# US 7,611,909 B1 · Near infrared chemiluminescent acridinium compounds and uses thereof

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- Number: US 7,611,909 B1
- Document ID: US7611909B1
- Status: Granted
- Kind: B1
- Application number: 11/266,902
- Filed: 2005-11-04
- Granted: 2009-11-03
- Earliest claimed: 1998-08-11
- Assignees: Siemens Healthcare Diagnostics Inc.
- Inventors: Anand Natrajan; Qingping Jiang; David Sharpe; Say-Jong Law
- Web page: https://patentgrep.com/patent/US7611909B1

## CPC

- G01N 33/582: {with fluorescent label}
- 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
- Y10S 436/80: Fluorescent dyes, e.g. rhodamine
- Y10S 436/815: Test for named compound or class of compounds
- Y10S 436/817: Steroids or hormones
- Y10S 530/812: Peptides or proteins is immobilized on, or in, an organic carrier
- Y10T 436/13: Tracers or tags
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A method for the detection or quantitation of unlabeled target analyte in a biological sample, the method comprising labeling a target analyte for a biological sample suspected of containing unlabeled target analyte with an acridinium compound to form a labeled target analyte and providing the labeled target analyte to the biological sample, or providing the labeled target analyte to the biological sample, wherein the acridinium compound comprises an acridinium nucleus having an electron-donating substituent directly attached to the acridinium nucleus, with the electron-donating substituent attached at the C2 position. Chemiluminescent acridinium compounds useful in the method have emission maxima close to or in the near infrared (NIR) region (>590 nm). These chemiluminescent acridinium compounds when used in conjunction with short wavelength-emitting acridinium esters (with emission maxima below 450 nm) can be highly useful labels for the simultaneous detection of multiple target analytes in a single assay.

## Claims

### Claim 1

An assay method for the detection or quantitation of a biological molecule in a biological sample comprises binding a binding partner with a tracer to form a binding complex between the binding partner and the tracer, and wherein chemiluminescence is triggered from said tracer following said binding, wherein the improvement comprising detecting a light signal emitted at a wavelength maximum longer than 590 nm from said binding complex, and relating said detected light signal to the presence or an amount of said biological molecule in said biological sample,
wherein said tracer is formed by covalently coupling, either directly or indirectly, a near-infrared acridinium compound to said biological molecule or analog, wherein said biological molecule is selected from the group consisting of small organic biomolecules, haptens and ligands,
wherein said near-infrared acridinium compound comprises an acridinium nucleus having an electron-donating substituent directly attached to the acridinium nucleus, with the electron-donating substituent attached at the C2 position, the near-infrared acridinium compound having the structure:
wherein
R1 is alkyl, alkenyl, alkynyl or aralkyl, or alkyl, alkenyl, alkynyl or aralkyl having up to 20 heteroatoms;
R2 and R3 are identical or different, and are selected from the group consisting of hydrogen, R, substituted aryl, unsubstituted aryl, halide, amino, hydroxyl, nitro, sulfonate, —CN, —COOH, —SCN, —OR, —SR, —SSR, —C(O)R, —C(O)OR, —C(O)NHR and —NHC(O)R;
R is alkyl, alkenyl, alkynyl, aryl or aralkyl, or alkyl, alkenyl, alkynyl, aryl or aralkyl having up to 20 heteroatoms;
alternatively, R2 and R3 can be bridged so as to form an additional ring fused to the acridinium nucleus;
C1 and C3 through C8 of the acridinium nucleus can be substituted with substituents as defined by R2 and R3, and wherein the substituents defined by R2 and R3 are identical or different if more than one substituent is used;
W is an electron-donating substituent that can donate an electron pair selected from the group consisting of OH, SH, NR′R″ and —CHp(EWG)m where p+m=3 and EWG is an electron withdrawing group selected from the group consisting of —NO2, —NO, —CN, —CHO, —C(O)R, +NR′R″R′″, —COOR, —COOH, —S(O)R, —SO2R, —SO2OR, —SO2NHR, —SO2NR′R″, —SO2OH and F;
R′, R″ and R′″ are hydrogen or low alkyl and can all be the same or different;
R3 can also be the same as W in which case the acridinium nucleus has two electron donating substituents instead of one;
a counter-ion to pair with the quaternary nitrogen of the acridinium nucleus, wherein said counter-ion may be part of R1 or may be a group A−;
X is nitrogen, oxygen or sulfur, such that when X is oxygen or sulfur, Z is omitted and Y is a polysubstituted aryl moiety of the formula:
where R4 and R8 are alkyl, alkenyl, alkynyl, alkoxyl (—OR), alkylthiol (—SR) or substituted amino groups that serve to stabilize the —COX— linkage between the acridinium nucleus and the Y moiety through steric and/or electronic effect;
R5 and R7 are any of R2 and R3 as defined above,
R6=—R9-R10, where R9 is not required but optionally can be a branched or straight-chained alkyl, substituted or unsubstituted aryl or aralkyl containing optionally up to 20 heteroatoms; and
R10 is selected from the group consisting of:
a halide, —COOH, —Q—R—Nu, —Q—R—(I)nNu—, —Q—Nu, —R—Nu and —Nu, where n is a integer of at least 1, Nu is a nucleophilic group, Q is a functional linkage, and I is an ionic or ionizable group; and wherein
R5 and R6 and/or R6 and R7 are interchangeable; and
when X is nitrogen, then Z is —SO2—Y′, and Y and Y′ are each the polysubstituted aryl moiety defined above, or Y′ is the polysubstituted aryl moiety defined above and Y is a branched or straight-chained alkyl containing optionally up to 20 carbon atoms, halogenated or unhalogenated, a substituted aryl or heterocyclic ring system.

### Claim 2 (depends on 1)

The method of claim 1, further comprising detection of a second biological molecule in a second binding complex by detecting a second light signal emitted at a different wavelength from said light signal emitted from said near-infrared acrindinium compound.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein
A− is selected from the group consisting of CH3SO4−, FSO3−, CF3SO4−, C4F9SO4−, CH3C6H4SO3−, halide, CF3COO−, CH3COO− and NO3−,
R4 and R8 are methyl or low alkyl, and
R1 is methyl or sulfoalkyl.

### Claim 4 (depends on 1, 2)

The method of claim 1 or 2, wherein the near-infrared acridinium compound is selected from the group consisting of 2-hydroxy-DMAE, NSB-2-hydroxy-DMAE, NSP-2-hydroxy-DMAE, 2-hydroxy-7-methoxy-DMAE, NSB-2-hydroxy-7-methoxy-DMAE, and NSP-2-hydroxy-7-methoxy-DMAE, and their corresponding ester, amide, carbonyl halide and anhydride derivatives of the free carboxylate group.

### Claim 5 (depends on 1, 2)

The method of claim 1 or 2, wherein the tracer is formed by covalently coupling either directly or indirectly, said near-infrared acridinium compound to said biological molecule or analog through a spacer.

### Claim 6 (depends on 5)

The method of claim 5, wherein the spacer is provided by a bifunctional cross-linker.

### Claim 7 (depends on 1)

The method of claim 1, wherein said small organic biomolecule is a biologically active molecule.

### Claim 8 (depends on 1)

The method of claim 1, wherein said hapten is a thyroid hormone, steroid, vitamin, antibiotic, enzyme cofactor, therapeutic drug, metabolite, lipid, neurotransmitter or controlled chemical substance.