Self-immolative probes for enzyme activity detection
Provided is a compound having the structure: (SIG)-(SI-MOD) m where 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, such as a cell, comprises an activator, such as a nitroreducase, using the compound. Further provided are methods of determining whether a mammalian cell is hypoxic using the compound where nitroreductase is the activator. A method of detecting a microorganism that comprises a nitroreductase using the compound where nitroreductase is the activator is also provided.
1. A compound for detecting lipase or esterase activity, said compound consisting of the structure:
wherein
q is an integer from 1 to 4,
X is an oxygen or sulfur,
R 7 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 in any of the foregoing groups can be substituted with an O atom, N atom, S atom, NH group, CO group, OCO group or CONR 3 group, an unsubstituted aromatic group or a substituted aromatic group,
m is an integer from 1 to 10,
SIG is a signaling moiety,
SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI, and
when the compound is modified by lipase or esterase activity, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal.
2. The compound of claim 1 , wherein SIG is a fluorescent moiety or a luminescent moiety.
3. The compound of claim 1 , wherein m is an integer from 2 to 10.
4. The compound of claim 3 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.
5. The compound of claim 1 , wherein m=1.
6. The compound of claim 5 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.
7. The compound of claim 1 , consisting of the structure:
8. The compound of claim 7 , wherein SIG is a fluorescent moiety or a luminescent moiety.
9. The compound of claim 7 , wherein m is an integer from 2 to 10.
10. The compound of claim 9 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.
11. The compound of claim 7 , wherein m=1.
12. The compound of claim 11 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.
13. A method for detecting lipase or esterase activity in a sample, the method comprising
(a) incubating the sample with the compound of claim 1 for a time and under conditions sufficient for the compound to be modified by a lipase or an esterase if present in the sample; and
(b) measuring the signal generated by SIG, wherein the amount of signal generated is proportional to the amount of lipase or esterase activity in the sample.
14. The method of claim 13 , wherein the sample is a fluid of an organism or a colony of organisms, or an extract thereof.
15. A method for detecting or esterase activity in a sample, the method comprising
(a) incubating the sample with the compound of claim 7 for a time and under conditions sufficient for the compound to be modified by a lipase or esterase if present in the sample; and
(b) measuring the signal generated by SIG, wherein the amount of signal generated is proportional to the amount of lipase or esterase activity in the sample.
16. The method of claim 15 , wherein the sample is a fluid of an organism or a colony of organisms, or an extract thereof.