Directed evolution of hemeproteins for selective cyclizations

用于选择性环化的血红素蛋白的定向进化

• 批准号: 313888180
• 负责人: Dr. Grzegorz Kubik
• 金额: --
• 依托单位: Division of Chemistry and Chemical Engineering
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/313888180?language=en
• 关键词: Directed; evolution; hemeproteins; selective; cyclizations

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.

总之，在本研究中，将创建用于生产多种杂环化合物的合成工具，这些杂环化合物在许多药剂学中具有高度重要性。该方法的原理是利用亲电试剂捕获类卡宾和类氮杂环戊烯的杂原子氢键插入过程中的中间叶立德。为此，首先通过细胞色素P450的定向进化产生在杂原子-氢键处的插入反应的所需范围。接下来，酶变体将被进一步工程化以促进捕获反应，采用三种不同的亲电基团。酶作为催化剂的益处由经常提供的高选择性和重新设计这些酶以获得选择性的可能性（例如将选择性从一种对映体切换到另一种对映体）来定义。此外，可以应用温和和环境友好的反应条件，这意味着最新方法的可持续替代品。在这个项目中，还可以提出对叶立德形成，稳定性和反应性的新见解，从而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，可以建立光谱高通量测定来跟踪反应。这将允许筛选在整个完整氨基酸序列中含有随机突变的酶文库。到目前为止，研究的P450文库主要集中在对催化口袋有贡献的位点处的突变，这是由于缺乏这样的测定。因此，在该项目中，可以揭示P450部分中不直接参与催化口袋的完全未知的结构-活性关系。