Probing hydrophobicity of cationic amphiphilic polyproline helices through side-chain variations

The cell membrane is an exceptionally efficient barrier, limiting the internalization of a variety of proteins and therapeutics due to their large size and hydrophilic nature. Even though methods have been developed to circumvent this issue, such as microinjection and electroporation, they are inefficient and can be highly cytotoxic. Thus there have been investigations on the use of cell-penetrating peptides (CPPs) for the intracellular delivery of proteins and other macromolecules as they have low cytotoxicity, demonstrate an affinity towards a variety of cell lines and are able to deliver diverse cargo. The basic domain from the HIV-1 Tat protein is one of the most studied CPPs to date. Utilizing information from this protein, it has been demonstrated that the addition of multiple cationic residues to proteins and small drugs enhances cellular uptake. Therefore we have designed a class of molecules termed cationic amphipilic polyproline helices (CAPHs) that have the ability to effectively cross the cell membrane. The polyproline backbone, which consists of trans-polyproline residues, provides the rigidity necessary to control the orientation of the hydrophobic and hydrophilic moieties to create an amphiphilic molecule. Presented herein are three CAPHs displaying a type II polyproline helical backbone that are functionalized to contain six cationic moieties and three distinctive hydrophobic functionalities, namely n-butyl (PLc4), n-pentyl (PLc5), or n-hexyl (P Lc6) groups. It was found that non-branched moieties (PLc4) introduced into a peptide facilitate greater cellular uptake than their branched counterparts (PL). It was also found that the introduction of hydrophobicity into the CAPHs is limited, as P11Lc5RR has the greatest uptake efficiency of all peptides studied at 15 μM, while the more hydrophobic peptide P11Lc6RR demonstrated less uptake efficiency than P11Lc5RR