US5442045A
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Biological conjugates of fluorescent rhodol dyes
Assignee: Molecular Probes Inc
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Filing: 1993-03-08
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Granted: 1995-08-15
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Expires: 2013-03-08
Abstract
Abstract Novel fluorescent dyes based on the rhodol structure are provided. The new reagents contain functional groups capable of forming a stable fluorescent product with functional groups typically found in biomolecules or polymers including amines, phenols, thiols, acids, aldehydes and ketones. Reactive groups in the rhodol dyes include activated esters, isothiocyanates, amines, hydrazines, halides, acids, azides, maleimides, aldehydes, alcohols, acrylamides and haloacetamides. The products are detected by their absorbance or fluorescence properties. The spectral properties of the fluorescent dyes are sufficiently similar in wavelengths and intensity to fluorescein or rhodamine derivatives as to permit use of the same equipment. The dyes, however, show less spectral sensitivity to pH in the physiological range than does fluorescein, have higher solubility in non-polar solvents and have improved photostability and quantum yields.
Inventors
- 👤 Richard P. Haugland
- 👤 James E. Whitaker
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 14:39
CPC Classifications
C; C07; C07D; C07D491/00; C07D491/12; C07D491/14; C07D311/00; C07D311/02; C07D311/78; C07D311/80; C07D311/82; C09; C09B; C09B11/00; C09B11/04; C09B11/10; C09K; C09K11/00; C09K11/06
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
225 words
A compound comprising a fluorescent dye having the structure ##STR2## wherein R 1 is H or CH 3 , and R 2 is H, CF 3 CO, CH 3 , or C 2 H 5 ; or R 1 in combination with R 2 is (CH 2 ) 3 ; R 3 is H, CH 3 , or C 2 H 5 , and R 4 is H; or R 3 in combination with R 4 is (CH 2 ) 3 ; R 5 is H, CH 2 NH 2 , CH 2 NHCOCH 2 Cl, CH 2 NHCOCH 2 I; CH 2 NHCOCH═CH 2 , or CH 2 NHCOC 6 H 4 N 3 ; R 6 is H, Cl, Br, CH 3 , or C 2 H 5 , or (CH 2 ) n CH 3 where n is an integer from 0 to 15, or (CH 2 ) n CO 2 H, where n is an integer from 0 to 11; R 7 is H, CO 2 H, ##STR3## NH 2 , NCS, CH 2 NHCOCH 2 Cl, or CH 2 NHCOCH 2 I; such that R 7 is not H if R 5 is H; conjugated to a reactive site on a biomolecule via reaction of a chemically reactive functional group on the dye with a reactive site on the biomolecule.
Claim 2
Dependent Claim
225 words
A compound comprising a fluorescent dye having the structure ##STR2## wherein R 1 is H or CH 3 , and R 2 is H, CF 3 CO, CH 3 , or C 2 H 5 ; or R 1 in combination with R 2 is (CH 2 ) 3 ; R 3 is H, CH 3 , or C 2 H 5 , and R 4 is H; or R 3 in combination with R 4 is (CH 2 ) 3 ; R 5 is H, CH 2 NH 2 , CH 2 NHCOCH 2 Cl, CH 2 NHCOCH 2 I; CH 2 NHCOCH═CH 2 , or CH 2 NHCOC 6 H 4 N 3 ; R 6 is H, Cl, Br, CH 3 , or C 2 H 5 , or (CH 2 ) n CH 3 where n is an integer from 0 to 15, or (CH 2 ) n CO 2 H, where n is an integer from 0 to 11; R 7 is H, CO 2 H, ##STR3## NH 2 , NCS, CH 2 NHCOCH 2 Cl, or CH 2 NHCOCH 2 I; such that R 7 is not H if R 5 is H; conjugated to a reactive site on a biomolecule via reaction of a chemically reactive functional group on the dye with a reactive site on the biomolecule.
Claim 3
Dependent Claim
13 words
A compound as claimed in claim 1, wherein R 7 is not H.
Claim 4
Dependent Claim
21 words
A compound, as claimed in claim 1, wherein one of R 5 and R 6 is a chemically reactive functional group.
Claim 5
Dependent Claim
153 words
A compound comprising a fluorescent dye having the structure ##STR4## wherein R 1 arid R 4 are independently hydrogen, alkyl, carboxyalkyl, aminoalkyl, halogen or alkoxy, the alkyl portions of which contain less than 4 carbons; R 2 and R 3 are independently hydrogen, methyl or ethyl, or trifluoroacetyl; or R 1 in combination with R 2 , or R 3 in combination with R 4 are (CH 2 ) 3 ; R 5 is H: R 6 is H, Cl, Br. CH 3 , or C 2 H 5 , and R 9 is COCH 3 ; or R 6 and R 9 in combination are (CH 2 ) 2 CO; ##STR5## such that R 7 is is not H if R 9 is COCH 3 ; conjugated to a reactive site on a biomolecule via reaction of a chemically reactive functional group on the dye with a reactive site on the biomolecule.
Claim 6
Dependent Claim
153 words
A compound comprising a fluorescent dye having the structure ##STR4## wherein R 1 arid R 4 are independently hydrogen, alkyl, carboxyalkyl, aminoalkyl, halogen or alkoxy, the alkyl portions of which contain less than 4 carbons; R 2 and R 3 are independently hydrogen, methyl or ethyl, or trifluoroacetyl; or R 1 in combination with R 2 , or R 3 in combination with R 4 are (CH 2 ) 3 ; R 5 is H: R 6 is H, Cl, Br. CH 3 , or C 2 H 5 , and R 9 is COCH 3 ; or R 6 and R 9 in combination are (CH 2 ) 2 CO; ##STR5## such that R 7 is is not H if R 9 is COCH 3 ; conjugated to a reactive site on a biomolecule via reaction of a chemically reactive functional group on the dye with a reactive site on the biomolecule.
Claim 7
Dependent Claim
31 words
A compound as claimed in claim 4, wherein the biomolecule is a peptide, a protein, a hormone, a drug a nucleotide, an oligonucleotide, a nucleic acid, a polysaccharide, or a lipid.
Claim 8
Dependent Claim
24 words
A compound as claimed in claim 4, where the fluorescent dye is conjugated to a biomolecule that is a nucleotide, oligonucleotide, or nucleic acid,
Claim 9
Dependent Claim
45 words
A compound as claimed in claim 4, wherein R 1 in combination with R 2 , and R 3 in combination with R 4 are (CH 2 ) 3 ; and R 7 is selected from the group consisting of CO 2 H and ##STR6##
Claim 10
Dependent Claim
62 words
A compound as claimed in claim 7, wherein R 1 in combination with R 2 , and R 3 in combination with R 4 are (CH 2 ) 3 ; R 5 is H; R 7 is selected from the group consisting of CO 2 H and ##STR7## and R 6 and R 9 in combination are (CH 2 ) 2 CO.
Claim 11
Dependent Claim
56 words
The compound of claim 1 formed when the fluorescent dye is conjugated to a reactive site on a biomolecule via reaction of the chemically reactive functional group with the reactive site on the biomolecule, the reactive site selected from the group consisting of amines, thiols, alcohols, phenols, aldehydes, ketones, carboxylic acids, alkyl halides, imidazoles, and olefins.
Claim 12
Dependent Claim
31 words
A compound, as claimed in claim 1, wherein the biomolecule is a peptide, a protein, a hormone, a drug, a nucleotide, an oligonucleotide, a nucleic acid, a polysaccharide, or a lipid.
Claim 13
Dependent Claim
26 words
The compound of claim 3, where the fluorescent dye is conjugated to a biomolecule that is a peptide, a protein, a nucleotide, oligonucleotide, or nucleic acid.
Claim 14
Dependent Claim
21 words
The compound of claim 4, where the fluorescent dye is conjugated to a biomolecule that is a peptide or a protein.
Claim 15
Dependent Claim
231 words
A compound of the formula; dye-biomolecule wherein said dye is fluorescent dye of the formula: ##STR8## that is covalently attached to said biomolecule through a linkage that is amine, amide, carboxamide, sulfonamide, propionamide, glycineamide, amidine, imine, imide, imidazole, hydrazide, hydrazone, urea, thiourea, urethane, ester, ether, or thiother at one of the positions R 1 through R 7 ; and at the positions where the biomolecule is not attached: R 1 is H or CH 3 and R 2 is H CF 3 CO CH 3 or C 2 H 5 ; or R 1 in combination with R 2 is (CH 2 ) 3 ; R 3 is H, CH 3 , or C 2 H 5 , and R 4 is H; or R 3 in combination with R 4 is (CH 2 ) 3 ; R 5 is H CH 2 NH 2 CH 2 NHCOCH 2 Cl, CH 2 NHCOCH 2 I; CH 2 NHCOCH═CH 2 , or CH 2 NHCOC 6 H 4 N 3 ; R 6 is H, Cl, Br, or (CH 2 ) n CH 3 where n is an integer from 0 to 15, or (CH 2 ) n CO 2 H, where n is an integer from 0 to 11; R 7 is H, CO 2 H, ##STR9## NH 2 NCS CH 2 NHCOCH 2 Cl, or CH 2 NHCOCH 2 I.
Claim 16
Dependent Claim
231 words
A compound of the formula; dye-biomolecule wherein said dye is fluorescent dye of the formula: ##STR8## that is covalently attached to said biomolecule through a linkage that is amine, amide, carboxamide, sulfonamide, propionamide, glycineamide, amidine, imine, imide, imidazole, hydrazide, hydrazone, urea, thiourea, urethane, ester, ether, or thiother at one of the positions R 1 through R 7 ; and at the positions where the biomolecule is not attached: R 1 is H or CH 3 and R 2 is H CF 3 CO CH 3 or C 2 H 5 ; or R 1 in combination with R 2 is (CH 2 ) 3 ; R 3 is H, CH 3 , or C 2 H 5 , and R 4 is H; or R 3 in combination with R 4 is (CH 2 ) 3 ; R 5 is H CH 2 NH 2 CH 2 NHCOCH 2 Cl, CH 2 NHCOCH 2 I; CH 2 NHCOCH═CH 2 , or CH 2 NHCOC 6 H 4 N 3 ; R 6 is H, Cl, Br, or (CH 2 ) n CH 3 where n is an integer from 0 to 15, or (CH 2 ) n CO 2 H, where n is an integer from 0 to 11; R 7 is H, CO 2 H, ##STR9## NH 2 NCS CH 2 NHCOCH 2 Cl, or CH 2 NHCOCH 2 I.
Claim 17
Dependent Claim
32 words
A compound, as claimed in claim 13, wherein the biomolecule X is a peptide, a protein, a hormone, a drug, a nucleotide, an oligonucleotide, a nucleic acid, a polysaccharide, or a lipid.
Claim 18
Dependent Claim
220 words
A compound, as claimed in claim 13, wherein said dye has the formula: ##STR10## wherein the biomolecule is attached at one position R 5 , R 6 , or R 7 ; and at the positions where the biomolecule is not attached: R 1 is H or CH 3 , and R 2 is H, CF 3 CO, CH 3 , or C 2 H 5 ; or R 1 in combination with R 2 is (CH 2 ) 3 ; R 3 is H, CH 3 , or C 2 H 5 , and R 4 is H; or R 3 in combination with R 4 is (CH 2 ) 3 ; R 5 is H, CH 2 NH 2 , CH 2 NHCOCH 2 Cl CH 2 NHCOCH 2 I; CH 2 NHCOCH═CH 2 or CH 2 NHCOC 6 H 4 N 3 ; R 6 is H, Cl, Br, or (CH 2 ) n CH 3 where n is an integer from 0 to 15, or (CH 2 ) n CO 2 H where n is an integer from 0 to 11; R 7 is H, CO 2 H ##STR11## NH 2 , NCS CH 2 NHCOCH 2 Cl, or CH 2 NHCOCH 2 I; such that R 7 is not H if R 5 is H.
Claim 19
Dependent Claim
25 words
A compound, as claimed in claim 15, wherein the biomolecule X is attached at one position R 5 or R 6 by an amide linkage.
Claim 20
Dependent Claim
23 words
A compound, as claimed in claim 15, wherein the biomolecule X is attached at R 7 by an amide, urea or thiourea linkage.
Claim 21
Dependent Claim
78 words
A compound, as claimed in claim 13, wherein the biomolecule X is attached at one position R 6 or R 7 ; and at the positions where the biomolecule is not attached: R 1 is H or methyl; R 2 and R 3 are independently H or (C 1 -C 2 ) alkyl; R 4 and R 5 are H; R 6 is hydrogen, Cl, Br, or (C 1 -C 16 ) alkyl; and R 7 is hydrogen.
Claim 22
Dependent Claim
76 words
A compound, as claimed in claim 13, wherein the biomolecule X is attached at one position R 6 or R 7 ; and at the positions where the biomolecule is not attached: R 1 in combination with R 2 and R 3 in combination with R 4 are each (CH 2 ) 3 ; R 5 is H; R 6 is hydrogen, Cl, Br, or (C 1 -C 16 ) alkyl; and R 7 is hydrogen.
Claim 23
Dependent Claim
91 words
A compound, as claimed in claim 13, wherein the biomolecule X is a peptide or protein that is attached at R 7 by an amide linkage; and at the positions where the biomolecule is not attached: R 1 and R 4 are independently hydrogen; R 2 and R 3 are independently hydrogen or (C 1 -C 2 ) alkyl; or R 1 in combination with R 2 and R 3 in combination with R 4 are each (CH 2 ) 3 ; and R 5 and R 6 are independently hydrogen.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-08-14 | 1999-02-13 | 1999-08-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-08-14 | 2003-02-13 | 2003-08-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-08-14 | 2007-02-13 | 2007-08-15 | $7700.00 | Window Closed / Lapsed |