新型十元环内酯活性位点形成及立体构型控制的生物合成研究

10-Membered ring lactiones (also known as nonanolides) were drawn extensive attention due to their special structure and significant biological activities.Cytospolides A-Q, new family of nonanolides, were recently isolated from an endophytic fungi Cytospora sp. The structures were elucidated by spectroscopic analysis, chemical interconversion, and single-crystal XRD. The specific chemical feature of a C-2 methyl group of cytospolides made them unique to all other nonanolides.Besides, the coexistance of C-2 stereoisomers of cytospolides were also found in these fungal metabolites. A cytotoxic screening in vitro against several tumor strains indicated that the stereochemistry of C-2 methyl played an important role in the cytotoxicity. In contrast to the negative cytotoxity of the most part of the cytospolides cytospolides E and P, the minor constitutes with a (2S) -configuration, were shown stronge cytotoxicity to the A549 tumor cells.The two fungal metabolites were also proved to significantly mediate G1 arrest in A549 tumor cells at 1 ug/mL. Based upon the occurrence of the unique C-2 methyl group in cytospolides and its stereochemically related bioactivity, we were interested to investigate the biosynthetic procress of cytospolides. Starting from cloning of the complete genomic cluster responsible for cytospolides biosynthesis, our project was intended to clarify their biosynthetic pathway, especially to reveal the biological mechanisms of the formation of C-2 methyl group and its stereoisomers. The biosynthetic knowledge of cytospolides will enable us to manipulate cytospolides analogues by rationally genetic modification. Our project enlarge will the understanding of fungal type I PKS, and will also promote to establish the structure-activity relationship by empolying cytospolides and its analogues as a chemical genomic probe.

十元环内酯因结构特殊、具有显著生物活性而受到关注，cytospolides是我们最近从内生真菌Cytospora sp.中获得的一类新骨架的十元环内酯，其结构特征是含有C-2甲基，这在已知的所有十元环内酯中均未发现；Cytospolides中同时存在着2R-和2S-甲基两种立体构型，2S-甲基型具有良好的抗肿瘤活性，而2R-甲基型无活性，这使2S-甲基成为十元环内酯的一个新活性位点。基于cytospolides的新活性位点、C2-甲基及其立体构型的特异性和构效关系研究需要，我们拟从克隆负责cytospolides生物合成的完整基因簇出发，通过体内基因中断、体外生化试验等生物合成途径研究，阐明C-2甲基及其立体异构体控制的酶学机制，探索通过生物合成途径的合理遗传修饰获得含2S-甲基类似物的新途径，该研究将揭示新骨架形成的遗传基础，促进以cytospolides及其类似物为探针的构效关系研究。

十元环内酯cytospolides是我们从内生真菌Cytospora sp.中获得的一类新骨架分子，其化学结构中的C2甲基是区别于所有已知十元环内酯的新颖点所在。C2甲基也是一个新活性位点，2S-甲基型cytospolides抗肿瘤活性良好，而2R-甲基型没有活性。在前期实验基础上我们开展了对cytospolides生物合成机制的研究，重点探索C2甲基形成及立体控制的酶学机制。C2甲基的形成机制我们做了两种推测：1）由真菌PKS基因簇中自带的C-甲基转移酶在聚酮链延伸过程中形成；2）在十元环内酯形成后通过甲基转移酶上载。针对上述问题，我们首先建立和优化了液体发酵和分析检测方法，目标主产物cytospolide A、D和E在该条件下均能够很好的发酵并被分离监测。进行同位素喂养实验，分别喂养[1,2,3-13C] propionate ，[3-13C] propionate和13C甲硫氨酸，经过对目标化合物的分离和13C NMR谱分析，发现[1,2,3-13C] propionate 和[3-13C] propionate喂养时未见有13C标记的参入，而13C甲硫氨酸喂养时C2-Me引入13C标记，δ 12.2处的C信号明显增强。证实cytospolides中的甲基不是由PKS基因簇自带的C-甲基转移酶途径引入的，而是由SAM依赖的甲基化引入。利用真菌PKS保守基因设计引物，筛选负责cytospolides生物合成的基因，体内敲除实验发现突变菌株不再产生cytospolides，证明该片段与cytospolides生物合成相关，通过染色体位移找到了完整的生物合成基因，由此我们可确定cytospolides的生物合成途径。此外，还从海参共附生真菌中获得了13个新颖的柔性大环内酯dendrodolides A-M；从海兔共附生真菌中获得了一个重排聚酮新骨架herbarone；从海绵中发现了一类具有6/6/5/7四环并环结构的新骨架甾体，swinhoeisterols A和B，并运用反向虚拟筛选技术成功发现了swinhoeisterol A是一种新型的(h)P300抑制剂。研究成果在Nat Chem Biol，Org Lett，J Org Chem，J Nat Prod，ChemBioChem等SCI期刊上发表论文7篇，申请专利2项。