Ziconotide (ω‐conotoxin MVIIA)—Efficient solid‐phase synthesis of a linear precursor peptide and its strategic native folding

Abstract

AbstractTo 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 μM) 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.