Weijin Wang

Aromatic d‐amino acids (d‐AAs) are valuable building blocks in drug discovery and peptide therapeutics, yet their direct and efficient biocatalytic synthesis remains a challenge. Here, we report the rational engineering of phenylalanine ammonia lyase from Planctomyces brasiliensis (PbPAL) to enable asymmetric hydroamination for the enantioselective synthesis of d‐aromatic amino acids. By targeting active‐site residue L205, we identified variants capable of highly d‐enantioselective hydroamination, with L205F enabling the transformation of electron‐deficient aryl acrylates with >99% enantiomeric excess (ee). The synthetic utility of this platform was demonstrated by gram‐scale synthesis of d‐benzoxazole and substituted 2‐pyridylalanines. Structural and mutational studies revealed distinct roles for the 4‐methylideneimidazole‐5‐one (MIO) prosthetic group and active‐site residues L205, Y64, and K397 in modulating enantioselectivity. These results enabled the identification of PbPAL variants with the opposite selectivity, such as L205V‐K397A, which preferentially produce l‐amino acids. This work broadens the utility of PALs as programmable biocatalysts for asymmetric synthesis.

Aromatic d‐amino acids (d‐AAs) are valuable building blocks in drug discovery and peptide therapeutics, yet their direct and efficient biocatalytic synthesis remains a challenge. Here, we report the rational engineering of phenylalanine ammonia lyase from Planctomyces brasiliensis (PbPAL) to enable asymmetric hydroamination for the enantioselective synthesis of d‐aromatic amino acids. By targeting active‐site residue L205, we identified variants capable of highly d‐enantioselective hydroamination, with L205F enabling the transformation of electron‐deficient aryl acrylates with >99% enantiomeric excess (ee). The synthetic utility of this platform was demonstrated by gram‐scale synthesis of d‐benzoxazole and substituted 2‐pyridylalanines. Structural and mutational studies revealed distinct roles for the 4‐methylideneimidazole‐5‐one (MIO) prosthetic group and active‐site residues L205, Y64, and K397 in modulating enantioselectivity. These results enabled the identification of PbPAL variants with the opposite selectivity, such as L205V‐K397A, which preferentially produce l‐amino acids. This work broadens the utility of PALs as programmable biocatalysts for asymmetric synthesis.

Artificial metalloenzymes (ArMs) enable the integration of abiotic cofactors within a native protein scaffold, allowing for non‐natural catalytic activities. Previous ArMs, however, have primarily relied on single cofactor systems, limiting them to only one catalytic function. Here we present an approach to construct ArMs embedding two catalytic cofactors based on the biotin‐streptavidin technology. By incorporating multiple catalytic cofactors into the four binding sites of streptavidin, we engineered programmable ArMs for tandem abiotic transformations including an enantioselective formal C−H hydroxylation and a photooxidation‐Michael addition. This work thus outlines a promising strategy for the development of ArMs embedding multiple cofactors.

Artificial metalloenzymes (ArMs) enable the integration of abiotic cofactors within a native protein scaffold, allowing for non‐natural catalytic activities. Previous ArMs, however, have primarily relied on single cofactor systems, limiting them to only one catalytic function. Here we present an approach to construct ArMs embedding two catalytic cofactors based on the biotin‐streptavidin technology. By incorporating multiple catalytic cofactors into the four binding sites of streptavidin, we engineered programmable ArMs for tandem abiotic transformations including an enantioselective formal C−H hydroxylation and a photooxidation‐Michael addition. This work thus outlines a promising strategy for the development of ArMs embedding multiple cofactors.

ConspectusLate-stage halogenation, targeting specific positions in complex substrates, has gained significant attention due to its potential for diversifying and functionalizing complex molecules such as natural products and pharmaceutical intermediates. Utilizing readily available halogenating reagents, such as hydrogen halides (HX), N-halosuccinimides (NXS), and dichloroethane (DCE) reagents for late-stage halogenation shows great promise for expanding the toolbox of synthetic chemists. However, the reactivity of haleniums (X⁺, X = Cl, Br, I) can be significantly hindered by the presence of various functional groups such as hydroxyl, amine, amide, or carboxylic acid groups. The developed methods of late-stage halogenation often rely on specialized activating reagents and conditions. Recently, our group (among others) has put great efforts into addressing these challenges and unlocking the potential of these readily available HX, NXS, and DCE reagents in complex molecule halogenation. Developing new methodologies, catalyst systems, and reaction conditions further enhanced their utility, enabling the efficient and selective halogenation of intricate substrates.With the long-term goal of achieving selective halogenation of complex molecules, we summarize herein three complementary research topics in our group: (1) Efficient oxidative halogenations: Taking inspiration from naturally occurring enzyme-catalyzed oxidative halogenation reactions, we focused on developing cost-effective oxidative halogenation reactions. We found the combination of dimethyl sulfoxide (DMSO) and HX (X = Cl, Br, I) efficient for the oxidative halogenation of aromatic compounds and alkenes. Additionally, we developed electrochemical oxidative halogenation using DCE as a practical chlorinating reagent for chlorination of (hetero)arenes. (2) Halenium reagent activation: Direct electrophilic halogenation using halenium reagents is a reliable method for obtaining organohalides. However, compared to highly reactive reagents, the common and readily available NXS and dihalodimethylhydantoin (DXDMH) demonstrate relatively lower reactivity. Therefore, we focused on developing oxygen-centered Lewis base catalysts such as DMSO, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and nitromethane to activate NXS or DXDMH, enabling selective halogenation of bioactive substrates. (3) Halogenation of inert substrates: Some substrates, such as electron-poor arenes and pyridines, are inert toward electrophilic functionalization reactions. We devised several strategies to enhance the reactivity of these molecules. These strategies, characterized by mild reaction conditions, the ready availability and stability of catalysts and reagents, and excellent tolerance for various functional groups, have emerged as versatile protocols for the late-stage aromatic halogenation of drugs, natural products, and peptides. By harnessing the versatility and selectivity of these catalysts and methodologies, synthetic chemists can unlock new possibilities in the synthesis of halogenated compounds, paving the way for the development of novel functional materials and biologically active molecules.

The achievement of directly activating and utilizing bulk small molecules has remained a longstanding objective in the field of chemical synthesis. The present work reports a catalytic activation method for bulk chemical nitromethane (MeNO₂). This method combines homogeneous Lewis acid with recyclable heterogeneous Brønsted acid catalysis, featuring practicality, sustainability, and low cost, thus solving the inherent drawbacks of previous Nef processes where stoichiometric reductants or activators were required. By combining the advantages of both homo‐ and heterogeneous catalysts, this chemistry may not only offer new opportunities for the further development of MeNO₂ as a nitrogen source for organic synthesis, but also promote the catalysis design in synthetic chemistry.

The achievement of directly activating and utilizing bulk small molecules has remained a longstanding objective in the field of chemical synthesis. The present work reports a catalytic activation method for bulk chemical nitromethane (MeNO₂). This method combines homogeneous Lewis acid with recyclable heterogeneous Brønsted acid catalysis, featuring practicality, sustainability, and low cost, thus solving the inherent drawbacks of previous Nef processes where stoichiometric reductants or activators were required. By combining the advantages of both homo‐ and heterogeneous catalysts, this chemistry may not only offer new opportunities for the further development of MeNO₂ as a nitrogen source for organic synthesis, but also promote the catalysis design in synthetic chemistry.

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth‐abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin‐streptavidin technology. By incorporating bio‐mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo‐genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional‐group tolerance. According to QM/MM calculations and X‐ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base‐meal cofactors for enantioselective organic synthesis.

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth‐abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin‐streptavidin technology. By incorporating bio‐mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo‐genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional‐group tolerance. According to QM/MM calculations and X‐ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base‐meal cofactors for enantioselective organic synthesis.