Coumarin-labeled nucleoside 5'-triphosphates

• US 5,587,472 A
• Filed: 06/02/1995
• Issued: 12/24/1996
• Est. Priority Date: 08/13/1991
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics GmbH (Siemens AG)
• Original Assignee: Bayer Corporation (Bayer AG)
• Inventors: Kocher, Jurgen, Dattagupta, Nanibhushan
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/460,027
• Time in Patent Office: 571 Days
• Field of Search: 536/24.31, 536/24.32, 536/26.2
• US Class Current: 536/26.2
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

A fluorescent label compound having the formula

A--B.sup.1 --B.sup.2 --B.sup.3 --C

wherein

A represents the residue of a natural or synthetic nucleotide or nucleoside or a derivative thereof;

B¹ represents a divalent spacer radical or a single bond;

B² represents a divalent spacer radical;

B³ represents a divalent spacer radical or a single bond; and

C represents a coumarin radical having the formula ##STR1## in which R¹ represents hydrogen or cyano;

R² represents phenyl or thiazolyl bonded in the 2-, 4- or 5-position, wherein the phenyl is unsubstituted or substituted by nitro, cyano, amino, --NH--C_1-4 -alkyl, --(CH₂)_1-4 --NH₂, --(CH₂)_1-4 --NH--(CH₂)_1-3 --CH₃, carboxy, C_1-4 -alkoxycarbonyl, C_1-4 -alkoxycarbonyloxy, hydroxy, C_1-4 -alkylaminocarbonyl, or C_1-4 -alkylcarbonylamino and either further unsubstituted or substituted by C_1-4 -alkyl, fluorine, chlorine or bromine, and where the thiazolyl is unsubstituted or monosubstituted or disubstituted by chlorine, cyano, carboxyl, or C_1-4 -alkoxycarbonyl, or the thiazolyl is fused in the 4- and 5-position with a benzene ring which is either unsubstituted or substituted by carboxyl, amino, or hydroxyl;

R³ represents hydrogen, C_1-4 -alkyl, C_1-4 -alkoxycarbonyl-C_1-4 -alkyl, or phenylsulphonyl, where the C_1-4 -alkyl is unsubstituted or substituted by hydroxyl, amino, carboxyl, or C_1-4 -alkoxycarbonyl, and where the phenylsulphonyl is unsubstituted or monosubstituted or disubstituted by chlorine, bromine, or C_1-4 -alkyl;

or where one of the radicals R² or R³ denotes or is substituted by a primary or secondary amino group, hydroxyl, carboxyl, or C_1-4 -alkoxycarbonyl, or can be converted into such a group by hydrolysis or hydrogenation.

Claims

1. A fluorescent label compound having the formula:

space="preserve" listing-type="equation">A--B.sup.1 --B.sup.2 --B.sup.3 --C

wherein

A represents a radical selected from the group consisting of dATP-8-yl and dUTP-5-yl;

B¹ --B² --B³ collectively represent a spacer moiety selected from the group consisting of;

space="preserve" listing-type="equation">--CH═

CH--CH.sub.2 --NH--CO--(CH.sub.2).sub.5 --NH--CO--(CH.sub.2).sub.3 --a

and

space="preserve" listing-type="equation">--NH--(CH.sub.2).sub.6 --NH--CO--(CH.sub.2).sub.5 --NH--CO--(CH.sub.2).sub.3 --a

wherein

"a" represents the point of attachment to A; and

C represents a coumarin radical having the formula;

##STR26##

2. The fluorescent compound according to claim 1 having the formula ##STR27##

3. The fluorescent label compound according to claim 1 having the formula ##STR28##

4. In a test for the presence of a particular nucleic acid sequence in a sample, wherein the sample is subjected to a labeled probe under hybridizing conditions and the sample is thereafter assayed for hybridization product, the improvement wherein the probe is labeled with a fluorescent label compound according to claim 1.

5. A diagnostic kit for use in detecting the presence of a particular nucleic acid sequence in a sample, said kit comprising a fluorescent label compound according to claim 1.