Helix formation in alpha,gamma- and beta,gamma-hybrid peptides: theoretical insights into mimicry of alpha- and beta-peptides

Alpha,gamma- and beta,gamma-hybrid peptides, which are composed of two different homologous amino acid constituents in alternate order, are suggested as novel classes of peptide foldamers. On the basis of a systematic conformational search employing the methods of ab initio MO theory, the possibilities for the formation of periodic secondary structures in these systems are described. The conformational analysis provides a great number of helix conformers widely differing in energy, which can be arranged into three groups: (i) helices with all hydrogen bonds formed in forward direction along the sequence, (ii) helices with all hydrogen bonds in backward direction, and (iii) helices with alternate hydrogen-bond directions (mixed or beta-helices). Most stable are representatives of beta-helices, but their stability decreases considerably in more polar environments in comparison to helix conformers from the other two classes. There is a great similarity between the overall topology of the most stable hybrid peptide helices and typical helices of peptides which are exclusively composed of a single type of homologous amino acids. Thus, the helices of the beta,gamma-hybrid peptides mimic perfectly those of the native alpha-peptides as, for instance, the well-known alpha-helix, whereas the most stable helix conformers of alpha,gamma-hybrid peptides correspond well to the overall structure of beta-peptide helices. The two suggested novel hybrid peptide classes expand considerably the pool of peptide foldamers and may be promising tools in peptide design and in material sciences.     

Asymmetric synthesis of beta-amino acid derivatives incorporating a broad range of substitution patterns by enolate additions to tert-butanesulfinyl imines

Addition of Ti(Oi-Pr)(3) ester enolates to tert-butanesulfinyl aldimines and ketimines provided beta-substituted, alpha,beta- and beta,beta-disubstituted, alpha,beta,beta- and alpha,alpha,beta-trisubstituted, and alpha,alpha,beta,beta-tetrasubstituted beta-amino acid derivatives in high yields and with high diastereoselectivites. The N-sulfinyl-beta-amino ester products were further employed as versatile intermediates for both standard solution-phase and solid-phase synthetic transformations, including the synthesis of beta-peptide foldamers.     

Residue requirements for helical folding in short alpha/beta-peptides: crystallographic characterization of the 11-helix in an optimized sequence

Design of functional foldamers requires knowledge of the conformational propensities of constituent residues. Here, we explore the effects of variations in both alpha-amino acid and beta-amino acid substitution on alpha/beta-peptide helicity. We also report the first X-ray crystal structure of a helical alpha/beta-peptide. We conclude that a certain amount of conformational preorganization in alpha/beta-peptides (via the inclusion of constrained beta-amino acids or alpha,alpha-disubstituted alpha-amino acids) is needed to promote helical folding; acyclic beta-amino acids and beta-branched alpha-amino acids are tolerated to only a limited extent.     

Gamma4-aminoxy peptides as new peptidomimetic foldamers

The conformational properties of peptides 1-3 of gamma-aminoxy acids have been investigated by using FT-IR, NMR spectroscopy, and X-ray crystallography. Diamide 1 consisting of unsubstituted gamma-aminoxy acid cannot form intramolecular hydrogen bond. A novel gamma N-O turn involving a 10-membered-ring intramolecular hydrogen bond between NHi+2 and COi is formed in gamma4-aminoxy peptides 2 and 3. Triamides 3 prefers a new helical structure featuring two consecutive gamma N-O turns. Therefore, gamma4-aminoxy peptides represent new peptidomimetic foldamers.     

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.     

Chimeric (alpha/beta + alpha)-peptide ligands for the BH3-recognition cleft of Bcl-XL: critical role of the molecular scaffold in protein surface recognition

Molecules that bind to specific surface sites on proteins are of great interest from both fundamental and practical perspectives. We are exploring a ligand development strategy that is based on oligomers with discrete folding propensities ("foldamers"); we target a specific cleft on the cancer-associated protein Bcl-xL because this system is well characterized structurally. In vivo, this cleft binds to alpha-helical segments (BH3 domains) of other proteins. We evaluated several types of helical foldamer, built entirely from beta-amino acid residues or from mixtures of alpha- and beta-amino acid residues, and ultimately identified foldamers in the latter class that bind very tightly to Bcl-xL. Our results suggest that combining different types of foldamer backbones will be an effective and general strategy for creating high-affinity and specific ligands for protein surface sites.     

Versatile Post-synthetic Modifications of Helical β-Peptide Foldamers Derived from a Thioether-Containing Cyclic β-Amino Acid

We introduce a novel cyclic β-amino acid, trans-(3S,4R)-4-aminotetrahydrothiophene-3-carboxylic acid (ATTC), as a versatile building block for designing peptide foldamers with controlled secondary structures. We synthesized and characterized a series of β-peptide hexamers containing ATTC using various techniques, including X-ray crystallography, circular dichroism, and NMR spectroscopy. Our findings reveal that ATTC-containing foldamers can adopt 12-helical conformations similar to their isosteres and offer the possibility of fine-tuning their properties via post-synthetic modifications. In particular, chemoselective conjugation strategies demonstrate that ATTC provides unique post-synthetic modification opportunities, which expand their potential applications across diverse research areas. Collectively, our study highlights the versatility and utility of ATTC as an alternative to previously reported cyclic β-amino acid building blocks in both structural and functional aspects, paving the way for future research in the realm of peptide foldamers and beyond.     

Anion-coordination-driven single–double helix switching and chiroptical molecular switching based on oligoureas

Synthetic foldamers with helical conformation are widely seen, but controllable interconversion amongst different geometries (helical structure and sense) is challenging. Here, a family of oligourea (tetra-, penta-, and hexa-) ligands bearing stereocenters at both ends are designed and shown to switch between single and double helices with concomitant inversion of helical senses upon anion coordination. The tetraurea ligand forms a right-handed single helix upon chloride anion (Cl⁻) binding and is converted into a left-handed double helix when phosphate anion (PO₄³⁻) is coordinated. The helical senses of the single and double helices are opposite, and the conversion is further found to be dependent on the stoichiometry of the ligand and phosphate anion. In contrast, only a single helix is formed for the hexaurea ligand with the phosphate anion. This distinction is attributed to the fact that the characteristic phosphate anion coordination geometry is satisfied by six urea moieties with twelve H-bonds. Our study revealed unusual single–double helix interconversion accompanied by unexpected chiroptical switching of helical senses.

Two-in-one switching of single–double helical forms and helicities is demonstrated using anion-coordination-driven oligourea foldamers.     

Photocontrol of cell adhesion processes: model studies with cyclic azobenzene-RGD peptides

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.     

(alpha/beta+alpha)-peptide antagonists of BH3 domain/Bcl-x(L) recognition: toward general strategies for foldamer-based inhibition of protein-protein interactions

The development of molecules that bind to specific protein surface sites and inhibit protein-protein interactions is a fundamental challenge in molecular recognition. New strategies for approaching this challenge could have important long-term ramifications in biology and medicine. We are exploring the concept that unnatural oligomers with well-defined conformations ("foldamers") can mimic protein secondary structural elements and thereby block specific protein-protein interactions. Here, we describe the identification and analysis of helical peptide-based foldamers that bind to a specific cleft on the anti-apoptotic protein Bcl-xL by mimicking an alpha-helical BH3 domain. Initial studies, employing a fluorescence polarization (FP) competition assay, revealed that among several alpha/beta- and beta-peptide foldamer backbones only alpha/beta-peptides intended to adopt 14/15-helical secondary structure display significant binding to Bcl-xL. The most tightly binding Bcl-xL ligands are chimeric oligomers in which an N-terminal alpha/beta-peptide segment is fused to a C-terminal alpha-peptide segment ((alpha/beta + alpha)-peptides)). Sequence-affinity relationships were probed via standard and nonstandard techniques (alanine scanning and hydrophile scanning, respectively), and the results allowed us to construct a computational model of the ligand/Bcl-xL complex. Analytical ultracentrifugation with a high-affinity (alpha/beta + alpha)-peptide established 1:1 ligand:Bcl-xL stoichiometry under FP assay conditions. Binding selectivity studies with the most potent (alpha/beta + alpha)-peptide, conducted via surface plasmon resonance measurements, revealed that this ligand binds tightly to Bcl-w as well as to Bcl-xL, while binding to Bcl-2 is somewhat weaker. No binding could be detected with Mcl-1. We show that our most potent (alpha/beta + alpha)-peptide can induce cytochrome C release from mitochondria, an early step in apoptosis, in cell lysates, and that this activity is dependent upon inhibition of protein-protein interactions involving Bcl-xL.     

Secondary structural preferences of 2,2-disubstituted pyrrolidine-4-carboxylic acid oligomers: beta-peptide foldamers that cannot form internal hydrogen bonds

We examine a new class of beta-peptides, 2,2-disubstituted pyrrolidine-4-carboxylic acid oligomers, and show that they manifest discrete conformational preferences despite the impossibility of internal hydrogen bonding. Numerous beta-peptide families have been described that display specific secondary structural preferences, but all of the conformations characterized in detail so far have contained internal hydrogen bonds. Internal hydrogen bonding is observed within the most common secondary structures of conventional peptides as well. Identifying foldamers in which shape control is independent of hydrogen bonding is significant in two ways. At a fundamental level, foldamers in this small but growing class are interesting because their shapes are controlled by distinctive networks of noncovalent forces. At a practical level, non-hydrogen bonded foldamers may be useful in biomedical applications because the low intrinsic polarity of their backbones may promote bioavailability.     

Application of tri- and tetrasubstituted alkene dipeptide mimetics to conformational studies of cyclic RGD peptides

The first application of a combination of novel ψ[(E)-CXCX]-type alkene dipeptide isosteres to conformation studies of cyclic bioactive peptides was carried out (X=H or Me). For exploration of bioactive conformations of Kessler's cyclic RGD peptides, cyclo(-Arg-Gly-Asp-d-Phe-Val-) 1 and cyclo(-Arg-Gly-Asp-d-Phe-N-MeVal-) 2, d-Phe-ψ[(E)-CXCX]-l-Val-type dipeptide isosteres were utilized having di-, tri- and tetrasubstituted alkenes containing the γ-methylated isosteres that have been reported to be potential type II′ β-turn promoters. All of the (E)-alkene pseudopeptides 3-6 exhibited higher antagonistic potency against α_vβ₃ integrin than 1, although potencies were slightly lower than 2. Detailed structural analysis using ¹H NMR spectroscopy revealed that representative type II′ β/γ backbone arrangements proposed for 1, were not observed in peptides 3-6. Rather on the basis of ¹H NMR data, the conformations of peptides 3-6 were estimated to be more analogous to those of the N-methylated peptide 2.     

Hydrogen-bonding-induced oligoanthranilamide foldamers. Synthesis, characterization, and complexation for aliphatic ammonium ions

The self-assembly of a novel series of intramolecular hydrogen bonding-driven foldamers have been described. Five linear aromatic amide oligomers 1-5, which bear two to six repeating benzoyl amide subunits, respectively, have been prepared by continuous amide-coupling reactions. The existence of three-centered hydrogen bonds in the oligomers and consequently, the folding conformation of the oligomers in the solid state and solution have been proved by the X-ray analysis (for 2) and the ¹H NMR and IR experiments. Molecular modeling reveals a planar and rigid conformation for the oligomers and a cavity of 0.86 nm in diameter for 6-mer 5. Fluorescent and ¹H NMR experiments have demonstrated that the new aromatic oligo-amide foldamers can bind primary and secondary alkyl ammonium ions in chloroform and the associated binding constants have been determined. It is revealed that 5-mer 4 exhibits the largest binding ability. A face-to-face binding mode has been proposed for the complexes.     

Pompanopeptins A and B, new cyclic peptides from the marine cyanobacterium Lyngbya confervoides

A new 3-amino-6-hydroxy-2-piperidone (Ahp) containing peptolide, pompanopeptin A (1), and a novel N-methyl-2-amino-6-(4′-hydroxyphenyl)hexanoic acid (N-Me-Ahpha) containing cyclic pentapeptide connected with a sixth amino acid residue via a rare ureido linkage, pompanopeptin B (2), were isolated from the marine cyanobacterium Lyngbya confervoides collected from the southeastern coast of Florida. Their planar structures were determined by a combination of NMR spectroscopic analysis and mass spectrometry. The absolute configurations were established using advanced Marfey's method and chiral HPLC analysis of the chemical degradation products. Compound 1 selectively inhibited trypsin over elastase and chymotrypsin, with an IC₅₀ value of 2.4 μM; selectivity is conferred by an arginine residue in the cyclic core.     

Largamides A-H, unusual cyclic peptides from the marine cyanobacterium Oscillatoria sp

Seven new depsipeptides, termed largamides A-G (1-7), and one new cyclic peptide, largamide H (8), have been isolated from the marine cyanobacterium Oscillatoria sp. Their structures were determined by NMR and ESI-MS techniques. The absolute configurations were assigned using LC-MS, chiral HPLC, and combined analysis of homonuclear and heteronuclear (2,3)J couplings, along with ROE data. Largamides, isolated from a single homogeneous cyanobacterial collection, represent three different structural classes of peptides. Largamides A-C (1-3) are characterized by the unusual occurrence of a senecioic acid unit, while largamides B (2) and C (3) possess in addition the rare 2-amino-5-(4'-hydroxyphenyl)pentanoic acid (Ahppa) and the novel 2-amino-6-(4'-hydroxyphenyl)hexanoic acid (Ahpha), respectively. Largamides D-G (4-7) are the first 3-amino-6-hydroxy-2-piperidone acid (Ahp)-containing depsipeptides reported with the rare Ahppa unit. Largamide H (8) is a unique cyclic peptide displaying a new 2,5-dihydroxylated beta-amino acid moiety, a methoxylated derivative of Ahppa, and two residues of the nonstandard 2,3-dehydro-2-aminobutanoic acid (Dab). Largamides D-G (4-7) inhibited chymotrypsin with IC(50) values ranging between 4 and 25 microM.     

Directed evolution of cyclic peptides for inhibition of autophagy

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

SUPR peptide mRNA display was used to evolve a cell-permeable, macrocyclic peptide for autophagy inhibition.     

α-Amino-iso-Butyric Acid Foldamers Terminated with Rhodium(I) N-Heterocyclic Carbene Catalysts

To investigate how remotely induced changes in ligand folding might affect catalysis by organometallic complexes, dynamic α-amino-iso-butyric acid (Aib) peptide foldamers bearing rhodium(I) N-heterocyclic carbene (NHC) complexes have been synthesized and studied. X-ray crystallography of a foldamer with an N-terminal azide and a C-terminal Rh(NHC)(Cl)(diene) complex showed a racemate with a chiral axis in the Rh(NHC) complex and a distorted 3₁₀ helical body. Replacing the azide with either one or two chiral L-α-methylvaline (L-αMeVal) residues gave diastereoisomeric foldamers that each possessed point, helical and axial chirality. NMR spectroscopy revealed an unequal ratio of diastereoisomers for some foldamers, indicating that the chiral conformational preference of the N-terminal residue(s) was relayed down the 1 nm helical body to the axially chiral Rh(NHC) complex. Although the remote chiral residue(s) did not affect the stereoselectivity of hydrosilylation reactions catalysed by these foldamers, these studies suggest a potential pathway towards remote conformational control of organometallic catalysts.     

All-cis cyclic peptides

Amide bonds -NH-CO- preferentially exist in trans conformations, the cis conformation being thermodynamically unfavored with respect to the trans by about 2 kcal/mol. Yet, the main reason most proteins or peptides cannot be made from cis-peptide plaques only lies in that connecting them into open chains appears to be sterically impracticable. It is possible, however, to build all-cis cyclic peptides in which all cis-plaques are efficiently locked. The present work examines, through quantum calculations, the structural and energetic issues associated with these peculiar arrangements. Systematic exploration at DFT-B3LYP level of the potential-energy surfaces for all-cis cyclopolyglycines cG(n)(c) (n = 2-10,15), and to a lesser extent, for all-cis cyclopolyalanines and all-cis cyclopolyphenylalanines confirms that all these structures are true minima. Optimal ring size occurs around eight peptide units, resulting in planar cG7(c), cG8(c), and cG9(c). In smaller systems, the ring strain is relieved through nonplanar cup-like distortions, particularly in cG6(c). From 10 peptide units and beyond, the ring framework distorts into a saddle-edge shape. These molecules disclose some molecular flexibility, as combinatorial tilting of the plaques may give sets of minima close in energy. Indexes based on isodesmic reactions are used to estimate the energy for joining all-cis or all-trans plaques into cyclic peptides. One of them, the mean plaque-junction energy (MPJE) suggests that within sensible sizes from six peptide units and beyond, all-cis plaque association is almost equally favorable as all-trans one. The frame of radiating cis-amide bonds can be considered as defining a new kind of peptidic material, endowed with specific self-assembling properties.     

An extended planar C5 conformation and a 310-helical structure of peptide foldamer composed of diverse alpha-ethylated alpha,alpha-disubstituted alpha-amino acids

Optically active peptide foldamers Tfa-[(S)-(alphaEt)Leu]-[(S)-(alphaEt)Nva]-Deg-[(S)-(alphaEt)Nle]-OEt (10) and Tfa-[(S)-(alphaEt)Val]-[(S)-(alphaEt)Leu]-[(S)-(alphaEt)Nva]-Deg-[(S)-(alphaEt)Nle]-OEt (11) composed of diverse alpha-ethylated alpha,alpha-disubstituted alpha-amino acids were synthesized. The dominant conformation of these peptides in solution was an unusual, fully extended planar conformation, and that in the crystal state was both right-handed (P) and left-handed (M) 3(10)-helical structures in 10 and a P 3(10)-helical structure in 11, respectively. The preferred planar C(5) conformation of the peptides prepared from chiral alpha-ethylated alpha,alpha-disubstituted alpha-amino acids was drastically different from the 3(10)-helical structure of the peptides prepared from chiral alpha-methylated alpha,alpha-disubstituted alpha-amino acids.     

Conformationally constrained aliphatic-aromatic amino-acid-conjugated hybrid foldamers with periodic beta-turn motifs

In this note, we describe the design, synthesis, and structural studies of novel hybrid foldamers derived from Aib-Pro-Adb building blocks that display repeat beta-turn structure motif. The foldamer having a conformationally constrained aliphatic-aromatic amino acid conjugate adopts a well-defined, compact, three-dimensional structure, governed by a combined conformational restriction imposed by the individual amino acids with which it is made of. Conformational investigations by single-crystal X-ray and solution-state NMR studies were undertaken to investigate the conformational preference of these foldamers with a hetero-backbone. Our findings suggest that constrained aliphatic-aromatic amino acid conjugates would offer new avenues for the de novo design of hybrid foldamers with distinctive structural architectures.