Synthesis and fluorescent properties of novel biotinylated labels

Two novel biotinylated fluorophores, (2-biotinyloxyethyl) acridine-9-carboxylate (4a) and (2-biotinylamidoethyl) acridine-9-carboxamide (4b) have been synthesized and their fluorescent properties examined, in the presence and absence of avidin. In aqueous solutions the biotinylated conjugates 4, as well as their precursors 3, exhibit intense fluorescence and can be detected down to nanomolar concentrations. A four-atom spacer, ethylene glycol, in the case of 4a and ethylene diamine, in the case of 4b was chosen in order to minimize steric repulsion between two biotin-acridine conjugates adjacently bound on avidin. In the presence of avidin, they show fast and stoichiometric binding to the tetrameric protein. In contrast to most known biotin-fluorophore conjugates, the novel fluorescent labels 4a and 4b not only retain their fluorescence after binding to avidin but fluorescence is also enhanced by 37 and 16.5%, respectively, even when four ligands are bound per avidin tetramer. Consequently, these novel biotin-fluorophore conjugates are prospective fluorescent labels in bioanalytical applications.