Novel green and orange fluorescent labels and their uses
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Abstract
The present invention provides novel fluorescent compounds and covalent attachment chemistries which facilitate the use of these compounds as labels for ultrasensitive and quantitative flourescent detection of low levels of biomolecules. In a preferred embodiment, the flourescent labels of this invention are novel derivatives of the hydroxy-pyrene trisulphonic and disulphonic acids which may be used in any assay in which radioisotopes, colored dyes or other fluorescent molecules are currently used. Thus, for example, any assay using labeled antibodies, proteins, oligonucleotides or lipids, including fluorescent cell sorting, fluorescence microscopy (including dark-field microscopy), fluorescence polarization assays, ligand, receptor binding assays, receptor activation assays and diagnostic assays can benefit from use of the compounds disclosed herein.
14 Citations
87 Claims
- 1. A fluorescent compound of the general structure:
- 13. A fluorescent compound of the structure:
- 25. A fluorescent compound of the structure
- 32. A kit comprising the fluorescent of the general structure:
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42. The kit, according to claim 325, wherein:
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R1=CH2—
(CH2)n, where n=1-15 andR2=—
SH, —
NH2, —
NCS, —
NCO, -Maleimide, —
COONHS, or —
NHNH2.
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- 44. A kit comprising a compound of the structure:
- 56. A kit comprising a fluorescent compound of the structure:
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63. A method for detecting biomolecules comprising:
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a) providing a fluorescent compound of the general structure where R2=—
OH, —
NH2, SO3—
or SH(NH2)2,M is one of the alkali metals (Li, Na, K), ammonium (NH4), or pyridinium (Py), R1 is a spacer comprised of 2 to 20 carbon, oxygen or nitrogen atoms, with the carbon containing sequences chosen from alkyl, alkenyl, arylalkyl, or alkoxy groups, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and, R′
=a terminal carboxyl, amino, sulfhydryl or biotinyl group; and
b) detecting the presence or absence of a fluorescence signal. - View Dependent Claims (64, 65, 66, 67, 68, 69, 70, 71)
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-
72. A method for detecting biomolecules comprising:
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a) providing a fluorescent compound of the general structure where R2=—
OH, —
NH2, SO3—
or SH(NH2)2,M is one of the alkali metals (Li, Na, K), ammonium (NH4), or pyridinium (Py), R1 is a spacer comprised of 2 to 20 carbon, oxygen or nitrogen atoms, with the carbon containing sequences chosen from alkyl, alkenyl, arylalkyl, or alkoxy groups, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
R′
= a succinimidyl ester, hydrazide, isothiocynato, isocyanato, maleimido, halo-acetamido, biotin or other homofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule; and
b) detecting the presence or absence of a fluorescence signal. - View Dependent Claims (73, 74, 75, 76, 77, 78, 79)
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80. A method for detecting biomolecules comprising:
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a) providing a fluorescent compound of the general structure R= is chosen from any of alky, alkenyl, alkylaryl, aromatic, or alkoxy groups which can be substituted by various constituents including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, R4 is chosen from —
H, —
CH3, alkyl, alkenyl, aromatic, or alkylaryl groups which can, in turn be substituted with any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties,X is chosen from one of (CH2)n, aryl-(CH2)n, PEG, DEXTRAN, (CH2)n—
CONH—
(CH2)n, (CH2)N—
CONH-Z, where n=1-15, Z=PEG, DEXTRAN or a polypeptide, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,Y is chosen from one of —
NH2, —
NCS, —
SH, —
OH, —
COOH, —
COONHS, —
NH2, -Maleimide, -hydrazide, CHO, biotinyl group, avidinyl group, NHCOCH2I or other monofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule; and
b) detecting the presence or absence of a fluorescence signal. - View Dependent Claims (81, 82, 83, 84, 85, 86, 87)
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Specification