Self‐Activation in De Novo Designed Mimics of Cell‐Penetrating Peptides

The unique ability of cell-penetrating peptides (CPPs), also known as protein transduction domains, to navigate across the nonpolar biological membrane has been under intense investigation. In vitro studies have shown that multiple mechanisms are available, with the precise details being dependent on the peptide and cell line studied. The several clearly demonstrated pathways include various forms of endocytosis, macropinocytosis, lipid-raft-dependent macropinocytosis, and protein-dependent translocation. In addition, an energy-independent pathway, or spontaneous translocation, has also been illustrated. 5] Perhaps the clearest example of an energy-independent pathway is the ability of CPPs, and their synthetic mimics, to cross model phospholipid bilayer vesicle membranes. General consensus in the literature suggests that hydrophobic counterions play an essential role in this transduction by complexation around the guanidinium-rich backbone, thus coating the highly cationic structure with lipophilic moieties. For example, an octamer of arginine in the presence of sodium laurate partitioned into octanol versus water with better than 95% efficiency. Separately, it was shown that the simple peptide nonaarginine ((Arg)9) does not in fact transverse membranes very effectively on its own. However, the presence of hydrophobic counterions “activates” this molecule, thus turning it into a potent transduction peptide. It was shown that n-alkyl chain surfactants were good “activators” and thus efficient at promoting the transport of oligoand polyarginines across biological membranes. 8] After the initial discovery that CPP-like behavior could be emulated in simple norbornene-based polymers, we wondered if the presence of covalently attached hydrophobic residues would increase their translocation activity. To evaluate this hypothesis, we designed and synthesized a series of norbornene-based guanidine-rich polymers, where the hydrophobic groups were introduced through a side chain rather than as counterions (Scheme 1). Remarkably, the guanidine polymers containing certain alkyl side chains exhibited significantly enhanced activity (by three orders of magnitude) without the need for any “counterion activator”. Monomers were prepared by either Mitsunobu coupling or nucleophilic substitution reactions (see the Supporting Information). Random copolymers G1–G12 with 50:50 mol% monomer distribution were targeted at two molecular weights (Mn) using ring-opening metathesis polymerization (ROMP; low Mn 2.9–3.9 kDa and high Mn 11.4– 13.6 kDa of the tert-butyloxycarbonyl (Boc)-protected polymers were obtained). Gel-permeation chromatography gave monomodal signals and narrow molecular-weight indices (1.05–1.15). The Boc-protected polymers were deprotected to obtain G1–G12, and their activities were studied in vesicle assays. Using the standard biophysical assay well-accepted in the CPP literature, the transport activities of G1–G12 were determined. Specifically, 5(6)-carboxyfluorescein (CF) was used as a fluorescent probe in egg yolk phosphatidylcholine large unilamellar vesicles (EYPC-LUVs). The activity of G1– G12 transporters increased with increasing polymer content at a constant vesicle concentration as detected by CF emission intensity, yielding plots of fluorescence intensity versus polymer concentration for the series G1–G12 (Supporting Information, Figure S1). Fitting the Hill equation (Y/ (c/EC50) ) to this data for each individual polymer revealed a nonlinear dependence of the fractional fluorescence intensity Yon the polymer concentration c. This analysis gave Ymax (maximal CF release relative to complete release by Triton X100), EC50 (effective polymer concentration needed to reach Ymax/2), and the Hill coefficient n (Supporting Information, Figure S2, Tables S1 and S2). For direct comparison, it is worth mentioning that the CPPs heptaarginine and polyarginine were inactive under these conditions; it is known that polyarginine needs counterions for activation. Figure 1 collects the EC50 values for this series of copolymers. Polymers with lower EC50 values are said to be Scheme 1. Guanidino copolymers G1–G12.