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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.

We have investigated how doubly selective synthetic mimics of antimicrobial peptides (SMAMPs), which can differentiate not only between bacteria and mammalian cells, but also between Gram-negative and Gram-positive bacteria, make the latter distinction. By dye-leakage experiments on model vesicles and complementary experiments on bacteria, we were able to relate the Gram selectivity to structural differences of these bacteria types. We showed that the double membrane of E. coli rather than the difference in lipid composition between E. coli and S. aureus was responsible for Gram selectivity. The molecular-weight-dependent antimicrobial activity of the SMAMPs was shown to be a sieving effect: while the 3000 g mol(-1) SMAMP was able to penetrate the peptidoglycan layer of the Gram-positive S. aureus bacteria, the 50000 g mol(-1) SMAMP got stuck and consequently did not have antimicrobial activity.

Antimicrobial peptides (AMPs) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogues of AMPs, such as the family of phenylene ethynylene antimicrobial oligomers (AMOs), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. Homologues in this family can be inactive, specifically active against bacteria, or nonspecifically active against bacteria and eukaryotic cells. Using synchrotron small-angle X-ray scattering (SAXS), we show that observed antibacterial activity correlates with an AMO-induced topological transition of small unilamellar vesicles into an inverted hexagonal phase, in which hexagonal arrays of 3.4-nm water channels defined by lipid tubes are formed. Polarized and fluorescence microscopy show that AMO-treated giant unilamellar vesicles remain intact, instead of reconstructing into a bulk 3D phase, but are selectively permeable to encapsulated macromolecules that are smaller than 3.4 nm. Moreover, AMOs with different activity profiles require different minimum threshold concentrations of phosphoethanolamine (PE) lipids to reconstruct the membrane. Using ternary membrane vesicles composed of DOPG:DOPE:DOPC with a charge density fixed at typical bacterial values, we find that the inactive AMO cannot generate the inverted hexagonal phase even when DOPE completely replaces DOPC. The specifically active AMO requires a threshold ratio of DOPE:DOPC = 4:1, and the nonspecifically active AMO requires a drastically lower threshold ratio of DOPE:DOPC = 1.5:1. Since most gram-negative bacterial membranes have more PE lipids than do eukaryotic membranes, our results imply that there is a relationship between negative-curvature lipids such as PE and antimicrobial hydrophobicity that contributes to selective antimicrobial activity.

The lessons learned from p-octiphenyl beta-barrel pores are applied to the rational design of synthetic multifunctional pore 1 that is unstable but inert, two characteristics proposed to be ideal for practical applications. Nonlinear dependence on monomer concentration provided direct evidence that pore 1 is tetrameric (n = 4.0), unstable, and "invisible," i.e., incompatible with structural studies by conventional methods. The long lifetime of high-conductance single pores in planar bilayers demonstrated that rigid-rod beta-barrel 1 is inert and large (d approximately 12 A). Multifunctionality of rigid-rod beta-barrel 1 was confirmed by adaptable blockage of pore host 1 with representative guests in planar (8-hydroxy-1,3,6-pyrenetrisulfonate, KD = 190 microM, n = 4.9) and spherical bilayers (poly-L-glutamate, KD < or = 105 nM, n = 1.0; adenosine triphosphate, KD = 240 microM, n = 2.0) and saturation kinetics for the esterolysis of a representative substrate (8-acetoxy-1,3,6-pyrenetrisulfonate, KM = 0.6 microM). The thermodynamic instability of rigid-rod beta-barrel 1 provided unprecedented access to experimental evidence for supramolecular catalysis (n = 3.7). Comparison of the obtained kcat = 0.03 min(-1) with the kcat approximately 0.18 min(-1) for stable analogues gave a global KD approximately 39 microM3 for supramolecular catalyst 1 with a monomer/barrel ratio approximately 20 under experimental conditions. The demonstrated "invisibility" of supramolecular multifunctionality identified molecular modeling as an attractive method to secure otherwise elusive insights into structure. The first molecular mechanics modeling (MacroModel, MMFF94) of multifunctional rigid-rod beta-barrel pore hosts 1 with internal 1,3,6-pyrenetrisulfonate guests is reported.

The pyrene-1,3,6-trisulfonate scaffold is introduced as an internal cofactor for histidine-rich p-octiphenyl s-barrels with catalytic and ion channel activity to mediate binding and conversion of otherwise inaccessible benzaldehyde substrates. Up to 170,000-fold accelerations of amide, ester, and carbonate hydrolysis, high substrate diversity, high chemoselectivity, and absence of enantioselectivity are reported.

Intermediate internal charge repulsion (ICR) is required to create synthetic pores with large, stable, transmembrane, and variably functionalized space. This conclusion is drawn from maximal transport and, in one case, catalytic activity of p-octiphenyl s-barrel pores with internal lysine, aspartate, and histidine residues around pH 7, 6, and 4.5, respectively. pKa Simulations corroborate the experimental correlation of intermediate ICR with activity and suggest that insufficient ICR causes pore implosion' and excess ICR pore explosion'. Esterolysis experiments support the view that the formation of stable space within multifunctional p-octiphenyl s-barrels requires more ICR in bilayer membranes than in H2O. Multivalency effects are thought to account for p-octiphenyl s-barrel expansion with increasing number of s-sheets, and proximity effects for unchanged pH profiles with increasing s-sheet length. Q-TOF-nano-ESI-MS barrel-denaturation experiments indicate that contributions from internal counterion effects are not negligible. The overall characteristics of p-octiphenyl s-barrel pores with internal lysine, aspartate, and histidine residues, unlike de novo a-barrels' and similarly to certain biological channels, underscore the usefulness of rigid-rod molecules to preorganize complex multifunctional supramolecular architecture.

A new series of aryl-based synthetic mimics of antimicrobial peptides (SMAMPs) with antimicrobial activity and selectivity have been developed via systematic tuning of the aromatic groups and charge. The addition of a pendant aromatic group improved the antimicrobial activity against Gram-negative bacteria, while the addition of charge improved the selectivity. SMAMP 4 with six charges and a naphthalene central ring demonstrated a selectivity of 200 against both Staphylococcus aureus and Escherichia coli , compared with a selectivity of 8 for the peptide MSI-78. In addition to the direct antimicrobial activity, SMAMP 4 exhibited specific immunomodulatory activities in macrophages both in the presence and in the absence of lipopolysaccharide, a TLR agonist. SMAMP 4 also induced the production of a neutrophil chemoattractant, murine KC, in mouse primary cells. This is the first nonpeptidic SMAMP demonstrating both good antimicrobial and immunomodulatory activities.

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

One member of a prototypical class of antimicrobial oligomers was used to study pore formation in cardiolipin rich membranes. Using both vesicle dye-leakage assays and small angle x-ray scattering, bilayer remodeling was studied. The results indicate that the presence of negative intrinsic curvature (NIC) lipids is essential for pore formation by this class of molecules: In Gram-positive bacteria, cardiolipin and divalent metal cations like Ca2+ and Mg2+ are needed. This is completely consistent with the role of phosphatidylethanolamine (PE) lipid in Gram-negative bacteria, where antimicrobial activity is dependent on the negative intrinsic curvature of PE, rather than a specific interaction with PE.

The usefulness of pyrene-1,3,6-trisulfonates as cofactors for p-octiphenyl ?-barrel ion channels to mediate recognition and conversion of otherwise inaccessible benzaldehyde substrates is described.