Zinc-Selective Fluorescent Probes for Emission-Ratiometric Imaging

Abstract

The compounds relate to zinc-sensitive fluorescent probes, compositions and methods utilizing the same. Such compounds provide an emission-ratiometric fluorescence response upon binding of an analyte. In some embodiments, compounds can be used for two-photon excitation microscopy or conventional fluorescence microscopy. The compounds described herein can also contain one or more functional groups to improve the emission-ratiometric fluorescence response.

1 . A compound according to Formula I: 
       
         
           
           
               
               
           
         
         wherein:
 Z is: 
 
       
       
         
           
           
               
               
           
         
         X 1  is —N═; 
         X 2  is —N═; 
         X 3  is —CH═; 
         X 4  is —CH═; 
         Y 1  is selected from —NR 4 — and —S—; 
         Y 2  is selected from —NR 5 —, —S—, and —O —; 
         R c  is Ar, —OR 4  or —N(CH 2 ) q —Ar) 2 ; 
         R 1  is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted heteroaryl, —OCH 3 , —OCH 2 COOR 9 , —OCH 2 CH 2 COOR 9 , —OCH 2 CH 2 CH 2 SO 2 R 9 , —NH 2 , —NHR 9  and —N(R 9 ) 2 ; 
         R 2  is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, and substituted or unsubstituted heteroaryl or in the alternative, R 1  and R 2  join together to form a moiety selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, and substituted or unsubstituted heteroaryl; 
         R 3  is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, and substituted or unsubstituted heteroaryl; or in the alternative, R 1  and R 3  join together to form a moiety selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, and substituted or unsubstituted heteroaryl; 
         R 4  is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted heteroaryl, —CO 2 H, —CO 2 Na and —CO 2 C 1-8 -alkyl; 
         R 5  is selected from C 1-3 -alkyl optionally substituted with —OR 4  or Ar; 
         R 9  is selected from —CH 3 , —C 2 H 5 , substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, and substituted or unsubstituted heteroaryl; 
         n is 1, 2, or 3; 
         q is 1, 2, or 3; 
         Ar is independently at each occurrence a substituted or unsubstituted six-membered heteroaryl selected from: 
       
       
         
           
           
               
               
           
         
         and 
         m is any integer from 0 to 3. 
       
     
     
         2 . The compound of  claim 1 , wherein is —NR 5 —. 
     
     
         3 . The compound of  claim 1 , wherein
 Z is:   
       
         
           
           
               
               
           
         
       
     
     
         4 . The compound of  claim 1 , wherein:
 Z is selected from:   
       
         
           
           
               
               
           
         
         and 
         s is 1, 2, or 3. 
       
     
     
         5 . The compound of  claim 1 , wherein:
 Z is selected from:   
       
         
           
           
               
               
           
         
       
     
     
         6 . The compound of  claim 1 , wherein:
 Ar is   
       
         
           
           
               
               
           
         
         and 
         m is any integer from 0 to 3. 
       
     
     
         7 . The compound of  claim 6 , wherein Ar is the same at each occurrence. 
     
     
         8 . The compound of  claim 1 , wherein Y 1  is —S—. 
     
     
         9 . The compound of  claim 1 , wherein Y 1  is —NR 4 —. 
     
     
         10 . The compound of  claim 1 , wherein:
 R 1  is selected from —OCH 3 , OCH 2 COOR 9 , OCH 2 CH 2 COOR 9 , OCH 2 CH 2 CH 2 SO 2 R 9 , NH 2 , NHR 9 , N(R 9 )2; and   R 9  is selected from —CH 3 , C 2 H 5 , substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, and substituted or unsubstituted heteroaryl.   
     
     
         11 . The compound of  claim 10 , wherein: R 1  is —OCH 3 . 
     
     
         12 . The compound of  claim 1 , wherein:
 R 1  and R 2  together form a moiety selected from substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, and substituted or unsubstituted heteroaryl; and   R 3  is hydrogen.   
     
     
         13 . The compound of  claim 1 , wherein Formula I is: 
       
         
           
           
               
               
           
         
         wherein:
 R 6  and R 8  are independently selected from hydrogen and CO 2 R 7 ; and 
 R 7  is hydrogen or a C 1 —C 8  alkyl. 
 
       
     
     
         14 . The compound of  claim 13 , wherein R 7  is selected from H, —CH 3 , and —C 2 H 5 . 
     
     
         15 . The compound of  claim 1 , wherein Formula I is selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         16 . A fluorescent complex comprising the compound of  claim 1  in a 1:1 stoichiometric ratio. 
     
     
         17 . The fluorescent complex of  claim 16 , wherein the fluorescent complex further comprises a ligand. 
     
     
         18 . The fluorescent complex of  claim 16  wherein a maximum absorption wavelength of the fluorescent complex is from about 15 nm to about 50 nm greater than a maximum absorption wavelength of the compound of Formula I. 
     
     
         19 . The fluorescent complex of  claim 16 , wherein a maximum emission wavelength of the fluorescent complex is from about 40 nm to about 60 nm greater than a maximum emission wavelength of the compound of Formula I. 
     
     
         20 . A method of detecting zinc in a sample comprising:
 treating the sample with the compound of Formula I according to  claim 1 ;   detecting a first wavelength emission intensity of the sample at a first wavelength and a second wavelength emission intensity of the sample at a second wavelength, wherein the first wavelength emission intensity is associated with the compound not being bonded to zinc and the second wavelength emission intensity is associated with the compound being bonded to zinc; and   comparing the first wavelength emission intensity to the second wavelength emission intensity to determine the concentration of zinc in the sample.   
     
     
         21 . The method of  claim 20 , wherein the first wavelength is from about 380 nm to about 700 nm. 
     
     
         22 . The method of  claim 20 , wherein the second wavelength is about 40 nm to about 80 nm greater than the first wavelength. 
     
     
         23 . The method of  claim 20 , wherein the sample is a biological sample.