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.     

Aib-rich peptides containing lactam-bridged side chains as models of the 3(10)-helix.

Aib-rich side chain lactam-bridged oligomers with n =1, 2, 3, were designed and synthesized as putative models of the 3(10)-helix. These peptides were conformationally characterized in aqueous solution containing SDS micelles by CD, NMR, and computer simulations. The lactam bridge between the side chains of L-Glu and L-Lys in (i) and (i+3) positions was introduced in order to enhance the conformational preference toward the right-handed 3(10)-helix. The NMR results clearly indicate that there is an increase of 3(10)-helix formation upon chain elongation. In the dimer and trimer (n = 2 and n = 3, respectively, in the structure reported above) the observed NOE connectivities are compatible with the 3(10)-helical arrangement, confirmed by the temperature coefficients of the amide proton resonances which suggest the presence of a hydrogen-bonded structure. The phi and psi dihedral angles of the structures obtained by molecular dynamics calculations are also compatible with the 3(10)-helix. Identification of the hydrogen-bond pattern indicate that C=O(i)- - -HN(i+3) hydrogen bonds, typical of the 3(10)-helical conformation, are highly probable in all low-energy structures. The CD spectra of these Aib-rich lactam-bridged oligopeptides, obtained in the same solvent system used for NMR experiments, provide important insight into the spectroscopic characteristics of the 3(10)-helix.     

Synthesis and 12-helical secondary structure of beta-peptides containing (2R,3R)-aminoproline

[structure: see text] (2R,3R)-Aminoproline, a pyrrolidine-based beta-amino acid, was synthesized and incorporated into hexa-beta-peptide 4. This residue confers water solubility when the ring nitrogen is protonated and allows for 12-helix formation in aqueous solution. Circular dichroism spectra display the 12-helical signature, and 12-helical structure was confirmed by 2D NMR analysis.     

Environment- and sequence-dependence of helical type in membrane-spanning peptides composed of β3-amino acids

Transmembrane (TM) β-peptides comprised of acyclic β(3)-amino acids demonstrate equilibrium between 12- and 14-helical structures in an environment- and sequence-dependent manner. Circular dichroism (CD) spectra of TM β(3)-peptides may be described as linear combinations of the 12- and 14-helical CD spectra. The apparent malleability of β(3)-substituted acyclic β-peptides has practical implications for foldamer design, as it suggests that both the 14-helix and 12-helix might be reasonable platforms for molecular recognition.     

The first water-soluble 3(10)-helical peptides

Two water-soluble 3(10)-helical peptides are synthesized and fully characterized for the first time. The sequence of these terminally blocked heptamers comprises two residues of the Calpha-trisubstituted alpha-amino acid 2-amino-3-[1-(1,4,7-triazacyclononyl)]propanoic acid and five residues of a Calpha-tetrasubstituted alpha-amino acid (either alpha-aminoisobutyric acid or isovaline). Using CD and NMR techniques we were able to show that both heptapeptides are well structured in water, and that the type of conformation adopted is indeed the ternary 3(10)-helix.     

Stapling of a 3(10)-helix with click chemistry

Short peptides are important as lead compounds and molecular probes in drug discovery and chemical biology, but their well-known drawbacks, such as high conformational flexibility, protease lability, poor bioavailability and short half-lives in vivo, have prevented their potential from being fully realized. Side chain-to-side chain cyclization, e.g., by ring-closing olefin metathesis, known as stapling, is one approach to increase the biological activity of short peptides that has shown promise when applied to 3(10)- and α-helical peptides. However, atomic resolution structural information on the effect of side chain-to-side chain cyclization in 3(10)-helical peptides is scarce, and reported data suggest that there is significant potential for improvement of existing methodologies. Here, we report a novel stapling methodology for 3(10)-helical peptides using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction in a model aminoisobutyric acid (Aib) rich peptide and examine the structural effect of side chain-to-side chain cyclization by NMR, X-ray diffraction, linear IR and femtosecond 2D IR spectroscopy. Our data show that the resulting cyclic peptide represents a more ideal 3(10)-helix than its acyclic precursor and other stapled 3(10)-helical peptides reported to date. Side chain-to-side chain stapling by CuAAC should prove useful when applied to 3(10)-helical peptides and protein segments of interest in biomedicine.     

14-helical folding in a cyclobutane-containing beta-tetrapeptide

The efficient synthesis of tetrapeptide 5 containing, in alternation, cyclobutane and beta-alanine residues is described. NMR experiments both at low temperature in CDCl(3) and at 298 K in DMSO-d(6) solutions show the contribution of a strong hydrogen bond in the folded major conformation of 5. Temperature coefficients and diffusion times point out a hydrogen bond involving the NH proton from the cyclobutane residue 1 whereas NOEs manifest the high rigidity of the central fragment of the molecule and are compatible with a 14-membered macrocycle. Theoretical calculations predict a most stable folded conformation corresponding to a 14-helix stabilized by a hydrogen bond between NH(10) in the first residue and OC(25) in the third residue. This structure remains unaltered during the molecular dynamics simulation at 298 K in chloroform. All these results provide evidence for a 14-helical folding and reveal the ability of cis-2-aminocyclobutane carboxylic acid residues to promote folded conformations when incorporated into beta-peptides.     

Conformational stability studies of a stapled hexa-β3-peptide library

A library of 14-helical hexa β(3)-peptides was synthesized in order to determine the influence of sequence variation as well as staple size and location on conformational stability. From this study we show that appropriately stapled hexa-β(3)-peptides can allow for a number of variations without significant perturbation of the 14-helix.     

Ring-closing metathesis of olefinic peptides: design, synthesis, and structural characterization of macrocyclic helical peptides

Heptapeptides containing residues with terminal olefin-derivatized side chains (3 and 4) have been treated with ruthenium alkylidene 1 and undergone facile ring-closing olefin metathesis (RCM) to give 21- and 23-membered macrocyclic peptides (5 and 6). The primary structures of peptides 3 and 4 were based upon a previously studied heptapeptide (2), which was shown to adopt a predominantly 3(10)-helical conformation in CDCl(3) solution and an alpha-helical conformation in the solid state. Circular dichroism, IR, and solution-phase (1)H NMR studies strongly suggested that acyclic precursors 3 and 4 and the fully saturated macrocyclic products 7 and 8 also adopted helical conformations in apolar organic solvents. Single-crystal X-ray diffraction of cyclic peptide 8 showed it to exist as a right-handed 3(10)-helix up to the fifth residue. Solution-phase NMR structures of both acyclic peptide 4 and cyclic peptide 8 in CD(2)Cl(2) indicated that the acyclic diene assumes a loosely 3(10)-helical conformation, which is considerably rigidified upon macrocyclization. The relative ease of introducing carbon-carbon bonds into peptide secondary structures by RCM and the predicted metabolic stability of these bonds renders olefin metathesis an exceptional methodology for the synthesis of rigidified peptide architectures.     

Crystallographic characterization of helical secondary structures in alpha/beta-peptides with 1:1 residue alternation

Oligomers that contain both alpha- and beta-amino acid residues in a 1:1 alternating pattern have recently been shown by several groups to adopt helical secondary structures in solution. The beta-residue substitution pattern has a profound effect on the type of helix formed and the stability of the helical conformation. On the basis of two-dimensional NMR data, we have previously proposed that beta-residues with a five-membered ring constraint promote two different types of alpha/beta-peptide helix. The "11-helix" contains i, i+3 CO...H-N hydrogen bonds between backbone amide groups; these hydrogen bonds occur in 11-atom rings. The alpha/beta-peptide "14/15-helix" contains i, i+4 CO...H-N hydrogen bonds, which occur in alternating 14- and 15-atom rings. Here we provide crystallographic data for 14 alpha/beta-peptides that form the 11-helix and/or the 14/15-helix. These results were obtained for a series of oligomers containing beta-residues derived from ( S,S)- trans-2-aminocyclopentanecarboxylic acid (ACPC) and alpha-residues derived from alpha-aminoisobutyric acid (Aib) or l-alanine (Ala). The crystallized alpha/beta-peptides range in length from 4 to 10 residues. Nine of the alpha/beta-peptides display the 11-helix in the solid state, three display the 14/15-helix, and two display conformations that contain both i, i+3 and i, i+4 CO...H-N hydrogen bonds, but not bifurcated hydrogen bonds. Only 3 of the 14 crystal structures presented here have been previously described. These results suggest that longer alpha/beta-peptides prefer the 14/15-helix over the 11-helix, a conclusion that is consistent with previously reported NMR data obtained in solution.     

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.     

Interpreting NMR data for beta-peptides using molecular dynamics simulations

NMR is one of the most used techniques to resolve structure of proteins and peptides in solution. However, inconsistencies may occur due to the fact that a polypeptide may adopt more than one conformation. Since the NOE distance bounds and (3)J-values used in such structure determination represent a nonlinear average over the total ensemble of conformers, imposition of NOE or (3)J-value restraints to obtain one unique conformation is not an appropriate procedure in such cases. Here, we show that unrestrained MD simulation of a solute in solution using a high-quality force field yields a conformational ensemble that is largely compatible with the experimental NMR data on the solute. Four 100 ns MD simulations of two forms of a nine-residue beta-peptide in methanol at two temperatures produced conformational ensembles that were used to interpret the NMR data on this molecule and resolve inconsistencies between the experimental NOEs. The protected and unprotected forms of the beta-peptide adopt predominantly a 12/10-helix in agreement with the qualitative interpretation of the NMR data. However, a particular NOE was not compatible with this helix indicating the presence of other conformations. The simulations showed that 3(14)()-helical structures were present in the ensemble of the unprotected form and that their presence correlates with the fulfillment of the particular NOE. Additionally, all inter-hydrogen distances were calculated to compare NOEs predicted by the simulations to the ones observed experimentally. The MD conformational ensembles allowed for a detailed and consistent interpretation of the experimental data and showed the small but specific conformational differences between the protected and unprotected forms of the peptide.     

Thermal stabilities of homopolymers of amino acids

In order to study the thermal stabilities of the α-helical polyamino acids in the solid state, measurements of the infrared spectra at high temperature, weight loss by thermogravimetry, and the expansion of the α-helix by x-ray diffractometry were carried out on poly(γ-methyl D -glutamate), poly(γ-benzyl L -glutamate), poly-L -alanine, poly(β-benzyl L -aspartate), poly-δ-carbobenzoxy-L -ornithine and poly-ε-carbobenzoxy-L -lysine. The thermal degradation temperatures of these polymers lie between 140°C and 230°C. The α-helical conformation is stable at high temperature in these polyamino acids, except for poly(β-benzyl L -asparatate), unless thermal degradation takes place. As temperature rises, the amide A and the amide I bands of the infrared spectra shift slightly to higher frequencies and the amide II band to lower frequencies. At the same time, the intensities of these amide bands decrease. These changes differ among the different molecules. From the x-ray measurement, it was found that the α-helix expands along the helical axis with temperature. It is expected that the intramolecular hydrogen bonds of the α-helix become weak with increasing temperature and that the state of the hydrogen bonds of the α-helices depends upon the molecules.     

Structural characterizations of oligopyridyl foldamers, α-helix mimetics

Protein-protein interactions are central to many biological processes, from intracellular communication to cytoskeleton assembly, and therefore represent an important class of targets for new therapeutics. The most common secondary structure in natural proteins is an α-helix. Small molecules seem to be attractive candidates for stabilizing or disrupting protein-protein interactions based on α-helices. In our study, we assessed the ability of oligopyridyl scaffolds to mimic the α-helical twist. The theoretical as well as experimental studies (X-ray diffraction and NMR) on conformations of bipyridines in the function of substituent and pyridine nitrogen positions were carried out. Furthermore, the experimental techniques showed that the conformations observed in bipyridines are maintained within a longer oligopyridyl scaffold (quaterpyridines). The alignment of the synthesized quaterpyridine with two methyl substituents showed that it is an α-helix foldamer; their methyl groups overlap very well with side chain positions, i and i + 3, of an ideal α-helix.     

Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency

Antimicrobial alpha-helical alpha-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of beta-amino acids ("beta-peptides") that can adopt an amphiphilic helix defined by 14-membered ring hydrogen bonds ("14-helix") are active against Escherichia coli [Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-12201]. We have created two series of cationic 9- and 10-residue amphiphilic beta-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short beta-peptides encourages 14-helical structure in aqueous solution [Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310]. Circular dichroism of the beta-peptides described here reveals a broad range of 14-helix population in aqueous buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin derivative. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin derivative. Previous studies with conventional peptides (alpha-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because alpha-helix stability can be varied to only a limited extent among linear alpha-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with beta-peptides. For example, it is very rare for a linear alpha-peptide to display significant alpha-helix formation in aqueous solution and manifest antibacterial activity, while the linear beta-peptides described here range from fully unfolded to very highly folded in aqueous solution. This study shows that beta-peptides can be unique tools for analyzing relationships between conformational stability and biological activity.     

Distinctive circular dichroism signature for 14-helix-bundle formation by beta-peptides

We identify a distinctive circular dichroism (CD) signature for self-assembled 14-helical beta-peptides. Our data show that self-assembly leads to a mimimum at 205 nm, which is distinct from the well-known minimum at 214 nm for a monomeric 14-helix. The onset of assembly is indicated by [theta]205/[theta]214>0.7. Our results will facilitate rapid screening for self-assembling beta-peptides and raise the possibility that far-UV CD will be useful for detecting higher-order structure for other well-folded oligoamide backbones.     

Design, synthesis and structural investigations of a beta-peptide forming a 314-helix stabilized by electrostatic interactions

Two different strategies have been employed for the synthesis of Fmoc-protected beta(3)-homoarginine; the Arndt-Eistert homologation of alpha-arginine and the guanidinylation of beta(3)-homoornithine. Solid-phase beta-peptide synthesis was used for the preparation of beta-heptapeptide 1, which was designed to form a helix stabilized by electrostatic interactions through positively (beta(3)hArg) and negatively charged (beta(3)hGlu) amino acid residues. CD measurements and corresponding NMR investigations in MeOH and aqueous solutions do indeed show that the beta-peptidic 3(14)-helix can be stabilized by salt-bridge formation.     

Structural Changes of β-Casein Induced by Temperature and pH Analysed by Nuclear Magnetic Resonance,Fourier-Transform Infrared Spectroscopy,and Chemometrics

This study investigated structural changes in β-casein as a function of temperature (4 and 20 °C) and pH (5.9 and 7.0). For this purpose, nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopy were used, in conjunction with chemometric analysis. Both temperature and pH had strongly affected the secondary structure of β-casein, with most affected regions involving random coils and α-helical structures. The α-helical structures showed great pH sensitivity by decreasing at 20 °C and diminishing completely at 4 °C when pH was increased from 5.9 to 7.0. The decrease in α-helix was likely related to the greater presence of random coils at pH 7.0, which was not observed at pH 5.9 at either temperature. The changes in secondary structure components were linked to decreased hydrophobic interactions at lower temperature and increasing pH. The most prominent change of the α-helix took place when the pH was adjusted to 7.0 and the temperature set at 4 °C, which confirms the disruption of the hydrogen bonds and weakening of hydrophobic interactions in the system. The findings can assist in establishing the structural behaviour of the β-casein under conditions that apply as important for solubility and production of β-casein.     

Relationship between salt-bridge identity and 14-helix stability of beta3-peptides in aqueous buffer

[STRUCTURE: SEE TEXT] We report a systematic analysis of the relationship between salt bridge composition and 14-helix structure within a family of model beta-peptides in aqueous buffer. We find an inverse relationship between side-chain length and the extent of 14-helix structure as judged by CD. Introduction of a stabilizing salt bridge pair within a previously reported beta-peptide ligand for hDM2 led to changes in structure that were detectable by NMR.     

Mechanism for the noncovalent chiral domino effect: new paradigm for the chiral role of the N-terminal segment in a 3(10)-helix

Recently, novel chiral interactions on 3(10)-helical peptides, of which the helicity is controlled by external chiral stimulus operating on the N-terminus, were proposed as a "noncovalent chiral domino effect (NCDE)" (Inai, Y.; et al. J. Am. Chem. Soc. 2000, 122, 11731. Inai, Y.; et al. J. Am. Chem. Soc. 2002, 124, 2466). The present study clarifies the mechanism for generating the NCDE. For this purpose, achiral nonapeptide (1), H-beta-Ala-(Delta(Z)Phe-Aib)(4)-OMe [Delta(Z)Phe = (Z)-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], was synthesized. Peptide 1 alone adopts a 3(10)-helical conformation in chloroform. On the basis of the induced CD signals of peptide 1 with chiral additives, chiral acid enabling the predominant formation of a one-handed helix was shown to need at least both carboxyl and urethane groups; that is, Boc-l-amino acid (Boc = tert-butoxycarbonyl) strongly induces a right-handed helix. NMR studies (NH resonance variations, low-temperature measurement, and NOESY) were performed for a CDCl(3) solution of peptide 1 and chiral additive, supporting the view that the N-terminal H-beta-Ala-Delta(Z)Phe-Aib, including the two free amide NH's, captures effectively a Boc-amino acid molecule through three-point interactions. The H-beta-Ala's amino group binds to the carboxyl group to form a salt bridge, while the Aib(3) NH is hydrogen-bonded to either oxygen of the carboxylate group. Subsequently, the free Delta(Z)Phe(2) NH forms a hydrogen bond to the urethane carbonyl oxygen. A semiempirical molecular orbital computation explicitly demonstrated that the dynamic looping complexation is energetically permitted and that the N-terminal segment of a right-handed 3(10)-helix binds more favorably to a Boc-l-amino acid than to the corresponding d-species. In conclusion, the N-terminal segment of a 3(10)-helix, ubiquitous in natural proteins and peptides, possesses the potency of chiral recognition in the backbone itself, furthermore enabling the conversion of the terminally acquired chiral sign and power into a dynamic control of the original helicity and helical stability.