Total Synthesis of Tagitinins, Goyazensolide and Related Furanoheliangolides and their Covalent Interaction with Importin-5 (IPO5)

Herein, we detail an extension of our research on the synthesis of a small library of furanoheliangolides and the characterization of the covalent interaction between goyazensolide and IPO5. Using a build-couple-pair strategy, we assembled a small library of germacrene-type lactones and diversified them into eight groups of structurally different analogues. The germacrene lactones were synthesized using Sonogashira coupling and Barbier-type macrocyclization, while the furanoheliangolides were further elaborated through gold-catalyzed transannulation followed by esterification. This synthetic approach enabled the generation of a goyazensolide alkyne-tagged cellular probe, which was used to identify the selective binding between goyazensolide and the oncoprotein importin-5 (IPO5). Mass spectrometry analysis of the proteolytic digest from the reaction between the goyazensolide probe and a recombinant IPO5 indicated a covalent engagement at Cys560 of IPO5, which was confirmed by site-directed mutagenesis.本文详述了本团队关于呋喃雅槛兰烷内酯（furanoheliangolides）小分子库合成，以及戈亚辛内酯（goyazensolide）与IPO5之间共价相互作用表征的研究拓展工作。本研究采用构建-偶联-配对（build-couple-pair）策略，组装了一类牻牛儿烯型内酯小分子库，并将其衍生为八组结构各异的类似物。牻牛儿烯内酯通过薗头（Sonogashira）偶联与巴比耶（Barbier）型大环化反应合成，而呋喃雅槛兰烷内酯则经金催化跨环化反应及后续酯化反应进一步衍生得到。该合成策略成功制备了炔基标记的戈亚辛内酯细胞探针，借此鉴定出戈亚辛内酯与癌蛋白输入蛋白5（importin-5, IPO5）之间的选择性结合作用。对戈亚辛内酯探针与重组IPO5反应后的蛋白水解产物进行质谱分析，结果显示IPO5的Cys560位点发生了共价结合，该结果通过定点诱变实验得到了验证。