Av 500 mhz spectrometer

The deep‐frozen serum samples were thawed at 4°C overnight and then were vortexed to remove any precipitates. To 300 μL of each serum sample, 150 μL phosphate buffer was added, which was dissolved in D₂O (0.2 M, Na₂HPO₄/NaH₂PO₄ and pH 7.0) containing 0.05% TSP‐d₄ as chemical shift reference. Samples were vortexed and then centrifuged at 12 000 g for 10 minutes at 4°C to afford 500 μL of supernatant.
Frozen tissue sections were weighed (ca. 250 mg), homogenized in precooled acetonitrile/water (vol/vol = 1:1, 5 mL/g tissue) kept in an ice/water bath and centrifuged (12 000 g, 10 minutes, 4°C).38 The supernatant was lyophilized and then reconstituted in 600 mL phosphate buffer dissolved in D₂O. After vortexing and centrifugation (12 000 g, 10 minutes, 4°C), a total of 550 mL of the supernatants was pipetted into 5 mm NMR tubes for analysis.
All the ¹H NMR spectra were recorded at 298 K on a Bruker AV‐500 MHz spectrometer. The water‐suppressed Carr‐Purcell‐Meiboom‐Gill (CPMG) spin‐echo pulse sequence (RD‐90°‐ (τ‐180°‐τ) n‐ACQ) with a total spin‐echo delay (2nτ) of 40 ms was used to attenuate broad signals from proteins and lipoproteins. Typically, 128 transients were acquired with 32 K data points for each spectrum with a spectral width of 10 kHz.

The ¹H NMR spectra of kidney and serum samples were recorded at 25 °C on a Bruker AV 500 MHz spectrometer at 300 K. A 1D NOESYPRESAT pulse sequence for each kidney tissue sample and the transverse relaxation‐edited Carr–Purcell–Meiboom–Gill (CPMG) spin‐echo pulse sequence (RD‐90°‐(τ‐180°‐τ) n‐ACQ) for each serum sample was used to suppress the residual water signal. Prior to Fourier transformation, an exponential window function with a line broadening of 0.5 Hz was used to the free induction decays, which were collected into 32 k data points over a spectral width of 10 000 Hz with an acquisition time of 2.04 s.

About 1 mL serum samples were deproteinized by methanol with the ratios of serum: methanol as 1: 2 (v/v). The mixtures were vortexed and incubated at − 20 °C for about 30 min, and then were centrifuged into pellet proteins at 12000 g for 30 min. The supernatants were transferred into fresh tubes and lyophilized. The dried samples were dissolved in 600 μL 99.8% D₂O phosphate buffer (0.2 M, pH at 7.0) containing 0.05% sodium salt of 3-trimethylsilylpropionic acid (TSP, w/v), vortexed, centrifuged and decanted to 5 mm NMR tubes.
All ¹H NMR spectra were recorded with a Bruker AV 500 MHz spectrometer. A transverse relaxation-edited Carr-Purcell-Meiboom-Gill sequence [recycle delay-90-(τ-180-τ)n-acquisition] with a total spin echo delay (2nτ) of 40 ms was used to attenuate broad signals from slowly tumbling molecules such as proteins, whereupon the signals of the micro-molecule metabolites were clearly observed. ¹H NMR spectra were measured with 128 scans into 64 K data points with a recycle delay of 3 s over a spectral width of 20 ppm. The spectra were Fourier-transformed after multiplication by an exponential window function corresponding to a line broadening of 0.5 Hz. Resonances were assigned by querying the database HMDB (http://www.hmdb.ca/), MMCD (http://mmcd.nmrfam.wisc.edu/), and were aided by the Chenomx NMR suite (version 8.0, Chenomx, Inc.).

All reagents and other materials were purchased from commercial sources and used without additional purification unless otherwise noted. A B-545 melting point instrument was used to determine the melting point, without calibration. A Bruker AV-400 or AV-500 MHz spectrometer was used to generate NMR spectra, with CDCl₃ as the solvent. An Agilent 6545 Q-TOF liquid chromatography-mass spectrometer was used for mass spectrometry.

All reagents and other materials were purchased from commercial sources and used without additional purification unless otherwise noted. A B-545 melting point instrument was used to determine melting point without calibration. A Bruker AV-400 or AV-500 MHz spectrometer was used to generate NMR spectra with DMSO-d₆ or CDCl₃ serving as solvents. An Agilent 6545 Q-TOF LCMS spectrometer was used for mass spectrometry. A Bruker D8 Venture diffractometer was utilized to collect crystallographic data.

The ¹H and ¹³C spectra and DEPT135, HSQC, HMBC, NOESY spectra of the SQWP-2 were recorded at 30 °C with AV-500 MHz spectrometer (Bruker, Rheinstetten, German). Tetramethylsilane was used as an internal standard.

All chemicals were purchased from Sigma-Aldrich Chemical Co. (Sigma-Aldrich Corp., St. Louis, MO, USA). All melting points were measured with a Stuart Scientific Co. Ltd apparatusare uncorrected. The IR spectra were recorded on a KBr disc on a Jasco FT/IR 460 plus spectrophotometer. The ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were measured on BRUKER AV 500 MHz spectrometer in DMSO-d₆ as a solvent, using tetramethylsilane (TMS) as an internal standard, and chemical shifts were expressed as δ (ppm). The Microwave apparatus used is Milestone Sr1, Microsynth. Themass spectra were determined on a Shimadzu GC/MS-QP5050A spectrometer. Elemental analysis was carried out at the Regional Centre for Mycology and Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt, and the results were within ± 0.25%. Reaction courses and product mixtures were routinely monitored by thin layer chromatography (TLC) on silica gel precoated F₂₅₄ Merck plates.