Dionex ultimate 3000 system

To monitor the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO, reversed-phase chromatography (RP HPLC) was applied using a HPLC Dionex UltiMate 3000 System equipped with UV-DAD detector. The chromatograms were recorded at 260 nm for monitoring the effluents. The Wakopak reverse-phase C18 column (4.6 mm × 150 mm; particle size = 5 µm) was used. Separations were performed using linear gradient from 0 to 50% of phase B in 25 min (A: 0.1 M ammonium acetate, pH 5.5; B: 80% AcN in water; flow rate = 1 mL/min).

Blood was collected in a heparin tube and centrifuged for 5 min at 5000 rpm. Plasma was separated from blood cells, and about 1 ml was diluted with 2 ml of 0.1 M hydrochoric acid and loaded onto a tC18 Sep-Pak cartridge, which was pre-activated by elution with 6 ml of methanol and 12 ml of water, respectively. The cartridge was washed with 3 ml water to collect the polar radioactive fraction. Thereafter, the tC18 Sep-Pak cartridge was eluted with 1 ml of methanol and 2 ml of water to collect the mixture of non-polar metabolites. This fraction was further analysed by HPLC on a Dionex Ultimate 3000 system (Dionex, Sunnyvale, CA, USA) and equipped with a 1-ml loop. As a stationary phase, a Phenonenex Gemini C18, 250 × 10 mm, 5 μm (Phenomenex, Torrance, CA, USA) was used. The mobile phase consisted of 75 % 0.1 % trifluoroacetic acid in water in acetonitrile. The eluent was collected with a fraction collector (Teledyne ISCO Foxy Jr., Lincoln, NE, USA), and the fractions were counted for radioactivity with a Wallac 1470 gamma counter (Perkin Elmer, Waltham, MA, USA).

UV spectra were recorded on a Perkin-Elmer Lambda 19 uv/vis spectrophotometer. Fluorescence spectra were recorded on a Cary Eclipse fluorimeter. ¹H and ¹³C NMR were recorded using a Varian Mercury-VX spectrometer at 400 MHz (¹H) and 100 MHz (¹³C) or a Bruker Avance III 500 at 500 MHz (¹H) and 125 MHz (¹³C). Chemical shift values are given in ppm (δ). J values are given in Hz. Analytical RP-HPLC was performed on a Dionex Ultimate 3000 system (Dionex, UK), with a VWD-3400 variable wavelength detector, and a RF-2000 fluorescence detector. Analyses were performed at 35 ± 0.1 °C on a Gemini 5 μ C18 110 A column, (150 × 4.6 mm - Phenomenex, UK), equipped with a Security Guard C18 (ODS) 4 × 3.0 mm ID guard column (Phenomenex, UK), at a flow rate of 1 mL/min. Mobile phase A was 0.1% aq. TFA, mobile phase B was 0.1% TFA in MeCN. (Gradient: 0.0–10.0 min 0–95% B, 10.0–20.0 min 95% B, 20.0–20.1 min at 95–5% B, 20.1–23.0 min 5% B). Preparative RP-HPLC was performed on a Dionex HPLC system equipped with a Phenomenex Gemini 5 μ C18 (250 × 10w mm) column at a flow rate of 2.5 mL/min. High resolution mass spectrometry was performed using a Bruker MicroTOF autospec ESI mass spectrometer.

Optical rotations were recorded on an A25700-T digital polarimeter (RUDOLPH, Hackettstown, NJ, USA). IR spectra were obtained using a Thermo Tensor Nicolet-6700 spectrometer (Thermo Optics, Inc., Billerica, MA, USA) with KBr pellets, and 1D and 2D NMR spectra were recorded on a Bruker DRX-600 instrument (600 MHz for ¹H and 150 MHz for ¹³C) with TMS as an internal standard, the deuterated solvent used to solubilize the samples were CD₃OD and CDCl₃.HR-ESI-MS was recorded on an UPLC-Q Exactive MS system (Thermo Fisher, Santa Clara, CA, USA). Silica gel (200–300 mesh (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech, Switzerland) were used for the chromatography column. Semi-preparative HPLC was performed on a DIONEX Ultimate 3000 system equipped with a diode array detector and a C18 column (250 mm × 10 mm, 5 μm, YMC Co. Ltd., Kyoto, Japan).

Column chromatography (CC) was performed using silica gel (200–300 mesh, 300–400 mesh, Qingdao Haiyang Chemical Group Co., Qingdao, China). Thin-layer chromatography was performed on silica gel GF254 (Qingdao Haiyang Chemical Group Co., Qingdao, China). MCI was purchased from Mitsubishi Chemical Group Co. (Tokyo, Japan) Semi-preparative HPLC was performed on a DIONEX Ultimate 3000 system equipped with a diode array detector and a C18 column (250 mm × 10 mm, 5 μm, YMC Co. Ltd., Kyoto, Japan). HR-EI-MS was measured on a Waters Autospec Premier 776 mass spectrometer (Waters, Milford, MA, USA). HR-ESI-MS was recorded on an Agilent G6230 TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were obtained on a Bruker DMX-500 spectrometer (Bruker, Karlsruher, Germany) using TMS as an internal reference. l-cysteine methyl ester and standard monosaccharides (d-glucose and d-xylose) used in HPLC experiments were purchased from Aladdin industrial Co. Ltd. (Shanghai, China). O-Tolyl-isothiocyanate was obtained from Sigma-Aldrich Co. Ltd (Sigma-Aldrich China, Shanghai, China). Other chemical reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. Shanghai, China.

Aliquots of RNA equivalent to 10 μg were dissolved in 40 mM ammonium acetate, pH 5.0, containing 3 mM zinc chloride. After addition of 0.25 U nuclease P1 (from Penicillium citrinum, Sigma, Taufkirchen, Germany) per μg RNA samples were incubated over night at 37°C. Thereafter, samples were made alkaline by the addition of TRIS-HCl, pH 8.3 (final concentration 15 mM), containing 1,5 mM magnesium acetate and treated with shrimp alkaline phosphatase (Sigma) and snake venom phosphatase (phosphodiesterase I, Type VI from Crotalus adamanteus, Sigma) at 0.25 U/μg RNA and 0.1 mU/μg RNA, respectively. After incubation at 37°C for 2 hours samples were centrifuged at 30.000 x g for 10 minutes and the supernatant was harvested. HPLC analysis of digests was performed on a Dionex Ultimate 3000 System using a Supelcosil LC18 reverse phase column (250 x 4.6 mm, 5 μm) protected with a C18 guard column (4 x 2 mm) both obtained from Sigma (Taufkirchen, Germany). The mobile phase was: eluent A, 5 mM ammonium acetate, pH 6.0; eluent B, 40% acetonitrile. After injection of the RNA digest separation started with 100% eluent A followed by a 66 minute gradient to 60% eluent B using a flow rate of 0.85 ml/min. Finally the column was subjected to isocratic elution with 100% eluent B for 20 minutes. Detection of the eluted nucleosides was continuously recorded by UV spectrometry at 254 nm.

Sample preparation was performed by dissolving in a mixture of water, acetonitrile, and formic acid (94.9, 5, 0.1% v/v/v). Immediately before HPLC analysis, samples were filtered through a 0.22 μm filter. The apparatus used was Dionex Ultimate 3000 System (Dionex Corporation, United States) equipped with LPG-3400SD pump, WPS-3000T(B) FC Analytical autosampler, TCC-3000SD column compartment and a DAD-3000 diode array detector variable. The chromatography column was a C18 column (Phenomenex, 250 mm × 4.6 mm, 5 μm, 110 Å). The following chromatographic conditions were applied: the mobile phase: solvent A—0.1% formic acid in water; solvent B—0.1% formic acid in acetonitrile. The program was as follows: 0 min—5% solvent B; 15 min—95% solvent B; 16 min—95% solvent B; 16.3 min—5% solvent B; 27.5 min—5% solvent B. The inject sample volume was 100 μl. The flow rate was 0.5 ml/min and the eluent was monitored at 254 nm at room temperature.