Peptide Synthesis and Purification

Peptide hairpins and controls were synthesized by standard Fmoc Solid Phase Peptide Synthesis methods on a CEM Liberty Blue microwave peptide synthesizer. Wang resin preloaded with C-terminal amino acids and Rink Amide-MBHA resin were employed. DIC/Oxyma were used as coupling reagents and 20% piperidine in DMF was used for deprotection of Fmoc groups. N-terminal acetylation or benzoylation was accomplished using a 1:10:10 ratio of peptide: anhydride (acetic anhydride or benzoic anhydride): triethylamine in 5ml DMF. The mixture underwent shaking for 1 hour at room temperature before cleavage. Peptide cleavage was done using 95:2.5:2.5 trifluoroacetic acid (TFA):triisopropylsilane (TIPS): water mixture where water and TIPS served as radical scavengers . Disulfide bonds were formed by diluting the peptide in minimal amounts of DMSO (500µL) and 3mL of water. The peptides were allowed to oxidize overnight at room temperature. All peptides were purified by reverse-phase HPLC (Varian ProStar 220 HPLC, Agilent 21.2 × 50 mm C18 column, 10 mL/min, elutent A: water with 0.1% TFA, elutent B: acetonitrile with 0.085% TFA) on a 10–60% gradient over 25 minutes and visualized at 215nm and 280nm. Fractions that were collected were lyophilized and purity was assessed using a Bruker Esquire Ion Trap ESI mass spectrometer. All peptides were purified to an excess of 97% pure before use in experiments.