Traceable Traceless Delivery Tags

This study elaborates on traceless tags for the thiol‐mediated uptake (TMU) of amine‐containing substrates of interest (SOIs). Traceless tags typically contain linkers that can be enzymatically cleaved to trigger the release of SOIs in their native form. To trace SOI release inside cells, the dual function of asparagusic acid (AspA) derivatives to enable TMU and to track the Golgi apparatus is explored. We tested two of the most popular cleavable linkers based on esterases and quinone reductases. Conjugated to AspA through alkyloxycarbonyloxymethyl (AOCOM) linkers, fluorescent SOIs entered cells by TMU and spread in the cytosol with or without a brief stop in the Golgi, depending on the structure. FRET probes further demonstrated that after arrival in the Golgi, SOIs are released from AspA into the lumen, where they linger before spreading to the cytosol and beyond. Quinone‐based trimethyl locks (TML) are also compatible with TMU, but reductive cleavage after TMU appears to be slower in the Golgi. These results validate AOCOM and TML linkers for constructing traceless TMU tags for amine‐containing SOIs and introduce AspA probes as dynamic‐covalent chemistry tools to trace traceless tags inside cells and to track Golgi lumen and Golgi membranes simultaneously with separately visualizable fluorescent probes.