Chemical probe compounds that become fluorescent upon reduction, and methods for their use

Abstract

Chemical stain compounds containing a fluorophore and a reducible quenching unit are disclosed. The reducible quenching unit quenches the fluorophore while in its oxidized state. Upon reduction, the quenching properties of the quenching unit are diminished or eliminated. The chemical compounds can be used in a variety of applications, including the detection of bacterial cells, monitoring the electron transport chain function of bacterial cells, monitoring the oxidation state of non-biological systems, and assaying the effectiveness of antibacterial or antimicrobial agents.

1 . A chemical compound having the structure FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU is a reducible quenching unit, and L is a linker unit. 
     
     
         2 . The compound of  claim 1 , having the structure FU-RQU. 
     
     
         3 . The compound of  claim 1 , having the structure FU-L-RQU. 
     
     
         4 . The compound of  claim 1 , wherein:
 the reducible quenching unit has an oxidized state and a reduced state;   the reducible quenching unit in its oxidized state partially or fully quenches the fluorescence of the fluorophore unit.   
     
     
         5 . The compound of  claim 1 , wherein the fluorophore unit is fluorescein, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; boron dipyromethene difluoride), a rhodamine, a cyanine, or a coumarin. 
     
     
         6 . The compound of  claim 1 , wherein the reducible quenching unit is quinone, naphthaquinone, anthraquinone, quinonemethine, copper (Cu(II)), sulfate (SO 4   2− ), or nitrate (NO 3   − ). 
     
     
         7 . The compound of  claim 1 , wherein the reducible quenching unit is quinone, naphthaquinone, and anthraquinone. 
     
     
         8 . The compound of  claim 1 , wherein the linker unit is a linear linker, a branched linker, a cyclic linker, an aromatic linker, a polycyclic aromatic linker, or an unsaturated linker. 
     
     
         9 . The compound of  claim 1 , wherein the linker unit comprises carbon atoms, hydrogen, oxygen, sulfur, chlorine, fluorine, iodine, phosphorous, silicon, nitrogen, other atoms, or combinations thereof. 
     
     
         10 . The compound of  claim 1 , having the structure FU-polyethylene glycol-RQU, FU—O—P(═O)—O—RQU, FU—S(═O)—RQU, or FU—S(═O) 2 —RQU. 
     
     
         11 . A chemical compound having the structure:
 compound (5) shown in  FIG. 1B ;   compound (10) shown in  FIG. 1D ;   compound (11) shown in  FIG. 1D ;   compound (18) shown in  FIG. 1G ;   compound (19) shown in  FIG. 1G ;   compound (22) shown in  FIG. 11 ; or   compound (24) shown in  FIG. 1J .   
     
     
         12 . A kit comprising a chemical compound having the structure FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU is a reducible quenching unit, and L is a linker unit; and
 a solvent.   
     
     
         13 . The kit of  claim 12 , wherein the solvent comprises water, DMSO, ethanol, methanol, dimethylacetamide, dimethylformamide (DMF), N-methyl pyrrolidinone (NMP), or mixtures thereof. 
     
     
         14 . A method of assaying the oxidative or reductive environment of a material, the method comprising:
 providing the material;   contacting the material with at least one chemical compound to form a test sample, wherein the chemical compound has the structure FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU is a reducible quenching unit, and L is a linker unit; and   determining the fluorescence of the test sample.   
     
     
         15 . The method of  claim 14 , wherein the material comprises one or more cells, one or more tissues, or one or more organisms. 
     
     
         16 . The method of  claim 14 , wherein the material comprises one or more bacterial cells. 
     
     
         17 . The method of  claim 14 , wherein the determining step comprises irradiating the test sample with light or energy of a suitable wavelength to excite the chemical compound. 
     
     
         18 . A method of assaying the efficacy of an antibacterial compound, the method comprising:
 providing at least one bacterial cell;   contacting the bacterial cell with at least one chemical compound to form a test sample, wherein the chemical compound has the structure FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU is a reducible quenching unit, and L is a linker unit;   determining the fluorescence of the test sample to provide an initial fluorescence;   contacting the test sample with at least one antibacterial compound to form a treated sample;   determining the fluorescence of the treated sample to provide a final fluorescence; and   determining the difference between the initial fluorescence and the final fluorescence to assay the efficacy of the antibacterial compound.   
     
     
         19 . A method of monitoring change in the oxidation state of a non-biological system, the method comprising:
 providing a non-biological system;   contacting the non-biological system with at least one chemical compound to form a test system, wherein the chemical compound has the structure FU-RQU or FU-L-RQU; where FU is a fluorophore unit, RQU is a reducible quenching unit, and L is a linker unit;   determining the fluorescence of the test system at a first time point to provide a first fluorescence;   determining the fluorescence of the test system at a second time point to provide a second fluorescence;   determining the difference between the first fluorescence and the second fluorescence to monitor change in the oxidation state of the non-biological system.