Fluorescent compounds
First Claim
1. A compound having a maximal fluorescence excitation wavelength, wherein the compound has a structure of Formula II:
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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:
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
35 Claims
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1. A compound having a maximal fluorescence excitation wavelength, wherein the compound has a structure of Formula II:
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2. The compound of claim 1, wherein when the two adjacent R1 and/or the two adjacent R2 form a fused 6-membered ring, the ring so formed is aromatic.
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3. The compound of claim 1, wherein Y is:
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4. The compound of claim 1, wherein at least one R of R1 and R2 is a sulfonate group.
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5. The compound of claim 1, wherein at least one R of R1 and R2 comprises a sulfonate group or a phosphonate group.
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6. The compound of claim 1, wherein each R of R1 and R2 comprises a sulfonate group or a phosphonate group.
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7. The compound of claim 1, wherein the water-soluble polymer is a polyethylene oxide.
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8. The compound of claim 1, wherein two adjacent (R1)a and the atoms in ring A to which it is attached are combined to form a carbocyclic ring.
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9. The compound of claim 8, wherein the carbocyclic ring is aromatic.
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10. The compound of claim 1, wherein two adjacent (R2)b and the atoms in ring B to which it is attached are combined to form a carbocyclic ring.
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11. The compound of claim 10, wherein the carbocyclic ring is aromatic.
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12. The compound of claim 1, wherein the compound has the formula:
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13. The compound of claim 1, wherein the compound has the formula:
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14. The compound of claim 1, wherein the compound has the formula:
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15. The compound of claim 1, wherein the reactive group is selected from the group consisting of an activated esters, a maleimide, an azide, an alkyne, an acyl azide, an isothiocyanate, a halotriazine, a haloacetamide, a sulfonate ester, a phosphoramidite, an alkylhalide, a hydrazide, an acrylamide, and an epoxide.
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16. The compound of claim 1, wherein at least two R of the (R)p-(L)q- of the compound is a water-soluble polymer which is a polyalkylene oxide radical.
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17. The compound of claim 1, wherein with the molecular weight of the water-soluble polymer ranges from 800 to 3000 daltons.
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18. 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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19. A biomolecule having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the biomolecule comprises a compound of claim 1, wherein the at least one reactive group of the compound has undergone a reaction which attaches the compound to the biomolecule.
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20. The biomolecule of claim 19, wherein the biomolecule comprises a polynucleotide or a polypeptide.
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21. The biomolecule of claim 20, wherein the polypeptide further comprises an antigen binding site.
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22. The biomolecule of claim 20, wherein the polypeptide is a whole immunoglobulin or a Fab fragment.
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23. An immunoglobin having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the immunoglobin comprises a compound of claim 1, wherein the at least one reactive group of the compound has undergone a reaction which attaches the compound to the immunoglobin, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.
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24. An immunoglobulin having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the immunoglobin is labeled with a fluorescent compound comprising a polyalkylene oxide and a fluorophore that has an absorption maximal wavelength equal to or greater than 685 nm, wherein the fluorescent compound is a compound of claim 1.
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25. The immunoglobulin of claim 24, wherein the immunoglobulin retains binding specificity to a target upon conjugation to the fluorescent compound.
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26. The immunoglobulin of claim 25, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.
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27. A polypeptide having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the polypeptide is 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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28. 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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29. The method of claim 28, wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof.
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30. 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 19, 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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31. The method of claim 30, 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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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 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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33. The method of claim 32, wherein the binding agent is an antibody.
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34. The method of claim 33, 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 34, 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).
Specification