title: Thiolate and phosphorothioate functionalized fluoresceins and their use as fluorescent labels.
abstract: We report the syntheses of two new fluorescein derivatives, 3',6'-dihydroxy-3-oxo-2-[(phosphonothio)-acetyl]spiro[isobenzof uran- 1(3H),9'-9H-xanthene]-6-carboxylic acid hydrazide, disodium salt, a phosphorothioate fluorescein, and 3',6'-dihydroxy-3-oxo-2-(mercaptoacetyl)spiro[isobenzofuran-1(3H), 9'-9H- xanthene]-6-carboxylic acid hydrazide, a mercaptoacetyl fluorescein. The latter is derived from the first compound by hydrolysis of the phosphate. Direct nonenzymatic labeling of the maleimide-derivatized IgG molecule by the novel mercaptoacetyl fluorescein is discussed. We also present a new method of bioconjugating phosphorothioate-functionalized fluorophores to a maleimide-derivatized protein, based on the alkaline phosphatase-catalyzed hydrolysis of the S-P bond of the phosphorothioate and the concomitant liberation of the fluorophore thiolate. This last species reacts in situ with the maleimide on the protein. A high degree of conjugation control is achieved in that modulation of the stoichiometry of the label and enzyme results in incorporation from seven to eight fluorophores per protein, depending on the ratio of the phosphorothioate fluorescein to alkaline phosphatase. The quantum yield of the mercaptoacetyl fluorescein relative to 6-carboxyfluorescein is 0.22 and lambda exc = 494 nm and lambda em = 517 nm.
authors.affiliation: Abbott Laboratories, Department of Immunochemistry, Abbott Park, North Chicago, Illinois 60064-3500.
authors.forename: C
authors.initials: C
authors.lastname: Bieniarz
authors.forename: D F
authors.initials: DF
authors.lastname: Young
authors.forename: M J
authors.initials: MJ
authors.lastname: Cornwell
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000276:immunology; D005452:Fluoresceins / Q000138:chemical synthesis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006041:Goats; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis; D008301:Maleimides / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D006063:Chorionic Gonadotropin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D008301:Maleimides; C087610:3',6'-dihydroxy-3-oxo-spiro(isobenzofuran-1(3H),9'-9H-xanthene)-6-carboxylic acid 2-((phosphonothio)acetyl)hydrazide; C087611:3',6'-dihydroxy-3-oxospiro-(isobenzofuran-1(3H),9'-9H-xanthene)-6-carboxylic acid 2-(mercaptoacetyl)hydrazide; D000469:Alkaline Phosphatase