Xanthene dyes

Abstract

The invention provides a novel class of xanthene dyes, some of which are functionalized to allow their coupling to conjugation partners of interest, e.g. biomolecules, drugs, toxins and the like. Also provided are conjugates of the dyes, methods of preparing and using the dyes and their conjugates and kits including the dyes and their conjugates.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a Continuation of U.S. patent application Ser. No. 10 / 824,175, filed on Apr. 13, 2004 which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 60 / 541,686, filed on Feb. 3, 2004, the disclosures of which are incorporated by reference herein in their entirety for all purposes.FIELD OF INVENTION[0002]The present invention relates generally to the synthesis of fluorescent compounds that are analogues of xanthene dyes. The compounds of the invention are fluorophores that are derivatized to allow their facile attachment to a carrier molecule.BACKGROUND[0003]There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of nucleic acids, peptides, saccharides, pharmaceuticals, metabolites, microo...

AI Technical Summary

Benefits of technology

[0023]The inventors have prepared a class of xanthene-based fluorophores modified with a versatile linker arm, the structure of which is readily alterable, allowing the conjugation of the label to a variety of positions, through diverse functional groups, on a carrier molecule. The xanthene-based labels are readily attached to a carrier molecule using techniques well known in the art, or modifications of such techniques that are well within the abilities of those of skill in the art. The versatility of the labels set forth herein provides a marked advantage over currently utilized xanthene labels, probes assembled using these labels and methods relying upon such labels and probes. Moreover, the present invention provides a class of chemically versatile labels in which the fluorophore can be engineered to have a desired light emission profile.

[0024]In an exemplary embodiment, the fluorescent nucleus is functionalized through the ortho carboxylic acid group of the phenyl moiety attached to the xanthene nucleus, requiring fewer chemical synthesis steps and eliminating the need to separate structural isomers. Additionally, many xanthene dyes possessing the ortho carboxy functional group are commercially available at low cost for use as starting materials.

Problems solved by technology

For example, radioactive labels are quite versatile, and can be detected at very low concentrations, such labels are, however, expensive, hazardous, and their use requires sophisticated equipment and trained personnel.

Despite the limited availability of FET probes, techniques incorporating their use are rapidly displacing alternative methods.

When a complementary target sequence is present, hybridization of the probe to the complementary target sequence disrupts the hairpin structure and causes the probe to adopt a conformation where the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule.

However, a number of methods for monitoring the amplification process without prior separation of primer have been described; all of them are based on FET, and none of them detect the amplified product directly.

The incorporation of such reactive chemical functionality into xanthene dyes usually requires additional synthetic steps and / or difficult to implement purification methods.

In particular, separation of structural isomers of fluoresceins and rhodamines, which are the most commonly used xanthene labeling reagents for biological and medical applications, is tedious and is to be avoided if possible.

The carboxylic acid group has not been widely utilized as a site for conjugation of the dye to substrates due to its low reactivity and due to side reactions that render the dye non-fluorescent.

Although the carboxylic acid can be activated and reacted with alcohols to form esters or with amines to form amides, the ester linkage is of insufficient stability to be useful when preparing compounds that are stably labeled with a fluorophore.

The sulfonate esters are not stable under aqueous conditions and are of little use as linker functionality for preparing oligonucleotides.

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