for its biocontrol application in plant diseases: (1) By implementing the investigation on cell-wall degradation, cell-membrane damage, the inhibition on macromolecule biosynthesis and the interference on energy metabolism system, it is expected to illuminate the inhibition mechanism of Agricultural antibiotics N2 on phytopathogenic fungi. (2) According to the chemical structure characteristics of Agricultural antibiotics N2, the derivatization reactions with Agricultural antibiotics N2 as the direct skeleton will be carried out on the reduction of the double bound in N-hetercycle, the reduction of eater carbonyl, the hydrolytic ring-opening of the lactone group, the alkylation and acylation of the amine in N-hetercycle, respectively. By performing the antibacterial activity evaluation together with the analysis of structure-activity relationship for these derivatives, it is expected to reveal the key active functional groups of Agricultural antibiotics N2 for its antifungal activities.

本课题组筛选到一株对植物病原真菌具有较强广谱拮抗作用的链霉菌新种N2，发现其所产活性物质系新型的四元氮桥环内酯类化合物（1-甲基-3-氧代-6-氮杂二环[3.1.1]-5(7)-庚烯-2-酮，暂命名农抗N2）。与常见的有机合成类五、六元氮杂环农用杀菌剂相比，该活性物质的抑菌作用机制及构效关系有怎样的相似或独特之处，目前尚不清晰。为此，本项目以诱发水稻常见病害的水稻纹枯病菌和稻瘟病菌为供试菌，系统开展农抗N2抑菌机理及其构效关系研究，为其生防应用提供理论基础：1）通过开展农抗N2对供试菌细胞壁合成、细胞膜透性、菌体生物大分子合成、菌体能量代谢系统等方面的影响，阐明其拮抗植物病原真菌的作用机理；2）紧扣农抗N2化学结构特征，直接以其为母体结构进行氮杂环双键还原、酯羰基还原、内酯基水解开环、杂环胺基烷基化和酰基化等衍生反应，通过开展各衍生物的抑菌活性评价，揭示农抗N2起抑菌作用的关键活性官能团。

农抗N2（1-甲基-3-氧代-6-氮杂二环[3.1.1]-5(7)-庚烯-2-酮）系由链霉菌N2所产生的新型四元氮桥环内酯类化合物，对多种植物病原真菌均具有显著拮抗作用，展现出良好的植物病害防治潜力。与常见的有机合成类五、六元氮杂环农用杀菌剂相比，农抗N2抑菌作用机制及构效关系有怎样的相似或独特之处尚不清晰。本项目系统开展了农抗N2抑菌机理及其构效关系研究，旨在为其生防应用提供理论基础。.（1）通过开展农抗N2对植物病原真菌细胞壁合成、细胞膜通透性、菌体生物大分子合成、菌体能量代谢系统影响以及诱导抗性等方面的研究，阐释了农抗N2拮抗植物病原真菌的作用机制：一方面通过诱发植物病原真菌几丁质酶活的提高，促进细胞壁几丁质降解产生N-乙酰葡萄糖胺，从而干扰细胞壁合成，并通过抑制麦角甾醇的合成致使细胞膜功能受损，引起膜透性显著增加；另一方面，通过影响植物病原真菌电子传递和中间产物转化而抑制呼吸能量代谢，并破坏菌体抗氧化防御系统，加剧细胞脂质过氧化作用，造成菌体内活性氧大量积累，干扰并影响菌体的正常生理代谢；同时通过间接诱导水稻幼苗提高活性氧清除、酚类物质代谢和病程相关蛋白代谢能力，增强宿主对植物病原真菌的拮抗特性。.（2）开展了农抗N2衍生物的合成、抑菌活性评价及其构效分析。根据农抗N2的分子结构特征，分别进行杂环C=C双键还原、2位酯羰基还原、2,3位内酯基水解开环、杂环胺基烷基化与酰基化等四种类型的化学反应，共制备获得19个农抗N2衍生物。通过对比各衍生物与农抗N2的抑菌活性，研究发现内酯基系农抗N2的关键活性基团，其次为杂环C=C双键、2位酯羰基。另外，尽管各衍生物的抑菌活性较农抗N2均出现显著下降，但是酰基化和糖基化利于衍生物抑菌活性增强。.（3）开展了农抗N2对意大利青霉、指状青霉的拮抗与诱导抗性作用机制研究。从农抗N2能够破坏意大利青霉及指状青霉细胞膜及细胞壁的合成，增强膜透过性，加剧脂质过氧化，破坏抗氧化系统，抑制菌体呼吸能量代谢等角度，进一步验证了农抗N2对植物病原真菌的抑制作用机理。.本研究通过开展链霉菌N2所产新型抗真菌活性物质——农抗 N2 拮抗植物病原真菌的作用机理和构效关系分析，为其生防应用以及后续的产物结构修饰和新药成药性等研究提供了理论依据。