Development of Cyclooctyne-Nitrone Based Click Release Chemistry for Bioorthogonal Prodrug Activation both <i>In Vitro</i> and <i>In Vivo</i>

The advancement of bioorthogonal cleavage platforms has emerged as a critical frontier in chemical biology, offering precise molecular liberation through physiologically compatible activation mechanisms. Despite its significant potential, ensuring efficacy <i>in vivo</i> typically requires rapid reaction kinetics, high-efficiency payload release, and stable reactants; however, relevant reports remain sparse. Herein, we developed a strain-promoted alkyne-nitrone cycloaddition (SPANC)-based click-release chemistry through installation of a carbamate-linked release moiety at the propargyl position of cyclooctyne, triggering a spontaneous elimination following click cycloaddition to achieve efficient payload liberation. This bioorthogonal click-release system demonstrates rapid reaction kinetics and high-yield payload liberation while maintaining exceptional stability under both <i>in vitro</i> and cellular conditions. Furthermore, we established a facile synthesis of highly click-reactive nitrones, enabling the construction of a systematic substrate library and validating the broad applicability of this strategy. In the context of implementation, our developed bioorthogonal anticancer prodrugs utilize nitrone-mediated activation to unmask anticancer activity. In 4T1 murine breast cancer models, this bioorthogonal prodrug activation strategy demonstrated significant tumor suppression with favorable safety profiles, validating the feasibility of this click-release chemistry for <i>in vivo</i> use.