Reaction paths to pure 5- and 6-aminofluorescein. Pitfalls encountered in conventional synthesis procedures

Abstract

Previously unrecognized pitfalls in the published procedure for the preparation of 5- and 6-isothiocyanatofluoresceins were examined. Hydrolysis of 5-nitrofluorescein diacetate with hot ethanol saturated with sodium hydroxide gave varying amounts of 5,5″-azoxydifluorescein in addition to 5-nitrofluorescein. The use of methanol and sodium hydroxide was found to give pure 5- and 6-nitrofluorescein from the corresponding diacetate. Further, reduction of 5-nitrofluorescein with hydrogen and W-2 Raney nickel gave a mixture of 5-aminofluorescein and 9-(4-amino-2-carboxyphenyl)-3,6-dihydroxyxanthene. A sodium sulphide–sodium hydrosulphide reduction system reduced 5-nitrofluorescein cleanly to the amine without reductive lactone cleavage. Synthesis and characterization of 5,5″-azoxydifluorescein, 9-(4-amino-2-carboxyphenyl)-3,6-dihydroxyxanthene, and related fluorescein and xanthene derivatives are described.