Fluorescein 3′,6′-diacetate → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

HRID 433041Notes

Reaction equation

HRID 433041 reaction equation
CC([O:4][C:5]1[CH:6]=[CH:7][C:8]2[C:18]3([O:27][C:25](=[O:26])[C:24]4[CH:23]=[CH:22][CH:21]=[CH:20][C:19]3=4)[C:17]3[CH:16]=[CH:15][C:14]([O:28]C(C)=O)=[CH:13][C:12]=3[O:11][C:9]=2[CH:10]=1)=O>CS(C)=O>[CH:21]1[CH:22]=[CH:23][C:24]([C:25]([OH:27])=[O:26])=[C:19]([C:18]2[C:8]3[CH:7]=[CH:6][C:5]([OH:4])=[CH:10][C:9]=3[O:11][C:12]3[C:17]=2[CH:16]=[CH:15][C:14]([CH:13]=3)=[O:28])[CH:20]=1

Participants

3

Reactant 1

HCID 65047Fluorescein 3′,6′-diacetate

Product 1

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

Reagents, catalysts & solvents 1

HCID 679Dimethyl SulfoxideSolvent

Conditions

Temperature
-20 °C

Procedure

Fluorescein diacetate (FDA) (Sigma Chemical Co., St. Louis, Mo.), or 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Molecular Probes, Inc., Eugene, Oreg.) was dissolved in dimethyl sulfoxide (DMSO) to produce a 1 mg/ml stock solution. The stock was filtered through 0.8 μm nylon, aliquoted into 1.5 ml microfuge tubes and stored frozen at −20° C. in the dark. For individual assays, an intermediate stock was prepared by thawing and diluting an aliquot with RPMI-1640+10% Fetal Calf Serum (FCS) such that adding 50 μl of dye solution to a well would produce a final concentration of 8 μg/ml. To minimize reaction with endogenous enzymes in the FCS (which could increase the background fluorescence by cleaving ester groups to form free fluorescein), intermediate dilutions were prepared immediately prior to loading each individual plate.

Workup

  1. customto produce a 1 mg/ml stock solution
  2. filtrationThe stock was filtered through 0.8 μm nylon
  3. customFor individual assays, an intermediate stock was prepared by thawing
  4. additiondiluting an aliquot with RPMI-1640+10% Fetal Calf Serum (FCS) such that
  5. customwould produce a final concentration of 8 μg/ml
  6. customreaction with endogenous enzymes in the FCS (which
  7. temperaturecould increase the background fluorescence

Sources & evidence

ORD record
ord-ddc4957015ec40468e02a4a583b75244
Source dataset
uspto-grants-2003_12
Patent number
US06665430B2

Reaction notes

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