4.6. Formation of Disulfide Bonds

First, the disulfide bonds were formed between cysteine residues protected by trityl groups (removed during the cleavage conditions). After purification, the peptide was dissolved in a mixture of H₂O and methanol (1:9, v:v), at a concentration of 40 mg/L. The pH was adjusted and maintained between 8 and 9 using ammonia while stirring the solution at room temperature for 7 days in the presence of atmospheric oxygen. The solvents were then evaporated and the peptides were lyophilized. Reaction progress was verified using analytical RP-HPLC and LC ESI–IT–TOF MS (conditions: see peptide purification). Next, the second disulfide bonds were formed, and the peptides were dissolved in a solution of acetic acid, methanol, and H₂O (1:9:1, v:v:v) at a concentration of 40 mg/L. Iodine (25 to 50 fold excess) was dissolved in methanol and was added to the solution, which was then stirred at room temperature for 7 days. The mixture was filtered by using a Dowex ion exchange resin, and the excess iodine was removed. The progress of the reaction was monitored by analytical RP-HPLC and LC ESI-IT–TOF MS (conditions: see peptide purification). The solvents were then evaporated and the peptides were lyophilized. The peptides were repurified using RP-HPLC under conditions previously described.