MODIFIED CARBOCYANINE DYES AND THEIR CONJUGATES
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Abstract
Chemically reactive carbocyanine dyes incorporating an indolium ring moiety that is substituted at the 3-position by a reactive group or by a conjugated substance, and their uses, are described. Conjugation through this position results in spectral properties that are uniformly superior to those of conjugates of spectrally similar dyes wherein attachment is at a different position. The invention includes derivative compounds having one or more benzo nitrogens.
128 Citations
63 Claims
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1-17. -17. (canceled)
- 18. A compound of Formula:
- 34. A compound of Formula:
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41. A carbocyanine dye according to the formula:
A-BRIDGE-Bwherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and
BRIDGE is a polymethine linker. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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46. The carbocyanine dye of claim 41, wherein B is:
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47. The carbocyanine dye of claim 45, wherein R3 and R4 are methyl.
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48. The carbocyanine dye of claim 46, wherein R13 is -L-Rx or -L-Sc.
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49. The carbocyanine dye of claim 48, wherein R14 is methyl.
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50. The carbocyanine dye of claim 41, wherein BRIDGE is:
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51. The carbocyanine dye of claim 50, wherein a is 1 and b is 0.
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52. The carbocyanine dye of claim 50, wherein each of R21, R22, R23, R24, R25, R26, and R27 are H,
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53. A method of staining a biological sample, comprising:
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combining a dye solution comprising a compound having the formula;
wherein, δ
is 0 or 1;
n is 1, 2 or 3;
W represents the atoms necessary to form one fused aromatic ring having 6 atoms, which atoms are selected from —
CH and —
CR1′
;
each R1′
is sulfo;
R2 is C1-C6 alkyl, which is substituted by sulfo;
R3 is C1-C6 alkyl;
R4 is C1-C6 alkyl;
R6 is H or sulfo;
R7 is H or sulfo;
R8 is H or sulfo;
R9 is H or sulfo;
R12 is -L-Rx or -L-Sc, or a C1-C6 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;
R13 is -L-Rx or -L-Sc;
R14 is C1-C6 alkyl;
Rx is the reactive group;
Sc is a conjugated substance; and
L is a covalent linkage;
with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions. - View Dependent Claims (54, 55)
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56. A method of staining a biological sample, comprising:
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combining a dye solution comprising a compound having the formula;
A-BRIDGE-Bwherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and
BRIDGE is a polymethine linker;
with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.
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57. A method for forming a dye-conjugate of a protein, peptide or a nucleic acid polymer, said method comprising:
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combining a dye solution comprising a compound of the formula wherein, δ
is 0 or 1;
n is 1, 2 or 3;
W represents the atoms necessary to form one fused aromatic ring having 6 atoms, which atoms are selected from —
CH and —
CR1′
;
each R1′
is sulfo;
R2 is C1-C6 alkyl, which is substituted by sulfo;
R3 is C1-C6 alkyl;
R4 is C1-C6 alkyl;
R6 is H or sulfo;
R7 is H or sulfo;
R8 is H or sulfo;
R9 is H or sulfo;
R12 is -L-Rx or -L-Sc, or a C1-C6 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;
R13 is -L-Rx or -L-Sc;
R14 is C1-C6 alkyl;
Rx is the reactive group;
Sc is a conjugated substance; and
L is a covalent linkage;
with a sample comprising a protein, peptide or a nucleic acid polymer whereby a dye conjugate is formed.
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58. A method for forming a dye-conjugate of a protein, peptide or a nucleic acid polymer, said method comprising:
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combining a dye solution comprising a compound of the formula
A-BRIDGE-Bwherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and
BRIDGE is a polymethine linker;
with a sample comprising a protein, peptide or a nucleic acid polymer whereby a dye conjugate is formed.
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59. A kit for fluorescent labeling of a biological sample, comprising:
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a dye solution comprising a compound having the formula;
wherein, δ
is 0 or 1;
n is 1, 2 or 3;
W represents the atoms necessary to form one fused aromatic ring having 6 atoms, which atoms are selected from —
CH and —
CR1′
;
each R1′
is sulfo;
R2 is C1-C6 alkyl, which is substituted by sulfo;
R3 is C1-C6 alkyl;
R4 is C1-C6 alkyl;
R6 is H or sulfo;
R7 is H or sulfo;
R8 is H or sulfo;
R9 is H or sulfo;
R12 is -L-Rx or -L-Sc, or a C1-C6 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;
R13 is -L-Rx or -L-Sc;
R14 is C1-C6 alkyl;
Rx is the reactive group;
Sc is a conjugated substance; and
L is a covalent linkage; and
a buffer.
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60. A kit for fluorescent labeling of a biological sample, comprising:
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a dye solution comprising a compound having the formula;
A-BRIDGE-Bwherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and
BRIDGE is a polymethine linker; and
a buffer.
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61. A carbocyanine dye comprising at least one substituted indolium ring system wherein a substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance.
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62. The carbocyanine dye of claim 62, comprising two substituted indolium ring systems wherein a substituent on the 3-carbon of one of the indolium ring contains a chemically reactive group or a conjugated substance.
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63. A compound comprising 3-(5-carboxypentyl)-2,3-dimethyl-5-sulfoindolium or a salt thereof.
Specification