Release kinetics were studied by serial HPLC analysis of the reactions of tetrazines 5 and 6 with rTCO-probe 7 in PBS at 37 °C (Fig. ). Based on the respective k2 values (cf. Fig. ) we selected reactant concentrations (10 µM 7, 20 µM Tz) that facilitated complete (>99%) click reaction within minutes (t0.99 = 5 min for Tz 5, 11 min for Tz 6), thereby enabling temporal resolution of the release process. One-phase exponential fitting of the data (up to a reaction time of t0.99) and extrapolation to t = 0 min indicates an initial phase of instantaneous release of approx. 50%, which we hypothesize is due to immediate formation of the releasing 1,4-DHP tautomer upon click reaction with Tz 5 or 6. Due to the competition of both directing OH groups, 50% of the initial click product (4,5-DHP) reacts to the non-releasing 2,5-DHP tautomer, leading to a second phase of release (via 2,5-DHP ⇌ 4,5-DHP ⇌ 1,4-DHP) with first-order kinetics. This elimination proceeds significantly faster upon click reaction with h2P2 ( ), with a first-order rate constant (k1) of 0.036 min -1 , overall achieving 98% release in ~100 min (Fig. ). In comparison, 7 hours are required for the reaction with Tz 6 to reach a matching extent of release (k1 = 0.009 min -1 ). ) and h4P2 ( ) in PBS at 37 °C (serial HPLC measurements, mean ± SD, n = 3).