Derivatives of 6,8-difluoro-7-hydroxycoumarin

• US 5,830,912 A
• Filed: 11/15/1996
• Issued: 11/03/1998
• Est. Priority Date: 11/15/1996
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard P., Gee, Kyle R., Sun, Wei-Chuan
• Primary Examiner(s): Trinh, Ba K.
• Application Number:
  US08/749,684
• Time in Patent Office: 718 Days
• Field of Search: 549/289, 549/283, 549/285, 549/287, 514/457
• US Class Current: 514/457
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 311/54 substituted in the carbocyclic ring
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C09B 57/02 Coumarine dyes

Abstract

The present invention comprises 6,8-difluoro-7-hydroxycoumarins and derivatives of 6,8-difluoro-7-hydroxycoumarins, including reactive dyes, dye-conjugates and enzyme substrates. These fluorine-substituted fluorescent dyes typically possess greater photostability and lower pH sensitivity in the physiological pH range than their nonfluorinated analogs, exhibit less fluorescence quenching when conjugated to a substance, possess absorption and emission spectra that closely match those of their nonfluorinated analogs, and also exhibit higher quantum yields than their nonfluorinated analogs.

Claims

1. A compound having the formula ##STR47## wherein R³ is H, C₁ -C₈ alkyl, C₁ -C₁₈ perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C═

O)--NR¹ R² where R¹ and R² are independently H, C₁ -C₆ alkyl, aryl, or R¹ and R² taken in combination are --(CH₂)₂ --M--(CH₂)₂ -- where M is a single bond, --O--, --CH₂ --, or --NR⁶ --, where R⁶ is H or C₁ -C₆ alkyl;

or R³ is --L--R_X or --L--S_C ;

R⁴ is H, OH, CN, C₁ -C₁₈ is alkyl, C₁ -C₁₈ perfluoroalkyl, sulfomethyl, biologically compatible salt of sulfomethyl, halomethyl, aryl, --L--R_X or --L--S_C ;

R⁵ is H or C₁ -C₆ alkoxy;

R⁷ 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 R⁷ 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, C₁ -C₆ alkyl, or C₁ -C₆ 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 R³ and R⁴ is --L--R_X or --L--S_C ;

or that R⁷ is not hydrogen;

or both.

2. A compound, as claimed in claim 1, wherein at least one of R³ and R⁴ is --L--R_X.

3. A compound, as claimed in claim 2, wherein R_X is an acrylamide, a carboxylic acid, an activated ester of a carboxylic acid, an acyl azide, an acyl halide, hydroxy, an aldehyde, an alkyl halide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a carbodiimide, a diazoalkane, an epoxide, a glycol, a haloacetamide, a halotriazine, a hydrazine, a hydroxylamine, an imido ester, an isothiocyanate, a ketone, a maleimide, a sulfonyl halide, or a thiol group.

4. A compound, as claimed in claim 3, wherein R_X is a carboxylic acid, an activated ester of a carboxylic acid, an amine, a maleimide, an iodoacetamide, an isothiocyanate, a dichloro-s-triazine, or a halomethyl.

5. A compound, as claimed in claim 2, wherein R³ is --L--R_X and R_X is a succinimidyl ester.

6. A compound, as claimed in claim 1, wherein R⁴ is sulfomethyl, salt of sulfomethyl, halomethyl, C₁ -C₁₈ alkyl or C₁ -C₁₈ perfluoroalkyl.

7. A compound, as claimed in claim 1, wherein at least one of R³ and R⁴ is --L--Sc and R⁷ is H.

8. A compound, as claimed in claim 7, wherein S_C is an amino acid, a peptide, a protein, a monosaccharide, a polysaccharide, an ion-complexing moiety, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a toxin, a lipid, a phospholipid, a lipoprotein, a lipopolysaccharide, a liposome, a lipophilic polymer, a non-biological organic polymer, a polymeric microparticle, an animal cell, a plant cell, a bacterium, a yeast, or a virus.

9. A compound, as claimed in claim 8, wherein S_C is an amino acid, a peptide, a protein, a nucleotide, an oligonucleotide, a nucleic acid, a monosaccharide, a polysaccharide, a drug, a toxin, or a tyramide.

10. A compound, as claimed in claim 9, wherein S_C is a peptide or a protein.

11. A compound, as claimed in claim 9, wherein S_C is a nucleoside, nucleotide or oligonucleotide.

12. A compound, as claimed in claim 7, wherein S_C is further substituted by one or more dyes that are the same or different.

13. A compound, as claimed in claim 1, wherein R³ is aryl or heteroaryl.

14. A compound, as claimed in claim 1, wherein

R³ is H;

R⁴ is methyl, sulfomethyl, salt of sulfomethyl, chloromethyl, or a C₁₁ -C₁₈ alkyl; and

R⁵ is H.

15. A compound, as claimed in claim 14, wherein R⁷ is a monovalent moiety derived by removal of a hydroxy group from a phosphate or a salt of phosphate.

16. A compound, as claimed in claim 14, wherein R⁷ is a monovalent moiety derived by the removal of a hydroxy group from an aliphatic carboxylic acid having 1-18 carbons, guanidinobenzoic acid, or sulfuric acid;

or a biologically compatible salt thereof.

17. A compound, as claimed in claim 14, wherein R⁷ is a monovalent moiety derived by removal of a hydroxy group from an alcohol having 1-6 carbons;

or a monovalent moiety derived by removal of a hydroxy group from a mono- or polysaccharide.

18. A compound, as claimed in claim 17, wherein the monosaccharide is β

-D-galactose, β

-D-glucose, β

-D-glucuronic acid, N-acetylglucosamine, or N-acetylgalactosamine.

19. A compound, as claimed in claim 1, wherein

R³ has the formula ##STR48## wherein n=0-5;

and

K is H, sulfonic acid, or a salt of sulfonic acid;

R⁴ is H or methyl; and

R⁵ is H.

20. 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 R³ is H, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C═

O)--NR¹ R² where R¹ and R² are independently H, C₁ -C₆ alkyl, aryl, or R¹ and R² taken in combination are --(CH₂)₂ --M--(CH₂)₂ -- where M is a single bond, --O--, --CH₂ --, or --NR⁶ --, where R⁶ is H or C₁ -C₆ alkyl;

or R³ is --L--S_C ;

R⁴ is H, OH, CN, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, sulfomethyl, salt of sulfomethyl, halomethyl, aryl, or --L--S_C ;

R⁵ is H or C₁ -C₆ alkoxy;

R⁷ 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, C₁ -C₆ alkyl, or C₁ -C₆ 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 R⁷ -oxygen bond; and

b) detecting the resulting fluorescence either qualitatively or quantitatively.

21. A method, as claimed in claim 20, wherein the enzyme is a glycosidase.

22. A method, as claimed in claim 21, wherein R⁷ is a monovalent moiety derived by removal of a hydroxy group from a monosaccharide that is β

-D-galactose, β

-D-glucose β

-D-glucuronic acid, N-acetylglucosamine, or N-acetylgalactosamine.

23. A method, as claimed in claim 20, wherein the enzyme is a phosphatase.

24. A method, as claimed in claim 20, wherein said enzyme is present as a result of expression of a transfected gene, or as a result of cell secretion.

25. A method, as claimed in claim 20, wherein

R³ is H, aryl, heteroaryl, or --L--S_C ;

R⁴ is CH₃, CF₃, sulfomethyl, salt of sulfomethyl, halomethyl, C₁₁ -C₁₈ alkyl, or --L--S_C ;

R⁵ is H.

26. A method, as claimed in claim 25 wherein R⁴ is halomethyl.

27. A method, as claimed in claim 25 wherein S_C is a tyramine and the enzyme is a peroxidase.

28. 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 R³ is H, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl;

or R³ is --L--S_C ;

R⁴ is H, OH, CN, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, sulfomethyl, salt of sulfomethyl, aryl, or --L--S_C ;

R⁵ is H or C₁ -C₆ alkoxy;

R⁷ 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 R⁷ 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, C₁ -C₆ alkyl, or C₁ -C₆ 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 R³ and R⁴ 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.

29. A method, as claimed in claim 28, wherein the optical response is a fluorescence response.

30. A method, as claimed in claim 28, wherein the first member of the specific binding pair is a peptide, a protein, a nucleotide, an oligonucleotide, a nucleic acid polymer, a drug, a toxin, or a polysaccharide.

31. A method, as claimed in claim 28, wherein the first member of the specific binding pair comprises an antibody, an antibody fragment, an avidin, a streptavidin, a lectin or an enzyme.

32. A method, as claimed in claim 28, wherein the complementary member is present in a cell, bacteria, virus or yeast cell, or is immobilized on a polymer, polymeric membrane or polymeric particle.

33. A method, as claimed in claim 29, further comprising distinguishing the fluorescence response from that of a second fluorophore having detectably different optical properties.

34. A method, as claimed in claim 29, wherein the fluorescence response is detected using a flow cytometer, further comprising sorting said complex based on the fluorescence response.

35. A compound having the formula ##STR51## wherein R³ is H, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, CN, formyl, aryl, heteroaryl, arylcarbonyl, or --(C═

O)--NR¹ R² where R¹ and R² are independently H, C₁ -C₆ alkyl, aryl, or R¹ and R² taken in combination are --(CH₂)₂ --M--(CH₂)₂ -- where M is a single bond, --O--, --CH₂ --, or --NR⁶ --, where R⁶ is H or C₁ -C₆ alkyl;

R⁴ is H, OH, CN, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, sulfomethyl, biologically compatible salt of sulfomethyl, halomethyl, or aryl;

R⁵ is;

H or C₁ -C₆ alkoxy;

R⁷ 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, C₁ -C₆ alkyl, or C₁ -C₆ alkoxy.