Biocatalytic dealkylation toolbox for organic synthesis

用于有机合成的生物催化脱烷基化工具箱

• 批准号: 492770259
• 负责人: Professor Dr. Uwe T. Bornscheuer
• 金额: --
• 依托单位: Institut für Angewandte Synthesechemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/492770259?language=en
• 关键词: Biocatalytic; dealkylation; toolbox; organic; synthesis

Complex organic synthesis generally requires the use of orthogonal protecting groups. For this, methyl ethers and related alkyl derivatives are highly relevant. They are easily introduced, cheap, chemically robust and fulfill almost all criteria for a perfect protection of hydroxy groups. However, their removal requires strongly acidic and/or nucleophilic conditions, substantially limiting their actual usability in the synthesis of complex molecules. We aim to establish a generic biocatalytic solution based on redox-active enzymes for O dealkylation. They catalyze this transformation under mild conditions utilizing molecular oxygen as oxidant. The aim is to create a versatile toolbox for organic chemists to remove O-alkyl side chains in a regio-, and chemoselective fashion. We will address two major research questions: (1) “How much chemical space can be covered by enzymatic O-dealkylation?” and (2) “Can biocatalysts be identified and engineered towards a promiscuous, chemo- and regioselective efficient dealkylation tool for the organic chemist?” Dealkylation will be catalyzed by P450-monooxygenases, which upon hydroxylation of alkyl ethers yield unstable hemi-acetals, decomposing into the free hydroxy group and an aldehyde. We will use established tools for in silico-based enzyme discovery (i.e, BLAST search), a wide range of protein engineering methods (e.g. structure-guided rational design, directed evolution) in combination with adapted in-house- developed high throughput assays (HTA) to develop and improve the biocatalysts for synthetic applications. A smart synthetic library of compounds will be established and biocatalysis will be followed by organic analysis, purification and structural elucidation (prep. & anal. HPLC-MS, 1D/2D-NMR spectroscopy, HRMS-analysis). We aim to establish novel O-dealkylating enzymes as a general means for the cleavage of alkyl ethers and closely related stable O-protecting groups. We will provide biochemical data of yet unexplored proteins and focus on the scope and limitations of cytochrome P450s from the BM3 family and the enzymes recently discovered by the Bornscheuer group in marine bacteria which naturally catalyze the demethylation of 6-O-methyl-D-galactose. As the X-ray structure of one P450 enzyme has been solved, rational protein design approaches are highly feasible to alter their substrate scope. We will explore and study further candidate enzymes to enlarge the toolbox of suitable P450s with extended substrate scopes. The core team combines expertise from different scientific disciplines, namely synthetic chemistry (Christian Stanetty, TU Wien), biocatalysis (Florian Rudroff, TU Wien) and enzyme discovery, protein engineering & biocatalysis (Uwe Bornscheuer, University of Greifswald) in Germany. This scientific symbiosis complements the necessary know-how to accomplish the planned research.

复杂的有机合成通常需要使用正交保护基。为此，甲基醚和相关的烷基衍生物是高度相关的。它们易于引入，价格便宜，化学稳定，几乎满足所有完美保护羟基的标准。然而，它们的去除需要强酸性和/或亲核条件，基本上限制了它们在合成复杂分子中的实际可用性。我们的目标是建立一个通用的生物催化解决方案的基础上氧化还原活性酶O脱烷基化。它们利用分子氧作为氧化剂在温和条件下催化这种转化。目的是为有机化学家创造一个多功能的工具箱，以区域选择性和化学选择性的方式去除O-烷基侧链。我们将解决两个主要的研究问题：（1）“有多少化学空间可以覆盖酶的O-脱烷基化？”和（2）“生物催化剂能否被鉴定和设计成有机化学家的一种混杂的、化学选择性和区域选择性的有效脱烷基化工具？”脱烷基化将由P450-单加氧酶催化，其在烷基醚的羟基化后产生不稳定的半缩醛，分解成游离羟基和醛。我们将使用已建立的基于计算机的酶发现工具（即，BLAST搜索），广泛的蛋白质工程方法（例如，结构指导的合理设计，定向进化）与适应的内部开发的高通量测定（HTA）相结合，以开发和改进用于合成应用的生物催化剂。将建立一个智能的化合物合成库，生物催化之后将进行有机分析、纯化和结构鉴定（制备和分析）。HPLC-MS，1D/2D-NMR光谱，HRMS分析）。我们的目标是建立新的O-脱烷基化酶作为烷基醚和密切相关的稳定的O-保护基团的裂解的一般手段。我们将提供尚未探索的蛋白质的生化数据，并专注于从BM 3家族和最近发现的海洋细菌中的Bornscheuer组天然催化6-O-甲基-D-半乳糖的脱甲基化的酶的细胞色素P450的范围和限制。随着一种P450酶的X射线结构的解决，合理的蛋白质设计方法是非常可行的，以改变其底物范围。我们将探索和研究进一步的候选酶，以扩大合适的P450工具箱，并扩大底物范围。核心团队结合了来自不同科学学科的专业知识，即合成化学（Christian Stanetty，TU Wien），生物催化（Florian Rudroff，TU Wien）和酶发现，蛋白质工程和生物催化（Uwe Bornscheuer，德国格赖夫斯瓦尔德大学）。这种科学共生补充了必要的专门知识，以完成计划的研究。