# US 12,215,124 B2 · Fluorinated nucleotides and uses thereof

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- Number: US 12,215,124 B2
- Document ID: US12215124B2
- Status: Granted
- Kind: B2
- Application number: 16/969,879
- Filed: 2019-02-20
- Published: 2022-09-22
- Granted: 2025-02-04
- Earliest claimed: 2018-02-21
- Assignees: Singular Genomics Systems, Inc.
- Applicants: Singular Genomics Systems, Inc.
- Inventors: Ronald Graham; Andrew Spaventa; Eli Glezer
- Web page: https://patentgrep.com/patent/US12215124B2

## CPC

- A61P 37/00: Drugs for immunological or allergic disorders
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/14: Pyrrolo-pyrimidine radicals
- C12Q 1/6869: Methods for sequencing
- C12Q 2600/16: Primer sets for multiplex assays
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- 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

## Abstract

Disclosed herein, inter alia, are compounds, compositions, and methods of use thereof in the sequencing a nucleic acid.

## Claims

### Claim 1

A nucleotide analogue having the formula:
wherein
B is a base or analogue thereof;
L2B, L2C, L2D, and L2E are independently a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, wherein at least one of L2A, L2B, L2C, L2D, and L2E is not a bond;
L4 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;
R4 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R5 is a detectable label, an anchor moiety, or an affinity anchor moiety; and
X1 is hydrogen, halogen, —N3, or —CN.

### Claim 2 (depends on 1)

The nucleotide analogue of claim 1, having the formula:

### Claim 3 (depends on 1)

The nucleotide analogue of claim 1, wherein X1 is —F.

### Claim 4 (depends on 1)

The nucleotide analogue of claim 1, wherein X1 is hydrogen.

### Claim 5 (depends on 1)

The nucleotide analogue of claim 1, wherein X1 is —Cl.

### Claim 6 (depends on 1)

The nucleotide analogue of claim 1, wherein X1 is —Br.

### Claim 7 (depends on 1)

The nucleotide analogue of claim 1, wherein X1 is —I.

### Claim 8 (depends on 1)

The nucleotide analogue of claim 1, wherein L4 is substituted or unsubstituted C1-C8 alkylene, substituted or unsubstituted 2 to 8 membered heteroalkylene, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted 3 to 8 membered heterocycloalkylene, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

### Claim 9 (depends on 1)

The nucleotide analogue of claim 1, wherein L4 is a bond or an unsubstituted C1-C4 alkylene.

### Claim 10 (depends on 1)

The nucleotide analogue of claim 1, wherein R4 is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3 to 6 membered heterocycloalkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted 5 to 6 membered heteroaryl.

### Claim 11 (depends on 1)

The nucleotide analogue of claim 1, wherein R4 is substituted C1-C6 alkyl.

### Claim 12 (depends on 1)

The nucleotide analogue of claim 1, wherein R4 is unsubstituted C1-C6 alkyl.

### Claim 13 (depends on 1)

The nucleotide analogue of claim 1, wherein R4 is substituted phenyl.

### Claim 14 (depends on 1)

The nucleotide analogue of claim 1, wherein R4 is unsubstituted phenyl.

### Claim 15 (depends on 1)

The nucleotide analogue of claim 1, wherein R5 is a detectable label.

### Claim 16 (depends on 1)

A nucleic acid polymerase complex, wherein said nucleic acid polymerase is bound to a nucleotide analogue of claim 1.

### Claim 17 (depends on 1)

A method for sequencing a nucleic acid, comprising:
(i) incorporating in series with a nucleic acid polymerase, within a reaction vessel, one of four different labeled nucleotide analogues into a primer to create an extension strand, wherein said primer is hybridized to said nucleic acid and wherein each of the four different labeled nucleotide analogues comprise a unique detectable label;
(ii) detecting said unique detectable label of each incorporated nucleotide analogue, so as to thereby identify each incorporated nucleotide analogue in said extension strand, thereby sequencing the nucleic acid;
wherein each of said four different labeled nucleotide analogues is of the structure of claim 1.

### Claim 18 (depends on 1)

A method of incorporating a nucleotide analogue into a primer, the method comprising combining a polymerase, a primer hybridized to nucleic acid template and a nucleotide analogue within a reaction vessel and allowing said polymerase to incorporate said nucleotide analogue into said primer thereby forming an extended primer, wherein said nucleotide analogue is of the structure of claim 1.