微囊藻毒素衍生物的化学酶法合成及活性评价  

The Chemoenzymatic Synthesis and Bioactivity Study of Microcystin Analogue

作　　者：方剑[1] 何魁芳 季赛[1] 朱鹏[1] 金海晓[1] 严小军[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,浙江宁波315211

出　　处：《中国生物化学与分子生物学报》2014年第7期706-713,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.40906080);浙江省海洋生物技术产业科技创新团队(No.2010R50029);海洋公益性行业科研专项(No.201305013);宁波大学学科项目(No.xkc11003);宁波大学科研基金(No.XYL11014)~~

摘　　要：微囊藻毒素是一类环七肽蓝藻环肽化合物,目前自然界已发现70多种,其中microcystins-LR型因其独特的化学结构特征以及特异的磷酸酶结合模式,越来越受到药物化学家的关注.但因其天然结构中含有2个稀有氨基酸(Adda和Mdha),这使其化学全合成的难度增加,同时使其具有极高肝脏代谢毒性,严重影响了microcystin-LR作为新型的磷酸酶抑制剂的开发前景.本文以蛋白磷酸酶PP2A特异性抑制活性的micorcystin-LR为模板化合物,通过构效分析和GOLD 5.0分子对接结果,用L-Phe和L-炔丙基甘氨酸分别替代Adda和Mdha,设计出1个结合模式与模板较为一致的micorcystin类似物,并利用铜绿微囊藻(Microcystis aeruginosa)NRPS/PKS生物合成基因簇中TE结构域,在体外成功催化合成.该microcystin类似物的体外蛋白磷酸酶抑制实验显示有一定的抑制效果,虽与microcystin-LR本身还存在一定差距,但该microcystin类似物由于将引起肝脏毒性的主要残基Adda进行了取代,因此可以预见本研究最终合成的microcystin衍生物其对磷酸酶抑制活性降低同时,也将相应的降低对肝脏的毒性.这为开发新的低毒的磷酸酶抑制剂提供了一个新的选择对象,也为今后micorcystin衍生物的合成提供了一个新的合成思路.There are 70 more kinds of micorcystins, which are cyclic heptapeptides, found in cyanobacteria. Among them, the microcystin-LR has been receiving more and more attention from pharmaceutical chemists on account of its unique chemical structure and protein phosphatase combination mode. Nonetheless, two rare amino acids in its structure, Adda and Mdha, make it difficult to get microcystins-LR in complete chemical synthesis, and cause high hepatotoxicity, which severely limits its product development as a new protein phosphatase inhibitor. In this paper, micorcystin analogues benzyl mercaptan （MA-BMT） was structurally designed by GOLD 5.0 molecular docking and synthesized using specific inhibitor of protein phosphatase 2A, micorcystin-LR, as the template. It＇s shown from the result of molecular docking that the binding mode of microcystins analogue is consistent with microcystin-LR. We describe a novel chemoenzymatic method for the synthesis of microcystins analogue, which toxicityrelated rare amino acids were replaced by L-Phe and L-propargylglycine, respectively. Moreover, head- to-tail cyclisation was achieved using the thioesterase （TE） domain of microcystlns NRPS/PKS synthetase C （Mcy C ） from Microcystis aeruginosa to form micorcystin analogues. The in vitro protein phosphatase inhibitory experiment of this microcystin analogue shows that it has some certain effect but not good enough to be compared with mcrocystin-LR. But we replace the Adda residue, which results in hepatotoxicity, in this microcystin analogue and makes double benefits in synthesizing this compound much easier and bringing down its poison to liver. The novel method of designing this microcystin analogue is greatly prospected in the future industrial application.

关 键 词：微囊藻毒素 环肽 生物催化 蛋白磷酸酶抑制剂 

分 类 号：Q556[生物学—生物化学]