Sulfonated derivatives of 7-aminocoumarin

A method of detecting a complementary member of a specific binding pair in a sample, comprising:

• a) adding to said sample a dye-conjugate of a first member of a specific binding pair for which there is a complementary member, where said first member of a specific binding pair is attached to one or more dye molecules, which may be the same or different, having the formula ##STR47## wherein R.sup.5 is H;
• R.sup.6 is H, methyl, halogen, or SO.sub.3 X;
• R.sup.7 is NR.sup.1 R.sup.2 ;
• wherein
• R.sup.1 and R.sup.2 are independently H, C.sub.1 -C.sub.18 alkyl;
• or R.sup.1 in combination with R.sup.2 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;
• or R.sup.1 is a 2-nitrobenzyloxycarbonyl of the formula ##STR48## wherein R.sup.9 and R.sup.10 are H, C.sub.1 -C.sub.6 alkoxy, or R.sup.9 and R.sup.10 taken in combination are --O--CH.sub.2 --O--;
• and
• R.sup.11 is H, CH.sub.3, a carboxylic acid or a biologically compatible salt of a carboxylic acid;
• R.sup.8 is H, halogen or SO.sub.3 X;
• X is H, or a biologically compatible cation;
• R.sup.3 is H, SO.sub.3 X, halogen, CN, NO.sub.2, or --L--S.sub.C ;
• R.sup.4 is H, CN, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, CH.sub.2 SO.sub.3 X;
• or --L--S.sub.C ;
• wherein
• each L is a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds;
• and
• S.sub.C is a conjugated substance that is said first member of a specific binding pair;
• provided that at least one of R.sup.3, R.sup.6 or R.sup.8 is SO.sub.3 X;
• and
• provided that one of R.sup.3 and R.sup.4 is --L--S.sub.C ;
• b) allowing sufficient time for the dye-conjugate to form a complex with the complementary member, said complex exhibiting a detectable optical response;
• and
• c) detecting said complex to locate the complementary member.

A method of staining a sample, comprising:

• a) adding to said sample a compound of the formula ##STR49## wherein R.sup.5 is H;
• R.sup.6 is H, methyl, halogen, or SO.sub.3 X;
• R.sup.7 is NR.sup.1 R.sup.2 ;
• wherein
• R.sup.1 and R.sup.2 are independently H, C.sub.1 -C.sub.18 alkyl;
• or R.sup.1 in combination with R.sup.2 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;
• or R.sup.2 is H and R.sup.1 is a radical formed by a removal of the hydroxyl group from the carboxylic acid of an amino acid or from the carboxylic acid of the carboxy-terminal amino acid of a linear peptide consisting of 2-6 amino acids;
• or R.sup.1 is a 2-nitrobenzyloxycarbonyl of the formula ##STR50## wherein R.sup.9 and R.sup.10 are H, C.sub.1 -C.sub.6 alkoxy, or R.sup.9 and R.sup.10 taken in combination are --O--CH.sub.2 --O--;
• and
• R.sup.11 is H, CH.sub.3, a carboxylic acid or a biologically compatible salt of a carboxylic acid;
• R.sup.8 is H, halogen or SO.sub.3 X;
• X is H, or a biologically compatible cation;
• R.sup.3 is H, SO.sub.3 X, halogen, CN, NO.sub.2, or --L--S.sub.C ;
• R.sup.4 is H, CN, C.sub.1 -C.sub.18 alkyl, C.sub.1 -C.sub.18 perfluoroalkyl, CH.sub.2 SO.sub.3 X;
• or --L--S.sub.C ;
• wherein
• each L is a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds;
• and
• S.sub.C is a conjugated substance;
• provided that at least one of R.sup.3, R.sup.6 or R.sup.8 is SO.sub.3 X;
• in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength.