4'-去甲基表鬼臼毒素的酶法糖基化

Enzymatic glycosylation of 4'-demethylepipodophyllotoxin

4'-去甲基表鬼臼毒素(4'-demethylepipodophyllotoxin,DMEP)的糖苷衍生物具有多种药理活性,但其化学合成面临位置及立体选择性和基团的保护与脱保护等诸多挑战。该研究从库拉索芦荟Aloe barbadensis中克隆得到1个新颖糖基转移酶(glycosyltransferase,GT)基因Ab GT5,并成功进行了外源表达及蛋白纯化。重组Ab GT5能够催化4'-去甲基表鬼臼毒素进行糖基化,获得的产物经MS,~1H-NMR,^(13)C-NMR,HSQC以及HMBC等波谱技术鉴定为4'-去甲基表鬼臼毒素-4'-O-β-D-葡萄糖苷。酶学性质研究发现AbGT5的最适反应温度为20℃,最适pH 9.0,且不依赖金属离子。在最适反应条件下,4'-去甲基表鬼臼毒素的转化率可达80%。该研究表明利用新颖糖基转移酶AbGT5可实现4'-去甲基表鬼臼毒素的高效酶法糖基化,为其糖基化提供新方法。

The glycosides of 4＇-demethylepipodophyllotoxin （DMEP） possess various pharmacological activities; however, the chemical synthesis of these glycosides faces challenges in regioselectivity, stereoselectivity, and the protection and de-protection of functional groups. In this work, a novel glycosyhransferase （GT） gene AbGT5 from Aloe barbadensis was successfully cloned, heteroge- neously expressed and purified. Recombinant AbGT5 was able to catalyze the glycosylation of DMEP and the glycosylated product, which was separated from the preparative scale reaction, was characterized as DMEP 4＇-O-β-D-glucoside via MS, I H-NMR, 13 C-NMR, HSQC and HMBC. According to the investigations of enzyme properties, AbGT5 show the highest activity around 20 ℃ in the buffer of pH 9. 0, and it was independent of divalent metal ions. Under the optimum conditions, the conversion rate of DMEP can reach 80%. Above all, in this work the enzymatic glycosylation of DMEP was achieved with high efficiency by the novel GT AbGTS.