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

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Reaction Steps

Each of the HA-DETA, HA-DAHP, and HA-DAP obtained in Example 4-2 and Example 4-3-1 was dissolved in distilled water (Milli Q water), so as to prepare a solution of a concentration of 20.0 mg/ml. Thereafter, a 0.2 M carbonate buffer (pH 9.0) was added to the prepared solution, resulting in a concentration of 10.0 mg/ml. Thereafter, fluorescein isothiocyanate that was 0.07 mole times to an HA unit was dissolved in DMSO that was 1/10 the volume of a modified HA 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 was dissolved in DMSO that was 1/10 the volume of the 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 30 minutes. Thereafter, each sample was dialyzed against large excess amount of 0.5 M NaCl aqueous solution for purification, and the external dialysis solution was then exchanged with distilled water (Milli Q water), followed by further dialysis and purification (Spectrapore 4; molecular weight cut off (MWCO): 12 k-14 kDa). The obtained aqueous solution was freeze-dried, so as to obtain a fluorescein-labeled modified HA.

Provenance & Evidence