高碘酸氧化-还原胺化法对尿酸酶的聚乙二醇化研究  

Research on PEGylated uricase by periodate oxidation and reductiveamination

作　　者：陈阳建[1,2] 陈新齐 郑祥云[1] 宋潇达[2] 

机构地区：[1]浙江医药高等专科学校生物与制药学院,宁波315100 [2]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国药科大学学报》2015年第3期364-370,共7页Journal of China Pharmaceutical University

基　　金：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)~~

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 k D的单甲氧基聚乙二醇氨基盐酸盐(m PEG20000-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-m PEG20000,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-m PEG20000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-m PEG20000与尿酸酶物质的量比为2∶1,在溶液p H 5.0条件下,于25℃反应6 h。PEGylated uricase was prepared with the N-terminal amino site-specific modification by periodate oxidation followed by reduetive-amination. A monomethoxy poly（ ethylene glycol） intermediate was synthesized by amidation from monomethoxy poly（ ethylene glycol） amine hydrochloride 20000 （mPEG20000-NH2-HC1） 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 trifluoroaeetic 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 ultrafil- tration, and the modification conditions to uricase by Ser-mPEG20000 were optimized. The structures of poly （ ethyl- ene 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 uriease 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 ℃ and the reaction time was 6 h.

关 键 词：聚乙二醇衍生物 N端定点修饰 尿酸酶 高碘酸氧化 还原胺化 

分 类 号：O632.3[理学—高分子化学]