Enzymatic synthesis of new terpenes from terpene synthase substrate analogs

本项目旨在萜烯合酶的底物类似物的合成及其酶转化。计划的结构修饰包括具有移位双键的底物类似物，以实现预期的反应性变化，具有氢化双键（阻止反应性，预期导致脱轨产物），以及去除甲基（缓冲反应性）。此外，一系列骨架延伸1到4个碳的底物类似物将被合成，从而可以研究二萜和二萜之间，或二萜和三萜合成酶之间的功能切换。本项目中获得的所有化合物将被分离，其结构将通过光谱方法加以阐明。绝对构型将通过先前在我们实验室建立的立体选择性氘化策略来确定。如果由于空间原因，某些酶-底物组合不起作用，则通过位点定向诱变获得活性位点空腔扩大的酶变体。与Bernd Goldfuss教授博士（科隆大学）合作的DFT计算将加深对有趣的循环机制的理解。总体而言，该项目的目的是广泛展示萜烯合成酶在底物类似物转化中的适用性，从而使无法从天然底物中获得的新萜烯易于获得。

This project aims at the synthesis of substrate analogs of terpene synthases and their enzymatic conversions. The planned structural modifications include substrate analogs with shifted double bonds for an expected change of reactivity, with hydrogenated double bonds (blocked reactivity, expectedly leading to derailment products), and removed methyl groups (buffered reactivity). Furthermore, a series of substrate analogs with skeletal extensions by one to four carbons will be synthesised that allow to study functional switches between diterpene and sesterterpene, or between sesterterpene and triterpene synthases. All compounds obtained in this project will be isolated and their structures will be elucidated by spectroscopic methods. Absolute configurations will be determined through a stereoselective deuteration strategy previously established in our laboratory. If for steric reasons certain enzyme-substrate combinations fail to work, enzyme variants with enlarged active site cavities will be obtained by site-directed mutagenesis. DFT calculations in collaboration with Prof. Dr. Bernd Goldfuss (University of Cologne) will add to a deeper understanding of interesting cyclisation mechanisms. Overall, the aim of this project is to broadly show the applicability of terpene synthases in the conversion of substrate analogs to make new terpenes accessible that cannot be obtained from the natural substrates.