Extending these measurements, we went on to inves=gate the release kine=cs of iTCO with the remaining Tz classes. For DMT (10), release is no longer the rate-limi=ng step, as the observed overall rate of click-torelease tracks closely with the calculated click kine=cs under these condi=ons (Fig. ). Similarly, MeBAm (11) exhibits fast release with a 95% yield aUer 10 min, aligning with the calculated click kine=cs, which predict In contrast, under matched condi6ons, rTCO showed limited release, reaching 78% with the bis-alkyl-Tz DMT (10) but just ~5-25% with the more reac6ve Tz 1, 11 and 12. (c) Stopped-flow spectrophotometry (PBS, 37 °C, n > 4, SD = 6%) revealed comparable second-order rate constants (k2) for iTCO-PEG4 ( ) and rTCO-PEG4 ( ), with iTCO reaching the benchmark of 10 4 M -1 s -1 with Pyr2 (1). (d) Click-to-release of iTCO with Pyr2 at micromolar concentra6ons is extraordinarily fast and already complete at the one-minute 6me point. In contrast, the rTCO probe manages negligible release even auer 30 minutes (mean ± SD, n = 3). (e) Release kine6cs vs. Tz: at the experimental concentra6ons (10 µM iTCO + 20 µM Tz), DMT (10) triggers release at a pace that tracks closely with the expected click kine6cs, consistent with instantaneous release. For the alkyl-aryl-Tz 11 (MeBAm), the click-to-release reac6on reaches ≥95% comple6on within 10 min (first 6me point). In contrast, the more reac6ve H-Tz 12 (HBAm) reaches 99% click within 50 s but exhibits a half-life of release (t1/2) of 30 minutes yet resul6ng in near-complete cleavage (mean ± SD, n = 3). Hence, while Pyr2 (1) and HBAm (12) show similar click reac6vity with iTCO, they offer different release kine6cs. 95% comple=on of the click reac=on at this =me. In contrast, the release half-life of HBAm ( ) is considerably longer (t½ = 30 min) even though the click reac=on of this H-Tz reaches 99% comple=on within 50 seconds. We hypothesize that the decreased release rate is due to mechanisms that compete with the directed tautomeriza=on (e.g., addi=on of water to the 4,5-DHP); the underlying dynamics are a topic of ongoing inves=ga=on and will be reported separately.