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

This example illustrates a method of preparing an embodiment of the compound of formula II, namely, fluorescein diphosphate tetraethyl ester. 2.5 g of fluorescein (7.5 mmol) is suspended in THF (60 mL) and anhydrous CH2CI2 (mL). 3.1 g of 1H-tetrazole 3.1 g.(44 mmol) is added and stirred at room temperature for ˜1.5 hours or until the reaction mixture becomes a transparent dark yellowish solution. The resulting solution is cooled to 0° C. and 5.0 g of diethyl N. N-diisopropylphosphoramidite (23 mmol) is added dropwise over a period of 3-4 minutes to yield a very light yellow solution. The cooling bath is removed and the reaction stirred at room temperature until TLC shows no starting material remains (˜5 minutes). The reaction is cooled back to 0° C. Rf (fluorescein diphosphite, tetraethyl ester)=0.7-0.75, a quenching spot that becomes fluorescent yellow upon heating; hexanes/EtOAc (3:2) A solution of 3-chloroperoxybenzoic acid (MCPBA) (5.5 g, 32 mmol) in CH2Cl2/CHCl3 (9:1, 50 mL) is prepared, and washed with saturated NaCl (1×50 mL), followed by drying over Na2SO4. The dried solution is added to the 0° C. reaction mixture to yield a cloudy yellowish solution. The cooling bath is removed and the reaction stirred at room temperature until TLC indicates completion (˜10 minutes). The solvent is removed from the reaction mixture in vacuo and the resulting yellow gum is dissolved in EtOAc (˜100 mL). The solution is washed with 10-20% sodium thiosulfate/H2O (1×100 mL), saturated NaHCO3 (1×100 mL), and saturated NaCl (1×100 mL), and dried over Na2SO2. The solvent is removed in vacuo. At this point, TLC in hexanes/EtOAc (3:2) shows two spots: Rf-0.65-0.7, a quenching spot that becomes fluorescent upon heating; and Rf-0.95-1.0, a quenching spot that does not fluoresce upon heating. 50 mL of methanol are added to the gum to precipitate the high-Rf material. The solid is removed by filtration, and methanol is removed from the filtrate in vacuo. The resulting product is purified by column chromatography under the following conditions:

1. addition(44 mmol) is added
2. temperatureThe resulting solution is cooled to 0° C.
3. customto yield a very light yellow solution
4. customThe cooling bath is removed
5. stirringthe reaction stirred at room temperature until TLC
6. custom(˜5 minutes)
7. temperatureThe reaction is cooled back to 0° C
8. temperatureupon heating
9. washwashed with saturated NaCl (1×50 mL)
10. dry with materialby drying over Na2SO4
11. additionThe dried solution is added to the 0° C. reaction mixture
12. customto yield a cloudy yellowish solution
13. customThe cooling bath is removed
14. stirringthe reaction stirred at room temperature until TLC
15. customcompletion (˜10 minutes)
16. customThe solvent is removed from the reaction mixture in vacuo
17. dissolutionthe resulting yellow gum is dissolved in EtOAc (˜100 mL)
18. washThe solution is washed with 10-20% sodium thiosulfate/H2O (1×100 mL), saturated NaHCO3 (1×100 mL), and saturated NaCl (1×100 mL)
19. dry with materialdried over Na2SO2
20. customThe solvent is removed in vacuo
21. temperatureupon heating
22. temperatureupon heating
23. addition50 mL of methanol are added to the gum
24. customto precipitate the high-Rf material
25. customThe solid is removed by filtration, and methanol
26. customis removed from the filtrate in vacuo
27. customThe resulting product is purified by column chromatography under the following conditions

ORD record

ord-41875742fda14aca806b5f4b20217a41

Source dataset

uspto-grants-2008_05

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07374900B2