Synthesis of NB and HA-NB

Methyl 4-(4-(hydroxymethyl)−2-methoxy-5-nitrophenoxy)butanoate (mNB) was synthesized as follows¹⁷: 4-hydroxy-3-methoxybenzaldehyde (vaniline) (8.90 g, 58.5 mmol, 1.06 eq.), methyl 4-bromobutanoate (9.89 g, 55.0 mmol, 1.0 eq.), and potassium carbonate (10.2 g, 73.8 mmol, 1.34 eq.) were dissolved in N, N-Dimethylformamide (DMF) (40 mL). The mixture was stirred at ambient temperature for 16 h, after which time the resulting solution was poured into chilled water (200 mL) and allowed to precipitate for 15 min at 0 °C. The solid was filtered off, washed with water, redissolved in dichloromethane, and dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a white solid (methyl 4-(4-formyl-2-methoxyphenoxy)butanoate, 12.2 g, 48.4 mmol, 88 %). Methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (9.4 g, 37.3 mmol, 1 eq.) was added slowly to a pre-cooled (−2 °C) solution of nitric acid (70%, 140 mL) and stirred at −2 °C for 3 h. It is important to note that—depending on the temperature of the nitration reaction—ipso substitution of the formyl moiety occurs. The resulting solution was poured into chilled water (500 mL) and allowed to precipitate for 15 min at 0 °C (note: as saponification of the product can occur under these conditions, the precipitation time should be kept as short as possible). The product was filtered, washed with water, and dissolved in dichloromethane. The organic layer was dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a slightly yellow powder (methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate, 7.7 g, 25.9 mmol, 69%). Sodium borohydride (1.50 g, 39.7 mmol, 1.5 eq.) was slowly added at 0 °C to a solution of methyl -(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (7.7 g, 25.9 mmol, 1.0 eq.) in EtOH/THF 1:1 v/v (100 mL). After 3 h, all solvents were removed in vacuo and the residue was suspended in water (50 mL) and dichloromethane (50 mL). The aqueous layer was extracted two times with dichloromethane (2 × 50 mL) and the combined organic layers were dried over magnesium sulfate. The solvent was removed under reduced pressure. In order to increase the overall yield and to remove partially saponified products, methanol (100 mL) and tosylic acid (50 mg) were added to the residue. The solution was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was suspended in water (50 mL) and dichloromethane (50 mL). The aqueous layer was extracted two times with dichloromethane (2 × 50 mL) and the combined organic layers were dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a yellow solid as a raw product, which was purified by column chromatography on silica gel using hexane/ethyl acetate = 1:1 (Rf = 0.6) and finally 5.22 g (4.81 mmol, 75%) of a slightly yellow powder mNB were obtained.

Then mNB (0.5 g, 1.8 mmol) and ethylenediamine (1.1 mL, 2 mmol, Sigma-Aldrich) were dissolved in methanol. The mixture was refluxed overnight until the starting individual components were undetectable by thin layer chromatography. After the reaction was complete, the solvent was evaporated under vacuum. The crude precipitate was dissolved in methanol and re-precipitated three times using ethyl acetate. The filter cake was then dried for 12 h at 30 °C under vacuum until NB appeared as light yellow powder (0.4 g, 1.2 mmol, 66.7%). HA-NB was synthesized according to a previous report¹⁷. Briefly, HA (408 mg, 1 mmol of disaccharide unit, Dongyuan Biotech, Zhenjiang) was dissolved in 50 mL deionized water at room temperature and NB (224 mg, 0.69 mmol) was added followed by HOBt (153 mg, 1 mmol, Sigma-Aldrich). The pH of the mixed solution was adjusted to pH 4.5, after which 1-(3-Dimethylaminopropyl)–3-ethylcarbodimide hydrochloride (200 mg, 1.04, Sigma-Aldrich) was added to the mixture and stirred for 48 h at room temperature. The solution was loaded into dialysis tubing (Molecular Weight (MW) cutoff 3500, Spectrum®) and dialyzed against diluted HCl (pH 3.5) containing 0.1 M NaCl for 2 days, then dialyzed against deionized water for a further 2 days. The solution was lyophilized and HA-NB was obtained in powder form. The substitution degree of nitrobenzyl groups (3% of HA disaccharide units) was verified by ¹H-nuclear magnetic resonance (NMR).