Methanol d4

An Atago AP-300 digital polarimeter with a 1 dm microcell and a sodium lamp (589 nm) was used to obtain optical rotations. A Bruker DRX-600 NMR spectrometer (Bruker BioSpin GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI at 300 K was employed to run NMR data. Data processing was carried out with Topspin 3.2 software. All 2D NMR spectra were acquired in methanol-d₄ (99.95%, Sigma-Aldrich, Milano, Italy), and standard pulse sequences and phase cycling were used for COSY, HSQC, HMBC, 1D-TOCSY, and ROESY spectra. HRESIMS data were obtained in the positive ion mode on a Q Exactive Plus mass spectrometer, an Orbitrap-based FT-MS system, equipped with an ESI source (Thermo Fischer Scientific Inc., Bremen, Germany). Column chromatography was performed over silica gel (70–220 mesh, Merck). A Shimadzu LC-8A series pumping system equipped with a Shimadzu RID-10A refractive index detector and Shimadzu injector (Shimadzu Corporation, Kyoto, Japan) on a Waters XTerra Semiprep MS C₁₈ column (300 mm × 7.8 mm i.d.) and a mobile phase consisting of a MeOH-H₂O mixture at a flow rate of 2.0 mL/min were employed to purify the molecules. TLC was performed on silica gel 60 F₂₅₄ (0.20 mm thickness) plates (Merck, Darmstadt, Germany) as a spray reagent, and Ce(SO₄)₂/H₂SO₄ (Sigma-Aldrich, Milano, Italy) was used [60 (link)].

p-Nitrophenyl phosphate (pNPP), dimethyl sulfoxide (DMSO), sodium phosphate monobasic dihydrate, sodium phosphate dibasic, sodium hydroxide, sodium azide, tris-(hydroxymethyl)-aminomethane (Tris), bis-(2-hydroxyethyl)-imino-tris-(hydroxymethylmethane) (Bis-Tris), N,N,N′,N′-ethylenediaminetetraacetate (EDTA), dithiothreitol (DTT), methanol-d₄, and HPLC grade acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA), recombinant human PTP1B (BML-SE332-0050, EC 3.1.3.48) was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA), and formic acid was purchased from Merck (Darmstadt, Germany). Water was purified by deionization and filtration through a 0.22 μm membrane (Millipore, Billerica, MA, USA).

Methanol-d₄, tetramethylsilane, quercetin, linoleic acid, soybean 15-lipoxygenase (15-LO), hypoxanthine, xanthine oxidase (XO) from bovine milk, acarbose, 22-S-hydroxycholesterol (22-SHC), 4-nitrophenyl α-d-glucopyranoside (PNP-G), 2-chloro-4-nitrophenyl-α-d-maltotrioside (CNPG3), quercetin and gallic acid were from Sigma-Aldrich (St. Louis, MO, USA), Folin Ciocalteu reagent was from Merck (Darmstadt, Germany). Dulbecco‘s modified Eagle’s medium (DMEM-Glutamax™, 5.5 mM), DMEM, fetal bovine serum, Ultroser G, penicillin–streptomycin–amphotericin B and trypsin-EDTA were obtained from Gibco Life Technologies (Paisley, UK). d-[¹⁴C(U)]glucose (1 μCi/mL, 100 μM) was purchased from ARC (American Radiolabeled Chemicals, St. Louis, MO, USA). Corning CellBIND tissue culture plates were obtained from Corning Life-Sciences (Amsterdam, The Netherlands). The protein assay reagent was obtained from BioRad (Copenhagen, Denmark). All other reagents were of the highest purity available. All solvents were of analytical grade and water was purified by a MilliQ system (Millipore, Bedford, MA, USA).

All the high purity organic solvents were purchased from Merk. Certified reference materials of ethyl paraben (GBW(E) 100064) with a certificate value of 99.7% as an internal standard for qNMR measurement and chloramphenicol (GBW(E)060907) with a certificate value of 99.8% were supplied by National Institute of Metrology (NIM), China. Methanol-D₄ was obtained from Sigma-Aldrich. The powder of chloramphenicol material with a labeled purity of 99.8% was purchased from Tokyo Chemical Industry (TCI) Co., Ltd., Japan.

Ethanol (99.5%) used for the extraction of fomentariol was obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany), while dichloromethane (99.8%) was obtained from Honeywell Riedel-de Haën (Seelze, Germany). Acetonitrile (99.8%) and formic acid (98%) were also purchased from Sigma-Aldrich Chemie GmbH, as well as mEthanol-d4 (99.9%). Toluene (99.5%) obtained from POCH (Gliwice, Poland) was used for quantitative NMR determination. Purified water was obtained from a Simplicity 185 purification system (Millipore, Billerica, MA, USA). Before use, the sample was filtered through 0.22 µm nylon membranes (Agilent Technologies, Santa Clara, USA). All reagents used were of analytical grade except water and Acetonitrile, which were of HPLC grade.

Optical rotation of compound 11 was measured on an Autopol IV (Rudolph Research Analytical, Hackettstown, NJ, USA) polarimeter. IR measurements were carried out on a Bruker IFS-48 spectrometer. UV spectra were obtained on a Beckman DU 670 spectrometer. NMR experiments were acquired in methanol-d₄ (99.95%, Sigma-Aldrich, Milan, Italy) on a Bruker DRX-600 spectrometer (Bruker BioSpin GmBH, Rheinstetten, Germany), equipped with a Bruker 5 mm TCI CryoProbe at 300 K. Data processing was carried out with Topspin 3.2 software (Bruker BioSpin, Rheinstetten, Germany). The ROESY spectra were acquired with t_mix = 400 ms. Size exclusion chromatography was performed using Sephadex LH-20 (GE Healthcare, Sigma Aldrich, Milan, Italy).