Avance 3 hd 600 nmr spectrometer

The method to synthesize HA-SH was modified according to our earlier publications (Li et al., 2017 (link)). In brief, 1.0 g HA was dissolved in 250 ml deionized water, EDAC and NHS was then added at a final concentration of 50 mmol/L. 1 mol/L HCl was dropwise added to adjust the pH of the reaction solution to 5∼6 and stirred for 30 min. Afterwards, 2.0 g L-cysteine hydrochloride monohydrate (L-cys) was added to the reaction system and 1 mol/L NaOH was added dropwise into the mixture until the pH was adjusted to 5.0. After reacting for 5 h under stirring, the product was dialyzed (MWCO 14 kDa) against HCl solution (pH = 5, containing 1 wt% NaCl), and finally freeze-dried. ¹H NMR spectra of HA and HA-SH were obtained on an AVANCE III HD 600 NMR spectrometer (Bruker, Germany). The composition of the HA-SH and composite hydrogel with different precursor concentration was characterized by FT-IR with an attenuated total reflectance (ATR) accessory. The free sulfhydryl content of HA-SH prepared by Ellman’s reagent was determined by reference method (Li et al., 2017 (link)).

The analyses and structural identification of metabolites were performed by the combinational use of HPLC, high resolution electrospray ionization mass spectroscopy (HR-ESI-MS), and nuclear magnetic resonance (NMR), as described previously [3 (link),11 (link),23 (link),25 (link),28 (link)]. Briefly, analytical HPLC was performed in an Agilent HPLC 1200 system (Agilent, Waldbronn, Germany) equipped with a SilGreen C18 column (250 × 4.6 mm id, 5 μm particle size). The mobile phase was composed of 0.1% trifluoroacetic acid in H₂O (solvent A) and acetonitrile (solvent B). The gradient elution was as follows: 0–5 min, 15% B; 5–20 min, 50% B; 20–28 min, 100% B. The flow rate was 1.0 mL/min. The injection volume was 50 μL and the effluents were monitored at 25 °C by a DAD detector at 243 nm. Glucosylated products were collected on an SEP LC-52 system (SEP. Co. Ltd., Beijing, China) with a YMC C18 preparative column (250 × 10.0 μm ID, 5 μm; YMC Co. Ltd., Kyoto, Japan). The data collection for HR-ESI-MS were carried out on a Thermo Scientific Exactive Orbitrap LC-Mass spectrometer (Thermo Scientific, Waltham, MA, USA). ¹H-NMR (600 MHz), ¹³C-NMR (151 MHz), and 2D-NMR spectrometric data were recorded with AVANCE III HD 600 NMR spectrometer (Bruker, Rheinstetten, Germany). Chemical shifts are given in δ (ppm) with the solvent (CD₃OD-d4) peaks as references.

Dissolving 1 g HA in 100 ml DI water, add 5 ml 0.5 M sodium periodate, stir at 250 rpm for 2 h at RT, and protect from light. 1 ml of ethylene glycol was then added to deactivate the unreacted periodate. The samples were dialyzed using DD water for 3 days. The sample was removed from the completed dialysis, freeze-dried, and AHA was obtained. Afterward, dissolve 1 g AHA in 100 ml DI water and stir until completely dissolved. Add 1 ml of methacrylate and react for 12 h at pH = 8.0–8.5. The whole process is carried out on the ice to ensure a cryogenic environment. After the reaction, the solution was dialyzed for 2 days and freeze-dried to obtain AHAMA. 1H nuclear magnetic resonance (1H NMR, 500 MHz) spectra were recorded on an AVANCE III HD 600 NMR spectrometer (Bruker, Germany).