Ltq orbitrap xl hybrid ms

Leaves were weighed and frozen in liquid nitrogen. Immediately after freezing tissue was homogenized in a paint shaker for 20 sec. Following homogenization, frozen tissue was extracted as previously described (Casteel et al., 2015 (link)). For analysis, 5 mL of each extract was analyzed using a Thermo Electron LTQ-Orbitrap XL Hybrid MS. Samples were separated on a Zorbax Extend-C18 HPLC column (Phenomenex Kinetex, 5 μm, 100 × 2.10 mm) using 0.1% formic acid in water (Solvent A) and 0.1% (v/v) formic acid in acetonitrile (Solvent B) at a flow rate of 300 μL min–1. The gradient used was 0–0.5 min, 20% B; 0.5–13.5 min, linear gradient to 100% B; 13.6–17 min, linear gradient to 30% B; 17.1–18 min, linear gradient to 20% B.

All reagents and solvents were purchased from commercial suppliers and were used directly without further purification. All reactions were carried out at room temperature unless otherwise specified. Reactions were monitored by thin-layer chromatography (TLC) on Merck F₂₅₄ silica gel 60 aluminum sheets, and spots were revealed with UV light (254 mm), potassium permanganate or Ninhydrin stains. ¹H NMR spectra were recorded on a 400 MHz Bruker Avance III HD Nanobay Spectrometer with deuterated chloroform (CDCl₃; δ = 7.24 ppm) or deuterated dimethyl sulfoxide (DMSO-d₆) containing TMS as internal standard. ¹³C NMR spectra were recorded on a Bruker Avance III HD Nanobay Spectrometer at 100 MHz. The percent purity of the inhibitors reported in this manuscript were determined by HPLC-UV using Agilent 1200 series HPLC system equipped with Phenomenex Luna2 C18 reverse phase column (C18, 4.6 mm × 150 mm, 5 μm) coupled with Agilent G1314 UV–vis detector (detection at 200, 210, 254 and 360) with solvent gradient acetonitrile/water 20 to 95% over 15 min and they are expressed as percent total OD at 254 and 360 nm. The purity of all final compounds was above 95%. HRMS spectra were recorded on Thermo Electron LTQ-Orbitrap XL Hybrid MS in ESI mode.

Many compounds tested as inhibitors or required for synthesis are commercially available and were purchased from one of the following commercial sources: Sigma Aldrich Chemical Co. (Milwaukee, WI), Fisher Scientific (Houston, TX), Eanmine LLC (Monmouth Jct, NJ), Oakwood Chemical (Estill, SC), Chem-Impex Inc (Wood Dale, IL) or Combi-Blocks (San Diego, CA). All reactions were carried out under an atmosphere of dry nitrogen. All chemicals purchased from commercial sources were used as received without further purification. Analytical thin layer chromatography (TLC) was performed on Merck TLC silica gel 60 F254 plates. TLC plates predominantly indicated a single product, following exposure under 254 nm UV light or development using a potassium permanganate stain. Flash chromatography was performed on silica gel (230–400 mesh) from Macherey Nagel. NMR spectra were recorded on Varian VNMRS 600, Inova 400 or Bruker Avance III 800 MHz instruments. Multiplicity is described by the abbreviations b = broad, s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet. Chemical shifts are given in ppm. ¹H NMR spectra were referenced to the residual solvent peak at δ = 7.26 (CDCl₃). ¹³C NMR spectra were referenced to the solvent peak at δ = 77.16 (CDCl₃). HRMS spectra were recorded on Thermo Electron LTQ-Orbitrap XL Hybrid MS in ESI.