Fluorescein 5-isothiocyanate → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

HRID 36559

Reaction equation

HRID 36559 reaction equation
[CH:1]1[C:6](N=C=S)=[CH:5][C:4]2[C:10]([O:12][C:13]3([C:23]4[CH:24]=[CH:25][C:26]([OH:28])=[CH:27][C:22]=4[O:21][C:15]4[CH:16]=[C:17]([OH:20])[CH:18]=[CH:19][C:14]3=4)[C:3]=2[CH:2]=1)=[O:11].O[N:30]1[C:51](=[O:52])[NH:50][C@H:49]2[C@@H:31]1[CH2:32][S:33][C@H:34]2[CH2:35][CH2:36][CH2:37][CH:38](N1C(=O)CCC1=O)[C:39](=[O:41])[OH:40]>OP([O-])(O)=O.OP([O-])([O-])=O.[Na+].[Na+].[Na+].[Cl-].[Cl-].[K+].[K+].CN(C)C=O.CS(C)=O>[OH:41][C:39]([CH2:38][CH2:37][CH2:36][CH2:35][C@H:34]1[C@@H:49]2[C@@H:31]([NH:30][C:51]([NH:50]2)=[O:52])[CH2:32][S:33]1)=[O:40].[CH:1]1[CH:6]=[CH:5][C:4]([C:10]([OH:12])=[O:11])=[C:3]([C:13]2[C:14]3[CH:19]=[CH:18][C:17]([OH:20])=[CH:16][C:15]=3[O:21][C:22]3[C:23]=2[CH:24]=[CH:25][C:26]([CH:27]=3)=[O:28])[CH:2]=1

Participants

7

Reactant 2

HCID 18730Fluorescein 5-isothiocyanateHCID 129676281N-Hydroxysuccinimido-d-biotin

Product 2

HCID 33832-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acidHCID 171548Biotin

Reagents, catalysts & solvents 3

HCID 679Dimethyl SulfoxideSolventHCID 6228DimethylformamideSolventHCID 24978514Phosphate Buffered SalineSolvent

Procedure

Biotin and fluorescein labeled insulin was prepared. To 1 ml of a 1 mg/ml solution of insulin protein in phosphate buffered saline was added simultaneously 100 μl of a 1 mg/ml solution of fluorescein isothiocyanate (FITC) in dimethylformamide (DMF) and 100 μl of a 1 mg/ml solution of N-hydroxysuccinimido biotin in dimethylsulfoxide (DMSO). The two labeling reagents were allowed to react at room temperature for 4 hours, after which the unreacted reagents were quenched with 10 μl ethanolamine. The quenched reaction mixture was then dialyzed against double distilled water until unreacted fluorescein derivatives no longer dialyzed into the water. The covalent attachment of biotin and fluorescein to the desired protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis.

Workup

  1. customto react at room temperature for 4 hours
  2. customafter which the unreacted reagents were quenched with 10 μl ethanolamine
  3. customThe quenched reaction mixture
  4. customwas then dialyzed
  5. distillationagainst double distilled water until unreacted fluorescein
  6. customderivatives no longer dialyzed into the water

Sources & evidence

ORD record
ord-58ea642946594e2098e864896dd7741c
Source dataset
uspto-grants-1995_05
Patent number
US05416016

Linked notes

Loading…