The Pyridyl-Tz Challenge Aiming for a TCO equipped with a tautomeriza=on-direc=ng group (TDG) capable of efficient release across all Tz classes, we considered the ques=on of why the highly reac=ve and broadly used 3,6-bis(2-pyridyl)-Tz scaffold fails to trigger release with both rTCO and other exis=ng cleavable TCOs. Although this liability is circumvented with second genera=on aryl-Tz scissors, it remains unknown why the reac=on of rTCO with bispyridyl-Tz becomes trapped in the post-click tautomeriza=on network (Supplementary Figure ). We thus turned to unraveling the mechanism through NMR and HPLC monitoring of the reac=on of water-soluble Nmethylated rTCO-PEG4 and Pyr2 (1) under physiological condi=ons (37°C, pH 7.4), with the objec=ve of differen=a=ng failures in tautomeriza=on from failures in release (Fig. ). Serial sampling confirmed negligible release (<6% at 72 hours), matching previous reports . In addi=on, no 1,4-DHP was detected; instead, an equilibrium between the ini=al 4,5-DHP (71%) and a water adduct (23%) was observed (Supplementary Figure ). Given the known accelera=ng effects of low pH, we completed the same analysis under acidic condi=ons (pH 4), finding that slow tautomeriza=on of the 4,5-DHP favored the forma=on of stable non-releasing 2,5-DHP, with just 33% release (via 1,4-DHP). In this context, the molecular agenda for efficient release intrinsically directed by the TCO becomes clear: drive tautomeriza=on to the kine=cally controlled 1,4-DHP.