Derivatives of 6,8-difluoro-7-hydroxycoumarin

A method of detecting the activity of an enzyme, comprising:

• a) combining a sample suspected of containing the enzyme, with a substrate of the formula:
• ##STR49## wherein R.sup.3 is H, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C.dbd.O)--NR.sup.1 R.sup.2 where R.sup.1 and R.sup.2 are independently H, C.sub.1 -C.sub.6 alkyl, aryl, or R.sup.1 and R.sup.2 taken in combination are --(CH.sub.2).sub.2 --M--(CH.sub.2).sub.2 -- where M is a single bond, --O--, --CH.sub.2 --, or --NR.sup.6 --, where R.sup.6 is H or C.sub.1 -C.sub.6 alkyl;
• or R.sup.3 is --L--S.sub.C ;
• R.sup.4 is H, OH, CN, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, sulfomethyl, salt of sulfomethyl, halomethyl, aryl, or --L--S.sub.C ;
• R.sup.5 is H or C.sub.1 -C.sub.6 alkoxy;
• R.sup.7 is a monovalent moiety derived by removal of a hydroxy group from a phosphate, a thiophosphate, a sulfate, or a biologically compatible salt thereof;
• or a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aliphatic or aromatic carboxylic acid;
• or a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a mono- or polysaccharide;
• wherein
• aryl is an aromatic substituent having 6 conjugated carbon atoms that is optionally and independently substituted by H, halogen, cyano, sulfo, biologically compatible salts of sulfo, carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino or alkylamido;
• heteroaryl is a 5- or 6-membered aromatic heterocycle that is optionally fused to additional six-membered aromatic rings, or is fused to one 5- or 6-membered heteroaromatic ring, said heteroaromatic rings containing at least 1 and as many as 3 heteroatoms that are selected from the group consisting of O, N and S in any combination, that is attached by a single bond, and is optionally and independently substituted by H, halogen, C.sub.1 -C.sub.6 alkyl, or C.sub.1 -C.sub.6 alkoxy;
• each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon--carbon bonds, carbon-nitrogen bonds, nitrogen--nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds;
• S.sub.C is a conjugated substance
• under conditions suitable for cleavage of the R.sup.7 -oxygen bond;
• and
• b) detecting the resulting fluorescence either qualitatively or quantitatively.

A method of detecting a complementary member of a specific binding pair in a sample, comprising:

• a) adding to said sample a dye-conjugate of a first member of a specific binding pair for which there is a complementary member, to which is attached one or more dye molecules, which may be the same or different, having the formula ##STR50## wherein R.sup.3 is H, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl;
• or R.sup.3 is --L--S.sub.C ;
• R.sup.4 is H, OH, CN, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, sulfomethyl, salt of sulfomethyl, aryl, or --L--S.sub.C ;
• R.sup.5 is H or C.sub.1 -C.sub.6 alkoxy;
• R.sup.7 is H, or a monovalent moiety derived by removal of a hydroxy group from a phosphate, a thiophosphate, a sulfate, or a biologically compatible salt thereof;
• or a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aliphatic or aromatic carboxylic acid or of an amino acid, protected amino acid, peptide, or protected peptide;
• or a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a mono- or polysaccharide;
• or R.sup.7 is a photolabile caging group;
• wherein
• aryl is an aromatic substituent having 6 conjugated carbon atoms that is optionally and independently substituted by H, halogen, cyano, sulfo, biologically compatible salts of sulfo, carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino or alkylamido;
• heteroaryl is a 5- or 6-membered aromatic heterocycle that is optionally fused to additional six-membered aromatic rings, or is fused to one 5- or 6-membered heteroaromatic ring, said heteroaromatic rings containing at least 1 and as many as 3 heteroatoms that are selected from the group consisting of O, N and S in any combination, that is attached by a single bond, and is optionally and independently substituted by H, halogen, C.sub.1 -C.sub.6 alkyl, or C.sub.1 -C.sub.6 alkoxy;
• each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon--carbon bonds, carbon-nitrogen bonds, nitrogen--nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds;
• provided that at least one of R.sup.3 and R.sup.4 is --L--S.sub.C ;
• where S.sub.C is a conjugated substance that is the first member of the specific binding pair;
• b) allowing sufficient time for the dye-conjugate to form a complex with the complementary member, said complex exhibiting a detectable optical response;
• and
• c) detecting the complex to locate the complementary member.

A compound having the formula ##STR51## wherein R.sup.3 is H, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C.dbd.O)--NR.sup.1 R.sup.2 where R.sup.1 and R.sup.2 are independently H, C.sub.1 -C.sub.6 alkyl, aryl, or R.sup.1 and R.sup.2 taken in combination are --(CH.sub.2).sub.2 --M--(CH.sub.2).sub.2 -- where M is a single bond, --O--, --CH.sub.2 --, or --NR.sup.6 --, where R.sup.6 is H or C.sub.1 -C.sub.6 alkyl;

• R.sup.4 is H, OH, CN, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, sulfomethyl, biologically compatible salt of sulfomethyl, halomethyl, or aryl;
• R.sup.5 is;
• H or C.sub.1 -C.sub.6 alkoxy;
• R.sup.7 is H;
• wherein
• aryl is an aromatic substituent having 6 conjugated carbon atoms that is optionally and independently substituted by H, halogen, cyano, sulfo, biologically compatible salts of sulfo, carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino or alkylamido;
• heteroaryl is a 5- or 6-membered aromatic heterocycle that is optionally fused to additional six-membered aromatic rings, or is fused to one 5- or 6-membered heteroaromatic ring, said heteroaromatic rings containing at least 1 and as many as 3 heteroatoms that are selected from the group consisting of O, N and S in any combination, that is attached by a single bond, and is optionally and independently substituted by H, halogen, C.sub.1 -C.sub.6 alkyl, or C.sub.1 -C.sub.6 alkoxy.