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US 11,884,825 B2Granted
Exocyclic amine substituted coumarin compounds and uses as fluorescent labels
AssigneeIllumina Cambridge Limited
InventorsNikolai Nikolaevich Romanov, Carole Anastasi, Patrick McCauley
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Abstract
The present application relates to exocyclic 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.
Claims21
A method of sequencing different template polynucleotides, comprising:
- (a) incorporating different types of nucleotides to growing polynucleotide chains in the presence of a DNA polymerase, wherein the growing polynucleotide chains are at least partially complementary to the different template polynucleotides to be sequenced, and wherein each type of nucleotide comprises a 3′ blocking group covalently attached to a 2-deoxyribose sugar of the nucleotide;
- and
- (b) imaging and detecting fluorescent signals from the nucleotides incorporated into the growing polynucleotide chains;
- wherein at least one type of nucleotide is labeled with a compound of Formula (I):
- wherein:
- X is O, S, or NRn, wherein Rn is H, C1-6 alkyl, or C6-10 aryl;
- ring A is
- R, R1, R2, and R4 are each independently H, halo, —CN, —CO2H, amino, —OH, C-amido, N-amido, —NO2, —SO3H, —SO2NRaRb, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
- each R3 is independently —CO2H, —(CH2)p—CO2Rc, —SO3H, or C1-4 alkyl substituted with —CO2H or —SO3H;
- p is 1, 2, 3 or 4;
- each R5 is independently halo, —CN, —CO2Rf, amino, —OH, C-amido, N-amido, —NO2, —SO3H, —SO2NRaRb, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
- each Ra and Rb is independently H or optionally substituted C1-6 alkyl;
- each Rc, and Rf is independently H, optionally substituted C1-6 alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
- m is 0, 1, 2, 3, or 4;
- and
- n is 1, 2 or 3.
13 more elements
A method of preparing different growing polynucleotide chains complementary to respective template single-stranded polynucleotides, comprising:
- (a) incorporating different types of nucleotides to polynucleotide chains that are at least partially complementary to the respective template single-stranded polynucleotides in the presence of a DNA polymerase, wherein each nucleotide comprises a 3′ blocking group covalently attached to a 2-deoxyribose sugar of the nucleotide;
- and
- (b) removing the 3′ blocking group from the incorporated nucleotides;
- wherein at least one nucleotide is labeled with a compound of Formula (I):
- wherein:
- X is O, S, or NRn, wherein Rn is H, C1-6 alkyl, or C6-10 aryl;
- ring A is
- R, R1, R2, and R4 are each independently H, halo, —CN, —CO2H, amino, —OH, C-amido, N-amido, —NO2, —SO3H, —SO2NRaRb, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
- each R3 is independently —CO2H, —(CH2)p—CO2Rc, —SO3H, or C1-4 alkyl substituted with —CO2H or —SO3H;
- p is 1, 2, 3 or 4;
- each R5 is independently halo, —CN, —CO2Rf, amino, —OH, C-amido, N-amido, —NO2, —SO3H, —SO2NRaRb, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted aminoalkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
- each Ra and Rb is independently H or optionally substituted C1-6 alkyl;
- each Rc, and Rf is independently H, optionally substituted C1-6 alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
- m is 0, 1, 2, 3, or 4;
- and
- n is 1, 2 or 3;
- and
- wherein steps (a) and (b) are repeated multiple cycles.
15 more elements
Dependencies are inferred from the claim text.