# US 12,110,546 B2 · Tertiary amine substituted coumarin compounds and uses as fluorescent labels

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- Number: US 12,110,546 B2
- Document ID: US12110546B2
- Status: Granted
- Kind: B2
- Application number: 16/805,125
- Filed: 2020-02-28
- Published: 2020-09-03
- Granted: 2024-10-08
- Earliest claimed: 2019-03-01
- Assignees: Illumina Cambridge Limited
- Applicants: Illumina Cambridge Limited
- Inventors: Nikolai Nikolaevich Romanov; Patrick McCauley; Niall Hynes
- Web page: https://patentgrep.com/patent/US12110546B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C07D 405/04: directly linked by a ring-member-to-ring-member bond
- C07D 413/04: directly linked by a ring-member-to-ring-member bond
- C07D 417/04: directly linked by a ring-member-to-ring-member bond
- C07H 19/06: Pyrimidine radicals
- C07H 19/16: Purine radicals
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C09B 57/02: Coumarine dyes
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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)

## Abstract

The present application relates to tertiary amine substituted coumarin derivatives and their uses as fluorescent labels. These compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

## Claims

### Claim 1

A nucleotide defined by the formula:
wherein B is a nucleobase;
OR′ is a triphosphate;
R″ is a 3′ blocking group;
R′″ is H;
L is a linker moiety; and
Dye comprises:

### Claim 2 (depends on 1)

The nucleotide of claim 1, wherein B is uracil, thymine, cytosine, deaza adenine, or deaza guanine.

### Claim 3 (depends on 2)

The nucleotide of claim 2, wherein the Dye is attached to the C5 position of the uracil, the thymine or the cytosine, or the C7 position of the deaza purine or the deaza guanine through the linker moiety.

### Claim 4 (depends on 1)

The nucleotide of claim 1, wherein R″ is —OCH2N3.

### Claim 5 (depends on 1)

The nucleotide of claim 1, wherein the linker moiety comprises:
wherein X is O, Y is O, and * indicates where the moiety is connected to the remainder of the nucleotide.

### Claim 6 (depends on 1)

The nucleotide of claim 1, wherein the linker moiety comprises:

### Claim 7 (depends on 1)

The nucleotide of claim 1, wherein the linker moiety comprises:

### Claim 8 (depends on 1)

The nucleotide of claim 1, wherein the linker moiety comprises:

### Claim 9 (depends on 1)

A kit comprising a first type of labeled nucleotide according to claim 1.

### Claim 10 (depends on 9)

The kit of claim 9, further comprises a second type of labeled nucleotide, wherein the second labeled nucleotide is labeled with a different compound than the first labeled nucleotide.

### Claim 11 (depends on 10)

The kit of claim 10, wherein the first and second type of labeled nucleotides are excitable using a single laser wavelength.

### Claim 12 (depends on 10)

The kit of claim 10, further comprising a third type of nucleotide and a fourth type of nucleotide, wherein each of the second, third, and fourth type of nucleotides is labeled with a different compound, wherein each label has a distinct absorbance maximum that is distinguishable from the other labels.

### Claim 13 (depends on 9)

The kit of claim 9, wherein the kit comprises four types of nucleotides, wherein a second type of nucleotide carries a second label, a third type of nucleotide carries a third label, and a fourth type of nucleotide is unlabeled (dark).

### Claim 14 (depends on 9)

The kit of claim 9, wherein the kit comprises four types of nucleotides, wherein a second type of nucleotide carries a second label, a third type of nucleotide comprises a mixture of third type of nucleotides with two different labels, and a fourth type of nucleotide is unlabeled (dark).

### Claim 15 (depends on 9)

The kit of claim 9, wherein the first type of labeled nucleotide is excitable by a blue light having a wavelength between about 450 nm to about 460 nm.

### Claim 16 (depends on 14)

The kit of claim 14, wherein the first type of labeled nucleotide is excitable by a blue light having a wavelength between about 450 nm to about 460 nm.

### Claim 17 (depends on 16)

The kit of claim 16, wherein the second type of labeled nucleotide is excitable by a green light having a wavelength between about 520 nm to about 560 nm, or about 532 nm.

### Claim 18 (depends on 17)

The kit of claim 17, wherein the third type of labeled nucleotide is excitable by both the blue light and the green light.

### Claim 19 (depends on 1)

A method of sequencing comprising incorporating a labeled nucleotide according to claim 1 in a sequencing assay.

### Claim 20 (depends on 1)

An oligonucleotide comprising the nucleotide of claim 1 incorporated thereof.

### Claim 21 (depends on 20)

The oligonucleotide of claim 20, wherein the oligonucleotide is at least partially hybridized to a template polynucleotide.

### Claim 22 (depends on 21)

The oligonucleotide of claim 21, wherein the template polynucleotide is immobilized to a solid support.

### Claim 23 (depends on 22)

The oligonucleotide of claim 22, wherein the solid support comprises a plurality of immobilized template polynucleotides.