The click reactivity of bis-hydroxypyridyl-tetrazines 5 and 6 with cTCO was investigated by stopped-flow spectrophotometry using water-soluble cTCO-PEG4 (10). Measurements were conducted under pseudo-first-order conditions (see Supporting Information) in PBS at 37 °C. The observed second-order rate constants revealed that 5 (3400 M -1 s -1 ) and 6 (1100 M -1 s -1 ) react with cTCO (Fig. ) approx. twice as fast as with rTCO (cf. Fig. ). Click-to-Release of rTCO vs. cTCO (turn-on fluorescence) Initial experiments to assess cTCO-release kinetics using cTCO-AF594 (9) indicated that elimination is also faster than for rTCO. We thus adopted an alternative approach using TCO-caged 7amino-4-methylcoumarin (AMC) probes to study the click-torelease reaction. rTCO-AMC (11) and cTCO-AMC ( ) were reacted with Tz 5 and 6 and compared to DMT (1) and 2Pyr2 (3). The resulting elimination of fluorescent AMC allowed for real-time monitoring of the reaction through fluorescence measurement (Fig. ). While rTCO-release matched our previous findings using rTCO-AF594 (7) -except for the initial phase due to lower concentrations and consequently an extended duration to achieve complete click reaction (cf. Fig. ) -we observed a significantly faster cleavage of cTCO, achieving 95% release with h2P2 ( ) and approaching completion in less than 5 minutes (Fig. ). Click-torelease of cTCO-AMC ( ) triggered by reaction with h4P2 (6) proceeded slower, reaching 91% after 20 min. This difference can mostly be attributed to the different IEDDA reactivity. In fact, cTCO-release with 5 and 6 closely tracks with the click reaction (i. e., ~4 min for 5 and ~12 min for 6 to reach 99% click conversion at the experimental concentrations). Hence, the hydroxypyridyl groups appear to trigger near-instantaneous release (which is no longer the rate-determining step), such that cTCO-cleavage is controlled by click kinetics only. In comparison, click-to-release of caged AMC-probes 11 and 12 by reaction with DMT (1) is slow due to limited kinetics of both the IEDDA and the elimination step, resulting in 33% rTCO-release after 300 minutes and only 7% cTCO-release after 20 minutes, respectively (Fig. ). No detectable release of AMC was observed within these time frames upon reaction of 11 and 12 with 2Pyr2 (3), clearly demonstrating the release-enhancing effect of the hydroxypyridyl substituents.