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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., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

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 (ω i + 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 C 13 -member pseudocycle (α-turn) in linear and cyclic peptides., (© 2023 European Peptide Society and John Wiley & Sons Ltd.)

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 N α -alkylated four-membered ring and C α -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 β-turns or in γ-turns depending on the experimental conditions., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

Liraglutide is a new generation lipopeptide drug used for the treatment of type II diabetes. In this work, we describe new approaches for its preparation fully by chemical methods. The key step of these strategies is the synthesis in solution of the Lys/γ-Glu building block, Fmoc-Lys-(Pal-γ-Glu-OtBu)-OH, in which Lys and Glu residues are linked through their side chains and γ-Glu is N(α) -palmitoylated. This dipeptide derivative is then inserted into the peptide sequence on solid phase. As liraglutide is obtained with great purity and high yield, our approach can be particularly attractive for an industrial production. We also report here the results of a circular dichroism conformational analysis in a membrane mimetic environment that offers new insights into the mechanism of action of liraglutide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

A set of three analogs of the 10-residue, membrane-active lipopeptaibiotic trichogin GA IV, labeled with the promising 4-nitrophenylalanine IR absorption probe for local polarity, was synthesized by the solid-phase methodology, chromatographically purified, and extensively characterized. A single residue modification was inserted near the N-terminus, in the central region, or at the C-terminus. A solution conformational analysis, carried out by FT-IR absorption, CD, and 2D-NMR combined with molecular dynamics calculations, indicates that the mono-labeled analogs maintain the overall helical properties of the parent compound. Membrane permeabilization measurements and antimicrobial tests revealed that they possess membrane-modifying properties and in vitro antibacterial activities analogous to those of the natural lipopeptaibiotic. Our IR absorption and attenuated total reflectance investigations in various environments, from which water was excluded for solubility reasons, showed that the 1350 cm(-1) 4-nitrobenzyl band is a reporter group of rather limited sensitivity., (Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.)

A set of analogues of the 14-residue peptaibol tylopeptin B, containing the stable free-radical 4-amino-1-oxyl-2,2,6,6,-tetramethylpiperidine-4-carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid-phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane-mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α-helix or the 3(10) -helix type depending upon the nature of the solvent. In aqueous environment, the spin-labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N-terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC(8) - and TOAC(13) -tylopeptin B, showed that the labeled peptides exhibit membrane-modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium-length peptaibiotics., (Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.)

The medium-length peptaibiotics are characterized by a primary structure of 14-16 amino acid residues. Despite the interesting antibiotic and antifungal properties exhibited by these membrane-active peptides, their exact mechanism of action is still unknown. Here, we present our results on heptaibin, a 14-amino acid peptaibiotic found to exhibit antimicrobial activity against Staphylococcus aureus. We carried out the very challenging synthesis of heptaibin on solid phase and a detailed conformational analysis in solution. The peptaibiotic is folded in a mixed 3₁₀-/α-helix conformation which exhibits a remarkable amphiphilic character. We also find that it is highly stable toward degradation by proteolytic enzymes and nonhemolytic. Finally, fluorescence leakage experiments using small unilamellar vesicles of three different compositions revealed that heptaibin, although uncharged, is a selective compound for permeabilization of model membranes mimicking the overall negatively charged surface of Gram-positive bacteria. This latter finding is in agreement with the originally published antimicrobial activity data., (Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.)

The antibacterial and hemolytic activities of the amphiphilic helical, membrane-active, lipopeptaibol trichogin GA IV and its [Leu(11)-OMe] analogue were compared to those of the partially helical or non-helical 8-meric or 4-meric, C-terminal short sequences, respectively. The study on trichogin GA IV was extended to several methicillin-resistant Staphylococcus aureus strains. Using a large set of enzymes, we also evaluated the resistance to proteolysis of all of the four peptides., (Copyright 2009 European Peptide Society and John Wiley & Sons, Ltd.)

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide, exhibiting a wide spectrum of biological activities in mammals. However, the clinical applications of VIP are mainly hampered because of its rapid degradation in vivo. Peptide glycosylation, a procedure frequently used to increase peptide resistance to proteolytic degradation and consequently increase peptide metabolic stability, has not been performed yet on VIP. The presence of three N-glycosylation sites on VIP receptor type 1 (VPAC1) was previously demonstrated. Therefore, glycosylation of the VIP ligand could potentially increase its receptor affinity because of glyco-glyco interactions between the ligand and the receptor. In order to enhance VIP's metabolic stability and to increase its ligand-receptor binding/activation, eight glycosylated VIP derivatives were successfully synthesized by the solid-phase procedure. Each VIP analog was monoglycosylated by a monosaccharide addition to one amino-acid residue along the sequence. Glycosylation did not affect the alpha-helical structure shown by the native VIP in organic environment. Few glycosylated VIP analogs displayed highly potent VPAC1 receptor binding and cAMP-induced activation; only 4-6 fold lower in comparison to the native VIP. Furthermore, the peptide analog glycosylated on Thr11 ([11Glyc]VIP) showed a significantly enhanced stability toward trypsin enzymatic degradation in comparison to VIP. Analysis of the degradation products of [11Glyc]VIP showed that differently from VIP, incubation of the peptide [11Glyc]VIP with trypsin resulted in no cleavage at the Arg12-Leu13 peptide bond, suggesting that VIP glycosylation may lead to enhanced metabolic stability.

Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.