Diversity-oriented synthesis of second generation guanidinium-rich transporters toward cell-selective penetration

Cell-penetrating peptides (CPPs) hold significant promise for intracellular delivery of various cargo molecules such as therapeutics. However, the lack of selectivity remains a critical challenge and limits the clinical application of CPPs. Using an automated peptide synthesizer, we generated a diversity-oriented library of 256 peptidomimetics containing four modified peptoid guanidine-bearing monomers incorporated alternatively with four α-amino acids. These α-amino acids were chosen to enhance lipophilic interactions with the cell membrane (Phe, 2Nal) or to bear pH-sensitive properties (His), which could enhance cancer cell selectivity. The synthesized library exhibits selective internalization, with an average selectivity index (SI) of 1.49 for HeLa cells in comparison to non-cancerous HEK293 cells. Compounds 155 and 187, containing three His residues and either Phe or 2Nal, show high cellular uptake in HeLa cells (64.6% and 75.7%, respectively) and possess an SI of 2.7 and 2.9, respectively, at the tested dose of 5 μM. Altogether, these findings highlight the use of diversity-oriented library synthesis to identify cell-permeable candidates as well as their potential for targeted cellular delivery and enhanced specificity.