Synthesis of a fluorescent xanthenic derivative useful for labeling amine residues

The 2,5-dioxopyrrolidin-1-yl-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)-3- methylbenzoate has been synthesized as an amine-reactive derivative able to yield stable covalently labeled biopolymers. The new derivative has been used to label polyribocytidilic acid (5′), poly(C), amine residues. TG-II-poly(C) exhibits monoexponential decay at the physiological pH range. In addition, both steady-state fluorescence intensity and fluorescence decay are also sensitive to solution pH. The large decrease in steady-state fluorescence upon hybridization allows it to be used as a nucleic acid probe in a homogeneous assay format. In summary, we report an efficient synthesis to obtain labeled RNA from commercially available materials in excellent yields.