Structure–Uptake Relationship Study of DABCYL Derivatives Linked to Cyclic Cell‐Penetrating Peptides for Live‐Cell Delivery of Synthetic Proteins

Abstract Modifying cyclic cell‐penetrating deca‐arginine (cR10) peptides with 4‐(4‐dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time‐lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL‐R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure–uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell‐uptake efficiency when conjugated to mono‐Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.