With an excep=onally promising por‚olio of chemical characteris=cs established, we next explored the applica=on of iTCO in cellular environments. To inves=gate release kine=cs with clinically relevant compounds, we selected three cytotoxic agents-monomethyl aurista=n E (MMAE), doxorubicin (Dox) and combretasta=n A-4 (CA-4)-and prepared a matched set of conjugates for comparison with rTCO (Fig. ). MMAE and Dox were directly conjugated to the TCO; for CA4, we used N,N'-dimethylethylenediamine (DMEDA) as a minimal self-immola=ve linker between the TCO and drug . Following the condi=ons established for the AF594-based experiments, we reacted the TCO-caged drugs with Pyr2 (15), given its prime performance with iTCO, and with MeBAm (13), as this Tz scaffold is currently employed in clinical trials .