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

doi:10.6023/A21120580

Acta Chimica Sinica ›› 2022, Vol. 80 ›› Issue (4): 444-452.DOI: 10.6023/A21120580 Previous Articles Next Articles

Article

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)

1. .pdf(829KB)

Abstract

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

Cite this article

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.

Export EndNote|Reference Manager|ProCite|BibTeX|RefWorks

share this article

Fig. & Tab. 7

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

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

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

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

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

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.

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

References 15

[1]	(a) Liu, T.; Xu, S. L.; Zhao, J. F. Chin. J. Org. Chem. 2021, 41, 873. (in Chinese) doi: 10.6023/cjoc202011022
	(刘涛, 许泗林, 赵军锋, 有机化学, 2021, 41, 873.) doi: 10.6023/cjoc202011022
	(b) Chang, H. N.; Liu, B. Y.; Qi, Y. K.; Zhou, Y.; Chen, Y. P.; Pan, K. M.; Li, W. W.; Zhou, X. M.; Ma, W. W.; Fu, C. Y.; Qi, Y. M.; Liu, L.; Gao, Y. F. Angew. Chem., Int. Ed. 2015, 54, 11760. doi: 10.1002/anie.201506225
	(c) Guo, Y.; Zhou, P. P.; Zhang, S. Y.; Fan, X. W.; Dou, Y. W.; Shi, X. L. Med ChemComm 2018, 9, 1226.
	(d) Zhou, X. M.; Zuo, C.; Li, W. Q.; Shi, W. W.; Zhou, X. W.; Wang, H. F.; Chen, S. M.; Du, J. F.; Chen, G. Y.; Zhai, W. J.; Zhao, W. S.; Wu, Y. H.; Qi, Y. M.; Liu, L.; Gao, Y. F. Angew. Chem., Int. Ed. 2020, 59, 15114. doi: 10.1002/anie.202002783
	(e) Song, H.; Liu, C.; Wu, Y. J.; Hu, H. G.; Yan, F. Acta Chim. Sinica 2018, 76, 95. (in Chinese) doi: 10.6023/A17100473
	(宋慧, 刘超, 吴仪君, 胡宏岗, 阎芳, 化学学报, 2018, 76, 95.) doi: 10.6023/A17100473
	(f) Cui, T. T.; Chen, J. Y.; Zhao, R.; Guo, Y. Y.; Tang, J. H.; Li, Y. L.; Li, Y. M.; Bierer, D.; Liu, L. Chin. J. Chem. 2021, 39, 2517. doi: 10.1002/cjoc.202100272
[2]	(a) Guo, Y.; Fu, L. L.; Fan, X. W.; Shi, X. L. Chin. J. Org. Chem. 2018, 38, 1267. (in Chinese) doi: 10.6023/cjoc201710033
	(郭叶, 傅莉莉, 范晓文, 史宣玲, 有机化学, 2018, 38, 1267.) doi: 10.6023/cjoc201710033
	(b) Qi, Y. K.; Qu, Q.; Bierer, D.; Liu, L. Chem. Asian J. 2020, 15, 2793. doi: 10.1002/asia.202000609
	(c) Guo, Y.; Fu, L. L.; Fan, X. W.; Shi, X. L. Chin. Chem. Lett. 2018, 29, 1167. doi: 10.1016/j.cclet.2018.03.024
[3]	(a) Muramatsu, W.; Yamamoto, H. J. Am. Chem. Soc. 2021, 143, 6792. doi: 10.1021/jacs.1c02600 pmid: 29185032
	(b) Hu, L.; Xu, S.; Zhao, Z.; Yang, Y.; Peng, Z.; Yang, M.; Wang, C.; Zhao, J. J. Am. Chem. Soc. 2016, 138, 13135. doi: 10.1021/jacs.6b07230 pmid: 29185032
	(c) Jaradat, D. M. M. Amino Acids 2018, 50, 39. doi: 10.1007/s00726-017-2516-0 pmid: 29185032
[4]	Wang, Z.; Wang, X.; Wang, P.; Zhao, J. J. Am. Chem. Soc. 2021, 143, 10374. doi: 10.1021/jacs.1c04614
[5]	(a) Albericio, F.; El-Faham, A. Org. Process Res. Dev. 2018, 22, 760. doi: 10.1021/acs.oprd.8b00159 pmid: 30900880
	(b) Isidro-Llobet, A.; Kenworthy, M. N.; Mukherjee, S.; Kopach, M. E.; Wegner, K.; Gallou, F.; Smith, A. G.; Roschangar, F. J. Org. Chem. 2019, 84, 4615. doi: 10.1021/acs.joc.8b03001 pmid: 30900880
	(c) Subiros-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F. Chem. Eur. J. 2009, 15, 9394. doi: 10.1002/chem.200900614 pmid: 30900880
[6]	(a) Wang, F.; Xu, L.; Chu, G.; Shi, J.; Guo, Q. Chin. J. Org. Chem. 2016, 36, 218. (in Chinese) doi: 10.6023/cjoc201505014
	(王风亮, 许玲, 储国超, 石景, 郭庆祥, 有机化学, 2016, 36, 218.) doi: 10.6023/cjoc201505014
	(b) Pan, M.; Gao, S.; Zheng, Y.; Tan, X.; Lan, H.; Tan, X.; Sun, D.; Lu, L.; Wang, T.; Zheng, Q.; Huang, Y.; Wang, J.; Liu, L. J. Am. Chem. Soc. 2016, 138, 7429. doi: 10.1021/jacs.6b04031
	(c) Qi, Y.-K.; Si, Y.-Y.; Du, S.-S.; Liang, J.; Wang, K.-W.; Zheng, J.-S. Sci. China Chem. 2019, 62, 299. doi: 10.1007/s11426-018-9401-8
	(d) Qi, Y. K.; Ai, H. S.; Li, Y. M.; Yan, B. Front. Chem. 2018, 6, 19. doi: 10.3389/fchem.2018.00019
	(e) Huang, Y. C.; Guan, C. J.; Tan, X. L.; Chen, C. C.; Guo, Q. X.; Li, Y. M. Org. Biomol. Chem. 2015, 13, 1500. doi: 10.1039/C4OB02260B
	(f) Ben Haj Salah, K.; Inguimbert, N. Org. Lett. 2014, 16, 1783. doi: 10.1021/ol5003253
[7]	(a) Qu, Q.; Pan, M.; Gao, S.; Zheng, Q. Y.; Yu, Y. Y.; Su, J. C.; Li, X.; Hu, H. G. Adv. Sci. 2018, 5, 1800234. doi: 10.1002/advs.201800234
	(b) Qu, Q.; Gao, S.; Wu, F.; Zhang, M. G.; Li, Y.; Zhang, L. H.; Bierer, D.; Tian, C. L.; Zheng, J. S.; Liu, L. Angew. Chem., Int. Ed. 2020, 59, 6037. doi: 10.1002/anie.201915358
	(c) Zuo, C.; Shi, W.-W.; Chen, X.-X.; Glatz, M.; Riedl, B.; Flamme, I.; Pook, E.; Wang, J.; Fang, G.-M.; Bierer, D.; Liu, L. Sci. China Chem. 2019, 62, 1371. doi: 10.1007/s11426-019-9513-2
[8]	Liang, L. J.; Chu, G. C.; Qu, Q.; Zuo, C.; Mao, J.; Zheng, Q.; Chen, J.; Meng, X.; Jing, Y.; Deng, H.; Li, Y. M.; Liu, L. Angew. Chem., Int. Ed. 2021, 60, 17171. doi: 10.1002/anie.202105870
[9]	(a) Ma, Y.; Liu, Y.; Wang, J.; Chen, X.; Yin, H.; Chi, Q.; Jia, S.; Du, S.; Qi, Y.; Wang, K. Chin. J. Org. Chem. 2022, 42, 10.6023/cjoc202109003. (in Chinese)
	(马艳楠, 刘雅妮, 王金艳, 陈西同, 尹昊, 迟巧娜, 贾世玺, 杜姗姗, 齐昀坤, 王克威, 有机化学, 2022, 42, 10.6023/cjoc202109003.)
	(b) Chen, X. T.; Wang, J. Y.; Ma, Y. N.; Dong, L. Y.; Jia, S. X.; Yin, H.; Fu, X. Y.; Du, S. S.; Qi, Y. K.; Wang, K. J. Pept. Sci. 2022, 28, e3368.
[10]	Qiao, Z.; Luo, J.; Tang, Y. Q.; Zhou, Q.; Qi, H.; Yin, Z.; Tang, X.; Zhu, W.; Zhang, Y.; Wei, N.; Wang, K. J. Med. Chem. 2021, 64, 16282. doi: 10.1021/acs.jmedchem.1c01579
[11]	King, J. V. L.; Emrick, J. J.; Kelly, M. J. S.; Herzig, V.; King, G. F.; Medzihradszky, K. F.; Julius, D. Cell 2019, 178, 1362. doi: 10.1016/j.cell.2019.07.014
[12]	(a) Wang, J.; Dong, L.; Liu, Y.; Chen, X.; Ma, Y.; Yin, H.; Du, S.; Qi, Y.; Wang, K. Chin. J. Org. Chem. 2021, 41, 2800. (in Chinese) doi: 10.6023/cjoc202102045
	(王金艳, 董黎颖, 刘雅妮, 陈西同, 马艳楠, 尹昊, 杜姗姗, 齐昀坤, 王克威, 有机化学, 2021, 41, 2800.) doi: 10.6023/cjoc202102045
	(b) Qi, Y. K.; He, Q. Q.; Ai, H. S.; Guo, J.; Li, J. B. Chem. Commun. 2017, 53, 4148. doi: 10.1039/C7CC01721A
[13]	(a) Shi, J.; So, L. Y.; Chen, F.; Liang, J.; Chow, H. Y.; Wong, K. Y.; Wan, S.; Jiang, T.; Yu, R. J. Pept. Sci. 2018, 24, e3087. doi: 10.1002/psc.3087
	(b) Guo, Y.; Sun, D. M.; Wang, F. L.; He, Y.; Liu, L.; Tian, C. L. Angew. Chem., Int. Ed. 2015, 54, 14276. doi: 10.1002/anie.201500699
[14]	(a) Zheng, J. S.; Tang, S.; Qi, Y. K.; Wang, Z. P.; Liu, L. Nat. Protoc. 2013, 8, 2483. doi: 10.1038/nprot.2013.152
	(b) Li, J. B.; Qi, Y. K.; He, Q. Q.; Ai, H. S.; Liu, S. L.; Wang, J. X.; Zheng, J. S.; Liu, L.; Tian, C. Cell Res. 2018, 28, 257. doi: 10.1038/cr.2017.157
	(c) Zheng, J. S.; Liang, J.; Shi, W. W.; Li, Y.; Hu, H. G.; Tian, C. L.; Liu, L. Sci. Bull. 2021, 66, 1542. doi: 10.1016/j.scib.2021.03.010
[15]	Wang, N.; Xie, G.; Liu, C.; Cong, W.; He, S.; Li, Y.; Fan, L.; Hu, H. G. Front. Chem. 2020, 8, 616147. doi: 10.3389/fchem.2020.616147

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

RichHTML

PDF

Supporting Info.

Knowledge

Abstract

Cite this article

share this article

Fig. & Tab. 7

References 15

Related Articles 7

Recommended Articles

Metrics

Comments

[1]	Song Hui, Liu Chao, Wu Yijun, Hu Honggang, Yan Fang. Efficient Synthesis of Bicyclic Peptide BI-32169 Utilizing a Novel Aryl Boronate Ester Protecting Group [J]. Acta Chimica Sinica, 2018, 76(2): 95-98.
[2]	Gao Xiao-Fei, He Peng, Chen Huanwen. Study on the Interaction Between Water Radical Cations and Bis(2-hydroxyethyl) Disulfide at Ambient Temperature and Pressure Using Mass Spectrometry [J]. Acta Chim. Sinica, 2018, 76(10): 802-806.
[3]	Ma Fei, Chen Jianxin. Novel Method for Synthesis of Unsymmetrical α-Organyl-α-hydroxymalonamide Derivatives [J]. Acta Chimica Sinica, 2013, 71(08): 1118-1120.
[4]	TANG Qian-Qian, YUAN Li, YANG Dong, HU Jian-Hua. Preparation and Characterization of Disulfide Bond Functionalized Mesoporous Silica Microspheres [J]. Acta Chimica Sinica, 2010, 68(18): 1925-1929.
[5]	. Thermodynamic Study on Interaction of Oxymatrine with Bovine Serum Albumin [J]. Acta Chimica Sinica, 2009, 67(18): 2155-2158.
[6]	LIU Hong-Ni, WANG Yan, GONG Bo-Lin, GENG Xin-Du*. Study on Refolding of Urea-Reduced/Denatured Lysozyme by High-Performance Weak-cation Exchange Chromatography [J]. Acta Chimica Sinica, 2005, 63(7): 597-602.
[7]	ZHAO Ting-You,CAO Ying,DAI Xian-Dong,FAN Chong-Xu*,CHEN Ji-Sheng. Purification, Sequence and Disulfide Bonding Pattern of a Novel Conotoxin BtIIIB [J]. Acta Chimica Sinica, 2005, 63(2): 163-168.