Sulfonated derivatives of 7-aminocoumarin

• US 5,696,157 A
• Filed: 11/15/1996
• Issued: 12/09/1997
• 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: Wang, Hui-Ying, Leung, Wai-Yee, Mao, Fei
• Primary Examiner(s): Trinh, Ba K.
• Application Number:
  US08/749,753
• Time in Patent Office: 389 Days
• Field of Search: 514/457, 549/285, 549/288, 549/289
• US Class Current: 514/457
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links

Abstract

The present invention describes 7-aminocoumarin dyes that are substituted one or more times at the 3-, 6- and/or 8-positions by a sulfonic acid or a salt of a sulfonic acid, said dyes being useful as fluorescent probes or in the preparation of enzyme substrates, caged probes, or adducts with reducing sugars. The dyes of the invention optionally possess a reactive group useful for preparing fluorescent conjugates, which conjugates and methods for their preparation are described herein.

Claims

1. A compound of the formula ##STR44## wherein R⁵ is H;

R⁶ is H, methyl, halogen, or SO₃ X;

R⁷ is NR¹ R² ;

wherein

R¹ and R² are independently H, C₁ -C₁₈ alkyl, C₆ -C₁₈ aryl, C₁ -C₁₈ alkanoyl, C₇ -C₁₈ arylalkanoyl;

or R¹ in combination with R² forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;

or R² is H and R¹ is a radical formed by a removal of the hydroxyl group from the carboxylic acid of an amino acid or from the carboxylic acid of the carboxy-terminal amino acid of a linear peptide consisting of 2-6 amino acids;

or R¹ is a 2-nitrobenzyloxycarbonyl of the formula ##STR45## wherein R⁹ and R¹⁰ are H, C₁ -C₆ alkoxy, or R⁹ and R¹⁰ taken in combination are --O--CH₂ --O--; and

R¹¹ is H, CH₃, a carboxylic acid or a biologically compatible salt of a carboxylic acid;

or R⁷ is a nitrogen atom that is linked by a single or double covalent bond to the anomeric carbon of a mono- or polysaccharide and formally replaces the anomeric oxygen atom of the mono- or polysaccharide;

R⁸ is H, halogen or SO₃ X;

X is H, or a biologically compatible cation;

R³ is H, SO₃ X, halogen, CN, NO₂, or --L--R_X or --L--S_C ;

R⁴ is H, CN, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, CH₂ SO₃ X;

or --L--R_X or --L--S_C ;

wherein

each L is 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; and

S_C is a conjugated substance;

provided that at least one of R³, R⁶ or R⁸ is SO₃ X.

2. A compound, as claimed in claim 1, wherein 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, a hydroxy, an aldehyde, an alkyl halide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carbodiimide, an epoxide, a glycol, a haloacetamide, a halotriazine, a hydrazine, a hydroxylamine, an isothiocyanate, a ketone, a maleimide, a sulfonyl halide, or a thiol group.

4. A compound, as claimed in claim 1, wherein R_X is a carboxylic acid, an activated ester of a carboxylic acid, an alcohol, an aldehyde, an alkyl halide, an amine, a haloacetamide, a halotriazine, a hydrazine, an isothiocyanate, a maleimide or a thiol.

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, halomethyl, C₁ -C₁₈ alkyl or C₁ -C₁₈ perfluoroalkyl.

7. A compound, as claimed in claim 1, wherein one of R³ and R⁴ is --L--S_C.

8. A compound, as claimed in claim 7, wherein S_C is an amino acid, a tyramine, a peptide, a protein, a monosaccharide, a polysaccharide, an ion-complexing moiety, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a lipid, a phospholipid, a lipoprotein, a lipopolysaccharide, a liposome, a lipophilic 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, or a drug.

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

11. A compound, as claimed in claim 7, wherein S_C is further substituted by one or more additional dye compounds, which may be the same or different.

12. A compound, as claimed in claim 1, wherein R⁷ is NR¹ R² and R¹ and R² are independently H or C₁ -C₁₈ alkyl.

13. A compound, as claimed in claim 1, wherein R⁷ is NR¹ R², where R² is H and R¹ is a radical formed by a removal of the hydroxyl group from the carboxylic acid of an amino acid or from the carboxylic acid of the carboxy-terminal amino acid of a linear peptide consisting of 2-6 amino acids.

14. A compound, as claimed in claim 1, wherein R⁷ is NR1R², where R² is H and R¹ is a 2-nitrobenzyloxycarbonyl of the formula ##STR46## wherein R⁹ and R¹⁰ are H, C₁ -C₆ alkoxy, or R⁹ and R¹⁰ taken in combination are --O--CH₂ --O--;

and

R¹¹ is H, CH₃, a carboxylic acid or a biologically compatible salt of a carboxylic acid.

15. A compound, as claimed in claim 1, wherein R⁷ is a nitrogen atom that is linked by a single or double covalent bond to the anomeric carbon of a mono- or polysaccharide and formally replaces the anomeric oxygen atom of the mono- or polysaccharide.

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

R³ is H, --L--R_X, or --L--S_C ;

R⁴ is CH₃, --L--R_X or --L--S_C

R⁷ is NH₂ ; and

R⁸ is H;

provided that at least one of R³, R⁶ and R⁸ is SO₃ X.

17. 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, where said first member of a specific binding pair is attached to one or more dye molecules, which may be the same or different, having the formula ##STR47## wherein R⁵ is H;

R⁶ is H, methyl, halogen, or SO₃ X;

R⁷ is NR¹ R² ;

wherein

R¹ and R² are independently H, C₁ -C₁₈ alkyl;

or R¹ in combination with R² forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;

or R¹ is a 2-nitrobenzyloxycarbonyl of the formula ##STR48## wherein R⁹ and R¹⁰ are H, C₁ -C₆ alkoxy, or R⁹ and R¹⁰ taken in combination are --O--CH₂ --O--; and

R¹¹ is H, CH₃, a carboxylic acid or a biologically compatible salt of a carboxylic acid;

R⁸ is H, halogen or SO₃ X;

X is H, or a biologically compatible cation;

R³ is H, SO₃ X, halogen, CN, NO₂, or --L--S_C ;

R⁴ is H, CN, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, CH₂ SO₃ X;

or --L--S_C ;

wherein

each L is 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; and

S_C is a conjugated substance that is said first member of a specific binding pair;

provided that at least one of R³, R⁶ or R⁸ is SO₃ X; and

provided that one of R³ and R⁴ is --L--S_C ;

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 said complex to locate the complementary member.

18. A method, as claimed in claim 17, 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.

19. A method, as claimed in claim 17, wherein the optical response is a fluorescence response.

20. A method, as claimed in claim 17, wherein the first member of the specific binding pair is an antibody, an antibody fragment, avidin, streptavidin, or a conjugate of an antibody, an antibody fragment, avidin, or streptavidin and the complementary member is a hapten having a molecular weight less than 1,000, an antigen or a biotin.

21. A method, as claimed in claim 17, wherein the complementary member is present in a cell, bacteria, virus, yeast cell or is immobilized on a polymer, polymeric membrane or polymeric particle.

22. A method, as claimed in claim 19, further comprising distinguishing said fluorescence response from that of a second fluorophore having detectably different optical properties.

23. A method of staining a sample, comprising:

a) adding to said sample a compound of the formula ##STR49## wherein R⁵ is H;

R⁶ is H, methyl, halogen, or SO₃ X;

R⁷ is NR¹ R² ;

wherein

R¹ and R² are independently H, C₁ -C₁₈ alkyl;

or R¹ in combination with R² forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;

or R² is H and R¹ is a radical formed by a removal of the hydroxyl group from the carboxylic acid of an amino acid or from the carboxylic acid of the carboxy-terminal amino acid of a linear peptide consisting of 2-6 amino acids;

or R¹ is a 2-nitrobenzyloxycarbonyl of the formula ##STR50## wherein R⁹ and R¹⁰ are H, C₁ -C₆ alkoxy, or R⁹ and R¹⁰ taken in combination are --O--CH₂ --O--; and

R¹¹ is H, CH₃, a carboxylic acid or a biologically compatible salt of a carboxylic acid;

R⁸ is H, halogen or SO₃ X;

X is H, or a biologically compatible cation;

R³ is H, SO₃ X, halogen, CN, NO₂, or --L--S_C ;

R⁴ is H, CN, C₁ -C₁₈ alkyl, C₁ -C₁₈ perfluoroalkyl, CH₂ SO₃ X;

or --L--S_C ;

wherein

each L is 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; and

S_C is a conjugated substance;

provided that at least one of R³, R⁶ or R⁸ is SO₃ X;

in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength.

24. A method, as claimed in claim 23, wherein said optical response is a fluorescence response.

25. A method, as claimed in claim 23, wherein said sample comprises cells.

26. A method, as claimed in claim 25, further comprising observing said cells using flow cytometry.

27. A method, as claimed in claim 24, further comprising distinguishing the fluorescence response from that of a second fluorophore having detectably different optical properties.