# US 10,246,410 B2 · Hydrazinyl and aminooxy compounds and their methods of use

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- Number: US 10,246,410 B2
- Document ID: US10246410B2
- Status: Granted
- Kind: B2
- Application number: 15/529,437
- Filed: 2015-12-03
- Published: 2017-12-28
- Granted: 2019-04-02
- Earliest claimed: 2014-12-03
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation
- Inventors: Wenjun Zhou; Kyle Gee; Yolanda Tennico; Peter Slade; Hee Chol Kang; Shaheer Khan; Brian Evans; James Stray
- Web page: https://patentgrep.com/patent/US10246410B2

## CPC

- C07C 309/47: having at least one of the sulfo groups bound to a carbon atom of a six-membered aromatic ring being part of a condensed ring system
- C07C 309/51: at least one of the nitrogen atoms being part of any of the groups [IMAGE cpc-sch-C07C-0976.gif], X being a hetero atom, Y being any atom
- C07D 311/84: 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 in position 9
- G01N 33/582: {with fluorescent label}
- C07C 2603/50: Pyrenes; Hydrogenated pyrenes
- G01N 2400/10: Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)

## Abstract

The present disclosure is directed dye compounds containing a hydrazinyl substituent and optionally, one or more negatively charged groups, such as sulfonate, phosphate, phosphonate, and/or carboxylate groups and dye compounds containing an aminooxy substitutent. The compounds are useful in the detection of analytes containing aldehyde and ketone groups, including, for example, glycans.

## Claims

### Claim 1

A compound selected from the group consisting of:
or a salt thereof.

### Claim 2 (depends on 1)

The compound of claim 1, wherein the salt comprises a potassium, sodium, ammonium, alkylammonium, tetralkylammonium, triethylammonium, tert-butylammonium, tetralkyl-phosphonium, tetraaryl-phosphonium, lithium or cesium ion.

### Claim 3 (depends on 1)

A composition comprising:
(a) an analyte; and
(b) a compound according to claim 1.

### Claim 4 (depends on 3)

The composition of claim 3, further comprising a buffer solution.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the analyte is selected from the group consisting of: an amino acid, a peptide, a protein, a carbohydrate, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a biotin-binding protein, a polymeric microparticle, a biological cell and a virus.

### Claim 6 (depends on 1)

A method for determining the presence of an analyte of interest in a sample, wherein the method comprises:
a) contacting the sample with a compound of claim 1;
b) incubating the sample and the compound or composition for a sufficient amount of time to form a complex between the compound and analyte;
c) illuminating the complex with an appropriate wavelength to form an illuminated complex; and
d) detecting emissions from the illuminated complex, thereby determining the presence of the analyte.

### Claim 7

A method for determining the presence of an analyte of interest in a sample, wherein the method comprises:
a) contacting the sample with a compound selected from the group consisting of:
or a salt thereof;
b) incubating the sample and the compound or composition for a sufficient amount of time to form a complex between the compound and analyte;
c) illuminating the complex with an appropriate wavelength to form an illuminated complex; and
d) detecting emissions from the illuminated complex, thereby determining the presence of the analyte.

### Claim 8 (depends on 7)

The method of claim 7, wherein the analyte and the compound are connected by a covalent bond.

### Claim 9 (depends on 8)

The method of claim 8, wherein the compound becomes fluorescent after formation of the complex.

### Claim 10 (depends on 9)

The method of claim 9, wherein the analyte is selected from the group consisting of: an amino acid, a peptide, a protein, a carbohydrate, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a biotin-binding protein, a polymeric microparticle, a biological cell and a virus.

### Claim 11 (depends on 7)

The method of claim 7, wherein the sample comprises live cells, intracellular fluids, extracellular fluids, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions biological fluids or chemical reactors.

### Claim 12 (depends on 7)

The method of claim 7, 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 13 (depends on 7)

The method of claim 7, 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, and on a microfluidic chip.

### Claim 14 (depends on 1, 3)

A method of detecting a glycan in a sample, the method comprising:
a) contacting the sample with a compound according to claim 1, or a composition according to claim 3;
b) incubating the sample and the compound or composition for a sufficient amount of time to form a complex between the compound and glycan;
c) illuminating the complex with an appropriate wavelength to form an illuminated complex; and
d) detecting emissions from the illuminated complex, thereby determining the presence of the glycan.

### Claim 15

A method of detecting a glycan in a sample, the method comprising:
a) contacting the sample with a [a] compound selected from the group consisting of:
or a salt thereof;
b) incubating the sample and the compound or composition for a sufficient amount of time to form a complex between the compound and glycan;
c) illuminating the complex with an appropriate wavelength to form an illuminated complex; and
d) detecting emissions from the illuminated complex, thereby determining the presence of the glycan.

### Claim 16 (depends on 15)

The method of claim 15, wherein the salt comprises a potassium, sodium, ammonium, alkylammonium, tetralkylammonium, triethylammonium, tert-butylammonium, tetralkyl-phosphonium, tetraaryl-phosphonium, lithium or cesium ion.

### Claim 17 (depends on 1, 3)

A method of labeling a glycan in a sample, the method comprising:
a) contacting the sample with a compound according to claim 1, or a composition according to claim 3;
b) incubating the sample and the compound for a sufficient amount of time to form a complex between the compound and the one or more glycans;
c) separating the sample using electrophoresis or chromatography, thereby determining the presence of the one or more glycans in the sample.

### Claim 18 (depends on 17)

The method of claim 17, wherein the separating step is performed using electrophoresis.

### Claim 19 (depends on 18)

The method of claim 18, wherein the electrophoresis is capillary electrophoresis.

### Claim 20 (depends on 9)

The method of claim 9, wherein the capillary electrophoresis is part of a glycan analysis system.

### Claim 21 (depends on 17)

The method of claim 17, wherein the separation step is performed using chromatography.

### Claim 22 (depends on 21)

The method of claim 21, wherein the chromatography is high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectroscopy (LC-MS).

### Claim 23 (depends on 1, 17)

A kit for labeling a glycan, wherein the kit comprises:
a) a compound according to claim 1; and
b) instructions for labeling the glycan according to the method of claim 17.

### Claim 24 (depends on 17)

A kit for labeling a glycan, wherein the kit comprises:
a) a compound selected from the group consisting of:
or a salt thereof; and
b) instructions for labeling the glycan according to the method of claim 17.

### Claim 25 (depends on 14)

A kit for detecting an analyte in a sample, wherein the kit comprises:
a) a compound selected from the group consisting of:
or a salt thereof; and
b) instructions for detecting the analyte according to the method of claim 14.

### Claim 26 (depends on 25)

The kit of claim 25, further comprising instructions for covalently bonding the compound to the analyte.

### Claim 27 (depends on 25)

The kit of claim 25, further comprising one or more of the following: a buffering agent, a purification medium, a vial comprising the analyte, an organic solvent, one or more reagents for releasing the glycan from a biomolecule, or optionally, one or more reagents to purify the released glycan from the reaction mixture.

### Claim 28 (depends on 27)

The kit of claim 27, wherein the reagent for releasing the glycan from a biomolecule is selected from a physical method, a chemical or an enzyme.

### Claim 29 (depends on 28)

The kit of claim 28, wherein the enzyme is PNGase F.

### Claim 30 (depends on 27)

The kit of claim 27, wherein the purification medium is selected from the group consisting of a resin, a bead, a cartridge, a solid support, a plate and a well.

### Claim 31 (depends on 30)

The kit of claim 30, wherein the bead is a magnetic bead.

### Claim 32 (depends on 1, 25)

The kit of claim 25, further comprising instructions for labeling glycans in a sample in preparation of glycan analysis, the method comprising:
treating the sample with a release reagent, such as PNGase F enzyme, with an appropriate buffer under conditions suitable for the release of the glycan from the biomolecule, thereby forming a reaction mixture;
adding beads and buffer to the reaction mixture;
separating the supernatant from the beads;
washing the beads with wash buffer;
eluting the glycans from the beads with elution buffer;
performing dye labeling of the glycans using one or more dye compounds according to claim 1, thereby forming a glycan-containing solution;
optionally, removing excess dye compound using fresh beads; washing beads, separating the beads from excess dye/wash solution; and eluting glycans from the beads; and
collecting the glycan-containing solution.