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

在这个项目中，将合成几个萜类单体的甲基化类似物焦磷酸二甲基烯丙基(DMAPP)和焦磷酸异戊烯(IPP)。得到的化合物将通过FPP或GGPP合成酶与香叶基二磷酸(GPP)、法尼基二磷酸(FPP)或IPP进行酶偶联，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额外甲基。产物应用磷酸酶去磷酸化，分离出来，其结构将通过核磁共振光谱分析确定。得到的甲基化的无环萜类前体将使用各种萜类合成酶转化为环状产物。形成的萜类化合物将被分离出来，其结构将被阐明。通过这种方法，自然可达的结构空间将被显着扩大，我们对萜烯合成酶的可塑性的理解将会加深。此外，现有的13C标记的萜类前体文库将被扩展，使所有单一的13C标记的甲基化萜类类似物的同位异构体都可以通过酶反应获得。利用这些标记化合物，可以研究人工萜类化合物的环化机理及其电喷雾质谱裂解机理。

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.