Work performed:
1. The non-canonical metal binding amino acid BpyAla was synthesized with an optimized synthetic route.
2. The artificial protein scaffolds with a genetic encoded metal binding amino acid BpyAla were expressed and purified.
3. The in-situ reduction strategy was explored, and different reductants were tested.
4. The Cu(I)-dependent ArMs-catalyzed carbene B-H insertion reaction was investigated. During the project, optimization of basic conditions, alanine scanning, position scanning, and directed evolution, and substrate scope exploration were done.
5. Mechanism research was conducted to characterize the generation of Cu(I) catalytic species.
Main achievements:
1. Developed A New Strategy for Constructing Artificial Copper(I) Enzymes: The applicant has, for the first time, combined gene-encoded unnatural amino acids with an in-situ reduction strategy to successfully construct an artificial copper(I) enzyme with catalytic activity.
2. Mechanism Research: The reduction process was characterized using electron paramagnetic resonance (EPR) and UV-visible absorption spectroscopy, demonstrating that the new strategy is independent of the protein scaffold and highly portable. This enables the rapid construction of copper(I) catalytic centers in various protein scaffolds or at different sites within the same scaffold, showcasing its universality and adaptability.
3. New Catalytic Mode: Unlike traditional copper enzymes, which rely on Lewis acidity and redox activity, this study is the first to generate highly active copper(I)-carbene species within a gene-encoded artificial enzyme scaffold. This achievement enables the asymmetric insertion of B-H bonds and the efficient, highly selective synthesis of a series of chiral boron compounds, advancing the development of new functionalities in artificial copper enzymes.
4. Development of a Novel Artificial Enzyme Scaffold: The introduction of a new artificial enzyme scaffold, coupled with directed evolution studies, provides an excellent scaffold choice with exceptional chiral control capabilities. This opens new possibilities for exploring novel reactions in the field of artificial enzymes and overcomes the limitations of solely relying on the modification of existing scaffolds.
