Luciferins

Abstract

Novel luciferins, methods of making luciferins, and uses of the same are disclosed.

1 . A method of imaging a sample, the method comprising:
 obtaining a sample comprising a luciferase;   contacting the sample with a compound of Structure (III) or (IV), or a salt or acid ester thereof:   
       
         
           
           
               
               
           
         
       
       wherein:
 R 1  and R 2  are each independently H, provided that R 1  and R 2  are not both H, C 1-12  alkyl optionally substituted by an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, I, or a near infrared fluorophore, optionally with a spacer; wherein 
 R 3  is H, OH, C 1-12  alkyl optionally substituted by an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, I, or a near infrared fluorophore, optionally with a spacer; wherein 
 R 4  and R 5  are each independently H, OH, or C 1-6  alkyl optionally substituted by an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, or I; wherein 
 R 6  and R 7  are each independently H, or C 1-8  alkyl optionally substituted by an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, or I; wherein 
 R 1 , R 2 , R 3 , or R 5  together with one or more of its immediate neighbors define one or more 5, 6, or 7-membered rings; wherein 
 the rings optionally include one or more groups selected from: an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl, an aryl group, F, Cl, Br, and I; and wherein 
 the spacer is C 1-24  alkyl optionally substituted by one or more groups selected from: an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl, an aryl group, F, Cl, Br, and I; and 
 detecting bioluminescent emission from the sample. 
 
     
     
         2 . The method of  claim 1 , wherein the spacer is a polymer fragment of a water-soluble polymer fragment. 
     
     
         3 . The method of  claim 1 , wherein R 3 , R 4 , R 5 , R 6  and R 7  are each hydrogen, or an alkyl group having fewer than 4 carbon atoms. 
     
     
         4 . A method of imaging a sample, the method comprising:
 obtaining a sample comprising a luciferase;   contacting the sample with a compound of Structure (V), or salts or acid esters thereof:   
       
         
           
           
               
               
           
         
       
       wherein
 R 4  is H, OH, or C 1-6  alkyl optionally substituted by an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, or I; wherein 
 R 6  and R 7  are each independently H or C 1-8  alkyl optionally substituted by an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, or I; wherein 
 R 1  and R 5  and R 2  and R 3  together with one or more of its immediate neighbors define one or more 5, 6, or 7-membered rings; and wherein 
 the rings optionally include one or more groups selected from: an amino group, an amide group, an imine group, a hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, an ether group, a thio-ester group, a thiol group, a thioether group, a phosphate group, a phosphonate group, a phosphine group, a phosphoramide group, an alkyl group, an alkenyl group, an alkynyl, an aryl group, F, Cl, Br, and I; and detecting bioluminescent emission from the sample. 
 
     
     
         5 . The methods of  claim 1 , wherein R 1  and/or R 2  are each C 1-12  alkyl linked to a near infrared fluorophore. 
     
     
         6 . The methods of  claim 1 , wherein R 3  is C 1-12  alkyl linked to a near infrared fluorophore. 
     
     
         7 . The method of  claim 1 , wherein the compounds are selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         8 . The method of  claim 1 , wherein the luciferase has a mutation at Serine 347 to an amino acid with a smaller side chain. 
     
     
         9 . The method of  claim 8 , wherein the luciferase has a mutation at Serine 347 to alanine