Isolated α-turns in peptides: a selected literature survey.

The results of classifying into various types the 68 examples of isolated α-turns in the X-ray diffraction crystal structures of peptides documented in the literature are presented and discussed in this review article. α-Turns characterized by the trans disposition of all ω torsion angles are common for the backbone linear peptides investigated. In contrast, the cis arrangement of the N-terminal (ωi + 1 ) torsion angle, among those generated by the three residues internal to the α-turn, is a peculiar feature of 65% of the cyclic peptides. Among linear and cyclic peptides featuring the all-trans disposition of the ω torsion angles, only one third of the α-turns display φ,ψ values not too far from those characterizing regular α-helices. In general, our findings, taken together, suggest that a significant conformational diversity is compatible with the formation of an intramolecularly H-bonded C13 -member pseudocycle (α-turn) in linear and cyclic peptides.

Helical versus Flat Bis-Ferrocenyl End-Capped Peptides: The Influence of the Molecular Skeleton on Redox Properties.

Despite the fact that peptide conjugates with a pendant ferrocenyl (Fc) have been widely investigated, bis-ferrocenyl end-capped peptides are rarely synthetized. In this paper, in addition to the full characterization of the Fc-CO-[L-Dap(Boc)]n-NH-Fc series, we report a comparison of the three series of bis-ferrocenyl homopeptides synthesized to date, to gain insights into the influence of α-amino isobutyric (Aib), 2,3-diamino propionic (Dap) and Cα,ß-didehydroalanine (ΔAla) amino acids on the peptide secondary structure and on the ferrocene redox properties. The results obtained by 2D NMR analysis and X-ray crystal structures, and further supported by electrochemical data, evidence different behaviors depending on the nature of the amino acid; that is, the formation of 310-helices or fully extended (2.05-helix) structures. In these foldamers, the orientation of the carbonyl groups in the peptide helix yields a macrodipole with the positive pole on the N-terminal amino acid and the negative pole on the C-terminal amino acid, so that oxidation of the Fc moieties takes place more or less easily depending on the orientation of the macrodipole moment as the peptide chain grows. Conversely, the fully extended conformation adopted by ΔAla flat peptides neither generates a macrodipole nor affects Fc oxidation. The utilization as electrochemical and optical (Circular Dichroism) probes of the two terminal Fc groups, bound to the same peptide chain, makes it possible to study the end-to-end effects of the positive charges produced by single and double oxidations, and to evidence the presence "exciton-coupled" CD among the two intramolecularly interacting Fc groups of the L-Dap(Boc) series.

Carbodiimide-Mediated Beckmann Rearrangement of Oxyma-B as a Side Reaction in Peptide Synthesis.

The suppression of side reactions is one of the most important objectives in peptide synthesis, where highly reactive compounds are involved. Recently, the violuric acid derivative Oxyma-B was introduced into peptide synthesis protocols as a promising additive to efficiently control the optical purity of the amino acids prone to racemization. However, we discovered a side reaction involving the Beckmann rearrangement of Oxyma-B during the coupling reaction, which compromises the yield and purity of the target peptides. Here, we present the investigation of the mechanism of this rearrangement and the optimization of the coupling reaction conditions to control it. These results can be taken into account for the design of novel efficient oxime-based coupling reagents.

Peptide-membrane binding is not enough to explain bioactivity: A case study.

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api8), able to selectively kill cancer cells, and Tri(Leu4), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu4), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions.

Is Cys(MTSL) the Best α-Amino Acid Residue to Electron Spin Labeling of Synthetically Accessible Peptide Molecules with Nitroxides?

Electron paramagnetic resonance spectroscopy, particularly its pulse technique double electron-electron resonance (DEER) (also termed PELDOR), is rapidly becoming an extremely useful tool for the experimental determination of side chain-to-side chain distances between free radicals in molecules fundamental for life, such as polypeptides. Among appropriate probes, the most popular are undoubtedly nitroxide electron spin labels. In this context, suitable biosynthetically derived, helical regions of proteins, along with synthetic peptides with amphiphilic properties and antibacterial activities, are the most extensively investigated compounds. A strict requirement for a precise distance measurement has been identified in a minimal dynamic flexibility of the two nitroxide-bearing α-amino acid side chains. To this end, in this study, we have experimentally compared in detail the side-chain mobility properties of the two currently most widely utilized residues, namely, Cys(MTSL) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, two double-labeled, chemically synthesized 20-mer peptide molecules have been adopted as appropriate templates for our investigation on the determination of the model intramolecular separations. These double-Cys(MTSL) and double-TOAC compounds are both analogues of the almost completely rigid backbone peptide ruler which we have envisaged and 3D structurally analyzed as our original, unlabeled compound. Here, we have clearly found that the TOAC side-chain labels are largely more 3D structurally restricted than the MTSL labels. From this result, we conclude that the TOAC residue offers more precise information than the Cys(MTSL) residue on the side chain-to-side chain distance distribution in synthetically accessible peptide molecules.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

Conformationally Constrained Peptides with High Affinity to the Vascular Endothelial Growth Factor.

The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114* has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, Cα-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.

Sustainable, Site-Specific Linkage of Antimicrobial Peptides to Cotton Textiles.

A new general method to covalently link a peptide to cotton via thiazolidine ring formation is developed. Three different analogues of an ultrashort antibacterial peptide are synthesized to create an antibacterial fabric. The chemical ligation approach to the heterogeneous phase made up of insoluble cellulose fibers and a peptide solution in water is adapted. The selective click reaction occurs between an N-terminal cysteine on the peptide and an aldehyde on the cotton matrix. The aldehyde is generated on the primary alcohol of glucose by means of the enzyme laccase and the cocatalyst 2,2,6,6-tetramethylpiperidine-1-oxyl. This keeps the pyranose rings intact and may bring a benefit to the mechanical properties of the fabric. The presence of the peptide on cotton is demonstrated through instant colorimetric tests, UV spectroscopy, IR spectroscopy, and X-ray photoelectron spectroscopy analysis. The antibacterial activity of the peptides is maintained even after their covalent attachment to cotton fibers.

A Temperature-Driven, Reversible, Helical-Handedness Inversion in Peptaibol Analogues Tuned by the C-Terminal Capping Moiety.

Trichogin is a natural peptide endowed with antimicrobial and antitumor activity. A member of the peptaibol family, trichogin possesses a C-terminal amino alcohol. In the past, this moiety was substituted for a methyl ester for synthetic purposes and it was observed that this apparently slight modification caused significant changes in the peptide bioactivity. With the aim of understanding the reasons behind such observations, a detailed spectroscopic study on a number of trichogin analogues has been performed. Herein, data obtained from synchrotron radiation circular dichroism, NMR spectroscopy, and fluorescence spectroscopy in organic solvents at cryogenic temperatures are compared with those independently acquired by means of EPR spectroscopy at 80 K. It is unambiguously revealed that the presence of a reversible, temperature-driven, screw-sense interconversion from a right- to left-handed helix is determined by the C-terminal capping moiety. Data demonstrate, for the first time, the key role of a C-terminal methyl ester in promoting peptide screw-sense inversion.

Rational Design of Antiangiogenic Helical Oligopeptides Targeting the Vascular Endothelial Growth Factor Receptors.

Tumor angiogenesis, essential for cancer development, is regulated mainly by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which are overexpressed in cancer cells. Therefore, the VEGF/VEGFR interaction represents a promising pharmaceutical target to fight cancer progression. The VEGF surface interacting with VEGFRs comprises a short α-helix. In this work, helical oligopeptides mimicking the VEGF-C helix were rationally designed based on structural analyses and computational studies. The helical conformation was stabilized by optimizing intramolecular interactions and by introducing helix-inducing Cα,α-disubstituted amino acids. The conformational features of the synthetic peptides were characterized by circular dichroism and nuclear magnetic resonance, and their receptor binding properties and antiangiogenic activity were determined. The best hits exhibited antiangiogenic activity in vitro at nanomolar concentrations and were resistant to proteolytic degradation.

Molecular Sponge: pH-Driven Reversible Squeezing of Stimuli-Sensitive Peptide Monolayers.

The cyclic change of structure, thickness, and density, with pH switching from acidic (pH = 3) to basic (pH = 11) condition, has been revealed for chemisorbed monolayers of the peptide Lipo-Aib-Lys-Leu-Aib-Lys-Lys-Leu-Aib-Lys-Ile-Lol, a trichogin GA IV-analogue carrying Lys residues instead of Gly ones at positions 2, 5, 6, and 9, while a homologous peptide not containing Lys residues does not show any response to pH changes. Experimental and theoretical results, obtained by means of quartz crystal microbalance with dissipation monitoring, surface plasmon resonance, nanoplasmonic sensing technique, Fourier transform infrared-reflection attenuated spectroscopy and dynamic force spectroscopy, and molecular dynamics simulations provide detailed information on the overall monolayer structure changes with pH, including the analysis of the intra- and interchain peptide dynamics, the structure of the peptide layer/water/solid interface, as well as the position and role of solvation and nonsolvation water. The observed stimuli-responsive behavior of L1 peptide monolayers is accounted in terms of the occurrence of a pH-induced wetting/dewetting process, due to the pH-induced switching of the hydrophilic character of charged lysine groups to hydrophobic one of the same uncharged groups, along the peptide chain. This behavior in turn promotes the collective change of the aggregation state of the peptide chains. The present results may pave the way to critically reexamine the mechanism of stimuli-responsive systems.

Peptide antibiotic trichogin in model membranes: Self-association and capture of fatty acids.

The antimicrobial action of peptides in bacterial membranes is commonly related to their mode of self-assembling which results in pore formation. To optimize peptide antibiotic use for therapeutic purposes, a study on the concentration dependence of self-assembling process is thus desirable. In this work, we investigate this dependence for peptaibol trichogin GA IV (Tric) in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane in the range of peptide concentrations between 0.5 and 3.3 mol%. Pulsed double electron-electron resonance (PELDOR) applied on spin-labeled peptide analogs highlights the onset of peptide dimerization above a critical peptide concentration value, namely ~ 2 mol%. Electron spin echo (ESE) envelope modulation (ESEEM) for D2O-hydrated bilayers shows that dimerization is accompanied by peptide re-orientation towards a trans-membrane disposition. For spin-labeled stearic acids (5-DSA) in POPC bilayers, the study of ESE decays and ESEEM in the presence of a deuterated peptide analog indicates that above the critical peptide concentration the 5-DSA molecules are attracted by peptide molecules, forming nanoclusters. As the 5-DSA molecules represent a model for the behavior of fatty acids participating in bacterial membrane homeostasis, such capturing action by Tric may represent an additional mechanism of its antibiotic activity.

Anticancer Gold(III) Peptidomimetics: From Synthesis to in vitro and ex vivo Biological Evaluations.

Five new AuIII -peptidodithiocarbamato complexes of the type [AuIII Br2 (dtc-AA1 -AA2 -OR] (in which AA1 =N-methylglycine (Sar), l/d-Pro; AA2 =l/d-Ala, α-aminoisobutyric acid (Aib); R=OtBu, triethylene glycol methyl ether), differing with regard to the amino acid sequence and/or the chiral amino acid configuration, were designed to enhance tumor selectivity and bioavailability. The gold(III)-based moiety was functionalized to exploit the targeting properties of the peptidomimetic ligand toward two peptide transporters (namely PEPT1 and PEPT2), which are upregulated in several tumor cells. The compounds were synthesized and fully characterized, mainly by means of elemental analysis, one- and two-dimensional NMR spectroscopy, FT-IR, and UV/Vis spectrophotometry. The crystal structures of three compounds were also solved by X-ray diffraction. In vitro cytotoxicity studies using a panel of human tumor cell lines (A549 [non-small-cell lung carcinoma], MCF-7 [breast cancer], A2780 [ovarian carcinoma], H1975 [non-small-cell lung carcinoma], H460 [large-cell lung carcinoma], and A431 [human epidermoid carcinoma]) showed the dtc-Pro-Aib-OtBu derivative to be very effective, with GI50 values much lower than those of cisplatin. This complex was thus selected for evaluating stability under physiological conditions and possible interactions with serum albumin, as well in PARP-1 enzyme inhibition assays and preliminary ex vivo toxicity experiments on healthy rat tissues.

Amphiphilic polypeptides with prolonged enzymatic stability for the preparation of self-assembled nanobiomaterials.

Due to their excellent biocompatibility and biodegradability, polypeptides have emerged as versatile bio-inspired scaffolds for the preparation of artificial biomaterials. In order to create self-assembled polypeptide nanoparticles with enhanced stability towards enzymatic degradation, we synthesized a series of random and block polypeptides based on lysine and α-aminoisobutyric acid (Aib) by the ring-opening polymerization of N-carboxyanhydrides (ROP NCA) of the corresponding amino acids. A conformational analysis carried out by means of FT-IR absorption and CD spectroscopies revealed a noticeable difference between random and block copolymers. In turn, the spatial organization of the polypeptide chains induced the formation of nanostructures of different types. The block copolymers self-assembled in vesicle-like structures, whereas polypeptides with randomly distributed monomers formed micelles. In contrast with the polymers with only natural amino acids, all nanoparticles based on Aib/Lys polypeptides showed strong resistance to proteolytic cleavage. The cytotoxicity and the kinetics of the cellular uptake of the prepared nanostructures were also studied. The results obtained could not only contribute to the understanding of long Aib polypeptide folding and self-assembling, but also pave the way to the design of nanomaterials with finely tuned properties in the fields of drug delivery and tissue engineering.

Nanotraps with biomimetic surface as decoys for chemokines.

A creation of nanotraps that could selectively recognize the chemotactic mediators of leukocyte adhesion and eliminate them from the bloodstream and tissue intercellular matrix is a promising approach for the treatment of various inflammatory and autoimmune diseases. We designed nanotraps as artificial decoy receptors based on poly(lactic acid) (PLA) nanoparticles covered by heparin and bearing on the surface two fragments of CCR5 receptor (N-terminal domain, Nt, and second extracellular loop, ECL2), responsible for chemokine binding. In order to attach Nt and ECL2 to the heparin shell, the corresponding peptides were modified with N- and/or C-terminal oligolysines. The presence of the nanotraps in the cell medium completely eliminated the activating effect of a CCR5 ligand, chemokine Rantes, while strongly decreasing the adhesion of monocytes to the human endothelial cells. We found that the modified ECL2 alone was also able to prevent monocyte adhesion, thus acting as a decoy receptor itself.

Integrated Computational Approach to the Electron Paramagnetic Resonance Characterization of Rigid 310-Helical Peptides with TOAC Nitroxide Spin Labels.

We address the interpretation, via an integrated computational approach, of the experimental continuous-wave electron paramagnetic resonance (cw-EPR) spectra of a complete set of conformationally highly restricted, stable 310-helical peptides from hexa- to nonamers, each bis-labeled with nitroxide radical-containing TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) residues. The usefulness of TOAC for this type of analysis has been shown already to be due to its cyclic piperidine side chain, which is rigidly connected to the peptide backbone α-carbon. The TOAC α-amino acids are separated by two, three, four, and five intervening residues. This set of compounds has allowed us to modulate both the radical···radical distance and the relative orientation parameters. To further validate our conclusion, a comparative analysis has been carried out on three singly TOAC-labeled peptides of similar main-chain length.

Intramolecular backbone···backbone hydrogen bonds in polypeptide conformations. The other way around: ɛ-turn.

In this study, we performed a detailed literature survey of the ɛ-turn in peptides and proteins. This three-dimensional structural feature is characterized by an eleven-membered pseudo-cycle closed by an intramolecular backbone…backbone H-bond. Interestingly, in this motif the direction of the N-H…O = C H-bond runs opposite to that of the much more popular and extensively investigated α-, ß-, and γ-turns. We did not authenticate unequivocally the ɛ-turn main-chain reversal topology in any linear short peptide. However, it is frequently observed in small cyclic peptides formed by four, five, and six amino acid residues with stringent geometric requirements. Rather surprisingly, ɛ-turns do occur in proteins, although to a relatively moderate extent, as an isolated feature or in the turn segment of hairpin motifs based on two antiparallel, pleated ß-strands. Moreover, the ɛ-turn may also host not only the seven-membered, intramolecularly H-bonded, pseudo-cycle termed γ-turn, either of the classic or inverse type, but also one (or even two) cis peptide bond(s) or a ß-bulge conformation. Based on their ϕ, ψ backbone torsion angles, we were able to classify the protein ɛ-turns in six different families. Conformational energy computations using the DFT methodology were also performed on the ɛ-turns adopted by the amino acid triplet -Gly-Gly-Gly- (Gly is the most commonly found residue at each of the three positions in our analysis of proteins). Again, in this computational study, six families of turns were identified, but only some of them resemble rather closely those extracted from our investigation on proteins.

En route towards the peptide γ-helix: X-ray diffraction analyses and conformational energy calculations of Adm-rich short peptides.

We performed the solution-phase synthesis of a set of model peptides, including homo-oligomers, based on the 2-aminoadamantane-2-carboxylic acid (Adm) residue, an extremely bulky, highly lipophilic, tricyclic, achiral, Cα -tetrasubstituted α-amino acid. In particular, for the difficult peptide coupling reaction between two Adm residues, we took advantage of the Meldal's α-azidoacyl chloride approach. Most of the synthesized Adm peptides were characterized by single-crystal X-ray diffraction analyses. The results indicate a significant propensity for the Adm residue to adopt γ-turn and γ-turn-like conformations. Interestingly, we found that a -CO-(Adm)2 -NH- sequence is folded in the crystal state into a regular, incipient γ-helix, at variance with the behavior of all of the homo-dipeptides from Cα -tetrasubstituted α-amino acids already investigated, which tend to adopt either the ß-turn or the fully extended conformation. Our density functional theory conformational energy calculations on the terminally blocked homo-peptides (n = 2-8) fully confirmed the crystal-state data, strongly supporting the view that this rigid Cα -tetrasubstituted α-amino acid residue is largely the most effective building block for γ-helix induction, although to a limited length (anti-cooperative effect). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Endothioxopeptides: A conformational overview.

Although thionamides would have been first prepared two centuries ago and their chemical and spectroscopic properties extensively investigated, only much more recently (since about 1985) a well deserved but still insufficient attention has been paid to their endothioxopeptide subfamily which nonetheless currently represents a rapidly emerging area of great scientific interest in the broader field of foldameric compounds based on biologically relevant building blocks. After two brief sections offering information on the unfortunately still limited number of endothioxopeptides discovered from natural sources but also on the impressive advancements registered in the last few years in their synthetic methods, this review article outlines the results of a detailed literature survey on the ongoing great, but not systematic, progress related to the conformational consequences generated by incorporating one (or more) thionamide group(s) into a polypeptide chain. Finally, a short discussion of the growing, but still in its infancy, class of the endoselenoxopeptide congeners is also presented.