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

doi:10.7503/cjcu20141107

Abstract

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

CLC Number:

O625.31⁺6

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2015, 36(7): 1321.

Figures/Tables 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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