Creating Artificial Metallo-Enzymes for C-H Activation Chemistry

大自然使用由遗传密码指定的20种氨基酸的标准字母表来创造能够催化多种复杂转化的酶。因此，我们现有的酶生产和工程策略完全依赖于这些天然氨基酸构建块，其功能有限，不适合创造具有真正新颖活性的人工酶。在这个项目中，我们将利用最先进的蛋白质工程技术，将“化学编程”的配体和/或贵金属辅助因子安装到现有的金属酶支架中，以创造出用于选择性“催化剂控制”非活化C-H键功能化的人工酶。这种策略结合了小分子和酶催化的优点，允许优化催化中心周围的局部协调环境，同时保持选择性底物取向和稳定反应中间体所需的可调蛋白质环境。最近发现的多糖单加氧酶（LPMOs）将被利用作为宿主模板，以纳入新的化学程序化活性位点环境。LPMOs利用不寻常的铜配位环境来实现通常惰性的C-H键的氧化，其中n端甲基化残基充当催化金属中心的双齿配体。为了确定C-H活化化学的合适“活性位点”环境，我们将首先创建含有短合成肽的小分子铜和贵金属（例如Ir， Rh, Ru）配合物作为功能配体，以模拟在LPMOs中发现的双齿配位环境。这些“酶模拟物”的催化性能将被评估为一系列具有合成价值的C-H功能化，以生成C-C， C-N， C-O和C-X （X = F, Cl, Br， I,）键。实验数据将与DFT计算结果进行比较，探讨非标准氨基酸配体对这些过渡金属配合物催化性能的影响。最后，我们将利用遗传密码扩展技术将优化的活性位点环境安装到LPMO蛋白支架中，以创建用于“催化剂控制”C-H功能化的人工酶。值得注意的是，这些第一代生物催化剂很容易使用已建立的蛋白质工程技术进行进一步优化。这是一个高度跨学科的项目，处于酶设计和工程研究的前沿，将为学生提供有机合成、分子生物学、蛋白质表达/纯化、生化分析、生物无机化学和计算酶学方面的专业知识。

Nature uses a standard alphabet of 20 amino acids, specified by the genetic code, to create enzymes capable of catalyzing a diverse array of complex transformations. As a consequence, our existing enzyme production and engineering strategies rely exclusively on these natural amino acid building blocks, which contain limited functionality and are not suitable for the creation of artificial enzymes with truly novel activities. Within this project, we will exploit state-of-the-art techniques in protein engineering to install 'chemically programmed' ligands and/or noble metal co-factors into existing metallo-enzyme scaffolds, in order to create artificial enzymes for selective 'catalyst controlled' functionalizations of unactivated C-H bonds. This strategy combines the benefits of small molecule- and enzyme- catalysis by allowing optimization of the local co-ordination environment surrounding the catalytic centre whilst maintaining the tunable protein environment required for selective substrate orientation and stabilization reactive intermediates. The recently discovered lytic polysaccharide monooxygenases (LPMOs) will be exploited as host templates to incorporate new chemically programmed active site environments. LPMOs utilize an unusual copper co-ordination environment to achieve the oxidation of normally inert C-H bonds, in which an N-terminal methylated residue serves as a bidentate ligand to the catalytic metal center. To identify suitable 'active site' environments for C-H activation chemistry, we will initially create small molecule copper and noble metal (e.g. Ir, Rh, Ru) complexes containing short synthetic peptides as functional ligands to mimic the bidentate co-ordination environment found in LPMOs. The catalytic properties of these 'enzyme mimics' will be evaluated towards a range of synthetically valuable C-H functionalizations to generate C-C, C-N, C-O and C-X (X = F, Cl, Br, I,) bonds. The experimental data will be compared with DFT calculations to explore the effects of non-standard amino acid ligands on the catalytic properties of these transition metal complexes. Finally we will exploit genetic code expansion technology to install optimized active site environments into LPMO protein scaffolds to create artificial enzymes for 'catalyst controlled' C-H functionalization. Significantly, these first generation biocatalysts are readily amenable to further optimization using established protein engineering technologies. This is a highly interdisciplinary project at the cutting edge of enzyme design and engineering research, and will provide the student with expertise in organic synthesis, molecular biology, protein expression / purification, biochemical assays, bioinorganic chemistry and computational enzymology.