高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (7): 1321-1327.doi: 10.7503/cjcu20141107

• 有机化学 • 上一篇    下一篇

β-葡萄糖醛酸苷酶荧光底物试卤灵葡萄糖醛酸苷的高效制备

王欣欣1, 吕侠3, 葛广波3, 冯磊3, 辛红3, 李耀光3, 曹云峰3, 韩冠英2, 郭斌2()   

  1. 1. 辽宁医学院药学院, 2. 附属第一医院, 锦州 121000
    3. 中国科学院大连化学物理研究所, 大连 116023
  • 收稿日期:2014-12-18 出版日期:2015-07-10 发布日期:2015-05-29
  • 作者简介:联系人简介: 郭 斌, 男, 博士, 教授, 主要从事海洋药用资源开发研究. E-mail:jyguobin@126.com
  • 基金资助:
    国家“九七三”计划项目(批准号: 2013CB531800)和国家自然科学基金(批准号: 81473181, 81273590)资助

Highly Efficient Preparation of Resorufin-β-D-glucuronide

WANG Xinxin1, LÜ Xia3, GE Guangbo3, FENG Lei3, XIN Hong3, LI Yaoguang3, CAO Yunfeng3, HAN Guanying2, GUO Bin2,*()   

  1. 1. Department of Medicine, Liaoning Medical University,2. The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, China
    3. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
  • Received:2014-12-18 Online:2015-07-10 Published:2015-05-29
  • Contact: GUO Bin E-mail:jyguobin@126.com
  • Supported by:
    † Supported by the National Basic Research Program of China(No.2013CB531800) and the National Natural Science Foundation of China(Nos.81473181, 81273590)

摘要:

以猴肝微粒体(CyLM)为酶源, 采用生物制备法实现了荧光底物试卤灵(Resorufin)向试卤灵葡萄糖醛酸苷(Resorufin β-D-glucuronide)的高效转化, 同时借助新型色谱分离材料C18WAX及固相萃取技术实现了Resorufin β-D-glucuronide的高效富集及选择性洗脱, 最终获得纯度大于98%的目标产物. 所得产物结构经LC-MS, 1H NMR和13C NMR等手段进行了表征. 在此基础上, 以该葡萄糖醛酸产物为探针底物建立了β-葡萄糖醛酸苷酶活性检测及抑制剂高通量筛选的方法.

关键词: 试卤灵, 试卤灵葡萄糖醛酸苷, 固相萃取, β-葡萄糖醛酸苷酶, 抑制剂筛选

Abstract:

Resorufin-β-O-glucuronide(REG) is a substrate of the β-glucuronidase enzyme which exhibited almost no fluorescence, upon addition of β-glucuronidase a high fluorescent compound resorufin will be released. Resorufin, is a fluorescent compound possessing good optical properties. On the basis of the off-on type fluorescent reaction, REG can be served as the good fluorescent substrate of β-glucuronidase. However, commercial available REG is rather expensive due to the difficulty for preparation of REG and the studies on REG are very limited. This study aimed to adopt the mild biosynthesis approach to efficiently prepare REG, based on resorufin can be extensively metabolized to REG by UDP-glucuronosyltransferases(UGTs). REG was prepared using resorufin as the starting material and liver microsomes from cynomolgus monkeys(CyLM) as the enzyme source. Resorufin(RE, 100 μmol/L) was incubated in Tris-HCl(50 mmol/L, pH=7.4, with 1% DMSO) with CyLM(0.5 mg/mL) at 37 ℃ for 4 h. After the optimization of the incubation conditions, the conversion of REG was more than 80%. A unique solid-phase extraction column(SPE) packed with C18WAX was used to enrich and purify the product REG with high yield. The product was then identified by both LC-MS and NMR techniques. Finally, a high throughput screening method for β-glucuronidase inhibitors was well established, based on the accurate kinetic parameters of REG towards β-glucuronidase and the absorption and emission spectrum of REG.

Key words: Resorufin, Resorufin-β-O-glucuronide, Solid-phase extraction, β-Glucuronidase, Inhibitor screening

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