Discovery of an Ultrasound-Responsive Arylurea-Dihydrotetrazine Architecture for Ultrasound-Programmed Bioorthogonal Chemistry

Abstract Ultrasound-responsive chemical scaffolds capable of operating under clinically relevant conditions remain rare, limiting the development of programmable ultrasound-controlled molecular systems. Here, we report the discovery of a stable yet ultrasound-responsive arylurea-dihydrotetrazine (ArU-DHTz) scaffold that undergoes efficient activation under 1 MHz therapeutic ultrasound without requiring exogenous sonosensitizers. Structure–responsiveness studies revealed that activation arises from a unique combination of a monosubstituted arylurea motif and an appropriately tuned DHTz core, while mechanistic investigations identified cavitation-derived hydroxyl radicals as the primary activating species. Ultrasound activation simultaneously triggers cargo release and tetrazine generation, representing, to our knowledge, the first example of ultrasound-triggered tetrazine formation. Leveraging tetrazine as a chemical relay, we developed a “Release–Relay–Click” strategy that converts a single ultrasound input into dual molecular outputs: therapeutic payload release and downstream click-to-release activation. Representative prodrug D6 showed ultrasound-dependent activation, translating into antitumor efficacy in vitro and in vivo, together with fluorescence self-reporting through tetrazine-mediated bioorthogonal chemistry.