Semi preparative hplc

A Bruker DRX 400 or 500 MHz spectrometer (Bruker-Biospin, Karlsruhe, Germany) was used for the analysis of NMR signals using methanol-d₄ as a solvent. The UV and IR spectra were obtained using Jasco UV-550 (JASCO, Tokyo, Japan) and Perkin–Elmer model LE599 (Perkin–Elmer, Waltham, MA, USA) spectrometers, respectively. ESIMS and HRESI-TOF-MS data were obtained with LCQ Fleet and maXis 4G mass spectrometers (Bruker Daltonics, Bremen, Germany), respectively. Semi-preparative HPLC (Waters, Milford, MA, USA) was performed using a Waters 515 HPLC pump with a 996-photodiode array detector, and Waters Empower software using a Gemini-NX ODS-column (150 × 10.0 mm and 150 × 21.2 mm). Column chromatography procedures were performed using silica gel (200–400 mesh, Fisher Scientific, Waltham, MA, USA) and Sephadex LH-20 (25–100 µm, Pharmacia Fine Chemical Industries Co., Uppsala, Sweden). Thin-layer chromatography (TLC) was performed using aluminum plates precoated with Kieselgel 60 F₂₅₄ (0.25 mm, Merck, Darmstadt, Germany).

Large scale fermentation (50 L) and the extraction of XY-FW47 were obtained as the described method of small scale. The EA crude extract of XY-FW47 was separated by reverse phase C18 chromatography with water and methanol solvent mixtures of H₂O–MeOH (7:3), H₂O–MeOH (5:5), H₂O–MeOH (3:7), H₂O–MeOH (1:9), and 100% MeOH. The fractions of H₂O–MeOH (3:7, v/v) was evaporated and labeled Fraction (Fr.) 70%. The fraction was subjected to Sephadex LH-20 by mixtures of chloroform/methanol (1:1) yielding 16 fractions and marked Fr.70%-1 to Fr.70%-16. Then, Fr.70%-10 to Fr.70%-14 were purified by the elution of MeCN–H₂O (70:30, (v/v), flow rate: 2 mL/min) through the semi-preparative HPLC (Waters, Parsippany, NJ, USA) using analytical and semi-preparative reverse-phase phenomenex biphenyl columns (5 μm, 250 × 4.6 mm and 5 μm, 250 × 10 mm in size), and finally afforded pure Compounds 1 (2.6 mg), 2 (12 mg), 3 (7 mg) and 4 (1 mg) at a retention time of 24.0 min, 25.0 min, 26.0 min and 27.4 min, respectively. The further ¹H, ¹³C and 2D NMR spectral data were determined on a Bruker DRX 600 MHz NMR Spectrometers.
4,5-Dihydro-17-O-demethylgeldanamycin (1): yellow amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +42.0° (c 0.18, CHCl₃); UV (MeOH) λ_max 305 nm, HR-ESI-MS m/z 547.2636 [M − H]⁻ (calcs for C₂₈H₄₀N₂O₉ 547.2656); ¹H and ¹³C NMR data, see Table 4.

The NMR spectra were obtained with a Bruker 500 spectrometer ((Bruker, Bremen, Germany)) operating at 500 MHz for ¹H and 125 MHz for ¹³C, respectively. Chemical shifts were reported in parts per million on the δ scale with TMS as the internal standard. The optical rotations were measured on a JASCO P-1020 Optical Rotation Apparatus (Jasco, Tokyo, Japan). ESI-HRMS spectra were measured on an Agilent 1100 LC/MSD TOF mass spectrometer (Agilent, California, USA). The CD spectrum were obtained on a JASCO810 spectropolarimeter (Jasco, Tokyo, Japan). Compounds 1–7 were purified by semi preparative HPLC (Waters, Milford, USA) using a Waters 600 liquid chromatograph with a Alltech 2000Esc ELSD detector (110 °C, flow rate of the condensed air, 3.1 mL min⁻¹) and with a Phenomenex HPLC column (4 μm, 4.6 × 250 mm, Phenomenex Hydro-RP 80R).