Self-immolative probes for enzyme activity detection
A compound comprising the structure: (SIG)-(SI-MOD) m . is provided in which SIG is a signaling molecule, SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI, MOD is a moiety bound to SI that is subject to modification by an activator, and m is an integer from 1 to about 10. When MOD is modified by an activator, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal. Also provided are methods of determining whether a sample comprises an activator such as a nitroreductase, and methods of determining whether a mammalian cell is hypoxic using a compound of the formula (SIG)-(SI-MOD) m .
1. A compound selected from:
wherein Z is a reducible nitrogen-containing group selected from the group consisting of a nitro group (NO 2 ), an azo group (—N═N), a hydrazo group (NH—NH), a nitroso group (NO), and a hydroxylamino group (NHOH), or an amino group with an electron-deficient moiety, or Z is amino group with an electron-deficient moiety selected from the group consisting of carbonyl (C═O), phosphoryl (PO 3 2− ) and sulfonyl (SO 3 − ); and
wherein L is nothing when Z is a reducible nitrogen-containing group, or L is an unsubstituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group, a substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups are substituted by an O atom, N atom, S atom, or NH group, an unsubstituted or substituted aromatic group, or a linear or branched sequence of amino acids.
2. A compound of claim 1 wherein L is nothing and Z is NO 2 .
3. A method of determining whether a cell comprises a nitroreductase, the method comprising
(a) incubating the cell with a compound of claim 2 for a time and under conditions sufficient for the compound to enter the cell and be exposed to a nitroreductase if present in the cell; and
(b) determining if a signal greater than the signal generated by the compound when not exposed to a nitroreductase is generated,
wherein a greater signal indicates that the cell comprises the nitroreductase.
4. A method of determining whether a mammalian cell is hypoxic, the method comprising
(a) incubating the cell with a compound of claim 2 for a time and under conditions sufficient for the compound to enter the cell and be exposed to a nitroreductase if present in the cell, wherein the nitroreductase is indicative or hypoxia in the cell; and
(b) determining if a signal greater than the signal generated by the compound when not exposed to a nitroreductase is generated,
wherein a greater signal indicates that the cell is hypoxic.
5. A method of detecting a microorganism that comprises a nitroreductase, the method comprising
(a) incubating the microorganism with a compound of claim 2 for a time and under conditions sufficient for the compound to enter the cell and be exposed to a nitroreductase if present in the microorganism; and
(b) determining if a signal greater than the signal generated by the compound when not exposed to a nitroreductase is generated,
wherein a greater signal indicates that the microorganism comprises a nitroreductase.
6. A method of identifying nitroreductase in a sample, the method comprising
(a) incubating the sample a the compound of claim 2 , and
(b) determining if a signal greater than the signal generated by the compound when not exposed to a nitroreductase is generated,
wherein a greater signal indicates that the sample comprises a nitroreductase.
7. A method of detecting hydrosulfite in a sample, the method comprising
(a) combining the sample with a compound of claim 2 , then
(b) determining if a signal greater than the signal generated by the compound when not exposed to a nitroreductase is generated,
wherein a greater signal indicates that the sample comprises a hydrosulfite.