Click‐Cyclized Cell Penetrating Peptides Containing Hydrophobic Proline Derivatives for Efficient Intracellular Delivery

Abstract Targeting intracellular organelles with macromolecules is challenging due to cell membrane impermeability. Innovative approaches to direct intracellular delivery have been developed, including nanoparticles, as a promising method for protein delivery; however, with limited efficiency. Cell‐penetrating peptides (CPPs) provide a versatile alternative for intracellular cargo delivery. We suggest substituting hydroxy‐ or amino prolines in β‐turns in cyclic CPPs with hydrophobic residues to facilitate cell penetration. We investigated the influence of substituted amine and ether derivatives of trans ‐4‐hydroxy‐ or amino‐ l ‐proline on MCF‐7 and other cell lines for the import of the fluorophore ATOTA. Flow cytometry and spinning disk confocal microscopy on 17 linear and click‐cyclic peptides revealed that particularly cyclic cell penetrating peptide 15 (cCPP 15 ) exhibited remarkable permeability. When conjugated with enhanced green fluorescent protein (EGFP), cCPP 15 efficiently delivered the protein into MCF‐7 and HeLa cells. cCPP 15, comprising two endocyclic and two exocyclic d ‐arginines and a trans amino‐L‐proline with a 2‐naphthylmethylene modification in a typeIIβ‐turn, significantly enhanced intracellular penetration. The efficiency of cCPP 15 in intracellular delivery provides a valuable addition to the growing field of CPPs that can deliver macromolecules to the interior of the cell and possibly even cross the blood‐brain barrier with proteins or drugs.