In contrast to the symmetric tetrazines 7 and 8, IEDDA ligation of PymK ( ) and rTCO leads to formation of two initial click products (Fig. ). Depending on the click orientation, the NH3 +directing group (K) is either on the same (head-to-head) or the opposite side (head-to-tail) of the leaving group in allylic position of the TCO (Fig. ). Similar to unsymmetric Tz acids, as we have recently reported, NH3 + in head-to-head position accelerates TCO-release via B1, while the head-to-tail orientation leads to irreversible formation of a stable and therefore non-releasing B2 (Fig. ). Ammonium-functionalized Tz were first described by van Kasteren et al., who moreover discovered a pre-click interaction of the NH3 + moiety with the TCO-carbamate (Fig. ). As a result, click in head-to-head orientation is favored, thereby improving the efficiency of the overall bond-cleavage process. In addition, the release step upon click with ammoniumfunctionalized Tz was reported to be instantaneous (when NH3 + is in head-to-head position) and independent from pH. We exploited those explorations by using acidic HPLC conditions to achieve better separation of the intermediates (all bearing an NH3 + moiety) formed upon reaction of 6 with PymK ( ) without provoking any pseudo-release (for details see the Supporting Information). As expected, we observed immediate post-click tautomerization (no A detected) and 1,4-elimination (no B1 detected) leading to formation of the SIL-intermediate S and stable B2 in a ratio of 7/3, which confirms favored click orientation with NH3 + in head-to-head position (Fig. ). The uncaged phenol was obtained in a yield of 70% after a total reaction time of approx. pre-click interaction favors formation of A with K in head-to-head position N