Triplet quenchers for energy-transfer photobiocatalysis

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Engineering protein catalysts represents an attractive approach for enantioselective energy transfer photochemistry. Now, combining a genetically encoded photosensitizer in the protein catalyst and a judiciously selected triplet quenchers to suppress racemic background reaction in the solution, photobiocatalytic [2+2] cycloaddition offered improved enantiocontrol in triplet sensitization catalysis.

Enantioselective energy transfer catalysis represents a powerful strategy for developing asymmetric photochemistry¹. Previously, enantioselective energy transfer catalysis has been elegantly achieved by Bach and Yoon using tailored chiral photosensitizers with a substrate recognition moiety, engaging substrates through non-covalent interactions for catalyst-controlled excited-state [2+2] cycloaddition reactions (Figure 1a)^1–3. Alternatively, chiral Lewis acid binding and activation have also enabled photochemical [2+2] cycloaddition reactions to occur with excellent stereocontrol (Figure 1b)^4,5. However, the development of catalytic asymmetric triplet sensitization reactions is often hampered by the presence of racemic background reactions due to substrate excitation in the absence of chiral catalysts.

Figure 1. Energy transfer photobiocatalysis for asymmetric [2+2] cycloaddition reactions using a genetically encoded photosensitizer in protein and a designer triplet quencher in solution.

a, Chiral photosensitizer with a substrate recognition hydrogen bonding moiety for asymmetric [2+2] cycloaddition. b, Chiral Lewis acid activation via triplet energy (E_T’) lowering for asymmetric [2+2] cycloaddition. c, Protein compartmentalization combining a genetically encoded photosensitizer in the active site and a triplet quencher in solution to enhance the stereocontrol in biocatalytic [2+2] cycloaddition. d, Directed evolution of RamR1.0. Active-site illustration is made based on PDB ID 3VVX. S₀ = ground state; T₁ = excited triplet state; E_T = triplet state energy; EnT = energy transfer.

Now, writing in Nature Catalysis, Chen, Wu, Zhong and co-workers report an improved strategy for energy transfer photobiocatalysis by using triplet quenchers in solution and genetically encoded sensitizers in protein catalysts to accomplish [2+2] cycloaddition reactions with enhanced enantioselectivity (Figure 1c)⁶. The inclusion of judiciously selected triplet quenchers in the solution effectively suppressed racemic photochemical reactions of unbound substrates, affording a potentially general approach to streamlining enantioselective energy transfer protein catalysis.

At the outset of this research, the team constructed an artificial photoenzyme library by genetically incorporating the synthetic triplet photosensitizer 4-benzoylphenylalanine (BpA)^7,8 into previously evaluated protein scaffolds at different residues. Protein scaffold evaluation led to the identification of RmaR1.0 (RmaR_F155BpA) as the best starting template⁹, affording the desired cycloaddition product in 37% yield and 30% enantiomeric excess (e.e.) under ultraviolet light irradiation (λ = 365 nm) (Figure 1d). The authors opted to investigate a model substrate featuring an overlapping UV absorption window as the benzophenone triplet photosensitizer. They found that while the presence of molecular oxygen enhanced the stereocontrol of this [2+2] cycloaddition, undesired side products also formed due to singlet oxygen oxidation processes.

To overcome this issue, the researchers assessed several triplet quenchers with different steric and charge properties to suppress the racemic uncatalyzed [2+2] photocycloaddition. They hypothesized that the anionic triplet quencher (E)-4-styrylbenzoic acid would not enter the protein active site due to electrostatic repulsion with the negatively charged protein residues, thus not interfering with stereoselective protein catalysis while still effectively inhibiting racemic background photochemistry. Indeed, the addition of (E)-4-styrylbenzoic acid increased the enantioselectivity (from 30% e.e. to 62% e.e.) and maintained the reaction mass balance (Figure 1d), indicating the utility of this triplet quencher strategy to augment enantioselective energy transfer biocatalysis.

The triplet sensitization protein catalyst RamR1.0 was next engineered via site-saturation mutagenesis and screening to further increase its enantioselectivity and catalytic activity. Single site-saturation mutagenesis targeting 12 active-site residues followed by combination of beneficial mutations furnished a triple mutant RamR3.0 (RamR1.0 K63R S67W S134A), allowing the product to form in 67% yield and 86% e.e. under a nitrogen atmosphere. Further enhanced enantiocontrol (70% yield and 94% e.e.) was observed when (E)-4-styrylbenzoic acid was included (Figure 1d). Moreover, X-ray crystallography of RamR3.0 in complex with the model substrate demonstrated the essential role of substrate-protein active site interactions in stereocontrol.

Engineered RamR3.0 mutants were found to be compatible with a range of substrates for this enantioselective [2+2] photocycloaddition. Naphthoates with a range of aromatic and O-substituents were transformed with good enantioselectivities. Methyl 2-naphthoate and methyl naphthyl ketone substrates were also tolerated. The addition of three equivalent (E)-4-styrylbenzoic acid consistently improved the enantioselectivity for all these substrates.

The utility of improved protein catalyst scaffold and triplet quencher was further demonstrated in whole-cell biocatalysts and enantioselective energy transfer photochemical [2+2] cycloaddition reactions of several other substrate designs with competing racemic background reactions. The authors also demonstrated laboratory scale photobiocatalytic synthesis by using a continuous flow photoreactor on a 100 mg scale on a model substrate.

In summary, through protein active site confinement, the use of triplet quenchers in conjunction with a protein catalyst incorporating a genetically encoded triplet sensitizer provided an effective means to enhance the enantiocontrol of energy transfer photobiocatalysis. While the presence of a carefully selected quencher in the solution suppressed racemic reactions via direct excitation of unbound substrates, protein-sensitized [2+2] cycloaddition within the engineered active site occurred with excellent enantiocontrol. This strategy is potentially general for other photobiocatalytic energy transfer processes involving a broad range of substrates with an absorption profile similar to the photosensitizer. The use of 96-well LED photoreactors and high-throughput screening may inspire the development of a wider range of photobiocatalytic reactions with enhanced efficiency and stereoselectivity. With the advent of new modes of activation, a broader range of synthetically useful photobiocatalytic transformations will be developed in the near future.