Efficient synthesis of cleavable CPP‐cargo conjugate via low‐equivalent of CPP activated by 2,2’‐dithiodipyridine

Cell‐penetrating peptides (CPPs) are favored for protein delivery due to their efficiency, rapidity, and low toxicity. However, conjugation of CPPs to proteins often requires significant amounts of CPPs to ensure yields, which also may result in increased proteins dimer formation. Here, we report that the use of low equivalents 2,2’‐dithiodipyridine (DPDS)‐activated CPP for conjugation cargo allows for high‐conversion‐rate CPP‐cargo conjugates. Using this strategy, we obtained high‐conversion‐rate conjugates of cyclic deca‐arginine peptide (cR10) with ubiquitin (Ub) and UbcH7 using only low equivalents of cR10. Furthermore, we successfully conjugate three CPPs to cargo via DPDS and confirmed its successful cytosolic delivery through fluorescence imaging.