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

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Reaction Steps

The HA-AM obtained in the aforementioned (Example 5-1) 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 (hereinafter also referred to as “FITC”; manufactured by Pierce) that was 0.07 mole times 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 (manufactured by Wako Pure Chemical Industries, Ltd.) 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, the resultant was roughly purified by gel filtration using a PD-10 column (manufactured by Amersham Biosciences), and it was then dialyzed against large excess amount of 0.5 M NaCl aqueous solution for purification (Spectrapore 4; molecular weight cut off (MWCO): 12 k-14 kDa). Furthermore, the external dialysis solution was exchanged with large excess amount of 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.

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