Phosphate-bound polyazaindacene derivatives of nucleotides

Details Phosphate-bound polyazaindacene derivatives of nucleotides Phosphate-bound polyazaindacene derivatives of nucleotides

2000-09-18

2001-11-27

Gitomer, Ralph (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S051000, C536S024300, C536S024320, C435S006120, C548S110000, C424S009100, C424S009341

Reexamination Certificate

active

06323186

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to dipyrrometheneboron difluoride-substituted nucleotides useful in protein binding assays and as fluorescent or fluorogenic substrates for enzyme-catalyzed cleavage.
BACKGROUND
A variety of dipyrrometheneboron difluoride dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes) have previously been described (U.S. Pat. Nos. 4,774,339 to Haugland et al.; U.S. Pat. No. 5,274,113 to Kang et al.; U.S. Pat. No. 5,274,113 to Kang et al., U.S. Pat. No. 5,248,782 to Haugland et al.; U.S. Pat. No. 5,187,288 to Kang et al.; U.S. Pat. No. 5,338,854 to Kang et al.; U.S. Pat. No. 5,433,896 to Kang et al.; and application Ser. No. 08/856,422 by Wu et al., filed May. 14, 1997; all incorporated by reference). The above patents describe fluorescent dyes and conjugates of fluorescent dyes based on the dipyrrometheneboron difluoride core structure.
The present invention relates to dipyrrometheneboron difluoride derivatives of nucleotides. Nucleotides fill critically important roles in a variety of cellular processes, Binding and hydrolysis of nucleotides regulate transcription, translation, secretion, signal transduction, and cellular decisions that relate to programmed cell death and neoplastic transformation. The present invention describes dipyrrometheneboron difluoride-labeled nucleotides that are useful in the study the physiological effects of the free nucleotide in biological systems, including cells, cell extracts, cell homogenates, and purified, reconstituted or synthetic protein and enzyme systems.
In particular, the binding of nucleotides to selected proteins is readily investigated using the instant materials. For example, G proteins are a family of intracellular proteins that mediate plasma membrane, translocational, secretory and other types of signaling via nucleotide-binding. The GTP derivatives of the invention are bound by G proteins, and possess utility for studies of G protein binding, activity and inhibition by fluorescence methods.
Fhit is a member of the histidine triad superfamily of nucleotide-binding proteins. Fhit binds and cleaves dinucleoside polyphosphates, and has been identified as a tumor-suppressor that is often inactivated early in many common human cancers. The fluorescent or fluorogenic nucleotides of the invention possess utility for studies of binding, activity and inhibition of the Fhit enzyme and other enzymes. (U.S. Pat. No. 5,928,884 (1999), incorporated by reference).
While a variety of fluorescent dye conjugates of dipyrrometheneboron difluoride dyes have been described, the selection of fluorescent labels for enzyme substrates or in protein binding assays is typically problematic, as the electronic and spatial requirements of the binding site of the protein of interest are difficult to predict a priori. For example, it was determined that the S-terminus bound GTP-&ggr;-S conjugate of OREGON GREEN dye (Molecular Probes, Inc.) was found to be stable with respect to hydrolysis by the Fhit enzyme.
While dipyrrometheneboron difluoride conjugates of nucleotides have been described previously, the fluorophore has been conjugated to either the base subunit, or the sugar subunit (some examples have been sold under the trademark CHROMATIDE fluorescent nucleotides by Molecular Probes, Inc.). Nucleotid,es labeled on the base have not been useful for analyzing protein interactions, and while nucleotides labeled on the sugar moiety are quickly bound by G-proteins, the nucleotide is then rapidly hydrolyzed and dissociates from the protein.
Nucleotide conjugates wherein the dipyrrometheneboron difluoride label is bound to the phosphate chain have not previously been described. The compounds of the invention have been found to possess utility for assays related to binding or enzymatic cleavage of nucleotides.


REFERENCES:
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