ASYMMETRIC BENZOXANTHENE DYES

A class of asymetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having structure (I) wherein Y1 and Y2 are individually hydroxyl, amino, imminium, or oxygen, R1-R8 are hydrogen, fluorine, chlorine, alkyl, alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and R9 is 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 genera l structure (II) where substituents R3-R7 correspond to like-referenced substituents in the structure of described above, Y2 is 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 reagent s labeled with the asymmetric bezoxanthene 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.