ABSTRACT Accurate delivery of small molecules to specific cell compartments or organelles offers great potential for bioimaging and targeted therapy but remains challenging to predict and control. In this study, we evaluate a modular strategy for organelle‐directed delivery based on organelle‐targeting groups combined with tetrazine‐ trans ‐cyclooctene bioorthogonal chemistry. We demonstrate that organelle‐targeting probes bearing tetrazine or trans ‐cyclooctene can efficiently undergo inverse‐electron‐demand Diels‐Alder reactions within various cellular compartments, including the plasma membrane, mitochondria, nucleus, endoplasmic reticulum, and lysosomes. Using this method, we localize fluorophores and the cytotoxic drug doxorubicin to specific organelles in living cells. We further show that tetrazine‐ or trans ‐cyclooctene‐modified doxorubicin prodrugs restore cytotoxic activity upon click‐to‐release activation inside cells. However, direct comparisons of different probe architectures revealed that organelle targeting and reaction efficiency are highly sensitive to structural context, cargo type, and subcellular environment, and cannot be predicted solely from targeting motifs. Overall, these findings establish a framework for comparing organelle‐targeted bioorthogonal chemistry and offer practical guidance for designing probes and prodrugs that enable precise spatiotemporal control of small‐molecule activity in living cells.
Durydivka et al. (Mon,) studied this question.