Fluorescent compounds
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Abstract
The present invention relates to fluorescent dyes. The present invention provides a wide range of fluorescent dyes and kits containing the same, which are applicable for labeling a variety of biomolecules, cells and microorganisms. In one aspect, the invention provides a compound having a maximal fluorescence excitation wavelength, wherein the compound has a structure of Formula II:
F—Y=Ψ Formula II
and wherein Z— is a counterion, Y is a bridge unit permitting electron delocalization between F and Ψ, and F is a moiety having the structure:
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.
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Citations
30 Claims
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1. A compound of any one of the formulae:
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2. A kit comprising:
i) the compound of claim 1;
ii) a buffer;
iii) materials or devices for purifying conjugation products; and
iv) instructions instructing the use of the compound.
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3. A biomolecule having an amine, a thiol, a hydroxyl, or an aldehyde functional group comprising a label having a structure of a Formula of claim 1, wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the biomolecule.
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4. The biomolecule comprising a label of claim 3 wherein the biomolecule comprises a polynucleotide.
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5. The biomolecule comprising a label of claim 3 wherein the biomolecule comprises a polypeptide.
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6. The biomolecule of claim 5, wherein the polypeptide further comprises an antigen binding site.
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7. The biomolecule of claim 5, wherein the polypeptide is a whole immunoglobulin.
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8. The biomolecule of claim 5, wherein the polypeptide is a Fab fragment.
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9. An immunoglobulin having an amine, a thiol, a hydroxyl, or an aldehyde functional group labeled with a compound of claim 1.
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10. The immunoglobulin of claim 9, wherein the immunoglobulin retains binding specificity to a target upon conjugation to the fluorescent compound.
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11. The immunoglobulin of claim 10, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.
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12. The immunoglobin of claim 11 wherein the antibody binds to erb2.
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13. An immunoglobin having an amine, a thiol, a hydroxyl, or an aldehyde functional group comprising a label having a structure of a Formula of claim 1 wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the immunoglobin, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.
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14. The immunoglobin of claim 13 wherein the antibody binds to erb2.
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15. A polypeptide having an amine, a thiol, a hydroxyl, or an aldehyde functional group labeled with a fluorescent compound, the polypeptide exhibiting a serum half-life no shorter than that of a corresponding polypeptide that lacks the fluorescent compound, wherein the fluorescent compound is a compound of claim 1.
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16. A method of preparing a labeled biomolecule comprising reacting a compound of claim 1 and a substrate biomolecule under conditions sufficient to effect crosslinking between the compound and the substrate biomolecule.
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17. The method of claim 16, wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof.
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18. The method of claim 17, wherein the substrate biomolecule is a polynucleotide.
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19. A method of labeling a polypeptide comprising:
- forming a complex that comprises the polypeptide and a binding agent, wherein the binding agent comprises a fluorescent label having a structure of formula of claim 1 wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the binding agent.
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20. The method of claim 19 wherein the binding agent is an antibody.
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21. The method of claim 20 wherein the complex comprises (a) a primary antibody that binds to the polypeptide, and (b) the binding agent which functions as a secondary antibody exhibiting binding capability to the primary antibody.
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22. The method of claim 21, wherein the labeling occurs on a solid substrate.
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23. The method of claim 21 that labels a polypeptide intracellularly.
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24. The method of claim 21, wherein the complex yields a signal to noise ratio greater than about 100, wherein the signal to noise ratio is calculated by the formula:
(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).
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25. The method of claim 21, wherein the complex yields a signal to noise ratio greater than about 250, wherein the signal to noise ratio is calculated by the formula:
(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).
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26. The method of claim 21, wherein the complex yields a signal to noise ratio greater than about 270, wherein the signal to noise ratio is calculated by the formula:
(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).
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27. A method for labeling a cell within a population of cells whereby the cell is differentially labeled relative to neighboring cells within the population, the method comprising contacting the cell with a biomolecule of claim 3, wherein the biomolecule comprises a targeting moiety that binds to a binding partner that is indicative of the cell, and thereby differentially labeling the cell relative to neighboring cells within the population.
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28. The method of claim 27, further comprising the step of imaging the cell, the imaging step comprising:
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i) directing exciting wavelength to the cell; and ii) detecting emitted fluorescence from the cell.
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29. The method of claim 27, wherein the labeling takes place in vitro.
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30. The method of claim 27, wherein the labeling takes place in vivo.
Specification