Directed evolution of hemeproteins for selective cyclizations

总而言之，在这项研究中，将创造用于生产多种杂环化合物的合成工具，这些化合物在许多制药学中具有非常重要的作用。该方法的原理是利用酶促杂原子氢键插入类卡宾和类硝基化合物中的亲电性来捕获中间体叶立德。为此，首先将通过细胞色素P450的定向进化产生所需范围的杂原子-氢键插入反应。接下来，将进一步设计酶变体以促进捕获反应，使用三个不同的亲电基团。酶作为催化剂的好处在于经常提供的高选择性，以及重新设计这些酶以获得选择性的可能性，例如将选择性从一个对映体切换到另一个对映体。此外，可以应用温和和环境友好的反应条件，这意味着可以作为最新方法的可持续替代。在这个项目中，还可以对叶立德的形成、稳定性和反应性提出新的见解，以便详细了解如何切换反应性。一个非常有趣的方面是，对于一些捕获反应，可以建立高通量光谱分析来跟踪反应。这将允许在整个氨基酸序列中筛选包含随机突变的酶文库。到目前为止，由于缺乏这样的分析方法，所研究的P450文库主要集中在有助于催化口袋的位点的突变。因此，在这个项目中，完全未知的结构-活性关系在P450中不直接对催化口袋起作用的部分可能会被揭示。

In summary, within this study synthetic tools for the production of a large variety of heterocyclic compounds, which are of high importance in many pharmaceutics, will be created. The principle of this method is to trap intermediate ylides with electrophiles in enzymatic heteroatom-hydrogen bond insertion of carbenoids and nitrenoids. For this, at first the required scope of insertion reactions at heteroatom-hydrogen bonds will be generated by directed evolution of Cytochrome P450. Next the enzyme variants will be further engineered to facilitate the trapping reaction, employing three different electrophilic groups. The benefits of enzymes as catalysts are defined by the high selectivity that is provided frequently and the possibility to reengineer those enzymes to obtain selectivities of choice, for example switching the selectivity from one enantiomer to the other. Furthermore mild and environment friendly reaction conditions can be applied, meaning a sustainable alternative to up to date methods. Within this project also new insights into ylide formation, stability and reactivity may be raised that allow to understand in detail how to switch reactivity. One very interesting aspect is that for some of the trapping reactions a spectroscopic high throughput assay can be set up to track the reaction. This will allow for screening of enzyme libraries containing random mutations throughout the complete amino acid sequence. Till now the investigated P450 libraries mainly focused on mutations at sites contributing to the catalytic pocket due to the lack of such an assay. So within this project completely unknown structure-activity relations within parts of P450 not contributing directly to the catalytic pocket could be disclosed.