Q extractive orbitrap mass spectrometer

The LC-MS-based metabolic analyses were carried out using a UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters) coupled to a Q Extractive™ Orbitrap Mass Spectrometer (Thermo Fisher Scientific, USA) in ESI positive (POS) and negative (NEG) ion modes. Formic acid (0.1%) and ammonium acetate (5 mM) in water were used as the mobile phase A for positive and negative ion modes, respectively. Acetonitrile was used as the mobile phase B. The injection volume was 1 µL and the flow rate was 500 µL min^− 1 through a 12 min elution gradient (0 min, 1% B; 1 min, 1% B; 8 min, 99% B; 10 min, 99% B; 10.1 min, 1% B; 12 min, 1% B). An information-dependent acquisition (IDA) mode of the mass spectrometer was used to acquire MS/MS spectra, and the acquisition software (Xcalibur 4.0.27, Thermo) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. ESI source conditions were set as follows: the spray voltages were 3.8 kV (POS) and 3.1 kV (NEG), sheath gas flow rate was 45 Arb, aux gas flow rate was 15 Arb, capillary temperature was 320 °C, full ms resolution was 70,000, MS/MS resolution was 175 00, and the collision energy was 20/40/60 eV in NCE model, respectively [43 (link)].

All reactions with moisture-sensitive reagents were carried out under an Ar atmosphere in anhydrous solvents, which were prepared using standard procedures. Commercially available reagents were used as received. Reaction monitoring was done by thin-layer chromatography (TLC) on precoated silica gel plates (Merck TLC Silica gel 60 F₂₅₄). Spots were visualized by UV light (254 nm or 366 nm) or by staining with aqueous KMnO₄, vanillin, or phosphomolybdic acid (PMA). Silica gel (40–63 μm) was used for column chromatography. Melting points were measured on a Büchi M-565 melting point apparatus and are uncorrected. Optical rotations were recorded through 5 continuous repetitions on a Jasco P-2000 polarimeter (1 dm cell) at 589 nm and concentrations close to 1.0% in chloroform or methanol. Infrared spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer. NMR spectra were measured on Bruker Avance III HD 300 and 500 instruments and calibrated using the residual non-deuterated solvent as an internal reference. High resolution mass spectrometry (HRMS) was performed on a ThermoFischer Scientific Q-Extractive (Orbitrap) mass spectrometer using electrospray ionization (ESI). Compounds 12aa–ad were synthesized in 92–96% yield according to literature.^27,28