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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2023, Vol. 43 Issue (1): 35-41    DOI: 10.13523/j.cb.2209002
技术与方法     
一种多肽固相合成方法与纯化策略研究
王宇航,陈学明,刘俗生,阮志军,张敏,宋春丽,尹丰*(),李子刚*()
深圳湾实验室坪山生物医药研发转化中心 深圳 518118
A Solid-phase Synthesis Method and Purification of Polypeptides
WANG Yu-hang,CHEN Xue-ming,LIU Su-sheng,RUAN Zhi-jun,ZHANG Min,SONG Chun-li,YIN Feng*(),LI Zi-gang*()
Shenzhen Bay Laboratory Pingshan Translational Medicine Center, Shenzhen 518118, China
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摘要:

目的: 多肽与小分子化学药物相比,具有生物活性高、特异性强、不容易产生耐药性等特点,是目前新型药物研发的重点领域。多肽的合成直接影响到多肽药物的作用机制以及药物效果,因此需要建立一种更加便捷、高效的多肽合成方法。方法: 采用Fmoc固相合成法合成多肽HF01,通过比较氨基酸连接的反应体系以及氨基酸脱保护的反应体系,从中确定最优体系。利用乙酰化基团进行肽链末端保护,经肽链剪切制备干燥的粗肽,最后采用高效液相色谱仪与高分辨质谱仪联用对粗肽进行纯化。结果: 确定多肽合成的连接和脱保护反应体系,并获得纯度高达98.3%的线性多肽。结论: 建立了一种高效、便捷的多肽合成及纯化方法,提高了实验室合成多肽的效率,为多肽类药物的研发提供技术支撑。

关键词: 多肽Fmoc固相合成氨基酸连接液相色谱串联质谱纯化    
Abstract:

Objective: Compared with small molecule chemical drugs, peptides have the advantages of high biological activity, strong specificity, and drug resistance, and thus, they become one of the priorities of new drug research and development. Peptide synthesis is the premise of the research and development of polypeptide drugs. Therefore, it is very important to establish a more convenient and efficient synthesis method. Methods: Fmoc solid phase synthesis was used for HF01 synthesis of polypeptides. First, the optimal system was determined by comparing the amino acid linked reaction system and the amino acid deprotection reaction system, and the acetylation group was applied for peptide N-Term. Then, the peptides were cut off from the resin. Finally, the obtained peptides were purified using both an HPLC and a mass spectrometer. Results: The ligation and deprotection reaction system for polypeptide synthesis was determined, and the polypeptide was successfully synthesized and purified with high purification efficiency of 98.3%. Conclusion: Above all, the operation is simple and affordable. This method is commonly used for polypeptide synthesis and provides feasibility for polypeptide drug structure studies. It is expected that this reliable synthesis method will provide help for more laboratory researchers.

Key words: Polypeptide    Fmoc solid-phase synthesis    Amino acid connection    Liquid chromatography tandem mass spectrometry (LC-MS/MS)    Purification
收稿日期: 2022-09-02 出版日期: 2023-02-14
ZTFLH:  Q816  
通讯作者: *尹丰 电子信箱:yinfeng@szbl.ac.cn;李子刚 lizg@szbl.ac.cn   
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王宇航
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刘俗生
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张敏
宋春丽
尹丰
李子刚

引用本文:

王宇航, 陈学明, 刘俗生, 阮志军, 张敏, 宋春丽, 尹丰, 李子刚. 一种多肽固相合成方法与纯化策略研究[J]. 中国生物工程杂志, 2023, 43(1): 35-41.

WANG Yu-hang, CHEN Xue-ming, LIU Su-sheng, RUAN Zhi-jun, ZHANG Min, SONG Chun-li, YIN Feng, LI Zi-gang. A Solid-phase Synthesis Method and Purification of Polypeptides. China Biotechnology, 2023, 43(1): 35-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2209002        https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I1/35

图1  多肽合成路线
反应体系 反应时间
DIPEA-HATU 30 min+30 min
DIPEA-HATU 1 h
HOBt-DIC 45 min~1 h
表1  两种氨基酸连接反应体系的比较
反应体系 反应时间
50%吗啡啉、50%DMF 30 min+30 min
20%哌啶、80%DMF 5 min+15 min[12]
20%哌啶、80%DMF 5 min+5 min
表2  不同脱保护基的反应体系的比较
多肽 理论分子量 理论分子离子峰 肽序
HF01 1 910.2 637.7, 956.1 GSRALCYSQWWRRLK
表3  多肽HF01的高效液相和质谱检测数据
图2  高效液相检测结果
图3  质谱检测结果
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