For STD-NMR spectra, protein buffer was exchanged against an NMR buffer with 20 mM HEPES pH 7.5, 100 mM KCl, 2 mM EDTA, 1 mM DTT, 66 mM NDSB-195, D20 (10%), 10% glycerol using two cycles of 10-fold dilution, followed by concentration on Amicon Ultra (regenerated cellulose 3kDa, Millipore, Ireland) at 4000 g for 60 min. NMR tubes were prepared in 3 mm capillaries (165 μL) containing 14 μM of C-ter Lig4 protein and 1mM of ligands from 100% DMSO-d6 stock solutions. On-resonance irradiation of the protein was set to −600.13 Hz and off-resonance at 18000 Hz. The selective irradiation was performed with a gaussian pulse set at 86 Hz with a 50 ms length. NMR spectra were recorded at 283 K (10 °C) on a Bruker Avance 600 spectrometer equipped with a TXI cryoprobe. NMR data were processed and analyzed using Topspin 3.2 NMR software (Bruker). For TR-NOESY spectra, protein buffer was the same as for STD-NMR experiments. Samples were prepared with 165 μL in 3 mm capillaries containing 1 mM of ligands from 100% DMSO d6 stock solutions and with or without 14 μM of C-ter Lig4 protein. Experiments were measured with 8 scans and a mixing time of 200 ms. NMR spectra were recorded at 298 K (25 °C) using a Bruker Avance 600 spectrometer equipped with a TXI cryoprobe. NMR data were processed and analyzed using Topspin 3.2 NMR software (Bruker).
Avance 600 spectrometer
Manufactured by Bruker
Sourced in Germany, United States, Switzerland
The Avance 600 spectrometer is a high-performance nuclear magnetic resonance (NMR) instrument designed for advanced analytical applications. It operates at a magnetic field strength of 14.1 Tesla, providing high-resolution data for a wide range of sample types. The Avance 600 spectrometer is capable of performing various NMR experiments to analyze the structural and chemical properties of materials.
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Lab products found in correlation
Topspin 3, by Bruker (7 mentions)
Jms dx 303 spectrometer, by JEOL (5 mentions)
Silica gel 60 0.063 0 2 mm, by Merck Group (5 mentions)
J 815 spectrometer, by Jasco (4 mentions)
3100 ft ir excalibur series spectrometer, by Agilent Technologies (4 mentions)
Silica gel, by Qingdao Marine Chemical (4 mentions)
Topspin, by Bruker (3 mentions)
Autopol 3 polarimeter, by Rudolph Research Analytical (3 mentions)
Sephadex lh 20, by Cytiva (3 mentions)
Uhr tof maxis impact instrument, by Bruker (3 mentions)
Nicolet 6700 ftir spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Merck precoated silica gel plates kieselgel 60 f254, by Merck Group (3 mentions)
Topspin software package, by Bruker (2 mentions)
Impact 2 instrument, by Bruker (2 mentions)
Topspin software, by Bruker (2 mentions)
Q exactive hf orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Avance 500 spectrometer, by Bruker (2 mentions)
Avance 400 spectrometer, by Bruker (2 mentions)
Avance 500 mhz spectrometer, by Bruker (2 mentions)
Drx 500 spectrometer, by Bruker (2 mentions)
141 protocols using avance 600 spectrometer
1
Protein-Ligand Interaction Study by STD-NMR and TR-NOESY
2
600 MHz 1H NMR Spectroscopy of Compounds
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1H NMR spectra were measured at 600.13 MHz using an Avance 600 Bruker spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a Bruker 5 mm double resonance BBI probe. CD3OD and CDCl3 were used for internal lock purposes. Spectra of GLJ-C and GLJ-W/M were acquired using the zg30 sequence. Sixty-four scans were recorded, resulting in a total acquisition time per sample of 5 min.
3
Characterization of Novel Compounds
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Unless otherwise noted, all reagents were obtained from commercial suppliers and used without further purification. All starting materials were commercially available. 1H-NMR spectra were documented on AVANCE300 or AVANCE600 Bruker spectrometer (Leipzig, Bruker Company, Germany), and chemical shifts were documented in ppm downfield from tetrame-thylsilane (TMS). The coupling constants (J) were documented in Hz. Moreover, the mass spectra (MS) were determined on a Waters Acquity UPLC. TLC assessment was conducted on silica gel plates GF254 (Qingdao Haiyang Chemical, China). Besides, silica gel column chromatography was accomplished using Silica gel 60 G (Qingdao Haiyang Chemical, China). The purities of assayed compounds were greater than 95%, as estimated by reversephase (RP) HPLC analysis.
4
UV-vis, CD, and NMR Spectroscopic Analysis
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The UV-vis absorption spectra were recorded using an Agilent-8453 spectrophotometer (Santa Clara, CA, USA) equipped with a Peltier effect heated cuvette holder in a 10 mm quartz cell. All the CD spectra were recorded on a JASCO J-815 spectrophotometer (Tokyo, Japan) in a 10 mm quartz cell at room temperature. All CD spectra were collected with a scan speed of 500 nm/min and a response time of 0.5 s between 200 nm and 350 nm with three scans averaged.
The 1H-NMR titration spectra were carried out by a Bruker Avance 600 spectrometer (Fällanden, Switzerland) equipped with a BBI probe. The stocking solution of TG4T was prepared by dissolving in PBS [10 mM K2PO4/KH2PO4, 70 mM KCl, 10% D2O/90% H2O (v/v)]. The spectra was recorded immediately by addition of TC-P4 dissolved in DMSO-d6. Bruker pulse program p3919 gp was used to suppress water peak.
The 1H-NMR titration spectra were carried out by a Bruker Avance 600 spectrometer (Fällanden, Switzerland) equipped with a BBI probe. The stocking solution of TG4T was prepared by dissolving in PBS [10 mM K2PO4/KH2PO4, 70 mM KCl, 10% D2O/90% H2O (v/v)]. The spectra was recorded immediately by addition of TC-P4 dissolved in DMSO-d6. Bruker pulse program p3919 gp was used to suppress water peak.
5
NMR Analysis of Recombinant Mouse PrP
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For NMR measurements, recombinant mouse PrP 121-231 uniformly labeled with 15 N was prepared in 50 mM acetate-d3 buffer (pH 4.8) containing 1 mM NaN3 and 1 M DSS dissolved in 99% H2O/1% D2O. NMR spectra were recorded at 25.0ºC on a Bruker Avance 600 spectrometer (Bruker BioSpin, Rheinstetten, Germany) at Gifu University.
The spectrometer operated at a 1 H frequency of 600.13 MHz and a 15 N frequency of 60.81 MHz. A 5-mm 1 H inverse detection probe with triple-axis gradient coils was used for all measurements. 1 H-15 N HSQC spectra were acquired with 2048 complex points covering 9615 Hz for 1 H and 256 complex points covering 1521 Hz for 15 N. NMR data were processed using the TOPSPIN software package (Bruker BioSpin, Rheinstetten, Germany).
The spectrometer operated at a 1 H frequency of 600.13 MHz and a 15 N frequency of 60.81 MHz. A 5-mm 1 H inverse detection probe with triple-axis gradient coils was used for all measurements. 1 H-15 N HSQC spectra were acquired with 2048 complex points covering 9615 Hz for 1 H and 256 complex points covering 1521 Hz for 15 N. NMR data were processed using the TOPSPIN software package (Bruker BioSpin, Rheinstetten, Germany).
6
NMR and Mass Spectrometry Characterization
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1H and 13C NMR spectra were recorded on a Bruker Avance 600 spectrometer (Bruker, Germany) at 600 (1H) and 150 (13C) MHz, respectively. High-resolution ESI-MS mass spectra were carried out on a LTQ-Orbitrap XL instrument (Thermo, USA). Semi-preparative HPLC was performed on a Shimadzu SPD-20A instrument (Shimadzu, Japan), using an YMC-Pack ODS-A column (250 × 10 mm, 5 µm). Silica gel (200–300 mesh, Haiyang Co., Qingdao, China), and Sephadex LH-20 gel (Amersham Biosciences, USA) were used for column chromatography. Pre-coated silica GF254 plates (Haiyang Co., Qingdao, China) were used for TLC analysis.
7
NMR, Mass Spectral, and IR Characterization
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The nuclear magnetic resonance (NMR) spectra were measured using the Bruker Avance 600 spectrometer (Bruker, Billerica, MA, USA) in acetone-d6 solvents. Chemical shifts (δ) are reported in ppm and J values in Hz. Multiplicity is designated as singlet (s), doublet (d), triplet (t) and multiplet (m). High-resolution mass spectral analysis (HR-MS) was recorded using the Bruker Impact II instrument (Bruker, Billerica, MA, USA). The theoretical molecular weight calculations of compounds were performed using “The Exact Mass Calculator, Single Isotope Version” tool (http://www.sisweb.com/referenc/tools/exactmass.htm , accessed on 1 June 2023) (Ringoes, NJ, USA). Infrared (IR) spectra (Shimadzu, Kyoto, Japan) were performed using the IRXross spectrophotometer equipped with the attenuated total reflection (ATR) mode. Melting points were measured by the Electro-thermal IA 9300 (Electrothermal, London, UK) melting point apparatus.
8
NMR Spectroscopy Characterization Protocol
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The NMR spectra were recorded at a temperature of 25 °C using a Bruker Avance 600 spectrometer (Bruker, Billerica, MA, USA). The nuclear magnetic hydrogen spectrum (1H-NMR), nuclear magnetic carbon spectrum (13C-NMR and DEPT 135°) and two-dimensional nuclear magnetic correlation spectrum (13C-1H HSQC) were measured. The dried samples were dissolved in deuterium oxide and placed in an NMR tube with an inner diameter of 0.5 mm for testing. The test frequency for 1H-NMR was 600 MHz, and for 13C-NMR and DEPT 135° it was 150 MHz [67 (link)]. The number of sample collection points for each type of NMR is 64 K. The number of scans for 1H-NMR was 128, while the number of scans for other NMRs was 16. The recovery delay was 2 s. The free induction decay (FID) signal measured by the NMR instrument was imported into MestReNova 14.0.1 software (Mestrelab Research, Bajo, Santiago de Compostela, Spain) for Fourier transformation, and the NMR spectra were obtained after phase correction and baseline correction [68 (link)]. The spectra were then saved as ASCII files and imported into SpecAlign 2.4.1 software (University of Oxford, Oxford, England) for peak matching [69 (link)].
9
NMR Spectra Acquisition and Analysis
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A Bruker Avance-600 spectrometer (600 MHz) was used to record NMR spectra. Standard techniques were used to acquire 2D spectra: COSY (phase sensitive correlated spectroscopy), TOCSY (total correlated spectroscopy), and NOESY (nuclear Overhauser enhancement spectroscopy). Water signal suppression was achieved by presaturation or Watergate [64 (link)]. Mixing times of 60 ms were used to record the TOCSY spectra while 150 ms were used for the NOESY. 1H-13C HSQC (heteronuclear single quantum coherence spectroscopy) were acquired at 13C natural abundance. The IUPAC-IUB recommended 1H/13C chemical shift ratio was employed to indirectly referenced the 13C chemical shifts [65 (link)]. Depending on the experimental conditions, peptide samples were tested at 5 and/or 25 °C. Data processing was accomplished using the TOPSPIN software (Bruker Biospin, Karlsruhe, Germany).
10
NMR Spectroscopy in D2O and DMSO
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1H NMR studies were carried out by a Bruker AVANCE 600 spectrometer (Bruker, Germany) using D2O or DMSO-d6 as the solvent.
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