The bioorthogonal activation of 12 by reaction with tetrazines 7 and 9 was monitored by HPLC-MS as described above. The relative abundance of all intermediates and products was calculated based on the UV absorbance at 254 nm and confirmed via quantification of released CA4 by external calibration (see Supporting Information). In agreement with data previously reported, we observed a significantly faster Tz-triggered elimination of cTCO compared to rTCO (cf. Fig. ). Reacting prodrug 12 with DMT (7) resulted in 96% release of CA4 already after 2 hours, reaching an endpoint of 98% CA4 and of the respective oxidized dead-end after a total reaction time of approx. 4 hours (Fig. , left). In contrast, IEDDA ligation of 12 and PymK (9) did not lead to favored formation of the initial click product with the NH3 + -substituent (K) in head-to-head position, as indicated by an overall CA4 release of 50%. Accordingly, 50% of nonreleasing stable B2 with K in head-to-tail position was detected (Fig. , right). Nevertheless, this experiment allowed analysis of the self-immolation kinetics, revealing accelerated cyclization of the DMEDA-carbamate of CA4 with a half-life of 22 min (k1 = 1.9 h -1 , one-phase exponential fitting, R 2 = 0.9993), which we hypothesize is due to CA4 being a better leaving group than the tyrosine-BODIPY conjugate P (cf. Fig. and Fig. ).