Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing
The present application relates to dyes containing fused tetracyclic bis-boron containing heterocycle and their uses as fluorescent labels. These dyes may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.
1 . A compound of Formula (I):
a salt or a mesomeric form thereof,
wherein each of R 1 , R 2 , R 3 , R 6 , R 7 and R 8 is independently H, unsubstituted or substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, unsubstituted or substituted C 2 -C 6 alkenyl, unsubstituted or substituted C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 1 -C 6 haloalkoxy, C 1 -C 6 hydroxyalkyl, (C 1 -C 6 alkoxy)(C 1 -C 6 alkyl), unsubstituted or substituted amino, halo, cyano, carboxyl, hydroxy, nitro, sulfonyl, sulfino, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, —C(═O)NR 9 R 10 , —X—R 11 , unsubstituted or substituted C 3 -C 10 carbocyclyl, unsubstituted or substituted C 6 -C 10 aryl, unsubstituted or substituted 5 to 10 membered heteroaryl, or unsubstituted or substituted 3 to 10 membered heterocyclyl;
each of R 4 and R 5 is independently H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted C 3 -C 10 carbocyclyl, unsubstituted or substituted C 6 -C 10 aryl, unsubstituted or substituted 5 to 10 membered heteroaryl, or unsubstituted or substituted 3 to 10 membered heterocyclyl;
each of R 9 and R 10 is independently H, or unsubstituted or substituted C 1 -C 6 alkyl;
X is —O—, —S—, —CH═CH— or —C≡C—;
each of R 11 is independently unsubstituted or substituted C 3 -C 10 carbocyclyl, unsubstituted or substituted C 6 -C 10 aryl, unsubstituted or substituted 5 to 10 membered heteroaryl, or unsubstituted or substituted 3 to 10 membered heterocyclyl;
each of R a , R b , R c and R d is independently halo, cyano, unsubstituted C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, unsubstituted or substituted C 6 -C 10 aryl, unsubstituted or substituted C 6 -C 10 aryloxy, or —OC(═O)R 12 ;
R 12 is unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted C 2 -C 6 alkenyl, unsubstituted or substituted C 6 -C 10 aryl, or unsubstituted or substituted 5 to 10 membered heteroaryl;
alternatively, when both R a and R b are —OC(═O)R 12 , the two R 12 together with the atoms to which they are attached form an unsubstituted or substituted 6 to 10 membered heterocyclyl;
alternatively, when both R c and R d are —OC(═O)R 12 , the two R 12 together with the atoms to which they are attached form an unsubstituted or substituted 6 to 10 membered heterocyclyl; and
provided that at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 comprises a carboxyl group.
2 . The compound of claim 1 , wherein each of R 1 , R 2 and R 3 is H or unsubstituted C 1 -C 6 alkyl.
3 . The compound of claim 1 , wherein two of R 1 , R 2 and R 3 are H or unsubstituted C 1 -C 6 alkyl, and one of R 1 , R 2 and R 3 is halo, carboxyl, —C(═O)NR 9 R 10 , C 1 -C 6 alkyl substituted with carboxyl, carboxylate, sulfo or sulfonate, unsubstituted or substituted phenyl, unsubstituted or substituted 5 to 6 membered heteroaryl, or unsubstituted or substituted 5 to 6 membered heterocyclyl.
4 . The compound of claim 3 , wherein R 1 and R 2 are H, R 3 is phenyl optionally substituted with carboxyl, or R 1 and R 3 are H, R 2 is phenyl optionally substituted with carboxyl.
5 . The compound of claim 1 , wherein two of R 1 , R 2 and R 3 are H or unsubstituted C 1 -C 6 alkyl, and one of R 1 , R 2 and R 3 is —X—R 11 , and wherein X is —O—, —S— or —C≡C—.
6 . The compound of claim 5 , wherein R 1 and R 2 are independently H or unsubstituted C 1 -C 6 alkyl, and R 3 is —X—R 11 , or wherein R 1 and R 3 are independently H or unsubstituted C 1 -C 6 alkyl, and R 2 is —X—R 11 .
7 . The compound of claim 5 , wherein R 11 is phenyl, 5 or 6 membered heterocyclyl, or 5 or 6 membered heteroaryl, each either unsubstituted or substituted with one or more substituents selected from the group consisting of halo, unsubstituted C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, carboxyl, carboxylate, sulfo, sulfonate, or —C(O)NR 13 R 14 , and wherein each R 13 and R 14 is independently H, unsubstituted C 1 -C 6 alkyl, or C 1 -C 6 alkyl substituted with carboxyl, carboxylate, sulfo or sulfonate.
8 . The compound of claim 7 , wherein R 11 is phenyl, either unsubstituted or substituted with halo, methoxy, carboxyl, or —C(O)NHR 14 , wherein R 14 is C 1 -C 6 alkyl substituted with carboxyl, carboxylate, sulfo or sulfonate.
9 . The compound of claim 1 , wherein each of R 6 , R 7 and R 8 is independently H or unsubstituted C 1 -C 6 alkyl.
10 . The compound of claim 1 , wherein two of R 6 , R 7 and R 8 are H or unsubstituted C 1 -C 6 alkyl, and one of R 6 , R 7 and R 8 is halo, carboxyl, —C(═O)NR 9 R 10 , C 1 -C 6 alkyl substituted with carboxyl, carboxylate, sulfo or sulfonate, unsubstituted or substituted phenyl, unsubstituted or substituted 5 to 6 membered heteroaryl, or unsubstituted or substituted 5 to 6 membered heterocyclyl.
11 . The compound of claim 10 , wherein R 6 and R 7 are H, and R 8 is phenyl optionally substituted with carboxyl, or R 6 and R 8 are H, and R 7 is phenyl optionally substituted with carboxyl.
12 . The compound of claim 1 , wherein two of R 6 , R 7 and R 8 are H or unsubstituted C 1 -C 6 alkyl, and one of R 6 , R 7 and R 8 is —X—R 11 , and wherein X is —O—, —S— or —C≡C—.
13 . The compound of claim 12 , wherein R 6 and R 7 and independently H or unsubstituted C 1 -C 6 alkyl, and R 8 is —X—R 11 , or wherein R 6 and R 8 and independently H or unsubstituted C 1 -C 6 alkyl, and R 7 is —X—R 11 .
14 . The compound of claim 12 , wherein R 11 is phenyl, 5 or 6 membered heterocyclyl, or 5 or 6 membered heteroaryl, each either unsubstituted or substituted with one or more substituents selected from the group consisting of halo, unsubstituted C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, carboxyl, carboxylate, sulfo, sulfonate, or —C(O)NR 13 R 14 .
15 . The compound of claim 14 , wherein R 11 is phenyl, either unsubstituted or substituted with halo, methoxy, carboxyl, or —C(O)NHR 14 , wherein R 14 is C 1 -C 6 alkyl substituted with carboxyl, carboxylate, sulfo or sulfonate.
16 . The compound of claim 1 , wherein each of R 4 and R 5 is H.
17 . The compound of claim 1 , wherein each of R a , R b , R c and R d is independently fluoro, cyano, methyl, trifluoromethyl, methoxy, or —OC(═O)R 12 , wherein R 12 is unsubstituted C 1 -C 6 alkyl, unsubstituted C 2 -C 6 alkenyl, or unsubstituted phenyl.
18 . The compound of claim 17 , wherein each of R a , R b , R c and R d is fluoro, methyl, trifluoromethyl, methoxy, ethoxy, —OAc (—OC(═O)CH 3 ), —OC(═O)CH═CH 2 or —OC(═O)Ph.
19 . The compound of claim 1 , wherein both R a and R b are —OC(═O)R 12 , and the two R 12 together with the atoms to which they are attached form a 6 membered heterocyclyl having the structure
or both R c and R d are —OC(═O)R 12 , and the two R 12 together with the atoms to which they are attached form a 6 membered heterocyclyl having the structure
20 . The compound of claim 1 , selected from the group consisting of:
and salts and mesomeric forms thereof.
21 . A nucleotide labeled with a compound according to claim 1 , wherein the compound is attached to the nucleotide via a carboxyl group of the compound of Formula (I).
22 . The labeled nucleotide of claim 21 , further comprising a 3′ blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.
23 . An oligonucleotide or polynucleotide comprising the labeled nucleotide according to claim 21 incorporated thereto.
24 . The oligonucleotide or polynucleotide of claim 23 , wherein the oligonucleotide or polynucleotide is at least partially complementary and hybridized to a target polynucleotide immobilized on a surface of a solid support, and wherein the solid support comprises an array of a plurality of immobilized target polynucleotides.
25 . A kit comprising a first type of labeled nucleotide according to claim 21 .
26 . The kit of claim 25 , further comprising a second type of labeled nucleotide, wherein the second type of labeled nucleotide is labeled with a different compound than the first type of labeled nucleotide.
27 . The kit of claim 26 , further comprising a third type of nucleotide and a fourth type of nucleotide, wherein each of the second, third, and fourth types of nucleotides are labeled with a different compound, wherein each label has a distinct absorbance maximum that is distinguishable from the other labels.
28 . The kit of claim 25 , wherein the kit comprises four types of nucleotides, wherein a first type of nucleotide is a labeled nucleotide according to claim 21 , a second type of nucleotide carries a second label, a third type of nucleotide carries a third label, and a fourth type of nucleotide is unlabeled (dark).
29 . The kit of claim 25 , wherein the kit comprises four types of nucleotides, wherein a first type of nucleotide is a labeled nucleotide according to claim 21 , a second type of nucleotide carries a second label, a third type of nucleotide comprises a mixture of third type of nucleotides carry two labels, and a fourth type of nucleotide is unlabeled (dark).
30 . A method of determining the sequences of a plurality of target polynucleotides, comprising:
(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;
(b) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, wherein at least one type of nucleotide is a labeled nucleotide of claim 22 , and wherein each of the one or more of four different types of nucleotides comprises a 3′ blocking group covalently attached to the deoxyribose sugar of the nucleotide;
(c) incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides;
(d) performing one or more fluorescent measurements of the extended copy polynucleotides;
(e) removing the 3′ blocking group from the nucleotides incorporated into the extended copy polynucleotides; and
(f) washing the solid support after the removing of the 3′ blocking group from the incorporated nucleotides.