Expanding the synthetic toolbox of ruthenium-based artificial metalloenzymes (Ru-ArMs)

尽管酶的特点是其精致的催化性能，从而使生物体中几乎所有重要的化学转化都能实现，但可控制的反应的数量仅限于自然进化过程中出现的过程。人工金属酶(ARM)是含有一种非生物辅因子的蛋白质的杂交体，这种金属络合物在自然界中不存在。在怀特赛兹和凯泽从20世纪70年代末的S开始的初步工作的基础上，ARM在过去几年中再次引起了极大的关注，这是因为重组蛋白生产方面的突破，丰富的多功能有机金属辅因子，以及对结合均相和酶催化的诱人特征的潜在好处的日益认识。这项技术将经典合成化学与生物技术相结合。为了优化ARM以适应不同的应用，辅因子将被综合修饰，而蛋白质宿主可以通过定向进化来调整。定向合成变异和随机突变都可以用来优化手臂以进行自然界本身无法提供的反应。在这个项目中，我们将制备一个基于Ru的臂库(Ru-Arm)，并筛选其在烯炔转化、二炔环化以及烯丙基和丙叉醇的异构化反应中的催化性能。基于Ru的辅因子被证明是非常通用的，健壮的，已经被应用于细胞的周质和细胞质中，因此为该项目的成功提供了巨大的前景。我们将努力扩大Ru-ARM目前狭窄的应用领域，用于体内应用，即在活体生物中应用，如大肠杆菌(大肠菌群)，从而产生自然界中没有的化学反应。RU-ARM将通过蛋白质宿主的合成修饰和突变来针对各自的应用进行优化。该领域和项目(如这里提出的研究计划)的先前进展可能为新的生化/医学应用奠定基础，例如在需要它们的(癌细胞)细胞中准确且仅在原位形成生物活性物质，从而不影响未受损的组织。此外，武器技术具有利用可再生原料生产商品化学品的潜力，并可能有助于我国以石油为基础的经济不可避免地向可持续生产道路过渡。

Although enzymes are characterized by their exquisite catalytic performance, thus enabling nearly all vital chemical transformations in organisms, the number of manageable reactions is limited to processes that have emerged during natural evolution. Artificial Metalloenzymes (ArMs) are hybrids of proteins that contain an abiotic cofactor, metal complexes which do not occur in nature. Building on preliminary works by Whitesides and Kaiser from the late 1970’s, ArMs have gained significant attention again in the past years, due to breakthroughs in recombinant protein production, an abundance of versatile organometallic cofactors, as well as an increased appreciation of the potential benefits of combining the attractive features of both homogeneous and enzymatic catalysis.This technology combines classical synthetic chemistry with biotechnology. To optimize ArMs for the respective application, the cofactors will be modified synthetically, while the protein host can be tuned by directed evolution. Both targeted synthetic variations and random mutagenesis can be used to optimize ArMs for reactions that nature itself can not provide.In this project, a library of ruthenium-based ArMs (Ru-ArMs) shall be prepared and screened for catalytic properties in enyne transformations, diyne cylizations as well as isomerization reactions of allylic and propargylic alcohols. Ruthenium-based cofactors proved to be very versatile, robust, have already been applied in the periplasm and cytoplasm of cells and thus provide great prospects for the success of the proposed project. We will try to expand the currently narrow field of application of Ru-ArMs for in vivo applications, i.e. in living organisms such as E. coli (coliform bacterium), and thus create chemical reactivities that are not found in nature. Ru-ArMs will be optimized for the respective application both by synthetic modification as well as mutagenesis of the protein host.Previous progress made in the field and projects such as the here proposed research plan could build the foundation for novel biochemical/medicinal applications such as the in situ formation of biologically active agents exactly and only in (cancer) cells where they are needed, thus not affecting undamaged tissue. Furthermore, the ArMs technology bears the potential of producing commodity chemicals from renewable feedstocks and might contribute to the inevitable transition of our petroleum-based economy towards sustainable production pathways.