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

HRID 1099116Notes

Reaction equation

HRID 1099116 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-isothiocyanateHCID 19660Carbonate IonHCID 86631038HCID 86631038, compound2×

Product 1

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

Reagents, catalysts & solvents 2

HCID 679Dimethyl SulfoxideSolvent4×HCID 962WaterSolvent

Procedure

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.

Workup

  1. customEach of the HA-AM as obtained above (Example 6-1)
  2. customresulting in 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. customThe 20% amine modified product was first dialyzed against large excess amount of DMSO
  6. customwas then dialyzed against large excess amount of 25% EtOH aqueous solution
  7. customOn 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)
  8. customThereafter, these solutions were dialyzed against a 0.5 M NaCl aqueous solution for purification
  9. distillationFurthermore, the external dialysis solution was exchanged with distilled water (Milli Q water), and dialysis
  10. customdark for purification
  11. customThe obtained aqueous solution was freeze-dried

Sources & evidence

ORD record
ord-7019ffacf53a49b5ac68c506b9292f27
Source dataset
uspto-grants-2012_03
Patent number
US08143391B2

Reaction notes

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