木霉属真菌被广泛应用于农业生物防治等领域，其次生代谢产物结构类型丰富，并且部分产物具有显著的生物活性。海洋环境中也有木霉分布，对其次生代谢产物的研究始于20世纪90年代，是近年来比较活跃的研究领域。海洋藻栖木霉是指宿主为海洋藻类植物的木霉菌株。海洋环境的特殊性及与藻类的共附生关系，可能赋予了藻栖木霉特殊的次生代谢途径和产物类型，但目前仅10株藻栖木霉的次生代谢产物被研究。因此，海洋藻栖木霉次生代谢产物化学结构与生物活性的研究具有重要的探索意义。

  基于化学和活性筛选，从130株海洋藻栖木霉中选取4株作为研究对象，包括2株棘孢木霉(Trichoderma asperellum) cf44-2、A-YMD-9-2和2株哈茨木霉(T. harzianum) A-YMD-2-2、X-5。综合运用各种现代色谱技术，包括硅胶柱、C18反相柱、Sephadex LH-20凝胶柱、制备薄层及半制备高效液相色谱等，分离次生代谢产物单体。应用各种现代波谱和质谱技术，包括一维/二维核磁共振谱、红外光谱、紫外光谱、电子圆二色谱、X射线单晶衍射谱、低分辨和高分辨质谱等，确定单体化合物的结构。从棘孢木霉cf44-2中分离鉴定了33个化合物，其中新化合物16个(A1‒A16)，包括1个新骨架甾体衍生物(A1)、1个三降二萜(A2)、1个没药烷氨基糖苷(A3)、1个降没药烷氨基糖苷(A4)、1个没药烷倍半萜(A5)、1个降没药烷倍半萜(A6)、3个环橙花烷倍半萜(A7–A9)、1个harziane二萜(A10)、2个单萜(A11和A12)、1个环戊烯酮(A13)、1个氯代环戊烯酮(A14)、1个二酮哌嗪(A15)及1个生物碱(A16)，化合物A2–A4为首次发现的结构类型。从棘孢木霉A-YMD-9-2中分离鉴定了42个化合物，其中30个新化合物(B1–B30)，包括10个含氮环橙花烷衍生物(B1–B10)、7个环橙花烷倍半萜(B11–B17)、4个降没药烷倍半萜(B18–B21)、3个降没药烷氨基糖苷(B22–B24)、2个杜松烷倍半萜(B25和B26)、1个harziane二萜(B27)、1个二酮哌嗪(B28)及2个多肽(B29和B30)，特别是首次发现2个萜类铁载体(B1和B2)，能与三价铁离子发生络合反应。从哈茨木霉A-YMD-2-2中分离鉴定了21个化合物，其中包括1个新环橙花烷倍半萜(C1)。从哈茨木霉X-5中分离鉴定了36个化合物，其中7个新化合物(D1–D7)，包括1个harziane二萜(D1)、1个proharziane二萜(D2)、3个环橙花烷倍半萜(D3–D5)、1个菖蒲烷倍半萜(D6)及1个sorbicillin衍生物(D7)，化合物D2的发现对harziane二萜的生源合成途径起到了很好的证实作用。

  对分离鉴定的部分单体化合物进行了抗微藻、抗菌和卤虫毒性评价。甾体衍生物A1对4种赤潮微藻表现出显著的抑制作用，半数抑制浓度(IC₅₀值)为0.3–0.6 μg/mL，还可抑制植物病原真菌围小丛壳的生长，最小抑菌浓度(MIC值)为12 μg/mL，并且对卤虫低毒。含氮环橙花烷B1和B2与三价铁离子的络合物对赤潮微藻抑制活性(IC₅₀值为0.99–10 μg/mL)较自身显著增强。没药烷氨基糖苷A3、A4和B22–B24对4种微藻具有抑制作用，IC₅₀值为3.7–24.8 μg/mL，同时还对4种弧菌均表现出一定的抑制作用，抑菌圈直径为6.1–7.5 mm。降没药烷倍半萜B19–B21对4种微藻也表现出较好的抑制活性(IC₅₀值为0.93–9.2 μg/mL)，分子中苯环的存在具有增强活性的作用。Harziane二萜B27和proharziane二萜D2 对4种微藻表现出较好的抑制活性，IC₅₀值为1.2–7.7 μg/mL。环戊烯酮A13和A14对造成海产养殖病害的鳗弧菌、哈维氏弧菌、副溶血弧菌和灿烂弧菌具有一定的抑制作用，其中卤代结构A14活性较好，抑菌圈直径为6.5–8.5 mm，但A13对4种微藻表现出较好的抑制活性，IC₅₀值为4.2–8.5 μg/mL。

  通过对四株海洋藻栖木霉次生代谢产物化学结构和生物活性的研究，总计分离鉴定次生代谢产物132个，其中新化合物54个，结构类型涵盖甾体、二萜、倍半萜、单萜、聚酮、二酮哌嗪、肽和生物碱等，并筛选出若干具有较好抗微藻和抗病原菌等生物活性的分子，丰富了海洋藻栖木霉次生代谢产物的结构多样性，为海洋藻栖木霉的在有害赤潮和病原菌防治方面的开发利用提供了一定的理论基础和科学依据。

  Fungi of the genus Trichoderma have been regarded as the most potential biocontrol agents in agriculture, and hundreds of secondary metabolites with various bioactivities have been discovered from them so far. Although Trichoderma is commonly considered as a terrestrial genus, halotolerant strains have been continuously reported from marine environments. Chemical investigations toward them have been performed since the 1990s, which have attracted great attention for natural product research recently. Marine algicolous Trichoderma spp. are the filamentous fungi inhabiting or associated with marine algae, and they possibly feature unique pathways and structures of secondary metabolites due to their special habitats. To date, only 10 marine algicolous Trichoderma strains have been chemically surveyed. It is meaningful to further explore the structures and bioactivities of secondary metabolites from marine algicolous Trichoderma fungi.

  Through chemical and biological screening of 130 marine algicolous Trichoderma isolates, four strains including Trichoderma asperellum cf44-2 and A-YMD-9-2 and Trichoderma harzianum A-YMD-2-2 and X-5 were selected as the objects. Their secondary metabolites were isolated by repeated column chromatography on silica gel, RP-18, and Sephadex LH-20 and preparative thin layer chromatography as well as semipreparative HPLC. Their structures were identified by spectroscopic methods, including 1D/2D NMR, IR, UV, ECD, X-ray, and MS. Effects on T. asperellum cf44-2 resulted in the isolation and identification of 33 compounds, with sixteen (A1–A16) including one highly transformed ergosterol derivative (A1) with a new carbon skeleton, one ethylated bisobolane diterpene (A2), two aminoglycosides of bisabolane sesquiterpenes (A3 and A4), one bisobolane sesquiterpene (A5), one norbisobolane sesquiterpene (A6), three cyclonerane sesquiterpenes (A7–A9), one harziane diterpene (A10), two naturally occurring monoterpenes (A11 and A12), one naturally occurring trichodenone (A13), one chlorine-containing trichodenone (A14), one diketopiperazine (A15), and one naturally occurring oxazole derivative (A16) being new ones. Among them, compounds A2–A4 are new structural types. 42 compounds including 30 new ones (B1–B30) were characterized from T. asperellum A-YMD-9-2. The new isolates comprise ten nitrogenous cyclonerane derivatives (B1–B10), seven cyclonerane sesquiterpenes (B11–B17), four norbisabolane sesquiterpenes (B18–B21), three norbisabolane aminoglycosides (B22–B24), two cadinane sesquiterpenes (B25 and B26), one harziane diterpene (B27), one diketopiperazine (B28), and two peptaibols (B29 and B30). Of those, compounds B1 and B2 featuring a cyclonerane backbone represent an unprecedented family of siderophores. On the other hand, 21 compounds including one new cyclonerane sesquiterpene (C1) were obtained from the culture of T. harzianum A-YMD-2-2. Additionally, T. harzianum X-5 gave 36 compounds, including seven new ones, i.e. harziane diterpene D1, proharziane diterpene D2, cyclonerane sesquiterpenes D3–D5, acorane sesquiterpene D6, and sorbicillin derivative D7. The discovery of D2 verifies the biogenetic pathways of harziane diterpenes.

  Some isolates were evaluated for antimicroalgal activity against Chattonella marina, Heterosigma akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense and antimicrobial activity mainly against Vibrio parahaemolyticus, V. anguillarum, V. harveyi, V. splendidus, Pseudoalteromonas citrea, and Glomerella cingulata as well as brine shrimp lethality against Artemia salina. The results showed that A1 could remarkably inhibit the four phytoplankton species, and the IC₅₀ values ranged from 0.27 to 0.59 μg/mL. Its antifungal activity against the phytopathogenic Glomerella cingulata was also assayed, and the MIC value was determined to be 12 μg/mL. Compared to nitrogenous cycloneranes B1 and B2, their ferric complexes exhibit high inhibition of the four phytoplankton species tested (IC₅₀, 0.99–10 μg/mL). Aminoglycosides A3, A4, and B22–B24 showed potent inhibition of the four microalgae (IC₅₀ 3.7–24.8 μg/mL) and four Vibrio bacteria (6.1–7.5 mm zones). Norbisabolanes B19–B21 with IC₅₀ values ranging from 0.93 to 9.2 μg/mL were more active to the four phytoplankton species than B18 and B22–B24, which might correlate with the presence of the phenyl group. Additionally, harziane diterpene B27 and proharziane diterpene D2 showed potent inhibition of the four phytoplankton species with IC₅₀ values 1.2–7.7 μg/mL. Trichodenones A13 and A14 exhibited growth inhibition of the four Vibrio bacteria, and the latter that contains a chlorine atom was more active (6.5–8.5 mm zones). The former could inhibit the four phytoplankton species tested, with IC₅₀ values ranging from 4.2 to 8.5 μg/mL.

  In this dissertation, studies on the structures and bioactivities of secondary metabolites from four marine algicolous Trichoderma strains are described. Totally, 132 secondary metabolites including 54 new ones were isolated and identified, comprising steroids, diterpenes, sesquiterpenes, monoterpenes, polyketides, diketopiperazines, peptaibols, and alkaloids. Some of them were evidenced to feature antimicroalgal and antimicrobial activities. The discovery of these new bioactive compounds greatly added to the structural diversity of secondary metabolites from marine algicolous Trichoderma, and it will greatly contribute to the development of utilization of marine algicolous Trichoderma in controlling harmful algal blooms and pathogenic microbes.

目 录

第1章 引言... 1

1.1 木霉次生代谢产物的研究进展... 1

1.1.1 聚酮类... 1

1.1.2 萜类及甾体类衍生物... 13

1.1.3 肽类... 20

1.1.4 生物碱及其它含氮化合物... 25

1.1.5 其它类... 30

1.2 藻栖木霉次生代谢产物的研究意义... 31

第2章 海洋藻栖木霉菌株分离与筛选... 35

2.1 菌株分离... 35

2.2 菌株发酵与产物提取... 36

2.3 菌株筛选... 37

2.3.1 化学筛选... 37

2.3.2 活性筛选... 37

2.4 菌株选取... 39

第3章 棘孢木霉cf44-2次生代谢产物的分离纯化与结构鉴定... 41

3.1 化合物结构鉴定... 41

3.1.1 新化合物结构鉴定... 41

3.1.2 已知化合物结构鉴定... 62

3.2 实验部分... 68

3.2.1 仪器与试剂... 68

3.2.2 菌株鉴定... 68

3.2.3 菌株发酵... 68

3.2.4 提取与分离... 69

3.2.5 化合物物理常数与波谱数据... 70

3.3 小结... 77

第4章 棘孢木霉A-YMD-9-2次生代谢产物的分离纯化与结构鉴定... 79

4.1 化合物结构鉴定... 79

4.1.1 新化合物结构鉴定... 79

4.1.2 已知化合物结构鉴定... 108

4.2 实验部分... 111

4.2.1 仪器与试剂... 111

4.2.2 菌株鉴定... 111

4.2.3 菌株发酵... 112

4.2.4 提取与分离... 112

4.2.5 化合物物理常数与波谱数据... 113

4.3 小结... 122

第5章 哈茨木霉A-YMD-2-2次生代谢产物的分离纯化与结构鉴定... 125

5.1 化合物结构鉴定... 125

5.1.1 新化合物结构鉴定... 125

5.1.2 已知化合物结构鉴定... 126

5.2 实验部分... 132

5.2.1 仪器与试剂... 132

5.2.2 菌株鉴定... 132

5.2.3 菌株发酵... 132

5.2.4 提取与分离... 133

5.2.5 化合物物理常数与波谱数据... 134

5.3 小结... 140

第6章 哈茨木霉X-5次生代谢产物的分离纯化与结构鉴定... 141

6.1 化合物结构鉴定... 141

6.1.1 新化合物结构鉴定... 141

6.1.2 已知化合物结构鉴定... 150

6.2 实验部分... 158

6.2.1 仪器与试剂... 158

6.2.2 菌株鉴定... 158

6.2.3 菌株发酵... 158

6.2.4 提取与分离... 159

6.2.5 化合物物理常数与波谱数据... 160

6.3 小结... 169

第7章 四株藻栖木霉次生代谢产物的生物活性评价... 171

7.1 抗微藻活性测试... 171

7.2 抑菌活性测试... 173

7.3 卤虫毒性测试... 176

7.4 小结... 177

第8章 结语... 179

8.1 结论... 179

8.2 创新点... 180

8.3 展望... 180

参考文献... 181

附录：化合物结构与部分化合物谱图... 205

致 谢... 219

作者简历及攻读学位期间发表的学术论文与研究成果... 221