# US 11,999,999 B2 · Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis

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- Number: US 11,999,999 B2
- Document ID: US11999999B2
- Status: Granted
- Kind: B2
- Application number: 17/373,295
- Filed: 2021-07-12
- Published: 2022-12-29
- Granted: 2024-06-04
- Earliest claimed: 2015-09-28
- Assignees: The Trustees of Columbia University in the City of New York
- Applicants: Jingyue Ju; Xiaoxu Li; Xin Chen; Zengmin Li; Shiv Kumar; Shundi Shi; Cheng Guo; Jianyi Ren; Min-Kang Hsieh; Minchen Chien; Chuanjuan Tao; Ece Erturk; Sergey Kalachikov; James J. Russo
- Inventors: Jingyue Ju; Xiaoxu Li; Xin Chen; Zengmin Li; Shiv Kumar; Shundi Shi; Cheng Guo; Jianyi Ren; Min-Kang Hsieh; Minchen Chien; Chuanjuan Tao; Ece Erturk; Sergey Kalachikov; James J. Russo
- Web page: https://patentgrep.com/patent/US11999999B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/14: Pyrrolo-pyrimidine radicals
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- 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
- 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)

## Abstract

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

## Claims

### Claim 1

A compound of the formula:
Wherein
B is a base;
L1 is covalent linker;
L2 is covalent linker;
R3 is —OH, monophosphate, polyphosphate or a nucleic acid;
R4A is hydrogen, —CH3, —CX13, —CHX12, —CH2X1, —CN, —Ph, 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;
R4B is hydrogen, —CH3, —CX23, —CHX22, —CH2X2, —CN, —Ph, 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 or anchor moiety;
R6 is hydrogen or a polymerase-compatible cleavable moiety and the polymerase-compatible cleavable moiety is —CH2—CH═CH2, —CH2N3 or
R8A is hydrogen;
R8B is hydrogen;
R9, R10, and R11 are each independently hydrogen or unsubstituted alkyl;
R7 is hydrogen or —OR7A, wherein R7A is hydrogen; and
X1 and X2 are independently halogen.

### Claim 2 (depends on 1)

The compound of claim 1, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, a divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a deaza-adenine or a derivative thereof, a deaza-guanine or a derivative thereof, a deaza-hypoxanthine or a derivative thereof, a divalent 7-methylguanine or a derivative thereof, a divalent 5,6-dihydrouracil or a derivative thereof, a divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof.

### Claim 3 (depends on 1)

The compound of claim 1, wherein B is

### Claim 4 (depends on 1)

The compound of claim 1, wherein
L1 is L1A-L1B-L1C-L1D-L1E; and
L1A, L1B, L1C, L1D and L1E are independently 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;
wherein at least one of L1A, L1B, L1C, L1D and L1E is not a bond.

### Claim 5 (depends on 1)

The compound of claim 1, wherein
L1 is L1A-L1B-L1C-L1D-L1E; and
L1A, L1B, L1C, L1D and L1E are independently a bond, 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;
wherein at least one of L1A, L1B, L1C, L1D and L1E is not a bond.

### Claim 6 (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.

### Claim 7 (depends on 1)

The compound of claim 1, wherein
L2 is L2A-L2B-L2C-L2D-L2E; and
L2A, L2B, L2C, L2D and L2E are independently 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;
wherein at least one of L2A, L2B, L2C, L2D and L2E is not a bond.

### Claim 8 (depends on 1)

The compound of claim 1, wherein
L2 is L2A-L2B-L2C-L2D-L2E; and
L2A, L2B, L2C, L2D and L2E are independently a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted C1-C20 alkylene, substituted or unsubstituted 2 to 20 membered heteroalkylene, substituted or unsubstituted C3-C20 cycloalkylene, substituted or unsubstituted 3 to 20 membered heterocycloalkylene, substituted or unsubstituted C6-C20 arylene, or substituted or unsubstituted 5 to 20 membered heteroarylene;
wherein at least one of L2A, L2B, L2C, L2D, and L2E is not a bond.

### Claim 9 (depends on 1)

The compound of claim 1, wherein R3 is triphosphate.

### Claim 10 (depends on 1)

The compound of claim 1, wherein R4A and R4B are each independently hydrogen or —CH3.

### Claim 11 (depends on 1)

The compound of claim 1, wherein R5 is a fluorescent dye.

### Claim 12 (depends on 1)

The compound of claim 1, wherein R7 is hydrogen.

### Claim 13 (depends on 1)

The compound of claim 1, wherein R6 is —CH2—CH═CH2 or —CH2N3.

### Claim 14 (depends on 1)

The compound of claim 1, wherein the compound has the formula:
wherein m is an integer from 1 to 4.

### Claim 15 (depends on 14)

The compound of claim 14, wherein m is 1 or 4.

### Claim 16 (depends on 1)

The compound of claim 1, wherein the compound has the formula:

### Claim 17 (depends on 1)

The compound of claim 1, wherein the compound has the formula:

### Claim 18 (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; and
wherein each of said four different labeled nucleotide analogues are of the structure of claim 1, wherein in the first of said four different labeled nucleotide analogues, B is a thymidine or uridine hybridizing base;
in the second of said four different labeled nucleotide analogues, B is an adenosine hybridizing base;
in the third of said four different labeled nucleotide analogues, B is a guanosine hybridizing base; and
in the fourth of said four different labeled nucleotide analogues, B is a cytosine hybridizing base.

### Claim 19 (depends on 16)

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; and
wherein each of said four different labeled nucleotide analogues are of the structure of claim 16, wherein
in the first of said four different labeled nucleotide analogues, B is a thymidine or uridine hybridizing base;
in the second of said four different labeled nucleotide analogues, B is an adenosine hybridizing base;
in the third of said four different labeled nucleotide analogues, B is a guanosine hybridizing base; and
in the fourth of said four different labeled nucleotide analogues, B is a cytosine hybridizing base.

### Claim 20 (depends on 19)

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; and
wherein each of said four different labeled nucleotide analogues are of the structure of claim 19, wherein
in the first of said four different labeled nucleotide analogues, B is a thymidine or uridine hybridizing base;
in the second of said four different labeled nucleotide analogues, B is an adenosine hybridizing base;
in the third of said four different labeled nucleotide analogues, B is a guanosine hybridizing base; and
in the fourth of said four different labeled nucleotide analogues, B is a cytosine hybridizing base.

### Claim 21

A compound of the formula:
Wherein
B is a base;
L1 is covalent linker;
L2 is covalent linker;
R3 is —OH, monophosphate, polyphosphate or a nucleic acid;
R4A is hydrogen;
R4B is hydrogen;
R5 is a detectable label or anchor moiety;
R6 is hydrogen or a polymerase-compatible cleavable moiety and the polymerase-compatible cleavable moiety is —CH2—CH═CH2, —CH2N3 or
R8A is hydrogen;
R8B is hydrogen;
R9, R10, and R11 are each independently hydrogen or unsubstituted alkyl; and
R7 is hydrogen or —OR7A, wherein R7A is hydrogen.