The Effect of Aza-Glycine Substitution on the Internalization of Dabcyl-Containing Short Oligoarginine

Background/Objectives: Longer oligoarginines are very effective cell-penetrating peptides. It has been shown that a minimal number of positively charged side chains is necessary for efficient cellular uptake. But a highly positively charged peptide may interact with its cargo molecule, thereby reducing its efficiency. Several chemical modifications were tested to improve the internalization of short tetraarginine derivatives. Aromatic groups, such as Dabcyl at the N-terminus, Trp in the sequence, and AMBA or PABA in the backbone, were used to improve internalization. The other useful modification was the aza-glycine substitution in the case of penetratin. Methods: In this study, the effect of aza-glycine insertion into the peptide Dabcyl-RRRRK(Cf) on internalization was studied and compared with that of the Trp-modified peptide Dabcyl-RRWRRK(Cf). To explain the noticed difference in the biological activity of peptides, DFT calculations and the prediction of membrane-binding free energy (ΔΔF) from a peptide sequence were performed. Results: It turned out that the position of the aza-glycine moiety does not have an influence on the cellular uptake. The aza-glycine-containing peptide showed higher internalization than the Dabcyl-RRRRK(Cf) peptide. Besides this, these peptides have similar or higher cellular uptake than that of octaarginine at lower concentrations (c < 2 µM). The aza-glycine affected not only cellular uptake but also the entry mechanism. The structure of peptides depended on the amino acids (Trp, Gly, or azaGly) in their sequences and their positions. Conclusions: These may result in the different amphiphilicity of peptides, and thus changes in the hydrophobic moment and in the binding affinity of peptides to the negatively charged membrane surface.