Tribute peptide synthesizer

All peptides were prepared in house by a solid phase peptide synthesis method using Fmoc chemistry on a Liberty microwave peptide synthesizer (CEM, Matthews, NC, USA) or a Tribute peptide synthesizer (Protein Technologies, Tucson, AZ, USA). Peptides were cleaved and deblocked using a reagent mixture of 95% trifluroacetic acid :2% water: 2% anisole:1% ethanedithiol and purified by reversed-phase HPLC using a water:acetonitrile:0.1% TFA gradient (90-95% purity). Correct peptide structures were confirmed by MALDI mass spectrometry. All peptides were dissolved in deionized water as a 1 mM stock solution and were stored at −70°C until further use.

Dawson (3-(Fmoc-amino)-4-aminobenzoyl) resin and HBTU were purchased from Novabiochem (San Diego, CA). 4-nitrophenylchloroformate, fluorescein-5-maleimide, Tris base and Guanidine Hydrochloride were purchased from Thermo Fisher Scientific (Waltham, MA). 4-mercaptophenylacetic acid was purchased from Sigma Aldrich (St. Louis, MO). The phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). Fmoc-(4, 4′)-Biphenylalanine (Fmoc-Bip) was purchased from Chem-Impex Int’l Inc (Wood Dale, IL). All other Fmoc-protected and Boc-protected amino acids were purchased from Advanced Chemtech (Louisville, KY) or Chem-Impex Int’l Inc (Wood Dale, IL). Peptide synthesis was carried out on a Tribute peptide synthesizer (Protein Technologies, Tuscon, AZ). Peptide concentration measurements were performed on a Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

Benzaldehyde-KGRGDS was synthesized using standard Fmoc chemistry and Rink Amide MBHA resin on a Protein Technologies Tribute Peptide Synthesizer (0.5 mmol scale), as previously described.^{[15 (link)]} After deprotection of the N-terminus, the resin was transferred to a peptide synthesis glass vessel and washed with DCM (3×10 mL). HATU (760.5 mg, 2.0 mmol), 4-Formylbenzoic acid (mg, 2.0 mmol), DIPEA (700 μL), DMF (10 mL), and DCM (3 mL) were added to the vessel, and the mixture was stirred at room temperature for 3 h. The solution was drained from the vessel under argon pressure and the resin was washed with DCM (5×10mL). Synthesized peptides were cleaved using, a peptide cleavage solution formed by dissolving dithiothreitol (DTT) and phenol (1:1) in a solution of 95% trifluoroacetic acid (TFA), 2.5% tri-isopropylsilane (TIPS), and 2.5% deionized water. The synthesized peptides were cleaved at room temperature for 2 h. Cleaved peptides were precipitated in cold diethyl ether, recovered by centrifugation, and desiccated overnight. The cleaved peptides were then purified by reverse-phase HPLC (Waters Delta Prep 4000) purification on a C₁₈ column using a linear acetonitrile:water gradient. The collected fractions of purified peptides were identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry.