Synthesis and Characterization of Self-Assembling Peptides

All reagents and solvents used for the peptide synthesis were purchased in highest quality commercially available and used without further purification. All peptides were synthesized with solid-phase Fmoc-based chemistry on Rink amide resin (0.19–0.56 mmol/g, 100–200 mesh) using a Liberty Blue System synthesizer (CEM Corp, Matthews, NC, Canada). Peptides were cleaved from resin by addition of a freshly prepared mixture containing 92.5% TFA, 2.5% H₂O, 2.5% DODt, 2.5% TIS. All synthesis were carried out on a 0.25 mmol in presence of a 0.2 M amino acid solution (in DMF), 1 M DIC (in DMF), and 1 M Oxyma (in DMF). The deprotection of Fmoc groups was determined by a 10% v/v of piperazine in 9:1 NMP/EtOH. The N-terminal acetylation (for CK₁ and pureHYDROSAP components) was performed using 20% v/v solution of Ac₂O (in DMF). The crude products were purified via reversed-phase chromatography by semi-preparative Waters binary HPLC (>96%) using a c18 RestekTM column and then lyophilized (Labconco, Kansas City, MO, USA). Purified peptides powder was subsequently dissolved in 0.1 M HCl to remove the presence of possible TFA salts. Three different SAPs were used for this study: pureHYDROSAP (Marchini et al., 2019 (link); Marchini et al., 2020 (link)), FAQ (NH₂-FAQRVPP-GGG-LDLKLDLKLDLK-CONH₂) (Gelain et al., 2012 (link)) and CK₁ (Ac-CGGLKLKLKLKLKLKGGC-CONH₂) (Pugliese et al., 2018c (link); Ciulla et al., 2022 (link)). As previously described, pureHYDROSAP is composed by linear SAPs Ac-(LDLK)₃-CONH₂, Ac-KLPGWSGGGG-(LDLK)₃-CONH₂ (Caprini et al., 2013 (link)) and Ac-SSLSVNDGGG-(LDLK)₃-CONH₂ (Gelain et al., 2012 (link)) and branched SAP tris(LDLK)₃-CONH₂ (Pugliese et al., 2018a (link)). For the experiments, pureHYDROSAP (abbrev. HYDROSAP), FAQ and CK₁ powders were dissolved respectively to a final concentration of 2% (w/v), 5% (w/v) and 5% (w/v) in distilled water (Gibco).