Carboxy X rhodamine analogs

Patent metadata

Field	Value
Publication number	US-10139400-B2
Application number	US-201514724111-A
Country	US
Kind code	B2
Filing date	May 28, 2015
Priority date	Nov 21, 2011
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

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.

First claim

The invention claimed is:

1. A method to detect a selected molecule in a sample, comprising:
a) contacting a sample suspected of containing the selected molecule with a composition comprising a dye conjugate according to formula (IIIa), (IIIb) or (IIIc) so as to yield a mixture:

wherein
R 11 is independently H or C 1-4 alkyl or L-C s ;
L is a covalent linkage that is linear or branched, cyclic or heterocyclic saturated or unsaturated, having 1-16 non hydrogen atoms such that the linkage contains any combination of ester, acid, amine, amide, alcohol, ether, thioether or halide groups or single, double, triple or aromatic carbon-carbon bond;
C s is a conjugated substance selected from the group consisting of solid supports, resin particles, beads, assay plates, proteins, nucleotides, polynucleotides, enzyme substrates, nanobodies, polypeptides, amino acids, lipids, carbohydrates, haptens, drugs, ion-complexing agents, microparticles, polymers, cells, viruses, fluorophores, chloroalkanes, and cyanobenzothiazoles;
R 2 and R 16 can be independently H, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
R 3 and R 4 are H, alkyl, L-C s , L-CO 2 H, L-SO 3 H or together form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;
alternatively, R 2 and R 3 and independently R 4 and R 16 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R 5 , R 12 , R 13 , R 14 and R 15 are independently H, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
R 20 , R 21 , R 22 and R 23 are independently H or C 1-6 alkyl or one or more of R 20 and R 21 , R 21 and R 22 , R 22 and R 23 , together form an aryl, heteroaryl, carbocyclic or heterocyclic ring;
alternatively R 11 and R 12 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R 6-10 are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
X is CHR 23 , O, S or NR 30 ; and
R 30 is H, C1-4 alkyl or —C(O)C1-4 alkyl; and
b) detecting the presence or amount of the dye conjugate in the mixture, thereby detecting the selected molecule in the sample.

2. The method of claim 1 , wherein at least one of R 2-16 is -L-C s .

3. The method of claim 1 , wherein C s is a polynucleotide.

4. A method of detecting the presence of a nucleic acid polymer in a sample comprising:
a) contacting a sample suspected of containing a nucleic acid polymer with a composition comprising a dye conjugate according to formula (IIIa), (IIIb) or (IIIc) so as to yield a mixture:

wherein
R 11 is independently H, C 1-4 alkyl, or L-C s ;
L is a covalent linkage that is linear or branched, cyclic or heterocyclic saturated or unsaturated, having 1-16 non hydrogen atoms such that the linkage contains any combination of ester, acid, amine, amide, alcohol, ether, thioether or halide groups or single, double, triple or aromatic carbon-carbon bond;
C s is an oligonucleotide;
R 2 and R 16 can be independently H, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
R 3 and R 4 are H, alkyl, L-C s , L-CO 2 H, L-SO 3 H or together form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;
alternatively, R 2 and R 3 and independently R 4 and R 16 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R 5 , R 12 , R 13 , R 14 and R 15 are independently H, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
R 20 , R 21 , R 22 and R 23 are independently H or C 1-6 alkyl or one or more of R 20 and R 21 , R 21 and R 22 , R 22 and R 23 , together form an aryl, heteroaryl, carbocyclic or heterocyclic ring;
alternatively, R 11 and R 12 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R 6-10 are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
X is CHR 23 , O, S or NR 30 ; and
R 30 is H, C 1-4 alkyl or —C(O)C 1-4 alkyl; and
b) detecting the presence or amount of the dye conjugate in the mixture, thereby detecting the presence of the nucleic acid polymer in the sample.

5. The method of claim 4 , wherein more than one nucleic acid polymer in a sample is detected in a single reaction.

6. The method of claim 5 , wherein the more than one nucleic acid polymer is detected by capillary electrophoresis.

7. A method of labeling a biomolecule comprising:
a) contacting a sample suspected of containing the biomolecule with a composition comprising a dye conjugate according to formula (IIIa), (IIIb) or (IIIc) so as to yield a mixture:

wherein
R 11 is independently H or C 1-4 alkyl, or L-C s ;
L is a covalent linkage that is linear or branched, cyclic or heterocyclic saturated or unsaturated, having 1-16 non hydrogen atoms such that the linkage contains any combination of ester, acid, amine, amide, alcohol, ether, thioether or halide groups or single, double, triple or aromatic carbon-carbon bond;
C s is a conjugated substance selected from the group consisting of solid supports, resin particles, beads, assay plates, proteins, nucleotides, polynucleotides, enzyme substrates, nanobodies, polypeptides, amino acids, lipids, carbohydrates, haptens, drugs, ion-complexing agents, microparticles, polymers, cells, viruses, fluorophores, chloroalkanes, and cyanobenzothiazoles;
R 2 and R 16 can be independently H, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
R 3 and R 4 are H, alkyl, L-C s , L-CO 2 H, L-SO 3 H or together form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;
alternatively, R 2 and R 3 and independently R 4 and R 16 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R 5 , R 12 , R 13 , R 14 and R 15 are independently H, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
R 20 , R 21 , R 22 and R 23 are independently H or C 1-6 alkyl or one or more of R 20 and R 21 , R 21 and R 22 , R 22 and R 23 , together form an aryl, heteroaryl, carbocyclic or heterocyclic ring;
alternatively R 11 and R 12 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R 6-10 are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl, CO 2 H, SO 3 H, L-CO 2 H, L-SO 3 H, or L-C s ;
X is CHR 23 , O, S or NR 30 ; and
R 30 is H, C 1-4 alkyl or —C(O)C 1-4 alkyl;
b) detecting the presence or amount of the dye conjugate, thereby detecting the presence or amount of the labeled biomolecule in the mixture.

8. The method of claim 7 , wherein the dye conjugate contains a cyanobenzothiazole.

9. The method of claim 7 , wherein the biomolecule is a protein.

10. The method of claim 9 , wherein the protein comprises an N-terminal cysteine.

11. The method of claim 10 , wherein the protein comprising an N-terminal cysteine is labeled with the dye conjugate comprising a cyanobenzothiazole.

12. The method of claim 4 , wherein L is —CO—, —SCH 2 CO—, or —SO 2 —.

13. The method of claim 12 , wherein L is —SCH 2 CO— and L is bonded to a nucleotide base in the oligonucleotide to form a group

Assignees

• Promega Corp

Inventors

• Kirkland Thomas A
• Mcdougall Mark G
• Dwight Stephen J

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• G01N33/582

  with fluorescent label · CPC title

• C07F9/65586

  at least one of the hetero rings does not contain nitrogen as ring hetero atom · CPC title

• G01N33/542 Primary

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C07D471/06

  Peri-condensed systems · CPC title