4.3. Synthesis of Fmoc-Gly-Pro-Cysteic Acid-Ile-Gly-NH2 Peptide

In order to obtain the target peptide, Fmoc-Gly-Pro-Cysteic acid-Ile-Gly-NH₂, first Fmoc-Gly-Pro-Cys-Ile-Gly-NH₂ peptide was synthesized (Fmoc, 9-fluorenylmethoxycarbonyl, Avidin Ltd., Szeged, Hungary). Reagents, otherwise not stated, were purchased from Sigma-Aldrich (St. Louis, MI, USA). Fmoc-strategy synthesis was carried out manually in a solid-phase vessel on Rink Amide ChemMatrix resin, the protected Fmoc-amino acids (3 equiv.) were coupled using DCC (dicyclohexylcarbodiimide 3 equiv.) and HOBt (1-hydroxybenzotriazole, 3 equiv.) in DMF (N,N-dimethylformamide) for 2 h at room temperature. The deprotection of the Fmoc-group was achieved with the treatment of the resin with 5% piperazine/DMF (1 × 5 min, 1 × 20 min). Washings between coupling and deprotection were performed with DMF (3 × 1 min), MeOH (methanol) (1 × 1 min) and DMF (3 × 1 min). The completion of the coupling was monitored using the Kaiser test. Following the final coupling, the resin was washed with DMF (3 × 1 min) and MeOH (3 × 1 min), and dried under a stream of air. The dry resin was treated with TFA/H₂O (trifluoroacetic acid/H₂O, 95:5) for 3 h at room temperature. The cleavage mixture was lyophilized, and the pellet was redissolved in MeOH/AcN (methanol/acetonitrile, 1:1) for Liquid chromatography-mass spectrometry (LC-MS) analysis (Agilent, Santa Clara, CA, USA). The LC-MS analysis found that Fmoc-Gly-Pro-Cys-Ile-Gly-NH₂ was obtained in 90% purity (linear gradient from 0% to 100% AcN over 30 min, t_R: 21.77 min). LC-MS observed [M + H]⁺ 667.2, required [M + H]⁺ 667.8. The obtained Fmoc-Gly-Pro-Cys-Ile-Gly-NH₂ was transformed into Fmoc-Gly-Pro-Cysteic acid-Ile-Gly-NH₂ peptide with no further purification. It was dissolved in acetone, cooled to 1 °C in a mixture of ice and water. First 30% aqueous H₂O₂ was added, then Na₂WO₄.2H₂O in catalytic amount. After 3 h, we did not detect the starting material (Fmoc-Gly-Pro-Cys-Ile-Gly-NH₂), it completely transformed into the desired Fmoc-Gly-Pro-Cysteic acid-Ile-Gly-NH₂ peptide. The LC-MS analysis found that Fmoc-Gly-Pro-Cysteic acid-Ile-Gly-NH₂ was obtained in 75% purity (linear gradient from 0% to 100% AcN over 30 min, t_R: 16.90 min). LC-MS observed [M + H]⁺ 715.2, required [M + H]⁺ 715.8. To purify the crude peptide, it was dissolved in AcN/MeOH/H₂O, then filtered, using a 0.45 μm nylon filter. Gradient elution was used, 70–100% AcN in 60 min at a 3 mL min-1 flow rate with detection at 220 nm. Pure fractions were collected and lyophilized to give a fluffy white material.