A designed photoenzyme for enantioselective [2+2] cycloadditions.

Area of Science:

• Biocatalysis and Synthetic Chemistry
• Protein Engineering and Biotechnology

Background:

• Genetic code expansion allows introducing noncanonical amino acids for novel protein functions.
• Triplet energy transfer (EnT) catalysis is a powerful synthetic tool not currently accessible via biocatalysis.

Purpose of the Study:

• To develop a photoenzyme capable of triplet energy transfer (EnT) catalysis.
• To engineer an efficient and enantioselective photocatalyst using protein scaffolds.

Main Methods:

• Utilized genetic code expansion to install a photosensitizer into a de novo Diels-Alderase (DA_20_00).
• Employed a directed evolution platform to optimize the photoenzyme for enhanced catalytic activity and selectivity.
• Determined the X-ray crystal structure of the photoenzyme-product complex.

Main Results:

• Created a photoenzyme (EnT1.0) for [2+2] cycloadditions via EnT catalysis.
• Developed an evolved variant (EnT1.3) achieving up to 99% enantiomeric excess (e.e.) for various cycloadditions.
• Demonstrated >300 turnovers, aerobic stability, and ambient temperature operation, outperforming small-molecule catalysts.
• Structural analysis revealed synergistic interactions within the protein active site.

Conclusions:

• Established a framework for creating a new generation of enantioselective photocatalysts based on engineered proteins.
• Opened new possibilities for excited-state chemistry within protein active sites.
• Demonstrated the potential of photoenzymes for challenging synthetic transformations.