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

doi:10.7503/cjcu20141107

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

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. 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 Bin^2,^*()

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/Abstract

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

中图分类号:

O625.31⁺6

TrendMD:

王欣欣, 吕侠, 葛广波, 冯磊, 辛红, 李耀光, 曹云峰, 韩冠英, 郭斌. 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. 高等学校化学学报, 2015, 36(7): 1321.

WANG Xinxin, LÜ Xia, GE Guangbo, FENG Lei, XIN Hong, LI Yaoguang, CAO Yunfeng, HAN Guanying, GUO Bin. Highly Efficient Preparation of Resorufin-β-D-glucuronide^†. Chem. J. Chinese Universities, 2015, 36(7): 1321.

图/表 7

Scheme 1 Biosynthesis of resorufin glucuronide by liver microsome from cynomolgus monkeys(CyLM)

Fig.1 HPLC-UV and MS spectra of resorufin and resorufin β-D-glucuronide(A) HPLC-UV profiles of resorufin and its metabolite in liver microsomes from different species; (B) MS spectrum of resorufin;(C) MS spectrum of resorufin β-D-glucuronide.

Fig.2 LC-UV chromatography of REG before and after purification(A) and hydrolysis(B)(A) LC-UV chromatography of the reaction mixture(a) and the aim metabolite after isolation and purification by SPE(b); (B) LC-UV profiles of REG(100 μmol/L) before(a) and REG(100 μmol/L) was incubated with β-glucosidase(1030 U/mL, complete hydrolysis) in at 37 ℃ for 15 min(b).

Fig.3 13C NMR spectrum of Resorufin-β-D-glucuronide(400 MHz, DMSO-d6)

Scheme 2 Hydrolysis of resorufin glucuronide by β-glucosidase

Fig.4 Emission spectrum(A) and calibration curve(B) of resorufin(A) Emission spectrum of REG after hydrolysis with β-glucosidase, the excitation wavelength was set at 500 nm; (B) resorufin calibration curve was obtained with a range of resorufin concentration, (0—20 μmol/L) in an equal volume of acetonitrile and PBS(total volume 400 μL), the excitation and emission wavelength were set at 577 and 620 nm, respectively.

Fig.5 Enzymatic kinetic and inhibition effect of REG hydrolysis in β-glucosidase(A) Enzymatic kinetic of REG hydrolysis in β-glucosidase, the abscissa is the concentration of resorufin glucuronide, the ordinate is the generation rate of resorufin. Eadie-Hofstee plot is shown as inset. Each point represents the average of two independent experiments in duplicate; (B) inhibitory effect of D-saccharic acid 1,4-lactone towards β-glucosidase with resorufin as the substrate IC50 value was obtained by fitting data to IC50 equation of GraphPad Prism 6.0.

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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^†

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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†

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Highly Efficient Preparation of Resorufin-β-D-glucuronide^†