Rp hplc system

The peptides were synthesized manually by SPPS using the Fmoc strategy on a Fmoc-SAL-PEG-PS resin (substitution: 0.23 mmol/g). Fmoc deprotection was performed 20% PPD/DMF (1×1 min, 1×15 min), followed by coupling of amino acids (5 equiv.) using DIC (5 equiv.) and Oxyma (5 equiv.) in DMF (60 min). To avoid the formation of deletion peptides, acetylation with 10% Ac₂O/DMF (10 min) after the coupling step was performed. The assembled peptides were deprotected and cleaved from the solid support with cleavage cocktail containing TFA/H₂O/TIS/EDT (94/2.5/1/2.5) at room temperature for 2 hours. Three rounds of extraction of the peptides were carried out using ice-cold diethyl ether. Crude peptides were analyzed and purified by RP-HPLC system (Hitachi) with a C18 column (250 × 10 mm, 5 μm, YMC) using linear gradients of 30 to 60% B (A: 0.1% TFA in water, B: 0.08% TFA in acetonitrile) over 30 min at a flow rate of 3.0 mL/min. Fractions were analyzed with MALDI-TOF-MS (AoutflexII, Bruker Daltnics). The disulfide bond formation was performed following procedures. The thiol free peptide (5 mg) was dissolved in 50 mL of 20 mM NH₄HCO₃ (pH 8.0) and stirred for 12 hours. After the reaction, the solution was lyophilized and purified by RP-HPLC. All peptides were obtained with a purity >95%.