Use of Novel Coumarins as Glutathione and Thiol Labels

Abstract

Fluorescent quinolizinocoumarin compounds substituted with electrophilic reactive groups that bind thiol compounds are described. The compounds are useful in detecting oxidative stress and processes associated therewith in live cells.

What is claimed is: 
     
         1 . A 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin. 
     
     
         2 - 3 . (canceled) 
     
     
         4 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of  claim 1 , further substituted with a cationic group. 
     
     
         5 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of  claim 1 , wherein the cationic group comprises a triphenylphosphonium ion. 
     
     
         6 . A compound of Formula (I) or a salt or tautomer thereof: 
       
         
           
           
               
               
           
         
         wherein, 
         X is an electrophilic reactive group that does not comprise a carbonyl group; or 
         X is a substituted thiol group. 
       
     
     
         7 - 10 . (canceled) 
     
     
         11 . The compound of  claim 6 , wherein X is a substituted thiol group. 
     
     
         12 - 32 . (canceled) 
     
     
         33 . A method for detecting or quantifying intracellular glutathione levels in a sample, the method comprising:
 (a) contacting the sample with a compound according to  claim 1 , to form a contacted sample;   (b) incubating the contacted sample for an appropriate amount of time to form an incubated sample;   (c) illuminating the incubated sample with an appropriate wavelength to form an illuminated sample; and   (d) detecting fluorescent emissions from the illuminated sample;   wherein the fluorescent emissions are used to detect or quantify the glutathione level in the sample.   
     
     
         34 . The method of  claim 33 , wherein the sample comprises cells. 
     
     
         35 . The method of  claim 33 , wherein the contacted sample is incubated for a sufficient amount of time for the compound to enter the cell. 
     
     
         36 . The method of  claim 33 , wherein the sample comprises live cells, intracellular fluids, extracellular fluids, sera, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions biological fluids or chemical reactors, blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles; vascular tissue, blood fluids, saliva, urine, water, soil, waste water, sea water; pharmaceuticals, foodstuffs or beverages. 
     
     
         37 . A method for binding and detecting target thiol compound in a live cell, the method comprising:
 (a) contacting a sample of live cells with a compound of  claim 1 ;   (b) incubating the sample and the compound for a sufficient amount of time to allow the compound to enter the live cells, thereby forming an incubated sample;   (c) illuminating the incubated sample with an appropriate wavelength to form an illuminated sample; and   (d) detecting fluorescent emissions from the illuminated sample;   wherein the fluorescent emissions are used to detect or quantify the target thiol compound in the live cell.   
     
     
         38 . The method of  claim 37 , wherein the compound is substituted with a cationic group and the glutathione that is detected or quantified is present in the mitochondria of the cell. 
     
     
         39 . The method of  claim 38 , wherein the cationic group comprises a triphenylphosphonium ion. 
     
     
         40 . A method for detecting or quantifying mitochondrial proteins under oxidative stress in a sample, the method comprising:
 (a) contacting the sample with the compound of  claim 1 , to form a contacted sample;   (b) incubating the contacted sample for an appropriate amount of time to form an incubated sample;   (c) illuminating the incubated sample with an appropriate wavelength to form an illuminated sample; and   (d) detecting fluorescent emissions from the illuminated sample;   wherein the fluorescent emissions are used to detect or quantify the mitochondrial proteins under oxidative stress in the sample.   
     
     
         41 . A kit for binding an intracellular metal ion in a sample, the kit comprising:
 the compound of  claim 1 ; and   one or more components selected from the group consisting of a calibration standard of an intracellular thiol compound, a fluorescent standard, an aqueous buffer solution and an organic solvent.   
     
     
         42 . A kit for binding a target thiol compound in a sample, the kit comprising:
 the compound of  claim 1 ; and   instructions for use thereof.