Artificial Photoenzymes Enable Selective Energy Transfer for Asymmetric Synthesis

Breakthrough in Enantioselective Photocatalysis

Researchers have developed a novel approach to enantioselective energy transfer catalysis using compartmentalized triplet photoenzymes, according to a recent study published in Nature Catalysis. The system reportedly addresses longstanding challenges in controlling stereochemistry during photochemical reactions by creating protected environments within engineered protein scaffolds.

Breakthrough in Enantioselective Photocatalysis
Overcoming Background Reaction Challenges
Engineering a Diverse Photoenzyme Library
RamR Emerges as Promising Scaffold
Designer Quenchers Enhance Performance
Evolution to High-Performance Variants
Broad Substrate Scope and Scalable Applications
Whole-Cell Photobiocatalysis Demonstration

Overcoming Background Reaction Challenges

The investigation began with 1-naphthol derivative substrates that compete with benzophenone-encoded triplet photoenzymes in light absorption, sources indicate. Analysis revealed that substantial background photocycloaddition occurred through direct excitation at the triplet hyperface, which could be inhibited by triplet quenchers including molecular oxygen. The report states that oxygen concentration inversely affected rac-2 yield, dropping from 37% under ultraviolet light to just 7% with increased oxygen presence.

According to the research team, this observation confirmed that background reactions proceed through triplet state pathways, making them susceptible to quenching strategies. However, oxygen also induced undesirable side reactions through singlet oxygen formation, compromising mass balance despite improved stereochemical control.

Engineering a Diverse Photoenzyme Library

To identify optimal catalysts, researchers established a primary library of artificial triplet photoenzymes with diversity in both protein scaffold and benzophenone insertion position. The library included homodimeric LmrR protein alongside TetR family regulators CgmR, RamR and QacR, selected based on their structural characteristics and hydrophobic chiral cavities. Two additional proteins, TM1030 and tHisF, were also utilized as scaffolds., according to industry news

Analysts suggest the variation in cavity size and aromatic residue composition enhanced the potential for identifying suitable hosts for binding non-native aromatic substrates. For each protein, four residue positions deep within cavity interiors were selected for benzophenone alanine insertion using genetic code expansion technology, creating a focused library of 24 mutants.

RamR Emerges as Promising Scaffold

Among the tested variants, RamR-derived photoenzymes demonstrated particularly favorable reactivity and enantioselectivity, according to the report. The RamR_F155BpA variant, designated RamR1.0, delivered the most promising initial results with 37% yield and 30% enantiomeric excess under anaerobic conditions. Notably, the research team observed that enantioselectivity improved under oxygen atmosphere, reaching 46% enantiomeric excess, which sources indicate aligns with their compartmentalization concept.

The report states that photoenzyme compartmentalization enabled spatial-selective quenching of excited substrates by ground-state oxygen in bulk solution while maintaining undisturbed photochemical processes within the protein cavity. Transient absorption spectroscopy studies reportedly supported this hypothesis, showing similar excited-state lifetimes for RamR1.0 in both oxygen and anaerobic conditions, unlike free benzophenone which was significantly influenced by oxygen.

Designer Quenchers Enhance Performance

Common triplet quenchers like (E)-1,2-diphenylethene and 2,5-dimethylhexa-2,4-diene successfully suppressed destructive side reactions and led to higher enantiomeric excess values but substantially reduced conversion, according to the findings. Researchers suggest this inhibitory effect may stem from these quenchers freely entering RamR’s hydrophobic pocket and disrupting energy transfer catalysis.

To address this limitation, the team designed two unique quenchers: (E)-4-styrylbenzoic acid and diphenylethene-functionalized bovine serum albumin. Under reaction pH conditions, (E)-4-styrylbenzoic acid is reportedly repelled from the photoenzyme due to negatively charged residues surrounding the active site entrance, while the large size of the albumin conjugate prevents enzyme cavity entry. Both quenchers improved energy transfer catalysis, with (E)-4-styrylbenzoic acid showing superior enantioselectivity and mass balance at optimized loading.

Evolution to High-Performance Variants

Through focused semirational site-saturation mutagenesis starting from RamR1.0, researchers evolved significantly improved photoenzyme variants. Leveraging structural knowledge and molecular docking studies, they targeted 12 amino acid residues near the benzophenone cofactor pointing toward the hydrophobic pocket. The report indicates position 67 emerged as crucial, with S67Y and S67W variants demonstrating superior stereochemical induction.

Combinatorial mutagenesis yielded RamR_F155BpA_S67W_K63R (RamR2.0), which produced 76% yield with 80% enantiomeric excess. Further optimization led to RamR_F155BpA_K63R_S67W_S134A (RamR3.0), facilitating cycloaddition in 67% yield with 86% enantiomeric excess. Crystal structure analysis of the RamR3.0-substrate complex revealed a spatial arrangement with benzophenone positioned 5.7 Å from the substrate’s naphthalene ring, ensuring efficient triplet energy transfer.

Broad Substrate Scope and Scalable Applications

The research team evaluated substrate scope for enantioselective [2+2] photocycloaddition using the RamR3.0-(E)-4-styrylbenzoic acid system, achieving >90% enantiomeric excess for most tested substrates. The system accommodated diverse 2-naphthoates bearing electron-donating or electron-withdrawing groups, phenol esters, benzyl ester homologues with varying chain lengths, and nitrogen heterocycles of potential biological interest.

Notably, researchers established a laboratory-built continuous-flow photoreactor system enabling scalable photoenzymatic synthesis. Under optimized continuous-flow conditions with (E)-4-styrylbenzoic acid, product 2m was isolated in 98% yield with 91% enantiomeric excess. The reactivity and stereoselectivity in continuous-flow mode were comparable to or higher than analytical-scale batch synthesis, representing an attractive platform for scalable enantioselective synthesis.

Whole-Cell Photobiocatalysis Demonstration

The compartmentalization strategy proved effective even in whole-cell photobiocatalysis systems, according to the report. Using Escherichia coli cells containing 4.3 mol% RamR resuspended in reaction buffer with naphthalene derivative, researchers achieved product formation with 73% yield and 82% enantiomeric excess despite the complex cytoplasmic environment. Flow cytometry analysis demonstrated that E. coli cells retained substantial viability with less than 3% cell death after photoreaction, suggesting efficient cellular uptake coupled with intracellular enantioselective photocycloaddition.

The research team suggests their photoenzyme compartmentalization strategy is原则上 applicable to other enantioselective energy transfer catalysis systems associated with competing background processes, as demonstrated with indole substrates and thioxanthone-photoenzymes. This approach establishes a versatile platform for developing enantioselective photochemical transformations with broad synthetic utility.