Hydrochloric Acid → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid
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].[Al].Cl.[OH-].[Na+]>>[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]=1Participants
Procedure
Into a scintillation vial was placed 180 mgs of BSA (2.6×10-6Moles) (Pentex, crystallized) dissolved in 6 mls H2O containing 180 mgs K2CO3. To this was added 18.3 mgs fluorescein isothiocyanate (FITC) (4.85×10-5Moles) and the mixture was stoppered, covered with aluminum foil then placed in the center hole of a vortex mixer and shaken gently overnight at room temperature. The following morning the reaction mixture was acidified to pH 4 with 1N HCl (heavy precipitate), then made basic to pH 8 with 0.1N NaOH, applied to a 2.5×30 cm column of Sephadex G-10 and eluted with 0.05M PO4, pH 8.0 at 5.4 mls/hr, 0.9 ml/fraction. Fractions 43-72 were pooled. Hapten number was calculated from the UV absorption of the conjugate at 493 mμ and the extinction coefficient for protein-bound fluorescein of 7.2×104. Hapten number was thus calculated to be 14.5.
Workup
- custom180 mgs of BSA (2.6×10-6Moles) (Pentex, crystallized)
- dissolutiondissolved in 6 mls H2O containing 180 mgs K2CO3
- washeluted with 0.05M PO4, pH 8.0 at 5.4 mls/hr, 0.9 ml/fraction
- customHapten number was calculated from the UV absorption of the conjugate at 493 mμ
Sources & evidence
- ORD record
- ord-9b7fee49f63c4825acb0167cbe7524f3
- Source dataset
- uspto-grants-1979_07
- Patent number
- US04160016
Reaction notes
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