Directed Evolution of Enzymes for Bioorthogonal Chemistry Using Acid Chloride Proximity Labeling.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Chemistry

Background:

• Localized catalysts are crucial for spatiotemporal control of molecular activity.
• Enzymes offer genetically targetable catalysis but are not always available for specific protecting group removal.
• Existing ester protecting groups can lack sufficient bioorthogonality.

Purpose of the Study:

• To develop a novel bioorthogonal protecting group with enhanced properties.
• To engineer enzymes capable of efficiently unmasking the new protecting group.
• To establish a platform for ultrahigh-throughput enzyme evolution for protecting group removal.

Main Methods:

• Yeast surface display combined with masked acylating probes for enzyme evolution.
• Introduction of the phenylcyclopropyl (pCP) ester protecting group.
• Directed evolution of BS2 esterase for improved pCP unmasking activity.

Main Results:

• The phenylcyclopropyl (pCP) ester demonstrates improved bioorthogonality.
• Evolved BS2 esterase mutants show up to 232-fold increased activity toward the pCP group.
• Successful application of pCP probe and evolved BS2 for spatially resolved RNA tagging in mammalian cells.

Conclusions:

• A new bioorthogonal protecting group (pCP ester) and engineered enzymes for its removal have been developed.
• The developed platform enables rapid engineering of enzymes for protecting group unmasking.
• This approach opens new avenues for molecular imaging, proximity tagging, and therapeutic applications.