Ecx 500 nmr spectrometer

Melting points of the compounds were recorded on an XT-4 binocular microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China), and are uncorrected. Proton nuclear magnetic resonance (NMR) spectra were determined at 500 and 125 MHz using an ECX 500 NMR spectrometer (JEOL, Tokyo, Japan) in CDCl₃ solvent, using TMS as an internal standard. Infrared (IR) spectra were obtained on a Vector 22 Fourier transform infrared (FTIR) spectrometer (Bruker, Karlsruhe, Germany) in KBr disks. Elemental analyses were determined on an Elementar Vario-III CHN analyzer (Elementar Analysensysteme GmbH, Frankfurt, Germany). High resolution mass spectrometer (HRMS) data was conducted using a Thermo Scientific Q Exactive (Thermo, Waltham, MA, USA). Reaction progress was monitored by thin-layer chromatography (TLC) on silica gel GF254. Column chromatographic purification was carried out using silica gel (200–300 mesh, Qingdao Bangkai Hi-Tech materials Co., Ltd. Qingdao, Shandong, China). All solvents and reagents were of analytical reagent grade or chemically pure, and the solvents were dried in advance and distilled before use.

Folic acid, purchased from Sigma Aldrich, was used for the NMR experiment. The purity of the sample was 99.98%. NMR experiments were performed on a JEOL ECX 500 NMR spectrometer. Nearly 50 mg Folic acid was tightly packed in 3.2 mm JEOL double resonance MAS probe for performing solid state NMR experiments. ¹³C CP-MAS and ¹⁵N CP-MAS experiments were done at magic angle spinning speed 10 kHz. The CP contact time was 2 ms to satisfy the state of cross-polarization (CP). SPINAL-64 ¹H decoupling was used during acquisition. External referencing for ¹⁵N was done by NH₄Cl. ¹³C spin-lattice relaxation time was measured by the Torchia CP method.³⁵ The number of scans for ¹³C CP MAS experiment scan was 32 768 and for ¹³C 2DPASS CP MAS experiment number of scans was 4030. For measuring ¹³C relaxation by the Torchia CP method, the number of scans was 2048.

The melting points of the target products were measured using a WRX-4 micro-melting-point apparatus (Shanghai Yice Apparatus & Equipment Co., Ltd., Shanghai, China). ¹H and ¹³C nuclear magnetic resonance (NMR) spectral analyses were performed on a JEOL-ECX 500 NMR spectrometer using CDCl₃ or DMSO-d6 as the solvent and tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry (HRMS) was conducted on a LTQ Orbitrap (Thermo Scientific, Missouri, USA). The CD spectrum of helicase was performed through a JASCO J-1500 spectropolarimeter from JASCO (Tokyo, Japan) Co., Ltd. Binding studies were performed on a nanotemper monolith NT.115 instrument (Nanotemper, Munich, Germany) for microscale thermophoresis (MST). The ATPase activity experiment used enzyme assay reagent kits purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All analytical reagents were used in the experiment obtained from Energy Chemical (Shanghai Saen Chemical Technology Co., Ltd., Shanghai, China), without further drying or purification.