Supramolecular systems composed of α-helical peptides

Hydrophobic helical peptides having alternating hydrophobic amino acid and Aib in the sequence were synthesized to construct supramolecular systems. Three types of supramolecular systems were constructed by the peptides and the derivatives in different environments. First, the dispersion of TFA · H-(Ala-Aib)₈-OBzl in water was studied by dynamic light scattering, which suggests the formation of a vesicular structure with an average diameter of 76 nm. We call the peptide assembly in water “peptosome”. Second, Boc-Ser(Ant)-(Ala-Aib)₈-OMe spanned the phospholipid bilayer membrane and formed a helix-bundle structure. The bundle structure was supported by ion-channel formation in the membrane. Third, Boc-(Ala-Aib)₈-OMe and Boc-(Leu-Aib)₈-OBzl formed a two-dimensional crystal at the air-water interface. Boc-(Ala-Aib)₁₂-OBzl also formed a monolayer in a solid state at the air-water interface, but the helix orientation was perpendicular to the interface, which presents a contrast to the parallel orientation of the former hexadecapeptides.     

Stabilization of β-hairpin structures via inter-strand π-π and hydrogen bond interactions in α-, β-, γ-hybrid peptides

Synthesis and conformational studies of α-, β-, γ-hybrid peptides containing a pyrrole amino acid (Paa, 1) and a furan amino acid (Faa, 2), namely Boc-β-Phe-Faa-d-Pro-Gly-Paa-β-HGly-Faa-OMe (3) and Boc-Paa-β-Phe-Faa-d-Pro-Gly-Paa-β-HGly-Faa-OMe (4), were carried out and they adopt β-hairpin structures stabilized via inter-strand π-π and hydrogen bonding interactions.     

Molecular dynamics studies of α-helix stability in fibril-forming peptides

Diseases associated with protein fibril-formation, such as the prion diseases and Alzheimer’s disease, are gaining increased attention due to their medical importance and complex origins. Using molecular dynamics (MD) simulations in an aqueous environment, we have studied the stability of the α-helix covering positions 15–25 of the amyloid β-peptide (Aβ) involved in Alzheimer’s disease. The effects of residue replacements, including the effects of Aβ disease related mutations, were also investigated. The MD simulations show a very early (2 ns) loss of α-helical structure for the Flemish (Aβ(A21G)), Italian (Aβ(E22K)), and Iowa (Aβ(D23N)) forms associated with hereditary Alzheimer’s disease. Similarly, an early (5 ns) loss of α-helical structure was observed for the Dutch (Aβ(E22Q)) variant. MD here provides a possible explanation for the structural changes. Two variants of Aβ, Aβ(K16A,L17A,F20A) and Aβ(V18A,F19A,F20A), that do not produce fibrils in vitro were also investigated. The Aβ(V18A,F19A,F20A) initially loses its helical conformation but refolds into helix several times and spends most of the simulation time in helical conformation. However, the Aβ(K16A,L17A,F20A) loses the α-helical structure after 5 ns and does not refold. For the wildtype Aβ(1–40) and Aβ(1–42), the helical conformation is lost after 5 ns or after 40 ns, respectively, while for the “familial” (Aβ(A42T)) variant, the MD simulations suggest that a C-terminal β-strand is stabilised, which could explain the fibrillation. The simulations for the Arctic (Aβ(E22G)) variant indicate that the α-helix is kept for 2 ns, but reappears 2 ns later, whereafter it disappears after 10 ns. The MD results are in several cases compatible with known experimental data, but the correlation is not perfect, indicating that multimerisation tendency and other factors might also be important for fibril formation.     

Synthesis of α-fluoroalkyl substituted peptides via enzymatic fragment condensation

A peptide library consisting of di- and tripeptide esters and tripeptide amides, respectively, containing α-Tfm amino acids in different positions was synthesized and tested for enzymatic fragment condensations catalyzed by the proteases α-chymotrypsin, trypsin and clostripain.     

Formation of α-helix-based twisted ribbon-like fibrils from ionic-complementary peptides

Ionic-complementary peptides (ICPs) are well known for their strong propensity to form amyloid-like β-sheet fibrils. Here, we present the first example that α-helical based ICPs can self-assemble into a highly ordered fibrillar structure. Intriguingly, the individual α-helices in such fibrils are arranged shoulder-to-shoulder, making them distinct from conventional coiled-coil-based fibrils.     

Syntheses and lipophilicity measurement of N/N-terminus-1,1-dihydroperfluoroalkylated α-amino acids and small peptides

(1,1-Dihydroperfluoroalkyl)phenyliodonium N,N-bis(trifluoromethylsulfonyl)imides (4, n = 0-2) were synthesized and used to transfer the corresponding 1,1-dihydroperfluoroalkyl groups to the α-amino group of (l)tyrosine. The obtained N^α-2,2,2-trifluoroethylated (l)tyrosine (6, n = 0) was further used as the N-terminus in the solid phase peptide synthesis of leucine enkephalin analogue. The lipophilicity of the N^α-1,1-dihydroperfluoroalkylated (l)tyrosines (6, n = 0-2) and N-terminus-2,2,2-trifluoroethylated leucine enkephalin analogue (7), as well as the corresponding parent compounds, was measured.     

Conformational studies on peptides containing α,α-disubstituted α-amino acids: chiral cyclic α,α-disubstituted α-amino acid as an α-helical inducer

Four types of α,α-disubstituted amino acids {i.e., α-aminoisobutyric acid (Aib), 1-aminocyclopentanecarboxylic acid (Ac(5)c), (3S,4S)-1-amino-(3,4-dimethoxy)cyclopentanecarboxylic acid [(S,S)-Ac(5)c(dOM)] and its enantiomer (R,R)-Ac(5)c(dOM)} were introduced into l-leucine-based hexapeptides and nonapeptides. The dominant conformations of eight peptides: Cbz-(L-Leu-L-Leu-dAA)(2)-OMe [dAA = 1: Aib; 2: Ac(5)c; 3: (S,S)-Ac(5)c(dOM); 4: (R,R)-Ac(5)c(dOM)] and Boc-(L-Leu-L-Leu-dAA)(3)-OMe [dAA = 5: Aib; 6: Ac(5)c; 7: (S,S)-Ac(5)c(dOM); 8: (R,R)-Ac(5)c(dOM)], were investigated by IR, CD spectra and X-ray crystallographic analysis. The CD spectra revealed that Aib hexapeptide 1 and Ac(5)c hexapeptide 2 formed right-handed (P) 3(10)-helices, while Ac(5)c(dOM) hexapeptides 3 and 4 formed a mixture of (P) 3(10)- and α-helices. The Aib nonapeptide 5 formed a (P) 3(10)-helix, the Ac(5)c nonapeptide 6 formed a mixture of (P) 3(10)- and α-helices, and the Ac(5)c(dOM) nonapeptides 7 and 8 formed (P) α-helices. X-Ray crystallographic analysis revealed that the Aib hexapeptide 1 formed a (P) 3(10)-helix, while (S,S)-Ac(5)c(dOM) hexapeptide 3 formed a (P) α-helix. In addition, the Ac(5)c nonapeptide 6 and (R,R)-Ac(5)c(dOM) nonapeptide 8 formed (P) α-helices. The Aib and achiral Ac(5)c residues have the propensity to form 3(10)-helices in short peptides, whereas the chiral Ac(5)c(dOM) residues have a penchant for forming α-helices.     

Modification of N-terminal α-amino groups of peptides and proteins using ketenes

A method of highly selective N-terminal modification of proteins as well as peptides by an isolated ketene was developed. Modification of a library of unprotected peptides XSKFR (X varies over 20 natural amino acids) by an alkyne-functionalized ketene (1) at room temperature at pH 6.3 resulted in excellent N-terminal selectivity (modified α-amino group/modified ε-amino group = >99:1) for 13 out of the 20 peptides and moderate-to-high N-terminal selectivity (4:1 to 48:1) for 6 of the 7 remaining peptides. Using an alkyne-functionalized N-hydroxysuccinimide (NHS) ester (2) instead of 1, the modification of peptides XSKFR gave internal lysine-modified peptides for 5 out of the 20 peptides and moderate-to-low N-terminal selectivity (5:1 to 1:4) for 13 out of the 20 peptides. Proteins including insulin, lysozyme, RNaseA, and a therapeutic protein BCArg were selectively N-terminally modified at room temperature using ketene 1, in contrast to the formation of significant or major amounts of di-, tri-, or tetra-modified proteins in the modification by NHS ester 2. The 1-modified proteins were further functionalized by a dansyl azide compound through click chemistry without the need for prior treatment.     

Interactions between octyl-β-D-glucoside and α-amino acids or small peptides

The interactions between octyl--D-glucoside and glycine in water have been investigated by surface tension, viscosity, and density measurements. The results show that the -amino acid causes an unexpected lowering of the critical micellar concentration of octyl--D-glucoside. Such a finding has been interpreted in temss of dipole-dipole interactions between the hydrophilic site of the surfactant and the peptidic cosluttes. From three to seven amino acid molecules have been estimated to be coordinated with each glucoside unity in the micellar state. The research has been extended to glycine oligopeptides and L-lysine. The latter compound has effects similar to those observed with glycine whereas diglycine and triglycine show weaker effects on the micellization process.     

Electron transfer across α-helical peptides: Potential influence of molecular dynamics

Three hydrophobic leucine-rich peptides Fc18L, Ac18L and 18LAc were prepared. These peptides are equipped with a cystein sulfhydryl group which enables the formation of thin films on gold surfaces. Using these peptides, two types of films of α-helical peptides have been prepared, in which the redox-active peptide Fc18L is diluted by Ac18L (SAM1) or by a mixture of Ac18L and 18LAc (SAM2). In SAM1, the dipole moments of the peptides are aligned in the same direction, whereas in SAM2, they are opposite. Reflection absorption infrared spectroscopy (RAIRS) revealed that the peptides are more vertically oriented in SAM2 compared to those in SAM1. The interaction among the macroscopic helix dipoles gives tighter packing of the peptides in SAM2. Importantly, the electron transfer properties in the two films are significantly different, which is rationalized by differences in the molecular dynamics of the two films.     

Multi-layer coarse-graining polarization model for treating electrostatic interactions of solvated α-conotoxin peptides

A multi-layer coarse-graining (CG) model is presented for treating the electrostatic interactions of solvated α-conotoxin peptides. According to the sensitivity to the electrostatic environment, a hybrid set of electrostatic parameters, such as secondary-structure- and residue-based dipoles, and atom-centered partial charges, are adopted. For the polarization "inert" secondary-structures and residues, the fragment dipole moments are distributed within narrow ranges with the magnitude close to zero. The coarse-graining fragment dipoles are parameterized from a large training set (10,000 configurations) to reproduce the electrostatic features of molecular fragments. In contrast, the electrostatically "sensitive" atoms exhibit large fluctuations of charges with the varied environments. The environment-dependent variable charges are updated in each energetic calculation. The electrostatic interaction of the whole chemical system is hence partitioned into several sub-terms coming from the fragment dipole-dipole, (fragment) dipole-(atom) charge, and atom charge-charge interactions. A large number of test calculations on the relative energies of cyclo-peptide conformers have demonstrated that the multi-layer CG electrostatic model presents better performance than the non-polarized force fields, in comparison with the density-functional theory and the fully polarized force field model. The selection of CG fragment centers, mass or geometric center, has little influence on the fragment-based dipole-dipole interactions. The multi-layer partition of electrostatic polarization is expected to be applied to many biologically interesting and complicated phenomena.     

Studies of β-turn opening with model peptides containing non-coded α-amino isobutyric acid

Single crystal X-ray diffraction studies show that among the three terminally protected model tripeptides I-III, Boc-Ile-Aib-Xx-OMe (Xx in peptide I: Val; II: Leu; III: Phe) with a centrally placed non-coded amino acid Aib (Aib: α-amino isobutyric acid), peptide I displays a conformational preference for β-turn, peptide II forms a hydrated β-turn representing the solvent mediated intermediate for the interconversion between β-turn and β-strand and peptide III adopts a completely unfolded β-strand like structure. By varying the steric bulk of the third residue, Xx(3), various conformations related to the structural interconversion between the β-turn and β-strand have been isolated. The peptide conformations in the solution phase have been probed by solvent dependent NMR titration and CD spectroscopy. Morphological studies with scanning electron microscopy (SEM) reveal that among the three peptides only peptide III can form filamentous fibrils in the solid state.     

New cyclic RGD peptides: synthesis,characterization, and theoretical activity towards αvβ3 integrin

Two new cyclic RGD peptides were prepared using a click chemistry approach. The linear RGDfV peptide was synthesized by solid-phase peptide synthesis using a 9-fluorenylmetoxicarbonyl (Fmoc) strategy and a 2-chlorotrityl chloride resin. After coupling 5-hexynoic acid the peptide was cleaved from the resin and linked to propargylamine. The bis-alkynyl RGDfV peptide was then reacted with two different bis-azides by treatment with copper iodide and triethylamine. These two cyclic RGD peptides were characterized by NMR and HRMS. In order to evaluate the interaction of these new compounds with integrin α_vβ₃ docking experiments were carried out and the results compared with those obtained with cyclo(RGDf[N–Me]V) (Cilengitide). The two new cyclic RGD peptides showed a higher affinity to the α_vβ₃ integrin when compared with Cilengitide thus representing two new potential integrin α_vβ₃ antagonists.     

Enantioselective epoxidation of α,β-unsaturated ketones catalyzed by stapled helical l-Leu-based peptides

Stapled helical l-leucine-based heptapeptides were synthesized and used as catalysts for the enantioselective epoxidation of α,β-unsaturated ketones. All N-terminal free stapled peptides were successfully used as chiral catalysts. Among them, the use of H-hS₃,₇hS-10 gave epoxide products with high enantioselectivities of up to 99% ee. Furthermore, the dominant conformations of the N-terminal protected stapled peptides R₃,₇R-10 and hS₃,₇hS-10 were investigated by ¹H NMR, IR, CD spectra, and X-ray crystallographic analysis. The peptide R₃,₇R-10 formed a right-handed (P) α-helix in solution and in the crystalline state, while hS₃,₇hS-10 formed a right-handed (P) 3₁₀-helix in solution.     

Preparation of modified peptides: direct conversion of α-amino acids into β-amino aldehydes

A direct method for the transformation of α-amino acids into β-amino aldehydes was developed, and applied to the modification of the C-terminal residue of peptides. The method takes place in good yields and under mild conditions. The application of this methodology to the preparation of small peptides with γ-amino alcohol units, which are precursors of analogues of peptaibol antibiotics, is also described.