Lcq fleet ion trap mass spectrometer

Identification of β-valinyl-CoA and methyl-crotonyl-CoA was performed in a LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific). Reaction components were separated by HPLC using an Alltech Altima HP C18 column (3 μm, 100 × 2.1 mm). The CoA adducts were eluted using eluent A (water, 0.1 % formic acid) with an elution gradient of eluent B (acetonitrile, 0.08 % formic acid; 0–2 min, 0–2 %; 2–20 min, 2–15 %; 20–32 min, 15–80 %; 32–37 min, 80 %; 37–38 min, 80 0 %; 38–48 min, 0 % eluent B). The samples were analyzed in a negative ion mode, and mass spectra were collected over a scan range of m/z 500–1000. Typical retention times were 13 min for β-valinyl-CoA (m/z 864; M–H) and 28 min for methyl-crotonyl-CoA (m/z 847; M–H).

Thin-layer chromatography was carried out using pre-coated silica Q-gel 60 F254 plates (0.25 mm, Merck). Column chromatography (CC) was conducted using silica gel (Kieselgel 60, 230–400 mesh, Merck), and Sephadex LH-20 (18–111 µm, GE Healthcare AB, Stockholm, Sweden). HPLC was performed using the Varian Prostar 210 system. A YMC-Pack ODS-A column (5 μm, 250 × 20 mm i.d., YMC, Kyoto, Japan) was used for preparative HPLC analysis with an 8 mL/min flow rate. ESI-MS was performed on an LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific, Madison, WI, USA). NMR spectral data were acquired by using a Varian 500-MHz NMR spectrometer (Inova 500 Spectrometer, Varian, Palo Alto, CA, USA). Tetramethylsilane was used as an internal standard, and chemical shifts data were expressed as δ value.

All commercial materials and solvents (> 95% purity grade) were purchased from Merck KGaA (Darmstadt, Germany) and used without further purification. All reactions were carried out under a nitrogen atmosphere, unless otherwise noted. All reactions were monitored by thin layer chromatography (TLC) on precoated silica gel 60 F254; spots were visualized with UV light, or by treatment with a 1% aqueous KMnO₄ solution, or with a hydroalcoholic curcumin solution (100 mg of curcumin in a 100 mL solution of ethanol with 2 N HCl (99:1 v/v)). Products were purified by flash chromatography (FC) on silica gel 60 (230–400 mesh). Yields refer to isolated compounds estimated to be >95% pure as determined by ¹H-NMR. NMR spectra were recorded on 300 or 400 MHz Bruker spectrometers, using tetramethylsilane (TMS) as the internal standard. Supplementary materials for ¹³C-NMR, the APT pulse sequence was adopted. Chemical shifts are reported in parts per million relative to the residual solvent. Multiplicities in ¹H-NMR are reported as follows: s = singlet, d = doublet, t = triplet, m = multiplet, br s = broad singlet. High-resolution MS spectra (HR-MS) were recorded with a Thermo Fisher LCQ Fleet ion trap mass spectrometer, equipped with an ESI source. The purity of compound 6e was confirmed to be >95% by means of elemental analysis on a CHN PerkinElmer 2400 instrument.