After we observed that the new TCO gave satisfactory payload release, we decided to synthesize a drug-conjugated TCO that would release the drug upon tetrazine ligation. This was examined with the release of the chemotherapeutic drug doxorubicin in a cellular environment. The doxorubicin conjugate 27 was synthesized from the 4-nitrophenyl activated TCO and purified with reversed-phase HPLC. The effectiveness of doxorubicin release was tested in HeLa cells through an EC50 assay (Figure ). The cells were incubated for three days at 37 °C with varying concentrations of the indicated compounds (tetrazine 20, doxorubicin, TCO-Dox (27), or TCO-Dox (27) + 10 equiv tetrazine 20). Addition of 10 equivalents of tetrazine 20 increased the cytotoxicity of the doxorubicin TCO conjugate 27 with a 21-fold (75.9 ± 12.08 nM vs 1.654 ± 0.418 μM) which indicates successful release of doxorubicin. This change cannot be attributed to toxicity of the tetrazine, as its EC50 was in the mid-micromolar range (29.81 ± 5.21 μM). Together, these findings confirmed that conjugation to the TCO reduces doxorubicin's toxicity and that the drug can effectively be released upon tetrazine activation. Besides, HPLC analysis of the same click reaction in solution revealed successful and rapid release of doxorubicin from the conjugate (see: Figure ).