Synthesis based on covalent capture and release (SCCR): a programmable strategy for automated preparation of protease-activatable molecules

ABSTRACT Enzyme-activatable chemical tools, including fluorogenic probes and prodrugs, are essential in chemical biology and targeted therapeutics but remain challenging to access in structurally diverse forms because their synthesis is often bespoke and difficult to standardize. Here, we introduce synthesis based on covalent capture and release (SCCR) as a programmable chemical strategy that enables the modular assembly of protease-activatable molecules through specifically designed protecting-group logic. The SCCR framework establishes a standardized capture–elongation– release workflow that decouples molecular diversification from individual synthetic optimization, thereby enabling automated preparation of complex libraries. Using this chemistry, we generated a diverse set of fluorogenic probes and applied them to single-molecule enzyme activity analyses to identify candidate activity-based biomarkers of liver diseases. The generality of the SCCR strategy was further demonstrated by extending the same chemical logic to the preparation of antibody–drug conjugate (ADC) linkers, allowing systematic evaluation of plasma stability and cytotoxic potential. By establishing a programmable capture–release chemistry for the synthesis of enzyme-activatable molecules, this work provides a generalizable chemical foundation for the scalable and automated construction of functional small-molecule tools across biological and translational research.