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

虽然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成为可能，但可控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（ArMs）是含有非生物辅助因子的蛋白质的杂交产物，这种金属配合物在自然界中不存在。在Whitesides和Kaiser从20世纪70年代末开始的初步工作的基础上，ArMs在过去几年中再次获得了显著的关注，由于重组蛋白生产的突破，丰富的多功能有机金属辅助因子，以及结合均相催化和酶催化的诱人特征的潜在好处的增加。这项技术结合了传统的合成化学和生物技术。为了优化各自的应用，将对辅助因子进行综合修饰，而蛋白质宿主可以通过定向进化进行调整。有针对性的合成变异和随机诱变都可以用来优化arm，以实现自然界本身无法提供的反应。本项目将制备钌基ArMs （Ru-ArMs）库，并对其在烯丙醇和丙炔醇的催化转化、二炔环化以及异构化反应中的催化性能进行筛选。钌基辅助因子被证明用途广泛、活性强，已在细胞外周质和细胞质中得到应用，为本项目的成功提供了广阔的前景。我们将努力扩大Ru-ArMs目前狭窄的体内应用领域，即在大肠杆菌（大肠菌群）等活生物体中应用，从而产生自然界中没有的化学反应。Ru-ArMs将通过对蛋白质宿主的合成修饰和诱变来优化各自的应用。以前在该领域取得的进展和项目，如这里提出的研究计划，可以为新的生化/医学应用奠定基础，例如生物活性物质的原位形成，精确地，只在（癌症）细胞中需要它们的地方，从而不影响未受损的组织。此外，ArMs技术具有从可再生原料生产商品化学品的潜力，并可能有助于我们以石油为基础的经济向可持续生产途径的不可避免的过渡。

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.