Photocleavable Linker Methods and Compositions

Abstract

Bifunctional linkers are provided that comprise a photocleavable moiety flanked by two different amine reactive moieties. In some embodiments the photocleavable moiety is a dimethoxynitrobenzyl moiety. In other embodiments the photocleavable moiety is 8-bromo-7-hydroxyquinoline. In other embodiments the photocleavable moiety is nitrodibenzofuran. In other embodiments the photocleavable moiety is 6-bromo-7-hydroxycoumarin-4-ylmethyl. The linkers find use in synthetic methods, including the generation of photocleavable oligonucleotides, e.g. caged morpholinos.

1 . A bifunctional linker having the structure: 
       
         
           
           
               
               
           
         
         where X is a photocleavable moiety; 
         R1 and R2 are different reactive moieties used for conjugation; 
         R3, R4 and R5 are independently selected from H and C1-C6 lower alkyls; and 
         and n is an integer from 0 to 10. 
       
     
     
         2 . The linker of  claim 1 , wherein X is a UV excited moiety. 
     
     
         3 . The linker of  claim 2 , wherein X is 1,2-dimethoxy-4-nitrobenzyl moiety; 
       
         
           
           
               
               
           
         
       
       a 1,2-dimethoxy-2-nitrobenzyl moiety, or a nitrobenzyl moiety. 
     
     
         4 . The linker of  claim 1 , wherein X is a multiphoton activated moiety. 
     
     
         5 . The linker of  claim 4 , wherein X is selected from 6-bromo-7-hydroxycoumarin-4-ylmethyl: 
       
         
           
           
               
               
           
         
       
       8-bromo-7-hydroxyquinolinyl: 
       
         
           
           
               
               
           
         
       
       or a nitrodibenzofuran moiety. 
       
         
           
           
               
               
           
         
       
     
     
         6 . The linker of  claim 1 , wherein R1 and R2 are independently selected from succinimide (NHS ester): 
       
         
           
           
               
               
           
         
       
     
     
         7 . A photocleavable oligonucleotide having the structure: 
       
         
           
           
               
               
           
         
         where X is a photocleavable moiety; 
         R3, R4 and R5 are independently selected from H and Cl-C6 lower alkyls; and 
         and n is an integer from 0 to 10. 
       
     
     
         8 . The photocleavable oligonucleotide of  claim 7 , wherein X is a UV excited moiety. 
     
     
         9 . The photocleavable oligonucleotide of  claim 8 , wherein X is 1,2-dimethoxy-4-nitrobenzyl moiety; 
       
         
           
           
               
               
           
         
       
       a 1,2-dimethoxy-2-nitrobenzyl moiety, or a nitrobenzyl moiety. 
     
     
         10 . The photocleavable oligonucleotide of  claim 7 , wherein X is a multiphoton activated moiety. 
     
     
         11 . The photocleavable oligonucleotide of  claim 10 , wherein X is selected from 6-bromo-7-hydroxycoumarin-4-ylmethyl: 
       
         
           
           
               
               
           
         
       
       8-bromo-7-hydroxyquinolinyl: 
       
         
           
           
               
               
           
         
       
       or nitrodibenzofuran moiety 
       
         
           
           
               
               
           
         
       
     
     
         12 . The photocleavable oligonucleotide of  claim 7 , wherein oligonucleotide 1 and oligonucleotide 2 are morpholino oligonucleotides. 
     
     
         13 . The photocleavable oligonucleotide of  claim 12 , wherein oligonucleotide 1 and oligonucleotide 2 are complementary sequences of different lengths.