Methods for detecting anionic and non-anionic compositions using carbocyanine dyes

• US 20050244976A1
• Filed: 02/22/2005
• Published: 11/03/2005
• Est. Priority Date: 02/20/2004
• Status: Abandoned Application

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Patton, Wayne Forrest, Gee, Kyle Richard
• Application Number:
  US11/063,707
• Publication Number:
  US 20050244976A1
• Time in Patent Office: Days
• US Class Current: 436/86
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: G01N 33/6839 involving dyes, e.g. Coomassie blue, bromcresol green
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Abstract

The present invention relates to methods of detecting anionic proteins in a sample with fluorescent carbocyanine dye compounds. The invention also describes methods of simultaneously detecting anionic and non-anionic proteins in a sample with discrete fluorescent signals produced by carbocyanine dye compounds. The invention is of use in a variety of fields including immunology, diagnostics, molecular biology and fluorescence based assays.

Claims

1. A method for determining the presence or absence of an anionic protein and the presence or absence of a non-anionic protein in a sample, the method comprising:

a) contacting the sample with a dye to form a labeled sample, wherein the dye has the following formula;

in which X is

wherein at most one of the H is replaced with

wherein

R¹ is hydrogen, alkyl or substituted alkyl;

R² is hydrogen, alkyl or substituted alkyl;

R⁷ is hydrogen, alkyl or substituted alkyl;

R⁸ is hydrogen, alkyl or substituted alkyl;

R¹³ is hydrogen, alkyl or substituted alkyl;

R¹⁴ is hydrogen, alkyl or substituted alkyl; and

R³ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁴ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁵ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁶ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁹ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁰ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹¹ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹² is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁵ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁶ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁷ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁸ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁹ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

or,

a member independently selected from

R³ in combination with R⁴;

R⁴ in combination with R⁵;

R⁵ in combination with R⁶;

R⁹ in combination with R¹⁰;

R¹⁰ in combination with R¹¹;

R¹¹ in combination with R¹²;

R¹⁵ in combination with R¹⁶;

R¹⁶ in combination with R¹⁷;

R¹⁷ in combination with R¹⁸

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

b) incubating the labeled sample for sufficient time to allow the dye to associate with an anionic protein and non-anionic protein to form an incubated sample;

c) illuminating the incubated sample with a first appropriate wavelength for observing the anionic proteins to form a first illuminated sample;

d) illuminating the incubated sample with a second appropriate wavelength for observing non-anionic proteins to form a second illuminated sample;

e) observing the first and the second illuminated sample whereby the presence of anionic and non-anionic proteins are determined.

2. The method according to claim 1, wherein the dye has the formula:

wherein R¹ is substituted alkyl or unsubstituted alkyl;

R² is substituted alkyl or unsubstituted alkyl;

R⁷ is substituted alkyl or unsubstituted alkyl;

R⁸ is substituted alkyl or unsubstituted alkyl; and

R⁴ is a halogen;

R⁵ is a halogen;

R¹⁰ is a halogen; and

,

R¹¹ is a halogen.

3. The method according to claim 1, wherein the dye is:

4. The method according to claim 1, wherein the dye further comprises a reactive group, carrier molecule or solid support.

5. The method according to claim 4, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group.

6. The method according to claim 4, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

7. The method according to claim 4, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus, or combinations thereof.

8. The method according to claim 4, wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

9. The method according to claim 4, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

10. The method according to claim 4, wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

11. The method according to claim 1, wherein said sample is in a cuvette.

12. The method according to claim 1, wherein the sample is immobilized on a solid or semi solid support.

13. The method according to claim 12, wherein the solid or semi-solid support is a polymeric microparticle, polymeric membrane, polymeric gel or glass slide.

14. The method according to claim 1, wherein the anionic protein is phosphoproteins, calcium-binding proteins, sulfoproteins, or sialoglycoproteins.

18. The method according to claim 11, wherein the sample is immobilized on a solid or semi solid support.

19. The method according to claim 18, wherein the solid or semi-solid support is a polymeric microparticle, polymeric membrane, polymeric gel or glass slide.

15. A method for detecting the presence or absence of an anionic protein in a sample, the method comprising the steps of:

a) contacting the sample with a dye to form a labeled sample, wherein the dye is;

wherein R¹ is a substituted alkyl or unsubstituted alkyl;

R² is a substituted alkyl or unsubstituted alkyl;

R⁷ is a substituted alkyl or unsubstituted alkyl;

R⁸ is a substituted alkyl or unsubstituted alkyl; and

,

R⁴ is a halogen;

R⁵ is a halogen;

R¹⁰ is a halogen; and

R¹¹ is a halogen;

b) incubating the labeled sample for a sufficient amount of time to allow the dye to associate with the anionic proteins to form an incubated sample;

c) illuminating the incubated sample with an appropriate wavelength to form an illuminated sample;

d) observing the illuminated sample whereby the presence or absence of the anionic proteins is determined.

16. The method according to claim 15, wherein the dye is:

17. The method of claim 15, wherein said sample is in a cuvette.

20. The method according to claim 15, wherein the anionic protein is phosphoproteins, calcium-binding proteins, sulfoproteins, or sialoglycoproteins.

21. A kit which comprises:

a) a compound having the formula;

wherein R¹ is a substituted alkyl or unsubstituted alkyl;

R² is a substituted alkyl or unsubstituted alkyl;

R⁷ is a substituted alkyl or unsubstituted alkyl;

R⁸ is a substituted alkyl or unsubstituted alkyl; and

,

R⁴ is a halogen;

R⁵ is a halogen;

R¹⁰ is a halogen; and

R¹¹ is a halogen; and

,

b) instructions for detecting the presence or absence of anionic proteins in a sample.

22. A compound having the formula:

in which X is

wherein at most one of the H is replaced with

wherein

R¹ is hydrogen, alkyl or substituted alkyl;

R² is hydrogen, alkyl or substituted alkyl;

R⁷ is hydrogen, alkyl or substituted alkyl;

R⁸ is hydrogen, alkyl or substituted alkyl;

R¹³ is hydrogen, alkyl or substituted alkyl;

R¹⁴ is hydrogen, alkyl or substituted alkyl; and

R³ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁴ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁵ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁶ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R⁹ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁰ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹¹ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹² is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁵ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁶ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁷ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁸ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

R¹⁹ is hydrogen, OH, NH₂, NO₂, —

SO₂NH₂, nitro, cyano, halogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted 3- to 7-membered cycloalkyl, unsubstituted 3- to 7-membered cycloalkyl, substituted 5- to 7-membered heterocycloalkyl, unsubstituted 5- to 7-membered heterocycloalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

or,

a member independently selected from

R³ in combination with R⁴;

R⁴ in combination with R⁵;

R⁵ in combination with R⁶;

R⁹ in combination with R¹⁰;

R¹⁰ in combination with R¹¹;

R¹¹ in combination with R¹²;

R¹⁵ in combination with R¹⁶;

R¹⁶ in combination with R¹⁷;

R¹⁷ in combination with R¹⁸

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ or R¹² is a reactive group.