Asymmetric benzoxanthene dye labelling reagents

摘要

A class of asymmetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having the structure ;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 the general structure ;where substituents R₃-R₇ correspond to like-referenced substituents in the structure of described above, and 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 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.