Agilent prep c18 column

Standard solid-phase N-αFmoc chemistry was used to synthesize meditope derivatives on CS136XT peptide synthesizer (CS Bio). Reagent K (trifluoroacetic acid (TFA)/water/phenol/thioanisole/1,2-ethanedithiol (EDT) = 82.5:5:5:5:2.5) was used to cleave the peptides from the resin. Crude peptides were collected by precipitation with cold ether. Oxidation using 20% DMSO in ammonium acetate buffer (pH 6) was performed for disulfide cyclization. All peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) (Agilent 1200 system with Agilent prep-C18 column, 21.2 × 150 mm, 5 µm) with a water (0.1% TFA)/acetonitrile (0.1% TFA) solvent system. Alexa647- and Cy5-labeled peptides were synthesized from Alexa647-NHS and Cy5-NHS and purified using RP-HPLC. All peptides were characterized by mass spectrometry.

Meditope peptides were synthesized at the Synthetic and Biopolymer Chemistry Core, City of Hope, Duarte, California, USA or by CS Bio Co., Menlo Park, California, USA. Specifically, standard solid-phase N-αFmoc chemistry was used to synthesize meditope derivatives using a CS136XT peptide synthesizer (CS Bio). Lactam peptides were synthesized starting from Fmoc-Asp(Wang resin LL)-Oall. After cleavage of the peptides from resin using reagent K (TFA:water:phenol:thioanisole:EDT in a 82.5:5:5:5:2.5 ratio), the crude peptides were collected by precipitation from cold ether. For disulfide-linked meditopes, a further oxidation using either 20% DMSO in ammonium acetate buffer pH 6 or iodine was performed. All peptides were purified using reverse-phase HPLC (Agilent 1200 system with Agilent Prep-C18 column; 21.2 × 150 mm, 5 µm) with a water (0.1% TFA)/acetonitrile (0.1% TFA) solvent system. All peptides were characterized by mass spectrometry.