Hydrophilic chemiluminescent acridinium labeling reagents

• US 7,297,555 B2
• Filed: 07/14/2005
• Issued: 11/20/2007
• Est. Priority Date: 08/20/2002
• Status: Expired due to Fees

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Evangelista, Ramon, Garrity, Martha
• Primary Examiner(s): Le; Long V.
• Assistant Examiner(s): Haq; Shafiqul
• Application Number:
  US11/181,531
• Publication Number:
  US 20050255528A1
• Time in Patent Office: 859 Days
• Field of Search: 546/104, 544/335, 544/405, 436/526, 436/546, 436/544, 436/172, 436/800, 435/7.5, 435/7.1, 435/968
• US Class Current: 436/546
• CPC Class Codes: C07D 219/04 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

In accordance with the present invention, it has been discovered that introduction of hydrophilic sulfoalkyl substituents and/or hydrophilic linkers derived from homocysteic acid, cysteic acid, glycine peptides, tetraethylene oxide, and the like, offset the hydrophobicity of the acridinium ring system to produce a more soluble label which can be attached to an antibody at higher loading before precipitation and aggregation problems are encountered. Additional compounds described herein contain linkers derived from short peptides and tetraethylene oxide which increase aqueous solubility due to hydrogen bonding with water molecules. The present invention also embraces reagents for multiple acridinium labeling for signal amplification composed of a peptide bearing several acridinium esters with sulfonate groups at regularly spaced intervals for increased solubility. The invention also embraces assays employing the above-described compounds.

Claims

1. A chemiluminescent compound having the structure:

2. A compound according to claim 1 wherein X is O.

3. A compound according to claim 1 wherein Y is O.

4. A compound according to claim 1 wherein Z has the structure:

—

(CR″

₂)_q—

SO₃⁻

,

wherein;

each R″

is independently H, lower alkyl or substituted lower alkyl; and

q=1-6.

5. A compound according to claim 4 wherein Z is sulfopropyl.

6. A compound according to claim 1 wherein Z has the structure:

—

(CR″

₂)_r—

C(R″

)═

C(R″

)—

(CR″

₂)_r—

SO₃⁻

,

wherein;

each R″

is independently H, lower alkyl or substituted lower alkyl; and

each r is independently 0-4.

7. A compound according to claim 1 wherein Ar is aryl.

8. A compound according to claim 7 wherein said aryl is 2,6-dialkyl substituted.

9. A compound according to claim 8 wherein Ar has the structure:

10. A compound according to claim 1 wherein Ar is heteroaryl.

11. A compound according to claim 10 wherein said heteroaryl is a pyridyl, pyrimidyl, pyrazinyl, triazolyl, isooxazolyl, isothioazolyl, or imidazolyl.

12. A compound according to claim 1 wherein the amine reactive group is a succinimidyl ester, N-hydroxyphthalimide ester, pentafluorophenyl ester, tetrafluorophenyl ester, 2-nitrophenyl ester, 4-nitrophenyl ester, dichlorotriazine, or isothiocyanate.

13. A compound according to claim 1 that is 2,3,4,5,6-pentafluorophenyloxy-1-carbonyl-4,7,10,13-tetraoxapentadecyl-(15-amino)-3′

-sulfonyl-2′

,6′

-dimethylphenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate or [2,5-dioxo-(1-pyrrolidinyl)oxy]-1-carbonyl-4,7,10,13-tetraoxapentadecyl-(15-amino)-3′

-sulfonyl-2′

,6′

-dimethylphenyl-N-methyl-acridinium-9-carboxylate.

14. A chemiluminescent conjugate comprising a chemiluminescent compound according to claim 1 conjugated with a specific binding material.

15. A conjugate according to claim 14 wherein said specific binding material is an antibody.

16. A chemiluminescent assay kit comprising a conjugate according to claim 15.

17. An assay for the presence of an analyte in a sample, said assay comprising:

contacting an analyte with a conjugate according to claim 14, wherein said conjugate specifically binds to the analyte,

inducing chemiluminescence of the conjugate specifically bound to the analyte in the presence of a peroxide or molecular oxygen, and

measuring chemiluminescence therefrom to assay the presence of the analyte.