Polygram sil g uv254

All reactions and chromatographic separations were monitored by analytical thin-layer chromatography (TLC) 0.25 mm Silica gel plastic sheets (Macherey–Nagel, Polygram^® SIL G/UV 254). TLC plates were analyzed under 254 nm UV light or using iodine vapor. Flash chromatography on Silica gel 60 (J. T. Baker, 40 mm average particle diameter) was used to purify the crude reaction mixtures. Microwave-assisted synthesis was performed in a CEM Discover reactor. NMR spectra were recorded at 400 MHz (¹H NMR) and 100 MHz (¹³C NMR) using a Brucker Avance NEO-400 spectrometer at 21 °C. Chemical shifts (δ) are reported as follows: chemical shift (multiplicity (s: singlet, d: doublet, t: triplet, q: quartet, quint: quintet, m: multiplet), coupling constant, integration) using TMS as reference. When two conformers are observed, (*) denotes the signal corresponding to the minor one. High-resolution mass spectrometry experiments (HRMS) were measured on a MicroTOF-Q-Bruker Daltronics using electrospray ionization. Melting points were determined using a Fisher-Jones Melting Point apparatus. All solvents were purified according to literature procedures. All yields refer to isolated compounds after the final purification process, unless otherwise stated.

Reactions were monitored by TLC using Polygram® SIL G/UV254 (Macherey-Nagel) plastic-backed plates (0.25 mm layer thickness), column chromatography was performed using silica gel 60 (40–63 µm). The yields are not optimised. Microwave-assisted reaction was carried out using a PC operated (Synergy software) CEM Discover apparatus, provided with a fibre optic temperature control. Melting points were determined with a Kofler hot-stage microscope (Reichert) and are uncorrected. IR spectra were recorded on a Bruker ALPHA FT-IR apparatus equipped with a Platinum ATR module. ¹H NMR spectra were recorded on Varian Gemini 200 spectrometer (200 MHz) or “Mars” 400 MHz Bruker Avance 4 Neo spectrometer, ¹³C NMR spectra were recorded on “Mars” 400 MHz Bruker Avance 4 Neo spectrometer with DMSO-d₆ as the solvent. The centre of the solvent multiplet (DMSO-d₆) was used as internal standard (chemical shifts in δ ppm), which was related to TMS with δ 2.49 ppm (¹H) and 39.5 ppm (¹³C). Coupling constants (J) were reported in hertz, and splitting patters were used as follows: s, singlet; br. s, broad singlet; d, doublet; dd, doublet of doublets; m, multiplet. Elemental analyses were performed by Mag. J. Theiner, “Mikroanalytisches Laboratorium,” Faculty of Chemistry, University of Vienna, Austria.

The NMR spectra were recorded on a Varian Inova-500 spectrometer at 599.737 MHz (¹H) or 150.818 MHz (¹³C), respectively. The chemical shifts are given in δ values with TMS as internal reference, and coupling constants are given in [Hz]. The ESI and ESI HR mass spectra were recorded on a Bruker micrOTOF mass spectrometer. Open column chromatography was done on silica gel 60 (0.063–0.20 mm), and PTLC was performed on silica gel P/UV₂₅₄ (both obtained from Macherey–Nagel, Düren, Germany). Size exclusion chromatography was performed on Sephadex LH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd., purchased from Sigma-Aldrich Chemie, Steinheim, Germany). Pre-coated silica gel plates (Polygram SIL G/UV₂₅₄, Macherey–Nagel & Co.) were used for TLC. Spots were visualized at 254 or 365 nm, and sprayed with an anisaldehyde/sulfuric acid reagent followed by heating.

Melting points are uncorrected. All ¹H NMR and ¹³C NMR spectra were recorded on spectrometer Varian Mercury Plus operating at 399.93 MHz and 100.57 MHz, respectively. Chemical shifts are given in ppm (δ) and coupling constants in Hertz; multiplicities are indicated by s (singlet), d (doublet), t (triplet), ddt (doublet doublet triplet), m (multiplet) or series of m (series of multiplet). CDCl₃, acetone-d6, were used as solvents as indicated below. Shifts are given in ppm relative to the remaining protons of the deuterated solvents used as internal standard (¹H, ¹³C). Elemental analyses were performed using an elemental analyser Perkin-Elmer model 240 C. Ethanol (EtOH) grade 96% was used, dry acetone was freshly distilled from CaCl₂ under N₂. All reagents were of commercial quality and used as purchased from various producers (Sigma-Aldrich, Merck). Magnolol 1 and honokiol 2 were purchased from Chemos GmbH, Germany. Flash chromatography was carried out with silica gel 60 (230–400 mesh, Kiesgel, EM Reagents) eluting with appropriate solution in the stated v:v proportions. Analytical thin-layer chromatography (TLC) was performed with 0.25 mm thick silica gel plates (Polygram^® Sil G/UV₂₅₄, Macherey-Nagel). The purity of all new compounds was judged to be >98% by ¹H-NMR spectral determination. Solvents were used without additional purification or drying, unless otherwise noted.

All solvents used for extraction and processing were purchased from Fisher Chemical UK while HPLC grade solvents were obtained from Rathburn Chemicals Ltd. (Walkerburn, UK). Column chromatography was performed on Acros Organics 0.03–0.20 mm mesh size silica gel. Thin layer chromatography (TLC) technique was used for preliminary identification and performed on pre-coated TLC sheets POLYGRAM^® SIL G/UV₂₅₄, Macherey-Nagel GmbH & Co., and samples were spotted using 5 μL micropipettes (Drummond Scientific Company, Broomall, PA, USA). Silica plates were visualized under UVGL Handheld UV lamp and para-Anisaldehyde (99% pure, Acros Organics, 2440 Geel, Belgium). Deuterated DMSO (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) was used for NMR measurements. The ingredients used to prepare fungal culture media were purchased from Oxiod, Hampshire RG24 8PW, UK.

⁶⁴Cu was produced at the in-house radiopharmacy of the Department of Preclinical Imaging and Radiopharmacy in Tübingen, using a PETtrace cyclotron (General Electric Medical Systems). ⁶⁴Cu was generated by 12.5 MeV proton irradiation of enriched ⁶⁴Ni metal (35–75 mg; Isoflex, >98% enrichment) electroplated on a platinum/iridium disk via the ⁶⁴Ni(p,n)⁶⁴Cu nuclear reaction as described previously (Alt et al., 2010 (link)). In brief, ⁶⁴Cu was separated from the bulk nickel target and other metallic impurities by acid dissolution followed by anion exchange chromatography (AG1 × 8, Bio-Rad) in aqueous hydrochloric acid media (Trace Select, Sigma-Aldrich). The fractions containing the ⁶⁴Cu product were dried under argon at 120°C.
For antibody radiolabeling, the dry ⁶⁴CuCl₂ was re-dissolved in 0.1 M HCl and the pH was adjusted to 5–6 using 0.5 M ammonium acetate. 1.3 μg of p-NCS-benzyl-NODAGA conjugated MC3 antibody was added per MBq of ⁶⁴Cu and incubated at 42°C for 60 min. Thin layer chromatography (Polygram SIL G/UV254, Macherey-Nagel, Düren, Germany, mobile phase: 0.1 M sodium citrate, pH 5.0) and radio-HPSEC (as described above) were conducted for quality control of the radiolabeled antibodies [radiochemical purity at least 95% (TLC), HPSEC elution profile corresponding to the unmodified antibody].