蝎子毒素多肽WaTx的高效化学合成及氧化折叠研究
收稿日期: 2021-12-24
网络出版日期: 2022-02-17
基金资助
国家自然科学基金(21807063); 国家自然科学基金(82003647); 国家自然科学基金(22177058); 国家自然科学基金(81870653)
Efficient Chemical Synthesis and Oxidative Folding Studies of Scorpion Toxin Peptide WaTx
Received date: 2021-12-24
Online published: 2022-02-17
Supported by
National Natural Science Foundation of China(21807063); National Natural Science Foundation of China(82003647); National Natural Science Foundation of China(22177058); National Natural Science Foundation of China(81870653)
相比于1-羟基-7-氮杂苯并三氮唑(HOAt)、1-羟基苯并三唑(HOBt)等传统偶联试剂, 新型多肽偶联试剂2-肟氰乙酸乙酯(Oxyma)具有安全、偶联效率高、消旋率低等优势, 在多肽合成特别是微波固相合成中得到广泛应用. 但是, 不同反应温度(例如28、50和75 ℃)对N,N'-二异丙基碳二亚胺(DIC)/Oxyma的偶联效率以及对甲硫氨酸(Met)等易氧化氨基酸的影响尚有待研究. 瞬时受体电位锚蛋白1(TRPA1)通道在感受温度、听觉和炎症痛中发挥重要作用. 蝎子毒素多肽芥末受体毒素(WaTx)是一种新型、非共价结合的TRPA1特异性激动剂. 本研究利用6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯(HCTU)/N,N'-二异丙基乙胺(DIEA)和DIC/Oxyma缩合体系, 首次探索了不同温度下线性WaTx的合成效率以及Met残基的氧化情况. 通过一次氧化折叠和两次氧化折叠策略, 实现了WaTx的体外快速复性折叠, 利用圆二色谱和钙荧光检测等技术评价WaTx的结构和活性. 本研究建立了WaTx的温和、高效合成以及复性折叠方法, 为固相多肽合成特别是手动固相合成WaTx等含有易氧化基团的二硫键构象锁定多肽提供了重要参考.
关键词： N,N'-二异丙基碳二亚胺(DIC); Oxyma; 固相多肽合成; 二硫键; 氧化折叠; 芥末受体毒素(WaTx); 多肽毒素
尹昊 , 陈西同 , 付邢言 , 马艳楠 , 徐以梅 , 张特 , 梁帅 , 杜姗姗 , 齐昀坤 , 王克威 . 蝎子毒素多肽WaTx的高效化学合成及氧化折叠研究[J]. 化学学报, 2022 , 80(4) : 444 -452 . DOI: 10.6023/A21120580
Coupling or condensation reagents that could be used to promote the condensation of carboxylic acids with amines to furnish amide bonds play crucial roles in solid phase peptide synthesis (SPPS) of peptides and peptide-based derivatives. Compared with traditional coupling reagents used in SPPS such as 1-hydroxy-7-azabenzotriazole (HOAt) and 1-hydroxybenzotriazole (HOBt), the novel N,N'-diisopropylcarbodiimide (DIC)/ethyl 2-cyano-2-(hydroxyimino) acetate (Oxyma) condensation system has advantages of inexpensiveness, safety, high coupling efficiency, low racemic rate, and compatibility with manual and automatic peptide synthesis represented by microwave-assisted SPPS. However, the effects of different reaction temperatures (e.g. 28, 50, and 75 ℃) on the coupling efficiency of DIC/Oxyma and on the easily oxidized amino acids such as methionine (Met) remain to be further investigated. The transient receptor potential ankyrin 1 (TRPA1) channel plays an important role in temperature perception, auditory perception, and inflammatory pain. As a novel non-covalently TRPA1-specific agonist, Wasabi Receptor Toxin (WaTx) that consists of 33 amino acid residues and two pairs of disulfide bonds is considered as an important molecular tool to study the opening mechanism and function of TRPA1. In this study, 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexa-fluorophosphate (HCTU)/N,N'-diisopropyl- ethylamine (DIEA) and DIC/Oxyma condensation systems were separately utilized to explore the synthetic efficiency of linear WaTx and the oxidative degree of Met residues at different temperatures. The robustness of DIC/Oxyma condensation system was validated by the rapid manual synthesis of linear WaTx. The one-step and two-step oxidative folding strategies were separately applied for the construction of two pairs of disulfide bridges, affording the active WaTx, which was further confirmed by circular dichroism and calcium fluorescence assay. In this study, a moderate, efficient synthesis and renaturation folding method of WaTx was established. Moreover, the effects of different reaction temperatures on the synthetic efficiency of DIC/Oxyma and on amino acid residues oxidization were compared for the first time. From the aspects of reaction efficiency (represented by the timescale of the overall Fmoc deprotection-washing-amino acid coupling SPPS cycle), amino acid residues oxidization and synthetic cost, n(Fmoc-amino acid):n(DIC):n(Oxyma)=3:6:3 under 50 ℃ should be the ideal reaction condition. This work provides both an imperative complement for SPPS and a particularly useful strategy for the manual efficient synthesis of disulfide-containing peptides with easily oxidized groups.
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