Sodium 3 trimethylsilyl propionate 2 2 3 3 d4 tsp

Nuclear magnetic resonance (NMR) data were acquired using a Bruker AVANCE III HD 400 MHz spectrometer (Bruker Corp., Billerica, MA, USA). A (S)-5-oxotetrahydrofuran-2-carboxylic acid sample was dissolved in CDCl₃ with 0.03% v/v tetramethylsilane (Sigma-Aldrich) and transferred to 5 mm NMR standard tubes. (S)-2HG was dissolved in double-distilled water. The (S)-2HG aqueous solution was analyzed inside a 5 mm NMR tube using a coaxial system (Wilmad^® coaxial insert, Wilmad-LabGlass, Vineland, NJ, USA), filled with D₂O with 0.75% sodium 3-(trimethylsilyl)propionate-2,2,3,3-d₄(TSP) (Sigma-Aldrich).
¹H and ¹³C-NMR spectra of (S)-5-oxotetrahydrofuran-2-carboxylic acid were obtained using standard acquisition parameters. The (S)-2HG proton spectrum was acquired using the noesypr1d pulse sequence for water signal suppression. Compounds were identified by comparing the obtained data with previously reported spectra (Bal & Gryff-Keller, 2002 (link)). DEPT-135, HMBC, and HSQC spectra were also acquired. Relative (S)-2HG purity was determined by the integration of all the signals of the ¹H-NMR spectrum, with exception of the ¹³C couplings and the TSP signal using a method similar to published chromatographic procedures (Pauli, Jaki & Lankin, 2007 (link)). The obtained data were processed and analyzed using Bruker TopSpin 3.2 software (Bruker Corp.) and used to confirm (S)-2HG synthesis.

Acetonitrile extraction was employed to quench cell metabolism and to extract low molecular weight compounds from A375 melanoma cells quantitatively [83 (link),88 (link)]. Following removal of acetonitrile via vacuum concentration, dried extracts were resuspended in 550 μL of D₂O (Sigma-Aldrich) containing 0.005% sodium 3-(trimethylsilyl)-propionate-2,2,3,3-d4 (TSP) (Sigma-Aldrich) used as both chemical shift reference and internal standard for metabolite quantification.
Information on the concentration of metabolites in individual samples was derived from volumes of corresponding signals in 1D 1H NMR spectrum. The assignment of signals in the NMR spectra of individual samples to a metabolite was achieved via a comparison of a sample spectrum with spectra of pure metabolites (Sigma-Aldrich). The 1D 1H spectra were measured at 700 MHz using a Bruker Avance III NMR spectrometer (Bruker, Billerica, MA, USA) equipped with a triple resonance room temperature probe using the zgpr pulse sequence (standard Bruker pulse program library). All spectra were acquired at 20 °C and processed using TopSpin 3.2 (Bruker). To make the comparison of metabolite concentration profiles among various samples possible, the signal intensities in individual samples were normalized to total protein concentration.

NaN₃, NaH₂PO₄·2H₂O, and K₂HPO₄·2H₂O (all in analytical grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). D₂O (99.9% in D) containing sodium 3-(trimethylsilyl)propionate-2,2,3,3-d₄ (TSP) as an internal standard for chemical shift reference was provided by Sigma-Aldrich (Sigma Chemical Corp., St. Louis, MO, USA). A buffer system containing 0.2% NaN₃ was prepared in D₂O consisting of TSP and 1.5 M K₂HPO₄/NaH₂PO₄ (molar ratio of 4 : 1) at pH 7.4 in order to prevent pH effect on chemical shift at different concentrations.