Synthesis of isotopically labelled terpene precursors for structure elucidation of terpenes and investigation of mechanistic aspects of their biosynthesis

本项目将合成萜烯单体二甲基丙烯基焦磷酸（DMAPP）和异戊烯基焦磷酸（IPP）的几个甲基化类似物。得到的化合物将使用FPP或GGPP合成酶与香叶基二磷酸（GPP）、法尼基二磷酸（FPP）或IPP偶联，得到带有一个额外甲基的FPP和香叶基二磷酸（GGPP）的甲基化类似物。随后，还将尝试加入一个以上的附加甲基。产物将被磷酸酶去磷酸化，分离并通过核磁共振波谱分析其结构。得到的甲基化的无环萜烯前体将通过各种萜烯合成酶转化为环产物。形成的萜烯将被分离出来，并对其结构进行分析。通过这种方法，自然可达的结构空间将大大扩大，我们对萜烯合酶的可塑性的理解将加深。此外，现有的13c标记的萜烯前体文库将得到扩展，使所有单一13c标记的甲基化萜烯类似物的同位素体都可以通过酶反应获得。利用所标记的化合物，可以研究人工萜烯的环化机理及其EI-MS断裂机理。

In this project several methylated analogs of the terpene monomers dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphat (IPP) will be synthesised. The obtained compounds will be coupled enzymatically to geranyl diphosphate (GPP), farnesyl diphosphate (FPP) or IPP using an FPP or GGPP synthase to obtain methylated analogs of FPP and geranylgeranyl diphosphate (GGPP) with one additional methyl group. Subsequently, also the incorporation of more than one additional methyl group will be tried. The products shall be dephosphorylated by a phosphatase, isolated and their structures will be elucidated by NMR spectroscopy. The obtained methylated acyclic terpene precursors will be converted into cyclic products using various terpene synthases. The formed terpenes will be isolated and their structures will be elucidated. Through this approach the naturally accessible structural space will be significantly expanded and our understanding of the plasticity of terpene synthases will be deepened. Furthermore, an already existing library of 13C-labelled terpene precursors will be extended to make all singly 13C-labelled isotopomers of methylated terpene analogs accessible by enzyme reactions. With the labelled compounds the cyclisation mechanisms of artificial terpenes and their EI-MS fragmentation mechanisms can be studied.