One-step solid-phase synthesis of activated cell-penetrating peptides for cytosolic delivery of protein

Abstract

Cell-penetrating peptides (CPP) enable to deliver large biomolecules (proteins, peptides, oligonucleotides, etc.) into cells. An important step for the conjugation of CPPs to target proteins is the acquisition of activated CPPs, however, it requires a multi-step isolation and purification process. Here, we report a facile strategy for the synthesis of activated cell-penetrating peptides via a one-step solid-phase synthesis through the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) with CPP on resins, which can then be treated with a TFA cocktails to directly obtain activated CPP (TNB-CPP). Using this strategy, we successfully obtained activated cyclic cell-penetrating deca-arginine peptide (TNB-cR10) and activated Tat (TNB-Tat) and efficient cytosolic delivery of ubiquitin (Ub) can be achieved by linking it to these CPPs.