Waters 2545 preparative system

Two distinct peptide substrates were designed and prepared for the FRET- and HPLC-based analyses, respectively. The sequences are reported in the section of Results. All peptides were synthesized on solid phase following the Fmoc (N-9-Fluorenylmethyloxycarbonyl) strategy and following an optimized protocol reported in literature [31 (link)]. The FRET substrate was acetylated at the N-terminus by treatment with a solution of acetic anhydride 30% and DIPEA 5% in DMF for 30 min. On the peptide substrate for the HPLC-based analysis the Fmoc group was left at the N-terminus to favor reverse phase retention and identification of products by the characteristic UV absorption spectrum. After cleavage from the resins and lyophilization peptides were purified by RP-HPLC on a WATERS 2545 preparative system (Waters, Milan) equipped with a WATERS 2489 UV/Vis detector set at at 214 nm and using a Nucleodur HTec C18 column (5 μm, 150 × 21 mm) operated at a flow rate of 12 ml/min. A linear gradient from 5 to 70% of solvent B (0.1% TFA in acetonitrile, ACN) in 15 min was applied to separate the peptides from impurities.

Protected amino acids, coupling agents (HATU, Oxyma), and Fmoc-Rink Amide AM resin used for peptide synthesis were purchased from IRIS Biotech GmbH (Marktrewitz, DE). Solvents, including acetonitrile (CH3CN) and dimethylformamide (DMF) were purchased from Carlo Erba reagents (Milan, Italy). Other products such as trifluoroacetic acid (TFA), sym-collidine, diisopropylethylamine (DIPEA), piperidine, were from Sigma-Aldrich (Milan, Italy). HPLC analyses for peptides characterization were performed on an Alliance HT WATERS 2795 system, equipped with a PDA WATERS detector 2996, whereas preparative purifications were carried out on a WATERS 2545 preparative system (Waters, Milan, Italy) fitted out with a WATERS 2489 UV/Visible detector.