Prep lc 4000

NMR spectra were recorded at 25 °C on either a 500 MHz Bruker Avance spectrometer, equipped with a triple resonance 5mm CPTCI cryo-probe or a 400 MHz Agilent Inova spectrometer equipped with a 5 mm OneNMR probe. NMR spectral processing and data interpretation was done using MestReNova software, version 8.0. Ultra-Performance Liquid Chromatography (UPLC) high resolution mass spectral (HRMS) data were acquired using on a Waters Acquity UPLC system coupled to a Waters LCT Premier TOF mass spectrometer with an electrospray ionization source. Preparative HPLC was run on a Waters Prep LC 4000 system, equipped with a Delta 600 pump and a 996-diode array detector, using a Phenomenex Kinetex C₈ or C₁₈ column [5 μ, 150 × 21.2 mm]. All solvents used for chromatography, UV, and MS were GC/LC-MS or HPLC grade, and the H₂O for preparative HPLC was Millipore Milli-Q PF filtered.

Peptides were synthesized using standard Fmoc solid-phase methods on a Peptide Machines Discovery-4 synthesizer on rink resin^{37 –41} . All coupling reactions were performed with ten-fold excess (vs. load capacity of the resin) of activated amino acid (Aapptec or Advanced Chemtech), using FMOC amino acids/HBTU/HoBt/DIEA (1:1:1:2.5) in DMF for 60 minutes. Deprotection of the FMOC group was accomplished in 20% v/v piperidine diluted in DMF for 30 minutes. Peptides used for ELISA experiments were acetylated at the N-terminus by reaction with 1:1:2 v/v acetic anhydride:DIEA:DMF. The peptides were cleaved by exposure to a 90:5:3:2 v/v mixture of trifluoroacetic acid, thioanisole, ethanedithiol, and anisole for two hours. Peptides were precipitated by addition of cold diethyl ether, lyophilized and purified by reverse-phase HPLC (Waters Prep LC 4000) equipped with a Waters 2487 detector and C18 column. Final purified fractions were lyophilized until further use. Isolation of the target peptide was confirmed by MALDI mass spectrometry.