Double Orthogonal Click Reactions for the Development of Antimicrobial Peptide Nanotubes.

A new class of amphipathic cyclic peptides, which assemble in bacteria membranes to form polymeric supramolecular nanotubes giving them antimicrobial properties, is described. The method is based on the use of two orthogonal clickable transformations to incorporate different hydrophobic or hydrophilic moieties in a simple, regioselective, and divergent manner. The resulting cationic amphipathic cyclic peptides described in this article exhibit strong antimicrobial properties with a broad therapeutic window. Our studies suggest that the active form is the nanotube resulted from the parallel stacking of the cyclic peptide precursors. Several techniques, CD, FTIR, fluorescence, and STEM, among others, confirm the nanotube formation.

Molecular Plumbing to Bend Self-Assembling Peptide Nanotubes.

Light-induced molecular piping of cyclic peptide nanotubes to form bent tubular structures is described. The process is based on the [4+4] photocycloaddition of anthracene moieties, whose structural changes derived from the interdigitated flat disposition of precursors to the corresponding cycloadduct moieties, induced the geometrical modifications in nanotubes packing that provokes their curvature. For this purpose, we designed a new class of cyclic peptide nanotubes formed by ß- and α-amino acids. The presence of the former predisposes the peptide to stack in a parallel fashion with the ß-residues aligned along the nanotube and the homogeneous distribution of anthracene pendants.

Parallel Versus Antiparallel ß-Sheet Structure in Cyclic Peptide Hybrids Containing γ- or δ-Cyclic Amino Acids.

Cyclic peptides with disc-shaped structures have emerged as potent building blocks for the preparation of new biomaterials in fields ranging from biological to material science. In this work, we analyze in depth the self-assembling properties of a new type of cyclic peptides based on the alternation of α-residues and cyclic δ-amino acids (α,δ-CPs). To examine the preferred stacking properties adopted by cyclic peptides bearing this type of amino acids, we carried out a synergistic in vitro/in silico approximation by using simple dimeric models and then extended to nanotubes. Although these new cyclic peptides (α,δ-CPs) can interact either in a parallel or antiparallel fashion, our results confirm that although the parallel ß-sheet is more stable, it can be switched to the antiparallel stacking by choosing residues that can establish favorable cross-strand interactions. Moreover, the subsequent comparison by using the same methodology but applied to α,γ-CPs models, up to the moment assumed as antiparallel-like d,l-α-CPs, led to unforeseen conclusions that put into question preliminary conjectures about these systems. Surprisingly, they tend to adopt a parallel ß-sheet directed by the skeleton interactions. These results imply a change of paradigm with respect to cyclic peptide designs that should be considered for dimers and nanotubes.

Tight Xenon Confinement in a Crystalline Sandwich-like Hydrogen-Bonded Dimeric Capsule of a Cyclic Peptide.

A cyclic hexapeptide with three pyridyl moieties connected to its backbone forms a hydrogen-bonded dimer, which tightly encapsulates a single xenon atom, like a pearl in its shell. The dimer imprints its shape and symmetry to the captured xenon atom, as demonstrated by 129 Xe NMR spectroscopy, single-crystal X-ray diffraction, and computational studies. The dimers self-assemble hierarchically into tubular structures to form a porous supramolecular architecture, whose cavities are filled by small molecules and gases.

Self-Assembling Molecular Capsules Based on α,γ-Cyclic Peptides.

A new capsule based on a ß-sheet self-assembling cyclic peptide with the ability to recognize and release several guests is described. The host structure is composed of two self-complementary α,γ-cyclic peptides bearing a Zn porphyrin cap that is used for the selective recognition of the guest. The two components are linked through two dynamic covalent bonds. The combination of binding forces, including hydrogen bonding, metal coordination, and dynamic hydrazone bonds, allows the reversible recognition of long bipyridine guests. The affinity for these ligands showed a strong dependence on the guest length. Delivery of the encapsulated ligand can be achieved by hydrolysis of hydrazones to disrupt the sandwich complex structure.

Anion Recognition and Induced Self-Assembly of an α,γ-Cyclic Peptide To Form Spherical Clusters.

A cyclic octapeptide composed of hydroxy-functionalized γ-amino acids folds in a "V-shaped" conformation that allows the selective recognition of anions such as chloride, nitrate, and carbonate. The process involves the simultaneous self-assembly of six peptide subunits and the recognition of four anions to form a tetrahedral structure, in which the anions are located at the corners of the resulting structure. Each anion is coordinated to three different peptides. The structure was fully characterized by several techniques, including NMR spectroscopy and X-ray diffraction, and the material was able to facilitate the transmembrane transport of chloride ions.

Molecular pom poms from self-assembling α,γ-cyclic peptides.

The hierarchical self-assembly properties of a dimer-forming cyclic peptide that bears a nicotinic acid moiety to form molecular pom-pom-like structures are described. This dimeric assembly self organizes into spherical structures that can encapsulate small organic molecules owing to its porosity and it can also facilitate metal deposition on its surface directed by the pyridine moiety.

Design of stable ß-sheet-based cyclic peptide assemblies assisted by metal coordination: selective homo- and heterodimer formation.

Metal-directed supramolecular construction represents one of the most powerful tools to prepare a large variety of structures and functions. The ability of metals to organize different numbers and types of ligands with a variety of geometries (linear, trigonal, octahedral, etc.) expands the supramolecular synthetic architecture. We describe here the precise construction of homo- and heterodimeric cyclic peptide entities through coordination of a metal (Pd, Au) and to ß-sheet-type hydrogen-bonding interactions. The selective coordination properties of the appropriate metal allow control over the cross-strand interaction between the two-peptide strands.

3D-Printing of Capsule Devices as Compartmentalization Tools for Supported Reagents in the Search of Antiproliferative Isatins.

The application of high throughput synthesis methodologies in the generation of active pharmaceutical ingredients (APIs) currently requires the use of automated and easily scalable systems, easy dispensing of supported reagents in solution phase organic synthesis (SPOS), and elimination of purification and extraction steps. The recyclability and recoverability of supported reagents and/or catalysts in a rapid and individualized manner is a challenge in the pharmaceutical industry. This objective can be achieved through a suitable compartmentalization of these pulverulent reagents in suitable devices for it. This work deals with the use of customized polypropylene permeable-capsule devices manufactured by 3D printing, using the fused deposition modeling (FDM) technique, adaptable to any type of flask or reactor. The capsules fabricated in this work were easily loaded "in one step" with polymeric reagents for use as scavengers of isocyanides in the work-up process of Ugi multicomponent reactions or as compartmentalized and reusable catalysts in copper-catalyzed cycloadditions (CuAAC) or Heck palladium catalyzed cross-coupling reactions (PCCCRs). The reaction products are different series of diversely substituted isatins, which were tested in cancerous cervical HeLa and murine 3T3 Balb fibroblast cells, obtaining potent antiproliferative activity. This work demonstrates the applicability of 3D printing in chemical processes to obtain anticancer APIs.

Self-assembling properties of all γ-cyclic peptides containing sugar amino acid residues.

In this study, a novel dimer-forming cyclic peptide composed exclusively by cyclic γ-amino acids with a saccharide-like outer surface is described. The antiparallel ß-sheet type hydrogen bonding interactions responsible for the large association constant in non-polar solvents constitute a suitable model for a novel class of self-assembling peptide nanotubes.

Macromolecular assembly and membrane activity of antimicrobial D,L-α-Cyclic peptides.

Antimicrobial peptides are viewed as a promising alternative to conventional antibiotics, as their activity through membrane targeting makes them less prone to resistance development. Among them, antimicrobial D,L-α-cyclic peptides (CPs) have been proposed as an alternative, specially due to their cyclic nature and to the presence of D-α-amino acids that increases their resistance to proteases. In present work, second generation D,L-α-cyclic peptides with proven antimicrobial activity are shown to form complex macromolecular assemblies in the presence of membranes. We addressed the CPs:membrane interactions through a combination of experimental techniques (DSC and ATR-FTIR) with coarse-grained molecular dynamics (CG-MD) simulations, aiming at understanding their interactions, macromolecular assemblies and eventually unveil their mechanism of action. DSC shows that the interaction depends heavily on the negatively charge content of the membrane and on lipid/peptide ratio, suggesting different mechanisms for the different peptides and lipid systems. CG-MD proved that CPs can self-assemble at the lipid surface as nanotubes or micellar aggregates, depending on the peptide, in agreement with ATR-FTIR results. Finally, our results shed light into possible mechanisms of action of the peptides with pending hydrocarbon tail, namely membrane extensive segregation and/or membrane disintegration through the formation of disk-like lipid/peptide aggregates.

Transmembrane Self-Assembled Cyclic Peptide Nanotubes Based on α-Residues and Cyclic δ-Amino Acids: A Computational Study.

Self-assembling cyclic peptide nanotubes have been shown to function as synthetic, integral transmembrane channels. The combination of natural and nonnatural aminoacids in the sequence of cyclic peptides enables the control not only of their outer surface but also of the inner cavity behavior and properties, affecting, for instance, their permeability to different molecules including water and ions. Here, a thorough computational study on a new class of self-assembling peptide motifs, in which δ-aminocycloalkanecarboxylic acids are alternated with natural α-amino acids, is presented. The presence of synthetic δ-residues creates hydrophobic regions in these α,δ-SCPNs, which makes them especially attractive for their potential implementation in the design of new drug or diagnostic agent carrier systems. Using molecular dynamics simulations, the behavior of water molecules, different ions (Li+, Na+, K+, Cs+, and Ca2+), and their correspondent counter Cl- anions is extensively investigated in the nanoconfined environment. The structure and dynamics are mutually combined in a diving immersion inside these transmembrane channels to discover a fascinating submarine nanoworld where star-shaped water channels guide the passage of cations and anions therethrough.

Membrane targeting antimicrobial cyclic peptide nanotubes - an experimental and computational study.

The search of new antibiotics, particularly with new mechanisms of action, is nowadays a very important public health issue, due to the worldwide increase of resistant pathogens. Within this effort, much research has been done on antimicrobial peptides, because having the membrane as a target, they represent a new antibiotic paradigm. Among these, cyclic peptides (CPs) made of sequences of D- and L-amino acids have emerged as a new class of potential antimicrobial peptides, due to their expected higher resistance to protease degradation. These CPs are planar structures that can form Self-assembled Cyclic Peptide Nanotubes (SCPNs), in particular in the presence of lipid membranes. Aiming at understanding their mechanism of action, we used biophysical experimental techniques (DSC and ATR-FTIR) together with Coarse-grained molecular dynamics (CG-MD) simulations, to characterize the interaction of these CPs with model membranes of different electrostatic charges' contents. DSC results revealed that the CPs show a strong interaction with negatively charged membranes, with differences in the strength of interactions depending on peptide and on membrane charge content, at odds with no or mild interactions with zwitterionic membranes. ATR-FTIR suggested that the peptides self-assemble at the membrane surface, adopting mainly a ß-structure. The experiments with polarized light showed that in most cases they lie parallel to the membrane surface, but other forms and orientations are also apparent, depending on peptide structure and lipid:peptide ratio. The nanotube formation and orientation, as well as the dependence on membrane charge were also confirmed by the CG-MD simulations. These provide detail on the position and interactions, in agreement with the experimental results. Based on the findings reported here, we could proceed to the design and synthesis of a second-generation CPs, based on CP2 (soluble peptide), with increased activity and reduced toxicity.

Alpha,gamma-cyclic peptide ensembles with a hydroxylated cavity.

Here we describe a self-assembling alpha,gamma-cyclic tetrapeptide that contains the 4-amino-3-hydroxytetrahydrofuran-2-carboxylic acid, in which the hydroxy group is pointing towards the inner cavity of the resulting dimers.

Versatile symport transporters based on cyclic peptide dimers.

We present the synthesis and transmembrane transport properties of a new family of tris-pyridine-decorated cyclic peptides. These molecules are designed to self-assemble into dimeric shuttles in nonpolar media, which act as symport ionophores in which, apparently, the tris-pyridine scaffold complexes both cations and anions with high potency and efficacy.

Novel synthesis of platinum complexes and their intracellular delivery to tumor cells by means of magnetic nanoparticles.

Platinum-based drugs are popular in clinics as chemotherapeutic agents to treat solid tumors. However, severe side effects such as nephro- and neurotoxicity impose strict dosage limitations that can lead to the development of drug resistance and tumor relapse. To overcome these issues Pt(iv) prodrugs and platinum delivery systems might represent the next generation of platinum-based drugs. In this study four novel Pt(ii) complexes (namely, PEG-Glu-Pt-EDA, PEG-Glu-Pt-DACH, PEG-Mal-Pt-EDA and PEG-Mal-Pt-DACH) were synthesized and a general strategy to covalently bind them to iron oxide nanoparticles was developed. The intracellular uptake and cell distribution studies of Pt-tethered magnetic nanoparticles on breast and ovarian cancer cell line models indicate that binding of the Pt complexes to the nanoparticles facilitates, for all the complexes, cellular internalization. Moreover, the magnetic nanoparticles (MNPs), as shown in a magnetofection experiment, enhance the uptake of MNP-Pt conjugates if a magnet is placed beneath the culture dish of tumor cells. As shown by a Pt release experiment, intranuclear platinum quantification and TEM analysis on cell sections, the presence of a pH-sensitive dicarboxylic group coordinating the Pt complex, triggers platinum dissociation from the NP surface. In addition, the triazole moiety facilitates endosomal swelling and the leakage of platinum from the endosomes with intranuclear localization of platinum release by the NPs. Finally, as assessed by MTT, caspase, calcein/ethidium bromide live/dead assays, among the four NP-Pt conjugates, the NP-Glu-Pt-EDA complex having a glutamate ring and ethylenediamine as a chelating amine group of the platinum showed higher cytotoxicity than the other three MNP-platinum conjugates.

Multicatalysis Combining 3D-Printed Devices and Magnetic Nanoparticles in One-Pot Reactions: Steps Forward in Compartmentation and Recyclability of Catalysts.

A tricatalytic compartmentalized system that immobilizes metallic species to perform one-pot sequential functionalization is described: a three-dimensional (3D)-printed palladium monolith, ferritic copper(I) magnetic nanoparticles, and a 3D-printed polypropylene capsule-containing copper(II) loaded onto polystyrene-supported 1,5,7-triazabicyclo[4.4.0]dec-5-ene (PS-TBD) allowed the rapid synthesis of diverse substituted 1-([1,1'-biphenyl]-4-yl)-1H-1,2,3-triazoles. The procedure is based on the Chan-Lam azidation/copper alkyne-azide cycloaddition/Suzuki reaction strategy in the solution phase. This catalytic system enabled the efficient assembly of the final compounds in high yields without the need for special additives or intermediate isolation. The monolithic catalyst-containing immobilized palladium species was synthesized by surface chemical modification of a 3D-printed silica monolith using a soluble polyimide resin as a key reagent, thus creating an extremely robust composite. All three immobilized catalysts described here were easily recovered and reused in numerous cycles. This work exemplifies the role of 3D printing in the design and manufacture of devices for compartmented multicatalytic systems to carry out complex one-pot transformations.

A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution.

The ability to monitor DNA polymerase activity with single-nucleotide resolution has been the cornerstone of a number of advanced single-molecule DNA sequencing concepts. Toward this goal, we report the first observation of the base-by-base DNA polymerase activity with single-base resolution at the single-molecule level. We describe the design and characterization of a supramolecular nanopore device capable of detecting up to nine consecutive DNA polymerase-catalyzed single-nucleotide primer extensions with high sensitivity and spatial resolution (

New self-assembling peptide nanotubes of large diameter using δ-amino acids.

Here we show that 4-aminocyclohexanecarboxylic acid is a rigid stretcher building block for the preparation of cyclic peptides that self-assemble to form peptide nanotubes with large diameter and hydrophobic pores. The hydrophobic properties of the resulting nanotubes provided by the two methylene groups per δ-residue allow the encapsulation of C60 moieties forming a new type of bionanopeapod structure.

Self-assembling Venturi-like peptide nanotubes.

We describe the design and synthesis of self-assembling peptide nanotubes that have an internal filter area and whose length and internal diameters, at the entrance and in the constricted area, are precisely controlled.