Relevance of amphiphilicity and helicity on the antibacterial action of a histatin 5-derived peptide.

Peptide dhvar4, derived from the active domain of our salivary peptide histatin 5, bears a Phe residue in the middle of its hydrophilic face when folded into an α-helix. We then synthesized an analog with this Phe replaced by Lys and two analogs preserving Phe but bearing two and three α-aminoisobutyric acid (Aib) residues to stabilize the helical structure. The aim of this design was to verify which of the two features is more favorable to the biological activity. We performed a conformational study by means of circular dichroism and nuclear magnetic resonance, made antibacterial tests, and assessed the stability of the peptides in human serum. We observed that amphiphilicity is more important than helix stability, provided a peptide can adopt a helical conformation in a membrane-mimetic environment.

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.

Oxime and thiazolidine chemoselective ligation reactions: a green method for cotton functionalization.

During the last years, the need to create textile materials provided with peculiar properties has grown significantly. In particular, new textiles are studied to be a first protection in the prevention of living organisms from pathogens. In this regard, modifying a textile material with biologically active compounds, such as antibacterial or antiviral peptides would be useful for many applications. Our work shows a study on the possibility of modifying cotton fabrics with peptides using thiazolidine and oxime chemoselective ligations. For this purpose, an enzymatic oxidation of cellulose in a heterogeneous phase and the possibility to reuse the oxidation solution for multiple times was successfully applied. Model peptides have been designed and synthesized in order to set up the conditions for conjugating peptides to cotton via either thiazolidine or oxime bond. A systematic study of the time, pH, and quantities needed for the best reaction conditions has been conducted. The efficiency and stability of the two chemoselective ligation bonds have been studied and compared. Supplementary Information: The online version contains supplementary material available at 10.1007/s10570-023-05253-1.

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.

Targeted Amino Acid Substitutions in a Trichoderma Peptaibol Confer Activity against Fungal Plant Pathogens and Protect Host Tissues from Botrytis cinerea Infection.

Fungal species belonging to the Trichoderma genus are commonly used as biocontrol agents against several crop pathogens. Among their secondary metabolites, peptaibols are helical, antimicrobial peptides, which are structurally stable even under extreme pH and temperature conditions. The promise of peptaibols as agrochemicals is, however, hampered by poor water solubility, which inhibits efficient delivery for practical use in crop protection. Using a versatile synthetic strategy, based on green chemistry procedures, we produced water-soluble analogs of the short-length peptaibol trichogin. Although natural trichogin was inactive against the tested fungal plant pathogens (Botrytis cinerea, Bipolaris sorokiniana, Fusarium graminearum, and Penicillium expansum), three analogs completely inhibited fungal growth at low micromolar concentrations. The most effective peptides significantly reduced disease symptoms by B. cinerea on common bean and grapevine leaves and ripe grape berries without visible phytotoxic effects. An in-depth conformational analysis featuring a 3D-structure-activity relationship study indicated that the relative spatial position of cationic residues is crucial for increasing peptide fungicidal activity.

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers.

Trichogin GA IV is a short peptaibol with antimicrobial activity. This uncharged, but amphipathic, sequence is aligned at the membrane interface and undergoes a transition to an aggregated state that inserts more deeply into the membrane, an assembly that predominates at a peptide-to-lipid ratio (P/L) of 1:20. In this work, the natural trichogin sequence was prepared and reconstituted into oriented lipid bilayers. The 15 N NMR chemical shift is indicative of a well-defined alignment of the peptide parallel to the membrane surface at P/Ls of 1:120 and 1:20. When the P/L is increased to 1:8, an additional peptide topology is observed that is indicative of a heterogeneous orientation, with helix alignments ranging from around the magic angle to perfectly in-plane. The topological preference of the trichogin helix for an orientation parallel to the membrane surface was confirmed by attenuated total reflection FTIR spectroscopy. Furthermore, 19 F CODEX experiments were performed on a trichogin sequence with 19 F-Phe at position 10. The CODEX decay is in agreement with a tetrameric complex, in which the 19 F sites are about 9-9.5 Šapart. Thus, a model emerges in which the monomeric peptide aligns along the membrane surface. When the peptide concentration increases, first dimeric and then tetrameric assemblies form, made up from helices oriented predominantly parallel to the membrane surface. The formation of these aggregates correlates with the release of vesicle contents including relatively large molecules.

Heterochiral Ala/(αMe)Aze sequential oligopeptides: Synthesis and conformational study.

α-Amino acid residues with a ϕ,ψ constrained conformation are known to significantly bias the peptide backbone 3D structure. An intriguing member of this class of compounds is (αMe)Aze, characterized by an Nα -alkylated four-membered ring and Cα -methylation. We have already reported that (S)-(αMe)Aze, when followed by (S)-Ala in the homochiral dipeptide sequential motif -(S)-(αMe)Aze-(S)-Ala-, tends to generate the unprecedented γ-bend ribbon conformation, as formation of a regular, fully intramolecularly H-bonded γ-helix is precluded, due to the occurrence of a tertiary amide bond every two residues. In this work, we have expanded this study to the preparation and 3D structural analysis of the heterochiral (S)-Ala/(R)-(αMe)Aze sequential peptides from dimer to hexamer. Our conformational results show that members of this series may fold in type-II ß-turns or in γ-turns depending on the experimental conditions.

A novel peptide conformation: the γ-bend ribbon.

Unlike the extensively investigated relationship between the peptide ß-bend ribbon and its prototypical 310-helix conformation, the corresponding relationship between the narrower γ-bend ribbon and its regular γ-helix counterpart still remains to be studied, as the latter 3D-structures have not yet been experimentally authenticated. In this paper, we describe the results of the first characterization, both in the crystal state and in solution, of the γ-bend ribbon conformation using X-ray diffraction and FT-IR absorption, electronic CD and 2D-NMR spectroscopies applied to an appropriate set of synthetic, homo-chiral, sequential dipeptide oligomers based on (S)-Ala and the known γ-bend inducer, Cα-tetrasubstituted, N-alkylated α-amino acid residue (S)-Cα-methyl-azetidine-carboxylic acid.

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs.

In this work, an extensive set of spectroscopic and biophysical techniques (including FT-IR absorption, CD, 2D-NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14-mer peptaibiotic chalciporin A, characterized by a relatively low (≈20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in-depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical-containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC-labeled analogs in view of the poorly reactive α-amino function of this very bulky residue and the specific requirements of its free-radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, α-helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class.

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR.

Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C19F3-labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19F-19F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

The peculiar N- and (-termini of trichogin GA IV are needed for membrane interaction and human cell death induction at doses lacking antibiotic activity.

Peptaibiotics, non-ribosomally synthetized peptides from various ascomycetes, are uniquely characterized by dialkylated a-amino acids, a rigid heli cal conformation, and membrane permeation properties. Although generally considered as antimicrobial peptides, peptaibiotics may display other toxicological properties, and their function is in many cases unknown. With the goal to define the biological activity and selectivity of the peptaibiotictrichogin GA IV from the human opportunist Trichodenna longibrachiatum we analyzed its membrane interaction,cytotoxic activity and antibacterial effect. Trichogin GA IV effectively killed several types of healthy and neoplastic human cells at doses (EC 50%= 4-6 ~) lacking antibiotic effects on both Gram- and Gram+ bacteria(MIC > 64 ~ ). The peptaibiotic distinctive (-terminal primary alcohol was found to cooperate with theN-terminal n-octanoyl group to permeate the membrane phospholipid bilayer and to mediate effective binding and active endocytosis of trichogin GA IV in eukaryotic cells, two steps essential for cell death induction.Replacement of one Gly with Lys plus the simultaneous esterification of the (-terminus, strongly increased trichogin GA IV anti-Gram+ activity (MIC 1-4 ~ ). but further mitigated its cytotoxicity on human cells.

Helical Foldamers Incorporating Photoswitchable Residues for Light-Mediated Modulation of Conformational Preference.

An E unsaturated fumaramide linkage may be introduced into Aib peptide foldamer structures by standard coupling methods and photoisomerized to its Z (maleamide) isomer by irradiation with UV light. As a result of the photoisomerization, a new hydrogen-bonded contact becomes possible between the peptide domains located on either side of the unsaturated linkage. Using the fumaramide/maleamide linker to couple a chiral and an achiral fragment allows the change in hydrogen bond network to communicate a conformational preference, inducing a screw sense preference in the achiral domain of the maleamide-linked foldamers that is absent from the fumaramides. Evidence for the induced screw sense preference is provided by NMR and CD, and also by the turning on by light of the diastereoselectivity of a peptide chain extension reaction. The fumaramide/maleamide linker thus acts as a "conformational photodiode" that conducts stereochemical information as a result of irradiation by UV light.

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.

Peptide δ-Turn: Literature Survey and Recent Progress.

Among the various types of α-peptide folding motifs, δ-turn, which requires a central cis-amide disposition, has been one of the least extensively investigated. In particular, this main-chain reversal topology has been studied in-depth neither in linear/cyclic peptides nor in proteins. This Minireview article assembles and critically analyzes relevant data from a literature survey on the δ-turn conformation in those compounds. Unpublished results from recent conformational energy calculations and a preliminary solution-state analysis on a small model peptide, currently ongoing in our laboratories, are also briefly outlined.

Handedness preference and switching of peptide helices. Part II: Helices based on noncoded α-amino acids.

In this second part of our review article on the preferred screw sense and interconversion of peptide helices, we discuss the most significant computational and experimental data published on helices formed by the most extensively investigated categories of noncoded α-amino acids. They are as follows: (i) N-alkylated Gly residues (peptoids), (ii) C(α) -alkylated α-amino acids, (iii) C(α,ß) -sp(2) configurated α-amino acids, and (iv) combinations of residues of types (ii) and (iii). With confidence, the large body of interesting papers examined and classified in this editorial effort will stimulate the development of helical peptides in many diverse areas of biosciences and nanosciences.

4-Cyano-α-methyl-l-phenylalanine as a spectroscopic marker for the investigation of peptaibiotic-membrane interactions.

Two analogs of the ten-amino acid residue, membrane-active lipopeptaibiotic trichogin GA IV, mono-labeled with 4-cyano-α-methyl-L-phenylalanine, a potentially useful fluorescence and IR absorption probe of the local microenvironment, were synthesized by the solid-phase methodology and conformationally characterized. The single modification was incorporated either at the N-terminus (position 1) or near the C-terminus (position 8) of the peptide main chain. In both cases, the replaced amino acid was the equally helicogenic α-aminoisobutyric acid (Aib) residue. We performed a solution conformational analysis by use of FT-IR absorption, CD, and 2D-NMR spectroscopies. The results indicate that both labeled analogs essentially maintain the overall helical propensity of the naturally occurring lipopeptaibiotic. Peptide-membrane interactions were assessed by fluorescence and ATR-IR absorption techniques. Analogies and differences between the two peptides were highlighted. Taken together, our data confirm literature results that some of the spectroscopic parameters of the 4-cyanobenzyl chromophore are sensitive markers of the local microenvironment.

Enhancement of the helical content and stability induced in a linear oligopeptide by an i, i+4 intramolecularly double stapled, overlapping, bicyclic [31, 22, 5]-(E)ene motif.

Two consecutive i, i+4 intramolecular, side chain-to-side chain, macrocyclizations of different type carried out on a preformed, partially helical peptide result in a largely predominant, double stapled, overlapping, bicyclic [31,22,5]-(E)ene motif. A detailed ECD and NMR conformational study revealed a significant enhancement of the original helical content and stability, accompanied by an increase of the α-helix amount over that of the 3(10)-helix.

The 2.05-helix in hetero-oligopeptides entirely composed of C(α,α)-disubstituted glycines with both side chains longer than methyls.

The existence of the very uncommon, but potentially quite interesting, multiple, consecutive fully-extended conformation (2.05-helix) has been already clearly demonstrated in homo-oligopeptides based on quaternary α-amino acids with both side chains longer than methyls, but not cyclized on the α-carbon atom. To extend the scope of this research, in this work we investigated the occurrence of this flat 3D-structure in hetero-oligopeptides, each composed of two or three different residues of that class. The synthesis of a terminally protected peptide series to the tetrapeptide level was carried out by solution methods. The resulting oligomers were chemically and conformationally characterized. The data obtained point to an overwhelming population of the fully-extended conformation in CDCl3. However, a solvent-driven switch to a predominant 310-helical structure was seen in CD3CN. A delicate, local balance between these two conformations is confirmed to occur in the crystalline state. Molecular dynamics simulations in CHCl3 on a hetero-tetrapeptide converged to the fully-extended conformation even starting from the 310-helical structure.

Cotton functionalized with peptides: characterization and synthetic methods.

Three approaches for the chemical ligation of peptides to cotton fibers are described and compared. This investigation was encouraged by the need to create peptide-decorated natural textiles, furnished with useful properties (e.g. antimicrobial activity). IR absorption spectroscopy is proved to be an easy and fast method to check the covalent anchorage of a peptide to cotton, whereas for a quantitative determination, a UV absorption method was employed. We also analyzed the usefulness of electron paramagnetic resonance spectroscopy to characterize our peptide-cotton conjugates.

Handedness preference and switching of peptide helices. Part I: Helices based on protein amino acids.

In this article, we review the relevant results obtained during almost 60 years of research on a specific aspect of stereochemistry, namely handedness preference and switches between right-handed and left-handed helical peptide structures generated by protein amino acids or appropriately designed, side-chain modified analogs. In particular, we present and discuss here experimental and theoretical data on three categories of those screw-sense issues: (i) right-handed/left-handed α-helix transitions underwent by peptides rich in Asp, specific Asp ß-esters, and Asn; (ii) comparison of the preferred conformations adopted by helical host-guest peptide series, each characterized by an amino acid residue (e.g. Ile or its diastereomer aIle) endowed with two chiral centers in its chemical structure; and (iii) right-handed (type I)/left-handed (type II) poly-(Pro)n helix transitions monitored for peptides rich in Pro itself or its analogs with a pyrrolidine ring substitution, particularly at the biologically important position 4. The unique modular and chiral properties of peptides, combined with their relatively easy synthesis, the chance to shape them into the desired conformation, and the enormous chemical diversity of their coded and non-coded α-amino acid building blocks, offer a huge opportunity to structural chemists for applications to bioscience and nanoscience problems.