To assess the performance of iTCO and the func=onal impact of the OH-TDG, we aimed to measure release yields with different broadly used Tz scaffolds, benchmarked against the reference standard of rTCO. Given the complex influence of environmental condi=ons on tautomeriza=on and release, we prepared a set of fluorescent and non-fluorescent probes op=mized for solubility in fully aqueous, physiologically-relevant condi=ons (Fig ). We began by reac=ng the matched TCO-Alexa Fluor 594 (AF594) probes 8 and 9 with Tz 1 and 10-12 in phosphate buffered saline (PBS) at 37 °C (Fig. ); endpoints for cleavage of rTCO-AF594 ( ) and iTCO-AF594 (9) were determined aUer 48 hours via HPLC analysis with fluorescence detec=on. In alignment with previous findings , incomplete rTCO-release was observed with the bis-alkyl tetrazine 10 (3,6-dimethyl-1,2,4,5-tetrazine, DMT), a consequence of tricyclic dead-end forma=on from the 4,5-DHP in the absence of an N-methylated carbamate (Supplementary Fig. ). In contrast, iTCO exhibits 98% cleavage with DMT, obvia=ng the need for N-methyla=on and thus enabling near-quan=ta=ve release of primary amines. Reac=ng cleavable TCOs with alkyl-aryl Tz typically gives a maximum release of ~50%, the result of a random click orienta=on and lack of release when the aryl subs=tuent is in the head-to-head posi=on, i.e., adjacent to the releasing carbamate . Consistent with this report and poten=al dead-end forma=on, the methyl-phenyl Tz 11 (MeBAm) triggers less than 30% release with rTCO. With iTCO, however, we again observed ≥95% release, implying that the TCO-anchored TDG is able to drive forma=on of the cri=cal releasing 1,4-DHP (Fig. ).