Derivatives of 6,8-difluoro-7-hydroxycoumarin

Claim 1

A compound having the formula ##STR47## wherein R 3 is H, C 1 -C 8 alkyl, C 1 -C 18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C═O)--NR 1 R 2 where R 1 and R 2 are independently H, C 1 -C 6 alkyl, aryl, or R 1 and R 2 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M is a single bond, --O--, --CH 2 --, or --NR 6 --, where R 6 is H or C 1 -C 6 alkyl; or R 3 is --L--R X or --L--S C ; R 4 is H, OH, CN, C 1 -C 18 is alkyl, C 1 -C 18 perfluoroalkyl, sulfomethyl, biologically compatible salt of sulfomethyl, halomethyl, aryl, --L--R X or --L--S C ; R 5 is H or C 1 -C 6 alkoxy; R 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 a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a mono- or polysaccharide; or R 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 1 -C 6 alkyl, or C 1 -C 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; R X is a reactive group; S C is a conjugated substance; provided that at least one of R 3 and R 4 is --L--R X or --L--S C ; or that R 7 is not hydrogen; or both.

Claim 21

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 3 is H, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C═O)--NR 1 R 2 where R 1 and R 2 are independently H, C 1 -C 6 alkyl, aryl, or R 1 and R 2 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M is a single bond, --O--, --CH 2 --, or --NR 6 --, where R 6 is H or C 1 -C 6 alkyl; or R 3 is --L--S C ; R 4 is H, OH, CN, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, sulfomethyl, salt of sulfomethyl, halomethyl, aryl, or --L--S C ; R 5 is H or C 1 -C 6 alkoxy; R 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 1 -C 6 alkyl, or C 1 -C 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 C is a conjugated substance under conditions suitable for cleavage of the R 7 -oxygen bond; and b) detecting the resulting fluorescence either qualitatively or quantitatively.

Claim 22

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 3 is H, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C═O)--NR 1 R 2 where R 1 and R 2 are independently H, C 1 -C 6 alkyl, aryl, or R 1 and R 2 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M is a single bond, --O--, --CH 2 --, or --NR 6 --, where R 6 is H or C 1 -C 6 alkyl; or R 3 is --L--S C ; R 4 is H, OH, CN, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, sulfomethyl, salt of sulfomethyl, halomethyl, aryl, or --L--S C ; R 5 is H or C 1 -C 6 alkoxy; R 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 1 -C 6 alkyl, or C 1 -C 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 C is a conjugated substance under conditions suitable for cleavage of the R 7 -oxygen bond; and b) detecting the resulting fluorescence either qualitatively or quantitatively.

Claim 30

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 3 is H, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl; or R 3 is --L--S C ; R 4 is H, OH, CN, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, sulfomethyl, salt of sulfomethyl, aryl, or --L--S C ; R 5 is H or C 1 -C 6 alkoxy; R 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 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 1 -C 6 alkyl, or C 1 -C 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 3 and R 4 is --L--S C ; where S 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.

Claim 31

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 3 is H, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl; or R 3 is --L--S C ; R 4 is H, OH, CN, C 1 -C 18 alkyl, C 1 -C 18 perfluoroalkyl, sulfomethyl, salt of sulfomethyl, aryl, or --L--S C ; R 5 is H or C 1 -C 6 alkoxy; R 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 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 1 -C 6 alkyl, or C 1 -C 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 3 and R 4 is --L--S C ; where S 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.