4.2. Manual Fmoc solid‐phase peptide synthesis

The peptides H‐Cys‐Gly‐Phe‐OH (CGF), H‐Cys‐Leu‐Phe‐OH (CLF), H‐Cys‐Glu‐Phe‐OH (CEF), H‐Cys‐Lys‐Phe‐OH (CKF), and H‐Cys‐Ser‐Phe‐OH (CSF) were synthesized on a 2.5‐mmol scale by first loading Fmoc‐Phe‐OH onto 2‐chlorotrityl chloride resin (10‐mmol scale, substitution = 1.3 mmol/g) and then splitting the resin into four lots for the subsequent divergent coupling reactions. Loading of the first amino acid derivative was carried out using Fmoc‐Phe‐OH (1.0 eq., 10 mmol) and DIPEA (1.0 eq., 10 mmol) in dimethylformamide (DMF). Coupling of the second amino acid derivative was performed using TCTU/DIPEA (1.9 eq., 4.8 mmol/2.5 eq., 6.3 mmol) in DMF. Coupling of Fmoc‐Cys (Trt)‐OH was performed using DIC/HOBt (6.2 eq, 15.4 mmol/2.3 eq, 5.7 mmol). All reactions were performed at room temperature. For Fmoc removal, 20% (v/v) piperidine in DMF was used. Cleavage of the peptidyl‐resin to obtain crude peptides was performed using TFA/TIS/water/EDT (92.5:2.5:2.5:2.5 v/v). Crude peptides were analyzed by reversed‐phase UHPLC (Waters Acquity HSS T3 100 Å, 1.8 μm, 2.1 × 150 mm) using a linear gradient of 0.1% TFA in acetonitrile. Peptides were purified by preparative reversed‐phase HPLC using Luna ® C18(3) 10‐μm particle size media packed in an axial compression column (ModCol ®, 25 × 5 iD). After purification, the peptides were lyophilized to give white powders. No correction for TFA and water content was performed prior to weighing out peptides for the maleimide conjugation experiments.