Ziconotide (ω-conotoxin MVIIA)——线性前体肽的高效固相合成及其策略性天然折叠,Peptide Science

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Ziconotide (ω-conotoxin MVIIA)——线性前体肽的高效固相合成及其策略性天然折叠
Peptide Science ( IF 1.5 ) Pub Date : 2021-02-18 , DOI: 10.1002/pep2.24223
Gopalakrishnan Aridoss ₁ , Dong‐Min Kim ₁ , Jae Il Kim ₁ , Jae Eun Kang ₁

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为了开发目标肽1的有效合成，对前体肽2（线性 25 聚体）的固相肽合成 (SPPS)及其水相折叠过程进行了系统优化研究。DIC/HOBt（16-25-mer）和 HBTU/NMM（1-15-mer）在 DMF 中的组合证明是提供线性前体肽2的最佳偶联策略，其在粗品 (81.3%) 和纯化 (99.4%) 中具有更高的纯度%) 形式，此外还提高了产量 (35.5%)。对于前体2，具有短链有机助溶剂（如 20% EtOH 或 MeOH）的修饰的胱氨酸-半胱氨酸氧化还原系统促进了天然折叠，而表面活性剂、离液剂和其他添加剂不是必需的。值得注意的是，在 1 mg/20 mL (18.9 μM) 的浓度下，折叠效率在 24 小时内显着提高。然而，延长反应时间并没有进一步改善错误折叠产物向其精确折叠对应物的异构化。采用修改后的氧化条件，成功地进行了放大研究。通过制备型HPLC纯化和冻干得到纯度为98.3%的海绵状白色固体。核磁共振波谱得到的溶液状态结构信息与文献报道一致，证实了我们合成衍生的齐考诺肽（1 ) 具有正确的二硫键连接和三维构象。

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Ziconotide (ω-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 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.

更新日期：2021-02-18

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