Enzymatic synthesis of new terpenes from terpene synthase substrate analogs

本项目旨在合成萜烯合成酶的底物类似物及其酶促转化。计划中的结构修改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(受阻的反应性，预计会导致脱轨产物)，以及去除甲基(缓冲反应性)。此外，还将合成一系列骨架延伸了一到四个碳的底物类似物，这些底物可以研究二萜和七萜之间的功能开关，或者七萜和三萜合成酶之间的功能开关。在这个项目中得到的所有化合物都将被分离出来，它们的结构将通过光谱分析方法来确定。绝对构型将通过我们实验室先前建立的立体选择性重氢策略来确定。如果由于空间原因，某些酶-底物组合不起作用，将通过定点突变获得活性部位腔扩大的酶变体。与科隆大学Bernd Goldfuss教授合作的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，以使无法从天然底物获得的新的萜类化合物变得容易获得。

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.