In addition to our experiments confirming bioorthogonal activation of prodrug 20, with the reaction predominantly occurring in media, we aimed to demonstrate enhanced click-to-release inside live cells. Therefore, U2OS osteosarcoma cells were incubated with the mitochondria-targeting fluorogenic probe 21 (rTCO-TPP-Reso), previously developed by Vrabel et al., followed by treatment with DMT (1), 2Pyr2 (3), or h2P2 ( ), leading to intramitochondrial click reaction and -depending on the Tzsubsequent release of fluorescent resorufin (Fig. ). The rate of release is influenced by two factors: the Tz-triggered cleavage of rTCO and the elimination of the self-immolative ethylenediamine linker, to which the mitochondria-targeting triphenylphosphonium moiety (TPP) is attached. Hence, high turn-on of fluorescence is achieved only upon fast and efficient click-to-release, with the self-immolation being the rate-determining step. While reaction with DMT (1) results in near-quantitative cleavage of 21, the kinetics of both the click and the release step are not sufficient at a Tz concentration of 10 µM to allow significant accumulation of fluorescence signal. Hence, when monitoring the cells by fluorescence microscopy, we observed only a weak signal after 60 minutes (Fig. ). 2Pyr2 (3) exhibits a >100-fold higher click reactivity compared to 1 (cf. Fig. ) but, due to a release efficiency of less than 10% (cf. Fig. ), gives no detectable fluorescence after 1 hour. In contrast, treatment with the bishydroxypyridyl-Tz 5 resulted in accelerated release of resorufin and thus a significant turn-on of fluorescence already after 15 min (Fig. , see Supporting Information for panels of serial images taken every minute). These findings not only demonstrate the superior click-to-release performance of h2P2 ( ) compared to its non-hydroxylated analog 2Pyr2 (3) and commonly used DMT (1), but also confirm adequate live cell permeability.