Geduran si 60

The phytocompound, yatein, was isolated from C. formosana leaves extracts. In brief, C. formosana leaves were extracted using methanol at room temperature (RT) for one week (twice) to obtain a methanolic extract. The dried samples were further divided to n-hexane, ethyl acetate (EtOAc), n-butanol, and H₂O fractions using liquid–liquid partition. The n-hexane fraction was further fractionated into ten subfractions using normal phase column chromatography (Geduran Si-60, Merck, Darmstadt, Germany). Yatein was isolated and purified from the subfraction 4 by semipreparative high-performance liquid chromatography using a PU-2080 pump (Jasco, Tokyo, Japan) equipped with an RI-2031 detector (Jasco) and a 5 μm Luna silica column (250 mm × 10.0 mm internal diameter; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 30% of EtOAc and 70% of n-hexane (v/v), and the flow rate was 4 mL/min. The retention time of yatein in HPLC analysis was 18.0 min. The purity and the structure elucidation of yatein was conducted by ¹H and ¹³C NMR, and all spectrum data were consistent with literature [34 (link)].

Reactions were carried out in oven-dried glassware under an atmosphere of argon unless otherwise indicated. Thin-layer chromatography (TLC) was conducted on aluminum plates coated with silica gel (60 F254, Merck, Merck Life Science S.L.U., Madrid, Spain). Column chromatography was performed using silica gel (Geduran Si 60 from Merck (Merck Life Science S.L.U., Madrid, Spain), particle size 0.040–0.063 mm) as a stationary phase.

Sephadex LH-20 (Merck KGaA, Darmstadt, Germany), silica gel 60 (Merck KGaA) and Geduran Si 60 (Merck KGaA) were used for column chromatography. TLC plates (Silica Kiesel 60 F254) were from Merck KGaA. Jasco V-530 ultraviolet spectrophotometer (Jasco International Co., Ltd, Tokyo, Japan) was used to measure UV spectra. IR spectra were obtained on an FT-IR-4100 Jasco spectrophotometer (Jasco). Optical rotations were achieved by a Jasco P-2000 digital polarimeter (Jasco). NMR spectra were obtained by JEOL JNM ECS 400 MHz. Electrospray ionization mass spectrometry (ESIMS) data were collected on a Waters micromass ZQ mass spectrometer (Waters Corporation, Milford, MA, USA). High-resolution ESIMS data was accomplished by a Bruker APEX II spectrometer (FT-ICR/MS, FTMS) (Bruker Daltonics Inc., Billerica, MA, USA). Dulbecco’s modified Eagle’s medium (high glucose) powder (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), p-nitrophenyl-N-acetyl-d-glucosaminide (p-NAG), penicillin and streptomycin, dexamethasone, calcium ionophore A23187, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT, USA). Mouse anti-DNP IgE antibody was a generous gift from Dr. Daniel H. Conrad (Virginia Commonwealth University, Richmond, VA, USA).

The
structural characterization of compounds was conducted by monodimensional
(¹H and ¹³C), bidimensional (¹H–¹H COSY, ¹H–¹³C HSQC, and ¹H–¹³C HMBC), and NOE NMR experiments, along
with mass spectrometry, specific rotation measurement, and Fourier-transform
infrared spectroscopy (FTIR). NMR spectra were recorded on Agilent
spectrometers at 500 MHz using CDCl₃ (Merck) as solvent
and using its residual peak as internal reference (δ 7.26 ppm
in ¹H and δ 77.0 ppm in ¹³C NMR). Exact
masses were obtained by ultraperformance liquid chromatography coupled
with quadrupole time-of-flight mass spectrometry operating in electrospray
ionization mode on a Waters SYNAPT G2 high-resolution mass spectrometer.
Mass spectra were recorded in the positive-ion mode in the range m/z 100–2000 Da, with a mass resolution
of 20,000 and an acceleration voltage of 0.7 kV. Optical rotation
values were measured in CHCl₃ on a JASCO P-2000 polarimeter.
FTIR spectra were obtained on a PerkinElmer Spectrum Two IR spectrophotometer.
Major absorptions are given as wavenumbers in cm^–1.
The purification of compounds was performed by column chromatography
on silica gel (Merck, Geduran Si 60, 0.063–0.200 mm).

External standards were obtained after purification of crude extracts from sample A by flash chromatography over silica gel (Geduran^® Si 60, Merck), with an elution gradient of methanol from 3% to 15% in chloroform [29 (link)]. Maximal absorbance wavelengths were 250 nm and 224 nm for Compounds 1 and 2, respectively, whereas Compound 3 absorbed up to 220 nm. The purity of the compounds was determined by HPLC with an Altima RP C18 column and gradient system containing water as solvent A and MeOH as solvent B, using solvent B from 20% to 100% over 45 min, at 1 mL·min⁻¹. Using UV and ELSD detection and washing the column for 10 min with MeOH, purity of each compound has been estimated to be superior to 99%. Further identification by NMR spectroscopy (¹H, ¹³C, 1D, 2D) and LC-ESI-MS ([M + H]⁺, [M − H]⁻) were necessary to confirm the structures, and the results are in agreement with the literature and listed in the Results and Discussion section (Section 3.4). Semi-synthetic compounds from 4 to 7 (Figure 4) were obtained by treatment of 1 according to the procedure described in the literature [30 (link)] (Scheme 1).
Compound 7 came from the standard isomerization of Compound 5 and the semi-synthetic Compounds 8 and 9 were obtained after treatment of 1 with MeONa in MeOH [31 (link)] at room temperature (Scheme 2) according to the mechanism proposed in Scheme 3.

All synthetic procedures were performed under an inert atmosphere of nitrogen with the use of standard Schlenk or glovebox techniques. All chemicals were purchased commercially (Table S1 in the Supporting Information) and used without further purification unless otherwise noted. Solvents were purified under nitrogen atmosphere via distillation from CaH₂ or sodium/benzophenone ketyl radical. Some copper salts [49 , 50 ] and hybrid guanidine ligand TMGbenza [40 (link)] were synthesized according to literature procedures. Triethylamine was purified by distillation from CaH₂. Molecular sieves (3 Å, AppliChem) were flame-dried prior to use. Thin-layer chromatography sheets were purchased from MACHEREY–NAGEL (SiO₂, layer thickness 0.20 mm, fluorescent indicator). Column chromatography was performed on Geduran Si 60 (40–63 µm, Merck).