We report the synthesis of new ferrocene (Fc)-peptide conjugates, namely, N-Fc-succinamide-Gly-Ala-OEt, N-Fc-glutaramide-Gly-Ala-OEt, N-Fc-pimelamide-Gly-Ala-OEt, and N-Fc-glutaramide-Gly-Gly-Ala-OEt. They were designed on the base of four components: (i) a Fc unit as an electroactive element; (ii) the linker -NH-CO- to adjust the oxidation potential of Fc towards the physiological conditions; (iii) spacers of different lengths between Fc and the peptide; (iv) a peptide unit to drive Fc to selected biological targets. Fc-peptide conjugates have already been explored as potential anticancer drugs. It is reported that Fc displays the anti-proliferative activity through the production of reactive oxygen species (ROS) as its redox potential (0.40 V vs. SCE) is compatible with the intracellular potential which varies from +0.40 V to −0.44 V. However, the way Fc binds to the peptide significantly influences its redox potential. So far, this issue was not deeply addressed in the literature. Therefore, in this contribution we aimed at investigating the influence of the linker, and of the length of spacer and peptide on the Fc oxidation potential. Noteworthy, we linked Fc to the remaining part of the molecule via an amide bond, but with N-end attached to Fc and not the carbonyl, as reported in the literature so far. The cyclic voltammetry measurements we performed revealed that the transition from an electron-withdrawing (Fc-CO-NH-) to an electron-donating group (Fc-NH-CO-) significantly affects the Fc redox potential. On the contrary, spacer and peptide lengths display a moderate effect. We also carried out a conformational study in the crystal state (X-ray diffraction), and in solution (2D-NMR) on three intermediate molecules. Interestingly, the tripeptide Boc-Gly-Gly-Ala-OEt adopts a β-turn structure in all environments. This finding help to explain its resistance to the enzymatic hydrolysis. Enzymatic degradation tests in human serum were performed on the other conjugates as well, highlighting that the Fc unit acts as a protector of the peptide portion.

The growing number of peptides approved as drugs is pushing the introduction of synthetic methodologies alternative to the solid‐phase peptide synthesis (SPPS), currently the most exploited method for large‐scale peptide production. Indeed, SPPS is easily scalable, can be automated, and allows the synthesis of long peptides in short times. However, it also has some disadvantages, as it uses large excesses of reagents, huge amounts of solvents (often not “green"), a non‐biodegradable solid support, and features a heterogeneous reaction environment in which amino acid couplings are more difficult than in a homogeneous environment. For this reason, in recent years there has been growing interest in developing better synthetic methods. In this work, we present a new dendrimeric support based on lysine (Lys) and short polyethylene glycol (PEG) chains. Its main advantages are (i) the possibility of using more eco‐friendly solvents that cannot be used in standard SPPS, (ii) the replacement of polystyrene‐based supports with a PEG‐lysine hybrid one, and (iii) the formation of a homogeneous reaction medium that will help to reduce the excess of reagents and solvents. The results obtained in the synthesis of a model peptide are promising and amenable to transfer to an industrial scale.

The results of an analysis on the presence of π-turns, characterized by an i ← i + 5 C=O···H-N intramolecular hydrogen bond, in the X-ray diffraction structures of peptides are discussed. The survey returned a total of 55 π-turn occurrences in linear and cyclic peptides. π-Turns characterized by a helical conformation for residue i + 4, but with a screw sense opposite to that of the three preceding residues, are largely prevailing. They are often found at the C-end of incipient or fully developed α-helices, 310-helices, and mixed α-/310-helices, thus acting as a C-capping motif. However, the structures of two linear peptides and 15 cyclopeptides indicate that these types of π-turns can exist in isolation, without the support of a preceding helix. The frequent presence of additional intramolecular hydrogen bonds internal to the π-turn is also investigated. Cyclopeptides offered examples of two types of π-turns that have no parallel in the structures of proteins. Differently from proteins, π-turns characterized by helical ϕ, ψ sets of the same screw sense for all internal residues are hitherto unreported in the X-ray diffraction structures of peptides. A suggestion for the rational design in peptides/peptidomimetics of a π-turn featuring the screw-sense reversal of residue i + 4 is proposed.

This study explores the use of a novel polymeric mesoporous support (pDVB) for solid-phase peptide synthesis (SPPS), with the aim of improving the efficiency and sustainability of the process. The pDVB support, functionalized with the Fmoc-Rink amide linker, offers advantages over conventional supports based on gel-type, lightly crosslinked polymer skeletons, particularly with regard to reduced reliance on swelling capacity, which allows the use of a wider range of solvents. The work focuses on greener and eco-friendly solvents such as TEP, ACN, IPA, and their mixtures with DMSO to replace toxic solvents such as DMF. The synthesis of two model peptide sequences, Fmoc-LLVF-NH2 and ACP(65-74), showed that pDVB-Rink performs better than a conventional-type Rink Amide MBHA support, especially when using environmentally friendly solvents. These results suggest that mesoporous pDVB-Rink is a promising solid support for SPPS to reduce the use of toxic solvents and to improve sustainability.

Glucagon-like peptide-1 analogs are receiving exponential attention for the treatment of type II diabetes and controlling weight due to their unique structure with non-natural lipidated side chains. However, the industrial-scale manufacturing of such peptides poses noticeable challenges due to the gel-like appearance of the peptide intermediates used to form the branched structure. Here, we found an efficient approach for the complexation of these protected peptide building blocks with magnesium chloride, which allows for obtaining free-flowing stable solids suitable for the more process-friendly synthesis of the peptides. A screening of the solvents and magnesium chloride content allowed us to determine the optimal parameters for the preparation of the complexes. NMR spectral analysis showed the involvement of amide groups in the interaction with the magnesium salt. Lastly, the efficiency of the coupling of the Mg-complexed lipidated side-chain building blocks was confirmed for the synthesis of tirzepatide and semaglutide fragments, showing a comparable performance with respect to the noncomplexed derivatives.

Antimicrobial resistance represents a significant global health threat, prompting the exploration of alternative therapeutic strategies. Antimicrobial peptides (AMPs) and lipopeptides are promising candidates due to their unique ability to disrupt bacterial cell membranes through mechanisms distinct from conventional antibiotics. These peptides are typically enhanced by motifs involving cationic amino acids, positive charge, and aromatic residues. Additionally, the conjugation of acyl chains to the N-terminus of AMPs has been shown to improve their antimicrobial activity and selectivity. However, the susceptibility of peptides to enzymatic degradation presents a major limitation. To address this, we investigated the incorporation of non-coded amino acids (NCAAs) to enhance peptide stability. Specifically, we synthesized the NCAA 2-amino-3-(1H-imidazol-1-yl)propanoic acid [His*], producing both enantiomers with high yield and optical purity. We then designed various analogs of ultra-short AMPs by inserting His* at specific positions, evaluating their antimicrobial properties with different acyl chain lengths (C16 and C12) at the N-terminus and the C-terminus. We were able to identify a very promising candidate for applications (P8) characterized by resistance to proteolysis and enhanced biological effectiveness.

Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (similar to 10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.

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.

This work reports single-crystal X-ray diffraction (XRD), Scanning Tunneling Microscopy (STM), and quantum mechanics calculations of the 310-helical peptide Z-(Aib)2-L-Dap(Boc)-Aib-NHiPr (Aib, α-aminoisobutyric acid; Dap, 2,3-diaminopropionic acid; Z, benzyloxycarbonyl; Boc, t-butoxycarbonyl). The peptide forms a double-helical superstructure, studied by XRD and STM. Such architecture is rare in short peptides. Here, we show, by combining XRD and STM that this intriguing conformational feature is not driven by crystal packing; rather, it is an intrinsic property of this peptide. Indeed, the double helix is clearly detected also by STM, where crystal packing cannot be invoked. XRD reveals that intermolecular H-bonds stabilize two left-handed supra-helices (tertiary structure) that develop around a 6-fold screw axis. Then, two supra-helices are intertwined in a quaternary structure as a left-handed, double supra-helix, where C-H⋯π interactions play a crucial role. STM images show the formation of long, isolated "necklaces" (>110 nm). They are of left and right helical handedness. Their size agrees with the XRD finding. DFT calculations allowed us to weigh the contribution of the different intermolecular interactions in the two single supra-helices and the supramolecular double-helix. Interestingly, we were able to conclude that the contribution of the C-H⋯π interactions to the binding energies is close to 50 %.

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, Cα -tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol-membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure-activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes.

In this work, we developed two new polyfunctional hybrid systems in which the presence of Fc redox “antennas” on peptide scaffolds allows for a modulation of their electronic properties. Specifically, we synthesized two helical hexapeptides containing four Aib (α-amionoisobutyric acid) and two L-Dap (2,3-diamino propionic acid) residues. L-Dap side chains were then functionalized with Fc moieties. The structures of the two 310 helical peptides, namely Z-Aib-L-Dap(Fc)-Aib-Aib-L-Dap(Fc)-Aib-NH-iPr and Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, were investigated by X-ray diffraction, 2D-NMR, CD and IR spectroscopies. Due to the helical conformation, in Z-Aib-L-Dap(Fc)-Aib-Aib-L-Dap(Fc)-Aib-NH-iPr, the Fc groups are located on the same face of the helix, but in Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, they are located on opposite faces. Surprisingly, two bands were found through DPV for Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, indicating an electrostatic interaction between the Fc groups despite their longer reciprocal distance with respect to that in Z-Aib-L-Dap(Fc)-Aib-Aib-L-Dap(Fc)-Aib-NH-iPr. CD experiments at different concentrations evidenced aggregation for Z-Aib-L-Dap(Fc)-Aib-L-Dap(Fc)-Aib-Aib-NH-iPr, even at high dilutions, thus suggesting that the Fc-Fc electrostatic interaction could be of an intermolecular nature.

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.