Synthesis of HiBiT Peptide for Functionalization

The HiBiT peptide (CGSSG-VSGWRLFKKIS-NH₂) synthesis with additional N-terminal CGSSG, for further functionalization, if necessary, was performed on a 0.25 mmol scale using Amphispheres 40 RAM resin, and 0.46 mmol/g loading (Agilent Technologies, Waldbronn, Germany) via Fmoc-based solid phase peptide synthesis. The peptide was synthesized on an automated platform with microwave assistance using the LibertyBlue^® (CEM, Matthews, NC, USA). Fmoc-deprotection was performed with 20% piperidine (v/v) and 0.1 M ethyl 2-cyano-2-(hydroxyimino)acetate (oxyma) (Carbolution, St. Ingbert, Germany) in N,N-dimethylformamide (DMF) (Carl Roth, Karlsruhe, Germany). Amino acid activation and coupling was performed with N,N-diisopropylcarbodiimide (DIC) (Carbolution) and oxyma in DMF. The peptide was cleaved from the resin by treatment of the resin with trifluoracetic acid (TFA) (Carl Roth)/triethyl silane (TES) (Sigma–Aldrich)/Anisole (Thermo Fischer Scientific)/H₂O (94/2/2/2) for 3 h and precipitated in cold diethyl ether (Honeywell, Charlotte, NC, USA). The peptide was dissolved in water, lyophilized, and purified by preparative reversed phase-high performance liquid chromatography (RP-HPLC). The product was analyzed via liquid-chromatography mass-spectrometry (LC-MS) and analytical RP-HPLC. A white product with a yield 167.2 mg (39%) was obtained.