Chiralcel od h column

The products were separated, at first, by NP-HPLC on two successively connected Separon SIX columns (5 µm; 3.2 × 150 mm; Tessek, Prague, Czech Republic) under isocratic conditions using the solvent mixture hexane/propan-2-ol/acetic acid 98.4:1.5:0.1 (by volume), at a flow rate of 0.4 mL/min. Peaks of separate products were collected and then subjected to CP-HPLC analysis on a Chiralcel OD-H column (5 µm; 4.6 × 250 mm; Daicel Chemical Industries, France) under isocratic conditions using the solvent mixture hexane/propan-2-ol 97:3 (by volume) at a flow rate of 0.4 mL/min. UV spectra of compounds being purified by HPLC were recorded on line using an RSD 2140 diode array detector and Wavescan software (LKB, Bromma, Sweden).

The PUFA substrates LA, AA and EPA were purchased from TCI chemicals (Tokyo, Japan). DHA was obtained from Thraustochytrid microalgae (Schizochytrium sp. SH103)^{14 (link)}. The HFA standards 17S-HDHA and resolvin D1-5 were obtained from Cayman Chemical (Ann Arbor, MI, USA). The pfu DNA polymerase premix was purchased from Bioneer Inc. (Daejeon, Korea). Restriction enzymes were supplied by New England Biolabs (Beverly, MA, USA). The epoxide hydrolase (EH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The E. coli DH5α strain used for gene cloning was purchased from Real Biotech Corporation (Banqiao, Taiwan). The plasmid pET-28a and E. coli BL21 (DE3) were supplied by Novagen (Madison, WI, USA). HiTrap Talon crude (5 mL) and Superdex 200 pg (16/600) were purchased from GE Healthcare (Madison, WI, USA). The SUPELCOSIL LC-DIOL column (25 cm × 3 mm, 5 μm) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The HECTOR-M C18 column (25 cm × 4.6 mm, 5 μm) was purchased from RS Tech (Cheongju, Korea). The CHIRALPAK IB column (25 cm × 4.6 mm, 5 μm) and CHIRALCEL OD-H column (25 cm × 4.6 mm, 5 μm) were obtained from Daicel (Tokyo, Japan). Diaion HP20 resin was obtained from Mitsubishi Chemical (Tokyo, Japan). All solvents for high-performance liquid chromatography (HPLC) analysis were from DaeJung Chemical (Siheung, Korea). All reagents used in this study were extra pure grade.

All reactions were performed in a Schlenk system under an argon atmosphere unless otherwise indicated. All commercial reagents were used directly, whereas solvents were purified following the standard strategies before use. Polarimetric measurements were taken on a Perkin–Elmer PE-341 polarimeter. Enantiomeric excesses were determined by an Agilent 1200 HPLC system with a Daicel Chiralcel OD-H column with the eluents of n-hexane and isopropanol. ¹H and ¹³C NMR spectra were recorded on a Bruker DP-X500 MHz spectrometer. Chemical shifts were reported in ppm relative to tetramethylsilane for ¹H NMR and CDCl₃ (77.16 ppm) for ¹³C NMR. High resolution mass spectra were collected on Waters LCT Premier™ with an ESI mass spectrometer. Low-resolution mass spectra were obtained from an Exactive GC-MS (EI).

High performance liquid chromatography (HPLC) experiments were carried out on an Agilent 1200 high-performance liquid chromatograph (Agilent Technology, Santa Clara, CA, USA) equipped with a G1322A degasser, a G1310A isocratic pump, a G1314B UV detector set at 225 nm, a G1328B manual injector of 20 μL and an Agilent 1200 chemical analytical workstation. Chromatographic separation was conducted at 25 °C, and the flow rate was maintained at 1.0 mL/min. Chiral analysis was conducted according to the enantioselective HPLC method CIPAC/4907 provided from Bayer, using a Chiralcel OD-H column (Daicel Chemical Industries Ltd. Japan, 250 × 4.6 mm i.d., 5 μm particle size), protected with a guard column of the same phase. A mixture of n-hexane/isopropanol (95:5 v/v) was used as eluent, whereas the samples were dissolved in n-hexane [39 (link)].

The chromatography system consisted of SpectraSystem (Thermo Electron S.A., Waltham, MA, USA) P1000-010XR2 isocratic pump and a dual wavelength SpectraSystem UV2000 detector. T data acquisition system was performed with an IBM PC/computer using Azur 3.0 (Datalys, Saint-Martin d’Héres, France) as chromatography sofware. Chromatography was performed on the Chiralcel OD-H column (250 × 4.6 mm, Daicel Chemical Industries Ltd, Tokyo, Japan) packed with 5 µm silica gel coated by cellulose tris(3,5-dimethylphenylcarbamate). A Rheodyne 9125 injector with a 20 μL sample loop was used. The mobile phases used were: (A) Acetonitrile, 100; (B) acetonitrile/diethylamine, 100:0.1 (v/v); (C) ethanol/diethylamine, 100:0.1 (v/v); (D) methanol/diethylamine, 100:0.1 (v/v); (E) n-hexane/ethanol/diethylamine, 60:40:0.1 (v/v/v); (F) n-hexane/methanol/ethanol/diethylamine, 75:15:10:0.1 (v/v/v/v). Solvents were of HPLC quality (Carlo Erba, Val de Reuil, France). The flow rate was 1.1 mL/min and the injection volume was 20 μL. The detection wavelength was 250 nm. The column temperature was at 25–30 °C. The sample concentration was 10 μg/mL in mobile phase. This method was used to separate the enantiomers of compound 8g.