Silica gel 60 f254 plate

Chemicals were obtained from Aldrich Chemical Company (St. Louis, MO, USA) and were used without further purification unless otherwise stated. Unmodified nucleoside phosphoramidites and 5′-dimethoxytrityl-2′-propargyluridine 3′-O-(N,N-diisopropyl-2-cyanoethyl) phosphoramidite were purchased from ChemGenes Corporation (Wilmington, MA, USA). [(3,3′-Iron-1,2,1′,2′-dicarbollide)(−1)]ate cesium salt was bought from Katchem (Režn/Prague, Czech Republic). Flash chromatography was performed on silica gel 60 (230–400 mesh, Aldrich Chemical Company). R_f values refer to analytical thin layer chromatography (TLC) performed using pre-coated silica gel 60 F₂₅₄ plates purchased from Sigma-Aldrich (Steinheim, Germany) and developed in the solvent system indicated. Compounds were visualized with UV light (at λ_max = 254 nm). The yields are not optimized.

The procedures described below were used for the synthesis of compounds 1–14. The rest of substrates as well as all solvents were purchased and used without further purification. Thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC) were performed on silica gel 60 F254 plates purchased from Sigma Aldrich. Column chromatography was conducted using silica gel 60 (70–230 mesh) produced by Sigma Aldrich. Melting points were measured using a Stuart Scientific SMP30 apparatus. ¹H NMR, ¹³C NMR and ³¹P NMR data were obtained on a Varian Gemini spectrometer (400 MHz for ¹H, 125 MHz for ¹³C and 202 MHz for ³¹P NMR). Chemical shifts (δ) are reported in ppm relative to the residual peak of chloroform as a solvent (¹H 7.26 ppm and ¹³C 77.16 ppm). A total proton decoupling was applied for ¹³C NMR spectra. For ¹H NMR description, the following symbols are used: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sext (sextet), sept (septet), m (multiplet); and the coupling constant values (J) are determined in Hertz. MS spectra were measured on matrix-assisted laser desorption/ionization-time on flight mass spectrometry (MALDI-TOF MS, Biflex III Bruker).

The fraction containing the active antibacterial compounds was purified and separated using silica gel 60 F254 plates (1.5 mm thickness; Sigma-Aldrich, MA, USA). A mobile phase composed of dichloromethane:ethyl acetate:hexane (5:20:75) was used for the chromatographic separation. Fractions exhibiting antibacterial activity were collected by scraping with a scraper and extracted with ethyl acetate. The resulting solution was filtered through 0.45 filter (Labfil, China). The purified samples were subjected to vacuum desiccation to eliminate residual moisture. The sample was subsequently dissolved in acetone and transferred to an NMR sample tube for further structural analysis using NMR spectroscopy.

NMR spectra were recorded using Avance DRX-400 and DPX-200 spectrometers (Bruker, Milan, Italy) operating at frequencies of 400 MHz (¹H) and 100 MHz (¹³C) and 200 MHz (¹H) and 50 MHz (¹³C), respectively The spectra were measured in CDCl₃. The ¹H- and ¹³C-NMR chemical shifts (δ) are expressed in ppm with reference to the solvent signals (CDCl₃, δ_H 7.26 and δ_C 77.1). Coupling constants are given in Hz. NOESY (2D- NOE) experiments were executed on the Bruker Avance DRX-400 instrument. Preparative TLC was performed using pre-coated silica gel 60 F-254 plates (10 × 20 cm, Merck, Sigma-Aldrich, Milan, Italy) using n-hexane-acetone 8.5:1.5 as the eluent. Spots were visualized under UV light. Compounds were recovered from the stationary phase by washing five times with CH₂Cl₂ (DCM). Column chromatography was performed using MN Kiesegel 60 (70–230 mesh, Macherey-Nagel, Fisher Scientific, Milan, Italy). Fractions were monitored by TLC (Silica gel 60 F254; Merck), and spots on TLC were visualised under UV light and after staining with p-anisaldehyde-H₂SO₄-EtOH (1:1:98) followed by heating at 110 °C. All solvents used were of analytical grade and were purchased from VWR (VWR, Milan, Italy). Anhydrous Na₂SO₄ was purchased from Scharlau S.L. (Milan, Italy).

Solvents, reagents, standards, silica gel 60 F254 plates, and silica gel 60 were purchased from Sigma-Aldrich (Milan, Italy). Hydroxytyrosol was prepared by the IBX oxidation of tyrosol as already reported [19 (link)]. A patented procedure based on membrane technology was applied to obtain hydroxytyrosol-enriched extracts from Olea europaea by-products [45 ]. The vegetal material was extracted with water and then subject to microfiltration (MF), nanofiltration (NF), and reverse osmosis (RO). Microfiltration was carried out with tubular ceramic membranes in titanium oxide; nanofiltration and reverse osmosis were carried out using spiral wound module membranes of poly(ether sulfone) [45 ]. The collected fraction was concentrated using a heat pump evaporator (Vacuum Evaporators—Scraper Series, C&G Depurazione Industriale Srl, Firenze, Italy) [3 (link),4 (link)]. High Performance Liquid Chromatographic (HPLC) analysis of the resulting extracts was carried out at λ = 280 nm using a diode array detector (DAD) to identify the polyphenolic compounds found in the sample. The quantitative data were referred to pure standards (tyrosol, hydroxytyrosol, oleuropein, and caffeic acid).

All chemicals (analytical grade), silica gel 60 F254 plates, and silica gel 60 were purchased from Sigma-Aldrich (Milan, Italy). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH₂-DA) was supplied by Molecular Probes (Eugene, OR, USA). Fresh pure hydroxytyrosol was synthetized in our laboratory as previously reported [37 (link)]. L6 myoblasts from rat skeletal muscle and THP-1 human leukemic monocytes were purchased from the American Type Culture Collection (Rockville, MD, USA). DMEM (Dulbecco’s modified Eagle’s medium), RPMI 1640, streptomycin (100 mg/mL), penicillin (100 U/mL), d-glucose, and sterile plasticware for cell culture were from Falcon (San Diego, CA, USA). Fetal bovine serum was from GIBCO (Grand Island, NY, USA).

Silica Gel 60-F254 plates (Sigma Aldrich) were used for TLC analyses.