A mixture of 3',6'-dihydroxy-6-nitrospiro[2-benzofuran-3,9'-xanthene]-1-one (also known as 5-nitrofluorescein) and 3',6'-dihydroxy-5-nitrospiro[2-benzofuran-3,9'-xanthene]-1-one may be prepared by reacting 4-nitrophthalic acid (or 4-nitrophthalic anhydride) with benzene-1,3-diol (resorcinol) in methanesulphonic acid. Preferably, the methanesulphonic acid is substantially pure (being not less than 95% w/w), and in a ratio of 2 to 10 v/w relative to the nitrophthalic acid/anhydride. The reaction may be conducted at 60° to 120°C and quenched by adding it to a solvent, such as water, to precipitate the product. The ratio of water to methanesulphonic acid is preferably 4.5 w/w or less, the temperature of the water is preferably 0° to 10°C before quenching and the aqueous phase is maintained no higher than 30°C during quenching. The product may be isolated by filtration or centrifugation, and dried in vacuum at up to 70°C. The isolated product may be repeatedly heated in water at 80° to 100°C with agitation in order to reduce the level of phenolic esters of methanesulphonic acid (e.g. to below 5%) by hydrolysis of such esters. The thus obtained nitrofluoresceins may be used to prepare fluorescein 5-isothiocyanate and fluorescein lisicol (cholyl-lysylfluorescein, CLF).
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