Precoated silica gel 60 f254 plates

Precoated silica gel 60 F₂₅₄ plates (Merck) for thin layer chromatography (TLC) were used. TLC spots were visualized under UV (254 nm) and sprayed with convenient spray reagent. Column chromatography (CC) was carried out using silica gel (Si) 60 mesh of 35–60 and 60–120 (E. Merck, Darmstadt, Germany). Electrothermal digital apparatus and Gallenkamp electrothermal melting point apparatus were used. UV analyses for pure samples were recorded, separately, in methanol solutions on UV spectra and were measured using a Shimadzu UV 240 spectrophotometer (P/N 240–58000). Electrospray ionization mass spectrometry (ESIMS) measurement was run on a double focusing Mat 95 sector field mass spectrometer (Finnigan, Bremen, Germany). Also, ESIMS and high-resolution electrospray ionization mass spectrometry (HRESIMS) were measured with Orbital XL (Thermo Fisher, San Jose, CA, USA) and electron ionization mass spectrometry (EIMS) with a Finnigan MAT 8500 was used. The NMR spectra were recorded at 300 (¹H) and 75 (¹³C) MHz, on a Varian Mercury 300 (Varian, UK) NMR spectrometer and δ-values are reported as ppm relative to TMS in the convenient solvent.

All reagents were of the highest purity grade from commercial suppliers: Merck (St Louis, MO, USA) or VWR (Radnor, PA, USA).
Laccase from Trametes versicolor was from Sigma-Aldrich. The enzyme was used based on their respective activities evaluated according to literature assay based on the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as model substrate. [32 (link)]
Biotransformations were performed in a G24 Environmental Incubator New Brunswick Scientific Shaker (Edison, USA) or in a Thermomixer Comfort (Eppendorf, DE).
Reactions were monitored by thin-layer chromatography (TLC) (precoated silica gel 60 F254 plates (Merck, DE)); development with UV lamp, Komarovsky reagent (1 mL 50% ethanolic H₂SO₄ with 10 mL 2% methanolic 4-hydroxybenzaldehyde), a 20% solution of H₂SO₄ in ethanol or a molybdate reagent ((NH₄)₆Mo₇O₂₄·₄H₂O, 42 g; Ce(SO₄)₂, 2 g; H₂SO₄ conc., 62 mL; made up to 1 L of deionized water). Flash chromatography: silica gel 60 (70–230 mesh, Merck, DE).
NMR spectra were recorded with a Bruker AC spectrometer (400 or 500 MHz) in [D₄]MeOH, [D₆]DMSO or [D₁]CHCl₃. Mass spectra were recorded with a Bruker Esquire 3000 Plus spectrometer.
High-resolution mass spectra (HRMS) were conducted on FT-Orbitrap mass spectrometer in positive electrospray ionization (ESI).

The ¹H and ¹³C NMR spectra were obtained in DMSO-d₆ on a Bruker Avance III HD 400 spectrometer at 400 and 100 MHz, respectively, using TMS as internal standard. The chemical shift values (δ) are reported in ppm units, and the coupling constants (J) are in Hertz (Hz). UV spectra were recorded on a Jasco V-530 spectrophotometer (Jasco, Tokyo, Japan). HRESIMS was performed on a Waters Synapt G2 QTOF mass spectrometer (Waters, Milford, MA, USA). TLC analyses were carried out on precoated silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). The developing system used was CHCl₃:MeOH solution, and visualization of TLC plates was performed under UV light (254 and 365 nm) or performed with anisaldehyde-H₂SO₄ spray reagent. The adsorbent used for open column chromatography was silica gel 60–230 mesh. HPLC was performed on a Waters 1525 Binary HPLC pump (Waters Corp., Milford, MA, USA) connected to a Waters 996 Photodiode Array detector using Zorbax RX-C18 (21.2 × 250 mm) and Waters Symmetry C18 (4.6 × 150 mm) columns with HPLC-grade methanol and water.

Column chromatography was performed using a Sephadex LH-20 column (10–25 μm; GE Healthcare Bio-Science AB, Uppsala, Sweden) or MCI CHP 20P column (75–150 μm; Mitsubishi Chemical, Tokyo, Japan). Daisogel (40–60 μm; Daiso, Osaka, Japan) and Toyopearl HW-40F (30–60 μm; Tosoh Corp., Tokyo, Japan) were used in the stationary phase in a middle-pressure liquid chromatography (MPLC) system (Gilson, Seoul, Korea). For monitoring of each fraction, thin layer chromatography (TLC) was carried out using pre-coated silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany) that were developed with chloroform, methanol and water (6:4:1 volume ratio), or benzene, ethylformate and formic acid (1:7:1 or 1:7:2 volume ratio). The spots were detected using ultraviolet radiation (254 nm) and by spraying with a FeCl₃ solution and 10% H₂SO₄ followed by heating. Structural identification was by nuclear magnetic resonance (NMR; Varian, Palo Alto, CA, USA), high resolution fast atom bombardment mass spectrum (HRFAB-MS; JEOL, Tokyo, Japan) and circular dichroism (CD; Jasco, Tokyo, Japan).