To explore iTCO-release in progressively stringent biological environments, we next reacted iTCO-AMC ( ) with Pyr2 (1) in cell growth medium (FluoroBrite DMEM + 10% fetal bovine serum) and in human plasma, employing low-micromolar concentra=ons to mimic realis=c biological scenarios (Fig. ). The half-life of the overall click-to-release reac=on in cell growth medium was 30 s and in human plasma 3.3 min. To dis=nguish the contribu=ons of click and release steps, we determined the click kine=cs of iTCO-PEG4 ( ) with Pyr2 (1) in human plasma and DMEM/FBS. The twofold reduc=on in the reac=vity of Pyr2 in human plasma (5,400 M - 1 s -1 ) compared to PBS, intermediate kine=cs in DMEM/FBS (7,600 M -1 s -1 ), and the comparable reac=vi=es of DMT, HBAm and MeBAm in all three media (Table ), suggest that hydrophobic interac=ons between Pyr2 and plasma proteins may lower its effec=ve concentra=on. In comparison, cTCO exhibited significantly lower reac=vi=es with all Tz in plasma vs. PBS, which we abribute to its greater hydrophobicity and stronger protein interac=ons. This finding underscores the beneficial effect of iTCO's reduced lipophilicity, imparted by the addi=onal OH func=onality. Despite the observed decelera=on of the release step, which we hypothesize is due to compe=ng mechanisms or macromolecular interac=ons arising from increasing biological complexity, Pyr2-triggered iTCO-release remained excep=onally fast and complete in <20 min at single-digit micromolar concentra=ons. ), with a half-life of 4.8 s (n = 3; SD error bars depicted as a gray-shaded region above and below the line connec6ng the mean data points). In striking contrast, the cTCO probe 17 showed <1% release within this 6meframe. (b) While complex biological matrices (cell growth medium DMEM (FluoroBrite) including 10% fetal bovine serum (FBS); human plasma) slightly lower the rate constants for both click and release, the overall reac6on s6ll proceeds excep6onally fast at single-digit micromolar concentra6ons, with a half-life of 3.3 min in human plasma (n = 3; SD error bars depicted as a gray-shaded region above and below the line connec6ng the mean data points). (c) Incuba6on of iTCO-PEG4 ( ) in PBS and human plasma followed by quan6fica6on of intact compound via 6tra6on with Pyr2 (1) revealed a remarkable stability of iTCO in both media, with <3% degrada6on auer 24 hours (mean ± SD, n = 3). Based on these findings, we evaluated the stability of iTCO under physiological condi=ons by incuba=ng iTCO-PEG4 ( ) in PBS and human plasma at 37°C (Fig. ). The TCO concentra=on was determined by =tra=on with Pyr2 (1) and quan=fica=on of excess Tz via UV-Vis measurement at the characteris=c Tz wavelength (520 nm). iTCO demonstrated remarkable stability in both PBS and human plasma, with less than 3% degrada=on observed aUer 24 hours, further emphasizing its robustness and reliability as a next-level bioorthogonal tool.