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

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Reaction Steps

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:

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