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各种复杂转化的酶。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结构单元，其包含有限的功能并且不适合于产生具有真正新颖活性的人工酶。在这个项目中，我们将利用蛋白质工程中最先进的技术，将“化学编程”配体和/或贵金属辅因子安装到现有的金属酶支架中，以创造人工酶，用于选择性“催化剂控制”未活化的C-H键的功能化。该策略通过允许优化催化中心周围的局部配位环境，同时保持选择性底物取向和稳定反应性中间体所需的可调蛋白质环境，结合了小分子催化和酶催化的益处。最近发现的溶解性多糖单加氧酶（LPMO）将被利用作为主机模板，以纳入新的化学编程活性位点环境。LPMO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-末端甲基化残基用作催化金属中心的双齿配体。为了确定合适的C-H活化化学的“活性位点”环境，我们将首先创建含有短合成肽作为功能配体的小分子铜和贵金属（例如Ir、Rh、Ru）络合物，以模拟在LPMO中发现的双齿配位环境。这些“酶模拟物”的催化性质将针对一系列合成上有价值的C-H官能化进行评估，以产生C-C、C-N、C-O和C-X（X = F、Cl、Br、I）键。将实验数据与DFT计算进行比较，以探索非标准氨基酸配体对这些过渡金属配合物的催化性能的影响。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易于使用已建立的蛋白质工程技术进行进一步优化。这是酶设计和工程研究前沿的高度跨学科项目，将为学生提供有机合成，分子生物学，蛋白质表达/纯化，生物化学测定，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.