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 - 6 . (canceled)
7 . A compound represented by the structure
wherein
L-Z is MOD and is ortho or para to the benzyl group, wherein
Z is a reducible nitrogen-containing group, or an amino group with an electron-deficient moiety, and
L is nothing when Z is a reducible nitrogen-containing group, otherwise 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 with 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;
each R 1 is independently a hydrogen, a halogen, a Z, a cyano group (CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 3 ), a sulfate group (OSO 3 R 3 ), a carboxyl group (CO 2 H), a carbonyl group (COR 3 ), an ester group (CO 2 R 3 or OCOR 3 ), an amido group (CONR 3 2 or NR 3 COR 3 ), a carbamate group (NR 3 CO 2 R 3 ), a phosphate group (OPO 3 R 3 3 ), a phosphonate group (PO 3 R 3 2 ), an amino group (NR 3 2 ), are alkoxy group (OR 3 ), a thiol group (SR 3 ), a sulfoxy group (SOR 3 ), a sulfone group (SO 2 R 3 ), a sulfonamide group (SO 2 NR 3 2 ), a phosphino group (PR 3 2 ), or a silane group (SiR 3 3 );
each R 3 is independently a hydrogen, 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 with an O atom, N atom, S atom, or NH group, or an unsubstituted or substituted aromatic group; and
n is 0, 1 2, 3 or 4.
8 . The compound of claim 7 , 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).
9 . The compound of claim 7 , wherein Z is a nitro group (NO 2 ).
10 . The compound of claim 7 , wherein Z is an amino group with an electron-deficient moiety selected from the group consisting of carbonyl (C═O), phosphoryl (PO 3 2− ) and sulfonyl (SO 3 − ).
11 . The compound of claim 7 , wherein Z is an amino group with an electron-deficient moiety and L is an amino acid sequence that is a substrate for an activator that is an enzyme, wherein the enzyme is capable of cleaving L from Z, leading to self-cleavage of SI and releasing SIG-NR 2 .
12 - 15 . (canceled)
16 . The compound of claim 7 , wherein m>1.
17 . The compound of claim 7 , wherein m=1.
18 . The compound of claim 17 , wherein SIG further comprises at least one blocker moiety that blocks sites of potential SI-MOD attachment during synthesis of the compound, wherein the moiety does not substantially interfere with the SIG signal.
19 . The compound of claim 18 , wherein the blocker moiety is R 4 2 N—CO—NR 4 and the compound is
wherein each R 4 is independently a hydrogen, 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.
20 . The compound of claim 19 , wherein two or more R 4 groups are fused to form a ring, the ring comprising one or more heteroatoms, wherein the heteroatoms are the same heteroatoms or different heteroatoms.
21 . The compound of claim 20 , wherein the at least one blocker moiety is
22 . The compound of claim 7 , wherein SIG is a chromophore, a fluorophore, a luminescent moiety, an enzyme, a catalytic antibody, a ribozyme or a pro-enzyme.
23 . The compound of claim 7 , wherein SIG is a fluorophore.
24 . The compound of claim 23 , wherein the fluorophore comprises a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, coumarin or an iminocoumarin.
25 . The compound of claim 23 , wherein the fluorophore comprises a xanthene.
26 . The compound of claim 25 , wherein the xanthene is a rhodamine or rhodamine derivative.
27 . The compound of claim 25 , wherein the xanthene is a rosamine or rosamine derivative.
28 . The compound of claim 25 , wherein the xanthene is a rhodol or rhodol derivative.
29 . The compound of claim 25 , wherein the xanthene comprises the structure:
wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), art isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3, where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), a sulfoxy group (SOR 6 ), a sulfone group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), an phosphino group (PR 6 2 ), a silane group (SiR 6 3 ), an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group; and
wherein each R 6 is independently hydrogen, an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group.
30 . The compound of claim 25 , wherein the xanthene comprises the structures:
wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R 6 or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), a sulfoxy group (SOR 6 ), a sulfone group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), a phosphino group (PR 6 2 ), a silane group (SiR 6 3 ), an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group; and
wherein each R 6 is independently hydrogen, an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group.
31 . The compound of claim 23 , wherein the fluorophore comprises a coumarin.
32 . The compound of claim 31 , wherein the coumarin comprises the structure:
wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), tare isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R 6 or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), to sulfoxy group (SOR 6 ), a still:one group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), a phosphine group (PR 6 2 ), a silane group (SiR 6 3 ), an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group; and
wherein each R 6 is independently hydrogen, an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group.
33 . The compound of claim 31 , wherein the coumarin is an iminocoumarin.
34 . The compound of claim 33 , wherein the iminocoumarin comprises the structure:
wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), a carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R 6 or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), a sulfoxy group (SOR 6 ), a sulfone group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), a phosphine group (PR 6 2 ), a silane group (SiR 6 3 ), an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group; and
wherein each R 6 is independently hydrogen, an optionally substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an optionally substituted aromatic group.
35 . The compound of claim 7 , wherein SIG is a luminescent moiety.
36 . (canceled)
37 . The compound of claim 7 , wherein the activator is an enzyme.
38 . The compound of claim 37 , wherein the enzyme is a nitroreductase, kinase, an aminopeptidase, an esterase, a lipase, a protease, a peptidase, to phosphatase, a sulfatase, a sulfotransferase, a carboxylase, a decarboxylase, a glycosylase, an amidase, deamidase, a aminase, a deaminase, a acetyltransferase, a methylase, a deacetylase, a demethylase, or an acetylase.
39 - 40 . (canceled)
41 . The compound of claim 37 , wherein the enzyme is a nitroreductase and the compound has the structure:
42 . The compound of claim 41 , wherein the compound is
43 - 44 . (canceled)
45 . The compound of claim 37 , wherein the enzyme is involved in addition or removal of a posttranslational modification of a protein.
46 . The compound of claim 45 , wherein the enzyme is involved in acylation, alkylation, amidation, amino acid addition, diphthamide formation, gamma-carboxylation, glycosylation, glypiation, addition of a heme moiety, hydroxylation, iodination, attachment of nucleotide moiety, nitrosylation, S-gluthathionylation, oxidation, phosphopantetheinylation, phosphorylation, pyroglutamate formation, sulfation, selenoylation, SUMOylation, or ubiquitination.
47 - 55 . (canceled)
56 . A method of determining whether a sample comprises an activator, the method comprising
(a) incubating the sample with a compound according to claim 7 for a time and under conditions sufficient for MOD to be modified by the activator; and
(b) determining whether SIG generates a greater signal than the signal generated by the compound without the activator,
wherein a greater signal indicates that the sample comprises the activator.
57 . The method of claim 56 , wherein the sample is a fluid of an organism or a colony of organisms, or an extract thereof.
58 . The method of claim 57 , wherein the organism or colony of organisms comprises microorganisms.
59 . The method of claim 57 , wherein the organism or colony of organisms comprises a prokaryote or an archaea.
60 . The method of claim 57 , wherein the organism or colony of organisms comprises a eukaryote.
61 . The method of claim 60 , wherein the eukaryote is a multicellular organism.
62 . The method of claim 61 , wherein the sample is an extract of a cell, tissue or organ of the multicellular organism.
63 . The method of claim 62 , wherein the multicellular organism is a mammal.
64 . The method of claim 56 , wherein the sample comprises a living cell.
65 . The method of claim 64 , wherein the living cell is a cell of a prokaryote or an archaea.
66 . The method of claim 64 , wherein the living cell is a eukaryotic
67 . The method of claim 66 , wherein the eukaryotic cell is a mammalian cell.
68 . The method of claim 66 , wherein the eukaryotic cell is a human cell.
69 . The method of claim 56 , wherein the sample comprises cells.
70 - 80 . (canceled)
81 . A method of determining whether a cell comprises a nitroreductase, the method comprising
(a) incubating the cell with a compound according to claim 41 , 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 whether SIC generates a greater signal than the signal generated by the compound when not exposed to a nitroreductase,
wherein a greater signal indicates that the cell comprises the nitroreductase.
82 - 83 . (canceled)
85 . The method of claim 80 , wherein the cell is a mammalian cell.
86 . The method of claim 85 , wherein the mammalian cell is hypoxic.
87 . The method of claim 80 , wherein the cell is a microorganism.
88 . The method of claim 87 , wherein the microorganism is a bacterium.
89 . The method of claim 80 , wherein SIG is a fluorophore.
90 . (canceled)
91 . The method of claim 80 , wherein the nitroreductase is DT-diaphorase [NQO1; E.C.1.6.99.2]; cytochrome P450-reductase [CYPOR; E.C.1.6.2.4]; inducible nitric oxide synthase [NOS2A; E.C.1.14.13.39]; cytochrome B5 reductase [DIAL; E.C.1.6.2.2]; xanthine oxidase [XO; E.C.1.17.3.2]; xanthine dehydrogenase [XDH; E.C.1.17.1.4]; adrenodoxin oxidoreductase [FDXR; E.C.1.18.1.2]; methionine synthase reductase [MTRR; E.C.1.16.1.8]; aldose reductase [ALDR1; E.C.1.1.1.21]; aldehyde reductase [AKR1B10; E.C.1.1.1.2] or thioredoxin reductase [TXNRD; E.C.1.8.1.9].
92 . A method of determining whether a mammalian cell is hypoxic, the method comprising
(a) incubating the cell with a compound according to claim 41 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 whether SIG generates a greater signal than the signal generated by the compound when not exposed to a nitroreductase,
wherein a greater signal indicates that the cell is hypoxic.
93 - 108 . (canceled)
109 . A method of detecting a microorganism that comprises a nitroreductase, the method comprising
(a) incubating the microorganism with a compound according to claim 41 , 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 whether SIG generates a greater signal than the signal generated by the compound when not exposed to a nitroreductase,
wherein a greater signal indicates that the microorganism comprises a nitroreductase.
110 . The method of claim 109 , wherein the microorganism is in a tissue or fluid sample of a vertebrate infected with the microorganism.
111 . The method of claim 109 , wherein the microorganism is in an environmental sample.
112 . A method of identifying nitroreductase in a sample, the method comprising
(a) incubating the sample with a compound according to claim 41 , then
(b) determining whether SIG generates a greater signal than the signal generated by the compound when not exposed to a nitroreductase,
wherein a greater signal indicates that the sample comprises a nitroreductase,
113 - 115 . (canceled)
116 . The method of claim 112 , wherein the nitroreductase is quantified in the sample by comparing the fluorescence of the compound after the incubation with fluorescence of a known quantity of nitroreductase incubated with the compound under the same conditions.
117 . A method of detecting hydrosulfite in a sample, the method comprising
(a) combining the sample with a compound according to claim 41 , then
(b) determining whether SIC generates a greater signal than the signal generated by the compound when not exposed to a hydrosulfite,
wherein a greater signal indicates that the sample comprises a hydrosulfite.
118 . The method of claim 117 , wherein the hydrosulfite is sodium hydrosulfite.
119 . The method of claim 117 , wherein the sample is a food.