Synthesis Based on Covalent Capture and Release Enables Purification-free Fluorogenic Probe Libraries for...

Area of Science:

• Chemical Biology
• Biochemistry
• Molecular Diagnostics

Background:

• Direct enzyme activity measurement offers biological insights but is hindered by limited scalable strategies for generating diverse, assay-ready fluorogenic probes.
• Current probe synthesis methods, reliant on chromatography, impede rapid, large-scale exploration of enzyme substrate space.

Purpose of the Study:

• To introduce a novel, purification-free chemical strategy for the scalable generation of fluorogenic probe libraries.
• To enable high-throughput enzyme activity profiling and substrate discovery using these probes.

Main Methods:

• Developed covalent capture and release (SCCR), a strategy embedding a capture handle in a removable protecting group for purification-free probe synthesis.
• Established a standardized capture-elongation-release workflow decoupling diversification from purification, compatible with liquid-phase synthesis.
• Automated the preparation of high-purity fluorogenic probe libraries.

Main Results:

• Generated a library of over 100 fluorogenic probes using the SCCR platform.
• Successfully profiled protease activities at the single-molecule level, facilitating substrate discovery.
• Identified disease-associated enzymatic signatures in blood samples based on enzyme activity.

Conclusions:

• SCCR provides a scalable route for generating functional enzyme probes, expanding possibilities for single-molecule activity profiling.
• This platform accelerates the discovery of enzyme substrates and biomarkers for disease diagnostics.