# US 8,772,487 B2 · Fluorogenic hydrazine-substituted compounds

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- Number: US 8,772,487 B2
- Document ID: US8772487B2
- Status: Granted
- Kind: B2
- Application number: 12/864,038
- Filed: 2009-01-23
- Published: 2010-11-18
- Granted: 2014-07-08
- Earliest claimed: 2008-01-24
- Assignees: Life Technologies Corporation
- Applicants: Aimei Chen; Kyle Gee; Hee Chol Kang
- Inventors: Aimei Chen; Kyle Gee; Hee Chol Kang
- Web page: https://patentgrep.com/patent/US8772487B2

## CPC

- G01N 33/64: involving ketones
- C07D 311/82: Xanthenes
- C07D 471/06: Peri-condensed systems
- C07F 5/022: {without C-boron linkages}
- C07F 9/5442: {Aromatic phosphonium compounds (P-C aromatic linkage)}
- C07F 9/65522: {condensed with carbocyclic rings or carbocyclic ring systems}
- C09B 11/24: Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes}
- C09K 11/06: containing organic luminescent materials
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)}
- C09K 2211/10: Non-macromolecular compounds
- C09K 2211/1007: Non-condensed systems
- C09K 2211/1014: bridged by heteroatoms, e.g. N, P, Si or B
- C09K 2211/1022: bridged by heteroatoms, e.g. N, P, Si or B
- C09K 2211/1033: with oxygen
- C09K 2211/1055: with other heteroatoms
- C09K 2211/1088: containing oxygen as the only heteroatom
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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)
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present disclosure is directed to fluorogenic schiff base-forming dyes capable of detecting analytes containing aldehyde and ketone groups. The dyes contain nucleophilic hydrazinyl appendages and are capable of binding and detecting analytes in situ.

## Claims

### Claim 1

A kit for detecting an analyte in a sample, wherein the analyte contains an aldehyde group or a ketone group, wherein the kit comprises:
a compound of Formula I that binds the analyte:
or a stereoisomer, tautomer, hydrate, solvate, or salt thereof;
wherein,
X is H;
R1 is selected from the group consisting of H, alkyl and substituted alkyl;
R2, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO3−, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;
R3 is selected from the group consisting of alkoxy, substituted alkoxy, amino, substituted amino, hydrazinyl, alkyl, substituted alkyl, acyl, acylamino, acyloxy, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO3−, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or
R2 and R3, R3 and R4, or R2, R3 and R4 are taken together to form a fused heterocyclyl group, a fused substituted heterocyclyl group, a fused aryl group, a fused substituted aryl group, a fused heteroaryl group or a fused substituted heteroaryl group; and
instructions for detecting the analyte comprising the steps of:
contacting the sample with the compound;
incubating the sample and the compound for a sufficient amount of time to form a complex between the compound and analyte;
illuminating the complex with an appropriate wavelength to form an illuminated complex; and
detecting emissions from the illuminated complex;
wherein the hydrate or solvate thereof is a hydrate or solvate of an aqueous solution, an aqueous miscible solution, or a water-miscible organic solvent.

### Claim 2 (depends on 1)

The kit of claim 1, further comprising instructions for covalently bonding the compound to the analyte.

### Claim 3 (depends on 1)

The kit of claim 1, further comprising one or more of the following: a buffering agent, a purification medium, a vial comprising the analyte, or an organic solvent.

### Claim 4 (depends on 1)

The kit of claim 1, wherein the analyte is malondialdehyde or 4-hydroxynonenal.

### Claim 5 (depends on 1)

The kit of claim 1, wherein the analyte comprises an aldehyde group.

### Claim 6 (depends on 1)

The kit of claim 1, wherein the sample comprises live cells, intracellular fluids, extracellular fluids, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions, or biological fluids.

### Claim 7 (depends on 1)

The kit of claim 1, wherein the sample comprises blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles, vascular tissue, blood fluids, saliva, urine, water, soil, waste water, sea water, pharmaceuticals, foodstuffs or beverages.

### Claim 8 (depends on 1)

The kit of claim 1, wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, or on a microfluidic chip.