Succinic anhydride → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

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Reaction Steps

Each of the HA-AM as obtained above (Example 6-1) was dissolved in distilled water (Milli Q water), resulting in a concentration of 20.0 mg/ml. Thereafter, a 0.2 M carbonate buffer (pH 9.0) was added to the obtained solution, resulting in a concentration of 10.0 mg/ml. Thereafter, fluorescein isothiocyanate that was 0.07 mole times (69% and 90.5% amine-modified HA solutions), 0.175 mole times (54.5% amine-modified HA solution), 0.28 mole times (36.5% amine-modified HA solution), and 0.63 mole times (20.0% amine-modified HA solution) to an HA unit (1 unit=a repeating unit, N-acetylglucosamine-glucuronic acid) was dissolved in DMSO that was 1/10 the volume of an HA-AM solution. The DMSO solution was then added to the above solution, and the obtained mixture was then stirred in the dark at room temperature for 1 hour. Thereafter, succinic anhydride that was 40 mole times to the HA unit (1 unit=a repeating unit, N-acetylglucosamine-glucuronic acid) was dissolved in DMSO that was 1/10 the volume of the HA-AM solution. The DMSO solution was then added to the above reaction solution, and the obtained mixture was then stirred in the dark at room temperature for 30 minutes. The 20% amine modified product was first dialyzed against large excess amount of DMSO, and was then dialyzed against large excess amount of 25% EtOH aqueous solution. On the other hand, other samples were directly dialyzed against large excess amount of 25% EtOH aqueous solution (Spectrapore 4; molecular weight cut off (MWCO): 12 k-14 kDa). Thereafter, these solutions were dialyzed against a 0.5 M NaCl aqueous solution for purification. Furthermore, the external dialysis solution was exchanged with distilled water (Milli Q water), and dialysis was carried out in the dark for purification. The obtained aqueous solution was freeze-dried, so as to obtain fluorescein-labeled HA-AM-SUC.

Provenance & Evidence