The use of 6-carboxynaphthofluorescein phosphoramidite in the automated synthesis of quencher-dye oligonucleotide probes (QDOPs)

Real-time detection and quantitation of specific amplicons have been achieved using quencher and dye-labeled oligonucleotides such as molecular beacons. The molecular beacon molecule has a fluorescent reporter dye at the 5′-end and a quencher at the 3′-end. When the closed molecular beacon is excited by irradiation, the reporter fluorescence is greatly reduced by quenching through the process of fluorescence resonance energy transfer. When the molecular beacon hybridizes to the target, the stem loop opens making the fluorophore and quencher spatially distinct, thus increasing the reporter dye fluorescence intensity. Labeling of dyes to 5′-end of oligonucleotides has been done typically using manual methods, it is possible to do manual coupling at the milligram scale. Described here is the development of a scalable process for oligonucleotide labeling, which is robust, and has been achieved for 6-carboxynaphthofluorescein by connecting it to a corresponding phosphoramidite [Theisen, P.; McCollum, C.; Upadhya, K.; Jacobson. K.; Vu, H.; Andrus, A.; Tetrahedron Lett. 1992, 33, 5033–5036].