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

SELF-ASSEMBLY OF MINIMALISTIC PHENYLALANINE DERIVATIVES INTO SUPRAMOLECULAR HYDROGELS

Nel corso degli anni, i materiali nanostrutturati costituiti da piccoli peptidi e con una buona biodegradabilità hanno suscitato un notevole interesse nella ricerca. Grazie ai numerosi vantaggi che presentano rispetto ad altri tipi di materiali (ad es. biocompatibilità, versatilità chimica di composizione e semplicità di preparazione con bassi costi e su grande scala), si è assistito ad un crescente ed attivo interesse verso lo sviluppo di biomateriali di natura peptidica a partire da sequenze il più semplici e brevi possibili, quali singoli amminoacidi o dipeptidi, secondo un approccio di tipo minimalista. Questa tesi di dottorato, per prima cosa, descrive la capacità di un derivato dell’amminoacido fenilalanina, protetta all’N-terminale in posizione para con un gruppo nitrobenzoile, di auto-assemblarsi in condizioni fisiologiche per formare un idrogel trasparente. Studi preliminari in vitro, hanno rivelato per questo composto una promettente attività antimicrobica ed una buona biocompatibilità verso colture cellulari di mammifero. Successivamente, è stato investigato l’effetto della sostituzione del gruppo N-terminale della fenilalanina con un altro amminoacido di natura idrofobica, quale fenilalanina, leucina, isoleucina o valina, considerando questa sostituzione come una strategia promettente per ottenere biomateriali a partire da dipeptidi capaci di auto-organizzarsi. È stata inoltre esplorata l’influenza della chiralità nei singoli amino acidi, sulla capacità di questi dipeptidi di formare idrogeli. L’eterochiralità si è rivelata una promettente strategia sia per guidare l’organizzazione gerarchica di piccole molecole quali i dipeptidi, specialmente nel caso della difenilalanina, sia per favorirne la loro auto-organizzazione in acqua, aumentando l’idrofobicità della sequenza peptidica. Poichè il fenomeno di auto-organizzazione è un processo cooperativo, anche minime differenze strutturali nella sequenza peptidica, quali la diversa ramificazione della catena alifatica nei tre regioisomeri leucina, isoleucina e norleucina, può essere amplificata e portare a significative differenze a livello sopramolecolare. In questa tesi, infine, è stata investigata anche la possibile influenza dell’alogenazione sull’organizzazione sopramolecolare della difenilalanina eterochirale. I peptidi sono stati sintetizzati in fase solida, purificati mediante HPLC in fase inversa e caratterizzati a livello molecolare tramite 1H-NMR, 13C-NMR e studi di massa ESI-MS. La capacità di ogni dipeptide sintetizzato di auto-organizzarsi e formare degli idrogeli è stata testata in condizioni fisiologiche (es. soluzioni di tampone fosfato) ed il comportamento sopramolecolare è stato investigato tramite l’utilizzo di diverse tecniche, quali analisi di reologia, dicroismo circolare (CD), spettroscopia infrarossa (FT-IR), saggi di fluorescenza (Th-T) amiloide, microscopia ottica ed a trasmissione elettronica (TEM), diffrazione a raggi X (XRD) su singolo cristallo e su polvere e spettroscopia visibile ed UV-Raman. Infine, la biocompatibilità di questi composti è stata testata in vitro attraverso saggi di citotossicità sia di tipo qualitativo (live/dead) che quantitativo (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) su culture cellulari di mammifero. Complessivamente, questa tesi di dottorato consente di definire le regole chiave che guidano l’auto-organizzazione di semplici e piccole molecole, costituite da 1 o 2 amminoacidi, per ottenere biomateriali con ideali caratteristiche di biodegradabilità e biocompatibilità. Inoltre, molti dei sistemi studiati in questa tesi dimostrano la loro capacità di organizzarsi in maniera ordinata a formare interessanti strutture a canale ed idrogel, con promettenti e potenziali applicazioni future nel campo della biomedicina.Biodegradable nanomaterials based on short-peptide building blocks have raised an increasing interest in research over the years. They present several advantages (i.e., biocompatibility, chemical diversity, low cost) relative to other types of materials, and there is a large scope to study minimalistic short sequences as active building blocks for biomaterials, with single amino acids and dipeptides standing out as ideal candidates, athough prediction of their supramolecular behaviour is very challenging. This PhD thesis aims to fill this gap firstly describing the self-assembly of a N-capped phenylalanine (Phe) derivative into a transparent gel under physiological conditions with promising antimicrobial activity and biocompatibility in vitro. Next, the substitution of the N-cap with another hydrophobic amino acid (i.e., Phe, Leu, Ile, or Val) has been explored as a preferable strategy towards biocompatible self-assembling dipeptides, and effects of amino acid chirality over gelation has been studied. Heterochirality revealed to be a successful strategy to control the hierarchical assembly of dipeptides, especially in the case of Phe-Phe, and to promote self-assembly in water through increased peptide hydrophobicity. As self-assembly is a cooperative process, small structural sequence differences (i.e., branching of the aliphatic amino acid sidechain for the regioisomers Leu and Ile) can be amplified and lead to significant supramolecular differences. Furthermore, halogenation was studied for its influence on the supramolecular organization of D-Phe-L-Phe. Peptides were synthesized by Fmoc-based solid-phase peptide synthesis, purified by reverse-phase HPLC and characterized by 1H-NMR, 13C-NMR and ESI-MS. Self-assembly was probed under physiological conditions (i.e., phosphate buffered solutions) and the supramolecular behaviour was investigated by means of rheology, circular dichroism (CD), Fourier-transformed infrared spectroscopy (FT-IR), Thioflavin T fluorescence, optical imaging, transmission electron microscopy (TEM), powder and single-crystal X-ray diffraction (XRD), visible and UV-resonant Raman spectroscopy. Finally, biocompatibility was assayed qualitatively (live/dead) and quantitatively (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Overall, this PhD thesis allows to delineate design rules for the self-assembly of very simple building blocks based on 1-2 amino acids into biocompatible biomaterials and outlines several examples of supramolecular water-channels and hydrogels with potential applications in medicine

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

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

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 H-1- and C-13-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 121 (0.7\u201310 \u3bcM), where a lower limit of quantification (LLOQ) of 400 ng mL 121 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\u201352.8%. 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

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

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

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

10N-(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 pK(a) 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 degrees C, thus opening the way to its potential use as a biomaterial.reservedmixedGarcia, Ana M; Lavendomme, Roy; Kralj, Slavko; Kurbasic, Marina; Bellotto, Ottavia; Cringoli, Maria C; Semeraro, Sabrina; Bandiera, Antonella; De Zorzi, Rita; Marchesan, SilviaGarcia, Ana M; Lavendomme, Roy; Kralj, Slavko; Kurbasic, Marina; Bellotto, Ottavia; Cringoli, Maria C; Semeraro, Sabrina; Bandiera, Antonella; De Zorzi, Rita; Marchesan, Silvi

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 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 (Au NP) formation, with the supramolecular hydrogel being key to control and stabilize Au NPs, yielding a nanocomposite that catalyzed the photodegradation of a dye. Importantly, the Au NP formation occurred without the use of reductants other than the peptide, and the redox chemistry was investigated by LC-MS and NMR. This study provides important insights for the rational design of simple peptides as minimalistic and green building blocks for functional nanocomposites