To overcome the limitations arising from the lack of control of the Tz/TCO click orientation, we have previously developed the C2symmetric cleavable linker C2TCO, featuring carbamate leaving groups at both allylic positions of the TCO scaffold (Fig. ). C2TCO was shown to be cleaved completely and instantaneously upon reaction with unsymmetric Tz equipped with a directing group (CO2H, NH3 + ). Although the introduction of an additional leaving group led to increased steric hindrance, resulting in reduced IEDDA reactivity compared to rTCO and cTCO, we have demonstrated that the less sterically demanding alkyl-H-Tz 13 (HPA) and 14 (HK, Fig. ) enable efficient cleavage of C2TCO. To accelerate the click-triggered bioorthogonal disassembly of C2TCO-conjugates we hypothesized that highly reactive aryl-H-Tz -previously shown to lead to incomplete or even negligible release -such as 3-phenyl-1,2,4,5-tetrazine (Ph, 15) can be transformed into bioorthogonal scissors by introduction of an OH group on the aryl substituent. Therefore, we prepared the hydroxyaryl-H-Tz 16 and 17 (Fig. ) starting from the respective nitriles applying a procedure developed by Audebert et al. (see Supporting Information). To determine the cleavage efficiency, ) HK ( ) Ph ( ) hPh ( ) h2P ( fluorescent probe 18 (AF594-C2TCO-AF594, 25 µM, Fig. ) was reacted with Tz 13-17 (50 µM) in buffered aqueous solution (pH 7.4) at 37 °C (Fig. ). HPLC analysis after 1 hour revealed nearquantitative C2TCO cleavage upon reaction with HPA (13) and HK (14), and the hydroxyaryl-H-Tz 16 and 17, while only limited release (<50%) was observed for the phenyl-H-Tz 15. Moreover, 16 (hPh) achieved >98% cleavage after a reaction time of 10 min, in contrast to approx. 16% with its non-hydroxylated analog 15, clearly demonstrating the effect of the OH group (Fig. ). Consistent with our previous findings regarding compounds 13 and 14, we did not detect any post-click intermediates, even at lower concentrations, upon reaction with tetrazines equipped with a directing group. Hence, the hydroxyaryl substituent facilitates instantaneous elimination (Fig. ), making the initial IEDDA reaction the rate-determining step.