DOTA-benzene-p-SCN (2 mg; 2.91 µmol) or HEHA-benzene-p-SCN (3 mg; 3.1 µmol) was dissolved in 1 mL of 0.2 M NaHCO3 buffer at pH 9.5, and then PD-L1-i ligand (6 mg; 3.2 µmol) was added. The mixture was incubated at 95 °C for 30 min. After cooling, 100 µL of ethanol was added. Finally, the conjugates were purified by preparative HPLC in line with a UV-vis detector (Waters) (Epic C18 Column, 5 μm, L:250 mm, Diam. Int: 20 mm, Perkin Elmer) using a linear gradient (rate flow of 4 mL/min) of CH3CN-0.1% TFA (B)/H2O-TFA (A), from 100% to 10% of A in 30 min. The fractions containing the conjugates (absorbance: 283 nm) were lyophilized.
Mass spectroscopy analysis (+Q1: 0.168 to 0.503 min, turbo spray, centroided) for the PD-L1-i peptide precursor was performed in an LC/MS system (Agilent 6460 series instrument) (Agilent Technologies, Santa Clara, CA, USA).
The IR spectra of DOTA-PD-L1-i and HEHA-PD-L1-i were acquired on an Agilent Technologies FT-IR 660 spectrometer (from 50 scans at 0.4 cm−1, from 400 to 4000 cm−1).
The UV-vis spectra (Thermo Fisher Genesys 10S spectrometer) of the PD-L1-i conjugates (1 mg/mL) were obtained from 210 to 300 nm.
Mass spectroscopy analysis (+Q1: 0.168 to 0.503 min, turbo spray, centroided) for the PD-L1-i peptide precursor was performed in an LC/MS system (Agilent 6460 series instrument) (Agilent Technologies, Santa Clara, CA, USA).
The IR spectra of DOTA-PD-L1-i and HEHA-PD-L1-i were acquired on an Agilent Technologies FT-IR 660 spectrometer (from 50 scans at 0.4 cm−1, from 400 to 4000 cm−1).
The UV-vis spectra (Thermo Fisher Genesys 10S spectrometer) of the PD-L1-i conjugates (1 mg/mL) were obtained from 210 to 300 nm.