Lyotropic liquid crystalline phases from helical β-peptides as alignment media

Lyotropic liquid crystalline (LLC) phases from helical β-peptides are proposed as novel water-based alignment media. In contrast to α-peptides, β-peptides form LLCs at very short chain lengths and at concentrations as low as 1 percent. Spectra obtained in these LLC phases are artefact-free and lead to RDCs of the desired size. First indications towards enantiodiscrimination are provided.     

Design and synthesis of glycosylated beta3-peptides capable of folding into the 3(14)-helical conformation in water

Herein, we describe the synthesis of seven glycosylated beta(3)-peptides, 1-7, which were designed to adopt stable 3(14)-helical conformations in aqueous solution. Such molecules are representative for a novel class of functionalized foldamers in which a natural post-translational modification is attached to an unnatural peptidomimetic backbone. Conformational studies by CD spectroscopic measurements were performed in methanol and in water (pH 7). Additionally, the influence of temperature, pH, and concentration on the ability of glycosylated beta(3)-peptides to adopt stable helical conformations were investigated. The first NMR-derived solution state structure of a glycosylated beta(3)-peptide in water is also presented.     

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.     

Synthesis and structural study of novel dimethylcyclobutyl β-peptides

The synthesis of some representative compounds of a new class of cyclobutane-containing β-peptides starting from (?)-verbenone as a chiral precursor is presented. In these products, the cyclobutane moiety is not a part of the peptide backbone but a bulky substituent at the β³-position. These compounds have been carefully characterized and studied on the basis of the combined use of several experimental techniques together with molecular modeling by means of theoretical calculations. In the solid state, the non-cyclic β-peptides adopt a hairpin-like molecular folding ruled by intermolecular hydrogen bonds in the crystal packing.     

‘Mixed’ β-peptides: A unique helical secondary structure in solution. Preliminary communication

β-Hexapeptides 1–5 and a β-dodecapeptide 6 with sequences containing two different types of β-amino acids (aliphatic proteinageous side chains in the 2- or in the 3-position) have been prepared. CD (Fig. 1) and NMR measurements indicate that, with one exception, the secondary structures formed by these new β-peptides differ from those of isomers studied previously. Detailed NMR analysis of the β-hexapeptide 5 (with alternating β²,β³-building blocks) and molecular-dynamics simulations have produced a minimum energy conformation (Fig. 2,b)which might be described as a novel irregular helix containing ten- and twelve-membered H-bonded rings. This demonstrates the great structural variability of β-peptides, since three different helical secondary structures have been discovered to date.     

Supramolecular Self-Assembly of β3-Peptides Mediated by Janus-Type Recognition Units

To gain mechanistic insights, natural systems with biochemical relevance are inspiring for the creation of new biomimetics with unique properties and functions. Despite progress in rational design and protein engineering, folding and intramolecular organization of individual components into supramolecular structures remains challenging and requires controlled methods. Foldamers, such as β-peptides, are structurally well defined with rigid conformations and suitable for the specific arrangement of recognition units. Herein, we show the molecular arrangement and aggregation of β³-peptides into a hexameric helix bundle. For this purpose, β-amino acid side chains were modified with cyanuric acid and triamino-s-triazine as complementary recognition units. The pre-organization of the β³-peptides leads these Janus molecule pairs into a hexameric arrangement and a defined rosette nanotube by stacking. The helical conformation of the subunits was indicated by circular dichroism spectroscopy, while the supramolecular arrangement was detected by dynamic light scattering and confirmed by high-resolution electrospray ionization mass spectrometry (ESI-HRMS).     

Probing the Helical Secondary Structure of Short-Chain β-Peptides

Structural prerequisites for the stability of the 3₁ helix of β-peptides can be defined from inspection of models (Figs. 1 and 2): lateral non-H-substituents in 2- and 3-position on the 3-amino-acid residues of the helix are allowed, axial ones are forbidden. To be able to test this prediction, we synthesized a series of heptapeptide derivatives Boc-(β-HVal-β-HAla-β-HLeu-Xaa-β-HVal-β-HAla-β-HLeu)-OMe 13–22 (Xaa = α- or β-amino-acid residue) and a β-depsipeptide 25 with a central (S)-3-hydroxybutanoic-acid residue (Xaa = –OCH(Me)CH₂C(O)–) (Schemes 1 3). Detailed NMR analysis (DQF-COSY, HSQC, HMBC, ROESY, and TOCSY experiments) in methanol solution of the β-hexapeptide H(-β-HVal-β-HAla-β-HLeu)₂-OH ( 1 ) and of the β-heptapeptide H-β-HVal-β-HAla-β-HLeu-(S,S)-β-HAla(αMe)-β-HVal-β-HAla- β-HLeu-OH ( 22 ), with a central (2S,3S)-3-amino-2-methylbutanoic-acid residue, confirm the helical structure of such β-peptides (previously discovered in pyridine solution) (Fig.3 and Tables 1–5). The CD spectra of helical β-peptides, the residues of which were prepared by (retentive) Arndt-Eistert homologation of the (S)- or L -α-amino acids, show a trough at 215 nm. Thus, this characteristic pattern of the CD spectra was taken as an indicator for the presence of a helix in methanol solutions of compounds 13–22 and 25 (including partially and fully deprotected forms) (Figs.4–6). The results fully confirm predicted structural effects: incorporation of a single ‘wrong’ residue ((R)-β-HAla, β-HAib, (R,S)-β-HAla(α Me), or N-Me-β-HAla) in the central position of the β-heptapeptide derivatives A (see 17, 18, 20 , or 21 , resp.) causes the CD minimum to disappear. Also, the β-heptadepsipetide 25 (missing H-bond) and the β-heptapeptide analogs with a single α-amino-acid moiety in the middle ( 13 and 14 ) are not helical, according to this analysis. An interesting case is the heptapeptide 15 with the central achiral, unsubstituted 3-aminopropanoic-acid moiety: helical conformation appears to depend upon the presence or absence of terminal protection and upon the solvent (MeOH vs. MeOH/H₂O).     

Lyotropic liquid crystals formed from ACHC-rich β-peptides

We have examined the effect of β-peptide modifications on the propensity of these helical molecules to form lyotropic liquid crystalline (LC) phases in water. All of the β-peptides we have examined contain 10 residues. In each case, at least three residues are derived from trans-2-aminocyclohexanecarboxylic acid (ACHC), which strongly promotes folding to a 14-helical conformation. The structural features varied include the number of ACHC residues, the nature and spatial arrangement of charged side chains (cationic vs anionic), and the identity of groups at the β-peptide termini. We found that relatively small changes (e.g., swapping the positions of a cationic and an anionic side chain) could have large effects, such as abrogation of LC phase formation. The trends revealed by sequence-property studies led to the design of LC-forming β-peptides that bear biomolecular recognition groups (biotin or the tripeptide Arg-Gly-Asp). Structural analysis via circular dichroism and cryo-transmission electron microscopy revealed the existence of two different types of self-associated species, globular aggregates and nanofibers. Nanofibers are the predominant assembly formed at concentrations that lead to LC phase formation, and we conclude that these nanofibers are the functional mesogens. Overall, these studies show how the modularity of β-peptide oligomers enables elucidation of the relationship between molecular structure and large-scale self-assembly behavior.     

Sheet-like assemblies of charged amphiphilic α/β-peptides at the air-water interface

There is growing interest in the design of molecules that undergo predictable self-assembly. Bioinspired oligomers with well-defined conformational propensities are attractive from this perspective, since they can be constructed from diverse building blocks, and self-assembly can be directed by the identities and sequence of the subunits. Here we describe the structure of monolayers formed at the air-water interface by amphiphilic α/β-peptides with 1:1 alternation of α- and β-amino acid residues along the backbone. Two of the α/β-peptides, one a dianion and the other a dication, were used to determine differences between self-assemblies of the net negatively and positively charged oligomers. Two additional α/β-peptides, both zwitterionic, were designed to favor assembly in a 1:1 molar ratio mixture with parallel orientation of neighboring strands. Monolayers formed by these α/β-peptides at the air-water interface were characterized by surface pressure-area isotherms, grazing incidence X-ray diffraction (GIXD), atomic force microscopy and ATR-FTIR. GIXD data indicate that the α/β-peptide assemblies exhibited diffraction features similar to those of β-sheet-forming α-peptides. The diffraction data allowed the construction of a detailed model of an antiparallel α/β-peptide sheet with a unique pleated structure. One of the α/β-peptide assemblies displayed high stability, unparalleled among previously studied assemblies of α-peptides. ATR-FTIR data suggest that the 1:1 mixture of zwitterionic α/β-peptides assembled in a parallel arrangement resembling that of a typical parallel β-sheet secondary structure formed by α-peptides. This study establishes guidelines for design of amphiphilic α/β-peptides that assemble in a predictable manner at an air-water interface, with control of interstrand orientation through manipulation of Coulombic interactions along the backbone.     

Relationship between side-chain branching and stoichiometry in β(3)-peptide bundles

The stability and stoichiometry of β(3)-peptide bundles is influenced by side-chain identity. β(3)-peptides containing β(3)-homoleucine on one helical face assemble into octamers, whereas those containing β(3)-homovaline form tetramers. From a structural perspective, the side chains of β(3)-homoleucine and β(3)-homovaline differ in terms of both side-chain length and γ-carbon branching. To evaluate the extent to which these two parameters control β(3)-peptide bundle stoichiometry, we synthesized the β(3)-peptide Acid-3Y, which contains β(3)-homoisoleucine in place of β(3)-homoleucine or β(3)-homovaline. Acid-3Y assembles into a stable tetramer whose stability resembles that of the previously characterized Acid-VY tetramer. These results suggest that β(3)-peptide bundle stoichiometry is dominated by the presence or absence of γ-carbon branching on core side chains.     

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.     

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.     

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.     

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.     

Water-Insensitive Synthesis of Poly-β-Peptides with Defined Architecture

Biocompatible and proteolysis-resistant poly-β-peptides have broad applications and are dominantly synthesized via the harsh and water-sensitive ring-opening polymerization of β-lactams in a glovebox or using a Schlenk line, catalyzed by the strong base LiN(SiMe₃)₂. We have developed a controllable and water-insensitive ring-opening polymerization of β-amino acid N-thiocarboxyanhydrides (β-NTAs) that can be operated in open vessels to prepare poly-β-peptides in high yields, with diverse functional groups, variable chain length, narrow dispersity and defined architecture. These merits imply wide applications of β-NTA polymerization and resulting poly-β-peptides, which is validated by the finding of a HDP-mimicking poly-β-peptide with potent antimicrobial activities. The living β-NTA polymerization enables the controllable synthesis of random, block copolymers and easy tuning of both terminal groups of polypeptides, which facilitated the unravelling of the antibacterial mechanism using the fluorophore-labelled poly-β-peptide.     

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.     

A new peptide motif in the formation of supramolecular double helices

The single crystal X-ray diffraction studies of a new tripeptide motif Boc-Tyr-Aib-Xaa-OMe (Xaa = Leu/Ile/Ala) reveal that the peptides adopt β-turn conformations which self-assemble to form a supramolecular double helical structure using various non-covalent interactions in the solid state and the peptides exhibit a type-III N(2) sorption isotherm.     

Molecular modeling study for a novel structured oligomer subunit selection: the example of 2-aminomethyl-phenyl-acetic acid

Oligomers derived from dipeptide mimics were selected by computational study for their suitability to fold in ordered structures. After selection of a monomeric unit, short oligomers were synthesized and analyzed by NMR and IR. Oligomers built from 2-aminomethyl-phenyl-acetic acid were shown to adopt a helical structure stabilized by 10-membered ring hydrogen bonds.     

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.