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高碘酸氧化-还原胺化法对尿酸酶的聚乙二醇化研究

陈阳建, 陈新齐, 郑祥云, 宋潇达

陈阳建, 陈新齐, 郑祥云, 宋潇达. 高碘酸氧化-还原胺化法对尿酸酶的聚乙二醇化研究[J]. 中国药科大学学报, 2015, 46(3): 364-370. DOI: 10.11665/j.issn.1000-5048.20150318
引用本文: 陈阳建, 陈新齐, 郑祥云, 宋潇达. 高碘酸氧化-还原胺化法对尿酸酶的聚乙二醇化研究[J]. 中国药科大学学报, 2015, 46(3): 364-370. DOI: 10.11665/j.issn.1000-5048.20150318
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CHEN Yangjian, CHEN Xinqi, ZHENG Xiangyun, SONG Xiaoda. Research on PEGylated uricase by periodate oxidation and reductive- amination[J]. Journal of China Pharmaceutical University, 2015, 46(3): 364-370. DOI: 10.11665/j.issn.1000-5048.20150318
Citation: CHEN Yangjian, CHEN Xinqi, ZHENG Xiangyun, SONG Xiaoda. Research on PEGylated uricase by periodate oxidation and reductive- amination[J]. Journal of China Pharmaceutical University, 2015, 46(3): 364-370. DOI: 10.11665/j.issn.1000-5048.20150318
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高碘酸氧化-还原胺化法对尿酸酶的聚乙二醇化研究

  • 1.

    浙江医药高等专科学校生物与制药学院

  • 2.

    中国药科大学生命科学与技术学院

基金项目: 宁波市自然科学基金资助项目(No.2014A610208)；浙江省大学生科技创新活动计划暨新苗人才计划项目(No.2014R433003)

Research on PEGylated uricase by periodate oxidation and reductive- amination

摘要

    摘要
  • 摘要: 采用高碘酸氧化-还原胺化法对尿酸酶的N端氨基进行聚乙二醇修饰研究。首先，以相对分子质量为20 kD的单甲氧基聚乙二醇氨基盐酸盐(mPEG20000-NH2·HCl)和N-叔丁氧羰基-L-丝氨酸(Boc-Ser-OH)为原料通过酰胺化反应制备聚乙二醇中间体，然后由三氟乙酸(TFA)作用脱去Boc基团获得目标产物Ser-mPEG20000，该化合物经高碘酸钠氧化生成具有较高活性的聚乙二醇醛类衍生物，经超滤处理后即可用于蛋白质N端氨基的定点修饰，并对其修饰尿酸酶的条件进行了优化。聚乙二醇中间体和目标产物的结构通过IR和1H NMR进行表征，目标产物的总收率达72.8%。对尿酸酶的初步修饰研究表明，化合物Ser-mPEG20000具有较好的修饰蛋白的能力，对尿酸酶的最佳修饰条件为:Ser-mPEG20000与尿酸酶物质的量比为2∶1，在溶液pH 5.0条件下，于25 ℃反应6 h。
    Abstract: PEGylated uricase was prepared with the N-terminal amino site-specific modification by periodate oxidation followed by reductive-amination. A monomethoxy poly(ethylene glycol)intermediate was synthesized by amidation from monomethoxy poly(ethylene glycol)amine hydrochloride 20000(mPEG20000-NH2 ·HCl)with the relative molecular mass of 20 kD and N-(tert-butoxycarbonyl)-L-serine(Boc-Ser-OH), and then the Boc group of the intermediate was removed by trifluoroacetic acid(TFA)to produce the desired product Ser-mPEG20000. This compound could be oxidated by periodate to obtain a new poly(ethylene glycol)aldehyde derivative with high activity, which could be used to modify proteins with the N-terminal amino site-specific PEGylation after ultrafiltration, and the modification conditions to uricase by Ser-mPEG20000 were optimized. The structures of poly(ethylene glycol)intermediate and the target product were characterized by IR and 1H NMR, and the overall yield of the target product was 72. 8%. The preliminary modification to uricase indicated that the desired product Ser-mPEG20000 could modify proteins easily and efficiently. The optimal modification conditions of uricase PEGylated by Ser-mPEG20000 were obtained as follows: the molar ratio of Ser-mPEG20000 to uricase was 2 ∶1; the pH value of solution was 5. 0; the reaction temperature was 25 °C and the reaction time was 6 h.
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    • 参考文献(18)
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  • 刊出日期:  2015-06-24

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