Stereochemical Behavior of Pyrrolo-Pyrazole Peptidomimetics Promoting Phase-Selective Supramolecular Organogels.

Supramolecular gels were developed by taking advantage of an assembly of small dipeptides containing pyrrolo-pyrazole scaffolds. The dipeptides were prepared through a robust and ecofriendly synthetic approach from the commercially available starting materials of diazoalkanes and maleimides. By playing with the functionalization of the scaffold, the choice of the natural amino acid, and the stereochemistry, we were able to obtain phase-selective gels. In particular, one peptidomimetic showed gelation ability and thermoreversibility in aromatic solvents at very low concentrations. Rheology tests showed a typical viscoelastic solid profile, indicating the formation of strong gels that were stable under high mechanical deformation. NMR studies were performed, allowing us to determine the conformational and stereochemical features at the base of the supramolecular interactions.

Phosphine-Catalyzed Domino Regio- and Stereo-Selective Hexamerization of 2-(Bromomethyl)acrylates to 1,2-Bis(cyclohexenyl)ethenyl Derivatives.

A phosphine-catalyzed domino assembly of six units of 2-bromomethyl acrylates afforded polyalkenyl adducts containing two cyclohexenyl rings. This reaction occurs under mild conditions providing the final product by formation of seven carbon-carbon bonds and four stereocenters. Experimental and computational studies support an initial dimerization of the substrate, which in turn trimerizes involving two totally regio- and stereocontrolled Diels-Alder cycloadditions. The yield of the hexamerization of the 2-bromomethyl acrylates depends on the size of the ester function. The protocol has also proved to be practicable on a gram scale.

Highly efficient morpholine-based organocatalysts for the 1,4-addition reaction between aldehydes and nitroolefins: an unexploited class of catalysts.

Many studies have demonstrated how the pyrrolidine nucleus is more efficient than the corresponding piperidine or morpholine as organocatalysts in the condensation of aldehydes with electrophiles via enamine. Focussing on morpholine-enamines, their low reactivity is ascribed to the presence of oxygen on the ring and to the pronounced pyramidalisation of nitrogen, decreasing the nucleophilicity of the enamine. Thus, the selection of efficient morpholine organocatalysts appears to be a difficult challenge. Herein, we reported on the synthesis of new organocatalysts belonging to the class of ß-morpholine amino acids that were tested in a model reaction, i.e., the 1,4-addition reaction of aldehydes to nitroolefins. Starting from commercially available amino acids and epichlorohydrin, we designed an efficient synthesis for the aforementioned catalysts, controlling the configuration and the substitution pattern. Computational studies indeed disclosed the transition state of the reaction, explaining why, despite all the limitations of the morpholine ring for enamine catalysis, our best catalyst works efficiently, affording condensation products with excellent yields, diastereoselection and good-to-exquisite enantioselectivity.

Non-Conventional Peptide Self-Assembly into a Conductive Supramolecular Rope.

Structures composed of alternating α and ß amino acids can give rise to peculiar secondary structural motifs, which could self-assemble into complex structures of controlled geometries. This work describes the self-assembly properties of an α,ß-peptide, containing three units of syn H2-(2-F-Phe)-h-PheGly-OH, able to self-organize on surfaces into a fascinating supramolecular rope. This material was characterized by AFM, electronic conduction and fluorescence measurements. Molecular dynamics simulations showed that this hexapeptide can self-assemble into an antiparallel ß-sheet layer, stabilized by intermolecular H-bonds, which, in turn, can self-assemble into many side-by-side layers, due to π-π interactions. As a matter of fact, we demonstrated that in this system, the presence of aromatic residues at the intramolecular interface promoted by the alternation of α,ß-amino-acids in the primary sequence, endorses the formation of a super-secondary structure where the aromatic groups are close to each other, conferring to the system good electron conduction properties. This work demonstrates the capability and future potential of designing and fabricating distinctive nanostructures and efficient bioelectronic interfaces based on an α,ß-peptide, by controlling structure and interaction processes beyond those obtained with α- or ß-peptides alone.

Ultrashort Peptides and Gold Nanoparticles: Influence of Constrained Amino Acids on Colloidal Stability.

Poor colloidal stability of gold nanoparticles (AuNPs) in physiological environments remains one of the major limitations that contribute to their difficult translation from bench to clinic. For this reason, an active research field is the development of molecules able to hamper AuNPs aggregation tendency in physiological environments. In this context, synthetic peptides are gaining an increased interest as an alternative to the use of biomacromolecules and polymers, due to their easiness of synthesis and their profitable pharmacokinetic profile. In this work, we reported on the use of ultrashort peptides containing conformationally constrained amino acids (AAs) for the stabilization of AuNPs. A small library of non-natural self-assembled oligopeptides were synthesized and used to functionalize spherical AuNPs of 20 nm diameter, via the ligand exchange method. The aim was to investigate the role of the constrained AA, the anchor point (at C- or N-terminus) and the peptide length on their potential use as gold binding motif. Ultrashort Aib containing peptides were identified as effective tools for AuNPs colloidal stabilization. Furthermore, peptide coated AuNPs were found to be storable as powders without losing the stabilization properties once re-dispersed in water. Finally, the possibility to exploit the developed systems for binding proteins via molecular recognition was also evaluated using biotin as model.

Exploiting Ultrashort α,ß-Peptides in the Colloidal Stabilization of Gold Nanoparticles.

Colloidal gold nanoparticles (GNPs) have found wide-ranging applications in nanomedicine due to their unique optical properties, ease of preparation, and functionalization. To avoid the formation of GNP aggregates in the physiological environment, molecules such as lipids, polysaccharides, or polymers are employed as GNP coatings. Here, we present the colloidal stabilization of GNPs using ultrashort α,ß-peptides containing the repeating unit of a diaryl ß2,3-amino acid and characterized by an extended conformation. Differently functionalized GNPs have been characterized by ultraviolet, dynamic light scattering, and transmission electron microscopy analysis, allowing us to define the best candidate that inhibits the aggregation of GNPs not only in water but also in mouse serum. In particular, a short tripeptide was found to be able to stabilize GNPs in physiological media over 3 months. This new system has been further capped with albumin, obtaining a material with even more colloidal stability and ability to prevent the formation of a thick protein corona in physiological media.

New Perspectives in the Antimicrobial Activity of the Amphibian Temporin B: Peptide Analogs Are Effective Inhibitors of Candida albicans Growth.

Temporin B (TB) is a short, positively charged peptide secreted by the granular glands of the European frog Rana temporaria. While the antibacterial and antiviral efficacy of TB and some of its improved analogs are well documented, nothing is known about their antifungal potency so far. We dedicated this study to characterize the antifungal potential of the TB analog TB_KKG6K and the newly designed D-Lys_TB_KKG6K, the latter having the L-lysines replaced by the chiral counterpart D-lysines to improve its proteolytic stability. Both peptides inhibited the growth of opportunistic human pathogenic yeasts and killed planktonic and sessile cells of the most prevalent human pathogen, Candida albicans. The anti-yeast efficacy of the peptides coincided with the induction of intracellular reactive oxygen species. Their thermal, cation, pH and serum tolerance were similar, while the proteolytic stability of D-Lys_TB_KKG6K was superior to that of its template peptide. Importantly, both peptides lacked hemolytic activity and showed minimal in vitro cytotoxicity in primary human keratinocytes. The tolerance of both peptides in a reconstructed human epidermis model further supports their potential for topical application. Our results open up an exciting field of research for new anti-Candida therapeutic options based on amphibian TB analogs.

In vitro angiogenesis inhibition with selective compounds targeting the key glycolytic enzyme PFKFB3.

Abnormal glycolytic metabolism contributes to angiogenic sprouting involved in atherogenesis. We investigated the potential anti-angiogenic properties of specific 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) inhibitors in endothelial cells (ECs). ECs were treated with PFKFB3 inhibitors (named PA-1 and PA-2) and their effects on metabolic and functional characteristics of ECs were investigated. The anti-glycolytic compound 3-(pyridinyl)- 1-(4-pyridinyl)- 2-propen-1-one (3PO) was used as reference compound. PFKFB3 expression and activity (IC50 about 3-21 nM) was inhibited upon treatment with both compounds. Glucose uptake and lactate export were measured using commercial assays and showed a partial reduction up to 40%. PFKFB3 inhibition increased intracellular lactate accumulation, and reduced expression of monocarboxylate transporters-1 (MCT1) and MCT4. Furthermore, endothelial cell migration and proliferation assays demonstrated significant reduction upon treatment with both compounds. Matrix- metalloproteinase (MMP) activity, measured by gelatin zymography, and expression was significantly reduced (up to 25%). In addition, PA compounds downregulated the expression of VCAM-1, VE-cadherin, VEGFa, VEGFR2, TGF-ß, and IL-1ß, in inflamed ECs. Finally, PA-1 and PA-2 treatment impaired the formation of angiogenic sprouts measured by both morphogenesis and spheroid-based angiogenesis assays. Our data demonstrate that the anti-glycolytic PA compounds may affect several steps involved in angiogenesis. Targeting the key glycolytic enzyme PFKFB3 might represent an attractive therapeutic strategy to improve the efficacy of cancer treatments, or to be applied in other pathologies where angiogenesis is a detrimental factor.

Morpholino-based peptide oligomers: Synthesis and DNA binding properties.

The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization.

Peptide grafting strategies before and after electrospinning of nanofibers.

Nanofiber films produced by electrospinning currently provide a promising platform for different applications. Although nonfunctionalized nanofiber films from natural or synthetic polymers are extensively used, electrospun materials combined with peptides are gaining more interest. In fact, the selection of specific peptides improves the performance of the material for biological applications and mainly for tissue engineering, mostly by maintaining similar mechanical properties with respect to the simple polymer. The main drawback in using peptides blended with a polymer is the quick release of the peptides. To avoid this problem, covalent linking of the peptide is more beneficial. Here, we reviewed synthetic protocols that enable covalent grafting of peptides to polymers before or after the electrospinning procedures to obtain more robust electrospun materials. Applications and the performance of the new material compared to that of the starting polymer are discussed.

Nucleobase morpholino ß amino acids as molecular chimeras for the preparation of photoluminescent materials from ribonucleosides.

Bioinspired smart materials represent a tremendously growing research field and the obtainment of new building blocks is at the molecular basis of this technology progress. In this work, colloidal materials have been prepared in few steps starting from ribonucleosides. Nucleobase morpholino ß-amino acids are the chimera key intermediates allowing Phe-Phe dipeptides' functionalization with adenine and thymine. The obtained compounds self-aggregate showing enhanced photoluminescent features, such as deep blue fluorescence and phosphorescence emissions.

Rational Design of a User-Friendly Aptamer/Peptide-Based Device for the Detection of Staphylococcus aureus.

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment.

Self-assembled hydrophobic Ala-Aib peptide encapsulating curcumin: a convenient system for water insoluble drugs.

The exploitation of self-assembled systems to improve the solubility of drugs is getting more and more attention. Among the different types of self-assembled biomaterials, peptides and in particular peptides containing non-coded amino acids (NCAPs) are promising because their use opens the door to more stable materials inducing increased stability to proteolysis. New classes of NCAP, Ac-Ala-X-Ala-Aib-AlaCONH2 (X = alpha-aminoisobutyric acid (Aib) or X = cyclopentane amino acid (Ac5c)) have been prepared and the correlation between the different secondary peptide structure and solvent (i.e. CD3CN, CD3OH, H2O/D2O) verified by NMR. Furthermore, the formation of a nanocolloidal system in water was deeply studied by DLS and the morphology of the obtained spherical aggregates with nanometric dimensions was assessed by TEM. Aib containing pentapeptide was selected for greater ease of synthesis. Its ability to encapsulate curcumin, as a model insoluble drug molecule, was investigated using fluorescence emission and confocal microscopy analyses. Two different approaches were used to study the interaction between curcumin and peptide aggregates. In the first approach peptide aggregates were formed in the presence of curcumin, while in the second approach curcumin was added to the already formed peptide aggregates. We succeeded in our challenge by using the second approach and 53.8% of added curcumin had been encapsulated.

Covalent Grafting of Antimicrobial Peptides onto Microcrystalline Cellulose.

The purpose of this work is to set up a general protocol for the production of antimicrobial materials based on cellulose and peptides. We exploited the chemical ligation reaction to achieve the conjugation of peptides to cellulose; to this aim, we produced thioester peptides and cysteine-modified cellulose. As the thioester handle can be inserted at any position of the peptide, the peptide can be immobilized onto the cellulose through its N- or C-terminal end or through any other position within the sequence. Our experiments performed on Escherichia coli cultures show that the cellulose conjugated to the peptides lasioglossin-III and TBKKG6A causes a significant reduction in the concentration of viable cells as compared to unmodified cellulose. In conclusion, antimicrobial peptides bound to cellulose through a covalent bond retain their activity and therefore have the potential to be used as active ingredients in antimicrobial materials.

Application of Biophysical Techniques to Investigate the Interaction of Antimicrobial Peptides With Bacterial Cells.

Gaining new understanding on the mechanism of action of antimicrobial peptides is the basis for the design of new and more efficient antibiotics. To this aim, it is important to detect modifications occurring to both the peptide and the bacterial cell upon interaction; this will help to understand the peptide structural requirement, if any, at the base of the interaction as well as the pathways triggered by peptides ending in cell death. A limited number of papers have described the interaction of peptides with bacterial cells, although most of the studies published so far have been focused on model membrane-peptides interactions. Investigations carried out with bacterial cells highlighted the limitations connected to the use of oversimplified model membranes and, more importantly, helped to identify molecular targets of antimicrobial peptides and changes occurring to the bacterial membrane. In this review, details on the mechanism of action of antimicrobial peptides, as determined by the application of spectroscopic techniques, as well as scattering, microscopy, and calorimetry techniques, to complex systems such as peptide/bacteria mixtures are discussed.

On-resin multicomponent 1,3-dipolar cycloaddition of cyclopentanone-proline enamines and sulfonylazides as an efficient tool for the synthesis of amidino depsipeptide mimics.

Depsipeptides are biologically active peptide derivatives that possess a high therapeutic interest. The development of depsipeptide mimics characterized by a chemical diversity could lead to compounds with enhanced features and activity. In this work, an on-resin multicomponent procedure for the synthesis of amidino depsipeptide mimics is described. This approach exploits a metal-free 1,3-dipolar cycloaddition of cyclopentanone-proline enamines and sulfonylazides. In this reaction, the obtained primary cycloadduct undergoes a ring opening and molecular rearrangement giving access to a linear sulfonyl amidine functionalized with both a peptide chain and a diazoalkane. The so-obtained diazo function "one pot" reacts with the carboxylic group of N-Fmoc-protected amino acids leading to amidino depsipeptide mimics possessing a C4 aliphatic chain. An important advantage of this procedure is the possibility to easily obtain amidino-functionalized derivatives that are proteolytically stable peptide bond bioisosteres. Moreover, the conformational freedom given by the alkyl chain could promote the obtainment of cyclic depsipeptide with a stabilized secondary structure as demonstrated with both in silico calculations and experimental conformational studies. Finally, labeled depsipeptide mimics can be also synthesized using a fluorescent sulfonylazide in the multicomponent reaction.

Fluoro-Aryl Substituted α,ß2,3-Peptides in the Development of Foldameric Antiparallel ß-Sheets: A Conformational Study.

α,ß2,3-Disteroisomeric foldamers of general formula Boc(S-Ala-ß-2R,3R-Fpg)nOMe or Boc(S-Ala-ß-2S,3S-Fpg)nOMe were prepared from both enantiomers of syn H-2-(2-F-Phe)-h-PheGly-OH (named ß-Fpg) and S-alanine. Our peptides show two appealing features for biomedical applications: the presence of fluorine, attractive for non-covalent interactions, and aryl groups, crucial for π-stacking. A conformational study was performed, using IR, NMR and computational studies of diastereoisomeric tetra- and hexapeptides containing the ß2,3-amino acid in the R,R- and S,S-stereochemistry, respectively. We found that the stability of peptide conformation is dependent on the stereochemistry of the ß-amino acid. Combining S-Ala with ß-2R,3R-Fpg, a stable extended ß-strand conformation was obtained. Furthermore, ß-2R,3R-Fpg containing hexapeptide self-assembles to form antiparallel ß-sheet structure stabilized by intermolecular H-bonds and π,π-interactions. These features make peptides containing the ß2,3-fluoro amino acid very appealing for the development of bioactive proteolytically stable foldameric ß-sheets as modulators of protein-protein interaction (PPI).

Electrospinning of pyrazole-isothiazole derivatives: nanofibers from small molecules.

We investigate the electrospinning of small molecules, specifically designed peptide derivatives of the pyrazole-isothiazole scaffold. Such non-natural peptides enhance the spectrum of fundamental materials used for electrospinning. Unlike standard electrospun materials, our peptides are not polymeric, but able to aggregate in solution and especially during processing. They contain donor/acceptor groups that can form hydrogen bonds, and groups that are able to generate π-stacking interactions, which are known as important requirements for assembly processes. The pyrazole-isothiazole derivatives were synthesized by means of a 1,3-dipolar cycloaddition reaction, which is completely regioselective, affording only one isomer. We demonstrate that our compounds can be electrospun from fluoroalcohol solution into solid, quasi-endless micro- and nanofibers. The electrospinnability varies substantially, depending on the amino acids linked to the scaffold. Some compounds provide only short fibers, while Fmoc-glycyl-(N-benzyl)-pyrazole-isothiazole-tert-butyl carboxylate-1,1-dioxide forms continuous, homogenous, and bead-free fibers (droplet-like beads are a common problem in electrospinning). We analyzed the compounds and the fibers with various spectroscopic techniques (MS, IR and Raman). Electrospinning does not change chemical composition and configuration, suggesting the monomeric form of the compounds even in the fibers. Interestingly, we found that the stereochemistry of the scaffold can affect the ability of the peptide to be electrospun.

Tetrahydro-4 H-(pyrrolo[3,4- d]isoxazol-3-yl)methanamine: A Bicyclic Diamino Scaffold Stabilizing Parallel Turn Conformations.

A tetrahydro-4 H-(pyrrolo[3,4- d]isoxazol-3-yl)methanamine scaffold was designed as a diamino derivative to stabilize parallel turn conformations. Its synthesis took advantage of a [1,3]-dipolar cycloaddition reaction between the nitrile oxide derived from the inexpensive enantiopure l -phenylalanine and N-benzyl-3-pyrroline. Two diastereoisomers were formed, whose distribution depends on the selected base. 3a R,6a S-Isomer is favored in organic bases, which formation is driven by π-interactions. However, the above interactions were significantly prevented using an inorganic base due to the chaotropic effect of the cation, decreasing the amount of the above isomer. Finally, we demonstrated that this isomer is able to stabilize parallel turn conformations when inserted in short peptide sequences.

Structure-activity relationships of ß-hairpin mimics as modulators of amyloid ß-peptide aggregation.

Aggregation of amyloid proteins is currently involved in more than 20 serious human diseases that are actually untreated, such as Alzheimer's disease (AD). Despite many efforts made to target the amyloid cascade in AD, finding an aggregation inhibiting compound and especially modulating early oligomerization remains a relevant and challenging strategy. We report herein the first examples of small and non-peptide mimics of acyclic beta-hairpins, showing an ability to delay the fibrillization of amyloid-ß (Aß1-42) peptide and deeply modify its early oligomerization process. Modifications providing better druggability properties such as increased hydrophilicity and reduced peptidic character were performed. We also demonstrate that an appropriate balance between flexibility and stability of the ß-hairpin must be reached to adapt to the different shape of the various aggregated forms of the amyloid peptide. This strategy can be investigated to target other challenging amyloid proteins.