Peptoid oligomers with alpha-chiral, aromatic side chains: sequence requirements for the formation of stable peptoid helices.

The achiral backbone of oligo-N-substituted glycines or "peptoids" lacks hydrogen-bond donors, effectively preventing formation of the regular, intrachain hydrogen bonds that stabilize peptide alpha-helical structures. Yet, when peptoids are N-substituted with alpha-chiral, aromatic side chains, oligomers with as few as five residues form stable, chiral, polyproline-like helices in either organic or aqueous solution. The adoption of chiral secondary structure in peptoid oligomers is primarily driven by the steric influence of these bulky, chiral side chains. Interestingly, peptoid helices of this class exhibit intense circular dichroism (CD) spectra that closely resemble those of peptide alpha-helices. Here, we have taken advantage of this distinctive spectroscopic signature to investigate sequence-related factors that favor and disfavor stable formation of peptoid helices of this class, through a comparison of more than 30 different heterooligomers with mixed chiral and achiral side chains. For this family of peptoids, we observe that a composition of at least 50% alpha-chiral, aromatic residues is necessary for the formation of stable helical structure in hexameric sequences. Moreover, both CD and 1H-13C HSQC NMR studies reveal that these short peptoid helices are stabilized by the placement of an alpha-chiral, aromatic residue on the carboxy terminus. Additional stabilization can be provided by the presence of an "aromatic face" on the helix, which can be patterned by positioning aromatic residues with three-fold periodicity in the sequence. Extending heterooligomer chain length beyond 12-15 residues minimizes the impact of the placement, but not the percentage, of alpha-chiral aromatic side chains on overall helical stability. In light of these new data, we discuss implications for the design of helical, biomimetic peptoids based on this structural motif.     

Structural and spectroscopic studies of peptoid oligomers with alpha-chiral aliphatic side chains

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.     

Simple, helical peptoid analogs of lung surfactant protein B

The helical, amphipathic surfactant protein, SP-B, is a critical element of pulmonary surfactant and hence is an important therapeutic molecule. However, it is difficult to isolate from natural sources in high purity. We have created and studied three different, nonnatural analogs of a bioactive SP-B fragment (SP-B(1-25)), using oligo-N-substituted glycines (peptoids) with simple, repetitive sequences designed to favor the formation of amphiphilic helices. For comparison, a peptide with a similar repetitive sequence previously shown to be a good SP mimic was also studied, along with SP-B(1-25) itself. Surface pressure-area isotherms, surfactant film phase morphology, and dynamic adsorption behavior all indicate that the peptoids are promising mimics of SP-B(1-25). The extent of biomimicry appears to correlate with peptoid helicity and lipophilicity. These biostable oligomers could serve in a synthetic surfactant replacement to treat respiratory distress syndrome.     

Tuning peptoid secondary structure with pentafluoroaromatic functionality: a new design paradigm for the construction of discretely folded peptoid structures

Peptoids, or oligomers of N-substituted glycine, are an important class of non-native polymers whose close structural similarity to natural alpha-peptides and ease of synthesis offer significant advantages for the study of biomolecular interactions and the development of biomimetics. Peptoids that are N-substituted with alpha-chiral aromatic side chains have been shown to adopt either helical or "threaded loop" conformations, depending upon solvent and oligomer length. Elucidation of the factors that impact peptoid conformation is essential for the development of general rules for the design of peptoids with discrete and novel structures. Here, we report the first study of the effects of pentafluoroaromatic functionality on the conformational profiles of peptoids. This work was enabled by the synthesis of a new, alpha-chiral amine building block, (S)-1-(pentafluorophenyl)ethylamine (S-2), which was found to be highly compatible with peptoid synthesis (delivering (S)-N-(1-(pentafluorophenyl)ethyl)glycine oligomers). The incorporation of this fluorinated monomer unit allowed us to probe both the potential for pi-stacking interactions along the faces of peptoid helices and the role of side chain electrostatics in peptoid folding. A series of homo- and heteropeptoids derived from S-2 and non-fluorinated, alpha-chiral aromatic amide side chains were synthesized and characterized by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Enhancement of pi-stacking by quadrupolar interactions did not appear to play a significant role in stabilizing the conformations of heteropeptoids with alternating fluorinated and non-fluorinated side chains. However, incorporation of (S)-N-(1-(pentafluorophenyl)ethyl)glycine monomers enforced helicity in peptoids that typically exhibit threaded loop conformations. Moreover, we found that the incorporation of a single (S)-N-(1-(pentafluorophenyl)ethyl)glycine monomer could be used to selectively promote looped or helical structure in this important peptoid class by tuning the electronics of nearby heteroatoms. The strategic installation of this monomer unit represents a new approach for the manipulation of canonical peptoid structure and the construction of novel peptoid architectures.     

Extraordinarily robust polyproline type I peptoid helices generated via the incorporation of α-chiral aromatic N-1-naphthylethyl side chains

Peptoids, or oligomers of N-substituted glycines, are a class of foldamers that have shown extraordinary functional potential since their inception nearly two decades ago. However, the generation of well-defined peptoid secondary structures remains a difficult task. This challenge is due, in part, to the lack of a thorough understanding of peptoid sequence-structure relationships and, consequently, an incomplete understanding of the peptoid folding process. We seek to delineate sequence-structure relationships through the systematic study of noncovalent interactions in peptoids and the design of novel amide side chains capable of such interactions. Herein, we report the synthesis and detailed structural analysis of a series of (S)-N-(1-naphthylethyl)glycine (Ns1npe) peptoid homo-oligomers by X-ray crystallography, NMR spectroscopy, and circular dichroism (CD) spectroscopy. Four of these peptoids were found to adopt well-defined structures in the solid state, with dihedral angles similar to those observed in polyproline type I (PPI) peptide helices and in peptoids with α-chiral side chains. The X-ray crystal structure of a representative Ns1npe tetramer revealed an all cis-amide helix, with approximately three residues per turn, and a helical pitch of approximately 6.0 ?. 2D-NMR analysis of the length-dependent Ns1npe series showed that these peptoids have very high overall backbone amide K(cis/trans) values in acetonitrile, indicative of conformationally homogeneous structures in solution. Additionally, CD spectroscopy studies of the Ns1npe homo-oligomers in acetonitrile and methanol revealed a striking length-dependent increase in ellipticity per amide. These Ns1npe helices represent the most robust peptoid helices to be reported, and the incorporation of (S)-N-(1-naphthylethyl)glycines provides a new approach for the generation of stable helical structure in this important class of foldamers.     

Toward the synthesis of artificial proteins: the discovery of an amphiphilic helical peptoid assembly

While nature exploits folded biopolymers to achieve molecular recognition and catalysis, comparable abiological heteropolymer systems have been difficult to create. We synthesized and identified abiological peptoid heteroploymers capable of binding a dye. Using combinatorial synthesis, we constructed a library of 3400 amphiphilic 15-mer peptoids on an ultra-high-capacity beaded support. Individual macrobeads, each containing a single peptoid sequence, were arrayed into plates, cleaved, and screened in aqueous solution to locate dye binding heteropolymer assemblies. Resynthesis and characterization demonstrated the formation of defined helical assemblies as judged by size-exclusion chromatography, circular dichroism, and analytical ultracentrifugation. Inspired by nature's process of sequence variation and natural selection, we identified rare abiological sequence-specific heteropolymers that begin to mimic the structure and functional properties of their biological counterparts.     

Peptoid oligomers with alpha-chiral, aromatic side chains: effects of chain length on secondary structure.

Oligomeric N-substituted glycines or "peptoids" with alpha-chiral, aromatic side chains can adopt stable helices in organic or aqueous solution, despite their lack of backbone chirality and their inability to form intrachain hydrogen bonds. Helical ordering appears to be stabilized by avoidance of steric clash as well as by electrostatic repulsion between backbone carbonyls and pi clouds of aromatic rings in the side chains. Interestingly, these peptoid helices exhibit intense circular dichroism (CD) spectra that closely resemble those of peptide alpha-helices. Here, we have utilized CD to systematically study the effects of oligomer length, concentration, and temperature on the chiral secondary structure of organosoluble peptoid homooligomers ranging from 3 to 20 (R)-N-(1-phenylethyl)glycine (Nrpe) monomers in length. We find that a striking evolution in CD spectral features occurs for Nrpe oligomers between 4 and 12 residues in length, which we attribute to a chain length-dependent population of alternate structured conformers having cis versus trans amide bonds. No significant changes are observed in CD spectra of oligomers between 13 and 20 monomers in length, suggesting a minimal chain length of about 13 residues for the formation of stable poly(Nrpe) helices. Moreover, no dependence of circular dichroism on concentration is observed for an Nrpe hexamer, providing evidence that these helices remain monomeric in solution. In light of these new data, we discuss chain length-related factors that stabilize organosoluble peptoid helices of this class, which are important for the design of helical, biomimetic peptoids sharing this structural motif.     

A CGenFF-based force field for simulations of peptoids with both cis and trans peptide bonds

Peptoids, or poly-n-substituted glycines, are peptide-like polymers composed of a flexible backbone decorated with diverse chemical side chains. Peptoids can form a variety of self-assembling structures based on the type and sequence of the side chains attached to their backbones. All-atom molecular dynamics simulations have been useful in predicting the conformational structures of proteins and will be valuable tools for identifying combinations of peptoid side chains that may form interesting folded structures. However, peptoid models must address a major degree of freedom not common in proteins – the cis/trans isomerization of the peptide bond. This work presents CHARMM general force field (CGenFF) parameters developed to accurately represent peptoid conformational behavior, with an emphasis on a correct representation of both the cis and trans isomers of the peptoid backbone. These parameters are validated against experimental and quantum mechanics data and used to simulate three peptoid side chains in explicitly solvated systems. © 2019 Wiley Periodicals, Inc.     

A Multifaceted Secondary Structure Mimic Based On Piperidine‐piperidinones

Minimalist secondary structure mimics are typically made to resemble one interface in a protein–protein interaction (PPI), and thus perturb it. We recently proposed suitable chemotypes can be matched with interface regions directly, without regard for secondary structures. Here we describe a modular synthesis of a new chemotype 1 , simulation of its solution‐state conformational ensemble, and correlation of that with ideal secondary structures and real interface regions in PPIs. Scaffold 1 presents amino acid side‐chains that are quite separated from each other, in orientations that closely resemble ideal sheet or helical structures, similar non‐ideal structures at PPI interfaces, and regions of other PPI interfaces where the mimic conformation does not resemble any secondary structure. 68 different PPIs where conformations of 1 matched well were identified. A new method is also presented to determine the relevance of a minimalist mimic crystal structure to its solution conformations. Thus dld ‐ 1 faf crystallized in a conformation that is estimated to be 0.91 kcal mol^?1 above the minimum energy solution state.     

Probing the structural requirements of peptoids that inhibit HDM2-p53 interactions

Many cellular processes are controlled by protein-protein interactions, and selective inhibition of these interactions could lead to the development of new therapies for several diseases. In the area of cancer, overexpression of the protein, human double minute 2 (HDM2), which binds to and inactivates the protein p53, has been linked to tumor aggressiveness and drug resistance. In general, inhibition of protein-protein interactions with synthetic molecules is challenging and currently remains a largely uncharted area for drug development. One strategy to create inhibitors of protein-protein interactions is to recreate the three-dimensional arrangement of side chains that are involved in the binding of one protein to another, using a nonnatural scaffold as the attachment point for the side chains. In this study, we used oligomeric peptoids as the scaffold to begin to develop a general strategy in which we could rationally design synthetic molecules that can be optimized for inhibition of protein-protein interactions. Structural information on the HDM2-p53 complex was used to design our first class of peptoid inhibitors, and we provide here, in detail, the strategy to modify peptoids with the appropriate side chains that are effective inhibitors of HDM2-p53 binding. While we initially tried to develop rigid, helical peptoids as HDM2 binders, the best inhibitors were surprisingly peptoids that lacked any helix-promoting groups. These results indicate that starting with rigid peptoid scaffolds may not always be optimal to develop new inhibitors.     

Fit to be tied: conformation-directed macrocyclization of peptoid foldamers

Covalent macrocyclic constraints can be readily installed on N-substituted glycine "peptoid" oligomer substrates. Cu(I)-catalyzed [3+2] cycloaddition reactions were conducted on solid support to ligate peptoid side chain azide and alkyne functionalities. Intramolecular macrocycle formation is facilitated by preorganizing the reactive groups across one turn of the helical secondary structure. These results confirm that conformational ordering can be exploited to assist the macrocyclization of folded oligomers.     

Synthesis and Characterization of a New Class of Oligomers with Nanosized Interior Cavities

Currently there is an intense interest in developing unnatural folding oligomers and polymers that may mimic or rival natural proteins^[1]. Most of the efforts in this field have been focused on designing helical structures that mimic the natural α-helix^[2], the best understood protein secondary structure.     

Stable helical peptoids via covalent side chain to side chain cyclization

Peptoids are oligomeric N-substituted glycines with potential as biologically relevant compounds. Helical peptoids provide an attractive fold for the generation of protein-protein interaction inhibitors. The generation of helical peptoid folds in organic and aqueous media has been limited to strict design rules, as peptoid-folding is mainly directed via the steric direction of alpha-chiral side-chains. Here a new methodology is presented to induce helical folds in peptoids with the aid of side chain to side chain cyclization. Cyclic peptoids were generated via solid-phase synthesis and their folding was studied. The cyclization induces significant helicity in peptoids in organic media, aids the folding in aqueous media, and requires the incorporation of only relatively few chiral aromatic side chains.     

Peptidic molecular brushes with enhanced chirality

Tethering oligopeptides through one end densely packed onto a linear polymer main chain will greatly reduce freedom of the peptide chains, which affords an easy access to investigate the secondary structure of peptides under constrained condition. Herein, molecular brushes with densely grafted monodispersed Cbz‐protected oligolysine were efficiently synthesized via free radical polymerization of the macromonomer‐bearing lysine octamer, and the secondary structures of the oligopeptide side chains in solutions were investigated. To examine the architecture effects on helical conformation, circular dichroism spectra from the polymer were compared with that from the corresponding macromonomer. To check the chemical structural effects on conformation of the oligopeptide, Cbz groups from the molecular brushes were deprotected, and the secondary structures of the polymers were compared before and after the deprotection. Conformation of the deprotected polymer was further explored by varying solution pH values. Complexation of the positively charged, deprotected polymer with anionic surfactant provides an alternative route to mediate the secondary structures of the short peptides in the constrained environment. It has been found that oligolysine side chains within the molecular brushes can adopt enhanced α‐helical conformation through the crowding structures or can form β‐sheet by hydrophobic interactions between the complexed surfactants. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Oxime Side‐Chain Cross‐Links in an α‐Helical Coiled‐Coil Protein: Structure,Thermodynamics, and Folding‐Templated Synthesis of Bicyclic Species

Covalent side‐chain cross‐links are a versatile method to control peptide folding, particularly when α‐helical secondary structure is the target. Here, we examine the application of oxime bridges, formed by the chemoselective reaction between aminooxy and aldehyde side chains, for the stabilization of a helical peptide involved in a protein–protein complex. A series of sequence variants of the dimeric coiled coil GCN4‐p1 bearing oxime bridges at solvent‐exposed positions were prepared and biophysically characterized. Triggered unmasking of a side‐chain aldehyde in situ and subsequent cyclization proceed rapidly and cleanly at pH 7 in the folded protein complex. Comparison of folding thermodynamics among a series of different oxime bridges show that the cross links are consistently stabilizing to the coiled coil, with the extent of stabilization sensitive to the exact size and structure of the macrocycle. X‐ray crystallographic analysis of a coiled coil with the best cross link in place and a second structure of its linear precursor show how the bridge is accommodated into an α‐helix. Preparation of a bicyclic oligomer by simultaneous formation of two linkages in situ demonstrates the potential use of triggered oxime formation to both trap and stabilize a particular peptide folded conformation in the bound state.     

Mimicking helical antibacterial peptides with nonpeptidic folding oligomers

Unnatural oligomeric scaffolds designed to adopt defined secondary structures (e.g., helices), while retaining the chemical diversity of amino acid side chains, are of practical value to elaborate functional mimetics of bioactive alpha-polypeptides. Enantiopure N,N'-linked oligoureas as short as seven residues long have been previously shown to fold into a stable helical structure, stabilized by 12- and 14-membered H-bonded rings. We now report that eight-residue oligoureas designed to mimic globally amphiphilic alpha-helical host-defense peptides are effective against both gram-negative and gram-positive bacteria (including methicillin-resistant Staphylococcus aureus [MRSA]) and exhibit selectivity for bacterial versus mammalian cells. Circular dichroism (CD) spectroscopy studies suggest enhanced helical propensity of oligoureas in the presence of phospholipid vesicles. The utility of this new class of nonpeptidic foldamers for biological applications is highlighted by high resistance to proteolytic degradation.     

Alpha- and beta-polypeptides show a different stability of helical secondary structure

β-Polypeptides are known to adopt helical secondary structure in organic solvents, even for rather short chain lengths. It is investigated whether a short α-polypeptide with amino-acid side chains that enable β-peptides to adopt helical structures, can maintain or adopt stable helical structure in methanol or in water. The molecular dynamics simulations do not predict a particular fold, which indicates an essential role for the additional methylene moiety in the backbone of β-peptides regarding helix stability.     

Secondary and Tertiary Structure of Polysaccharides in Solutions and Gels

Many polysaccharide chains can adopt ordered helical and ribbon-like secondary structures. It seems however that these chains are often so stiff and extended that the cooperative interactions necessary for stability in the solvent environment can only be achieved when inter-chain as well as intra-chain interactions are favorable. Hence we commonly find two-or more-stranded associations of helices, of ribbons, or of helices with ribbons. These can be regarded as tertiary and higher levels of structure. The ordered secondary structure characteristically requires a regular repeating sequence of sugar residues, and the termination of this sequence by insertion of a residue of different type may also terminate the secondary structure and hence the association in which it is involved. This is the mechanism by which native polysaccharides may link up to form three dimensional networks, or gels, in which state they perform their natural roles in maintaining the hydration and integrity of biological tissues. For several polysaccharides there is evidence that the mechanism of biological control over the fine topology and properties of the gel network is mediated by enzymes which modify sugar residues at the polymer level to change the pattern of “interrupting” sugar residues.     

New peptidomimetic polymers for antifouling surfaces

Exposure of therapeutic and diagnostic medical devices to biological fluids is often accompanied by interfacial adsorption of proteins, cells, and microorganisms. Biofouling of surfaces can lead to compromised device performance or increased cost and in some cases may be life-threatening to the patient. Although numerous antifouling polymer coatings have enjoyed short-term success in preventing protein and cell adsorption on surfaces, none have proven ideal for conferring long-term biofouling resistance. Here we describe a new biomimetic antifouling N-substituted glycine polymer (peptoid) containing a C-terminal peptide anchor derived from residues found in mussel adhesive proteins for robust attachment of the polymer onto surfaces. The methoxyethyl side chain of the peptoid portion of the polymer was chosen for its chemical resemblance to the repeat unit of the known antifouling polymer poly(ethylene glycol) (PEG), whereas the composition of the 5-mer anchoring peptide was chosen to directly mimic the DOPA- and Lys-rich sequence of a known mussel adhesive protein. Surfaces modified with this biomimetic peptide-peptoid conjugate exhibited dramatic reduction of serum protein adsorption and resistance to mammalian cell attachment for over 5 months in an in vitro assay. These new synthetic peptide based antifouling polymers may provide long-term control of surface biofouling in the physiologic, marine, and industrial environments.     

Sequence and Structure of Peptoid Oligomers Can Tune the Photoluminescence of an Embedded Ruthenium Dye

The understanding of structure–function relationships within synthetic biomimetic systems is a fundamental challenge in chemistry. Herein we report the direct correlation between the structure of short peptoid ligands—N-substituted glycine oligomers incorporating 2,2′-bipyridine groups—varied in their monomer sequence, and the photoluminescence of Ru^II centers coordinated by these ligands. Based on circular dichroism and fluorescence spectroscopy we demonstrate that while helical peptoids do not affect the fluorescence of the embedded Ru^II chromophore, unstructured peptoids lead to its significant decay. Transmittance electron microscopy (TEM) revealed significant differences in the arrangements of metal-bound helical versus unstructured peptoids, suggesting that only the latter can have through-space interactions with the ruthenium dye leading to its quenching. High-resolution TEM enabled the remarkable direct imaging of singular ruthenium-bound peptoids and bundles, supporting our explanation for structure-depended quenching. Moreover, this correlation allowed us to fine-tune the luminescence properties of the complexes simply by modifying the sequence of their peptoid ligands. Finally, we also describe the chiral properties of these Ru–peptoids and demonstrate that remote chiral induction from the peptoids backbone to the ruthenium center is only possible when the peptoids are both chiral and helical.