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  

Highly Efficient Preparation of Resorufin-β-D-glucuronide

作　　者：王欣欣[1] 吕侠[2] 葛广波[2] 冯磊[2] 辛红[2] 李耀光[2] 曹云峰[2] 韩冠英[3] 郭斌[3] 

机构地区：[1]辽宁医学院药学院,锦州121000 [2]中国科学院大连化学物理研究所,大连116023 [3]辽宁医学院附属第一医院,锦州121000

出　　处：《高等学校化学学报》2015年第7期1321-1327,共7页Chemical Journal of Chinese Universities

基　　金：国家“九七三”计划项目(批准号:2013CB531800);国家自然科学基金(批准号:81473181,81273590)资助~~

摘　　要：以猴肝微粒体(Cy LM)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(Resorufinβ-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Resorufinβ-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.Resorufin-β-O-glucuronide（REG） is a substrate of theβ-glucuronidase enzyme which exhibited almost no fluorescence, upon addition ofβ-glucuronidase a high fluorescent compound resorufin will be released. Resortffin, 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 ofβ-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 staaing material and liver microsomes from cynomolgus monkeys （CyLM） as the enzyme source. Resorufin （ RE, 100 μmol/L） was incubated in Tris-HC1 （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 LCMS and NMR techniques. Finally, a high established, based on the accurate kinetic throughput screening method forβ-glucuronidase inhibitors was well parameters of REG towardsβ-glucuronidase and the absorption and emission spectrum of REG.

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 

分 类 号：O625.316[理学—有机化学]