Solid-Phase Fmoc Peptide Synthesis

Each peptide was chemically synthesised via solid-phase Fmoc chemistry using a Tribute automated peptide synthesiser (Protein Technologies, Tucson, AZ, USA), which was described in the previous study [61 (link)]. Rink amide resin was employed as solid phase for the synthesis process. The synthetic peptides were released from the resin by adding a cleavage cocktail, which contains 94% trifluoroacetic acid (TFA), 2% water, 2% thioanisole (TIS), and 2% 1,2-ethanedithiol (EDT) for 2 h at room temperature and further washed by diethyl ether three times. After lyophilisation, the crude peptides were then purified by RP–HPLC (Phenomenex Aeris PEPTIDE 5 μm XB-C18 column, 250 mm × 21.2 mm, Macclesfield, Cheshire, UK) by gradient elution from 90% solution A (99.95% dd H2O and 0.05% TFA) and 10% solution B (80% acetonitrile, 19.95% dd H2O and 0.05% TFA) to 100% solution B at a flow rate of 8 mL/min. The purity of peptides was identified by RP–HPLC and MALDI-TOF mass spectrometry (Voyager DE, Perspective Biosystem, Foster City, CA, USA) in positive detection mode using CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix.