Synthesis and fluorescence properties of six fluorescein-nitroxide radical hybrid-compounds.

Abstract Six fluorescein-nitroxide radical hybrid-compounds ( 2ab , 3ab , 4 , and 5 ) were synthesized by the condensation of 5- or 6-carboxy-fluorescein and 4-amino-TEMPO ( 2ab ), 5- or 6-aminofluorescein and 4-carboxy-TEMPO ( 3ab ), and fluorescein and 4-carboxy-TEMPO ( 4 ), or by reaction of the 3-hydroxyl group of fluorescein with DPROXYL-3-ylmethyl methanesulfonate ( 5 ). Fluorescence intensities (around 520 nm) after reduction of the radical increased to 1.43-, 1.38-, and 1.61-folds for 2a , 2b and 3b respectively; 3a alone exhibited a decrease in intensity on reduction. Since 4 was readily solvolyzed in PBS or even methanol to afford fluorescein and 4-carboxy-TEMPO, its fluorescence change could not be measured. Hybrid compound 5 containing an ether-linkage between the fluorescein phenol and 3-hydroxymethyl-DPROXYL hydroxyl centers, was stable and on reduction, showed a maximum increase (3.21-fold) in relative fluorescence intensity in PBS (pH 5.0), despite its remarkably low absolute fluorescence intensity.