CTC resin with a capacity of 0.84 mmol/g was subjected to swelling reaction in a reactor containing DMF solvent. Then, 2 equivalents of Fmoc-C terminal amino acid and 2.5 equivalents of DIEA were added in DMF to the reactor and further subjected to reaction for 2 h. The termination of the reaction was confirmed with a Kaiser test kit (Sigma-Aldrich), and the resin was washed with DMF. The Fmoc was removed by adding 20% piperidine/DMF twice to the washed resin. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. Subsequently, the following process was repeated according to the sequence of amino acids in the C terminal to N terminal direction. After 2 equivalents of Fmoc-amino acid, 2 equivalents of HBTU, 2 equivalents of HOBt, and 2.5 equivalents of DIEA were added in DMF, the reaction was performed for 2 h. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. The Fmoc was removed from amino acid by adding 20% piperidine/DMF twice to the washed resin. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. In case of Pep-3, Ferulic acid conjugation was performed through the following procedure. After 2 equivalents of Ferulic acid, 2 equivalents of HBTU, 2 equivalents of HOBt, and 2.5 equivalents of DIEA were added in DMF, the reaction was performed for 2 h. The termination of the reaction was confirmed with the Kaiser test kit, and the resin was washed with DMF. After the final synthesis was completed, a cleavage solution (TFA:H₂O:thioanisole:phenol:EDT:TIS = 81.5:5:5:5:2.5:1) was added to separate the peptide from the resin. The peptide was then precipitated using diethyl ether and washed five times. Subsequently, drying was performed to recover the final peptide product.

The purity of the synthesized peptide was identified by HPLC (Thermo Fisher Scientific/U-3000) analysis, and it was detected at UV 214 nm under the flow rate of 1 ml/min under mobile phase 0.1% TFA in water/0.1% TFA in acetonitrile using a C18 (Agilent, Pursuit XRs, 250 × 4.6 mm, 5 μm, 100Å) column.

To identify the molecular weight, LC-MS/MS (AB SCIEX, 3200 Q-trap) analysis was performed, and it was detected by MS/MS at the flow rate of 0.25 ml/min under mobile phase 0.1% formic acid in water/0.1% formic acid in acetonitrile gradient using a C18 (Agilent, Pursuit XRs, 100 × 2.0 mm, 5 μm, 100Å) column. The MS/MS analysis conditions included ESI Positive mode, Source/Gas: CUR = 20, CAD = High, IS = 5500, TEM = 350, GS1 = 50, GS2 = 50/Compound DP = 50–80, EP = 10, CE = 10–50, and CES = 1–10 (Table 2).

Synthesized peptides

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