Biological conjugates of fluorescent rhodol dyes

• US 5,442,045 A
• Filed: 03/08/1993
• Issued: 08/15/1995
• Est. Priority Date: 04/16/1990
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Whitaker, James E., Haugland, Richard P.
• Primary Examiner(s): Lacey, David L.
• Assistant Examiner(s): EISENSCHENK, FRANK C
• Application Number:
  US08/028,319
• Time in Patent Office: 890 Days
• Field of Search: 544/150, 546/41, 548/417, 549/390, 549/391, 530/391.1, 530/391.3, 530/402, 536/25.32, 536/26.6
• US Class Current: 530/391.3
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C09B 11/10 Amino derivatives of triarylmethanes
• CPC Class Codes: C09K 11/06 containing organic luminescent materials

Abstract

Novel fluorescent dyes based on the rhodol structure are provided. The new reagents contain functional groups capable of forming a stable fluorescent product with functional groups typically found in biomolecules or polymers including amines, phenols, thiols, acids, aldehydes and ketones. Reactive groups in the rhodol dyes include activated esters, isothiocyanates, amines, hydrazines, halides, acids, azides, maleimides, aldehydes, alcohols, acrylamides and haloacetamides. The products are detected by their absorbance or fluorescence properties. The spectral properties of the fluorescent dyes are sufficiently similar in wavelengths and intensity to fluorescein or rhodamine derivatives as to permit use of the same equipment. The dyes, however, show less spectral sensitivity to pH in the physiological range than does fluorescein, have higher solubility in non-polar solvents and have improved photostability and quantum yields.

Claims

1. A compound comprising a fluorescent dye having the structure ##STR2## wherein R¹ is H or CH₃, and R² is H, CF₃ CO, CH₃, or C₂ H₅ ;

or R¹ in combination with R² is (CH₂)₃ ;

R³ is H, CH₃, or C₂ H₅, and R⁴ is H;

or R³ in combination with R⁴ is (CH₂)₃ ;

R⁵ is H, CH₂ NH₂, CH₂ NHCOCH₂ Cl, CH₂ NHCOCH₂ I;

CH₂ NHCOCH═

CH₂, or CH₂ NHCOC₆ H₄ N₃ ;

R⁶ is H, Cl, Br, CH₃, or C₂ H₅, or (CH₂)_n CH₃ where n is an integer from 0 to 15, or (CH₂)_n CO₂ H, where n is an integer from 0 to 11;

R⁷ is H, CO₂ H, ##STR3## NH₂, NCS, CH₂ NHCOCH₂ Cl, or CH₂ NHCOCH₂ I;

such that R⁷ is not H if R⁵ is H;

conjugated to a reactive site on a biomolecule via reaction of a chemically reactive functional group on the dye with a reactive site on the biomolecule.

2. A compound as claimed in claim 1, wherein R⁷ is not H.

3. A compound, as claimed in claim 1, wherein one of R⁵ and R⁶ is a chemically reactive functional group.

9. The compound of claim 1 formed when the fluorescent dye is conjugated to a reactive site on a biomolecule via reaction of the chemically reactive functional group with the reactive site on the biomolecule, the reactive site selected from the group consisting of amines, thiols, alcohols, phenols, aldehydes, ketones, carboxylic acids, alkyl halides, imidazoles, and olefins.

10. A compound, as claimed in claim 1, wherein the biomolecule is a peptide, a protein, a hormone, a drug, a nucleotide, an oligonucleotide, a nucleic acid, a polysaccharide, or a lipid.

11. The compound of claim 3, where the fluorescent dye is conjugated to a biomolecule that is a peptide, a protein, a nucleotide, oligonucleotide, or nucleic acid.

4. A compound comprising a fluorescent dye having the structure ##STR4## wherein R¹ arid R⁴ are independently hydrogen, alkyl, carboxyalkyl, aminoalkyl, halogen or alkoxy, the alkyl portions of which contain less than 4 carbons;

R² and R³ are independently hydrogen, methyl or ethyl, or trifluoroacetyl;

or

R¹ in combination with R², or R³ in combination with R⁴ are (CH₂)₃ ;

R⁵ is H;

R⁶ is H, Cl, Br. CH₃, or C₂ H₅, and R⁹ is COCH₃ ;

or R⁶ and R⁹ in combination are (CH₂)₂ CO;

##STR5## such that R⁷ is is not H if R⁹ is COCH₃ ;

conjugated to a reactive site on a biomolecule via reaction of a chemically reactive functional group on the dye with a reactive site on the biomolecule.

5. A compound as claimed in claim 4, wherein the biomolecule is a peptide, a protein, a hormone, a drug a nucleotide, an oligonucleotide, a nucleic acid, a polysaccharide, or a lipid.

6. A compound as claimed in claim 4, where the fluorescent dye is conjugated to a biomolecule that is a nucleotide, oligonucleotide, or nucleic acid,

7. A compound as claimed in claim 4, wherein

R¹ in combination with R², and R³ in combination with R⁴ are (CH₂)₃ ;

and

R⁷ is selected from the group consisting of CO₂ H and ##STR6##

8. A compound as claimed in claim 7, wherein

R₁ in combination with R², and R³ in combination with R⁴ are (CH₂)₃ ;

R⁵ is H;

R⁷ is selected from the group consisting of CO₂ H and ##STR7## and R⁶ and R⁹ in combination are (CH₂)₂ CO.

12. The compound of claim 4, where the fluorescent dye is conjugated to a biomolecule that is a peptide or a protein.

13. A compound of the formula;

dye-biomolecule wherein said dye is fluorescent dye of the formula;

##STR8## that is covalently attached to said biomolecule through a linkage that is amine, amide, carboxamide, sulfonamide, propionamide, glycineamide, amidine, imine, imide, imidazole, hydrazide, hydrazone, urea, thiourea, urethane, ester, ether, or thiother at one of the positions R¹ through R⁷ ; and

at the positions where the biomolecule is not attached;

R¹ is H or CH₃ and R² is H CF₃ CO CH₃ or C₂ H₅ ;

or R¹ in combination with R² is (CH₂)₃ ;

R³ is H, CH₃, or C₂ H₅, and R⁴ is H;

or R³ in combination with R⁴ is (CH₂)₃ ;

R⁵ is H CH₂ NH₂ CH₂ NHCOCH₂ Cl, CH₂ NHCOCH₂ I;

CH₂ NHCOCH═

CH₂, or CH₂ NHCOC₆ H₄ N₃ ;

R⁶ is H, Cl, Br, or (CH₂)_n CH₃ where n is an integer from 0 to 15, or (CH₂)_n CO₂ H, where n is an integer from 0 to 11;

R⁷ is H, CO₂ H, ##STR9## NH₂ NCS CH₂ NHCOCH₂ Cl, or CH₂ NHCOCH₂ I.

14. A compound, as claimed in claim 13, wherein the biomolecule X is a peptide, a protein, a hormone, a drug, a nucleotide, an oligonucleotide, a nucleic acid, a polysaccharide, or a lipid.

15. A compound, as claimed in claim 13, wherein said dye has the formula:

##STR10## wherein the biomolecule is attached at one position R⁵, R⁶, or R⁷ ; and

at the positions where the biomolecule is not attached;

R¹ is H or CH₃, and R² is H, CF₃ CO, CH₃, or C₂ H₅ ;

or R¹ in combination with R² is (CH₂)₃ ;

R³ is H, CH₃, or C₂ H₅, and R⁴ is H;

or R³ in combination with R⁴ is (CH₂)₃ ;

R⁵ is H, CH₂ NH₂, CH₂ NHCOCH₂ Cl CH₂ NHCOCH₂ I;

CH₂ NHCOCH═

CH₂ or CH₂ NHCOC₆ H₄ N₃ ;

R⁶ is H, Cl, Br, or (CH₂)_n CH₃ where n is an integer from 0 to 15, or (CH₂)_n CO₂ H where n is an integer from 0 to 11;

R⁷ is H, CO₂ H ##STR11## NH₂, NCS CH₂ NHCOCH₂ Cl, or CH₂ NHCOCH₂ I;

such that R⁷ is not H if R⁵ is H.

16. A compound, as claimed in claim 15, wherein the biomolecule X is attached at one position R⁵ or R⁶ by an amide linkage.

17. A compound, as claimed in claim 15, wherein the biomolecule X is attached at R⁷ by an amide, urea or thiourea linkage.

18. A compound, as claimed in claim 13, wherein the biomolecule X is attached at one position R⁶ or R⁷ ;

and at the positions where the biomolecule is not attached;

R¹ is H or methyl;

R² and R³ are independently H or (C₁ -C₂) alkyl;

R⁴ and R⁵ are H;

R⁶ is hydrogen, Cl, Br, or (C₁ -C₁₆) alkyl; and

R⁷ is hydrogen.

19. A compound, as claimed in claim 13, wherein the biomolecule X is attached at one position R⁶ or R⁷ ;

and at the positions where the biomolecule is not attached;

R¹ in combination with R² and R³ in combination with R⁴ are each (CH₂)₃ ;

R⁵ is H;

R⁶ is hydrogen, Cl, Br, or (C₁ -C₁₆) alkyl; and

R⁷ is hydrogen.

20. A compound, as claimed in claim 13, wherein the biomolecule X is a peptide or protein that is attached at R⁷ by an amide linkage;

and at the positions where the biomolecule is not attached;

R¹ and R⁴ are independently hydrogen;

R² and R³ are independently hydrogen or (C₁ -C₂) alkyl;

or R¹ in combination with R² and R³ in combination with R⁴ are each (CH₂)₃ ; and

R⁵ and R⁶ are independently hydrogen.