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

## TL;DR

Researchers developed a new method to evolve enzymes that can unmask bioorthogonal protecting groups, enabling precise control of molecular activity in cells.

## Contribution

A novel bioorthogonal protecting group (pCP) and a platform for enzyme evolution to unmask it are introduced.

## Key findings

- Evolved BS2 esterase mutants are up to 232-fold more active toward the pCP group.
- The pCP probe and evolved BS2 enabled spatially resolved RNA tagging in mammalian cells with high specificity.

## Abstract

Combining bioorthogonal protecting groups with localized
catalysts
that can unmask them is a powerful approach to spatially and temporally
modulate molecular activity. Enzymes are appealing catalysts in this
context because they are genetically targetable, but enzymes are not
always available to unmask a protecting group of interest. Here, we
report a platform for ultrahigh-throughput enzyme evolution by combining
yeast surface display with masked acylating probes, which selectively
label yeast cells based on target biocatalytic activity. We introduce
the phenylcyclopropyl (pCP) ester protecting group, which has improved
bioorthogonality compared to existing ester protecting groups, and
use our platform to evolve BS2 esterase for enhanced pCP unmasking.
Evolved BS2 mutants are up to 232-fold more active toward the pCP
group. Taking advantage of the enhanced bioorthogonality of the pCP
group, we applied a pCP probe together with evolved BS2 to perform
spatially resolved RNA tagging with high spatial specificity, including
in mammalian cell lines with high endogenous esterase activity. Overall,
this work delivers a new bioorthogonal protecting group and engineered
enzymes capable of unmasking it, and more broadly, it provides a platform
to rapidly engineer enzymes for protecting group removal, opening
opportunities in imaging, proximity tagging, induced cell signaling,
and therapeutics.