Jnm ecp 600

A HA–GA conjugate was synthesized from HA (70 kDa) and GA. GA in methanol was activated with DMT–MM to form active ester. After the rotary evaporation of methanol, the active ester was slowly added to ethylene diamine solution, and the mixture was stirred at room temperature overnight. The diamine-modified GA (GA-N) was purified through column chromatography. The HA–GA conjugate was synthesized by modifying GA-N to the backbone of HA in the presence of DMT–MM. After freeze-drying, the chemical structure of the GA-HA conjugate was determined via ¹H-NMR (JNMECP-600, JEOL, Tokyo, Japan), for which the sample was prepared by dissolving the conjugate in D₂O. The routes of synthesis are illustrated in Figure 1.
A HA–His conjugate was synthesized as previously described with some modifications [30 (link)]. In brief, HA in formylamine was activated by EDC and NHS (mol_HA:mol_EDC:mol_NHS = 1:1.2:1.2). Then, His (the molar ratio between the carboxyl group of HA and the amino group of His was 1:6) in N,N-dimethylformamide (DMF) was slowly added to the HA solution. The reaction mixture was stirred for 24 h at room temperature under nitrogen atmosphere. After filtration, the solution was freeze-dried to obtain the HA–His conjugate.

For FTIR analysis, the dried samples (1–2 mg) were mixed with 100 mg dried KBr and pressed to a transparent pellet and then analyzed with a Nicolet Nexus 470 FTIR spectrometer (Thermo Electron) under dry air at 400–4000 cm⁻¹. And For ¹H-NMR and ¹³C-NMR analyses, the sample was dissolved in D₂O (99.96%) and freeze-dried twice to replace all exchangeable protons with deuterium. Spectra were acquired at 25 °C using JNM-ECP 600 MHz equipment (JNM-ECP 600, Jeol, Japan). Chemical shift values were calibrated using acetone-d6 as an internal standard. The data were processed using the MestReNova software.