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

HRID 1055276Notes

Reaction equation

HRID 1055276 reaction equation
C(=O)([O-])[O-].[CH:5]1[C:10](N=C=S)=[CH:9][C:8]2[C:14]([O:16][C:17]3([C:27]4[CH:28]=[CH:29][C:30]([OH:32])=[CH:31][C:26]=4[O:25][C:19]4[CH:20]=[C:21]([OH:24])[CH:22]=[CH:23][C:18]3=4)[C:7]=2[CH:6]=1)=[O:15].C(N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1O)(=O)C.O=C[C@@H]([C@H]([C@@H]([C@@H](C(O)=O)O)O)O)O.C1(=O)OC(=O)CC1>O.CS(C)=O>[CH:5]1[CH:10]=[CH:9][C:8]([C:14]([OH:16])=[O:15])=[C:7]([C:17]2[C:18]3[CH:23]=[CH:22][C:21]([OH:24])=[CH:20][C:19]=3[O:25][C:26]3[C:27]=2[CH:28]=[CH:29][C:30]([CH:31]=3)=[O:32])[CH:6]=1

Participants

7

Reactant 4

HCID 7922Succinic anhydrideHCID 18730Fluorescein 5-isothiocyanate2×HCID 19660Carbonate IonHCID 86631038HCID 86631038, compound

Product 1

HCID 33832-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

Reagents, catalysts & solvents 2

HCID 679Dimethyl SulfoxideSolvent4×HCID 962WaterSolvent

Procedure

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.

Workup

  1. customThe HA-AM obtained in the aforementioned (Example 5-1)
  2. customso as to prepare a solution of a concentration of 20.0 mg/ml
  3. customresulting in a concentration of 10.0 mg/ml
  4. stirringthe obtained mixture was then stirred in the dark at room temperature for 30 minutes
  5. filtrationThereafter, the resultant was roughly purified by gel filtration
  6. customwas 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)
  7. distillationFurthermore, the external dialysis solution was exchanged with large excess amount of distilled water (Milli Q water), and dialysis
  8. customdark for purification
  9. customThe obtained aqueous solution was freeze-dried

Sources & evidence

ORD record
ord-92144f3505384b938df15ea86e95f48a
Source dataset
uspto-grants-2016_07
Patent number
US09394379B2

Reaction notes

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