Supramolecular Hydrogels and Water Channels of Differing Diameters from Dipeptide Isomers.

Dipeptides stereoisomers and regioisomers composed of norleucine (Nle) and phenylalanine (Phe) self-assemble into hydrogels under physiological conditions that are suitable for cell culture. The supramolecular behavior, however, differs as the packing modes comprise amphipathic layers or water channels, whose diameter is defined by either four or six dipeptide molecules. A variety of spectroscopy, microscopy, and synchrotron-radiation-based techniques unveil fine details of intermolecular interactions that pinpoint the relationship between the chemical structure and ability to form supramolecular architectures that define soft biomaterials.

Designing New Hybrid Antibiotics: Proline-Rich Antimicrobial Peptides Conjugated to the Aminoglycoside Tobramycin.

Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1-15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.

Self-Assembly of Unprotected Dipeptides into Hydrogels: Water-Channels Make the Difference.

Unprotected dipeptides are attractive building blocks for environmentally friendly hydrogel biomaterials by virtue of their low-cost and ease of preparation. This work investigates the self-assembling behaviour of the distinct stereoisomers of Ile-Phe and Phe-Ile in phosphate buffered saline (PBS) to form hydrogels, using transmission electron microscopy (TEM), attenuated total reflectance infrared spectroscopy (ATR-IR), circular dichroism (CD), and oscillatory rheometry. Each peptide purity and identity was also confirmed by 1 H- and 13 C-NMR spectroscopy and HPLC-MS. Finally, single-crystal XRD data allowed the key interactions responsible for the supramolecular packing into amphipathic layers or water-channels to be revealed. The presence of the latter in the crystal structure is a distinctive feature of the only gelator of this work that self-organizes into stable hydrogels, with fast kinetics and the highest elastic modulus amongst its structural isomers and stereoisomers.

Single-atom substitution enables supramolecular diversity from dipeptide building blocks.

Dipeptides are popular building blocks for supramolecular gels that do not persist in the environment and may find various applications. In this work, we show that a simple substitution on the aromatic side-chain of phenylalanine with either fluorine or iodine enables supramolecular diversity upon self-assembly at neutral pH, leading to hydrogels or crystals. Each building block is characterized by 1H- and 13C-NMR spectroscopy, LC-MS, circular dichroism, and molecular models. The supramolecular behaviour is monitored with a variety of techniques, including circular dichroism, oscillatory rheology, transmission electron microscopy, attenuated total reflectance Fourier-transformed infrared spectroscopy, visible Raman spectroscopy, synchrotron-radiation single-crystal X-ray diffraction and UV Resonance Raman spectroscopy, allowing key differences to be pinpointed amongst the halogenated analogues.

Dipeptide self-assembly into water-channels and gel biomaterial.

Dipeptides are convenient building blocks for supramolecular gel biomaterials that can be produced on a large scale at low cost and do not persist in the environment. In the case of unprotected sequences, hydrophobicity is a key requirement to enable gelation, with Phe-Phe standing out for its self-assembling ability. Conversely, more hydrophilic sequences such as homochiral dipeptides Phe-Val and Val-Phe neither fibrillate nor gel aqueous buffers and their crystal structures reveal amphipathic layers. In this work, we test emerging rules for the design of self-assembling dipeptides using heterochiral Phe-Val and Val-Phe. Each dipeptide is characterized by 1H- and 13C-NMR, LC-MS, circular dichroism, infrared and Raman spectroscopies, rheology, electron microscopy, and single-crystal X-ray diffraction. In particular, D-Phe-L-Val is the first heterochiral dipeptide to self-assemble into supramolecular water-channels whose cavity is defined by four peptide molecules arranged head-to-tail. This minimalistic sequence is devoid of amyloid character as probed by thioflavin T fluorescence and it displays excellent biocompatibility in vitro. The dataset provided, through comparison with the literature, significantly advances the definition of molecular design rules for minimalistic unprotected dipeptides that self-assemble into water-channels and biocompatible gels, to assist with the future development of supramolecular biomaterials with fine control over nanomorphological features for a variety of applications.

An SPR investigation into the therapeutic drug monitoring of the anticancer drug imatinib with selective aptamers operating in human plasma.

The anticancer drug imatinib is often involved in therapeutic drug monitoring (TDM) studies aimed at improving the treatment of several forms of leukemia and gastrointestinal stromal tumors (GIST). To further implement the TDM of imatinib in clinical practice, we developed a detection assay by using an ssDNA aptamer, which demonstrated excellent selectivity and was not affected by interference from the components of human plasma samples. The efficient binding of imatinib to the aptamer was demonstrated by means of surface plasmon resonance (SPR) analysis, which allowed the development of a quantitative assay in the concentration range between 400 and 6000 ng mL-1 (0.7-10 µM), where a lower limit of quantification (LLOQ) of 400 ng mL-1 was achieved. The precision of the assay was found to be within 12.0%, whereas the accuracy was in a range between 97.1 and 101.5%. The sample preparation procedure displayed a recovery in the range of 48.8-52.8%. Solid validation data were collected according to the regulatory guidelines and the method was compared with standard analytical techniques, leading to the development of a feasible aptasensor for the TDM of patients administered with imatinib.

Self-Assembly of an Amino Acid Derivative into an Antimicrobial Hydrogel Biomaterial.

N-(4-Nitrobenzoyl)-Phe self-assembled into a transparent supramolecular hydrogel, which displayed high fibroblast and keratinocyte cell viability. The compound showed a mild antimicrobial activity against E. coli both as a hydrogel and in solution. Single-crystal XRD data revealed packing details, including protonation of the C-terminus due to an apparent pKa shift, as confirmed by pH titrations. MicroRaman analysis revealed almost identical features between the gel and crystal states, although more disorder in the former. The hydrogel is thermoreversible and disassembles within a range of temperatures that can be fine-tuned by experimental conditions, such as gelator concentration. At the minimum gelling concentration of 0.63 wt %, the hydrogel disassembles in a physiological temperature range of 39-42 °C, thus opening the way to its potential use as a biomaterial.

Supramolecular hydrogels from unprotected dipeptides: a comparative study on stereoisomers and structural isomers.

Amino acid stereoconfiguration has been shown to play a key role in the self-assembly of unprotected tripeptides into hydrogels under physiological conditions. Dramatic changes were noted for hydrophobic sequences based on the diphenylalanine motif from the formation of amorphous aggregates in the case of homochiral peptides to nanostructured and stable hydrogels in the case of heterochiral stereoisomers. Herein, we report that by further shortening the sequence to a dipeptide, the overall differences between isomers are less marked, with both homo- and hetero-chiral dipeptides forming gels, although with different stability over time. The soft materials are studied by a number of spectroscopic and microcopic techniques, and single-crystal X-ray diffraction to unveil the supramolecular interactions of these hydrogel building blocks.

Peptide Stereochemistry Effects from pKa-Shift to Gold Nanoparticle Templating in a Supramolecular Hydrogel.

The divergent supramolecular behavior of a series of tripeptide stereoisomers was elucidated through spectroscopic, microscopic, crystallographic, and computational techniques. Only two epimers were able to effectively self-organize into amphipathic structures, leading to supramolecular hydrogels or crystals, respectively. Despite the similarity between the two peptides' turn conformations, stereoconfiguration led to different abilities to engage in intramolecular hydrogen bonding. Self-assembly further shifted the pKa value of the C-terminal side chain. As a result, across the pH range 4-6, only one epimer predominated sufficiently as a zwitterion to reach the critical molar fraction, allowing gelation. By contrast, the differing pKa values and higher dipole moment of the other epimer favored crystallization. The four stereoisomers were further tested for gold nanoparticle (AuNP) formation, with the supramolecular hydrogel being the key to control and stabilize AuNPs, yielding a nanocomposite that catalyzed the photodegradation of a dye. Importantly, the AuNP formation occurred without the use of reductants other than the peptide, and the redox chemistry was investigated by LC-MS, NMR, and infrared scattering-type near field optical microscopy (IR s-SNOM). This study provides important insights for the rational design of simple peptides as minimalistic and green building blocks for functional nanocomposites.

Nanotubes and water-channels from self-assembling dipeptides.

Dipeptides are attractive building blocks for biomaterials in light of their inherent biocompatibility, biodegradability, and simplicity of preparation. Since the discovery of diphenylalanine (Phe-Phe) self-assembling ability into nanotubes, research efforts have been devoted towards the identification of other dipeptide sequences capable of forming these interesting nanomorphologies, although design rules towards nanotube formation are still elusive. In this review, we analyze the dipeptide sequences reported thus far for their ability to form nanotubes, which often feature water-filled supramolecular channels as revealed by single-crystal X-ray diffraction, as well as their properties, and their potential biological applications, which span from drug delivery and regenerative medicine, to bioelectronics and bioimaging.

Polymer Conjugates of Antimicrobial Peptides (AMPs) with d-Amino Acids (d-aa): State of the Art and Future Opportunities.

In recent years, antimicrobial peptides (AMPs) have enjoyed a renaissance, as the world is currently facing an emergency in terms of severe infections that evade antibiotics' treatment. This is due to the increasing emergence and spread of resistance mechanisms. Covalent conjugation with polymers is an interesting strategy to modulate the pharmacokinetic profile of AMPs and enhance their biocompatibility profile. It can also be an effective approach to develop active coatings for medical implants and devices, and to avoid biofilm formation on their surface. In this concise review, we focus on the last 5 years' progress in this area, pertaining in particular to AMPs that contain d-amino acids, as well as their role, and the advantages that may arise from their introduction into AMPs.

Self-Assembly of Homo- and Hetero-Chiral Cyclodipeptides into Supramolecular Polymers towards Antimicrobial Gels.

There is an increasing interest towards the development of new antimicrobial coatings, especially in light of the emergence of antimicrobial resistance (AMR) towards common antibiotics. Cyclodipeptides (CDPs) or diketopiperazines (DKPs) are attractive candidates for their ability to self-assemble into supramolecular polymers and yield gel coatings that do not persist in the environment. In this work, we compare the antimicrobial cyclo(Leu-Phe) with its heterochiral analogs cyclo(D-Leu-L-Phe) and cyclo(L-Leu-D-Phe), as well as cyclo(L-Phe-D-Phe), for their ability to gel. The compounds were synthesized, purified by HPLC, and characterized by 1H-NMR, 13C-NMR, and ESI-MS. Single-crystal X-ray diffraction (XRD) revealed details of the intermolecular interactions within the supramolecular polymers. The DKPs were then tested for their cytocompatibility on fibroblast cells and for their antimicrobial activity on S. aureus. Overall, DKPs displayed good cytocompatibility and very mild antimicrobial activity, which requires improvement towards applications.

Cyclodipeptides: From Their Green Synthesis to Anti-Age Activity.

Cyclodipeptides (CDPs) or diketopiperazines (DKPs) are often found in nature and in foodstuff and beverages and have attracted great interest for their bioactivities, biocompatibility, and biodegradability. In the laboratory, they can be prepared by green procedures, such as microwave-assisted cyclization of linear dipeptides in water, as performed in this study. In particular, five CDPs were prepared and characterized by a variety of methods, including NMR and ESI-MS spectroscopies and single-crystal X-ray diffraction (XRD), and their cytocompatibility and anti-aging activity was tested in vitro, as well as their ability to penetrate the different layers of the skin. Although their mechanism of action remains to be elucidated, this proof-of-concept study lays the basis for their future use in anti-age cosmetic applications.

Peptide Gelators to Template Inorganic Nanoparticle Formation.

The use of peptides to template inorganic nanoparticle formation has attracted great interest as a green route to advance structures with innovative physicochemical properties for a variety of applications that range from biomedicine and sensing, to catalysis. In particular, short-peptide gelators offer the advantage of providing dynamic supramolecular environments for the templating effect on the formation of inorganic nanoparticles directly in the resulting gels, and ideally without using further reductants or chemical reagents. This mini-review describes the recent progress in the field to outline future research directions towards dynamic functional materials that exploit the synergy between supramolecular chemistry, nanoscience, and the interface between organic and inorganic components for advanced performance.

Nanoscale Assembly of Functional Peptides with Divergent Programming Elements.

Self-assembling peptides are being applied both in the biomedical area and as building blocks in nanotechnology. Their applications are closely linked to their modes of self-assembly, which determine the functional nanostructures that they form. This work brings together two structural elements that direct nanoscale self-association in divergent directions: proline as a ß-breaker and the ß-structure-associated diphenylalanine motif, into a single tripeptide sequence. Amino acid chirality was found to resolve the tension inherent to these conflicting self-assembly instructions. Stereoconfiguration determined the ability of each of the eight possible Pro-Phe-Phe stereoisomers to self-associate into diverse nanostructures, including nanoparticles, nanotapes, or fibrils, which yielded hydrogels with gel-to-sol transition at a physiologically relevant temperature. Three single-crystal structures and all-atom molecular dynamics simulations elucidated the ability of each peptide to establish key interactions to form long-range assemblies (i,e., stacks leading to gelling fibrils), medium-range assemblies (i.e., stacks yielding nanotapes), or short-range assemblies (i.e., dimers or trimers that further associated into nanoparticles). Importantly, diphenylalanine is known to serve as a binding site for pathological amyloids, potentially allowing these heterochiral systems to influence the fibrillization of other biologically relevant peptides. To probe this hypothesis, all eight Pro-Phe-Phe stereoisomers were tested in vitro on the Alzheimer's disease-associated Aß(1-42) peptide. Indeed, one nonfibril-forming stereoisomer effectively inhibited Aß fibrillization through multivalent binding between diphenylalanine motifs. This work thus defined heterochirality as a useful feature to strategically develop future therapeutics to interfere with pathological processes, with the additional value of resistance to protease-mediated degradation and biocompatibility.

Heterochirality and Halogenation Control Phe-Phe Hierarchical Assembly.

Diphenylalanine is an amyloidogenic building block that can form a versatile array of supramolecular materials. Its shortcomings, however, include the uncontrolled hierarchical assembly into microtubes of heterogeneous size distribution and well-known cytotoxicity. This study rationalized heterochirality as a successful strategy to address both of these pitfalls and it provided an unprotected heterochiral dipeptide that self-organized into a homogeneous and optically clear hydrogel with excellent ability to sustain fibroblast cell proliferation and viability. Substitution of one l-amino acid with its d-enantiomer preserved the ability of the dipeptide to self-organize into nanotubes, as shown by single-crystal XRD analysis, whereby the pattern of electrostatic and hydrogen bonding interactions of the backbone was unaltered. The effect of heterochirality was manifested in subtle changes in the positioning of the aromatic side chains, which resulted in weaker intermolecular interactions between nanotubes. As a result, d-Phe-l-Phe self-organized into homogeneous nanofibrils with a diameter of 4 nm, corresponding to two layers of peptides around a water channel, and yielded a transparent hydrogel. In contrast with homochiral Phe-Phe stereoisomer, it formed stable hydrogels thermoreversibly. d-Phe-l-Phe displayed no amyloid toxicity in cell cultures with fibroblast cells proliferating in high numbers and viability on this biomaterial, marking it as a preferred substrate over tissue-culture plastic. Halogenation also enabled the tailoring of d-Phe-l-Phe self-organization. Fluorination allowed analogous supramolecular packing as confirmed by XRD, thus nanotube formation, and gave intermediate levels of bundling. In contrast, iodination was the most effective strategy to augment the stability of the resulting hydrogel, although at the expense of optical transparency and biocompatibility. Interestingly, iodine presence hindered the supramolecular packing into nanotubes, resulting instead into amphipathic layers of stacked peptides without the occurrence of halogen bonding. By unravelling fine details to control these materials at the meso- and macro-scale, this study significantly advanced our understanding of these systems.