Synthesis of a New Tetradentate ß-Diketonate-Europium Chelate That Can Be Covalently Bound to Proteins in Time-Resolved Fluorometry

Abstract

A new tetradentate β-diketone chelator for europium: 1, 10-bis (4-chlorosulfo-1′, 1-Biphenyl-4′-yl)-4, 4, 5, 5, 6, 6, 7, 7-octafluorodecane-1, 3, 8, 10-tetraone (BCDOT), was synthesized. It forms a stable chelate with europium(III) and can be used as a fluorescence label for time-resolved fluorescence spectrometry. BCDOT can be covalently bound to a protein under relatively mild conditions, and forms a strongly fluorescent chelate having a lifetime of 260μs in Tris-HCl buffer and 290μs in carbonate buffer solution containing topo (trioctylphosphine oxide) and SDS (sodium dodecylsulfate), when complexed with europium(III). As a model reaction, bovine serum albumin (BSA) was labeled with BCDOT in carbonate buffer solution. Fluorescence spectrometric determination of the BSA(BCDOT)n-Eu3+ solution was carried out. Detection limits of 4.5×10-13M (n=47) in 0.1M Tris-HCl solution of pH 9.1, and 8.3×10-14M (n=47) in 0.1M carbonate buffer solution of pH 8.4 containing 1.0×10-5M topo and 0.05% SDS, for BSA were obtained by the method. These results show that a tetradentate chelate is superior to hitherto used bidentate ligands, giving ca. two orders of magnitude stronger fluorescence.