SP-PROTAC synthesis

All reagents and solvents used during the experiments were purchased from Titan Scientific Co., Ltd. Rink Amide MBHA resin was obtained from Tianjin Nankai Hecheng Science & Technology Co., Ltd. Rink amide MBHA resin was used as the solid phase support, and SPPS was used for the assembly of the linear peptide sequence. First, resin was soaked in dichloromethane (DCM) and dimethyl formamide (DMF) for 20 min and filtered. Fmoc deprotection was performed with piperidine (20% in DMF) for 5 min twice. Then Fmoc-AA-OH (1 mmol, 10 equivalents), Oxyma (1 mmol, 10 equivalents), DIC (2 mmol, 20 equivalents), and NMP (6 ml) were mixed, added to the resin, and shaken for 20 min at 60 °C to complete the coupling of the first amino acid. The peptide couplings of Fmoc-S5-OH and Fmoc-R8-OH were carried out over a single 2-h coupling cycle using two equivalents of the Fmoc protected amino acids at 60 °C. RCM reactions were performed through the addition of first-generation Grubbs' catalyst (20 mol% in DCM) for 2 h, which was repeated to ensure a complete reaction. After the RCM reaction was complete, a test cleavage was performed to ensure adequate yield. The target peptides were cleaved from the resin, purified by RP-HPLC, and verified by HR-MS.