Peptides synthesis and reagents

Temporin-SHa (SHa, FLSGIVGMLGKLF_NH2), its enantiomer D-temporin-SHa (D-SHa, corresponding to SHa in the all-D α-C configuration), and [K³]temporin-SHa (SHa with Ser³ residue replaced with Lys), were synthesized using solid-phase standard Fmoc chemistry protocols, as previously described⁵⁷ but with the following modifications. Synthesis was carried out on a CEM Liberty Blue automated microwave peptide synthesizer (CEM Corporation, Peptide Synthesis Platform, IBPS, Sorbonne University, Paris, France) using Protide Rink Amide LL resin (CEM Corporation, USA, 0.19 mmol/g substitution). Post-deprotection washing with N, N-dimethylformamide (DMF) was followed by coupling using a diisopropyl carbodiimide (DIC)/Oxyma activation method. The peptidyl resin was cleaved and deprotected by incubation (3 h at room temperature) with an acidic mixture containing 94% trifluoroacetic acid (TFA), 1% triisopropylsilane (TIS), 2.5% H₂O and 2.5% 1,2-ethanedithiol (EDT). Resin was removed by filtration and the peptide was precipitated in cold ether. The crude material was then subjected to semi-preparative RP-HPLC on a Phenomenex Luna C18(2) LC column (100 Å, 250 × 10 mm). Elution was performed at a flow rate of 5 mL/min by a 20–70% linear gradient of ACN (0.07% TFA) in 0.1% TFA/water (1% ACN/min). Peptide purity was assessed by RP-HPLC using an analytical Aeris PEPTIDE column (XB-C18, 3.6 µm, 4.6 ×. 250 mm, Phenomenex). Elution was performed at 0.75 mL/min using the same linear gradient of acetonitrile mentioned above, followed by MALDI-TOF analysis (Mass Spectrometry and Proteomics Platform, IBPS, Sorbonne University, Paris, France). Fluorescent temporin-SHa (SHa-Cy5) was obtained from Genscript (Piscataway, USA) by synthesizing the analog [C¹³]temporin-SHa (FLSGIVGMLGKLC_amide), and then by labeling thiol group of the Cys¹³ residue with maleimide-activated Cyanine5 dye. Control experiments were conducted with the Cyanine5 maleimide dye alone so it was purchased from abcam (Cambridge, United Kingdom).