Designed synthesis of short cell-penetrating peptides to explore structure-to-function insights in cellular uptake

Abstract

Cell-penetrating peptides (CPPs) are handy tools for protein/peptide delivery, thus helping in protein therapeutics, understanding versatile protein mechanisms etc. Indeed, CPP attached to the target protein, which is otherwise not cell permeable, can be delivered to the cell. To explore the role of CPPs in cell delivery, this work will investigate the effect of different CPP scaffolds on cellular uptake. Thus, a poly-arginine (Arg, R) based small CPP is synthesised. Linear R5 was chosen as the model CPP modified to K2R3 (Lys, K) to explore the influence of Arg-within the scaffold. G2R3 (Gly, G) probs the influence of charge as it failed to pass the membrane. To investigate the effect of aromaticity in cellular uptake, F2R3 (Phe, F) was synthesised, which was later modified to Y2R3 (Tyr, Y) to explore the effect of aromaticity along with active -OH groups. S2R3 (Ser, S) was designed to investigate the sole influence of -OH groups in cell delivery. Experiments suggest that R5 outperforms other peptides in cell delivery and was further examined at 37 °C and 4 °C. Successful cell delivery of R5 at 4 °C rules out chances of any energy-dependent cellular uptake pathway, including endocytosis. The probable structural influence of R5 and K2R3 was also investigated using DFT calculation.