Kinetic and optical properties of a new probe for sulfatase activity assay

A new probe for detecting sulfatase activity generated fluorescent N-methylisoindole when it was treated with sulfatase. Detailed synthetic procedures of reaction intermediates are described along with their spectral data. The Diels-Alder adduct of N-methylisoindole and N-phenylmaleimide was identified by LC–MS data. The fluorescence changes and kinetic values of the probe upon sulfatase treatment in the presence and absence of reducing agents were described. Inhibitory potency of EMATE was measured using the probe in the presence and absence of reducing agents. In addition, the fluorescence intensities of the probe upon sulfatase treatment were also monitored at pH 5 and 7 with and without reducing agents.


How data was acquired
UV/Vis spectrometry, NMR, mass spectrometry, microplate reader.

Data format Raw and analyzed Experimental factors
Before measurement of inhibitory potency, pre-incubation with EMATE and sulfatase was carried out at 37°C overnight.

Experimental features
Fluorescence measurements of the probe with sulfatase under various conditions were performed Data source location Data accessibility Within this article

Value of the data
The synthesis procedure will provide researchers with a general preparation method of sulfatase substrates.
The kinetic parameters of the probe and inhibitory potency showed the effects of reducing agents.
The fluorescence properties at pH 7 and pH 5 showed the utility of the probe under neutral conditions.

Data
A new probe to detect sulfatase activity was developed. When the probe was treated with sulfatase, it generated fluorescent N-methylisoindole which was influenced by reducing agents and pH conditions [1]. The fluorescence intensity, kinetic parameters and inhibitory potency under Tris buffer and reducing agents were described. Fluorescence intensities of the probe with sulfatase at pH 5 and 7 were compared.

Chemical synthesis
1 H and 13 C NMR spectra were collected on a Bruker Advance DPX-300. Fast atom bombardment mass spectrometry (FAB-MS) data were obtained using a JEOL JMS-AX505WA mass spectrometer with m-nitrobenzyl alcohol (NBA) as a matrix and the data were reported in units of mass to charge (m/z). Analytical thin layer chromatography was carried out using Kieselgel 60F-254 plates purchased from Merck and column chromatography was performed on Merck silica gel 60 (70-230 mesh). All chemical reagents purchased from either Sigma-Aldrich or TCI and used without any further purification.
A solution of sulfuryl chloride (1.62 ml, 20 mmol) in Et 2 O was cooled to À 75°C under nitrogen. A solution of neopentyl alcohol (1.76 g, 1 equiv.) and pyridine (1.62 ml, 1 equiv.) in Et 2 O was added dropwise to the cooled sulfuryl chloride solution for 1 h and stirred at room temperature for 2 h. The white precipitates were removed by filtration and the filtrate was concentrated in vacuo. The product was used for the next synthesis step without further purification.
Compound 7: To a solution of 4-hydroxybenzyl alcohol (1.7 g, 1 equiv.) in anhydrous THF was sodium hydride (437 mg, 1.1 equiv.) slowly added at 0°C. After 10 min, crude neopentyl chlorosulfate was added to the solution at 0°C and the reaction mixture was allowed to warm up to room temperature and stirring was continued overnight. Upon completion of the reaction, water was added to the reaction mixture to quench sodium hydride and THF was removed in vacuo. Ethyl acetate was added to the residue and the organic layer was washed with brine, then dried over Na 2 SO 4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (chloroform: acetone¼50:1) to give compound 7 (970 mg, 17% yield). 1  Compound 8: Pyridine (257 μl, 1 equiv.) was added to a solution of 4-nitrophenyl chloroformate (640 mg, 1.1 equiv.) in anhydrous THF at 0°C and the reaction mixture was stirred for 20 min. Then, compound 7 (970 mg, 3.54 mmol) dissolved in anhydrous THF was added dropwise within 10 min, and the mixture was stirred at room temperature for 16 h. After the reaction was complete, THF was removed in vacuo, the residue were dissolved in ethyl acetate. The resulting solution was washed with saturated aqueous NH 4 Cl solution several times and concentrated in vacuo. The crude product was purified by silica gel column chromatography (chloroform:acetone ¼100:1) to yield compound 8

Fluorescence changes upon addition of various concentrations of reducing agents
Sulfatases from Helix Pomatia (S9626) were purchased from Sigma-Aldrich. UV/vis spectra were collected on a Beckman DU-800 and fluorescence spectra were measured on a Jasco FP-6500 and SpectraMax M2 spectrophotometer.

Diels-Alder reactions and LC/MS data
A mixture of 0.25 mg/ml sulfatase and 20 μM probe 1 in 50 mM Tris buffer was incubated for 45 min to generate N-methylisoindole and then, the mixture was poured into N-phenylmaleimide solution in THF and stirred at 70°C overnight. The reaction mixture was extracted with CH 2 Cl 2 , dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was dissolved in 1 mL of methanol and subjected to liquid chromatography-mass spectrometry (LC/MS) analysis (Agilent technologies 1260 infinity) (Fig. 2) to monitor the formation of Diels-Alder adducts.
The same procedures were carried out in 1 mM GSH-containing buffer or 1 mM TCEP-containing buffer.

Determination of kinetic parameters
To measure kinetic parameters, 0.25 mg/mL sulfatase and various concentrations of probe 1 (50, 20, 10, 5, 2.4, 1.2 μM) in 50 mM Tris buffers with and without 1 mM reducing agents (pH 7.4, 37°C) were used. The rate of fluorescence enhancement at 415 nm when excited at 325 nm was measured to determine the kinetic values (K m and V max ) which were calculated by nonlinear fitting of the Michaelis-Menten equation using SigmaPlot 8.0 (Systat Software Inc.) (Fig. 3). The rate of decrease in fluorescence intensity at 415 nm was measured to determine the IC 50 of EMATE. The IC 50 values were calculated by fitting with the Hill equation using Sigmaplot 8.0 (Fig. 4).