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

HRID 2260197Notes

Reaction equation

HRID 2260197 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>CN(C=O)C.B([O-])([O-])[O-]>[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

5

Reactant 2

HCID 962WaterHCID 18730Fluorescein 5-isothiocyanate

Product 1

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

Reagents, catalysts & solvents 2

HCID 6228DimethylformamideSolventHCID 26574BorateSolvent

Procedure

A solution of fluorescein isothiocyanate (Aldrich) 10 μl of 10% solution in DMF) was added to the aminooligonucleotide (0.25 A260 units) in 0.2 M borate buffer, pH 8.6, 100 μl). The mixture was shaken in the dark overnight; then water (400 μl) was added and the mixture was desalted by gel filtration on a NAP-5 column (Pharmacia). The filtrate (1 ml) was collected, concentrated, and repurified by gel filtration. Reversed phase HPLC showed that the oligomers were converted essentially completely to the fluorescein conjugated derivatives: for the derivative of 3'-amino-oligomer 18, elution time 21.5 min (compared to 19.0 min for the starting amino-oligomer); for the derivative of 5'3'-diamino-oligomer 19, elution time 23.6 min (compared to 18.5 min for the starting diaminooligomer). The UV spectra show the presence of both the thymidine and fluorescein moieties: for derivative of 18, λmax 266, 456, 480 nm, λmin 240, 332, 468 nm; for derivative of 19, λmax 266, 460, 482, μmin 250, 332, 470 nm.

Workup

  1. filtrationthe mixture was desalted by gel filtration on a NAP-5 column (Pharmacia)
  2. customThe filtrate (1 ml) was collected
  3. concentrationconcentrated
  4. filtrationrepurified by gel filtration
  5. washfor the derivative of 3'-amino-oligomer 18, elution time 21.5 min (
  6. waitcompared to 19.0 min for the starting amino-oligomer
  7. washfor the derivative of 5'3'-diamino-oligomer 19, elution time 23.6 min (
  8. waitcompared to 18.5 min for the starting diaminooligomer

Sources & evidence

ORD record
ord-8c6cdc62263447318818bbadf1dc67f2
Source dataset
uspto-grants-1995_12
Patent number
US05476925

Reaction notes

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