Biological conjugates of fluorescent rhodol dyes
First Claim
1. A compound comprising a fluorescent dye having the structure ##STR2## wherein R1 is H or CH3, and R2 is H, CF3 CO, CH3, or C2 H5 ;
- or R1 in combination with R2 is (CH2)3 ;
R3 is H, CH3, or C2 H5, and R4 is H;
or R3 in combination with R4 is (CH2)3 ;
R5 is H, CH2 NH2, CH2 NHCOCH2 Cl, CH2 NHCOCH2 I;
CH2 NHCOCH═
CH2, or CH2 NHCOC6 H4 N3 ;
R6 is H, Cl, Br, CH3, or C2 H5, or (CH2)n CH3 where n is an integer from 0 to 15, or (CH2)n CO2 H, where n is an integer from 0 to 11;
R7 is H, CO2 H, ##STR3## NH2, NCS, CH2 NHCOCH2 Cl, or CH2 NHCOCH2 I;
such that R7 is not H if R5 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.
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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.
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Citations
20 Claims
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1. A compound comprising a fluorescent dye having the structure ##STR2## wherein R1 is H or CH3, and R2 is H, CF3 CO, CH3, or C2 H5 ;
- or R1 in combination with R2 is (CH2)3 ;
R3 is H, CH3, or C2 H5, and R4 is H;
or R3 in combination with R4 is (CH2)3 ;R5 is H, CH2 NH2, CH2 NHCOCH2 Cl, CH2 NHCOCH2 I;
CH2 NHCOCH═
CH2, or CH2 NHCOC6 H4 N3 ;R6 is H, Cl, Br, CH3, or C2 H5, or (CH2)n CH3 where n is an integer from 0 to 15, or (CH2)n CO2 H, where n is an integer from 0 to 11; R7 is H, CO2 H, ##STR3## NH2, NCS, CH2 NHCOCH2 Cl, or CH2 NHCOCH2 I;
such that R7 is not H if R5 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. - View Dependent Claims (2, 3, 9, 10, 11)
- or R1 in combination with R2 is (CH2)3 ;
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4. A compound comprising a fluorescent dye having the structure ##STR4## wherein R1 arid R4 are independently hydrogen, alkyl, carboxyalkyl, aminoalkyl, halogen or alkoxy, the alkyl portions of which contain less than 4 carbons;
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R2 and R3 are independently hydrogen, methyl or ethyl, or trifluoroacetyl;
orR1 in combination with R2, or R3 in combination with R4 are (CH2)3 ; R5 is H; R6 is H, Cl, Br. CH3, or C2 H5, and R9 is COCH3 ;
or R6 and R9 in combination are (CH2)2 CO;
##STR5## such that R7 is is not H if R9 is COCH3 ;
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. - View Dependent Claims (5, 6, 7, 8, 12)
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13. 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 R1 through R7 ; and
at the positions where the biomolecule is not attached;R1 is H or CH3 and R2 is H CF3 CO CH3 or C2 H5 ;
or R1 in combination with R2 is (CH2)3 ;R3 is H, CH3, or C2 H5, and R4 is H;
or R3 in combination with R4 is (CH2)3 ;R5 is H CH2 NH2 CH2 NHCOCH2 Cl, CH2 NHCOCH2 I;
CH2 NHCOCH═
CH2, or CH2 NHCOC6 H4 N3 ;R6 is H, Cl, Br, or (CH2)n CH3 where n is an integer from 0 to 15, or (CH2)n CO2 H, where n is an integer from 0 to 11; R7 is H, CO2 H, ##STR9## NH2 NCS CH2 NHCOCH2 Cl, or CH2 NHCOCH2 I. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20)
- dye-biomolecule wherein said dye is fluorescent dye of the formula;
Specification