Pyrenyloxysulfonic acid fluorescent agents
The invention provides a novel class of reactive fluorescent agents that are based on a pyrene sulfonic acid nucleus. The agents are readily incorporated into conjugates with other species by reacting the reactive group with a group of complementary reactivity on the other species of the conjugate. Also provided are methods of using the compounds of the invention to detect and/or quantify an analyte in a sample. In an exemplary embodiment, the invention provides multi-color assays incorporating the compounds of the invention.
1. A compound having structure VI:
wherein
designates a linker arm covalently attached to a reactive group; and
s and t are independently selected from the integers from 0 to 3, with the proviso that at least one of s and t is at least 1.
2. The compound of claim 1 , wherein the reactive group comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a sulfonyl halide, a thiol, a hydrazide, a silyl halide, a succinimidyl ester of a carboxylic acid, or a psoralen group.
3. The compound of claim 2 , wherein the reactive group comprises an activated ester of a carboxylic acid, a maleimide, or a succinimidyl ester of a carboxylic acid.
4. The compound of claim 3 , wherein the reactive group comprises an activated ester of a carboxylic acid comprising an aryloxy group substituted one or more times by nitro, fluoro, chloro, cyano, trifluoromethyl, or a combination thereof.
5. The compound of claim 1 , wherein the reactive group is an activated ester of a carboxylic acid comprising an aryloxy group substituted one or more times by fluoro.
6. The compound of claim 4 , wherein the reactive group comprises the structure:
7. The compound of claim 3 , wherein the reactive group comprises a maleimide.
8. The compound of claim 3 , wherein the reactive group comprises a succinimidyl ester of a carboxylic acid.
9. The compound according to claim 3 , wherein the linker arm comprises a single covalent bond or a series of stable covalent bonds incorporating 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, S and P.
10. The compound according to claim 3 , wherein the linker arm comprises a cleavable group.
11. The compound according to claim 3 , wherein the linker arm comprises —C(O)NH—, —C(O)O—, —NH—, —S—, or —O—.
12. The compound according to claim 3 , wherein s is 1, and t is 2.
13. A compound having structure VI:
wherein
s is 1;
t is 2; and
is a linker arm and the linker arm is covalently attached to a reactive group having the structure:
14. A fluorescently labeled conjugate resulting from covalent attachment of the compound of claim 3 and a component which is a member selected from 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 and a combination thereof.
15. A fluorescently labeled conjugate resulting from covalent attachment of the compound of claim 4 and a component which is a member selected from 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 and a combination thereof.
16. A fluorescently labeled conjugate resulting from covalent attachment of the compound of claim 7 and a component which is a member selected from 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 and a combination thereof.
17. A fluorescently labeled conjugate resulting from covalent attachment of the compound of claim 8 and a component which is a member selected from 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 and a combination thereof.
18. A composition comprising:
(a) a first conjugate according to claim 14 ; and
(b) a second conjugate, comprising a component covalently bonded to a second fluorophore having a structure different from said first fluorophore.
19. The composition according to claim 18 , wherein said second fluorophore comprises a moiety that is a member selected from a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and bimane.
20. The composition according to claim 18 , wherein said second fluorophore comprises a fluorescein moiety.
21. The composition according to claim 18 , wherein said first component and said second component have different structures.
22. The composition according to claim 18 , wherein said first conjugate is bound to a binding partner for said first component.
23. The composition according to claim 18 , wherein said second conjugate is bound to a binding partner for said second component.
24. A method for detecting an analyte in a sample, said method comprising:
(a) contacting said sample with a conjugate according to claim 14 wherein said component is a binding partner for said analyte;
(b) incubating said conjugate with said sample for a sufficient amount of time for said analyte and said component to interact, thereby forming a fluorescent analyte; and
(c) illuminating said fluorescent analyte with an appropriate wavelength whereby the presence of said analyte is determined in said sample.
25. The method according to claim 24 , further comprising, between steps (b) and (c):
(b1) separating said fluorescent analyte from said sample.
26. A method for detecting an analyte in a sample, said method comprising:
(a) contacting said sample with a conjugate according to claim 15 wherein said component is a binding partner for said analyte;
(b) incubating said conjugate with said sample for a sufficient amount of time for said analyte and said component to interact, thereby forming a fluorescent analyte; and
(c) illuminating said fluorescent analyte with an appropriate wavelength whereby the presence of said analyte is determined in said sample.
27. The method according to claim 26 , further comprising, between steps (b) and (c):
(b1) separating said fluorescent analyte from said sample.
28. A method for detecting an analyte in a sample, said method comprising:
(a) contacting said sample with a conjugate according to claim 16 wherein said component is a binding partner for said analyte;
(b) incubating said conjugate with said sample for a sufficient amount of time for said analyte and said component to interact, thereby forming a fluorescent analyte; and
(c) illuminating said fluorescent analyte with an appropriate wavelength whereby the presence of said analyte is determined in said sample.
29. The method according to claim 28 , further comprising, between steps (b) and (c):
(b1) separating said fluorescent analyte from said sample.
30. A method for detecting an analyte in a sample, said method comprising:
(a) contacting said sample with a conjugate according to claim 17 wherein said component is a binding partner for said analyte;
(b) incubating said conjugate with said sample for a sufficient amount of time for said analyte and said component to interact, thereby forming a fluorescent analyte; and
(c) illuminating said fluorescent analyte with an appropriate wavelength whereby the presence of said analyte is determined in said sample.
31. The method according to claim 30 , further comprising, between steps (b) and (c):
(b1) separating said fluorescent analyte from said sample.
32. A method for detecting a first analyte and a second analyte in a sample, said method comprising:
(a) incubating said sample with a composition according to claim 18 , wherein said first component is a binding partner for said first analyte and said second component is a binding partner for said second analyte, for a time sufficient for said first analyte to interact with said first conjugate and said second analyte to interact with said second conjugate, thereby forming a fluorescent first analyte and a fluorescent second analyte, respectively;
(b) illuminating said first fluorescent analyte with an appropriate wavelength whereby the presence of said first analyte is detected in said sample; and
(c) illuminating said second fluorescent analyte with an appropriate wavelength whereby the presence of said second analyte is detected in said sample.
33. The method according to claim 32 , wherein said first fluorescent analyte and said second fluorescent analyte are illuminated with said appropriate wavelength either simultaneously or sequentially.
34. A kit for the detection of an analyte in a sample, wherein said kit comprises a compound according to claim 3 .
35. A kit for the detection of an analyte in a sample, wherein said kit comprises a compound according to claim 4 .
36. A kit for the detection of an analyte in a sample, wherein said kit comprises a compound according to claim 7 .
37. A kit for the detection of an analyte in a sample, wherein said kit comprises a compound according to claim 8 .