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As a semipermeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs), because of their unique properties. In this Account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, we have generated scaffolds with biological potency exceeding that of the natural analogues. One of these compounds has progressed through a phase II clinical trial for pan-staph infections. Modern biophysical assays have highlighted the interplay between the synthetic scaffold and lipid composition: a negative Gaussian curvature is required both for pore formation and for the initiation of endosome creation. Although work remains to better resolve the complexity of this interplay between lipids, other bilayer components, and the scaffolds, significant new insights have been discovered. These results point to the importance of considering the various aspects of permeation and how these are related to "pore formation". More recently, our efforts have expanded toward protein transduction domains, or mimics of cell penetrating peptides. Using a combination of unique molecular scaffolds and guanidinium-rich side chains, we have produced an array of polymers with robust membrane (and delivery) activity. In this new area, researchers are just beginning to understand the fundamental interactions between these new scaffolds and the plasma membrane. Negative Gaussian curvature is also important in these systems, but the detailed relationships between molecular structure, self-assembly with lipids, and translocation will require more investigation. It has become clear that the combination of molecular design, biophysical models, and biological evaluation provides a robust approach to the generation and study of novel proteinomimetics.

Antimicrobial peptides and their synthetic analogues are well known to interact with the cell membrane, which has complex distributions of lipids. The phase behavior of DOPE/DOPG mixed lipids and the interaction between the lipids and several synthetic amphiphilic antimicrobial oligomers (AMOs) were studied by solid-state nuclear magnetic resonance (NMR). A phase diagram of the lipids over a broad window of water content was constructed. There are large areas in the phase diagram where multiple phases coexist, and the fraction of each phase at a given state is dependent on the sample’s preparation and thermal history. The comparable stability of the different phases implies that even slight changes in the lipid condition could result in substantial changes to the phase structure, which may be utilized by living organisms to achieve many membrane functions. Nuclear Overhauser spectroscopy (NOESY) and several other NMR experiments indicated that the AMO primarily resides in the head group region of the lipids and that DOPE, the negative intrinsic curvature lipid, does not selectively enrich in the inverted hexagonal phase.

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.

Synthetic mimics of antimicrobial peptides (SMAMPs) are known to disrupt cellular membranes in aqueous media. The impact of ionic strength, or the salt concentration, on membrane activity of these SMAMPs is an important issue since some AMPs are known to lose their activity at higher salt concentrations. In this report, the effect of salt concentration on membrane activity was evaluated using fluorescence dye leakage assays. Salt concentration did not affect the membrane activity of these SMAMPs significantly except for the 100% anionic vesicles (phosphatidylglycerol (PG) or phosphatidylserine (PS) only), where membrane activity decreased with increasing salt concentration. The results also indicated that the membrane activity of SMAMPs with monoamine side chains is independent on ionic strength against cardiolipin (CL) vesicles; however, SMAMPs containing bis- or trisamines exhibited salt concentration dependent membrane activity for CL liposomes.

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

Phenylene ethynylenes comprise a prototypical class of synthetic antimicrobial compounds that mimic antimicrobial peptides produced by eukaryotes and have broad-spectrum antimicrobial activity. We show unambiguously that bacterial membrane permeation by these antimicrobials depends on the presence of negative intrinsic curvature lipids, such as phosphatidylethanolamine (PE) lipids, found in high concentrations within bacterial membranes. Plate-killing assays indicate that a PE-knockout mutant strain of Escherichia coli drastically out-survives the wild type against the membrane-active phenylene ethynylene antimicrobials, whereas the opposite is true when challenged with traditional metabolic antibiotics. That the PE deletion is a lethal mutation in normative environments suggests that resistant bacterial strains do not evolve because a lethal mutation is required to gain immunity. PE lipids allow efficient generation of negative curvature required for the circumferential barrel of an induced membrane pore; an inverted hexagonal H II phase, which consists of arrays of water channels, is induced by a small number of antimicrobial molecules. The estimated antimicrobial occupation in these water channels is nonlinear and jumps from ≈1 to 3 per 4 nm of induced water channel length as the global antimicrobial concentration is increased. By comparing to exactly solvable 1D spin models for magnetic systems, we quantify the cooperativity of these antimicrobials.

Antimicrobial polynorbornenes composed of facially amphiphilic monomers have been previously reported to accurately emulate the antimicrobial activity of natural host-defense peptides (HDPs). The lethal mechanism of most HDPs involves binding to the membrane surface of bacteria leading to compromised phospholipid bilayers. In this paper, the interactions between biomimetic vesicle membranes and these cationic antimicrobial polynorbornenes are reported. Vesicle dye-leakage experiments were consistent with previous biological assays and corroborated a mode of action involving membrane disruption. Dynamic light scattering (DLS) showed that these antimicrobial polymers cause extensive aggregation of vesicles without complete bilayer disintegration as observed with surfactants that efficiently solubilize the membrane. Fluorescence microscopy on vesicles and bacterial cells also showed polymer-induced aggregation of both synthetic vesicles and bacterial cells. Isothermal titration calorimetry (ITC) afforded free energy of binding values (Delta G) and polymer to lipid binding ratios, plus revealed that the interaction is entropically favorable (Delta S0, Delta H0). It was observed that the strength of vesicle binding was similar between the active polymers while the binding stoichiometries were dramatically different.

In this study, amphiphilic polyoxanorbornene with different quaternary alkyl pyridinium side chains were synthesized. The biological efficiencies of these polymers, with various alkyl substituents, were determined by bacterial growth inhibition assays and hemolytic activity (HC 50 ) against human red blood cells (RBCs) to provide selectivity of these polymers for bacterial over mammalian cells. A series of polymers with different alkyl substituents (ethyl, butyl, hexyl, octyl, decyl and phenylethyl) and two different molecular weights (3 and 10 kDa) were prepared. The impact of alkyl chain length divided the biological activity into two different cases: those with an alkyl substituent containing four or fewer carbons had a minimum inhibitory concentration (MIC) of 200 μg. mL -1 and a HC 50 greater than 1650 μg. mL -1 , while those with six or more carbons had lower MICs < 12.5 μg. mL -1 and HC 50 ≤ 250 μg. mL -1 . Using MSI-78, the potent Magainin derivative which has an MIC=12.0 μg·mL -1 and HC 50 = 120 μg·mL -1 , as a comparison, the polymers with alkyl substituents ≤C 4 (four carbons) were not very potent, but did show selectivity values greater than or equal to MSI-78. In contrast, those with alkyl substituents ≥C 6 were as potent, or more potent, than MSI-78 and in three specific cases demonstrated selectivity values similar to, or better than, MSI-78. To understand if these polymers were membrane active, polymer induced lipid membrane disruption activities were evaluated by dye leakage experiments. Lipid composition and polymer hydrophobicity were found to be important factors for dye release.

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1–3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.

Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.

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.

Infectious disease is a critically important global healthcare issue. In the U.S. alone there are 2 million new cases of hospital-acquired infections annually leading to 90,000 deaths and 5 billion dollars of added healthcare costs. Couple these numbers with the appearance of new antibiotic resistant bacterial strains and the increasing occurrences of community-type outbreaks, and clearly this is an important problem. Our review attempts to bridge the research areas of natural host defense peptides (HDPs), a component of the innate immune system, and biocidal cationic polymers. Recently discovered peptidomimetics and other synthetic mimics of HDPs, that can be short oligomers as well as polymeric macromolecules, provide a unique link between these two areas. An emerging class of these mimics are the facially amphiphilic polymers that aim to emulate the physicochemical properties of HDPs but take advantage of the synthetic ease of polymers. These mimics have been designed with antimicrobial activity and, importantly, selectivity that rivals natural HDPs. In addition to providing some perspective on HDPs, selective mimics, and biocidal polymers, focus is given to the arsenal of biophysical techniques available to study their mode of action and interactions with phospholipid membranes. The issue of lipid type is highlighted and the important role of negative curvature lipids is illustrated. Finally, materials applications (for instance, in the development of permanently antibacterial surfaces) are discussed as this is an important part of controlling the spread of infectious disease.

The question of whether or not the surrounding lipid bilayer host contributes to structure and activity of included functional guests is a general topic of current scientific concern. We report that synthetic multifunctional pores are of use to address this elusive question, because the detection of their catalytic activity is membrane independent. According to their salt-rate profiles, unstable multifunctional supramolecules with permanent internal charges show highest membrane sensitivity, and the dependence of membrane sensitivity on the acidity of internal cations exceeds that on supramolecule stability. These results can, with all appropriate caution, be interpreted as indications for the existence of long-range EMP-ICR interactions (EMP: external membrane pressure, ICR: internal charge repulsion) between membrane hosts and functional guests that can, for instance, prevent the 'explosion' and promote the 'implosion' of over- and undercharged transmembrane barrel-stave supramolecules, respectively.

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 group of synthetic antimicrobial oligomers, inspired by naturally occurring antimicrobial peptides, were analyzed for the ability to modulate innate immune responses to Toll-like receptor (TLR) ligands. These synthetic mimics of antimicrobial peptides (SMAMPs) specifically reduced cytokine production in response toStaphylococcus aureusand theS. aureuscomponent lipoteichoic acid (LTA), a TLR2 agonist. Anti-inflammatory SMAMPs prevented the induction of tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-10 in response toS. aureusor LTA, but no other TLR2 ligands. We show that these SMAMPs bind specifically to LTAin vitroand prevent its interaction with TLR2. Importantly, the SMAMP greatly reduced the induction of TNF and IL-6in vivoin mice acutely infected withS. aureuswhile simultaneously reducing bacterial loads dramatically (4 log10). Thus, these SMAMPs can eliminate the damage induced by pathogen-associated molecular patterns (PAMPs) while simultaneously eliminating infectionin vivo. They are the first known SMAMPs to demonstrate anti-inflammatory and antibacterial activitiesin vivo.

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) and their synthetic analogs are of widespread interest. Here we report that guanidine rich small molecules can be potential membrane transporters in the presence of hydrophobic counteranion activators. To our knowledge, this is the first example of small molecules that mimic the anion-activated transport function of CPP.

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

Renee Miller RN, MSN, CPHRM, CIC, Infection Preventionist, Wellstar Windy Hill; Sarah Simmons MPH, Epidemiologist, Xenex Disinfection Services; Maria Rodriguez RN, BSN, Clinical Support Manager, Xenex Disinfection Services; Mark Stibich MHA, PhD, Chief Scientific Officer, Founder, Xenex Disinfection Services; Julie Stachowiak MIA, PhD, Chief Epidemiologist, Founder, Xenex Disinfection Services; Charles Dale BA, Science Intern, Xenex Disinfection Services

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.

Sum frequency generation (SFG) vibrational spectroscopy was used to study interactions between solid-supported lipid bilayers mimicking microbial and erythrocyte cellular membranes and synthetic antimicrobial arylamide oligomers named 2, 3, and 4, designed with the facial amphiphilicity common to naturally occurring antimicrobial peptides. The three compounds have the same backbone structure but varied side chains. The inherent interfacial sensitivity of SFG allowed for simultaneous monitoring of lipid ordering in the individual bilayer leaflets and orientation of 2, 3, and 4 upon interaction with the bilayer. Critical concentrations at which the inner leaflet is disrupted were determined for each oligomer. Spectral evidence of the oligomers' interaction with the bilayer below the critical concentrations was also found. Oligomers 2 and 3 tilted toward the bilayer surface normal, in agreement with previous experimental and simulation results. These oligomers selectively interact with microbial membrane models over erythrocyte membrane models, correlating well to previously published SFG studies on antimicrobial oligomer 1. It was shown that the oligomers interact with the lipid bilayers differently, indicating their different activity and selectivity. This research further shows that SFG is a particularly useful technique for the investigation of interaction mechanisms between cell membranes and membrane-active molecules. Additionally, SFG provides details of the specific interactions between these novel antimicrobials and lipid bilayers.

The current study is aimed at investigating the effect of fine-tuning the cationic character of synthetic mimics of antimicrobial peptides (SMAMPs) on the hemolytic and antibacterial activities. A series of novel norbornene monomers that carry one, two, or three Boc-protected amine functionalities was prepared. Ring-opening metathesis polymerization (ROMP) of the monomers, followed by deprotection of the amine groups resulted in cationic antimicrobial polynorbornenes that carry one, two, and three charges per monomer repeat unit. Increasing the number of amine groups on the most hydrophobic polymer reduced its hemolytic activity significantly. To understand the membrane activity of these polymers, we conducted dye leakage experiments on lipid vesicles that mimic bacteria and red blood cell membranes, and these results showed a strong correlation with the hemolysis data.

Stefan Matile and co-workers describe their recent results on synthetic multifunctional pores on p. 169. In Nature, pores are used to transport materials across cellular membranes. Synthetic pores, such as the one shown on the cover, can be used for molecular recognition, leading to applications such as hosting, sensing, or catalysis. The authors argue that demonstration of function is not enough to confirm that synthetic pores work as designed: structural studies and molecular modeling provide complementary, yet vital, information. This account summarizes five years of research devoted to the development of the concept of synthetic multifunctional pores. The objective is to complement a comprehensive graphical summary of molecular recognition with a survey of structural studies on the same topic. The relevance of the latter for research focusing on creation and application of supramolecular functional materials is discussed briefly in a subjective manner.

This account summarizes five years of research devoted to the development of the concept of synthetic multifunctional pores. The objective is to complement a comprehensive graphical summary of molecular recognition with a survey of structural studies on the same topic. The relevance of the latter for research focusing on creation and application of supramolecular functional materials is discussed briefly in a subjective manner.

We report that decreasing beta-sheet length in homologous multifunctional rigid-rod beta-barrels with internal histidines increases ion channel stability by three orders of magnitude, reduces binding activity by four orders of magnitude, and reduces esterase activity up to 22-times. These results are further used to evaluate methods employed to characterize suprastructure and activity of synthetic multifunctional pores formed by p-octiphenyl beta-barrels with emphasis on applicability of the Hille model to determine internal diameters and the Woodhull equation to locate internal active sites.

Studies on the usefulness of rigid-rod molecules to address pertinent questions of biological relevance are summarized. Emphasis is placed on (a) the supramolecular functional plasticity of p-octiphenyl ?-barrels expressed in molecular recognition (adaptable synthetic hosts), molecular translocation (adaptable synthetic ion channels) and molecular transformation (esterases, RNases), (b) molecular recognition of polarized membranes by rigid push-pull rods, as well as (c) the synthetic organic chemistry of rigid-rod molecules.

A family of synthetic mimic of antimicrobial peptides (SMAMP), amphiphilic meta-phenylene ethynylene (mPE) molecules show a wide range of antimicrobial activity and specificity. The interaction of a specifically active mPE molecule (AMO-2) with mixed DOPE/DOPG lipid was studied by solid-state NMR. The AMO-2 molecules do not preferentially interact more strongly with either lipid component, but rather are well dispersed in the lipid matrix. AMO-2 intimately interacts with all parts of lipid molecules, including head groups. Magic-angle spinning sideband analysis indicated that in samples with co-existing lamellar and inversed hexagonal phases (HII), neither lipid component aggregate in either phase. The presence of AMO-2 molecules causes dynamic disorder in lipid head groups, as evidenced by the broadening of both static and MAS 31P spectra. AMO-2 molecules do not massively transform the lamellar lipid into HII phase.