Asymmetric benzoxanthene dyes

Abstract

A class of asymmetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having the structure wherein Yand Yare individually hydroxyl, amino, imminium, or oxygen, R–Rare hydrogen, fluorine, chlorine, alkyl, alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and Ris acetylene, alkane, alkene, cyano, substituted phenyl, and combinations thereof. The invention further includes novel intermediate compounds useful for the synthesis of asymmetric benzoxanthene compounds having the general structure where substituents R–Rcorrespond to like-referenced substituents in the structure of described above, and Yis hydroxyl or amine. In another aspect, the invention includes methods for synthesizing the above dye compounds and intermediates. In yet another aspect, the present invention includes reagents labeled with the asymmetric benzoxanthene dye compounds, including deoxynucleotides, dideoxynucleotides, phosphoramidites, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.