Carboxy X rhodamine analogs

• US 10,215,751 B2
• Filed: 07/07/2016
• Issued: 02/26/2019
• Est. Priority Date: 11/21/2011
• Status: Active Grant

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Kirkland, Thomas A., McDougall, Mark G., Dwight, Stephen J.
• Primary Examiner(s): Jarrell, Noble E
• Application Number:
  US15/204,664
• Publication Number:
  US 20160320372A1
• Time in Patent Office: 964 Days
• CPC Class Codes: C07D 223/16 Benzazepines; Hydrogenated benzazepines
• CPC Class Codes: C07D 471/06 Peri-condensed systems
• CPC Class Codes: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• CPC Class Codes: C07D 491/16 Peri-condensed systems
• CPC Class Codes: C07D 491/22 in which the condensed system contains four or more hetero rings
• CPC Class Codes: C07F 9/65586 at least one of the hetero rings does not contain nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 2333/90241 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides novel fluorescent dyes and kits containing the same, which are useful for labeling a wide variety of biomolecules, cells and microorganisms. The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis and/or treatment of disease conditions.

Claims

1. A method to detect an interaction between a biomolecule and a protein of interest comprising:

a) contacting a sample suspected of containing the protein of interest with a composition comprising a dye conjugate according to formula (IIIa), (IIIb) or (IIIc); and

b) detecting the presence or amount of the dye conjugate, thereby detecting the interaction between the protein of interest and the biomolecule;

2. The method of claim 1, wherein the protein of interest is fused to a reporter protein.

3. The method of claim 2, wherein the reporter protein is a luciferase protein.

4. The method of claim 1, wherein the dye conjugate is a tracer.

5. The method of claim 1, wherein the interaction is detected by bioluminescence resonance energy transfer.

6. The method of claim 1, wherein the protein of interest is expressed in a cell.

7. The method of claim 3, wherein the interaction is detected by bioluminescence resonance energy transfer.

8. The method of claim 1, wherein the protein is an antibody.

9. The method of claim 1, wherein the polypeptide is a polypeptide-based toxin.

10. The method of claim 1, wherein L is —

CO—

, —

SCH₂CO—

, or —

SO₂—

.

11. The method of claim 1, wherein L is a self-immolative linker selected from the group consisting of

12. The method of claim 5, wherein

R¹¹ is independently H, C_1-4 alkyl, or L-C_S;

R² and R¹⁶ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, or L-C_S;

R³ and R⁴ are H, alkyl, L-C_S, L-CO₂H, L-SO₃H or together form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;

alternatively, R² and R³ and independently R⁴ and R¹⁶ together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;

R⁵, R¹², R¹³, R¹⁴ and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, or L-C_S;

R¹¹ and R¹² may together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;

R^6-10 are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, or L-C_S; and

the biomolecule is selected from the group consisting of proteins, nucleotides, polynucleotides, enzyme substrates, nanobodies, polypeptides, amino acids, lipids, carbohydrates, haptens, and drug compounds.

13. The method of claim 12, wherein the protein is an antibody.

14. The method of claim 12, wherein the polypeptide is a polypeptide-based toxin.

15. The method of claim 12, wherein the protein of interest is fused to a luciferase protein.

16. The method of claim 12, wherein the biomolecule is a drug compound.

17. The method of claim 12, wherein the biomolecule is a protein.

18. The method of claim 12, wherein the biomolecule is a polypeptide.

19. The method of claim 18, wherein the polypeptide is a polypeptide-based toxin.

20. The method of claim 17, wherein the protein is an antibody.