Enhanced Live‐Cell Delivery of Synthetic Proteins Assisted by Cell‐Penetrating Peptides Fused to DABCYL

Live‐cell delivery of a fully synthetic protein having selectivity towards a particular target is a promising approach with potential applications for basic research and therapeutics. Cell‐penetrating peptides (CPPs) allow the cellular delivery of proteins but mostly result in endosomal entrapment, leading to lack of bioavailability. Herein, we report the design and synthesis of a CPP fused to 4‐((4‐(dimethylamino)phenyl)azo)benzoic acid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic deca‐arginine (cR10) modified with a single lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin‐like modifier‐2 (SUMO‐2) scaffolds resulted in a threefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL‐assisted delivery of Ub and a Ub variant (Ubv) based activity‐based probes for functional studies of deubiquitinases in live cells. Efficient intracellular delivery of synthetic native proteins is a challenge due to endosomal entrapment and subsequent degradation. We report the enhanced cellular uptake of fluorescently labelled synthetic proteins by attaching a newly developed cell‐penetrating peptide (CPP) composed of cyclic deca‐arginine linked to DABCYL.