Stereoselective synthesis of 3-substituted 2-aminocyclopentanecarboxylic acid derivatives and their incorporation into short 12-helical beta-peptides that fold in water

A stereoselective synthetic route is reported for the introduction of side chains at the 3-position of trans-2-aminocyclopentanecarboxylic acid (ACPC). Ring opening of the aziridine 2-benzyloxymethyl-6-azabicyclo[3.1.0]hexane with selected nucleophiles occurs in a regioselective manner and provides ACPC precursors with functional groups at the 3-position, trans to the 2-amino group. Oligomers composed of the 3-substituted ACPC residues maintain the 12-helical conformation displayed by the nonsubstituted analogues, as shown by their similar circular dichroism signatures. The added diversity of the new residues provides good dispersion of NMR signals, allowing the assignment of nearly all the NOE signals of a selected hexamer in aqueous solution. The NOEs between protons on nonadjacent residues are characteristic of the 12-helix. 3-Substituted ACPC residues allow one to arrange specific functional groups in a geometrically defined fashion, which should facilitate the design of beta-peptides for biological applications.     

Tolerance of acyclic residues in the beta-peptide 12-helix: access to diverse side-chain arrays for biological applications

Oligomeric backbones with well-defined conformational propensities can serve as scaffolds for displaying sets of functional groups in specific three-dimensional arrangements. beta-Peptides are particularly interesting in this regard because several distinct secondary structures can be induced by appropriate choice of beta-amino acid substitution pattern.3 The beta-peptide 12-helix (defined by 12-membered ring C=O(i)- -H-N(i + 3) hydrogen bonds) is of particular interest because this helix resembles the alpha-helix. To date 12-helices have been observed in beta-peptides comprised exclusively of residues containing a five-membered ring constraint. Here we show that 12-helical propensity is maintained when some cyclic beta-amino acid residues are replaced with more flexible acyclic residues. This result is important because use of acyclic residues greatly facilitates introduction of diverse side chains at specific sites along the 12-helix. We demonstrate the utility of this advance in the context of antibiotic design.     

Helical beta-peptide inhibitors of the p53-hDM2 interaction

hDM2 is recognized in vivo by a short alpha-helix within the p53 trans-activation domain (p53AD). Disruption of the p53.hDM2 interaction is an important goal for cancer therapy. A functional epitope comprised of three residues on one face of the p53AD helix (F19, W23, and L26) contributes heavily to the binding free energy. We hypothesized that the p53AD functional epitope would be recapitulated if the side chains of F19, W23, and L26 were presented at successive positions three residues apart on a stabilized beta3-peptide 14-helix. Here, we report a set of beta3-peptides that possess significant 14-helix structure in water; one recognizes a cleft on the surface of hDM2 with nanomolar affinity. The strategy for beta3-peptide design that we describe is general and may have advantages over one in which individual or multiple beta-amino acid substitutions are introduced into a functional alpha-peptide, because it is based on homology at the level of secondary structure, not primary sequence.     

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.     

Synthesis of 4,4-disubstituted 2-aminocyclopentanecarboxylic acid derivatives and their incorporation into 12-helical beta-peptides

An enantioselective synthetic route is reported for trans-2-aminocyclopentanecarboxylic acids (ACPC) bearing geminal side chain pairs at the 4-position. Beta-peptides containing the 4,4-disubstituted ACPC residues adopt the 12-helical conformation, as demonstrated by 2D NMR analysis in aqueous solution. These 4,4-disubstituted ACPC residues display functional groups, including acidic and hydrogen bond donating groups, in a geometrically defined fashion, which should be useful for the design of beta-peptides for specific applications. [structure: see text]     

Synthesis and characterisation of helical beta-peptide architectures that contain (S)-beta3-HDOPA(Crown Ether) derivatives

A new set of beta-amino acids that carry various crown ether receptors on their side chains of the general formula (S)-beta(3)-HDOPA(crown ether) (HDOPA: homo-3,4-dihydroxyphenylalanine; (crown ether): [15]crown-5 ([15-C-5]), [18]crown-6 ([18-C-6]), [21]crown-7 ([21-C-7]), 1,2-Benzo-[24]crown-8 ([Benzo-24-C-8]) and (R)-Binol-[20]crown-6 ([(R)-Binol-20-C-6])) was prepared. Peptides that are based on these new crowned beta-amino acids combined with (1S,2S)-ACHC (2-aminocyclohexanecarboxylic acid), which is known to be a potent 3(14)-helix inducer, to the hexamer level, with two crowned residues at the i and i+3 positions of the main-chain, were synthesized in solution by stepwise coupling using Boc-N(alpha)-protection (Boc: tert-butoxycarbonyl) and the EDC/HOAt C-activation method. Their conformational analysis was performed by using FTIR absorption, NMR and CD spectroscopy techniques. Our results are in full agreement with a 3(14)-helix conformation.     

Design of a "new motif" with beta-amino acids and alpha-aminoxy acids: synthesis of hybrid peptides with 12/10-helix

Hybrid peptides are prepared from a C-linked carbo-beta-amino acid ester (R-beta-Caa) and an alpha-aminoxy acid (R-Ama) derived from S-lactic acid. Extensive NMR (in CDCl 3 solution), CD, and MD studies on the tetra- and hexapeptides led to identification of robust 12/10-mixed helices. The dipeptide repeat having an R-beta-Caa and an R-Ama thus provides a "new motif" to realize a 12/10-mixed helix, for the first time, in oligomers containing R-Ama. To understand the impact of side chains in the mixed helix formation, R-beta-Caa/Ama (with no substitution in Ama) and S-beta-hAla/R-Ama oligomers were investigated. NMR studies revealed the existence of 12/10-helices in these hybrid peptides, and the side chains of monomers were found to have a profound influence on their stabilities. These observations imply that the propensity of beta-amino acid to prefer a mixed 12/10-helix governs the structural behavior in these peptides. The structural consequences of the lone-pair repulsion between nitrogen and oxygen atoms result in a new and interesting structural motif which behaves like "pseudo" beta (3),beta(2)-peptides in generating 12/10-mixed helices.     

Negative ion fragmentations of deprotonated peptides: backbone cleavages directed through both Asp and Glu

The collision-induced spectra of [M - H](-) ions of a variety of natural and synthetic amphibian peptides containing Asp and/or Glu exhibit characteristic gamma backbone cleavage ions that identify the positions of these residues in the peptide. A theoretical study suggests that the Glu cleavage involves an S(N)i reaction of the carboxylate anion from the Glu alpha side chain to form a deprotonated cyclic lactone. The presence of either Asp or Glu or other residues that effect pronounced side-chain cleavages (e.g. Ser or Thr) results in the normal alpha and beta backbone cleavages being reduced in comparison to those cleavages which originate from side chains.     

Environment-independent 14-helix formation in short beta-peptides: striking a balance between shape control and functional diversity

We report a significant and unanticipated advance in the study of beta-amino acid-based foldamers: a small proportion of highly preorganized residues can impart high stability to a specific helical secondary structure in water. Most of the residues in these beta-peptides (2 and 3) are intrinsically flexible. Flexible beta-amino acids can be readily and enantiospecifically prepared in functionally diverse forms, but preorganized residues with side chains are rare and challenging to synthesize. Our findings demonstrate that interspersing a few copies of an unfunctionalized but rigid residue among a larger number of flexible residues with diverse side chains is a viable strategy for creating beta-peptides that adopt the 14-helix conformation and therefore display side chains in a predictable spatial arrangement. These results are significant because they enhance the prospects of developing beta-peptides with useful activities.     

Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH(2)O), Thr (-CH(3)CHO) and Asp (-H(2)O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert.     

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.     

Evaluation of diverse α/β-backbone patterns for functional α-helix mimicry: analogues of the Bim BH3 domain

Peptidic oligomers that contain both α- and β-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/β-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-x(L) and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/β-peptide homologues retain the side-chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → β(3)-residue substitutions. Previous work has shown that the ααβαααβ pattern, which aligns the β(3)-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here strengthen this conclusion. The present study provides the first evaluation of functional mimicry by ααβ and αααβ patterns, which cause the β(3)-residues to spiral around the helix periphery. We find that the αααβ pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/β-peptide bound to Bcl-x(L), affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααβ homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide.     

Functionalization of SWNTs to facilitate the coordination of metal ions, compounds and clusters

The carboxylate residues of the open ends of aryl-tert-butyl and arylsulfonic acid side-walled functionalized single walled carbon nanotubes (SWNTs) have been investigated for the complexation conditions of the iron-molybdenum cluster [H(x)PMo(12)O(40)CH(4)Mo(72)Fe(30)(O(2)CMe)(15)O(254)(H(2)O)(98)] ("FeMoC"). A range of alternative donor groups for the attachment of FeMoC have been investigated for piranha etched SWNTs, dodecyl side-walled functionalized SWNTs (DD-SWNTs) and ultra-short SWNTs (US-SWNTs), including include pyridines, thiols and phosphines, using coupling reactions to either the carboxylate or hydroxide residues of the SWNTs' open ends. The functionalized SWNTs have been characterized by XPS, uptake of Fe(3+) and, where appropriate, MAS (31)P NMR. The efficacy of binding is dependent on the presence and identity of the ligand moiety. TEM and AFM of the SWNT-FeMoC conjugates show the presence of a 2-3 nm spherical feature on the tip of individual SWNTs.     

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.     

Synthesis and Conformational Characterisation of Hexameric β‐Peptide Foldamers by Using Double POAC Spin Labelling and cw‐EPR

A selected set of terminally protected β‐hexapeptides, each containing two nitroxide‐based (3R,4R)‐4‐amino‐1‐oxyl‐2,2,5,5‐tetramethylpyrrolidine‐3‐carboxylic acid (POAC) residues combined with four (1S,2S)‐2‐aminocyclopentane‐1‐carboxylic acid (ACPC) residues, was synthesised by using solution methods and was fully characterised. The two POAC residues are separated in the sequences by different numbers of intervening ACPC residues. The conformational features of the doubly spin‐labelled β‐hexapeptides were examined in chloroform by FTIR absorption and continuous‐wave electron paramagnetic resonance spectroscopic techniques. In particular, the biradical exchange coupling (J) between two POAC residues within each peptide indicates unambiguously that the secondary structure overwhelmingly adopted is the 12‐helix. Taken together, these results support the view that POAC is an excellent β‐amino acid for exploring this type of helical conformation in doubly labelled β‐peptides.     

Effects of the alternating backbone configuration on the secondary structure and self-assembly of beta-peptides

Heterochiral homo-oligomers with alternating backbone configurations were constructed by using the different enantiomers of the cis- and trans-2-aminocyclopentanecarboxylic acid (ACPC) monomers. Molecular modeling and the spectroscopic techniques (NMR, ECD, and VCD) unequivocally proved that the alternating heterochiral cis-ACPC sequences form an H10/12 helix, where extra stabilization can be achieved via the cyclic side chains. The ECD and TEM measurements, together with molecular modeling, revealed that the alternating heterochiral trans-ACPC oligomers tend to attain a polar-strand secondary structure in solution, which can self-assemble into nanostructured fibrils. The observations indicate that coverage of all the possible secondary structures (various helix types and strand-mimicking conformations) can be attained with the help of cyclic beta-amino acid diastereomers. A relationship has been established between the backbone chirality pattern and the prevailing secondary structure, which underlines the role of stereochemical control in the beta-peptide secondary structure design and may contribute to future biological applications.     

Renin inhibitors. I. Synthesis and structure-activity relationships of transition-state inhibitors containing homostatine analogues at the scissile bond.

The synthesis and structure-activity relationships of transition-state renin inhibitors containing the homostatine analogues at the scissile bond are described. These inhibitors incorporate the amino acid side chains corresponding to positions 7-12 (P4-P2') of angiotensinogen. Ethyl, 2-hydroxyethyl and 3-hydroxypropyl groups at position 2 of the homostatine analogues (P1') are more effective for increasing potency than the isopropyl group. A combination of residues at P1, P3 and P4 is important for potency and this result suggests that S1, S3 and S4 form a huge hydrophobic core together in renin.     

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.     

Interplay among folding, sequence, and lipophilicity in the antibacterial and hemolytic activities of alpha/beta-peptides

Host-defense peptides inhibit bacterial growth but manifest relatively little toxicity toward eukaryotic cells. Many host-defense peptides adopt alpha-helical conformations in which cationic side chains and lipophilic side chains are segregated to distinct regions of the molecular surface ("globally amphiphilic helices"). Several efforts have been made to develop unnatural oligomers that mimic the selective antibacterial activity of host-defense peptides; these efforts have focused on the creation of molecules that are globally amphiphilic in the preferred conformation. One such endeavor, from our laboratories, focused on helix-forming alpha/beta-peptides, i.e., oligomers containing a 1:1 pattern of alpha- and beta-amino acid residues in the backbone [Schmitt, M. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2004, 126, 6848-6849]. We found, unexpectedly, that the most favorable biological activity profile was displayed by a "scrambled" sequence, which was designed not to be able to form a globally amphiphilic helix. Here we report new data, involving an expanded set of alpha/beta-peptides, from experiments designed to elucidate the origins of this surprising result. In addition, we evaluate the susceptibility of alpha/beta-peptides to proteolytic degradation. Our results support the hypothesis that the ability to adopt a globally amphiphilic helical conformation is not a prerequisite for selective antibacterial activity. This conclusion represents a significant advance in our understanding of the relationship among molecular composition, conformation, and biological activity. Our results should therefore influence the design of other unnatural oligomers intended to function as antibacterial agents.     

Hairpin folding behavior of mixed α/β-peptides in aqueous solution

The invention of new strategies for the design of protein-mimetic oligomers that manifest the folding encoded in natural amino acid sequences is a significant challenge. In contrast to the α-helix, mimicry of protein β-sheets is less understood. We report here the aqueous folding behavior of a prototype α-peptide hairpin model sequence varied at cross-strand positions by incorporation of 16 different β-amino acid monomers. Our results provide a folding propensity scale for β-residues in a protein β-sheet context as well as high-resolution structures of several mixed-backbone α/β-peptide hairpins in water.