Ascend 850 mhz

Optical rotations were measured on a JASCO DIP-370 digital polarimeter at 25 °C at the sodium D line (589 nm). UV spectra were recorded on a Hitachi 300 spectrometer (Hitachi High-Technologies Corporation, Kyoto, Japan). The ECD spectra were obtained on a JASCO J-810 spectropolarimeter with a 0.5 cm cell in MeOH. IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). 1D and 2D NMR spectra (chemical shifts in ppm, coupling constants in Hz) were recorded on Bruker Avance DRX 600 MHz (600 MHz for ¹H and 150 MHz for ¹³C NMR) (Bruker, Rheinstetten, Germany) and Bruker Ascend™ 850 MHz (850 MHz for ¹H and 213 MHz for ¹³C NMR) (Bruker BioSpin, Billerica, MA, USA) spectrometers using CDCl₃ or DMSO-d₆ as solvent. NMR spectra were referenced to the residual protonated solvent signals (for CHCl₃: 7.26 ppm for ¹H and 77.0 ppm for ¹³C; for DMSO: 2.51 ppm for ¹H and 39.6 ppm for ¹³C). Positive ion HRESIMS data were obtained with a Micromass Q-ToF equipped with leucine enkaphalin lockspray, using m/z 556.2771 [M + H]⁺ as a reference mass. For column chromatography, silica gel (Merck, 70-230 mesh ASTM) and Sephadex LH-20 (0.25–0.1 mm, Pharmacia) were used. Precoated silica gel 60 F-254 plates (Merck) were used for TLC. HPLC purifications were performed on a semi-preparative HPLC column (RP18, 5 μm, ARII Cosmosil, 250 × 10 mm, Waters).

NMR data were collected on a Bruker Ascend 850 MHz instrument fitted with a cryogenically cooled triple resonance 5 mm TCI probe with pulse filed gradients along the z-axis at 23 °C. The hydrolysis of NAD⁺ and NADP⁺ was measured by ¹H NMR using the pulse sequence zgesgppe allowing water suppression using excitation sculpting with pulse field gradients and perfect echo. The specta were acquired with eight scans and a recovery delay of 3.9 seconds. The reactions consisted of 500 µM NAD⁺ or NADP⁺ in NMR reaction buffer (25 mM sodium phosphate pH 5.8, 50 mM NaCl, 5 % (v/v) D₂O) in a final volume of 500 µl. Reference spectrums of NAD⁺ and NADP⁺ in the absence of AfNADase were acquired. The hydrolysis of NAD⁺ and NADP⁺ were measured by adding AfNADase to a final concentration of 0.2 nM, the time of enzyme addition was recorded, and spectra were acquired until the substrate resonances declined to the baseline level. The resulting spectra were manually phased and base line corrected. The resonances were assigned using standard correlation methods. The resonances of interest were integrated using the program Dynamics centre 2.5 (Bruker) and compared to the reference spectra of NAD⁺ and NADP⁺, respectively. The kinetic parameters of AfNADase were determined by progress curve analysis as described by Golicnik, M^{45 (link)} using MatLab.

The compounds were dissolved in NMR buffer (25 mM sodium phosphate, pH 5.8, and 5% (v/v) D₂O) and diluted to a final concentration of 500 µM. The samples were incubated at the indicated temperature for 10 min and chilled on ice for 5 min before measurement. NMR data were collected on a Bruker Ascend 850 MHz instrument fitted with a cryogenically cooled triple-resonance 5 mm TCI probe with pulsed-field gradients along the z-axis at 23 °C. The samples were measured by ¹H-NMR using the pulse sequence zgesgppe, allowing water suppression to be achieved using excitation sculpting with pulsed-field gradients and perfect echo. The spectra were acquired with 64 scans and a recovery delay of 3.9 s. The spectra were assigned using standard correlation methods. Resonances of interest were integrated using the program Dynamics Center 2.5 (Bruker; Bremen Germany) and compared to baseline spectra.