3′ protected nucleotides

Patent metadata

Field	Value
Publication number	US-11440933-B2
Application number	US-201917416186-A
Country	US
Kind code	B2
Filing date	Dec 16, 2019
Priority date	Dec 19, 2018
Publication date	Sep 13, 2022
Grant date	Sep 13, 2022

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.

First claim

The invention claimed is:

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;
R 1 is a bond, —CH 2 —, —C(O)—O—, —C(O)—NR a —, or —C(O)—R x —;
R 2 and R 3 are each independently H, a saturated or unsaturated C 1 -C 6 alkyl group, a C 5 -C 6 aryl or heteroaryl group, a halogen, —[(C(R a )(R b )) p —O] q (R a ), —C(O)—OR a , —C(O)—N(R a )(R b ), —(C(R a )(R b )) n —N + C − , —CN, or —NO 2 ;
R 4 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-;
R 5 is —(C(R a )(R b )) n —N 3 , —(C(R a )(R b )) 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:

Z 1 and Z 2 are independently H, a C 1 -C 4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups;
Q 1 and Q 3 are each independently a bond, —C(R e )(R f )—, or —C(O)—;
Q 2 is a bond, o-phenylene, or —[C(R e )(R f )] w —, where w is 1 or 2;
R a and R b are each independently H or a saturated C 1 -C 6 alkyl group;
R e and R f are independently H, methyl, ethyl, isopropyl, or a substituted or unsubstituted 5- or 6-membered aryl group;
R x 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 R 1 and R 4 are both bonds and when R 2 and R 3 are both H, then R 5 is not an azide.

2. The nucleotide or the salt thereof of claim 1 , wherein R 5 is:

3. The nucleotide or the salt thereof of claim 2 , wherein at least one of Z 1 or Z 2 is H.

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

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

6. The nucleotide or the salt thereof of claim 5 , wherein at least one of R 2 or R 3 is H.

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

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

9. The nucleotide or the salt thereof of claim 8 , wherein R 4 is a 6-membered aryl group.

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

11. The nucleotide or the salt thereof of claim 4 , wherein R 5 is —(C(R a )(R b )) n —N + C − .

12. The nucleotide or the salt thereof of claim 4 , wherein R 2 is —[(C(R a )(R b )) p —O] q —(R a ) or —C(O)—OR a —, and wherein at least one R a is a C 1 -C 6 alkyl group.

13. The nucleotide or the salt thereof of claim 1 , wherein R 1 is —C(O)—R x —, and wherein R 5 is —(C(R a )(R b )) n —N 3 .

14. The nucleotide or the salt thereof of claim 1 , wherein R 5 is derived from a substituted or unsubstituted 1,4-epoxy-1,4-dihydronaphthalene.

15. The nucleotide or the salt thereof of claim 1 , wherein R 1 is —CH 2 —, and R 5 is —B(OZ 1 )(OZ 2 ).

16. 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
Z 1 and Z 2 are independently H, a C 1 -C 4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups.

17. 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.

18. 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.

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;
R 1 is a bond, —CH 2 —, —C(O)—O—, —C(O)—NR a —, or —C(O)—R x —;
R 2 is H;
R 3 is a saturated or unsaturated C 1 -C 6 alkyl group, a C 5 -C 6 aryl or heteroaryl group, a halogen, —[(C(R a )(R b )) p —O] q —(R a ), —C(O)—OR a , —C(O)—N(R a )(R b ), —(C(R a )(R b )) n —N + C − , —CN, or —NO 2 ;
R 4 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-;
R 5 is —(C(R a )(R b )) n —N 3 , —(C(R a )(R b )) n —N + C − , —(C(R a )(R b )) 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:

Z 1 and Z 2 are independently H, a C 1 -C 4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups;
Q 1 and Q 3 are each independently a bond, —C(R e )(R f )—, or —C(O)—;
Q 2 is a bond, o-phenylene, or —[C(R e )(R f )] w —, where w is 1 or 2;
R a and R b are each independently H or a saturated C 1 -C 6 alkyl group;
R e and R f are independently H, methyl, ethyl, isopropyl, or a substituted or unsubstituted 5- or 6-membered aryl group;
R x 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 R 1 and R 4 are both bonds and when R 2 and R 3 are both H, then R 5 is not an azide.

20. A nucleotide or a salt thereof of Formula (IIIA):

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Bergmann Frank
• Crisalli Peter
• Hiendl Dieter
• Khakshoor Omid
• Taing Meng

Classifications

• C07H21/04 Primary

  with deoxyribosyl as saccharide radical · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C07H23/00

  Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12 (esters with inorganic acids C07H11/00) · CPC title

• C07H19/048

  Pyridine radicals · CPC title