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.     

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.     

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.     

Incorporation of unprotected heterocyclic side chains into peptoid oligomers via solid-phase submonomer synthesis

Peptoids (N-substituted glycines) are an important class of biomimetic oligomers that have made a significant impact in the areas of combinatorial drug discovery, gene therapy, drug delivery, and biopolymer folding in recent years. Sequence-specific peptoid oligomers are easily assembled from primary amines by the solid-phase submonomer method. However, most amines that contain heterocyclic nitrogens in the side chain do not incorporate efficiently. We present here a straightforward revision of the submonomer method that allows efficient incorporation of unprotected imidazoles, pyridines, pyrazines, indoles, and quinolines into oligomers as long as 15 monomers in length. This improved method uses chloroacetic acid instead of bromoacetic acid in the acylation step of the monomer addition cycle, and allows for the incorporation of new side chains that should enable the synthesis of peptoids with entirely new properties.     

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.     

Conformational rearrangements by water-soluble peptoid foldamers

Peptoids are a family of N-substituted glycine oligomers that are capable of forming stable helical structures. We seek peptoid monomers that can establish a strong folding propensity in aqueous conditions. Here we utilize L-phenylalanine tert-butyl ester as a readily available reagent for the synthesis of (S)-N-(1-carboxy-2-phenylethyl)glycine oligomers. The products form stable secondary structures in aqueous solution in which the conformation is dramatically responsive to variations in pH and solvent composition.     

Development and use of an atomistic CHARMM‐based forcefield for peptoid simulation

Peptoids are positional isomers of peptides: peptoid sidechains are attached to backbone nitrogens rather than α‐carbons. Peptoids constitute a class of sequence‐specific polymers resistant to biological degradation and potentially as diverse, structurally and functionally, as proteins. While molecular simulation of proteins is commonplace, relatively few tools are available for peptoid simulation. Here, we present a first‐generation atomistic forcefield for peptoids. Our forcefield is based on the peptide forcefield CHARMM22, with key parameters tuned to match both experimental data and quantum mechanical calculations for two model peptoids (dimethylacetamide and a sarcosine dipeptoid). We used this forcefield to demonstrate that solvation of a dipeptoid substantially modifies the conformations it can access. We also simulated a crystal structure of a peptoid homotrimer, H‐(N‐2‐phenylethyl glycine)₃‐OH, and we show that experimentally observed structural and dynamical features of the crystal are accurately described by our forcefield. The forcefield presented here provides a starting point for future development of peptoid‐specific simulation methods within CHARMM. © 2013 Wiley Periodicals, Inc.     

Introduction of a triazole amino acid into a peptoid oligomer induces turn formation in aqueous solution

Peptoids are a non-natural class of oligomers that are composed of repeating N-substituted glycine units and are capable of folding into helices that mimic peptide structure and function. In this letter, we report the concise synthesis of a 1,5-substituted triazole amino acid (Tzl) and its subsequent incorporation into a short peptoid. The Tzl amino acid was shown to induce turn formation in aqueous solution, thus expanding the structural repertoire available to peptoid chemists.     

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.     

Cyclization of peptoids by formation of boronate esters

Introduction of conformational constraints into peptoids (N-substituted oligoglycines) will enable new applications in molecular recognition and self-assembly. Peptoids that contain both a phenylboronic acid side chain and a vicinal diol cyclize by intramolecular condensation to form boronate esters. A fluorescent indicator of free boronic acid was used to assay esterification. A galactose moiety 2 to 5 monomer units away from a boronic acid side chain in a peptoid reacts with the boronic acid in competition with the indicator. The intramolecular reaction predominates in each case, with 80-90% of the peptoid cyclized. When the diol is a simple 2,3-dihydroxypropyl group, esterification is less favored but still appreciable.     

Helical peptoid mimics of lung surfactant protein C

Among the families of peptidomimetic foldamers under development as novel biomaterials and therapeutics, poly-N-substituted glycines (peptoids) with alpha-chiral side chains are of particular interest for their ability to adopt stable, helical secondary structure in organic and aqueous solution. Here, we show that a peptoid 22-mer with a biomimetic sequence of side chains and an amphipathic, helical secondary structure acts as an excellent mimic of surfactant protein C (SP-C), a small protein that plays an important role in surfactant replacement therapy for the treatment of neonatal respiratory distress syndrome. When integrated into a lipid film, the helical peptoid SP mimic captures the essential surface-active behaviors of the natural protein. This work provides an example of how an abiological oligomer that closely mimics both the hydrophobic/polar sequence patterning and the fold of a natural protein can also mimic its biophysical function.     

Heterocyclic amines for the construction of peptoid oligomers bearing multi-dentate ligands

Peptoids are oligomers of N-substituted glycine that can be readily assembled using haloacetic acids and primary amines as synthons. Here, we report the synthesis and characterization of three new heterocyclic amines, 2-(2,2′:6′,2″-terpyridine-4′-yloxy)ethylamine, 2-(1,10-phenanthroline-5-yloxy)ethylamine and 8-hydroxy-2-quinolinemethylamine, and their incorporation into a series of different peptoid oligomer sequences. Since the heterocycles are all known to coordinate metal ions, the peptidomimetic products are designed to bind metal species with the potential for applications in catalysis and materials science.     

Extended peptoids: a new class of oligomers based on aromatic building blocks

Peptoids (N-substituted polyglycines) represent a class of bioinspired oligomers that have unique physical and structural properties. Here, we report the construction of ‘extended peptoids’ based on aromatic building blocks, in which the N-alkylaminoacetyl group of the peptoid backbone has been replaced by an N-alkylaminomethylbenzoyl spacer. Both meta- and para-bromomethylbenzoic acids were synthesized, providing access to a new class of peptoids. Further, inclusion of hydrophilic side chains confers water solubility to these compounds, showing that, like simple peptoids, extended peptoids add an extra dimension to synthetic poly-amide oligomers with potential application in a variety of biological contexts.     

Kinetics and equilibria of cis/trans isomerization of backbone amide bonds in peptoids

The biological activities of N-substituted glycine oligomers (peptoids) have been the subject of extensive research. As compared to peptides, both the cis and trans conformations of the backbone amide bonds of peptoids can be significantly populated. Thus, peptoids are mixtures of configurational isomers, with the number of isomers increasing by a factor of 2 with each additional monomer residue. Here we report the results of a study of the kinetics and equilibria of cis/trans isomerization of the amide bonds of N-acetylated peptoid monomers, dipeptoids, and tripeptoids by NMR spectroscopy. Resonance intensities indicate the cis conformation of the backbone amide bonds of the peptoids studied is more populated than is generally the case for the analogous secondary amide bond to proline residues in acyclic peptides. Rate constants were measured by inversion-magnetization transfer techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. The rate of cis/trans isomerization by rotation around the amide bonds in the peptoids studied is generally slower than that around amide bonds to proline residues and takes place on the NMR inversion-magnetization transfer time scale only by rotation around the amide bond to the C-terminal peptoid residue.     

Unique β-Turn Peptoid Structures and Their Application as Asymmetric Catalysts

Peptoids, N-substituted glycine oligomers, represent an important class of peptidomimetics that can fold into three-dimensional structures in solution. Most of the folded peptoid structures, however, resemble helices, and this can limit their applications, specifically in asymmetric catalysis. In this work, for the first time, unique examples of pyrrolidine-based β-turn-like peptoids are described and characterized, both in the solid state, by single-crystal X-ray analysis, and in solution, by circular dichroism spectroscopy. Furthermore, their highly efficient and enantioselective catalytic activity for the production of γ-nitro aldehydes by asymmetric Michael reaction in water was demonstrated. The structural properties and DFT-D3 calculations of the new β-turn-like peptoids, as well as catalytic and spectroscopic studies on designed pyrrolidine-based helical peptoids, suggest that the β-turn structure plays a key role in the stereoselectivity of the catalytic reaction.     

A threaded loop conformation adopted by a family of peptoid nonamers

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.     

Photoresponsive peptoid oligomers bearing azobenzene side chains

N-Substituted glycine peptoid oligomers were synthesized to incorporate a photoresponsive azobenzene side chain. The ability of this side chain to undergo reversible photoisomerization was established, and the cis- to trans-azobenzene thermal isomerization of this side chain was investigated. Circular dichroism studies indicated that trans- to cis-azobenzene isomerization does not significantly alter the backbone conformation in a series of peptoids thought to have well-defined structures.     

N-Naphthyl peptoid foldamers exhibiting atropisomerism

We introduce peptoid oligomers incorporating N-(1)-naphthyl glycine monomers. Axial chirality was established due to restricted rotation about the C-N(aryl) bond. Atropisomerism of both linear and cyclic peptoids was investigated by computational analysis, dynamic HPLC, and X-ray crystallographic studies.     

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.     

Hydrazone- and hydrazide-containing N-substituted glycines as peptoid surrogates for expedited library synthesis: application to the preparation of Tsg101-directed HIV-1 budding antagonists

Replacing the Pro6 in the p6(Gag)-derived 9-mer "P-E-P-T-A-P-P-E-E" with N-substituted glycine (NSG) residues is problematic. However, incorporation of hydrazone amides ("peptoid hydrazones") can be readily achieved in library fashion. Furthermore, reduction of these hydrazones to N-substituted "peptoid hydrazides" affords a facile route to library diversification. This approach is demonstrated by application to Tsg101-binding compounds designed as potential HIV budding antagonists. [reaction: see text]