# US 8,541,555 B2 · Hydrazone-based and oxime-based fluorescent and chromophoric/pro-fluorescent and pro-chromophoric reagents and linkers

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- Number: US 8,541,555 B2
- Document ID: US8541555B2
- Status: Granted
- Kind: B2
- Application number: 11/787,932
- Filed: 2007-04-18
- Published: 2008-09-11
- Granted: 2013-09-24
- Earliest claimed: 2006-04-18
- Assignees: Solulink Biosciences, Inc.
- Applicants: David A. Schwartz; Leopoldo Mendoza
- Inventors: David A. Schwartz; Leopoldo Mendoza
- Web page: https://patentgrep.com/patent/US8541555B2

## CPC

- G01N 33/582: {with fluorescent label}
- C07D 303/02: Compounds containing oxirane rings
- 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)

## Abstract

Conjugationally extended hydrazine compositions of the formula (RR2)N(H)n(NH2)n, fluorescent hydrazone compositions of the formula (RR2)NN═C(R1R2), methods of the formation of hydrazones from the reaction of conjugationally extended hydrazines with conjugationally extended carbonyls and methods of their use in assays systems are described. Use of these conjugationally extended hydrazine and oxime compositions for direct calorimetric and fluorometric assays wherein a chromophore or the fluorophore is incorporated into the linker that is positioned between a reactive linking moiety and a biotin molecule. More specifically the linker comprises one molecule of a high affinity binding pair such as for example biotin of the biotin/avidin high affinity binding pair, connected to a spacer molecule such as for example a length of polyethyleneglycol followed by a pro-chromophoric, chromophoric, pro-fluorescent or fluorescent moiety connected to an amino-, thiol- or carbohydrate-reactive moiety such as for example succinimidyl, maleimido or aminoxy group respectively, that may covalently link to a biomolecule.

## Claims

### Claim 1

A spectrophotometrically quantifiable linker comprising of formula:
A-B-C-D
wherein;
A represents an amino reactive moiety, a thiol reactive moiety, or a carbohydrate reactive moiety, comprising: N-hydroxysuccinimidyl; p-nitrophenyl; pentafluorophenyl; N-hydroxybenzotriazolyl; maleimido; α-haloacetamido; pyridylsulfide; or aminooxy moiety;
B represents a chromophoric or fluorescent hydrazone-containing moiety, wherein the hydrazone-containing moiety fluoresces, emits light, or precipitates a colored insoluble product upon enzymatic processing;
C represents a flexible linker comprising PEG; and
D represents biotin or a receptor ligand.

### Claim 2 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the amino reactive moiety comprises: N-hydroxysuccinimidyl, p-nitrophenyl, pentafluorophenyl, or N-hydroxybenzotriazolyl moiety.

### Claim 3 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the thiol reactive moiety comprises: maleimido, α-haloacetamido, or pyridylsulfide moiety.

### Claim 4 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the carbohydrate reactive moiety comprises: aminooxy moiety.

### Claim 5 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the receptor ligand comprises: a digoxigenin, a peptide, a peptide-S, a protein, an antibody, an oligonucleotide, or a complimentary oligonucleotide.

### Claim 6 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein a biomolecule is bound to the spectrophotometrically quantifiable linker via:
i) the amino reactive moiety, thiol reactive moiety, or carbohydrate reactive moiety of said spectrophotometrically quantifiable linker; or
ii) the receptor ligand of said spectrophotometrically quantifiable linker.

### Claim 7 (depends on 6)

The spectrophotometrically quantifiable linker of claim 6, wherein the biomolecule comprises: a protein, a peptide, an oligonucleotide, a complimentary oligonucleotide, a polynucleotide, or combinations thereof.

### Claim 8 (depends on 6)

The spectrophotometrically quantifiable linker of claim 6, wherein the biomolecule and the biotin or receptor ligand of the biomolecule-bound spectrophotometrically quantifiable linker comprises:
i) a biotin/avidin pair;
ii) a digoxigenin/anti-digoxigenin pair;
iii) a peptide/anti-peptide antibody pair;
iv) a peptide-S/ribonuclease pair;
v) a complementary oligonucleotide pair; or
vi) an antibody/ligand pair.

### Claim 9 (depends on 6)

The spectrophotometrically quantifiable linker of claim 6, wherein the biomolecule is bound to the spectrophotometrically quantifiable linker via the amino reactive moiety, thiol reactive moiety, or carbohydrate reactive moiety of said spectrophotometrically quantifiable linker.

### Claim 10 (depends on 6)

The spectrophotometrically quantifiable linker of claim 6, wherein the biomolecule is bound to the spectrophotometrically quantifiable linker via the receptor ligand of said spectrophotometrically quantifiable linker.

### Claim 11 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein:
i) a first biomolecule is bound to the spectrophotometrically quantifiable linker via the amino reactive moiety, thiol reactive moiety, or carbohydrate reactive moiety of said spectrophotometrically quantifiable linker; and
ii) a second biomolecule is bound to the spectrophotometrically quantifiable linker via the biotin or receptor ligand of said spectrophotometrically quantifiable linker.

### Claim 12 (depends on 11)

The spectrophotometrically quantifiable linker of claim 11, wherein the first biomolecule, the second biomolecule, or both, comprises: a protein, a peptide, an oligonucleotide, a complimentary oligonucleotide, a polynucleotide, or combinations thereof.

### Claim 13 (depends on 11)

The spectrophotometrically quantifiable linker of claim 11, wherein the second biomolecule and the biotin or receptor ligand of the biomolecule-bound spectrophotometrically quantifiable linker comprises:
i) a biotin/avidin pair;
ii) a digoxigenin/anti-digoxigenin pair;
iii) a peptide/anti-peptide antibody pair;
iv) a peptide-S/ribonuclease pair;
v) a complementary oligonucleotide pair; or
vi) an antibody/ligand pair.

### Claim 14 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the flexible linker comprising PEG has no less than 8 carbon atoms and no more than 34 carbon atoms.

### Claim 15 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the linker is (5-(N′-{4-[2-(2,5-dioxo-pyrrolidin-1-yl)-2-oxo-acetyl]-benzylidene}-hydrazino)-pyridine-2-carboxylic acid {3-[2-(2-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-propoxy}-ethoxy)-ethoxy]-propyl}-amide).

### Claim 16 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the chromophoric or fluorescent hydrazone-containing moiety comprises: an aryl moiety, a heteroaryl moiety, a heterocyclic moiety, or combinations thereof.

### Claim 17 (depends on 16)

The spectrophotometrically quantifiable linker of claim 16, wherein the aryl moiety, the heteroaryl moiety, or the heterocyclic moiety comprises a ring fusion.

### Claim 18 (depends on 16)

The spectrophotometrically quantifiable linker of claim 16, wherein the aryl moiety, the heteroaryl moiety, or the heterocyclic moiety comprises: a phenyl moiety, a naphthylene moiety, an anthracenyl moiety, a pyridinyl moiety, a quinolinyl moiety, a benzimidazolyl moiety, a pyrrolyl moiety, a benzopyrrolyl moiety, a thiophenyl moiety, or a cytidinyl moiety.

### Claim 19 (depends on 16)

The spectrophotometrically quantifiable linker of claim 16, wherein the chromophoric or fluorescent hydrazone-containing moiety comprises:
i) an aryl-containing hydrazone-containing moiety;
ii) a heteroaryl-containing hydrazone-containing moiety;
iii) a bis-aryl-containing hydrazone-containing moiety;
iv) a bis-heteroaryl-containing hydrazone-containing moiety; or
v) an aryl-heteroaryl-containing hydrazone-containing moiety.

### Claim 20 (depends on 1)

The spectrophotometrically quantifiable linker of claim 1, wherein the chromophoric or fluorescent hydrazone-containing moiety comprises a conjugationally extended hydrazone-containing moiety.

### Claim 21 (depends on 20)

The spectrophotometrically quantifiable linker of claim 20, wherein the conjugationally extended hydrazone-containing moiety comprises: an aryl moiety, a heteroaryl moiety, a heterocyclic moiety, or combinations thereof.

### Claim 22 (depends on 20)

The spectrophotometrically quantifiable linker of claim 20, wherein the aryl moiety, the heteroaryl moiety, or the heterocyclic moiety comprises a ring fusion.

### Claim 23 (depends on 20)

The spectrophotometrically quantifiable linker of claim 20, wherein the aryl moiety, the heteroaryl moiety, or the heterocyclic moiety comprises: a phenyl moiety, a naphthylene moiety, an anthracenyl moiety, a pyridinyl moiety, a quinolinyl moiety, a benzimidazolyl moiety, a pyrrolyl moiety, a benzopyrrolyl moiety, a thiophenyl moiety, or a cytidinyl moiety.

### Claim 24 (depends on 20)

The spectrophotometrically quantifiable linker of claim 20, wherein the chromophoric or fluorescent conjugationally extended hydrazone-containing moiety comprises:
i) a conjugationally extended aryl-containing hydrazone-containing moiety;
ii) a conjugationally extended heteroaryl-containing hydrazone-containing moiety;
iii) a conjugationally extended bis-aryl-containing hydrazone-containing moiety;
iv) a conjugationally extended bis-heteroaryl-containing hydrazone-containing moiety; or
v) a conjugationally extended aryl-heteroaryl-containing hydrazone-containing moiety.