To verify release via Pc and subsequent intramolecular Michael addi=on, we monitored the reac=on of iTCO-PEG4 (14) and Pyr2 (1) by serial NMR measurements in deuterated water. Given the established reac=on kine=cs and the high concentra=ons needed for NMR, both click and release were calculated to be complete within 1 min. At the earliest experimental =me point (8 min), we observed complete release and a nearequimolar mixture of the elimina=on product Pi and the key intermediate Pc, dis=nguished by characteris=c downfield 1 H signals of the olefinic proton in release posi=on and the bridgehead H-atom (Fig. ). Sequen=al NMR analysis tracked the progressive conversion of Pc into the cyclized Pi, with a half-life of 6.2 min, followed by a very slow subsequent oxida=on to Pia (<30% aUer a week). Monitoring the reac=on by NMR in deuterated PBS, we observed the same events but accelerated to a pace that was less tractable to analysis, with an es=mated half-life of 2.6 min (see Supplementary Figure ). Theore=cal calcula=ons, performed in ORCA 6.0.0 47 using density func=onal theory at the M06-2X/def2-TZVP CPCM(water) level of theory, revealed a significantly lower free energy of ac=va=on (ΔG ⧧ ) for the intramolecular Michael addi=on upon click-torelease of iTCO with Pyr2 compared to DMT (see Supplementary Informa=on). This supports our hypothesis that an electron-withdrawing 2-pyridyl subs=tuent promotes the exclusive forma=on of Pi, whereas the reac=on with the bis-alkyl-Tz DMT (10) follows both post-release pathways (Fig. ), resul=ng in a mixture of Pa and Pia (upon oxida=on of Pi).