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Negli ultimi due decenni, i biomateriali a base di peptidi sono diventati molto popolari in diverse aree della ricerca come la diagnostica, la terapia e la medicina rigenerativa. In particolare, peptidi molto brevi (2-3 amminoacidi) sono molto interessanti per la loro semplicità di preparazione e basso costo. La loro auto-organizzazione può portare alla formazione di idrogel che sono per la loro natura tendenzialmente biocompatibili e biodegradabili. Purtroppo però predire quali sequenze di tripeptidi (privi di gruppi protettori) siano in grado di formare gel è estremamente difficile, come riportato in recenti studi. Tuttavia, un aspetto interessante è che recentemente si è visto che la sostituzione dell’amminoacido N-terminale con il suo D-enantiomero può risultare in tripeptidi che gelificano, a partire da L-analoghi idrofobici che non formano gel. Per questo motivo, una serie di 16 tripeptidi con stereoconfigurazione D-L-L è stata scelta come soggetto di studio per l’auto-organizzazione e gelificazione ed è descritta in questa tesi. I tripeptide sono stati sintetizzati in fase solida, purificati tramite RP-HPLC, caratterizzati a livello molecolare tramite 1H-NMR, 13C-NMR, ESI-MS e CD. I tripeptidi sono stati quindi testati per l’auto-organizzazione in idrogel in condizioni fisiologiche (tampone fosfato, pH 7.3). Il loro comportamento supramolecolare è stato quindi studiato tramite CD, FT-IR, microscopia a trasmissione elettronica (TEM), e diffrazione di raggi-X su singolo cristallo. 12 nuove sequenze in grado di formare idrogel sono state identificate e la loro concentrazione minima di gelificazione (MGC) è stata determinata. I sistemi sono stati studiati tramite reometria oscillatoria. Infine, sono state selezionate delle sequenze per effettuare delle prove di citotossicità in vitro su cultura cellular di fibroblasti. Questi esperimenti hanno rivelato una promettente performance per lo sviluppo futuro di biomateriali. In the last two decades, peptide-based biomaterials have become very popular in different research areas such as diagnostics, therapeutics and regenerative medicine. In particular, ultrashort (2-3 amino acids) peptides are particularly attractive for their ease of preparation and low-cost. Their self-assembly can yield supramolecular hydrogels that are inherently biocompatible and biodegradable. Unfortunately, prediction of self-assembly and hydrogelation of unprotected tripeptides is remarkably difficult, as shown in recent studies. Interestingly, it was reported that substitution of the N-terminal amino acid with its D-enantiomer can yield self-assembling tripeptides that gel, from hydrophobic L-analogues that do not. For this reason, a series of 16 tripeptides of stereoconfiguration D-L-L was chosen as a subject of study for self-assembly and hydrogelation and is described in this thesis. The tripeptides were synthesised by solid-phase, purified by RP-HPLC, characterised at the molecular level by 1H-NMR, 13C-NMR, ESI-MS, and CD. The tripeptides were probed for self-assembly and gelation at physiological conditions (i.e., phosphate buffer, pH 7.3). Their supramolecular behaviour was then investigated by means of CD, FT-IR, transmission electron microscopy (TEM), and single-crystal X-ray diffraction analyses. 12 new hydrogelators were identified and their minimum gelling concentration (MGC) was determined. The systems were investigated by means of oscillatory rheometry. Finally, selected sequences were evaluated for cytotoxicity in vitro on fibroblast cell culture, revealing a promising performance for future applications as biomaterials.

In this work, we present Thioflavin T fluorescence, transmission electron microscopy (TEM), circular dichroism (CD), Fourier-transformed infrared (FT-IR), and oscillatory rheometry studies applied to an antineoplastic drug, 5-fluorouracil (5-FU), embedded in a heterochiral tripeptide hydrogel to obtain a drug delivery supramolecular system. The release of 5-fluorouracil was monitored over time by reverse-phase high-performance liquid chromatography (HPLC) and its interaction with the tripeptide assemblies was probed by all-atom molecular dynamics simulations.

Dipeptides and their cyclized 2,5-piperazinedione (or diketopiperazine, DKP) derivatives are attractive building blocks for supra­molecular hydrogels. The Phe-Phe, (p-nitro)-Phe-Phe, and Phe-Val dipeptides and their corresponding DKPs are studied for self-assembly in water. The DKPs were obtained in high yields by microwave-assisted cyclization­ of the dipeptides in water, demonstrating that use of their methyl ester derivatives as reported in the literature is not necessary for successful cyclization. Single-crystal XRD structures are reported for two DKPs as well as stable hydrogels at neutral pH.

Self-assembling short peptides are attractive minimal systems for mimicking the constituents of living systems and building (bio)materials. The combination of both D- and L-amino acids into heterochiral sequences is a versatile strategy for building durable supramolecular architectures, especially when their homochiral analogs do not self-assemble. The reasons for this divergent behavior have remained obscure until now. Here, we elucidate how and why homochiral and heterochiral peptides behave differently. We identify a key spectroscopy signature and its corresponding molecular conformation, whereby an amphiphilic structure is uniquely enabled by the peptide stereochemistry. Importantly, we unravel the self-assembly process as a continuum from the conformation of single molecules to their organization into nano- and microstructures and through to macroscopic hydrogels, which are probed for cytotoxicity in fibroblast cell culture. In this way, (bio)material properties at the macro-scale can be linked to the chemical structure of their building blocks at the angstrom scale. Nature makes pervasive use of homochirality (e.g., D-sugars and L-peptides) to assemble biomolecules, whose interactions determine life processes. D-amino acids rarely occur, and their effects are not yet completely understood. For a long time, structural complexity (e.g., polypeptides and constrained molecules) was considered a requirement for achieving defined conformations that ultimately allow biomolecule recognition and function. Here, we detail how minimalist building blocks can adopt conformations with a characteristic spectroscopic signature, whereby substitution of just one L-amino acid for its D mirror image leads to a divergent path for assembly in water. Subtle molecular variations are amplified through increasing size scale all the way to macroscopic differences that are visible to the eye. Ultimately, the design of heterochiral (bio)molecules thus provides an alternative approach to shed new light on the supramolecular interactions that define life as we know it. This work explains why and how heterochiral and homochiral tripeptides differ in their assembly in water. A characteristic spectroscopic signature is assigned to molecular conformation. We monitor the process as a continuum from the molecular scale to the macroscopic biomaterials so that the final properties are linked to chemical structure of the building blocks. This work lays the foundation for the design of supramolecular hydrogel biomaterials based on short sequences of hydrophobic D- and L-amino acids.