USRE49362EExpiredUtility
Dye compounds and the use of their labelled conjugates
Est. expiryMay 18, 2026(expired)· nominal 20-yr term from priority
G01N 33/582C09B 11/24A61K 49/0041Y10S436/80A61K 49/0052
57
PatentIndex Score
0
Cited by
121
References
73
Claims
Abstract
Novel rhodamine dye compounds, labelled conjugates comprising the dyes are described, together with methods for their use. The dyes and labelled conjugates are useful as molecular probes in a variety of applications, such as in assays involving staining of cells, protein binding, and analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.
Claims
exact text as granted — not AI-modifiedThe invention claimed is:
1. A compound of the Formula (I):
wherein
X is SO 3 − ,
M + is a common counter ion,
k and m are independently integers from 1 to 6,
n is 3,
R 1 , R 4 , R 5 , R 6 , R 8 and R 9 are all H,
R 2 and R 3 are independently H or CH 3 ,
Z is carboxy-, carboxamido- or a substituted carboxamido group,
a is 1 and
R 7 is OH.
2. A compound of the Formula (I):
wherein
X is SO 3 H or SO 3 − ,
M + is a common counter ion,
k, n, m are independently integers from 1 to 6,
R 1 is linked to R 2 via a chain of CH 2 groups to form a ring, or
R 4 is linked to R 3 via a chain of CH 2 groups to form a ring,
R 5 and R 6 are independently H, alkyl or substituted alkyl group, halogen, hydroxy- or alkoy group,
R 8 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 1 is a carbon or heterosubstituted carbon chain forming a ring,
R 9 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 4 is a carbon or heterosubstituted carbon chain forming a ring,
R 7 is NR 11 R 12 where R 11 is H, alkyl or a substituted alkyl, and R 12 is a substituted alkyl,
Z is halogen, amino or substituted amino, hydroxyl- hydroxy-, alkoxy-, carboxy-, carboxamido- or a substituted carboxamido group, SO 3 H or SO 3 − , and
a is 0 to 4, wherein if a is 0, R 7 is NR 11 R 12 where R 11 and R 12 are independently H, alkyl or a substituted alkyl.
3. The compound of claim 2 , wherein
a is 0,
R 11 is H or alkyl,
R 12 is an alkyl substituted with COOR 13 , and
R 13 is H, alkyl or substituted alkyl group, halogen, hydroxy- or alkoxy group.
4. A compound of the Formula (I):
wherein
X is SO 3 − ,
M + is a common counter ion,
k is an integer from 1 to 6,
n=3,
m=3 and
R 1 is H or an alkyl or substituted alkyl group (CH 2 ) m SO 3 − ,
R 2 , R 3 , R 5 , R 6 , R 8 and R 9 are all H,
R 4 is H, or an alkyl or substituted alkyl group (CH 2 ) m SO 3 − ,
R 7 is OHor is joined to form aliphatic or aromatic cyclic or substituted aliphatic-, aromatic- or heterocyclic rings,
Z is halogen, amino or substituted amino, hydroxyl- hydroxy-, alkoxy-, carboxy-, carboxamido- or a substituted carboxamido group, SO 3 H or SO 3 − and
a is 0.
5. A compound of claim 4 , wherein R 1 and R 4 are (CH 2 ) m SO 3 − .
6. A nucleotide or oligonucleotide labelled with a compound according to claim 1 , 2 , 3 , or 4 .
7. A labelled nucleotide or oligonucleotide according to claim 6 wherein the label is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.
8. A labelled nucleotide or oligonucleotide according to claim 7 wherein said linker moiety comprises a moiety selected from the group consisting of:
wherein
X in said linker moiety is selected from the group comprising O, S, NH and NQ;
wherein Q is a C1-10 substituted or unsubstituted alkyl group,
Y is selected from the group comprising O, S, NH and N(allyl),
T is hydrogen or a C1-10 substituted or unsubstituted alkyl group.
9. A labelled nucleotide according to claim 7 , further comprising a 3′ OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.
10. A kit comprising two or more nucleotides wherein at least one nucleotide is a labelled nucleotide according to claim 6 .
11. A kit according to claim 10 wherein each nucleotide can be cleaved from an attached compound by chemical or enzymatic treatment.
12. A kit according to claim 11 wherein said chemical treatment comprises treating with a phosphine reagent.
13. A kit according to claim 10 wherein two of the labelled nucleotides are excited excitable using a single laser.
14. A kit according to claim 10 wherein
a first of four nucleotides is the labelled nucleotide and
the second, third, and fourth nucleotides are each labelled with a different compound,
wherein each compound has a distinct absorbance maximum and each of the compounds is distinguishable from the other three compounds.
15. A kit according to claim 14 wherein the second and third labelled nucleotides have a distinct absorbance maximum above 600 nm.
16. A kit according to claim 14 wherein
the first of four nucleotides is the labelled nucleotide and
a second and third nucleotide are each labelled with a different compound,
wherein each of the compounds of the second and third nucleotides can be excited using a single laser with a wavelength between 630-700 nm.
17. A kit according to claim 14 wherein
the first of four nucleotides is the labelled nucleotide and
a second, third, and fourth nucleotide are each labelled with a different compound,
wherein the compound of the second nucleotide can be excited using a laser with a wavelength at 532 nm and
each of the compounds of the third and fourth nucleotides can be excited using a single laser with a wavelength at 660 nm.
18. A kit according to claim 10 further comprising a DNA polymerase.
19. A method of synthesizing a compound of the Formula (I):
wherein
X is SO 3 H or SO 3 − ,
M + is a common counter ion,
k, n, m are independently integers from 1 to 6,
R 1 is H or an alkyl or substituted alkyl group,
R 2 is H, alkyl or substituted alkyl group, halogen, carboxy, carboxamide, hydroxy- or alkoxy group,
or R 2 together with R 1 or R 5 is a carbon or heterosubstituted chain forming a ring,
R 3 is H, alkyl or substituted alkyl group, halogen, carboxy, carboxamide, hydroxy- or alkoxy group
R 4 is H, or an alkyl or substituted alkyl group,
or R 3 together with R 4 or R 6 is a carbon or heterosubstituted chain forming a ring,
R 5 and R 6 are independently H, alkyl or substituted alkyl group, halogen, hydroxy- or alkoxy group,
R 8 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 1 is a carbon or heterosubstituted carbon chain forming a ring,
R 9 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 4 is a carbon or heterosubstituted carbon chain forming a ring,
R 7 is OH, OR 10 where R 10 is alkyl or substituted alkyl, or NR 11 R 12 where R 11 and R 12 are independently H, alkyl or a substituted alkyl, or is joined to form aliphatic or aromatic cyclic or substituted aliphatic-, aromatic- or heterocyclic rings,
Z is halogen, amino or substituted amino, hydroxyl- hydroxy-, alkoxy-, carboxy-, carboxamido- or a substituted carboxamido group, SO 3 H or SO 3 31 SO 3 − and
a is 0 to 4, wherein if a is 0, R 7 is NR 11 R 12 where R 11 and R 12 are independently H, alkyl or a substituted alkyl,
comprising the steps of:
(a) providing the following starting materials
(b) further providing substituted or unsubstituted phthalic anhydride derivatives, and
(c) allowing a condensation reaction to occur, thereby obtaining the compound.
20. The compound of claim 2 , wherein
X is SO 3 − ,
R 1 and R 2 form a six membered ring,
R 3 and R 4 form a six membered ring,
Z is carboxy-, carboxamido- or a substituted carboxamido group,
a is 1.
21. The compound of claim 2, wherein
R 8 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group; and R 9 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group.
22. The compound of claim 21, wherein
X is SO 3 − ; M + is selected from Na + , K + , Li + , NH 4 + , and N(Et) 3 H + ; R 1 is linked to R 2 to form a six membered aliphatic ring; R 4 is linked to R 3 to form a six membered aliphatic ring; R 5 , R 6 , R 8 , and R 9 are H; n and m are 3; k is 1 or 2; R 11 is H or alkyl; and R 12 is alkyl substituted with halo, cyano, SO 3 − , NR 13 R 14 , CONR 13 R 14 , COOH or COOR 13 , and where R 13 and R 14 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl or substituted aryl.
23. The compound of claim 22, wherein
R 11 is H or methyl; R 12 is alkyl substituted with —COOH; and a is 0.
24. The compound of claim 23, having the structure of:
25. The nucleotide or oligonucleotide according to claim 6, labeled with a compound of the Formula (I):
wherein
X is SO 3 H or SO 3 − ,
M + is a common counter ion,
k, n, m are independently integers from 1 to 6,
R 1 is linked to R 2 via a chain of CH 2 groups to form a ring,
R 4 is linked to R 3 via a chain of CH 2 groups to form a ring,
R 5 and R 6 are independently H, alkyl or substituted alkyl group, halogen, hydroxy- or alkoxy group,
R 8 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 1 is a carbon or heterosubstituted carbon chain forming a ring,
R 9 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 4 is a carbon or heterosubstituted carbon chain forming a ring,
R 7 is NR 11 R 12 where R 11 is H, alkyl or a substituted alkyl, and R 12 is a substituted alkyl,
Z is halogen, amino or substituted amino, hydroxy-, alkoxy-, carboxy-, carboxamido- or a substituted carboxamido group, SO 3 H or SO 3 − , and
a is 0 to 4.
26. The nucleotide or oligonucleotide according to claim 25, wherein the nucleotide or oligonucleotide is a nucleotide.
27. The nucleotide according to claim 26, 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 cleavable linker moiety, wherein the nucleotide comprises a deoxyribose sugar and a cleavable 3′ OH blocking group is covalently attached to the deoxyribose sugar of the nucleotide.
28. The nucleotide according to claim 27, wherein said linker moiety comprises:
wherein X in said linker moiety is O.
29. The nucleotide according to claim 27, wherein the 3′ OH blocking group comprises the structure of
wherein R 4 and R 5 in the 3′ OH blocking group are H.
30. The nucleotide according to claim 26, comprising a label moiety of the following structure:
31. The nucleotide according to claim 30, wherein the nucleotide has the structure of:
wherein
B is a nucleobase;
L is a linker moiety;
Dye is the label moiety;
R 14 is H, monophosphate, diphosphate, triphosphate, thiophosphate, or a phosphate ester;
R 15 is a cleavable 3′ OH blocking group; and
R 16 is H or OH.
32. The nucleotide according to claim 31, wherein
L comprises a moiety of the structure
wherein X in said linker moiety is O;
R 14 is triphosphate;
R 15 is a 3′ OH blocking group of the structure
wherein R 4 and R 5 in the 3′ OH blocking group are H; and
R 16 is H.
33. The nucleotide according to claim 32, where L comprises a moiety of the structure:
34. The nucleotide according to claim 33, wherein L has the structure:
35. The nucleotide according to claim 34, wherein the nucleobase is 7-deaza adenine or 7-deaza guanine, and L is attached to the 7-deaza adenine or 7-deaza guanine at the C7 position.
36. The nucleotide according to claim 34, wherein the nucleobase is cytosine and L is attached to the cytosine at the C5 position.
37. The nucleotide according to claim 34, having the structure:
38. The nucleotide or oligonucleotide according to claim 25, wherein the nucleotide or oligonucleotide is an oligonucleotide hybridized to a template polynucleotide.
39. The oligonucleotide of claim 38, wherein the template polynucleotide is immobilized on a solid support.
40. The oligonucleotide according to claim 39, wherein the solid support comprises an array of a plurality of immobilized template polynucleotides.
41. The oligonucleotide of claim 38, comprising a label moiety of the following structure:
42. The oligonucleotide of claim 41, wherein the label moiety is attached to a nucleobase in the oligonucleotide through a cleavable linker moiety.
43. The oligonucleotide of claim 42, wherein the cleavable linker moiety has the structure:
44. The kit according to claim 10, wherein the labelled nucleotide is a nucleotide labelled with a compound of the Formula (I):
wherein
X is SO 3 H or SO 3 − ,
M + is a common counter ion,
k, n, m are independently integers from 1 to 6,
R 1 is linked to R 2 via a chain of CH 2 groups to form a ring,
R 4 is linked to R 3 via a chain of CH 2 groups to form a ring,
R 5 and R 6 are independently H, alkyl or substituted alkyl group, halogen, hydroxy- or alkoxy group,
R 8 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 1 is a carbon or heterosubstituted carbon chain forming a ring,
R 9 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 4 is a carbon or heterosubstituted carbon chain forming a ring,
R 7 is NR 11 R 12 where R 11 is H, alkyl or a substituted alkyl, and R 12 is a substituted alkyl,
Z is halogen, amino or substituted amino, hydroxy-, alkoxy-, carboxy-, carboxamido- or a substituted carboxamido group, SO 3 H or SO 3 − , and
a is 0 to 4.
45. The kit according to claim 44, wherein the labelled nucleotide comprises a label moiety of the following structure:
46. The kit according to claim 45, wherein the labelled nucleotide has the structure of:
wherein
B is a nucleobase;
L is:
Dye is the label moiety;
R 14 is triphosphate;
R 15 is a 3′ OH blocking group of the structure
wherein R 4 and R 5 in the 3′ OH blocking group are H; and
R 16 is H.
47. The kit according to claim 45, wherein
a first of the nucleotides is the labelled nucleotide, and a second of the nucleotides is labelled with a different compound, wherein the label moiety and the different compound have a difference in absorbance maximum of at least 10 nm.
48. The kit according to claim 47, wherein the label moiety and one of the different compounds have a difference in absorbance maximum of between 20 nm and 50 nm.
49. The kit according to claim 47, wherein the different compound excites at 532 nm.
50. The kit according to claim 49, wherein the different compound is
51. The kit according to claim 49, wherein the second nucleotide has the structure of:
wherein:
B is selected from adenine, guanine, cytosine, and thymine;
L is a linker moiety;
R 14 is triphosphate;
R 15 is a cleavable 3′ OH blocking group; and
R 16 is H.
52. The kit according to claim 47, wherein the different compound is a dicarbocyanine dye.
53. The kit according to claim 47, wherein the second nucleotide has the structure of:
wherein:
B is selected from adenine, guanine, cytosine, and thymine;
L is a linker moiety;
Dye is a dicarbocyanine dye;
R 14 is triphosphate;
R 15 is a cleavable 3′ OH blocking group; and
R 16 is H.
54. The kit according to claim 53, comprising a third nucleotide having the structure of:
wherein:
B in the third nucleotide is selected from adenine, guanine, cytosine, and thymine;
L in the third nucleotide is a linker moiety;
Dye in the third nucleotide is
R 14 in the third nucleotide is triphosphate;
R 15 in the third nucleotide is a cleavable 3′ OH blocking group; and
R 16 in the third nucleotide is H.
55. The kit according to claim 47, comprising a third and fourth nucleotide labeled with a different compound, wherein the label moiety and each different compound has a different absorbance maximum and the label moiety and each of the different compounds are distinguishable from each other, wherein the third and fourth labelled nucleotides have distinct absorbance maximums above 600 nm.
56. The kit according to claim 55, wherein the third and fourth labelled nucleotides have distinct absorbance maximums above 640 nm.
57. The kit according to claim 45, further comprising a mutant DNA polymerase and one or more buffer compositions.
58. The kit according to claim 57, wherein the mutant DNA polymerase is mutant 9° N polymerase.
59. The kit according to claim 45, further comprising sodium ascorbate.
60. The kit according to claim 46, wherein
a first of the nucleotides is the labelled nucleotide, and a second of the nucleotides is labelled with a different compound, wherein the label moiety and the different compound have a difference in absorbance maximum of at least 10 nm.
61. The kit according to claim 60, wherein the label moiety and one of the different compounds have a difference in absorbance maximum of between 20 nm and 50 nm.
62. The kit according to claim 60, wherein the different compound excites at 532 nm.
63. The kit according to claim 62, wherein the different compound is
64. The kit according to claim 62, wherein the second nucleotide has the structure of:
wherein:
B in the second nucleotide is selected from adenine, guanine, cytosine, and thymine;
L in the second nucleotide is a linker moiety;
Dye in the second nucleotide is
R 14 in the second nucleotide is triphosphate;
R 15 in the second nucleotide is a cleavable 3′ OH blocking group; and
R 16 in the second nucleotide is H.
65. The kit according to claim 60, wherein the different compound is a dicarbocyanine dye.
66. The kit according to claim 60, wherein the second nucleotide has the structure of:
wherein:
B in the second nucleotide is selected from adenine, guanine, cytosine, and thymine;
L in the second nucleotide is a linker moiety;
Dye in the second nucleotide is a dicarbocyanine dye;
R 14 in the second nucleotide is triphosphate;
R 15 in the second nucleotide is a cleavable 3′ OH blocking group; and
R 16 in the second nucleotide is H.
67. The kit according to claim 66, comprising a third nucleotide having the structure of:
wherein:
B in the third nucleotide is selected from adenine, guanine, cytosine, and thymine;
L in the third nucleotide is a linker moiety;
Dye in the third nucleotide is
R 14 in the third nucleotide is triphosphate;
R 15 in the third nucleotide is a cleavable 3′ OH blocking group; and
R 16 in the third nucleotide is H.
68. The kit according to claim 60, comprising a third and fourth nucleotide labeled with a different compound, wherein the label moiety and each different compound has a distinct absorbance maximum and the label moiety and each of the different compounds are distinguishable from each other, wherein the second and third labelled nucleotides have a distinct absorbance maximum above 600 nm.
69. The kit according to claim 68, wherein the second and third labelled nucleotides have distinct absorbance maximums above 640 nm.
70. The kit according to claim 60, further comprising a DNA polymerase and one or more buffer compositions.
71. The kit according to claim 70, wherein the DNA polymerase is a mutant DNA polymerase.
72. The kit according to claim 71, wherein the mutant DNA polymerase is mutant 9° N polymerase.
73. The kit according to claim 70, comprising sodium ascorbate.Cited by (0)
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