Solid-phase peptide synthesis (SPPS)

All peptide synthesis reagents, as well as the Fmoc amino acid derivatives were purchased from GL Biochem (Shanghai) Ltd., Fmoc-β-Ala-OH was from Sigma Aldrich. C-terminal amide natural T4Ff peptides were synthesized following standard Fmoc-peptide synthesis protocols on a 0.1 mmol scale using a 0.5 mmol/g loading H-Rink amide ChemMatrix resin (35–100 mesh) from Sigma Aldrich with a Liberty Lite automatic microwave assisted peptide synthesizer from CEM Corporation. The amino acids were coupled in 5-fold excess using oxyme as an activating agent. Couplings were conducted for 4 min at 90°C. Deprotection of the temporal Fmoc protecting group was performed by treating the resin with 20% piperidine in DMF for 1 min at 75°C. Once the synthesis is finished, the peptide was acetylated with a solution of 0.8 ml AcOH, 2 ml of DIEA/DMF (0.2 M) and 3.2 ml of DMF. The last non-natural Fmoc-β-Ala-Bpy-OH residues were coupled by hand in 4-fold excess using HATU as activating agent. Each amino acid was activated for 1 min in DIEA/DMF 0.2 M before being added onto the resin. These manual couplings were conducted for 60 min. Deprotection of the temporal Fmoc protecting group was performed by treating the resin with 20% piperidine in DMF for 20 min. Cleavage and deprotection of the peptide were simultaneously performed using standard conditions by incubating the resin for 2.5 h with an acidic mixture containing 50 μL CH₂Cl₂, 25 μL of H₂O, 25 μL of TIS (triisopropylsilane), and 900 TFA μL. The resin was filtered, and the TFA filtrate was concentrated under a nitrogen stream to an approximate volume of 1 mL, and then added onto ice-cold diethyl ether (20 mL). After 10–30 min, the precipitate was centrifuged and washed again with 5 mL of ice-cold ether. The solid residue was dried under argon and redissolved in acetonitrile/water 1:1 (2–5 mL) and purified by semi-preparative RP-HPLC.

Peptides were purified by preparative RP-HPLC with an Waters 1500 series Liquid Chromatograph using a Sunfire Prep C18 OBD (5 μm, 19 × 150 mm) reverse-phase column from Waters. Standard conditions for analytical and preparative RP- HPLC consisted on an isocratic regime during the first 2 min, followed by a linear gradient from 15 to 75% of solvent B for 30 min (A: water 0.1% TFA, B: acetonitrile 0.1% TFA). Compounds were detected by UV absorption (222 nm) and by ESI/MS. The fractions containing the products were freeze-dried and their identity was confirmed by ESI/MS and MALDI-TOF. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was performed with a Bruker Autoflex MALDI/TOF model in positive scan mode by direct irradiation of the matrix-absorbed peptide.