Avance 3 hd 500 mhz spectrometer

Unless otherwise stated, all reagents and chemicals were purchased from qualified suppliers with required purities. All glassware was dried before use. Nuclear magnetic resonance spectra were measured on a Bruker AVANCE III HD 500-MHz spectrometer with tetramethylsilane as the internal standard. Mass spectrometry was performed on an Agilent Technologies 6530 quadrupole time-of-flight liquid chromatograph-mass spectrometer. UV-vis absorption spectra were performed on a UV-2550 scanning spectrophotometer (Shimadzu, Japan). Fluorescent spectra were recorded on a Hitachi F-2700 equipped with a 1-cm quartz cell. Dynamic light scattering measurements were performed at 25 °C on Zestier Nano ZS (Malvern Instruments Ltd, UK).

¹H-NMR spectra were measured on a Bruker AVANCE III 600 MHz spectrometer or on a Bruker AVANCE III HD 500 MHz spectrometer (Karlsruhe, Germany), equipped with 5 mm TCI cryogenic probes. The experiments were recorded at 298 K by using the Bruker library zg pulse sequence and the following parameters: number of scans 16, dummy scans 4, relaxation delay 12 s, spectral width 16 ppm, transmitter offset 4.7 ppm. After exponential multiplication (line broadening of 0.3 Hz), the spectra were Fourier transformed, phased, baseline corrected, and calibrated on the TSP signal.
The spectrum can be accepted only if the following test is satisfied: the width half height of TSP is ≤ 1.4 Hz.
The intensity of the following signals was measured:
The ratio r(1:2) between peaks 1 and 2, and the ratio r(3:2) between peaks 2 and 3 were calculated and used to distinguish the different heparin sources.

α-Syn samples were dissolved in Tris-HCl (20 mM, pH 7.4), and then mixed with CuCl₂ or EGCG aqueous solutions to obtain the protein concentration of 50 μM. The samples were incubated shakily at 37 °C for 58 h, and then D₂O was added in the ratio of 90% H₂O/10% D₂O for ¹H-NMR measurement at 25 °C. ¹H-NMR spectra were recorded on an AVANCE III HD 500 MHz spectrometer (Bruker, Karlsuhe, Germany). Water signal was suppressed using the WATER-GATE pulse sequence [58 (link)].

To produce sufficient amounts of involutin for NMR analysis, P. involutus was grown for 2 months on liquid malt extract (2%, w/v) in Petri dishes containing glass beads as described above. Metabolites were extracted from the culture filtrates and purified by HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific) using a Kinetex C₁₈ column and conditions as described above. The mass spectrum of the peak putatively assigned to involutin was recorded in negative mode (ESI) using an LTQx Orbitrap (Thermo Fisher Scientific). The ¹H NMR spectrum of the collected fraction was recorded on a Bruker Avance III HD 500 MHz spectrometer (Bruker, Billerica, Massachusetts, USA), equipped with a 5 mm broadband probe, optimized for ¹H observation. All spectra were recorded with the same settings, i.e., an acquisition time of 2 s, a relaxation delay of 6 s, and 512 scans. The involutin sample obtained from the HPLC purification was dissolved in methanol-d₄ (99.8%; Deutero GmbH, Kastellaun, Germany), and chemical shifts in standard ¹H experiments were referenced to the resonances of residual protons in deuterated methanol.

All reagents and solvents were purchased from commercial sources and used without further purification. MOM-CyHQ-CHO was prepared as previously described [27 (link)]. All reactions were carried out using glassware except for the MO couplings, which were performed in microcentrifuge tubes. Anhydrous reactions were performed at a positive pressure of dry nitrogen gas. Anhydrous THF was obtained by distillation over Na/benzophenone. Purifications were carried out using flash chromatography on an Isolera Spektra 4 with Biotage SNAP cartridges packed with KP-Sil silica. ¹H NMR and ¹³C NMR spectra were recorded using a Bruker Avance™ III HD 500 MHz spectrometer (Billerica, MA, USA). UV spectra for MO quantification were recorded on a Nanodrop 2000c. HPLC was performed on an Agilent Infinity series system with an autosampler and diode array detector. Separations were performed using Zorbax eclipse C-18 reverse phase columns 150 × 4.6 mm with 8 μm particle size and gradient elution at a flow rate of 0.5 mL min⁻¹: eluent A was acetonitrile and eluent B was water. The analysis started with 2% of eluent A, which was linearly increased up to 100% over 20 min, and then kept at 100% for 5 min. HRMS/LC-MS was performed on an Agilent 6540 HD Accurate Mass QTOF/LC/MS (Santa Clara, CA, USA) with electrospray ionization (ESI) using a fragmentor voltage of 175 V.