Self-Immolative Probes for Enzyme Activity Detection

Abstract

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.