To confirm the individual steps of the proposed mechanism, we first turned to the known fluorogenic postclick tautomeriza=on of bis-pyridyl-subs=tuted dihydropyridazines, where transforma=on from the nonfluorescent 4,5-DHP to fluorescent 1,4-DHP isomers offers a tool to monitor this cri=cal first step in real =me . In contrast to non-releasing TCOs, the 1,4-DHP becomes a short-lived intermediate in the case of cleavable TCOs, en route to elimina=on and subsequent events. However, as the post-release species such as the immediate elimina=on product Pc are expected to be fluorescent similar to the 1,4-DHP scaffold (see Supplementary Informa=on), the key readout-disappearance of the 4,5-DHP-remains intact (Fig. ). Accordingly, we monitored the reac=ons of Pyr2 (1) with iTCO-PEG4 ( ) and structurally similar cTCO-PEG4 (15), which includes an ester func=onality like iTCO but a methyl group instead of the direc=ng OH. A rapid increase in fluorescence upon click reac=on of Pyr2 with iTCO revealed fast conversion of the 4,5-DHP, in striking contrast to the minimal tautomeriza=on observed for cTCO under iden=cal condi=ons (3 µM Pyr2 + 7 µM TCO; 95% click in ~1 min) in the same =meframe, confirming the enhancing effect of the direc=ng OH group on post-click tautomeriza=on (Fig. ).