Assay for glutathiane transferase using polyhaloaryl-substituted reporter molecules

A method of detecting glutathione transferase activity in a sample, comprising:

• a) preparing a solution comprising
• i) a sample that possesses or is thought to possess glutathione transferase activity;
• ii) glutathione;
• and
• iii) a glutathione transferase substrate of the formula ##STR15## wherein R.sup.4 is F;
• at least two of R.sup.3, R.sup.5 and R.sup.6 are F;
• and the remaining of R.sup.3, R.sup.5 and R.sup.6 are independently H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy;
• R.sup.2 is H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy;
• or R.sup.2 is SO.sub.3 R.sup.7, (C.dbd.O)OR.sup.7, or (C.dbd.O)NR.sup.8 R.sup.9, where R.sup.7 is H, a suitable counterion, or a C.sub.1 -C.sub.6 alkyl, and R.sup.8 and R.sup.9 are independently H or a C.sub.1 -C.sub.6 alkyl;
• A is --(C.dbd.O)--, --(C.dbd.O)--O--, --(C.dbd.O)--NR.sup.10, --NR.sup.10 --(C.dbd.O)--, --(C.dbd.NR.sup.10)--, --CH.dbd.N--, --SO.sub.2 --, --SO.sub.2 --O--, --SO.sub.2 --NR.sup.10 --, or --NR.sup.10 --SO.sub.2 --, where R.sup.10 is H or C.sub.1 -C6 alkyl;
• or R.sup.2 in combination with A forms a 5-membered cyclic imide;
• or R.sup.2 in combination with A forms a 6-membered cyclic succinic hydrazide;
• SPACER is a single covalent bond, or SPACER 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;
• REPORTER is a covalently bound moiety that is H, or an organic residue having a definite molecular weight less than 2000 daltons;
• provided that all glutathione transferase substrates in the solution have the same chemical formula and the same isomeric configuration;
• b) incubating the solution for a time sufficient to form a glutathione adduct of said substrate if the sample possesses glutathione transferase activity;
• c) detecting the presence of said glutathione adduct.

A compound having the formula ##STR16## or the formula ##STR17## wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy;

• provided that at least two of R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are F;
• R.sup.2 is H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy;
• or R.sup.2 is SO.sub.3 R.sup.7, (C.dbd.O)OR.sup.7, or (C.dbd.O)NR.sup.8 R.sup.9, where R.sup.7 is H, a suitable counterion, or a C.sub.1 -C.sub.6 alkyl, and R.sup.8 and R.sup.9 are independently H or a C.sub.1 -C.sub.6 alkyl;
• A is --(C.dbd.O)--, --(C.dbd.O)--O--, --(C.dbd.O)--NR.sup.10, --NR.sup.10 --(C.dbd.O)--, --(C.dbd.NR.sup.10)--, --CH.dbd.N--, --SO.sub.2 --O--, --SO.sub.2 --NR.sup.10 --, or --NR.sup.10 --SO.sub.2 --, where R.sup.10 is H or C.sub.1 -C.sub.6 alkyl;
• or R.sup.2 in combination with A forms a 5-membered cyclic imide;
• or R.sup.2 in combination with A forms a 6-membered cyclic succinic hydrazide;
• SPACER is a single covalent bond, or SPACER 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;
• REPORTER is a covalently bound moiety that is H, or an organic residue having a definite molecular weight less than 2000 daltons.

A compound having the formula ##STR18## wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy;

• R.sup.2 is H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub. -C.sub.6 alkoxy, SO.sub.3 R.sup.7, (C.dbd.O)OR.sup.7, or (C.dbd.O)NR.sup.8 R.sup.9, where R.sup.7 is H, a suitable counterion, or a C.sub.1 -C.sub.6 alkyl, and R.sup.8 and R.sup.9 are independently H or a C.sub.1 -C.sub.6 alkyl;
• A is a single covalent bond, or A is --(C.dbd.O)--, --(C.dbd.O)--O--, --(C.dbd.O)--NR.sup.10, --NR.sup.10 --(C.dbd.O)--, --(C.dbd.NR.sup.10)--, --CH.dbd.N--, --SO.sub.2 --, --SO.sub.2 --O--, --SO.sub.2 --NR.sup.10 --, or --NR.sup.10 --SO.sub.2 --, where R.sup.10 is H or C.sub.1 -C.sub.6 alkyl;
• or R.sup.2 in combination with A forms a 5-membered cyclic imide;
• or R.sup.2 in combination with A forms a 6-membered cyclic succinic hydrazide;
• provided at least three of R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently F, Cl or Br;
• SPACER is a single covalent bond, or SPACER 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;
• REPORTER is a chromophore, a fluorophore, or a chemiluminescent precursor, or REPORTER has the formula DYE-(BLOCK).sub.a, wherein a=1 or 2;
• DYE is a chromophore, a fluorophore or a chemiluminescent precursor, and each BLOCK, which may be the same or different, is a monovalent moiety selected to be removable by action of an enzyme;
• such that when DYE is no longer bound to BLOCK by a DYE-BLOCK bond, DYE has spectral properties different from those of said compound;
• provided that A and SPACER are not both single covalent bonds.

A method of analyzing enzyme activity in a sample, comprising

• a) preparing a solution comprising a compound of the formula ##STR25## wherein R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy;
• R.sup.2 is H, F, Cl, Br, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy;
• or R.sup.2 is SO.sub.3 R.sup.7, (C.dbd.O)OR.sup.7, or (C.dbd.O)NR.sup.8 R.sup.9, where R.sup.7 is H, a suitable counterion, or a C.sub.1 -C.sub.6 alkyl, and R.sup.8 and R.sup.9 are independently H or a C.sub.1 -C.sub.6 alkyl;
• A is a single covalent bond, or A is --(C.dbd.O)--, --(C.dbd.O)--O--, --(C.dbd.O)--NR.sup.10, --NR.sup.10 --(C.dbd.O)--, --(C.dbd.NR.sup.10)--, --CH.dbd.N--, --SO.sub.2 --O--, --SO.sub.2 --NR.sup.10 --, or --NR.sup.10 --SO.sub.2 --, where R.sup.10 is H or C.sub.1 -C.sub.6 alkyl;
• or R.sup.2 in combination with A forms a 5-membered cyclic imide;
• or R.sup.2 in combination with A forms a 6-membered cyclic succinic hydrazide;
• provided at least three of R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently F, Cl or Br;
• SPACER is a single covalent bond, or SPACER 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;
• REPORTER is a chromophore, a fluorophore, or a chemiluminescent precursor, or REPORTER has the formula DYE-(BLOCK).sub.a, wherein a=1 or 2;
• DYE is a chromophore, a fluorophore or a chemiluminescent precursor, and each BLOCK, which may be the same or different, is a monovalent moiety selected to be removable by action of an enzyme;
• such that when DYE is no longer bound to BLOCK by a DYE-BLOCK bond, DYE has spectral properties different from those of said compound;
• provided that A and SPACER are not both single covalent bonds.
• b) adding the solution to a sample that contains or is thought to contain an enzyme capable of cleaving at least one DYE-BLOCK bond and producing a detectable product;
• c) incubating the sample for a time sufficient for the enzyme to cleave at least one DYE-BLOCK bond and produce said detectable product,
• d) observing the sample with means for detecting the detectable product;
• e) correlating the presence of the detectable product with the enzyme activity.