Chromenoquinoline dyes and uses in sequencing
The present application relates to chromenoquinoline dyes and their uses as fluorescent labels. For example, these dyes may be used to label nucleotides for nucleic acid sequencing.
1 . A compound of Formula (I), a salt or a mesomeric form thereof:
wherein ring A is a 4 to 10 membered heterocyclyl comprising at least one nitrogen atom, and ring A is optionally substituted with one or more R N ;
each R N is independently carboxyl, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, substituted C 1 -C 6 alkoxy, C 1 -C 6 haloalkyl, C 1 -C 6 haloalkoxy, (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, —O(C 1 -C 6 alkoxy)C 1 -C 6 alkyl, optionally substituted amino, amino(C 1 -C 6 alkyl), halo, cyano, hydroxy, hydroxy(C 1 -C 6 alkyl), nitro, sulfonyl, sulfo, sulfonate, S-sulfonamido, or N-sulfonamido;
each of R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10a and R 10b is independently H, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, substituted C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 1 -C 6 haloalkoxy, (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, —O—(C 1 -C 6 alkoxy)C 1 -C 6 alkyl, optionally substituted amino, amino(C 1 -C 6 alkyl), halo, cyano, hydroxy, hydroxy (C 1 -C 6 alkyl), nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido, N-sulfonamido, optionally substituted phenyl, optionally substituted 5 to 6 membered heteroaryl, optionally substituted C 3 -C 7 cycloalkyl, or optionally substituted 4 to 7 membered heterocyclyl; and
R 4 is C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl;
alternatively, R 5 and R 6 together with the atoms to which they are attached form an optionally substituted C 6 -C 10 aryl, an optionally substituted 3-10 membered carbocyclyl, an optionally substituted 5-10 membered heteroaryl or an optionally substituted 3-10 membered heterocyclyl;
alternatively, R 6 and R 7 together with the atoms to which they are attached form an optionally substituted C 6 -C 10 aryl, an optionally substituted 3-10 membered carbocyclyl, an optionally substituted 5-10 membered heteroaryl or an optionally substituted 3-10 membered heterocyclyl;
alternatively, R 7 and R 8 together with the atoms to which they are attached form an optionally substituted C 6 -C 10 aryl, an optionally substituted 3-10 membered carbocyclyl, an optionally substituted 5-10 membered heteroaryl or an optionally substituted 3-10 membered heterocyclyl;
provided that the compound of Formula (I) comprises a carboxyl group.
2 . The compound of claim 1 , wherein ring A is,
each optionally substituted with one R N , wherein each of R A and R B is independently H or C 1 -C 6 alkyl.
3 . The compound of claim 1 , wherein R 4 is C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl.
4 . The compound of claim 3 , wherein R 4 is C 1 -C 6 alkyl substituted with one or more substituents selected from the group consisting of carboxyl (—C(O)OH), carboxylate (—C(O)O − ), sulfo (—SO 3 H), sulfonate (—SO 3 ″), —C(O)OR a , and —C(O)NR b R c , wherein R a is optionally substituted C 1 -C 6 alkyl, optionally substituted C 6 -C 10 aryl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted C 3 -C 7 cycloalkyl, and wherein each of R b and R c is independently H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 6 -C 10 aryl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted C 3 -C 7 cycloalkyl.
5 . The compound of claim 1 , wherein each of R 1 , R 2 and R 3 is H.
6 . The compound of claim 1 , wherein at least one of R 5 , R 6 , R 7 and R 8 is independently C 1 -C 6 alkyl or C 1 -C 6 alkoxy.
7 . The compound of claim 1 , wherein at least one of R 5 , R 6 , R 7 and R 8 is substituted C 1 -C 6 alkyl or substituted C 1 -C 6 alkoxy, each substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(O)OR a or —C(O)NR b R c , and wherein each R b and R c is independently H or C 1 -C 6 alkyl substituted with carboxyl, carboxylate, —C(O)OR a , sulfo or sulfonate.
8 . The compound of claim 7 , wherein each of R 5 , R 6 and R 8 is H, and R 7 is substituted C 1 -C 6 alkyl or substituted C 1 -C 6 alkoxy, each substituted with carboxyl, carboxylate, sulfo, sulfonate, —C(O)OR a or —C(O)NR b R c .
9 . The compound of claim 8 , wherein ring A is
10 . The compound of claim 1 , wherein R 6 and R 7 together with the atoms to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl.
11 . The compound of claim 10 , wherein the optionally substituted 5 or 6 membered heterocyclyl is
12 . The compound of claim 1 , wherein R 7 and R 8 together with the atoms to which they are attached form an optionally substituted phenyl or six membered heteroaryl containing one or more nitrogen atoms.
13 . The compound of claim 1 , wherein R 9 is H, C 1 -C 6 alkyl, or phenyl.
14 . The compound of claim 1 , wherein each of R 10a and R 10b is H.
15 . The compound of claim 1 , selected from the group consisting of:
and salts and mesomeric forms thereof.
16 . The compound of claim 7 , wherein each of R 5 and R 7 is H, and each of R 6 and R 7 is methoxy.
17 . The compound of claim 16 , wherein ring A is
18 . A compound of claim 1 , covalently attached to a photo-protecting cyclooctatetraene moiety via an amide bond, wherein the photo-protecting cyclooctatetraene moiety comprises the structure of formula (III):
wherein Z is absent, optionally substituted C 2-6 alkenylene, or optionally substituted C 2-6 alkynylene;
each R x and R y is independently H, carboxyl, carboxylate, amino, sulfo, sulfonate, —C(O)OR a , or —C(O)NR b R c , or C 1 -C 6 alkyl substituted with amino, carboxyl, carboxylate, sulfo, sulfonate, —C(O)OR a , or —C(O)NR b R c ,
each R N1 and R N2 is independently H or C 1 -C 6 alkyl substituted with amino, carboxyl, carboxylate, sulfo, sulfonate, —C(O)OR a , or —C(O)NR b R c ;
R a is optionally substituted C 1 -C 6 alkyl, optionally substituted C 6 -C 10 aryl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted C 3 -C 7 cycloalkyl;
each of R b and R c is independently H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 6 -C 10 aryl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted C 3 -C 7 cycloalkyl;
the carbon atom to which R x and R y are attached in
is optionally replaced with O, S, or N, provided that when said carbon atom is replaced with O or S, then R x and R y are both absent; when said carbon atom is replaced with N, then R y is absent; and
m is an integral number between 1 and 10;
wherein the asterisk * indicates the attachment point of the photo-protecting cyclooctatetraene moiety to the carbonyl group of amide bond formed by the reaction of an amino group of the photo-protecting cyclooctatetraene moiety with the carboxyl group of the compound.
19 . The compound of claim 18 , wherein the photo-protecting moiety comprises the structure:
20 . The compound of claim 18 , having the structure:
or a salt or mesomeric form thereof.
21 . A nucleotide labeled with the compound according to claim 1 .
22 . The nucleotide of claim 21 , wherein the compound of Formula (I) is covalently attached to the nucleotide via a carboxyl group of ring A, or R 4 of Formula (I).
23 . The labeled nucleotide of claim 21 , wherein the compound is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.
24 . The labeled nucleotide of claim 21 , wherein the nucleotide is a nucleotide triphosphate comprising 2′ deoxyribose.
25 . An oligonucleotide or polynucleotide comprising the nucleotide of claim 24 incorporated therein.
26 . The oligonucleotide or polynucleotide of claim 25 , wherein the oligonucleotide or polynucleotide is hybridized to at least a portion of a target polynucleotide.
27 . The oligonucleotide or polynucleotide of claim 26 , wherein the target polynucleotide is immobilized on a solid support, and wherein the solid support comprises a plurality of different immobilized target polynucleotides.
28 . A kit comprising a first type of nucleotide, which is the labeled nucleotide according to claim 24 .
29 . The kit of claim 28 , further comprising a second type of labeled nucleotide, a third type of labeled nucleotide, and a fourth type of unlabeled nucleotide.
30 . The kit of claim 29 , wherein each of the first type, second type and the third type of nucleotide is excitable using the same light source with a single wavelength.
31 . The kit of claim 30 , the emissions of the first type of labeled nucleotide, the second type of labeled nucleotide and the third type of labeled nucleotide are detectable in two detection channels with different wavelengths.