基于DIC/Oxyma的蜘蛛毒素多肽GsMTx4的高效合成及活性评价

DIC/Oxyma Based Efficient Synthesis and Activity Evaluation of Spider Peptide Toxin GsMTx4

偶联试剂介导酰胺键形成,在固相多肽合成中发挥关键作用.新型DIC(N,N-二异丙基碳二亚胺)/Oxyma(2-肟氰乙酸乙酯)缩合体系具有廉价、操作安全、缩合效率高和抑制消旋等优势,在手动和自动多肽合成中得到广泛应用.但是,DIC/Oxyma缩合体系的理想反应比例和温和反应温度尚有待探索.Piezo通道是一种多功能的机械敏感阳离子通道,与多种遗传性疾病相关,蜘蛛毒素多肽GsMTx4是目前唯一的特异性靶向Piezo通道的抑制剂.本研究探索了DIC/Oxyma缩合体系在温和条件下的最佳反应比例,实现了线性Gs MTx4的高效手动固相合成.通过一次氧化折叠策略构建三对二硫键,得到活性的GsMTx4.利用圆二色谱和膜片钳技术等评价GsMTx4的结构和活性.本工作建立了基于DIC/Oxyma缩合体系的快速、稳健和安全的合成方法,为固相多肽合成特别是手动固相多肽合成提供了重要方法参考.

Coupling reagents mediate the formation of amide bonds and play a key role in solid phase peptide synthesis.The novel N,N-diisopropylcarbodiimide(DIC)/ethyl cyanoglyoxylate-2-oxime(Oxyma)condensation system has the advantages of low cost,operational safety,higher coupling efficiency and lower rate of racemization.The DIC/Oxyma condensation system has been widely used in manual and automated synthesis of peptides.However,the ideal reaction ratio in DIC/Oxyma condensation system at mild reaction temperature remains to be further investigated.GsMTx4 is a cysteine rich peptide toxin identified from the venom of Grammostola spatulata spider,which consists of 34 amino acid residues and three pairs of disulfide bonds.GsMTx4 is the only inhibitor specifically targeting piezo channel,which is a multifunctional mechanically sensitive cation channel and associated with a variety of hereditary diseases.Herein,the synthetic efficiencies of different reaction ratios of DIC and Oxyma at moderate reaction temperature were systematically evaluated.The efficiency and robustness of DIC/Oxyma condensation system were validated by the rapid manual synthesis of linear GsMTx4.The one-step oxidative folding strategy was applied for the construction of three pairs of disulfide bridges,affording the active GsMTx4.The circular dichroism and patch-clamp electrophysiology tests were conducted to evaluate the structure and activity of synthetic GsMTx4.In summary,a fast,robust and safe synthetic method based on DIC/Oxyma is established,which is particularly useful for the manual synthesis of peptides.