Modified carbocyanine dyes and their conjugates
3 Assignments
0 Petitions
Accused Products
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.
-
Citations
50 Claims
- 1. A compound of the formula:
-
18. A compound of the formula
-
24. A compound of the formula:
-
35. A compound of the formula:
-
36. A compound of the formula:
-
37. A compound of the formula:
-
38. A compound of the formula:
-
39. A compound of the formula:
-
40. A compound of the formula:
- 41. A compound of the formula:
-
43. A method of staining a biological sample, comprising:
-
combining a dye solution comprising a compound of the formula or its salts, wherein R3 is —
L—
Rx;
or —
L—
Sc; and
R4 is independently —
L—
Rx;
or —
L—
Sc;
or a C1-C22 alkyl or C7-C22 arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C1-C6 alkoxy, C1-C6 alkylamino, or C2-C12 dialkylamino, or C3-C18 trialkylammonium;
or R3 and R4 taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by —
L—
Rx;
or —
L—
Sc ; and
L is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O, and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;
or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds;
or aromatic or heteroaromatic bonds;
Rx is a reactive group;
Sc is a conjugated substance;
R6 through R9 are independently H, —
L—
Rx;
or —
L—
Sc;
or amino, sulfo, trifluoromethyl, or halogen;
or C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;
or any two adjacent substituents of R6 through R9 form a fused benzo ring that is optionally substituted one or more times by —
L—
Rx;
or by —
L—
Sc;
or by amino, sulfo, trifluoromethyl, or halogen;
or by C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C12 dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;
the atoms of W are selected from —
CH, —
C, —
CR1′
, and —
N(R12)β
′
, where β
′
is 1, but no more than one of such atoms is —
N(R12)β
′
; and
each R1′
is independently —
L—
Rx;
or —
L—
Sc;
or amino, sulfo, trifluoromethyl, or halogen;
or C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2C12 dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;
each R2 and R12 is independently —
L—
Rx;
or —
L—
Sc;
or a C1-C22 alkyl, which optionally incorporates up to six hetero atoms, and is optionally substituted one or more times by amino, carboxy, or sulfo;
δ
is 0 or 1, and δ
+all β
′
=1; and
δ
is 1 only when W contains no ring nitrogen atoms;
one of R13 and R14 is a C1-C6 alkyl and the other of R13 and R14 is —
L—
Rx;
or —
L—
Sc;
or a C1-C22 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C1-C6 alkoxy, C1-C6 alkylamino, or C2-C12 dialkylamino;
with a biological sample in a concentration sufficient to yield a detectable optical response under the desired conditions. - View Dependent Claims (44, 45, 46, 47, 48, 49)
-
-
50. A kit for fluorescent labeling of a biological sample, comprising
a dye solution comprising a compound of the formula or its salts, wherein R3 is — - L—
Rx;
or —
L—
Sc; and
R4 is C1-C6 alkyl; and
L is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O, and S, and incorporating the formula —
(CH2)d(CONH(CH2)e)z′
—
, where d is 0-5, e is 1-5 and z′
is 0 or 1, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;
or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds;
or aromatic or heteroaromatic bonds;
Rx is a reactive group that is an activated ester of a carboxylic acid, an amine, a carboxylic acid, a halotriazine, a hydrazide, a maleimide, a reactive platinum complex;
Sc is a conjugated substance that is an antibody, an amino acid, an angiostatin or endostatin, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a non-biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phospholipid-binding protein, a small-molecule drug, a structural protein, an NTA, or a tyramide; and
R7 is sulfo, and R6, R8, and R9 are H;
or R6 and R7 combine to form a fused benzo ring that is substituted one or more times by sulfo, and R8 and R9 are H;
the atoms of W are selected from —
CH, —
C, —
CR1′
, and —
N(R12)β
′
, where each β
′
is 1, but no more than one of such atoms is —
N(R12)β
′
, where each R1′
is sulfo or Br;
R2 and R12 are independently a C1-C6 alkyl or C1-C6 alkyl substituted by sulfo;
δ
is 0 or 1, and δ
+all β
′
=1; and
δ
is 1 only when W contains no ring nitrogen atoms;
R13 and R14 are independently C1-C6 alkyl;
each of R21, R22, and R23 are H;
or any two adjacent substituents of R21, R22, R23, when taken in combination, forms a 4-, 5-, or 6-membered saturated or unsaturated hydrocarbon ring that is optionally substituted one or more times by C1-C6 alkyl, halogen, or a carbonyl oxygen; and
a buffer.
- L—
Specification