Bioorthogonal Click‐to‐Release of Branched Pro‐Fluorophores

Bioorthogonal click-to-release prodrug activation strategies should be fast and high yielding. However, in strain-promoted alkene-azide click-to-release, a fast click-step translates to slow and inefficient drug release. To improve drug yield, branched dual-core scaffolds that increase drug release are reported. The dual-core consists of an exposed electron-deficient aryl azide (core-1) attached to an electron-rich self-immolating branched linker (core-2). Core-1, a tetrafluoroaryl azide with methyl-substitution at the benzylic carbon, facilitates a rapid click reaction with trans-cyclooctenes (TCOs). Core-2 had an N-methyl carbamate or ether linker and a 1,4/1,4- or 1,4/1,6-self-immolating scaffold. Various release rates of a fluorophore were observed across the series, with 1,4/1,6-self-immolating core-2, linked via the N-methyl carbamate, having sustained and enhanced fluorophore release. The second-order rate constant for cycloaddition of this 1,4/1,6-self-immolating analogue and d-TCO is, to the best of our knowledge, the fastest to date (22.0 M^-1 s^-1). A persistent intermediate was observed, leading to a long, sustained release of fluorophore from the branched scaffold, though activation under acidic conditions (pH = 5.5) led to ≈ 15% higher fluorophore release compared to activation at pH 7.4. Therefore, combinations of core-1 and core-2 could improve future pretargeted in vivo bioorthogonal click-to-release prodrug strategies, especially in the acidic tumor environment.