The present disclosure provides a compound of Formula (I), (II), or (III): ##STR00001##
an ionic derivative thereof, an isomer thereof, or a salt thereof. The present disclosure also provides conjugates of the compounds, and methods of using the compounds and the conjugates. The disclosure also provides the use of the compounds and conjugates in methods of sequencing nucleic acids.

The disclosed hemicyanine dyes contain an amino-chromene moiety as an electron donor, facilitating a variety of optical properties and uses in sensing applications.

The present disclosure is directed, in general, to synthesizing optionally substituted 2-dicyanomethylene-3-cyano-4-methyl-5-phenyl-5-perfluoromethyl-2,5-dihydrofuran comprising (i) reacting tributyl (1-ethoxyvinyl) tin with n-butyllithium at a temperature of between 30 C. and 10 C. to produce a first reaction product; (ii) reacting the first reaction product with 2,2,2-trifuoroacetophenone to produce a second reaction product: (iii) quenching the second reaction product with an acid to produce 3-hydroxy-3-phenyl-4,4,4-trifluoro-2-butanone; and (iv) reacting the 3-hydroxy-3-phenyl-4,4,4-trifluoro-2-butanone with malononitrile in the presence of base to produce 2-dicyanomethylene-3-cyano-4-methyl-5-phenyl-5-perfluoromethyl-2,5-dihydrofuran.

The present disclosure provides a compound of Formula (I), (II), or (III):

##STR00001##

an ionic derivative thereof, an isomer thereof, or a salt thereof. The present disclosure also provides conjugates of the compounds, and methods of using the compounds and the conjugates. The disclosure also provides the use of the compounds and conjugates in methods of sequencing nucleic acids.

A fluorescent dye, as well as a preparation method and uses thereof, wherein the fluorescent dye is sensitive and specific to viscosity and has low background fluorescence; it can also be used as a fluorescent activated and lighted probe used for fluorescent labeling, quantification or monitoring of protein, enzymes or nucleic acid.

The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.