Peptide synthesis

The peptides were prepared by solid phase peptide synthesis performed on an automated CS Bio 336 peptide synthesizer. The peptides were assembled on the PAL-PEG-PS resin (0.19 mmol/g, 200 mg) using standard Fmoc synthetic strategy. The resin was swollen with 5 mL of DMF (2 × 10 min). The Fmoc group on the resin was removed using 20% piperidine in DMF (5 mL, 2 × 10 min), and the resin washed with DCM/DMF (1:1, 10 × 30 sec). The desired Fmoc-protected amino acids (4 equiv) were coupled to the resin with PyBOP, HOBt (4 equiv each) and DIEA (10 equiv) in DMF (2 mL) for 2 h. The side chains of Lys, Gln, Arg and Tyr were protected with Boc, trityl (Trt), Pbf and tert-butyl, respectively. The Fmoc deprotection of the N-terminal amino acid on the resin was repeated followed by the next coupling cycle. The deprotection-coupling cycle was repeated until the desired peptide was assembled on the resin.

The peptide resins were treated with Reagent B⁴² (88% TFA, 5% phenol, 5% water, and 2% TIS, 5 mL) for 2 h with occasional shaking. Subsequently, the resins were filtered and the TFA was evaporated in vacuo. Water (20 mL) was added to the residues, and the solutions lyophilized to give the crude peptides.