蝎子毒素多肽WaTx的高效化学合成及氧化折叠研究

doi:10.6023/A21120580

化学学报 ›› 2022, Vol. 80 ›› Issue (4): 444-452.DOI: 10.6023/A21120580 上一篇下一篇

研究论文

蝎子毒素多肽WaTx的高效化学合成及氧化折叠研究

尹昊^a^,^c, 陈西同^a^,^c, 付邢言^a^,^c, 马艳楠^a^,^c, 徐以梅^a^,^c, 张特^a^,^c, 梁帅^a^,^c, 杜姗姗^b^,^*(), 齐昀坤^a^,^c^,^*(), 王克威^a^,^c

a 青岛大学药学院山东青岛 266073
b 青岛科技大学化工学院山东青岛 266042
c 青岛大学创新药物研究院山东青岛 266021

投稿日期:2021-12-24 发布日期:2022-02-17
通讯作者: 杜姗姗, 齐昀坤
作者简介:
† 共同第一作者
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

Hao Yin^a^,^c, Xitong Chen^a^,^c, Xingyan Fu^a^,^c, Yannan Ma^a^,^c, Yimei Xu^a^,^c, Te Zhang^a^,^c, Shuai Liang^a^,^c, Shanshan Du^b(), Yunkun Qi^a^,^c(), Kewei Wang^a^,^c

a School of Pharmacy, Qingdao University, Qingdao, Shandong 266073
b College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, Shandong 266042
c Institute of Innovative Drugs, Qingdao University, Qingdao, Shandong 266021

Received:2021-12-24 Published:2022-02-17
Contact: Shanshan Du, Yunkun Qi
About author:
† These authors contributed equally to this work.
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)

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1. Supporting Information-A21120580.pdf(829KB)

摘要/Abstract

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

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.

Key words: N,N'-diisopropylcarbodiimide (DIC), oxyma, solid phase peptide synthesis, disulfide bond, oxidative folding, Wasabi Receptor Toxin (WaTx), peptide toxin

引用此文

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.

Hao Yin, Xitong Chen, Xingyan Fu, Yannan Ma, Yimei Xu, Te Zhang, Shuai Liang, Shanshan Du, Yunkun Qi, Kewei Wang. Efficient Chemical Synthesis and Oxidative Folding Studies of Scorpion Toxin Peptide WaTx[J]. Acta Chimica Sinica, 2022, 80(4): 444-452.

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图/表 7

图1. (A) HOBt、Oxyma的结构式; (B)蝎子毒素WaTx的氨基酸序列(左)和结构(右, PDB: 6OFA); (C)线性WaTx的合成路线; (D)线性肽的一次氧化折叠策略和(E)线性肽(2,3-Cys被乙酰胺甲基保护)的两次氧化折叠策略

Figure 1. (A) The structures of HOBt and Oxyma. (B) The amino acid sequences (left panel) and structure (right panel, PDB: 6OFA) of scorpion toxin WaTx. (C) The synthetic route of linear WaTx. (D) The one-step oxidative folding strategy for the linear peptide. (E) The two-step oxidative folding strategy for the linear peptide with 2,3-Cys protected by the acetamidomethyl (Acm) group

Linear peptide^c	Reaction temperature/℃	Condensation system^a	Resin type	Retention time/ min	The oxidation ratio of Met ^b/%	HPLC purity/%	Isolated yield/%	Yield/mg
1	28	n(Aa):n(HCTU):n(DIEA)=3:2.8:6	Rink Amide AM	20.9	24	34	28	54
2	75	n(Aa):n(DIC):n(Oxyma)=3:6:3	Rink Amide AM	20.9	63	19	13	25
3	75	n(Aa):n(DIC):n(Oxyma)=3:6:3	Wang resin	19.6	74	41	36	70
4	28	n(Aa):n(HCTU):n(DIEA)=3:2.8:6	Rink Amide AM	21.8	Not detected	26	18	34
5	75	n(Aa):n(DIC):n(Oxyma)=3:6:3	Rink Amide AM	21.8	Not detected	6	2	4
6	50	n(Aa):n(DIC):n(Oxyma)=3:6:3	Rink Amide AM	21.8	Not detected	10	3	6
7	50	n(Aa):n(DIC):n(Oxyma)=3:6:3	Rink Amide AM	20.9	24	35	27	52
8	50	n(Aa):n(DIC):n(Oxyma)=3:6:3	Rink Amide AM	22.4	20	42	36	72

表1. 线性肽1~8的合成策略

Table 1. The synthetic strategies for linear peptides 1~8

图2. (A)多肽1、(B)多肽2、(C)多肽3、(D)多肽4、(E)多肽5、(F)多肽6、(G)多肽7和(H)多肽8的粗肽和分离纯化后多肽的分析型RP-HPLC色谱图和ESI-MS质谱图

Figure 2. Analytical RP-HPLC and ESI-MS spectra of crude and purified peptides (A) peptide 1, (B) peptide 2, (C) peptide 3, (D) peptide 4, (E) peptide 5, (F) peptide 6, (G) peptide 7, and (H) peptide 8 The amino acid sequences of peptides 2' and 3' were provided in table S1. RP-HPLC conditions: a linear gradient of 10%~60% buffer B (0.08%~0.1% trifluoroacetic acid (TFA) in CH3CN) in buffer A (0.1% TFA in water) over 30 min (10% for 2 min, then 10%~60% for 30 min) on a Vydac C18 column, λ=214 nm

图3. (A)肽1、(B)肽2、(C)肽3和(D)肽4/5/6 (肽4/5/6的混合物)、(E)肽7和(F)肽8的氧化折叠分析型RP-HPLC色谱图和ESI-MS质谱图

Figure 3. Analytical RP-HPLC and ESI-MS spectra of oxidative folding process of peptides (A) peptide 1, (B) peptide 2, (C) peptide 3, (D) peptides 4/5/6 (the mixtures of peptides 4, 5, and 6), (E) peptide 7 and (F) peptide 8 (A) (a)~(b) The RP-HPLC spectra of the folding solution for peptide 1 at 0 min and 12 h. (c) The RP-HPLC and ESI-MS spectra of purified peptide 9. (B) (d)~(e) The RP-HPLC spectra of the folding solution for peptide 2 at 0 min and 12 h. (f) The RP-HPLC and ESI-MS spectra of purified peptide 10. (C) (g)~(h) The RP-HPLC spectra of the folding solution for peptides 3 at 0 min and 12 h. (i) The RP-HPLC and ESI-MS spectra of purified peptide 11. (D) (j)~(k) The RP-HPLC spectra of the folding solution for peptides 4/5/6 at 0 min and 12 h. (l) The RP-HPLC and ESI-MS spectra of purified peptide 12. (E) (m)~(n) The RP-HPLC spectra of the folding solution for peptide 7 at 0 min and 12 h. (o) The RP-HPLC and ESI-MS spectra of purified peptide 13. (F) The analytical RP-HPLC spectra of two-step oxidative folding process of peptide 8. (p) The RP-HPLC spectrum of the folding solution for peptide 8 at 0 min. (q) The RP-HPLC and ESI-MS spectra of purified peptide 14 with one disulfide bond (observed mass of 3997.76 Da, calculated 3998.53 Da, average isotopes). (r) The RP-HPLC and ESI-MS spectra of purified peptide 15. RP-HPLC conditions: a linear gradient of 10%~60% buffer B in buffer A over 30 min (10% for 2 min, then 10%~60% for 30 min) on a Vydac C18 column, λ=214 nm. The amino acid sequences of peptides 9~15 were provided in table S1

Oxidation product	Precursor peptide	HPLC purity/%	Isolated yield/%	Yield/mg	Calculated mass^a/Da	Observed mass^b/Da
9	1	79	47	9.4	3854.34	3853.72
10	2	75	36	7.2	3854.34	3853.72
11	3	76	42	8.4	3871.33	3870.69
12	4/5/6	29	13	2.6	3836.31	3835.85
13	7	78	43	8.6	3854.34	3853.68
15	8	73	31	6.2	3854.34	3853.73

表2. WaTx (肽9~15)氧化折叠的色谱纯度、分离收率及ESI-MS数据

Table 2. The HPLC purity, isolated yield and ESI-MS data of oxidative folding products (WaTx, peptides 9~15)

图4. (A)肽9、10、13和15共进样的分析型RP-HPLC色谱图和高分辨ESI-MS质谱图; (B)肽1、9~13和15的圆二色谱图

Figure 4. (A) Analytical RP-HPLC and ESI-MS spectra of peptides 9, 10, 13 and 15 co-injections; (B) The circular dichroism (CD) spectra of peptides 1, 9~13 and 15 The ESI-MS spectrum gave an observed mass of 3853.70 Da (calculated 3854.34 Da, average isotopes). RP-HPLC condition: a linear gradient of 10%~60% buffer B in buffer A over 30 min (10% for 2 min, then 10%~60% for 30 min) on a Vydac C18 column, λ=214 nm.

图5. 利用钙荧光法评价肽1和9~15对TRPA1通道的激动作用

Figure 5. The agonistic effect of peptides 1 and 9~15 on TRPA1 channel was evaluated by calcium fluorescence assay (A) The time courses of calcium influx caused by 5 μmol/L peptides (1, 9~15), AITC (100 μmol/L) and vehicle (deionized H2O). Results are representative of 3 independent experiments. (B) Quantification of calcium influx by area under curve. n=3, mean±SD. One way ANOVA. ns, no significance

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蝎子毒素多肽WaTx的高效化学合成及氧化折叠研究

Efficient Chemical Synthesis and Oxidative Folding Studies of Scorpion Toxin Peptide WaTx

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