Protein Transduction Domain Mimics: The Role of Aromatic Functionality†

Cell-penetrating peptides (CPPs), or protein transduction domains (PTDs), are a special class of membrane-active proteins that can cross the cell membrane with unusual efficiency. They have attracted considerable attention because of their ability to readily cross biological membranes, in spite of their highly charged nature. While the exact mechanism of this transport remains under intense investigation, energy-independent pathways are known. Perhaps the clearest example is the ability of CPPs, and their synthetic mimics, to cross model phospholipid bilayer vesicle membranes. One suggested mechanism implies that, in fact, CPPs like polyarginine (pR) need assistance to cross the membrane. It suggests that hydrophobic counterions complex around the guanidinium-rich backbone, thus “coating” the highly cationic structure with lipophilic moieties. This process has been termed “activation”, in which the lipophilic anion acts as an activator. In a series of detailed studies it was shown that aromatic activators outperform aliphatic ones. For example, sodium 4-(pyren-1-yl)butane-1-sulfonate gave an EC50 (effective concentration to obtain 50% activity) of 6.7 mm whereas the value for sodium dodecane-1sulfonate was 16 mm. Among other activators studied, the larger aromatic counterion, coronene, was not better than pyrene; however, a fullerene analogue was surprisingly effective. While this work beautifully demonstrated the role of various counterions for pR activation, it was not clear if this better activation was due to general hydrophobicity or to the aromatic nature of these activators. There is good reason to think that aromatic functional groups may play a special role, beyond their general hydrophobicity. It is well recognized that membrane proteins are enriched in aromatic amino acids at the membrane surface. Their central hydrophobic core, composed mostly of aliphatic residues, is flanked on both sides by “aromatic belts”. Although this belt is predominantly composed of tryptophan and tyrosine, as opposed to phenylalanine, it was shown that aromatic residues, including N-methylindole, have favorable free energies of insertion into the bilayer interface. This rules out a dominant effect of hydrogen bonding. It was suggested that the flat-rigid shape, p-electronic structure, and associated quadrupolar moments provide unique and highly favorable interactions with the bilayer interface. Specific interactions that have been proposed include p-cation, electrostatic, dipole–dipole, and entropic factors related to bilayer perturbation. Even HIV-TAT, the original protein that initiated the field of small PTDs, requires tryptophan (Trp11) for translocation. [10] Moreover, an oligoarginine consisting of seven arginine residues with a C-terminal tryptophan (R7W) and a TAT48–60 peptide with residue 59 substituted with a tryptophan (TAT48–60P59W) exhibit cellular internalization through energy-independent pathways. Another classical CPP, penetratin (Pen), contains two tryptophan residues. Substitution of tryptophan by phenylalanine (Pen2W2F) did not significantly impact cell uptake. Among the aromatic amino acids, phenylalanine has the unique ability to partition at the interface and in the membrane core. In fact, aromatic residues, especially phenylalanine, are most effective at anchoring proteins in the membrane due to their “special ability” to form and stabilize essential interactions with the polar elements of the bilayer. As a result, aromatic functionality could be a critical element facilitating the interactions between CPPs and the bilayer during transduction. In the past few years, we and others have reported polymers designed to mimic the transduction activity of PTDs. More recently, we demonstrated that these protein transduction domain mimics (PTDMs) have “self-activation” properties when hydrophobic alkyl side chains were built into the copolymers. Here, a new series of PTDMs was designed to determine if an aromatic functionality provides better transduction efficiency than aliphatic ones, at the same relative hydrophobicity. Given the importance of aromatic amino acids in membrane proteins and their interactions with the bilayer, it was proposed that aromatic side chains would make better activators, given equal relative hydrophobicity. Although aromatic groups have been studied in peptidebased CPPs, demonstration of the importance of aromatic functionality in these synthetic analogues is critical to establishing them as appropriate mimics, or PTDMs. By using reversed-phase HPLC to determine side-chain hydrophobicity and EC50 values in a classic transduction experiment, it is demonstrated here that it was possible to differentiate between side-chain hydrophobicity and aromaticity. As shown in Table 1, a series of new PTDM polymers was prepared by ring-opening metathesis polymerization (see the Supporting Information for detailed synthesis and characterization of monomers and polymers). Reversed-phase HPLC, commonly used to evaluate relative hydrophobicity, was [*] Dr. A. Som, A. Reuter, Prof. G. N. Tew Polymer Science & Engineering Department University of Massachusetts 120 Governors Drive, Amherst, MA 01003 (USA) E-mail: tew@mail.pse.umass.edu Homepage: http://www.pse.umass.edu/gtew/index.html