Avance 3 nmr spectrometer

Serum samples (200 μL) were processed according to standard procedures for NMR metabolomics measurement [8 (link),9 (link),54 (link)]. Briefly, prior to NMR analysis, sera were thawed at room temperature, and an aliquot of 200 μL was added of 400 μL of saline buffer solution (NaCl 0.9%, 50 mM sodium phosphate buffer in D₂O containing TSP 0.05% wt for chemical shift calibration, pH 7.4) to minimize the variation in pH and transferred in a 5 mm NMR tube [54 (link),55 (link),56 (link)]. The NMR experiments were recorded on a Bruker Avance III NMR spectrometer (Bruker, Ettlingen, Germany), operating at 600.13 MHz for ¹H observation, equipped with a TCI cryoprobe (Triple Resonance inverse Cryoprobe), incorporating a z-axis gradient coil and automatic tuning-matching (Supplementary: Section S1).

The ¹H-NMR spectrawere recorded at 298 K using a Bruker 600-MHz AVANCE III NMR spectrometer (Bruker Biospin, Germany) and the noesygppr1d pulse sequence for water suppression. The ¹H-NMR spectrum for each sample consisted of 64 scans requiring 5 min of acquisition time with the following parameters: spectral width 12,345.7 Hz; spectral size 65,536 points; relaxation delay of 1.0 s; acquisition time of 2.654 s. All spectra were manually phased and baseline corrected using MestReNova software (Mestrelab Research, Santiago de Compostella, Spain). Chemical shifts were referenced to TSP at δ 0.00. Regions distorted by residual water (δ 4.5~5.0) were excluded in the subsequent analysis. Each spectrum was then segmented at 0.01-ppm intervals across the chemical shift 0.50~9.00; each data point was normalized to the sum of its row and then was exported as a text file for further multivariate statistical analysis.

2D NOESY experiments were carried out for PHEN solution in DMSO, methanol, aceto­nitrile and chloro­form at room temperature using a 600 MHz Bruker AVANCE III NMR spectrometer. 2D NOE spectra were measured with a standard pulse for both F1 and F2 dimensions. The number of F1 increments was 256, each with 65 536 data points in the F2 dimension. The NOE mixing time was optimized to 0.8 s by measuring NOE buildups. The number of scans and dummy scans were set to be 16 and 2, respectively.

The ³¹P-NMR spectra were recorded on a 400 MHz Bruker AVANCE III NMR spectrometer (Karlsruhe, Germany). Twenty-five milligrams of sample were completely dissolved in 400 μL of anhydrous pyridine and deuterated chloroform (1.6:1, v/v). Then 150 μL of cyclohexanol/chromium(III) acetylacetonate solution (4 mg·mL⁻¹/3.6 mg·mL⁻¹ in anhydrous pyridine and deuterated chloroform 1.6:1, v/v) was added as an internal standard and relaxation reagent, respectively. The mixture was reacted with 75 μL of phosphating reagent (2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane-tetramethyl-1,3,2-dioxaphospholane, TMDP) for about 5 min and then was transferred into a 5 mm NMR tube for subsequent NMR test.

NMR spectra were recorded on a Bruker Avance III NMR spectrometer with the magnetic field of 11.74 Tesla. HR-ESI-MS were acquired on a Bruker Q-TOF mass spectrometer. Preparative HPLC was performed on a Waters Delta Prep 4000 system equipped with a Waters 2487 dual λ absorbance detector. A Rainbow Kromasil-C₁₈ (10 mm × 250 mm, 10 μm) column was selected for preparative HPLC. Column chromatography was performed on silica gel (160–200 mesh) and TLC analysis was carried on silica gel G plates (Qingdao Marine Chemical Plant, China). Sephadex LH-20 was purchased from Amersham Pharmacia Biotech (Beijing, China). MCI GELCHP20P (75–150 μm) was supplied by the Kaiteki Company (Tokyo, Japan). Analytical grade solvents were produced by Beijing Chemical Factory (Beijing, China).The deuterated solvents (CDCl₃, CD₃OD, DMSO-d₆, deuterated ratio, 99.8%) with TMS as the internal referent were produced by Cambridge Isotope Labo-ratories, Inc. (Andover, MA, USA).

Cathepsin X inhibitors were selected from the in-house compound library or obtained from Enamine Ltd (Kiev, Ukraine) and ChemBridge (ChemBridge Corporation, San Diego, CA, USA). Reagents and solvents used were obtained from Acros Organics, Sigma and Merck. 1 H NMR and 13 C spectra were recorded at 400 MHz and 100 MHz, respectively on a Bruker Avance III NMR spectrometer (Bruker Corporation, Billerica, MA, USA) at 295 K. Chemical shifts (δ) are reported in parts per million (ppm) and are referenced to the deuterated solvent used. High-resolution mass measurements were performed on a VG Analytical Autospec Q Micromass mass spectrometer (Fisons, VG Analytical, Manchester, UK) at the Jozef Stefan Institute, Ljubljana, Slovenia. Analytical reversed-phase HPLC analyses were performed on an Agilent 1100 LC modular system (Agilent Technologies, Santa Clara, CA, USA) equipped with a G1365B UV-Vis detector, a G1316A thermostat, a G1313A auto-sampler, and a ChemStation data system. An Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 µm), thermostated at 25 °C, was used with a flow rate of 1.0 mL/min. The detector was set to 254 nm.

¹HNMR spectra of different concentrations were detected using a 600 MHz Bruker AVANCE III NMR spectrometer. First, 5 ml of DMSO-d6 and excessive 5-nitro­furazone (form α were chosen as an example) were added into a glass bottle at 20°C to prepare the suspension. The suspension was then stirred at 200 r min⁻¹ with a magnetic stirring for 30 min at 20°C to ensure sufficient dissolution. The suspension was withdrawn by syringes with organic membrane filters (0.22 µm, Tianjin Legg Technology Co. Ltd, Tianjin, China) to obtain the clarified saturated solution. Different contents (e.g. 500, 440, 380, 320, 260, 200, 140, 100, 80, 30 and 20 µl) of the saturated solution were put into different NMR tubes. And then, a certain amount of DMSO-d6 was added into the tube of 500 µl for¹HNMR analysis.