Click-to-release of 6 triggered by reaction with Tz was then studied by HPLC-MS. According to the current mechanistic understanding, IEDDA ligation leads to formation of an initial 4,5-dihydropyridazine (4,5-DHP) click product A that can tautomerize to the releasing 1,4-DHP isomer B1 and the nonreleasing 2,5-DHP isomer B2 (Fig. ). The N-methyl group of the DMEDA linker blocks undesired intramolecular cyclization of A, thereby preventing the formation of a tricyclic dead-end product that, as we have previously shown, could otherwise severely impact the outcome of the bioorthogonal bond-cleavage process. rTCO-release via 1,4-elimination of B1 yields the SILintermediate S that upon cyclization of the DMEDA-carbamate reacts to the uncaged phenol P. Depending on the chemical structure of the Tz, B2 is in equilibrium with A, but can oxidize to the non-releasing pyridazine Ox (Fig. ). Based on previous reports, we have selected 3,6-dimethyl-1,2,4,5-tetrazine (DMT, 7), the diacid PA2 ( ), and the ammonium-functionalized tetrazine PymK ( ) to investigate the IEDDA-triggered click-torelease of 6 (Fig. ). While second-order rate constants (k2) of <60 M -1 s -1 have been reported for the click reaction of rTCO with DMT (7) and PA2 ( ), pyrimidyl-alkyl-Tz such as 9 are known to be substantially more reactive, reaching k2 of >1000 M -1 s -1 . We have previously reported on the profound pH sensitivity of Tz/TCO-release and its impact on HPLC analysis, with routinely applied acidic conditions (e.g., formic acid) leading to 'pseudo-release' that impedes accurate reaction monitoring. Thus, buffered HPLC conditions (ammonium formate, 2.5 mM, pH 8.4) were used to investigate the reactions of 6 with Tz 7 and 8 in PBS at 37 °C. Despite a tailing peak for the SIL-intermediate S, all reactants, tautomers, and products could be separated and identified (Fig. ), allowing monitoring of the post-click reaction network by serial HPLC-MS measurements. We observed the formation of the initial click product A (at early time points synchronously to the consumption of 6), the non-releasing tautomer B2, and the S (as the product of TCOrelease) that upon self-immolation afforded phenol P with an overall yield of 89% after a reaction time of approx. 8 hours. In agreement with our previous findings, no detectable concentration of B1 was found due to rapid 1,4-elimination of this labile intermediate. The respective pyridazine dead-end Ox was identified as the only non-releasing byproduct (11%, Fig. ).