THIOLATE AND PHOSPHOROTHIOATE FUNCTIONALIZED FLUORESCEINS AND THEIR USE AS FLUORESCENT LABELS

被引:5
作者
BIENIARZ, C
YOUNG, DF
CORNWELL, MJ
机构
[1] Abbott Laboratories, Diagnostics Division, Department of Immunochemistry, North Chicago, Illinois 60064-3500, Abbott Park
关键词
D O I
10.1021/bc00025a005
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We report the syntheses of two new fluorescein derivatives, 3',6'-dihydroxy-3-oxo-2-[(phosphonothio)-acetyl]spiro[isobenzofuran-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.
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页码:31 / 39
页数:9
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