US 5,442,045 A · Biological conjugates of fluorescent rhodol dyes

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.

Claims20

Dependent claims

A compound comprising a fluorescent dye having the structure ##STR2## wherein R.sup.1 is H or CH.sub.3, and R.sup.2 is H, CF.sub.3 CO, CH.sub.3, or C.sub.2 H.sub.5 ;

or R.sup.1 in combination with R.sup.2 is (CH.sub.2).sub.3 ;
R.sup.3 is H, CH.sub.3, or C.sub.2 H.sub.5, and R.sup.4 is H;
or R.sup.3 in combination with R.sup.4 is (CH.sub.2).sub.3 ;
R.sup.5 is H, CH.sub.2 NH.sub.2, CH.sub.2 NHCOCH.sub.2 Cl, CH.sub.2 NHCOCH.sub.2 I;
CH.sub.2 NHCOCH.dbd.CH.sub.2, or CH.sub.2 NHCOC.sub.6 H.sub.4 N.sub.3 ;
R.sup.6 is H, Cl, Br, CH.sub.3, or C.sub.2 H.sub.5, or (CH.sub.2).sub.n CH.sub.3 where n is an integer from 0 to 15, or (CH.sub.2).sub.n CO.sub.2 H, where n is an integer from 0 to 11;
R.sup.7 is H, CO.sub.2 H, ##STR3## NH.sub.2, NCS, CH.sub.2 NHCOCH.sub.2 Cl, or CH.sub.2 NHCOCH.sub.2 I;
such that R.sup.7 is not H if R.sup.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.

6 more elements

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.sup.1 through R.sup.7 ;
and at the positions where the biomolecule is not attached:
R.sup.1 is H or CH.sub.3 and R.sup.2 is H CF.sub.3 CO CH.sub.3 or C.sub.2 H.sub.5 ;
or R.sup.1 in combination with R.sup.2 is (CH.sub.2).sub.3 ;
R.sup.3 is H, CH.sub.3, or C.sub.2 H.sub.5, and R.sup.4 is H;
or R.sup.3 in combination with R.sup.4 is (CH.sub.2).sub.3 ;
R.sup.5 is H CH.sub.2 NH.sub.2 CH.sub.2 NHCOCH.sub.2 Cl, CH.sub.2 NHCOCH.sub.2 I;
CH.sub.2 NHCOCH.dbd.CH.sub.2, or CH.sub.2 NHCOC.sub.6 H.sub.4 N.sub.3 ;
R.sup.6 is H, Cl, Br, or (CH.sub.2).sub.n CH.sub.3 where n is an integer from 0 to 15, or (CH.sub.2).sub.n CO.sub.2 H, where n is an integer from 0 to 11;
R.sup.7 is H, CO.sub.2 H, ##STR9## NH.sub.2 NCS CH.sub.2 NHCOCH.sub.2 Cl, or CH.sub.2 NHCOCH.sub.2 I.

8 more elements

Dependencies are inferred from the claim text.

Biological conjugates of fluorescent rhodol dyes

Life of this patent

Biological conjugates of fluorescent rhodol dyes

Life of this patent

Abstract

Claims20