Synthesis Based on Covalent Capture and Release Enables Purification-free Fluorogenic Probe Libraries for Single-Molecule Protease Activity Profiling.

Direct measurement of enzyme activities provides functional insights into complex biological systems; however, their broader application is limited by the lack of scalable strategies to generate diverse, assay-ready fluorogenic probes. In particular, conventional probe synthesis relies on chromatographic purification, preventing the rapid and parallel exploration of substrate space at the library scale. Here, we report synthesis based on covalent capture and release (SCCR), a general chemical strategy that enables purification-free generation of fluorogenic probe libraries. By embedding a covalent capture handle within a removable protecting group, SCCR establishes a standardized capture-elongation-release workflow that decouples molecular diversification from chromatographic purification while retaining the flexibility of liquid-phase synthesis. This approach enables automated preparation of high-purity probe libraries compatible with sensitive activity assays. Using this platform, we generated a library of over 100 fluorogenic probes and applied it to profile protease activities at the single-molecule level, enabling substrate discovery and the activity-based identification of disease-associated enzymatic signatures in blood samples. These results establish a scalable route to functional probe generation and expand the accessible space for single-molecule enzyme activity profiling in complex biological systems.