6230 tof lc ms system

The supernatant extract was applied to a reverse phase silica gel Polygoprep 100–50 C18 VFC (vacuum flash chromatography) system, using stepwise gradient elution from H₂O to MeOH. The active fraction (2.72 g) was eluted with H₂O-MeOH 1:9 and was subjected to preparative reversed-phase HPLC equipped with a Symmetry C18 column (19 × 150 mm, 7 μ) and using a linear gradient of H₂O/CH₃CN from 5% to 100% of CH₃CN in 60 min at a flow rate of 15 mL/min. The active fractions were further purified by a semi-preparative HPLC on a Symmetry C18 column (7.8 × 150 mm, 7 μ) using isocratic elution with H₂O/CH₃CN 55:45 at a flow rate of 3 mL/min to yield 80 mg of the pure compound 1, 94 mg of compound 2 and 44 mg of compound 3. NMR spectra were recorded on a Varian “Unity 500” spectrometer at 500/125 MHz (¹H/¹³C) and on a Varian Unity 400 spectrometer at 400/100 MHz (¹H/¹³C). Chemical shifts were reported in ppm using residual CDCl₃ (δ 7.26 ppm for ¹H and 77.0 ppm for ¹³C) as an internal reference. Two-dimensional experiments COSY, TOCSY, HSQC, and HMBC were performed using standard pulse sequences. Data were processed using MestReNova 14.0.1 software. (+)-ESIMS spectra obtained on an Agilent 1100 Series LC/MSD spectrometer. High-Resolution Mass Spectroscopy (HRMS) was performed on an Agilent 6230 TOF LC/MS system using the ESIMS technique.

All chemicals were ordered from Sigma Aldrich (Steinheim, Germany) and all solvents from Acros (Geel, Belgium). Prozomix (Haltwhistle, UK) provided the commercial TA kit. GDH was provided from DSM (7 U/mg), FDH from evocatal (Evo 1.1.230, 1.4 U/mg). TLC analysis was performed on pre‐coated silica gel 60F₂₅₄ and compounds were visualized by UV, ninhydrin, ceric ammonium molybdate or anisaldehyde staining. Flash column chromatography was carried out using Merck silica gel 60 (particle size 0.040 –0.063 mm). Optical rotation was measured at 20 °C on an Anton Paar MCP 5100 Polarimeter against the sodium D‐line. All NMR spectra were recorded using a Bruker NMR at 300 or 500 (1H) and 75 (13 C) MHz in CDCl₃ or d₄‐MeOH using the residual solvent peak as reference. Chemical shifts are reported in ppm and coupling constants (J) are given in Hz. High resolution MS measurements (HR‐MS) were carried out on an Agilent 6230 TOF LC/MS system in ESI⁺ mode. Purified recombinant l‐alanine dehydrogenase was prepared as described previously (11 U/mg).
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Optical rotations were determined using a Jasco P-1020 polarimeter (Oklahoma City, OK, USA). UV spectra were performed using an Agilent 8453 UV-VIS spectrometer (Santa Clara, CA, USA). IR spectra were obtained with a Perkin-Elmer Spectrum 100 FT-IR spectrometer (Waltham, MA, USA) with ATR sampling. NMR spectra were recorded on a Varian “Unity 500” spectrometer (Palo Alto, CA, USA) at 500/125 MHz (¹H/¹³C). Chemical shifts were reported in ppm using residual CDCl₃ (δ 7.26 ppm for ¹H and 77.0 ppm for ¹³C) and CD₃CN (δ 1.96 ppm for ¹H, and 118.3 and 1.8 ppm for ¹³C) as an internal reference. (+)-ESIMS were recorded using an Agilent 1100 Series LC/MSD spectrometer. High-resolution mass spectroscopy (HRMS) was performed with an Agilent 6230 TOF LC/MS system using the ESI-MS technique.