Peptoid microsphere coatings: The effects of helicity,temperature, pH,and ionic strength

Peptoids are peptidomimetic oligomers that predominantly harness similarities to peptides for biomimetic functionality. They have potential for use in biomedical applications and biosensors due to resistance to proteolytic degradation and low immunogenicity. The incorporation of chiral, aromatic side chains in the peptoid sequence allows for the formation of distinct secondary structures and self-assembly into supramolecular assemblies, including microspheres. Peptoid microspheres can be coated onto substrates for potential use in biosensor technologies, tissue engineering platforms, and drug-delivery systems. In order to be useful for these applications, the peptoid coatings must be robust under physiological conditions. In this study, we report the effects of various conditions on the peptoid microsphere coatings, including (i) helicity, (ii) temperature, (iii) pH, and (iv) ionic strength. These studies show that microsphere size decreases with increasing peptoid helicity and the positively charged side chains are positioned on the outside of the microspheres. The peptoid microsphere coatings are robust under physiological conditions but degrade in acidic conditions (pH < 7) and at low ionic strengths (<150 μM).     

Use of the environmentally sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide to study peptoid helix structures

Peptoids, oligomers of N-substituted glycine, have been valuable targets for study and diverse application as peptidomimetics and as nanomaterials. Their conformational heterogeneity has made the study of peptoid structures using high-resolution analyses challenging, limiting our understanding of the physiochemical features that mediate peptoid folding. Here, we introduce a new method for the study of peptoid structure that relies on the environmentally sensitive fluorescence properties of 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN). We have prepared a 4-DMN-functionalized primary amine that is compatible with the traditional submonomer peptoid synthesis methods and incorporated it sequence-specifically into 11 of 13 new peptoids. When included as a peptoid side chain modification, the fluorescence emission intensity of 4-DMN correlates with predictions of the fluorophore's local polarity within a putative structure. 4-DMN fluorescence is maximized when the fluorophore is placed in the middle of the hydrophobic face of an amphiphilic helical peptoid. When the fluorophore is placed near the peptoid terminus or on a polar face of an amphiphilic sequence, 4-DMN fluorescence is diminished. Disruption of the peptoid secondary structure or amphiphilicity also modulates 4-DMN fluorescence. The peptoids' helical secondary structures are moderately disrupted by inclusion of a 4-DMN-modified side chain as evaluated by changes in the peptoids' CD spectral features. This new method for peptoid structure evaluation should be a valuable complement to existing peptoid structural analysis tools.     

Transformation of the amyloidogenic peptide amylin(20-29) into its corresponding peptoid and retropeptoid: access to both an amyloid inhibitor and template for self-assembled supramolecular tapes

The highly amyloidogenic peptide sequence of amylin(20-29) was transformed into its corresponding peptoid and retropeptoid sequences to design a novel class of beta-sheet breaker peptides as amyloid inhibitors. This report describes the synthesis of the chiral peptoid building block of L-isoleucine, the solid phase synthesis of the peptoid and retropeptoid sequences of amylin(20-29), and the structural analysis of these amylin derivatives in solution by infrared spectroscopy, circular dichroism, and transmission electron microscopy. It was found that the peptoid sequence did not form amyloid fibrils or any other secondary structures and was able to inhibit amyloid formation of native amylin(20-29). Although the retropeptoid did not form amyloid fibrils it had only modest amyloid inhibitor properties since supramolecular tapes were formed.     

A VEGFR2 Antagonist and Other Peptoids Evade Immune Recognition

We have recently reported a peptoid (N-alkyl-oligoglycine) molecule that binds to the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) with high affinity and specificity. Moreover, this peptoid is capable of inhibiting VEGFR2 function in vivo (Udugamasooriya et al. J Am Chem Soc 130:5744–5745, 2008) and thus is a lead compound for anti-angiogenic agents. Moreover, the assay developed to identify this VEGFR2 inhibitor is likely to be a general route to peptoid antagonists or agonists of integral membrane receptors. Therefore, it is important to determine whether the VEGFR2-targeted peptoid, and indeed peptoids in general, are inherently immunogenic since an anti-peptoid immune response would significantly complicate their development as therapeutic candidates. In this study, the VEGFR2-targeted peptoid as well as other peptoids of varying lengths were injected into mice along with an immunostimulatory agent. We demonstrate that no significant anti-peptoid immune response is induced. It is further shown that this is not a trivial result of the lack of immunogenicity of a particular peptoid sequence, since conjugation of the peptoids to carrier proteins such as KLH prior to injection induces a robust anti-peptoid immune response. We conclude that free peptoid molecules are not immunogenic, probably due to a lack of T cell epitopes and that peptoid-based therapeutics are therefore not likely to be hindered by anti-peptoid antibody production in most cases.     

Aqueous solubility and membrane interactions of hydrophobic peptides with peptoid tags

Lysine tagging of hydrophobic peptides of parent sequence KKAAALAAAAALAAWAALAAAKKKK-NH(2) has been shown to facilitate their synthesis and purification through water solubilization, yet not impact on the intrinsic properties of the hydrophobic core sequence with respect to its insertion into membranes in an alpha-helical conformation. However, due to their positively charged character, such peptides often become bound to phospholipid head groups in membrane surfaces, which inhibits their transbilayer insertion and/or prevents their transport across cellular bilayers. We sought to develop more neutral peptides of membrane-permeable character by replacing most Lys residues with uncharged peptoid [N-(R)glycyl] residues, which might similarly confer water solubility while retaining membrane-interactive properties of the hydrophobic core. Several "peptoid-tagged" derivatives of the parent peptide were prepared with varying peptoid content, with five of the six Lys residues replaced with peptoids Nala and/or Nval. Conformations of these peptides measured by circular dichroism spectroscopy demonstrated that these water-soluble peptides retain the alpha-helix structure in micelles (lysophosphatidylcholine and sodium dodecyl sulfate) notwithstanding the known helix-breaking capacity of the peptoid tags. Blue shifts in Trp fluorescence spectra and quenching experiments with acrylamide confirmed that peptoid-tagged peptides insert spontaneously into micellar membranes. Results suggest that upon introduction of uncharged tags, the interaction between the membrane and the peptides is dominated by the hydrophobicity of the peptide core rather than the electrostatic interactions between the Lys and the head groups of the lipids. The overall findings indicate that peptoid residues are effective surrogates for Lys as uncharged water-solubilizing tags and, as such, provide a potentially valuable feature of design of membrane-interactive peptides.     

Effect of side chain hydrophobicity and cationic charge on antimicrobial activity and cytotoxicity of helical peptoids

Peptoids are peptidomimetic polymers that are resistant to proteolysis and less prone to immune responses; thus, they can provide a practical alternative to peptides. Among the various therapeutic applications that have been explored, cationic amphipathic peptoids have demonstrated broad-spectrum antibacterial activity, including activity towards drug-resistant bacterial strains. While their potency and activity spectrum can be manipulated by sequence variations, bacterial selectivity and systemic toxicity need to be improved for further clinical development. To this aim, we incorporated various hydrophobic or cationic residues to improve the selectivity of the previously developed antibacterial peptoid 1. The analogs with hydrophobic residues demonstrated non-specific cytotoxicity, while those with an additional cationic residue showed improved selectivity and comparable antibacterial activity. Specifically, compared to 1, peptoid 7 showed much lower hemolysis and cytotoxicity, while maintaining the antibacterial activity. Therefore, we believe that peptoid 7?has the potential to serve as a promising alternative to current antimicrobial therapies.     

D-Ala2]-Deltorphin I peptoid and retropeptoid analogues: synthesis, biological activity and conformational investigations.

The synthesis is described of a [D-Ala2]-deltorphin I peptoid analogue in which all amino acid residues have been substituted by the corresponding N-alkylglycine residues. The [D-Ala2]-deltorphin I retropeptoid was also prepared as well as [Ala1 ,D-Ala2]-deltorphin 1 and the corresponding peptoid. Structural investigations by FT-IR and fluorescence measurements were carried out on the synthetic analogues and on some [D-Ala2]-deltorphin 1 peptide-peptoid hybrids previously prepared. According to the fluorescence measurements the distance between the aromatic residues in the deltorphin I peptoid and retropeptoid is similar to that suggested for the delta- and micro-opioids, respectively. Measurements of CD in the presence of beta-cyclodextrin, and some preliminary pharmacological experiments were also performed. No dichroic bands are present in the spectrum of the [Ntyr1,D-Ala2]-deltorphin I, but an increasing dichroic effect appears in the spectra of both the deltorphin I peptoid and retropeptoid. Activity tests on isolated organ preparations showed that the modifications made produced a dramatic decrease in the agonistic activity of the synthetic derivatives.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Triple helical stabilities of guest-host collagen mimetic structures

The peptoid Nleu (N-isobutylglycine) has been successfully incorporated into a series of collagen mimetics composed of Gly-Pro-Nleu and Gly-Nleu-Pro sequences and has been able to maintain triple helices in appropriate structures. The achiral trimeric sequence Gly-Nleu-Nleu as a guest sequence in structures such as Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2 retains triple helicity. As an extension of this study, we report, in this paper, on a series of guest-host collagen mimetic structures in which Gly-Nleu-Pro sequences are employed as the host. The guest sequences for these guest-host structures include Gly-Nleu-Nleu and Gly-Nx-Pro sequences where Nx is composed of a variety of alkyl and aralkyl peptoid residues. From these guest-host collagen mimetic structures, we are able to elucidate the contributions of hydrophobic and steric effects on triple helix formation. The Gly-Nleu-Pro sequences have been shown to be effective in inducing triple helicity. Conformational characterization of the guest-host collagen mimetic structures was established by techniques such as temperature-dependent optical rotation measurements and circular dichroism (CD) spectroscopy.     

Application of ring-closing metathesis macrocyclization to the development of Tsg101-binding antagonists

HIV-1 viral budding involves binding of the viral Gag^p6 protein to the ubiquitin E2 variant domain of the human tumor susceptibility gene 101 protein (Tsg101). Recognition of p6 by Tsg101 is mediated in part by a proline-rich motif that contains the sequence ‘Pro-Thr-Ala-Pro’ (‘PTAP’). Using the p6-derived 9-mer sequence ‘PEPTAPPEE’, we had previously improved peptide binding affinity by employing N-alkylglycine (‘peptoid’) residues. The current study applies ring-closing metathesis macrocyclization strategies to Tsg101-binding peptide–peptoid hybrids as an approach to stabilize binding conformations and to observe the effects of such macrocyclization on Tsg101-binding affinity and bioavailability.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Peptoids advance multidisciplinary research and undergraduate education in parallel: Sequence effects on conformation and lipid interactions

Peptoids are versatile peptidomimetic molecules with wide-ranging applications from drug discovery to materials science. An understanding of peptoid sequence features that contribute to both their three-dimensional structures and their interactions with lipids will expand functions of peptoids in varied fields. Furthermore, these topics capture the enthusiasm of undergraduate students who prepare and study diverse peptoids in laboratory coursework and/or in faculty led research. Here, we present the synthesis and study of 21 peptoids with varied functionality, including 19 tripeptoids and 2 longer oligomers. We observed differences in fluorescence spectral features for 10 of the tripeptoids that correlated with peptoid flexibility and relative positioning of chromophores. Interactions of representative peptoids with sonicated glycerophospholipid vesicles were also evaluated using fluorescence spectroscopy. We observed evidence of conformational changes effected by lipids for select peptoids. We also summarize our experiences engaging students in peptoid-based projects to advance both research and undergraduate educational objectives in parallel.     

Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains

This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.     

Unconstrained peptoid tetramer exhibits a predominant conformation in aqueous solution

Conformational control in peptoids, N-substituted glycines, is crucial for the design and synthesis of biologically-active compounds and atomically-defined nanomaterials. While there are a growing number of structural studies in solution, most have been performed with conformationally-constrained short sequences (e.g., sterically-hindered sidechains or macrocyclization). Thus, the inherent degree of heterogeneity of unconstrained peptoids in solution remains largely unstudied. Here, we explored the folding landscape of a series of simple peptoid tetramers in aqueous solution by NMR spectroscopy. By incorporating specific ¹³C-probes into the backbone using bromoacetic acid-2-¹³C as a submonomer, we developed a new technique for sequential backbone assignment of peptoids based on the 1,n-Adequate pulse sequence. Unexpectedly, two of the tetramers, containing an N-(2-aminoethyl)glycine residue (Nae), had preferred conformations. NMR and molecular dynamics studies on one of the tetramers showed that the preferred conformer (52%) had a trans-cis-trans configuration about the three amide bonds. Moreover, >80% of the ensemble contained a cis amide bond at the central amide. The backbone dihedral angles observed fall directly within the expected minima in the peptoid Ramachandran plot. Analysis of this compound against similar peptoid analogs suggests that the commonly used Nae monomer plays a key role in the stabilization of peptoid structure via a side-chain-to-main-chain interaction. This discovery may offer a simple, synthetically high-yielding approach to control peptoid structure, and suggests that peptoids have strong intrinsic conformational preferences in solution. These findings should facilitate the predictive design of folded peptoid structures, and accelerate application in areas ranging from drug discovery to biomimetic nanoscience.     

Extreme stability of helices formed by water-soluble poly-N-substituted glycines (polypeptoids) with alpha-chiral side chains.

Poly-N-substituted glycines or "peptoids" are protease-stable peptide mimics. Although the peptoid backbone is achiral and lacks hydrogen-bond donors, substitution with alpha-chiral side chains can drive the formation of stable helices that give rise to intense CD spectra. To systematically study the solution properties and stability of water-soluble peptoid helices with alpha-chiral side chains, we have synthesized and characterized an amphipathic, 36-residue N-substituted glycine oligomer. CD was used to investigate effects of concentration and solvent environment on this helical peptoid. We saw no significant dependence of helical structure on concentration. Intense, "alpha-helix-like" CD spectra were observed for the 36-mer in aqueous, 2,2,2-trifluorethanol (TFE), and methanol solution, proving a relative insensitivity of peptoid helical structure to solvent environment. While CD spectra taken in these different solvents were fundamentally similar in shape, we did observe some interesting differences in the intensities of particular CD bands in the various solvents. For example, the addition of TFE to an aqueous solvent increases the degree of peptoid helicity, as is observed for polypeptide alpha-helices. Moreover, the helical structure of peptoids appears to be virtually unaffected by heat, even in an aqueous buffer containing 8 M urea. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role of steric forces in their structural stabilization. The structured polypeptoids studied here may have potential as robust mimics of helical polypeptides of therapeutic interest.     

Peptoid origins

Peptoid oligomers were initially developed as part of a larger basic research effort to accelerate the drug-discovery process in the biotech/biopharma industry. Their ease of synthesis, stability, and structural similarity to polypeptides made them ideal candidates for the combinatorial discovery of novel peptidomimetic drug candidates. Diverse libraries of short peptoid oligomers provided one of the first demonstrations in the mid-1990s that high-affinity ligands to pharmaceutically relevant receptors could be discovered from combinatorial libraries of synthetic compounds. The solid-phase submonomer method of peptoid synthesis was so efficient and general that it soon became possible to explore the properties of longer polypeptoid chains in a variety of areas beyond drug discovery (e.g., diagnostics, drug delivery, and materials science). Exploration into protein-mimetic materials soon followed, with the fundamental goal of folding a non-natural sequence-specific heteropolymer into defined secondary or tertiary structures. This effort first yielded the peptoid helix and much later the peptoid sheet, both of which are secondary-structure mimetics that are close relatives to their natural counterparts. These crucial discoveries have brought us closer to building proteinlike structure and function from a non-natural polymer and have provided great insight into the rules governing polymer and protein folding. The accessibility of peptoid synthesis to chemists and nonchemists alike, along with a lack of information-rich non-natural polymers available to study, has led to a rapid growth in the field of peptoid science by many new investigators. This work provides an overview of the initial discovery and early developments in the peptoid field.     

A peptoid antagonist of VEGF receptor 2 recognizes a 'hotspot' in the extracellular domain distinct from the hormone-binding site

Antagonists of VEGF-mediated angiogenesis are of great interest clinically for the treatment of solid tumors and certain forms of macular degeneration. We recently described a novel peptoid antagonist of VEGF Receptor 2 (VEGFR2) that binds to the extracellular domain of the receptor and inhibits VEGF-mediated autophosphorylation and subsequent downstream signaling. Given the structural similarities between peptides and peptoids, an obvious model for the mode of action of the peptoid is that it competes with VEGF for binding to VEGFR2. However, we present evidence here that this is not the case and that VEGF and the peptoid antagonist recognize non-overlapping surfaces located within the first three immunoglobulin-like subdomains of the receptor. These data argue that the peptoid inhibits receptor-mediated autophosphorylation by a novel allosteric mechanism that may prevent the receptor from acquiring the conformation necessary to propagate downstream signals.     

Synthesis of cyclic peptidosulfonamides as scaffolds for MC4 pharmacophoric groups

The synthesis of potential beta-turn mimetics based on cyclic sulfonamide peptoid/peptoid hybrids is described. These are readily synthesized using a solid phase protocol followed by cyclization in solution, and their suitability to combinatorial approaches is illustrated by the synthesis of a small but diversely functionalized library.     

Folding of a single-chain, information-rich polypeptoid sequence into a highly ordered nanosheet

The design and synthesis of protein-like polymers is a fundamental challenge in materials science. A means to achieve this goal is to create synthetic polymers of defined sequence where all relevant folding information is incorporated into a single polymer strand. We present here the aqueous self-assembly of peptoid polymers (N-substituted glycines) into ultrathin, two-dimensional highly ordered nanosheets, where all folding information is encoded into a single chain. The sequence designs enforce a two-fold amphiphilic periodicity. Two sequences were considered: one with charged residues alternately positive and negative (alternating patterning), and one with charges segregated in positive and negative halves of the molecule (block patterning). Sheets form between pH 5 and 10 with the optimal conditions being pH 6 for the alternating sequence and pH 8 for the block sequence. Once assembled, the nanosheets remain stable between pH 6 and 10 with observed degradation beginning to occur below pH 6. The alternating charge nanosheets remain stable up to concentrations of 20% acetonitrile, whereas the block pattern displayed greater robustness remaining stable up to 30% acetonitrile. These observations are consistent with expectations based on considerations of the molecules' electrostatic interactions. This study represents an important step in the construction of abiotic materials founded on biological informatic and folding principles.