Avance 3 hd 600 mhz spectrometer

NMR samples were prepared in 3 mm tubes with DNA concentrations ranging from 2 to 4 mM with 5% v/v deuterium oxide. NMR experiments were performed on a Bruker 850 MHz Avance III HD spectrometer equipped with a 5 mm TCI CryoProbe, and a Bruker 600 MHz Avance III HD spectrometer equipped with a Prodigy probe. NMR spectra were processed and analyzed using Bruker TopSpin 4.1, MestReNova 14.2, and Matlab 2019b.

¹⁹F NMR measurements were performed at 298 K using 3 mm tubes containing 160 μL of samples on a Bruker 600 MHz Avance III HD spectrometer equipped with a CP-QCI-F cryoprobe specifically designed for ¹⁹F detection. Both the ¹⁹F and ¹H NMR reference spectra were acquired on samples containing compound 12 at a concentration of 100 μM, in an NMR buffer containing 50 mM Tris-Cl pH 7.5, 250 mM NaCl, 1% DMSO-d₆, and 5% D₂O. Then, ¹⁹F and ¹H NMR spectra of the compounds were obtained in the presence of MabA at a concentration of 20 µM in the same NMR buffer. The parameters used for the ¹H experiments were TD = 16,384 points, NS = 64 scans, relaxation delay D1 = 1 s, carrier frequency O1P = 4.698 ppm, spectral window sw = 16 ppm, and with ¹⁹F decoupling. The acquisition time was about 2.5 min per sample. The parameters used for the ¹⁹F experiments were TD = 8192 points, NS = 512 scans, relaxation delay D1 = 2 s, carrier frequency O1P = −65.0 ppm, spectral window sw = 20 ppm, and with ¹H decoupling. The acquisition time was about 22 min per sample. The NMR data were processed using Bruker Topspin 4.06 software.

NMR spectra were acquired on either a Bruker 500 MHz AVANCE III spectrometer or a Bruker 600 MHz AVANCE III HD spectrometer, both with cryoprobes, on 60 µM protein samples in 20 mM deuterated TRIS buffer, pH 8.0, with 150 mM NaCl. For fragment screening, fragments were added from a concentrated DMSO stock to a concentration of 1 mM in the NMR tube. As described above, fragments were initially added as mixtures of 12 and those mixtures which induced significant changes in a 13C-HSQC spectrum of the protein were then deconvoluted to find the individual binders.

CDK5, PKCδ, ERK2, GRK5 and JNK2α2 were purchased from Upstate Merck Millipore (Tokyo, Japan) and CK1 and CK2 were purchased from New England Biolabs (Ipswich, MA, USA). For p53, 2.5 μg of each kinase was added to 100 μl of 50 μM¹³C/¹⁵N-labeled human p53-TAD. For XPC, 2.5 μg of CK2 was added to 150 μl of 50 μM¹³C/¹⁵N-labeled human XPC-AF. Phosphorylation was conducted in 20 mM potassium phosphate (pH 6.8), 1 mM ATP, 10 mM MgCl₂ and 10% D₂O and monitored at 25 °C on a Bruker AVANCE III HD 600 MHz spectrometer (Bruker, Rheinstetten, Germany) equipped with a triple-resonance TCI cryogenic probe. The ¹H,¹⁵N HSQC spectra shown in Figures 1c–i were taken at 1 day (CDK5), 1 day (PKCδ), 17 h (CK1), 9 h (ERK2), 1 day (CK2), 1 h (JNK2α2) and 21 h (GRK5) after addition of the respective kinase. The ¹H,¹⁵N HSQC spectrum shown in Figure 7a were taken at 1 h after the addition of CK2. Spectra were processed by NMRPipe^{48 (link)} and analyzed by NMRView.^{49 (link)}

Unless otherwise specified, all reagents and solvents were obtained from commercial suppliers and used without further purification.
Progression of the reaction was monitored by thin-layer chromatography [Merck silica gel 60 pre-coated plates with fluorescent indicator F254 (0.25 mm)]. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance III™ HD 600 MHz spectrometer at 600 and 151 MHz, respectively. HPLC/APCI-MS analysis were recorded using a Varian tandem mass spectrometer (Palo Alto, CA, USA) 1200 L triple quadrupole mass spectrometer with an APCI source. Data acquisition and processing were performed by a Varian MS workstation version 6.7 software. APCI ran in both positive and negative ion modes. The capillary potential was set at 75 V, the APCI torch at 450 °C and the shield at 600 V. Nitrogen at 48 mTorr was fixed at 400 °C. The range of 100−1000 amu, with scan time 0.75 amu, scan width 0.70 amu, and detector at 1450 V were set to obtain the full scan spectra. The atmospheric pressure ionization (API) housing was kept at 50 °C for APCI. The compounds were solubilized in a mixture of acetonitrile 90% (v/v) (A) and doubly distilled water 10% (v/v) (B), and then infused in the source at a rate of 0.05 mL/min.