# US 11,440,933 B2 · 3′ protected nucleotides

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- Number: US 11,440,933 B2
- Document ID: US11440933B2
- Status: Granted
- Kind: B2
- Application number: 17/416,186
- Filed: 2019-12-16
- Published: 2022-02-10
- Granted: 2022-09-13
- Earliest claimed: 2018-12-19
- Assignees: Roche Sequencing Solutions, Inc.
- Applicants: Roche Sequencing Solutions, Inc.
- Inventors: Frank Bergmann; Peter Crisalli; Dieter Hiendl; Omid Khakshoor; Meng Taing
- Web page: https://patentgrep.com/patent/US11440933B2

## CPC

- C07H 19/06: Pyrimidine radicals
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07H 19/048: Pyridine radicals
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 23/00: Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12 (esters with inorganic acids C07H11/00)
- C12Q 1/6869: Methods for sequencing
- 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

The present disclosure provides 3′ protected nucleotides, including those 3′ protected nucleotides having a detectable tag. Systems and methods of sequencing nucleic acids using the 3′ protected nucleotides are also disclosed, such as the sequencing of a nucleic acid using a nanopore or the sequencing of a nucleic acid via sequencing-by-synthesis.

## Claims

### Claim 1

A nucleotide or a salt thereof of Formula (IIIA):
wherein
X is a nucleobase or a tagged nucleobase;
Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH)—[O—P(O)(OH)]z—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5;
R1 is a bond, —CH2—, —C(O)—O—, —C(O)—NRa—, or —C(O)—Rx—;
R2 and R3 are each independently H, a saturated or unsaturated C1-C6 alkyl group, a C5-C6 aryl or heteroaryl group, a halogen, —[(C(Ra)(Rb))p—O]q (Ra), —C(O)—ORa, —C(O)—N(Ra)(Rb), —(C(Ra)(Rb))n—N+C−, —CN, or —NO2;
R4 is a bond, a substituted or unsubstituted 5- to 7-membered aryl group, —CH═CH—, a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group, or —O—C(O)-aryl-;
R5 is —(C(Ra)(Rb))n—N3, —(C(Ra)(Rb))n—N+C−, a 5- to 8-membered cycloalkyl group comprising two sulfur atoms positioned 1, 3 relative to each other, or a group having the structure:
Z1 and Z2 are independently H, a C1-C4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups;
Q1 and Q3 are each independently a bond, —C(Re)(Rf)—, or —C(O)—;
Q2 is a bond, o-phenylene, or —[C(Re)(Rf)]w—, where w is 1 or 2;
Ra and Rb are each independently H or a saturated C1-C6 alkyl group;
Re and Rf are independently H, methyl, ethyl, isopropyl, or a substituted or unsubstituted 5- or 6-membered aryl group;
Rx is a substituted or unsubstituted 5- or 6-membered aromatic group or heteroaromatic group;
n is 0 or an integer ranging from 1 to 3; and
p and q are each independently zero or an integer ranging from 1 to 3;
provided that when R1 and R4 are both bonds and when R2 and R3 are both H, then R5 is not an azide.

### Claim 2 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein R5 is:

### Claim 3 (depends on 2)

The nucleotide or the salt thereof of claim 2, wherein at least one of Z1 or Z2 is H.

### Claim 4 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein R1 is a bond.

### Claim 5 (depends on 4)

The nucleotide or the salt thereof of claim 4, wherein R4 is a bond.

### Claim 6 (depends on 5)

The nucleotide or the salt thereof of claim 5, wherein at least one of R2 or R3 is H.

### Claim 7 (depends on 4)

The nucleotide or the salt thereof of claim 4, wherein R4 is a 6-membered aryl group.

### Claim 8 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein R1 is —C(O)—O—.

### Claim 9 (depends on 8)

The nucleotide or the salt thereof of claim 8, wherein R4 is a 6-membered aryl group.

### Claim 10 (depends on 4)

The nucleotide or the salt thereof of claim 4, wherein R4 is —CH═CH—.

### Claim 11 (depends on 4)

The nucleotide or the salt thereof of claim 4, wherein R5 is —(C(Ra)(Rb))n—N+C−.

### Claim 12 (depends on 4)

The nucleotide or the salt thereof of claim 4, wherein R2 is —[(C(Ra)(Rb))p—O]q—(Ra) or —C(O)—ORa—, and wherein at least one Ra is a C1-C6 alkyl group.

### Claim 13 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein R1 is —C(O)—Rx—, and wherein R5 is —(C(Ra)(Rb))n—N3.

### Claim 14 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein R5 is derived from a substituted or unsubstituted 1,4-epoxy-1,4-dihydronaphthalene.

### Claim 15 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein R1 is —CH2—, and R5 is —B(OZ1)(OZ2).

### Claim 16 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein the nucleotide or the salt thereof is selected from the group consisting of:
where
X is a nucleobase or a tagged nucleobase;
Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH)—[O—P(O)(OH)]z—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5; and
Z1 and Z2 are independently H, a C1-C4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups.

### Claim 17 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein the nucleotide or the salt thereof is selected from the group consisting of:
where
X is a nucleobase or a tagged nucleobase; and
Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH)—[O—P(O)(OH)]z—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5.

### Claim 18 (depends on 1)

The nucleotide or the salt thereof of claim 1, wherein the nucleotide or the salt thereof is selected from the group consisting of:
where
X is a nucleobase or a tagged nucleobase; and
Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH)—[O—P(O)(OH)]—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5.

### Claim 19

A nucleotide or a salt thereof of Formula (IIIA):
wherein
X is a nucleobase or a tagged nucleobase;
Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH)—[O—P(O)(OH)]z—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5;
R1 is a bond, —CH2—, —C(O)—O—, —C(O)—NRa—, or —C(O)—Rx—;
R2 is H;
R3 is a saturated or unsaturated C1-C6 alkyl group, a C5-C6 aryl or heteroaryl group, a halogen, —[(C(Ra)(Rb))p—O]q—(Ra), —C(O)—ORa, —C(O)—N(Ra)(Rb), —(C(Ra)(Rb))n—N+C−, —CN, or —NO2;
R4 is a bond, a substituted or unsubstituted 5- to 7-membered aryl group, —CH═CH—, a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group, or —O—C(O)-aryl-;
R5 is —(C(Ra)(Rb))n—N3, —(C(Ra)(Rb))n—N+C−, —(C(Ra)(Rb))n—CN, a 5- to 8-membered cycloalkyl group comprising two sulfur atoms positioned 1, 3 relative to each other, or a group having the structure:
Z1 and Z2 are independently H, a C1-C4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups;
Q1 and Q3 are each independently a bond, —C(Re)(Rf)—, or —C(O)—;
Q2 is a bond, o-phenylene, or —[C(Re)(Rf)]w—, where w is 1 or 2;
Ra and Rb are each independently H or a saturated C1-C6 alkyl group;
Re and Rf are independently H, methyl, ethyl, isopropyl, or a substituted or unsubstituted 5- or 6-membered aryl group;
Rx is a substituted or unsubstituted 5- or 6-membered aromatic group or heteroaromatic group;
n is 0 or an integer ranging from 1 to 3; and
p and q are each independently zero or an integer ranging from 1 to 3;
provided that when R1 and R4 are both bonds and when R2 and R3 are both H, then R5 is not an azide.

### Claim 20

A nucleotide or a salt thereof of Formula (IIIA):
wherein
X is a nucleobase or a tagged nucleobase
Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH)—[O—P(O)(OH)]z—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5;
R1 is a bond, —CH2—, —C(O)—O—, —C(O)—NRa—, or —C(O)—Rx—;
R2 and R3 are independently a saturated or unsaturated C1-C6 alkyl group, a C5-C6 aryl or heteroaryl group, a halogen, —[(C(Ra)(Rb))p—O]q (Ra), —C(O)—ORa, —C(O)—N(Ra)(Rb), —(C(Ra)(Rb))n—N+C−, —CN, or —NO2;
R4 is a bond, a substituted or unsubstituted 5- to 7-membered aryl group, —CH═CH—, a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group, or —O—C(O)-aryl-;
R5 is —(C(Ra)(Rb))n—N3, —(C(Ra)(Rb))n—N+C−, —(C(Ra)(Rb))n—CN, a 5- to 8-membered cycloalkyl group comprising two sulfur atoms positioned 1, 3 relative to each other, or a group having the structure:
Z1 and Z2 are independently H, a C1-C4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups;
Q1 and Q3 are each independently a bond, —C(Re)(Rf)—, or —C(O)—;
Q2 is a bond, o-phenylene, or —[C(Re)(Rf)]w—, where w is 1 or 2;
Ra and Rb are each independently H or a saturated C1-C6 alkyl group;
Re and Rf are independently H, methyl, ethyl, isopropyl, or a substituted or unsubstituted 5- or 6-membered aryl group;
Rx is a substituted or unsubstituted 5- or 6-membered aromatic group or heteroaromatic group;
n is 0 or an integer ranging from 1 to 3; and
p and q are each independently zero or an integer ranging from 1 to 3;
provided that when R1 and R4 are both bonds and when R2 and R3 are both H, then R5 is not an azide.