Microwave-Assisted Synthesis of Peptide Therapeutics

A CEM microwave synthesizer (Liberty Blue) was used for solid phase peptide synthesis of PTs, as previously described [14 (link), 21 (link), 22 (link), 39 (link)]. Fmoc-4-azido-Proline used in the synthesis was prepared as previously reported [21 (link)]. All other Fmoc-, Boc- protected amino acids, N,Nʹ-diisopropylcarbodiimide, ethyl (hydroxyimino) cyanoacetate (OxymaPure) and rink amide resin (100–200 mesh size, 0.53 meq/g substitution) were purchased from Chem-Impex International, INC. Ethynylferrocene and CuI catalyst for the click reaction, and hydrazine, were purchased from Sigma Aldrich. HPLC purifications were performed using a Waters® HPLC system with reverse phase (RP) C18 prep columns. Purity verification of cPTs was carried out by analytical C18 RP-HPLC column, using a BeckmanCoulter® HPLC system. HPLC grade acetonitrile (ACN), Millipore-MilliQ water and 0.1% TFA were used as solvents for the HPLC purification. Mass was confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).