Periodic Reporting for period 1 - Cu-CATARMs (Asymmtric Copper(I) Catalysis in Artificial Metalloenzymes)
Reporting period: 2023-04-01 to 2025-03-31
The Cu-CATARMs project (Asymmetric Copper(I) Catalysis in Artificial Metalloenzymes) aims to design and develop a highly efficient artificial metalloenzyme featuring a low oxidation state copper center, Cu(I), which facilitates a range of enantioselective, new-to-nature transformations in a protein environment.
The Roelfes group has been engaged in creating LmrR-based artificial metalloenzymes (ARMs) for copper-catalyzed asymmetric unnatural reactions. They have developed reliable methods for efficiently incorporating copper (II) catalytic center into the artificial protein pocket, either by introducing metal-binding unnatural amino acids through in vivo stop codon suppression or via supramolecular assembling the metal complex, utilizing π–π stacking interactions between tryptophan residues.
Our approach to creating Cu(I)-dependent artificial metalloenzymes (Cu(I)-ArMs)involves the in situ reduction of the established copper(II) center to a copper(I) center using mild and efficient reductants (Objective 1). The created Cu(I)-ArM will be utilized to achieve the following objectives: Objective 2, asymmetric conjugate addition of alkynes; Objective 3, asymmetric intramolecular cyclopropanation; and Objective 4, the detailed mechanisms underlying these reactions will be investigated, aiming to provide theoretical insights for future related research.
In perspective, the successful development of the Cu-CATARMs project will offer a powerful strategy for utilizing low oxidation state transition metals in enzyme catalysis. The project’s outcomes will inspire the development of C–C and C–N bond formation reactions through copper(I) catalysis in ARMs, providing new strategies for the organic chemistry and materials science communities in designing synthetic routes for specific targets.
The Roelfes group has been engaged in creating LmrR-based artificial metalloenzymes (ARMs) for copper-catalyzed asymmetric unnatural reactions. They have developed reliable methods for efficiently incorporating copper (II) catalytic center into the artificial protein pocket, either by introducing metal-binding unnatural amino acids through in vivo stop codon suppression or via supramolecular assembling the metal complex, utilizing π–π stacking interactions between tryptophan residues.
Our approach to creating Cu(I)-dependent artificial metalloenzymes (Cu(I)-ArMs)involves the in situ reduction of the established copper(II) center to a copper(I) center using mild and efficient reductants (Objective 1). The created Cu(I)-ArM will be utilized to achieve the following objectives: Objective 2, asymmetric conjugate addition of alkynes; Objective 3, asymmetric intramolecular cyclopropanation; and Objective 4, the detailed mechanisms underlying these reactions will be investigated, aiming to provide theoretical insights for future related research.
In perspective, the successful development of the Cu-CATARMs project will offer a powerful strategy for utilizing low oxidation state transition metals in enzyme catalysis. The project’s outcomes will inspire the development of C–C and C–N bond formation reactions through copper(I) catalysis in ARMs, providing new strategies for the organic chemistry and materials science communities in designing synthetic routes for specific targets.
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.
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.
The applicant's research on artificial copper(I)-carbene insertion enzymes demonstrates multiple unique innovations. For example, a new strategy with strong portability and versatility has been proposed, which enables the in situ construction of a copper(I) catalytic center. This approach has successfully realized the first artificial copper enzyme-catalyzed carbene B-H insertion reaction. Additionally, a novel artificial enzyme scaffold with excellent compatibility and strong chiral control capabilities has been developed. These innovative achievements significantly advance the development of new functionalities in artificial enzymes. With the future publication of these results, it is expected to attract widespread attention in the fields of synthetic chemistry and synthetic biology and provide valuable guidance for future research in related areas.