In recent years, the field of bioorthogonal chemistry has expanded its molecular toolbox from ligation-only reactions ("click") to bond-cleavage reactions ("release"), enabling spatiotemporally controlled disassembly of biomolecules and in situ activation of therapeutics, such as prodrugs. Among these, the click-to-release reaction between tetrazines (Tz) and cleavable trans-cyclooctenes (TCOs) has emerged as a powerful tool due to its high reaction rates, versatility, and selectivity, enabling the first bioorthogonal therapeutic approach currently undergoing clinical trials.[1] An alternative but mechanistically related strategy inverts the roles: cleavable tetrazines serve as the cages for payloads, while highly reactive TCOs function as the bioorthogonal trigger.[2,3] This alternative design benefits from the use of more reactive strained TCOs with superior click reactivity, but broader application has been limited by slow and incomplete release. Specifically, efficient cleavage requires formation of the 2,5-dihydropyridazine (2,5-DHP) tautomer, which exists in equilibrium with other, non-releasing tautomeric forms such as 1,4-DHP. In many cases, this equilibrium is not sufficiently shifted toward the desired species, limiting the efficiency and speed of payload release (see figure a).[2] To address this challenge, we developed a new class of cleavable tetrazines by the introduction of an ortho-hydroxyaryl substituent, building on our group’s recent work demonstrating that such scaffolds can direct and accelerate tautomerization in Tz–TCO click products.[4] We hypothesized that the introduction of the hydroxy functionality would enable intramolecular control promoting the formation of the 2,5-DHP tautomer and thus enhancing the subsequent elimination of carbamate-linked payloads (see figure b). We began by synthesizing a set of known and newly designed ortho-hydroxylated cleavable tetrazine scaffolds, to evaluate their click and release performance. Stopped-flow measurements under physiological conditions (PBS, 37 °C, pH 7.4) revealed that ortho-hydroxy substitution had – as expected – no significant effect on click kinetics. To assess release efficiency, we prepared fluorogenic tetrazine conjugates and investigated the TCO-triggered cleavage reaction. Strikingly, the hydroxylated variant substantially outperformed its non-hydroxy analog, showing enhanced and faster payload release (see figure c), These improved release kinetics allowed efficient cleavage at low micromolar to submicromolar concentrations, offering a practical advantage for applications in chemical biology and drug delivery. Methodologically, these findings were supported by LC-MS as well as fluorescence measurements, confirming our hypothesis of the accelerated post-click tautomerization and efficient 1,4-elimination enabled by the hydroxy directing group. Building on these results, we designed cleavable tetrazine–caged prodrugs and demonstrated bioorthogonal activation in cellular environments with remarkable chemical performance. Overall, our results show that hydroxy-directed tautomerization provides a powerful strategy to boost the efficiency of the click-to-release chemistry of cleavable tetrazines, enabling bioorthogonal prodrug activation at submicromolar concentrations. This concept offers a broadly applicable approach to overcome current limitations in release efficiency and opens new opportunities for controlled molecular disassembly in biological systems. a) General mechanism of the click-to-release reaction using cleavable Tz. b) Novel ortho-hydroxy substituted cleavable Tz, enabling the control of tautomerization to the desired 2,5-DHP tautomer. c) Direct comparison of release performance under physiological relevant conditions References [1] Wu, K.; Yee, N. A.; Srinivasan, S.; Mahmoodi, A.; Zakharian, M.; Mejia Oneto, J. M.; Royzen, M., Click activated protodrugs against cancer increase the therapeutic potential of chemotherapy through local capture and activation. Chemical Science 2021, 12 (4), 1259-1271. [2] van Onzen, A. H. A. M.; Versteegen, R. M.; Hoeben, F. J. M.; Filot, I. A. W.; Rossin, R.; Zhu, T.; Wu, J.; Hudson, P. J.; Janssen, H. M.; ten Hoeve, W.; Robillard, M. S., Bioorthogonal Tetrazine Carbamate Cleavage by Highly Reactivetrans-Cyclooctene. Journal of the American Chemical Society 2020, 142 (25), 10955-10963. [3] Wang, Y.; Shen, G.; Li, J.; Mao, W.; Sun, H.; Feng, P.; Wu, H., Bioorthogonal Cleavage of Tetrazine-Caged Ethers and Esters Triggered by trans-Cyclooctene. Organic Letters 2022, 24 (29), 5293-5297. [4] Wilkovitsch, M.; Kuba, W.; Keppel, P.; Sohr, B.; Löffler, A.; Kronister, S.; del Castillo, A. F.; Goldeck, M.; Dzijak, R.; Rahm, M.; Vrabel, M.; Svatunek, D.; Carlson, J. C. T.; Mikula, H., Transforming Aryl-Tetrazines into Bioorthogonal Scissors for Systematic Cleavage of trans-Cyclooctenes. Angewandte Chemie International Edition 2025, 64 (5), e202411707