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 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of  claim 1 , wherein halo is bromo. 
     
     
         3 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of  claim 1 , wherein halo is chloro. 
     
     
         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 . The compound of  claim 6 , wherein X is halo. 
     
     
         8 . The compound of  claim 6 , wherein X is bromo. 
     
     
         9 . The compound of  claim 6 , wherein X is chloro. 
     
     
         10 . The compound of  claim 6 , wherein X is a tosyl, brosyl or mesyl group. 
     
     
         11 . The compound of  claim 6 , wherein X is a substituted thiol group. 
     
     
         12 . The compound of  claim 6 , wherein X is glutathione or a derivative thereof. 
     
     
         13 . A 2,3,6,7-tetrahydro-9-bromomethyl-1H,5H-quinolizino(9,1-gh)coumarin. 
     
     
         14 . A method for detecting or quantifying a thiol compound in a sample, the method comprising:
 (a) contacting the sample with 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin, 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 thiol compound in the sample.   
     
     
         15 . The method of  claim 14 , wherein the sample comprises cells. 
     
     
         16 . The method of  claim 15 , wherein the contacted sample is incubated for a sufficient amount of time for the 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin to enter the cell. 
     
     
         17 . The method of  claim 14 , 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. 
     
     
         18 . The method of  claim 14 , wherein the thiol compound reacts with 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin. 
     
     
         19 . The method of  claim 14 , wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip. 
     
     
         20 . The method of  claim 15 , wherein the 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin is substituted with a cationic group and the glutathione that is detected or quantified is present in the mitochondria of the cell. 
     
     
         21 . The method of  claim 20 , wherein the cationic group comprises a triphenylphosphonium ion. 
     
     
         22 . The method of  claim 14 , wherein the thiol compound comprises a cysteine residue. 
     
     
         23 . The method of  claim 14 , wherein the thiol compound is glutathione. 
     
     
         24 . The method of  claim 14 , further comprising a step of fixing the sample. 
     
     
         25 . The method of  claim 14 , further comprising a step of permeablizing the sample. 
     
     
         26 . The method of  claim 14 , further comprising a step of separating unbound 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin. 
     
     
         27 . The method of  claim 14 , further comprising a step of adding an antibody specific for the thiol compound after the incubating step. 
     
     
         28 . A method of synthesizing 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin comprising:
 contacting 8-hydroxyjulolidine with ethyl 4-haloacetoacetate to form a reaction mixture;   incubating the reaction mixture for a sufficient amount of time for 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin to form.   
     
     
         29 . The method of  claim 28 , wherein the incubating step comprises heating the reaction mixture to at least 100° C. 
     
     
         30 . The method of  claim 28 , wherein the reaction mixture further comprises a Lewis acid. 
     
     
         31 . The method of  claim 30 , wherein the Lewis acid is aluminum chloride (AlCl 3 ). 
     
     
         32 . A pro-fluor of a compound of Formula (I) or a salt or tautomer thereof: 
       
         
           
           
               
               
           
         
         wherein, 
         X is a substituted thiol group.