FLUORESCENT PYRENE COMPOUNDS
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Abstract
The present invention relates to fluorescent pyrene dyes in general. 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. 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
40 Claims
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1. A compound of Formula I:
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2. The compound of claim 1, wherein A is —
- O—
.
- O—
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3. The compound of claim 1, wherein A is —
- NR2—
.
- NR2—
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4. The compound of claim 1, wherein A is —
- NH—
.
- NH—
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5. The compound of claim 1, wherein a is between 2 and 10.
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6. The compound of claim 1, wherein a is 5.
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7. The compound of claim 1, wherein each R1 and R1′
- is H.
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8. The compound of claim 1, wherein L is a bond.
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9. The compound of claim 1, wherein L comprises a water-soluble moiety.
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10. The compound of claim 1, wherein Rx is a reactive group capable of forming a covalent bond by reacting with an amine group.
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11. The compound of claim 1, wherein Rx is an activated ester.
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12. The compound of claim 1, wherein Rx has the formula —
- CO-(Lg), wherein Lg is a leaving group.
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13. The compound of claim 1, wherein Rx is an N-hydroxysuccinimide ester.
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14. The compound of claim 1, wherein Rx is an aminooxy.
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15. The compound of claim 4, wherein Rx is an aminooxy.
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16. 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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17. A biomolecule comprising a label derived from a compound of Formula I, wherein the at least one reactive moiety of Formula I has undergone a reaction which attaches the label to the biomolecule.
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18. The biomolecule comprising a label of claim 17 wherein the biomolecule comprises a polynucleotide.
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19. The biomolecule comprising a label of claim 17 wherein the biomolecule comprises a polypeptide.
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20. The biomolecule of claim 19, wherein the polypeptide further comprises an antigen binding site.
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21. The biomolecule of claim 19, wherein the polypeptide is a whole immunoglobulin.
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22. The biomolecule of claim 19, wherein the polypeptide is a Fab fragment.
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23. An immunoglobin comprising a label derived from a compound of Formula I, wherein the at least one reactive moiety of Formula I 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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24. The immunoglobulin of claim 23, wherein the antibody binds to erb2.
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25. A method of preparing a labeled biomolecule comprising reacting a compound according to claim 1 and a substrate biomolecule under conditions sufficient to effect crosslinking between the compound and the substrate biomolecule.
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26. The method of claim 25, wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof.
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27. The method of claim 25, wherein the substrate biomolecule is a polynucleotide.
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28. 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 17, 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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29. The method of claim 28, further comprises the step of imaging the cell, the imaging step comprising:
- i) directing exciting wavelength to the cell; and
ii) detecting emitted fluorescence from the cell.
- i) directing exciting wavelength to the cell; and
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30. The method of claim 28, wherein the labeling takes place in vitro.
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31. The method of claim 28, wherein the labeling takes place in vivo.
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32. 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 derived from a compound according to claim 1, wherein the at least one reactive moiety of Formula I has undergone a reaction which attaches the label to the binding agent.
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33. The method of claim 32, wherein the binding agent is an antibody.
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34. The method of claim 32, 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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35. The method of claim 32, wherein the labeling occurs on a solid substrate.
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36. The method of claim 32, wherein the labeling occurs intracellularly.
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37. The method of claim 32, wherein the complex yields a signal to noise ratio greater than about 100.
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38. The method of claim 32, 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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39. The method of claim 32, 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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40. The method of claim 32, 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).
Specification