Ziconotide (Omega-Conotoxin Mviia)-Efficient Solid-Phase Synthesis Of A Linear Precursor Peptide And Its Strategic Native Folding

To develop an efficient synthesis for target peptide 1, systematic optimization studies were performed on solid-phase peptide synthesis (SPPS) of precursor peptide 2 (linear 25-mer) and its aqueous phase folding process. A combination of DIC/HOBt (16-25-mer) and HBTU/NMM (1-15-mer) in DMF proved the best coupling strategy to deliver linear precursor peptide 2 with enhanced purity in crude (81.3%) and purified (99.4%) forms, in addition to improved yield (35.5%). For precursor 2, a modified cystine-cysteine redox system with short-chain organic co-solvents such as 20% EtOH or MeOH promoted native folding, whereas surfactants, chaotropic agents, and other additives were not obligatory. Notably, folding efficiency was dramatically elevated at a concentration of 1 mg/20 mL (18.9 mu M) over a period of 24 hours. However, prolonging the reaction time did not further improve the isomerization of misfolded products to their precisely folded counterparts. Adopting the amended oxidative conditions, scale-up studies were successfully carried out. Purification by preparative HPLC and lyophilization afforded a spongy white solid with 98.3% purity. Solution state structural information derived from nuclear magnetic resonance spectroscopy was consistent with literature reports, and confirmed that our synthetically derived Ziconotide (1) possesses the correct disulfide connectivity and three-dimensional conformation.