Antimicrobial peptides (AMPs) and their mimics are emerging as promising antibiotic agents. We present a library of ''ampetoids'' (antimicrobial peptoid oligomers) with helical structures and biomimetic sequences, several members of which have low-micromolar antimicrobial activities, similar to cationic AMPs like pexiganan. Broad-spectrum activity against six clinically relevant BSL2 pathogens is also shown. This comprehensive structure-activity relationship study, including circular dichroism spectroscopy, minimum inhibitory concentration assays, hemolysis and mammalian cell toxicity studies, and specular x-ray reflectivity measurements shows that the in vitro activities of ampetoids are strikingly similar to those of AMPs themselves, suggesting a strong mechanistic analogy. The ampetoids' antibacterial activity, coupled with their low cytotoxicity against mammalian cells, make them a promising class of antimicrobials for biomedical applications. Peptoids are biostable, with a protease-resistant N-substituted glycine backbone, and their sequences are highly tunable, because an extensive diversity of side chains can be incorporated via facile solid-phase synthesis. Our findings add to the growing evidence that nonnatural foldamers will emerge as an important class of therapeutics.antibiotics ͉ peptidomimetics ͉ structure-activity studies N atural antimicrobial peptides (AMPs) defend a wide array of organisms against bacterial pathogens and show potential as supplements for or replacements of conventional antibiotics, because few bacteria have evolved resistance to them (1-3). Many AMPs kill bacteria by permeabilization of the cytoplasmic membrane, causing depolarization, leakage, and death (4), whereas others target additional anionic bacterial constituents (e.g., DNA, RNA, or cell wall components) (2, 5). Amphipathic secondary structures in which residues are segregated into hydrophobic and cationic regions ( Fig. 1 A and B) are the hallmark of most AMPs (6). Regardless of their final target of killing, AMPs must interact with the bacterial cytoplasmic membrane, and their amphipathicity is integral to such interactions (1, 2, 7). Additionally, their cationic nature imparts AMPs with some measure of selectivity, because mammalian cell membranes are largely zwitterionic. The precise nature of AMP-membrane interactions remains controversial and actively debated; a variety of mechanisms have been proposed, including the carpet (4), barrel-stave pore (4), toroidal pore (8), and aggregate (9) models. Nevertheless, a considerable number of structure-activity investigations have elucidated how the physicochemical properties of these molecules relate to their biological activities (4,7,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).Although AMPs have been actively studied for decades (23-25), they have yet to see widespread clinical use (1). This is due in part to the vulnerability of many peptide therapeutics to rapid in vivo degradation, which dramatically reduces their bioavailability. Nonnatural mimics of AMPs...

A series of peptoid oligomers were designed as helical, cationic, and facially amphipathic mimics of the magainin-2 amide antibacterial peptide. We used circular dichroism spectroscopy to determine the conformation of these peptoids in aqueous buffer and in the presence of bacterial membrane-mimetic lipid vesicles, composed of a 7:3 mol ratio of POPE:POPG. We found that certain peptoids, which displayed characteristically helical CD in buffer and lipid vesicles, exhibit selective (nonhemolytic) and potent antibacterial activity against both Gram-positive and Gram-negative bacteria. In contrast, peptoids that exhibit weak CD, reminiscent of that of a peptide random coil, were ineffective antibiotics. In a manner similar to the natural magainin peptides, we find a correlation between peptoid lipophilicity and hemolytic propensity. We observe that a minimum length of approximately 12 peptoid residues may be required for antibacterial activity. We also see evidence that a helix length between 24 and 34 A may provide optimal antibacterial efficacy. These results provide the first example of a water-soluble, structured, bioactive peptoid.

Substantial progress has been made in the synthesis and characterization of various oligomeric molecules capable of autonomous folding to well-defined, repetitive secondary structures. It is now possible to investigate sequence-structure relationships and the driving forces for folding in these systems. Here, we present detailed analysis by X-ray crystallography, NMR, and circular dichroism (CD) of the helical structures formed by N-substituted glycine (or "peptoid") oligomers with alpha-chiral, aliphatic side chains. The X-ray crystal structure of a N-(1-cyclohexylethyl)glycine pentamer, the first reported for any peptoid, shows a helix with cis-amide bonds, approximately 3 residues per turn, and a pitch of approximately 6.7 A. The backbone dihedral angles of this pentamer are similar to those of a polyproline type I peptide helix, in agreement with prior modeling predictions. This crystal structure likely represents the major solution conformers, since the CD spectra of analogous peptoid hexamers, dodecamers, and pentadecamers, composed entirely of either (S)-N-(1-cyclohexylethyl)glycine or (S)-N-(sec-butyl)glycine monomers, also have features similar to those of the polyproline type I helix. Furthermore, this crystal structure is similar to a solution NMR structure previously described for a peptoid pentamer comprised of chiral, aromatic side chains, which suggests that peptoids containing either aromatic or aliphatic alpha-chiral side chains adopt fundamentally similar helical structures in solution, despite distinct CD spectra. The elucidation of detailed structural information for peptoid helices with alpha-chiral aliphatic side chains will facilitate the mimicry of biomolecules, such as transmembrane protein domains, in a distinctly stable form.

Non-natural polymers with well-defined three-dimensional folds offer considerable potential for engineering novel functions that are outside the scope of biological polymers. Here we describe a family of N-substituted glycine or "peptoid" nonamers that folds into an unusual "threaded loop" structure of exceptional thermal stability and conformational homogeneity in acetonitrile. The structure is chain-length-specific and relies on bulky, chiral side chains and chain-terminating functional groups for stability. Notable elements of the structure include the engagement of the positively charged amino terminus by carbonyl groups of the backbone through hydrogen bonding interactions and shielding of polar groups from and near-complete exposure of hydrophobic groups to solvent, in a manner resembling a folded polypeptide globular domain turned inside-out. The structure is stable in a variety of organic solvents but is readily denatured in any solvent/cosolvent milieu with hydrogen bonding potential. The structure could serve as a scaffold for the elaboration of novel functions and could be used to test methodologies for predicting solvent-dependent polymer folding.