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.     

Toward beta-amino acid proteins: a cooperatively folded beta-peptide quaternary structure

Folded polymers in nature are assembled from simple monomers and adopt complex folded structures through networks of stabilizing noncovalent interactions. These interactions define secondary and tertiary structure and in most cases specify a unique three-dimensional architecture. Individual secondary or tertiary structures can also associate with one another to form multi-subunit quaternary structures. Nonnatural folded polymers have potential for similar structural versatility. Here we describe a pair of beta3-peptides whose sequences were designed to promote a 14-helix structure in water, favor hetero-oligomer formation, and disfavor nonspecific aggregation. These beta3-peptides assemble noncovalently into a well-defined hetero-oligomer characterized by a defined stoichiometry, a highly stabilized secondary structure, and a cooperative melting transition (TM > 55 degrees C). This work demonstrates that beta3-peptides can assemble into defined, cooperatively folded quaternary structures and constitutes an important step toward designing protein-like assemblies from nonnatural polymers.     

Helix macrodipole control of beta 3 peptide 14-helix stability in water

beta-Peptides have attracted considerable attention by virtue of their ability to populate helical secondary structures in methanol, even in the absence of stabilizing tertiary interactions. Recent efforts in beta-peptide design have produced few beta3-peptides that form stable 14-helices in water; those that do require stabilizing intramolecular salt bridges on two of three helical faces and therefore possess limited utility as tools in biological research. Here we show that favorable interactions with the 14-helix macrodipole significantly stabilize the 14-helix in water, alleviating the need for multiple salt bridges on two of three helical faces. We also report the previously unrecognized stabilization of 14-helix structure by gamma-branched beta3-amino acids. The most structured molecules we describe are highly heterogeneous at the primary sequence level, containing seven different beta3-amino acids within an 11-residue sequence. These results represent the essential first step toward the design of well-folded 14-helices that explore the interactions between beta3-peptides and biological macromolecules in vitro and in vivo.     

Relationship between side chain structure and 14-helix stability of beta3-peptides in water

Folded polymers are used in Nature for virtually every vital process. Nonnatural folded polymers, or foldamers, have the potential for similar versatility, and the design and refinement of such molecules is of considerable current interest. Here we report a complete and systematic analysis of the relationship between side chain structure and the 14-helicity of a well-studied class of foldamers, beta(3)-peptides, in water. Our experimental results (1) verify the importance of macrodipole stabilization for maintaining 14-helix structure, (2) provide comprehensive evidence that beta(3)-amino acids branched at the first side chain carbon are 14-helix-stabilizing, (3) suggest a novel role for side chain hydrogen bonding as an additional stabilizing force in beta(3)-peptides containing beta(3)-homoserine or beta(3)-homothreonine, and (4) demonstrate that diverse functionality can be incorporated into a stable 14-helix. Gas- and solution-phase calculations and Monte Carlo simulations recapitulate the experimental trends only in the context of oligomers, yielding insight into the mechanisms behind 14-helix folding. The 14-helix propensities of beta(3)-amino acids differ starkly from the alpha-helix propensities of analogous alpha-amino acids. This contrast informs current models for alpha-helix folding, and suggests that 14-helix folding is governed by different biophysical forces than is alpha-helix folding. The ability to modulate 14-helix structure through side chain choice will assist rational design of 14-helical beta-peptide ligands for macromolecular targets.     

Positional screening and NMR structure determination of side-chain-to-side-chain cyclized β3-peptides

Many β-peptides fold in a 14-helical secondary structure in organic solvents, but similar 14-helix formation in water requires additional stabilizing elements. Especially the 14-helix stabilization of short β-peptides in aqueous solution is critical, due to the limited freedom for incorporating stabilizing elements. Here we show how a single lactam bridge, connecting two β-amino acid side-chains, can lead to high 14-helix character in short β(3)-peptides in water. A comparative study, using CD and NMR spectroscopy and structure calculations, revealed the strong 14-helix inducing power of a side-chain-to-side-chain cyclization and its optimal position on the β(3)-peptide scaffold with respect to pH and ionic strength effects. The lactam bridge is ideally incorporated in the N-terminal region of the β(3)-peptide, where it limits the conformational flexibility of the peptide backbone. The lactam bridge induces a 14-helical conformation in methanol and water to a similar extent. Based on the presented first high resolution NMR 3D structure of a lactam bridged β(3)-peptide, the fold shows a large degree of high order, both in the backbone and in the side-chains, leading to a highly compact and stable folded structure.     

Stable helical beta 3-peptides in water via covalent bridging of side chains

An on-bead cyclization protocol of beta 3-peptides was developed, providing easy access to cyclic beta 3-peptides. With this methodology, a small library of helical cyclic beta 3-peptides was synthesized and investigated with CD spectroscopy. Covalent bridging of two side chains in beta 3-peptides significantly stabilized their helical conformation in aqueous solutions and turned out to be superior to the previously described electrostatic interactions.     

Sequence dependence of secondary-structure formation part IV Helix-forming potential of amphiphilic oligopeptides containing aib residues

The conformational properties of Aib-containing oligopeptides having the propensity to adopt amphiphilic helical conformations were investigated by CD spectroscopy in solution. The peptides CF₃COOH.H-Pro-Glu-[Ala-Aib-Glu-Aib-]₄Gly-OH ( I ), HCl.H-Pro-Ala-Aib-[Gul-Ala-Ala-Aib-]₂Glu-Ala-Aib-Gly-PEGM ( II ), and CF₃COOH.H-Ala-Aib-[Glu-Glu-Ala-Aib-]₃PEGM ( III ) were synthesized according to the general principles of the liquid-phase method for peptide synthesis. Peptides I-III exhibit helical conformations in CF₃CH₂OH, MeOH, and in H₂O at acidic pH; however, at pH 7, only II forms a stable helix, whereas I and III are predominantly in an unordered conformation. Some general features for the construction of amphiphilic helices are discussed in the light of the experimental data.     

(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.     

β-Peptides: Synthesis by Arndt-Eistert homologation with concomitant peptide coupling. Structure determination by NMR and CD spectroscopy and by X-ray crystallography. Helical secondary structure of a β-hexapeptide in solution and its stability towards pepsin

The β-hexapeptide (H-β-HVal-β-HAla-β-HLeu)₂-OH ( 2 ) was prepared from the component L -β-amino acids by conventional peptide synthesis, including fragment coupling. A cyclo-β-tri- and a cyclo-β-hexapeptide were also prepared. The β-amino acids were obtained from α-amino acids by Arndt-Eistert homologation. All reactions leading to the β-peptides occur smoothly and in high yields. The β-peptides were characterized by their CD and NMR spectra (COSY, ROESY, TOCSY, and NOE-restricted modelling), and by an X-ray crystal-structure analysis. β-Sheet-type structures (in the solid state) and a compact, left-handed or (M) 3₁ helix of 5-Å pitch (in solution) were discovered. Comparison with the analogous secondary structures of α-peptides shows fundamental differences, the most surprising one at this point being the greater stability of β-peptide helices. There are structural relationships of β-peptides with oligomers of β-hydroxyalkanoic acids, and dissimilarities between the two classes of compounds are a demonstration of the power of H-bonding. The β-hexapeptide 2 is stable to cleavage by pepsin at pH 2 in H₂O for at least 60 h at 37°, while the corresponding α-peptide H-(Val-Ala-Leu)₂-OH is cleaved instantaneously under these conditions. The implication of the described results are discussed.     

Conformation of N(alpha)-substituted hydrazino acetamides in CDCl3, the precious help of the analysis of Deltadelta between amidic hydrogens, and correlation to the conformation of aza-beta3-peptides

[structures: see text] We studied the conformation of a series of primary amides in a solution of chloroform. Classical NMR tools such as dilution experiments, influence of DMSO, and 2D-NOESY, together with X-ray diffraction, were combined with an analysis of the difference of the chemical shift Deltadelta between the geminal amidic protons. This study was addressed in order to understand the conformation adopted by hydrazino acetamides 1a and 1b as model compounds for aza-beta3-peptides. In this manner, it was possible to show that the amidic group of these compounds acts as a H-bond donor and interacts with two different H-bond acceptors. We concluded that the hydrazinoturn, a specific bifurcated H-bond system observed in the solid state, is also the preferred conformation of hydrazino acetamides 1a and 1b in solution. Our results show that the short-range interaction with the N(alpha)-nitrogen lone pair not only stabilizes the C8 pseudocycle but could also contribute to the folding process of aza-beta3-peptides. In light of this, it could explain why aza-beta3-peptides develop a different H-bond network in comparison to their isosteric beta3-peptides analogues. Our work is in keeping with the recent interest of hydrazino peptides as an extension of the beta-peptide concept.     

Methyl 3-amino-2,3,6,-trideoxy-L-hexopyranosides in DFT level theory conformational studies

Geometry optimizations at the B3LYP level of density functional theory are reported for the (1)C4 and (4)C1 conformations of four theoretically possible alpha and beta methyl 3-amino-2,3,6-trideoxy-L-hexopyranosides. The Gibbs free energies, relative Gibbs free energies, and geometry parameters are presented for all the optimized structures. Conformational analysis of the pyranose ring is performed for each stereoisomer on the basis of calculated rotamer populations. It is demonstrated that the alpha/beta-L-arabino, alpha/beta-L-lyxo, and alpha-L-ribo stereoisomers adopt the (1)C4 conformation, whereas beta-L-ribo and alpha/beta-L-xylo stereoisomers remain in (1)C4 <==> (4)C1 conformational equilibrium. The preference of the alpha over the beta anomers is due to the endo-anomeric effect. The factors affecting the stability of pyranose ring conformations are discussed, as is the influence of hydrogen bonds on the orientation of the hydroxyl and amino groups. Figures of the most stable conformers are presented.     

3‐Fluoro‐2,4‐dioxa‐7‐aza‐3‐phosphadecalin 3‐Oxides as Rigid Acetylcholine Mimetics: Conformational Analysis and Direct Evidence of the Anomeric Effect

Conformational analyses of the P(3)‐axially and P(3)‐equatorially F‐substituted (±)‐cis‐ and (±)‐trans‐2,4‐dioxa‐7‐aza‐3‐phosphadecalin 3‐oxides (3‐fluoro‐2,4‐dioxa‐7‐aza‐3‐phosphabicyclo[4.4.0]decane 3‐oxides) were performed. The results are based on independent studies in both solution and the solid state by ¹H‐ and ³¹P‐NMR experiments and computational and X‐ray crystallographic data. As expected, the axial epimers adopt neat double‐chair conformations in solution and in the crystal. Due to the anomeric effect of the electron withdrawing F‐substituent, the 2,4‐dioxa‐3‐phospha moiety in the equatorial epimers adopts a mixture of conformations in solution, mainly chair and twist‐boat; whereas a neat twist‐boat (trans‐isomer) and the unusual envelope conformation (cis‐isomer) were detected in the solid state. This is the first report of a straight visualization of these conformations and the impact of the anomeric effect in such systems.     

Synthesis and Secondary Conformations of Homochiral β-Oligopeptides Containing Aryl Side Chains

A series of novel homochiral β-oligopeptides bearing aryl side chains was designed and synthesized from (S)-β-phenyl-β-amino acid derivatives by solution-phase methods. By means of circular dichroism(CD), Fourier- transform infrared spectrometry(FTIR), powder X^-ray diffraction analysis(XRD) and density functional theory(DFT) calculations, we suggest that dipeptide P-2 and tripeptide P-3 adopt random coil-like conformations, pentapeptide P-5 and hexapeptide P-6 adopt stable 12-helix conformations in both solution and solid-state. Meanwhile, tetrapeptide P-4 adopt random coil-like conformation in solution and adopt 12-helix conformation in solid state.     

Competition between amide stacking and intramolecular H bonds in γ-peptide derivatives: controlling nearest-neighbor preferences

Resonant two-photon ionization (R2PI), IR-UV holeburning (IR-UV), and resonant ion-dip infrared spectroscopy (RIDIRS) have been used to record mass-selected, single-conformation ultraviolet and infrared spectra of three simple diamide derivatives of γ-amino acids as isolated molecules cooled in a supersonic expansion. This work builds on an earlier study of Ac-γ(2)-hPhe-NHMe (James, W. H., III, et al. J. Am. Chem. Soc. 2009, 131, 14243), which showed that this methyl-capped γ-peptide forms amide-stacked conformations that are similar in stability to H-bonded conformations containing a C9 ring and more stable than C7 H-bonded ring structures. Among the γ-peptides discussed here, Ac-γ(2)-hPhe-N(Me)(2) contains an additional methyl group relative to the previously studied Ac-γ(2)-hPhe-NHMe and therefore lacks the amide NH group responsible for C9 ring formation. Three conformations of Ac-γ(2)-hPhe-N(Me)(2) are observed, all of which are amide-stacked structures. In a second new molecule, Ac-γ(2)-hPhe-NH(iPr), the C-terminal NHMe group of Ac-γ(2)-hPhe-NHMe is replaced with an NH(iPr) group. Three conformations of Ac-γ(2)-hPhe-NH(iPr) are observed, all of which are C9 H-bonded structures. The dramatic difference between C-terminal NHMe and NH(iPr) reveals the delicate balance of noncovalent forces within these γ-peptides. The third molecule we examined is a gabapentin-derived diamide (designated 1), which contains a phenylacyl group at the N-terminus and an N(Me)(2) group at the C-terminus; the latter precludes C9 H bonding. Comparison of 1 with Ac-γ(2)-hPhe-N(Me)(2) allows us to examine the impact of the backbone substitution pattern (monosubstitution at carbon-2 vs disubstitution at carbon-3) on the competition between the C7 H-bonded and the amide-stacked conformation. In this case, only C7 rings are observed. The different gas-phase behaviors observed among the molecules analyzed here offer insight on the intrinsic conformational propensities of the γ-peptide backbone, information that provides a foundation for future foldamer design efforts.     

Solid-phase synthesis and CD spectroscopic investigations of novel beta-peptides from L-aspartic acid and beta-amino-L-alanine

[structure: see text] A solid-phase synthesis method for the preparation of novel beta3- and beta2-peptides derived from l-aspartic acid and beta-amino-l-alanine, respectively, is described. The methodology allows independent buildup of the beta-peptide backbone and the introduction of sequential side chain substitutions. Representative peptides from the two classes, an amino-substituted beta3-hexapeptide and an acyl-substituted beta2-hexapeptide, have been prepared, and their solution conformation is studied by circular dichroism (CD) spectroscopy.     

Crystal structures of aza-beta3-peptides, a new class of foldamers relying on a framework of hydrazinoturns

Crystals of aza-beta3-peptides have been obtained. This gives the first opportunity for hydrazino peptides, in the sense of oligomers built exclusively with alpha-hydrazinoacetic units, to be observed in the solid state. The structures make it clear that the H-bond network developed by aza-beta3-peptides differs radically from those of the corresponding beta3-peptides but strongly resembles that of the alpha-aminoxy peptides. Our study contributes to the current interest in hydrazino peptides as an extension of the beta-peptide concept.     

Cyclo-β-peptides: Structure and tubular stacking of cyclic tetramers of 3-aminobutanoic acid as determined from powder diffraction data

The solid-state structures of three stereoisomer, 1–3 , of the cyclic tetramer of 3-aminobutanoic acid are presented. These cyclo-β-peptides were found to be highly insoluble materials, and it proved to be impossible to grow crystals of sufficient quality for X-ray single-crystal analysis. The samples of 1–3 were, however, suitable candidates for structure determination from powder diffraction data (Fig. 1), and the application of this method is described. All three isomers have been found to adopt tubular structures (Figs. 2–4) in a fashion similar to those already observed for certain cyclo-α-peptides. The stacks of 16-membered rings are held together by four nonlinear C?O…?H? N H-bonds between pairs of molecules (Fig.5).     

Tris(1,2-dimethyl-3-hydroxy-4(1H)-pyridone)oxotantalum(v): a new water-soluble tantalum complex containing the [Ta=O]3+ core

The synthesis, the characterization and the X-ray crystal structure of a novel tantalum(v) complex tris(1,2-dimethyl-3-hydroxy-4(1H)-pyridone)oxotantalum(v) [TaO(C7H8O2N)3] 1 is reported. Starting from the tantalum pentaethoxide a two-step reaction was carried out observing rigorous anhydrous conditions in methanol in the first step, while the insertion of the oxo group was achieved using water as oxygen donor in the second step. X-Ray diffraction analysis of a crystal of 1 obtained upon evaporation of an aqueous solution shows a seven-coordinate monomeric complex containing the [Ta=O]3+ core in the triclinic space group P1, a = 7.282(3), b = 14.055(5), c = 16.987(6) A, alpha = 65.704(5), beta = 89.155(6), gamma = 75.270(6) degrees, V = 1525(1)A3, Z = 2. Spectroscopic investigation reveals that the complex is highly soluble and stable in water at physiological pH values; as a consequence the complex may represent a potential candidate for the development of new studies on aqueous tantalum(v) chemistry for radiopharmaceutical applications.     

Cadmium cyclam complexes: interconversion of cis and trans configurations and fixation of CO(2)

There is current interest in the antiviral activity of metal, especially zinc, cyclam (1,4,8,11-tetraazacyclotetradecane) complexes. Their biological activity appears to be dependent on recognition of membrane proteins (viral coreceptors) and therefore on their configurations. Here, we use Cd(II) as a probe for Zn(II) on account of its useful NMR properties. We have prepared and characterized Cd(II) complexes of cyclam, Cd(cyclam)(ClO(4))(2) (1), Cd(cyclam)Cl(2) (2), and [Cd(3)(cyclam)(3)(CO(3))](ClO(4))(4).3H(2)O (3), and have identified key markers for various configurations adopted by these complexes under a variety of solution conditions using 1D and 2D (1)H, (13)C, (15)N, and (111)Cd NMR spectroscopy, including Karplus-type analyses of (1)H, (1)H and (1)H, (111)Cd coupling constants. These complexes were stable at high pH (>8.2) but dissociated completely on lowering the pH to 5.3. Two major configurations of both 1 and 2 exist in aqueous solution: trans-I (R,S,R,S at nitrogen) and cis-V (R,R,R,R). (3)J((111)Cd, (1)H) coupling constants showed that the five-membered rings of the trans-I configuration adopt the eclipsed conformation, and the six-membered rings adopt chair conformations. The X-ray crystal structure of 3 shows that the cation adopts the unusual folded cis-I configuration in which all of the N-H bonds are oriented up (or down) in a novel tri-cadmium cluster. This complex contains triply bridged carbonate fixed from atmospheric CO(2). Each Cd(II) is bound by two cis oxygen atoms from CO(3)(2-) (Cd-O bond lengths 2.373 and 2.412 A) and four nitrogen atoms from cyclam (C-N bond lengths 2.270-2.323 A). The geometry can be described as trigonal bipyramidal with the two donor oxygen atoms occupying one of the apices of the in-plane triangle. In acetonitrile solution, complex 3 gives rise to only one configuration, trans-I, with eclipsed five-membered rings, and six-membered rings with chair conformations.     

Synthesis and characterisation of 3-aza-7-phosphabicyclo[3.3.1]nonan-9-ones

Novel 3-aza-7-phosphabicyclo[3.3.1]nonan-9-ones have been synthesised via the Mannich reaction of a phosphorinanone, a primary amine and formaldehyde. The new phosphorus-nitrogen (PN) compounds are rigid and adopt a twin-chair conformation both in solution and the solid states. The coordination properties of the PN compounds were explored and a stable platinum complex was synthesised in which the PN ligand was bonded through both donor atoms.