Synthetic Peptide Generation and Purification

The sequences and denotations of all peptides reported are given in Table 1. Peptides were synthesized manually by stepwise solid-phase peptide synthesis on a Rink Amide Resin with a substitution value of 0.7mmole gm⁻¹ (Novabiochem). All Fmoc-amino acid derivatives and coupling reagents were purchased from Chem-Impex International, Inc. Synthesis grade solvents were used in all procedures. 9-Fluorenylmethoxycarbonyl (Fmoc) group was employed for protection of the α-amino group during coupling steps. Side chain protecting groups were triphenylmethyl (Trt) for Asn, tert-butyl (tBu) for Ser and tert-butyloxycarbonyl (Boc) for Trp. Coupling of each residue was carried out using HBTU/HOBt in Dimethylformamide (DMF). Four equivalents of each Fmoc protected amino acid were used for coupling. Cleavage of the peptide from the resin was performed using a cleavage cocktail comprising 90:2.5:2.5:5(v/v) trifluoroaceticacid (TFA)/thioanisole/ethanedithiol/H₂O at room temperature for 2 hours. After the filtration and washing of resin by TFA, excess of solvent was removed under vacuum. Crude peptides were treated with ice-cold diethyl ether and then centrifuged at 3000 rpm for 10 minutes at 4 °C. Synthetic peptides were purified by HPLC (Beckman Coulter System Gold) using a semiPrep Vydac C-18 column with a 5 to 60% acetonitrile/water (0.1% TFA) gradient over 75 minutes at 3 mL min⁻¹ flow rate. All peptides were purified to ≥ 98% homogeneity as judged by analytical reverse phase HPLC on C-18. The integrity of purified peptides was confirmed by MALDI-TOF spectra. Analytical chromatograms and mass spectra for all peptides reported are given in the Supporting Material.