Autopol 4 automatic

Optical rotations were recorded at ambient temperature using a Rudolph Research Analytical Autopol IV automatic polarimeter. UV spectra were obtained in MeOH using a Hewlett-Packard 8453 UV/vis spectrometer, and ECD data were collected using an Olis Cary-17 spectrophotometer (1 cm path length cell). NMR spectra were acquired on a Bruker 400 MHz NMR spectrometer at 400 (¹H) and 100 MHz (¹³C) and a Bruker 500 MHz NMR spectrometer at 500 (¹H) and 125 MHz (¹³C), using the residual solvent as an internal standard. Multiplicity determinations (DEPT) and 2D-NMR spectra (HMQC, HMBC, NOESY) were obtained using standard Bruker pulse programs. HRESIMS data were acquired by direct injection using a Bruker Bioapex-FTMS with electrospray ionization (ESI). TLC was caonducted on precoated silica gel 60F254, 0.25 mm and reversed phase (RP) C₁₈ F254 silica gel (EMD Chemicals Inc., Darmstadt, Germany). Column chromatography was carried out on silica gel G60 (60–120 mesh, Merck, Darmstadt, Germany) and sephadex LH-20 (Mitsubishi Kagaku, Tokyo, Japan). Solid phase extraction (SPE) cartridges C₁₈ (Supelco Inc., Bellefonte, PA, USA) were used in the fractionation experiments. The compounds were visualized by spraying the TLC plates with 1% vanillin–H₂SO₄ spray reagent.

Optical rotation was measured on a Rudolph Research Analytical Autopol IV automatic polarimeter (MeOH, c in g/100 mL). The ECD spectrum was recorded in MeOH using an Applied Photophysics Chirascan ECD spectrometer. UV spectra were recorded using a Thermo Scientific Evolution 260 Bio spectrophotometer (Shanghai, China, designed in the USA). IR spectra were obtained on a Thermo Scientific Nicolet iS5 FT-IR spectrophotometer (Madison, WI, USA). NMR spectra were recorded on a Bruker Avance III NMR spectrometer with a CryoProbe (Germany). The proton (¹H) and carbon (¹³C) frequencies are 600.2 and 150.9 MHz, respectively. Chemical shifts (δ) in ppm were referenced using the corresponding solvent signals: δ_H at 2.05 and δ_C at 29.92 for acetone-d₆. Off-line FID processing was conducted with the Bruker TopSpin 3.2 software. HRESIMS data were obtained using a Waters SYNAPT (Milford, MA, USA) mass spectrometer. Silica gel 60, particle size 0.035−0.070 mm, supplied by Fluka, Switzerland (220−440 mesh), and silica gel, 230−400 mesh (Sorbent Technologies, GA, USA) were used for column chromatography. Analytical TLC analysis was carried out on silica gel plates (250 μm, 250 μM with UV254, Sorbent Technologies). Sephadex LH-20–100 was purchased from Sigma Chemical Company, USA. All procedures were carried out at room temperature using organic solvents of analytical grade (Sigma, USA).

Optical rotations were measured with an AUTOPOL IV automatic polarimeter (Rudolph Research Analytical). UV spectra were collected with a NanoDrop 2000C spectrophotometer (Thermo Scientific). Circular dichroism spectrum was measured with a J-815 CD spectrometer (JASCO). NMR data were recorded on a Bruker Ultra Shield 700 instrument. HRESI-MS analysis was carried on a Thermo Finnigan LTQ Orbitrap mass spectrometer. MPLC separation was conducted on Biotage Isolera One using a Biotage SNAP Cartridge HP-Sil column (25 g) column. HPLC was carried out on Varian semipreparative HPLC system using a Prostar 330 detector, and a GRACE Apollo C₁₈ column (250 mm × 4.6 mm, 5 μm) for analysis and an Alltima C₁₈ column (250 mm × 10.0 mm, 5 μm) for purification. Fermentation was carried out in a New Brunswick Scientific Innova 44 incubator shaker for small scale (50 mL in 250-mL baffled Erlenmeyer flasks) or New Brunswick BioFlo/CelliGen 115 fermentor for large scale (5 L in a 14-L vessel).