Pyrenyloxysulfonic acid fluorescent agents

• US 8,268,886 B2
• Filed: 05/31/2011
• Issued: 09/18/2012
• Est. Priority Date: 12/09/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard, Leung, Wai-Yee, Liu, Jixiang
• Primary Examiner(s): Wilson, James O
• Assistant Examiner(s): Sackey, Ebenezer O
• Application Number:
  US13/149,444
• Publication Number:
  US 20120045848A1
• Time in Patent Office: 476 Days
• Field of Search: 562/52, 530/391.3, 436/501, 514/553, 514/702
• US Class Current: 514/553
• CPC Class Codes: C07C 309/38 formed by at least three rings
• CPC Class Codes: C07D 207/16 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 211/56 Nitrogen atoms nitro radicals C07D211/38
• CPC Class Codes: C07D 211/60 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 211/62 attached in position 4
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07H 17/08 Hetero rings containing eight or more ring members, e.g. erythromycins
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07K 14/77 Ovalbumin
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C09B 57/001 Pyrene dyes
• CPC Class Codes: G01N 33/536 with immune complex formed in liquid phase

Abstract

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.

Claims

1. A compound having structure VI:

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.

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.

13. A compound having structure VI: