Peptide synthesis

All peptides used in this work were obtained from the Fmoc-based solid phase peptide synthesis. 2-Cl-(Trt)-Cl resin was treated with 5% NH₂NH₂ in N, N-Dimethylformamide (DMF, 30 min for twice) to prepare 2-Cl-(Trt)-NHNH₂ resin. For each step of amino-acid coupling, the resin was coupled with a pre-activated solution of protected amino acids (4 equiv.), of HATU (3.8 equiv.), the DIPEA (8 equiv.) was successively dissolved in DMF solvent. The coupling time should extend from 1 to 2 h in a constant temperature shaker at 30 °C until ninhydrin test showed no bare amino residue was left on resin, then the resin was washed with DMF, dichloromethane (DCM), DMF for five times alternately. To remove the Fmoc group, 20% piperidine in DMF was added to the resins for 15 min (twice: 5 min and 10 min, respectively), then the resin was washed with DMF, DCM, DMF for five times alternately. After the de-protection of the Fmoc of the final amino acid, cleavage reagent (Typically a trifluoroacetic acid (TFA) cocktail of TFA/phenol/water/TIPS (88/5/5/2)) was added to the dry resin pre-washed with DCM. After 2 h, the resin was washed with an equal volume of TFA once. Combined washing liquids were concentrated by blowing with N₂. The crude peptides were obtained by precipitating with cold ether and centrifugation at 5,000 r.p.m. The crude peptides were dissolved in 0.1% TFA containing co-solvent of acetonitrile and water, analysed by analytical HPLC plus ESI-MS and purified by semi-preparative HPLC and lyophilized immediately.