Azadioxatriangulenium (ADOTA) is a fluorescent triangulenium dye with a long fluorescence lifetime, highly polarized transitions and emission in the red part of the visible spectrum. These properties make the chromophore suited for application in fluorescence polarization/anisotropy assay. To be useful for these applications, reactive forms of the dyes must be available in significant quantities. Here, the synthesis and photophysical properties of amine-reactive NHS esters and a thiol-reactive maleimide derivate of ADOTA are reported. The synthesis involves two steps of nucleophilic bridge forming reactions starting from tris(2,6-dimethoxyphenyl) methylium tetrafluoroborate, which can readily be made on 100 gram scale. In the third and final step the reactive NHS or maleimide groups are formed. The beneficial photophysical properties of the ADOTA chromophore are maintained in these derivatives, and we conclude that these systems are ideal to study protein motion and protein-protein interactions for systems of up towards 1000 kDa.
Keywords: Bioconjugate chemistry; Fluorescent dyes; Nucleophilic aromatic substitution; Organic synthesis; Triangulenium.