Ultrasound-Controlled Activation of Tetrazine-TCO Click-to-release Chemistry Through Sonochemical Oxidation of Dihydrotetrazine

External control over click chemistry offers opportunities for the spatiotemporal regulation of bioorthogonal ligation and cleavage, but the limited tissue penetration of light constrains photoactivated approaches. Herein, we introduce a new mechanistic approach to ultrasound-controlled click chemistry based on the direct sonochemical oxidation of dihydrotetrazine (DHTz) precursors. The precursors require no additional cleavable masking group, and low-intensity therapeutic ultrasound activates their oxidation through sonochemically generated hydroxyl radicals without the need for added sensitizers. A pyridinyl-substituted precursor, DHTz2, exhibits limited background oxidation and efficient sonochemical activation, while the resulting tetrazine product shows good stability under ultrasound exposure. The ultrasound-activated tetrazine readily undergoes ligation reactions with TCOs, including a TCO-DOX conjugate in which a bioorthogonal click-to-release cascade releases free DOX and restores its cytotoxic activity against cancer cells. Importantly, ultrasound applied through a chicken breast tissue barrier remains effective in triggering bioorthogonal click-to-release and activating the prodrug. This work establishes a new mechanistic approach that couples the fundamental sonochemistry of acoustic cavitation with the selective activation of click chemistry and bioorthogonal cleavage, providing a new platform for the remote, non-optical control of bioorthogonal ligation and cargo release using tissue-penetrating biomedical ultrasound.