Q tof hplc ms

All solvents and reagents were commercially available and used without further purification unless otherwise stated. NMR spectra were recorded with a Varian 400 MHz instrument at room temperature with tetramethylsilane (TMS) as an internal standard. Mass spectra were performed on an Agilent Q-TOF HPLC-MS or VG (Micromass) 70-250-S Magnetic sector mass spectrometer employing the electrospray ionization (ESI) method or electron ionization (EI) method. High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan B-FC-1000 radiation detector. All procedures including anhydrous solvents were performed with rigorously dried glassware under inert atmosphere.

The synthesis of 1 was adapted from that reported.(Riel-Mehan and Shokat, 2014 (link)) ATP-triethylammonium salt (0.1 g, 0.11 mmol) was dissolved in anhydrous DMSO (3 mL) under nitrogen. A solution of methacrylic anyhydride (0.44 mmol), anhydrous DMSO (1 mL), dioxane (1 mL), anhydrous DMF (1 mL) was added, and the mixture was stirred at room temperature under nitrogen. After 4 d, the reaction was quenched with water (5 mL) and extracted with ethyl acetate (3 × 5 mL). The aqueous layer was collected, flash frozen and lyophilized. Crude 1 was then dissolved in water and purified by preparative reverse-phase HPLC using an Agilent 1260 Infinity HPLC equipped with a PrepHT XDB-C18 column (21.2 × 150 mm; 5 μm) at a flow rate of 20 mL/min using 100 % water as a mobile phase and detection at 254 nm. Fractions were analyzed off-line using an Agilent Q-TOF HPLC-MS. 1-containing fractions were pooled and lyophilized to dryness. Purified compound 1 (0.028 g) was subsequently dissolved in D₂O, and the stock concentration was determined by quantitative NMR using a 4,4-dimethyl-4-silapentane-1-sulfonic acid capillary normalized using calcium formate. Single-use aliquots (13.3 mM) were stored at −80 °C.

All solvents and reagents were commercially available and used without further purification unless otherwise stated. 7-Methylumbeliferone and 3-phenylphenol were obtained from Sigma-Aldrich; 4-Phenoxypyridine was purchased from TCI America and used directly as a precursor for radiolabeling. NMR spectra were recorded with a Varian 400 MHz instrument at room temperature with tetramethylsilane (TMS) as an internal standard. Mass spectra were performed on a Micromass LCT Time-of-Flight mass spectrometer or an Agilent Q-TOF HPLC-MS employing the electrospray ionization (ESI) method. High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan B-FC-1000 radiation detector.