Avance 500 spectrometer

Specific optical rotation was measured on a PerkinElmer 341 instrument at 25°C. Cary 5000 spectrophotometer was used to record UV spectra in MeOH. ECD data were obtained by Model 420SF CD spectrometer (Aviv Biomedical Inc). In KBr discs, IR spectra were obtained using Fourier infrared IS50 spectrometer. All NMR experiments were performed at room temperature on a Bruker AVANCE 500 spectrometer using the signals of residual solvent protons (CDCl₃: δ_H 7.26; CD₃OD: δ_H 3.31) and carbons (CDCl₃: δ_C 77.1; CD₃OD: δ_C 49.2). HRESIMS spectra were tested by Waters TQ-XS mass spectrometer. Column chromatography (CC) was conducted by silica gel (200–300 mesh, Yantai Huiyou Silica gel company) and Sephadex LH-20 (CHCl₂/MeOH, v/v 1:1) (Pharmacia Sweden). On silica gel plates, thin layer chromatography (TLC) was conducted (GF 254 Silica gel Thin Layer Plate Yantai Huiyou Silica company). The Typical Culture Preservation Committee Cell Bank, China provided RAW264.7 cells; the fetal bovine serum (FBS) was obtained by Gibco; ProCell provided Dulbecco's modified Eagle's medium (DMEM); Sigma supplied LPS and _L-NMMA; Shanghai Beyotime Biotechnology supplied the NO kit. Thermo Fisher Scientific (Shanghai, China) provided the primers for iNOS. Cell Signaling (Beverly, MA, USA) supplied all the antibodies.

NMR spectra were acquired by using an Avance-500 spectrometer (Bruker, Karlsruhe, Germany) equipped with a cryogenic probe that fits 5-mm-diameter NMR tubes (CPDUL). ¹H-NMR spectra were collected by using the Bruker pulse program, zgpr, which uses solvent pre-saturation to remove the residual-water signal. The acquisition was done in acquisition mode with a spectral width of 20 ppm, in digital quadrature detection, with a proton 90° pulse value of 15 μs, an offset frequency of 4.7 ppm, a 4 s relaxation delay, a 20 s increment delay, 65,536 data points, and 128 scans. Two-dimensional NMR spectra were recorded on an Avance-800 spectrometer with a cryogenic probe that fits 5-mm-diameter NMR tubes (CPTCI). ¹H-¹³C HSQC spectra were collected by using echo-anti-echo gradient selection (from the hsqcetgpsisp pulse program in the Bruker library) with 90° pulse values of 15 µs for protons and 15 µs for carbon, a 75 (f1) and 4.7 (f2) ppm offset frequency, a 2 s relaxation delay, a 160 (f1) and 12 (f2) ppm spectral width, 512 (f1) and 1,024 (f2) data points and 64 scans. When appropriate, to support metabolite annotation with the HSQC spectrum, other 2D NMR spectra (¹H-¹H DQF-COSY, TOCSY) were also recorded with the same instrument. The chemical shifts were calibrated by taking the signal of the DSS methyl group to be 0.00 ppm for ¹H and ¹³C.

Nuclear magnetic resonance (¹H-NMR) was accomplished with the Avance 500 spectrometer (Bruker Biospin, Rheinstetten, Germany), equipped with a 500 MHz magnet and a triple resonance inverse (TXI) probe. The experiment was carried out at room temperature with deuterated chloroform as solvent and tetramethylsilane as internal standard.

Aspiletrein A (AA), a dry powder, was isolated and characterized as previously described [25 (link)]. The structure was determined by NMR analysis on a Bruker Avance 500 spectrometer (Bruker, MA, USA), and the HRESIMS was recorded on an Agilent 6545 accurate-mass spectrometer (Agilent, CA, USA). AA: White amorphous powder [α]²⁵_D − 92.0 (c 0.1, MeOH); IR (KBr) ν_max (cm^− 1): 3422, 2928, 1632, 1454, 1379, 1344, 1221, 1165, 1049, 986, 918, 847, 810; ¹H NMR (500 MHz, pyridine-d₅) and ¹³C NMR (125 MHz, pyridine-d₅), HRESIMS m/z 891.4522 [M + Cl]⁻ (calcd. For C₄₄H₇₂O₁₆Cl, 891.4509) [25 (link)]. AA was dissolved in DMSO to yield a stock solution and further diluted in complete medium to the desired concentrations before use. The control samples in the experiments were incubated with 0.1% DMSO in the culture medium. The final concentration of DMSO was less than 0.1%, which showed no toxicity.
Hoechst 33342, propidium iodide, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). Rabbit anti-phosphorylated Akt (S473), rabbit anti-Akt and HRP-linked anti-rabbit IgG were purchased from Cell Signaling Technology (Beverly, MA, USA).

Each purified oligosaccharide sample (50 mg) with different degree of polymerization was loaded into nuclear magnetic tube and dissolved in D₂O at room temperature. The ¹H spectrum and ¹³C nuclear magnetic resonance (NMR) spectrum were recorded on a Bruker AVANCE 500 spectrometer (Bruker BioSpin Corporation, Billerica, MA, United States).