Site-specific chemoenzymatic protein modifications

Abstract

The present invention relates to methods and reagents for use in site-selective modification of proteins having lysine residues with functionalized peptides using a chemoenzymatic microbial transglutaminase-mediated reaction. The functionalized proteins may be used for study or therapeutic uses.

1 . A method for modifying a protein, comprising:
 providing a target protein having at least one lysine residue;   contacting the target protein with a modifying compound having the formula   R 1 -(Leu) x -Gln-(Gly) y -(A-W—B—R 2 ) z  in the presence of a microbial transglutaminase to form a modified protein;   wherein x is 0 or 1; y is 0 or 1; z is 0 or 1;   R 1  is selected from the group consisting of: acetyl,   
       
         
           
           
               
               
           
         
         wherein R 4  is selected from —H, —N 3 , and 
       
       
         
           
           
               
               
           
         
         W is selected from: C 1 -C 6  linear or branched alkyl or polyethylene glycol having a molecular weight of between about 40 and about 80,000 amu; 
         A is absent or selected from —O—, —NH—, and —S—; 
         B is absent or selected from —O—, —C(O)—, —NH—, —C(O)NH—, —NHC(O)—, —NHC(O)O—, —OC(O)NH—, —OC(O)O—, —C═N(OH)—, —S(O 2 )—, —NHS(O 2 )—, —S(O 2 )NH—, —S(O)—, —NHS(O)—, —S(O)NH—; —C(O)O—, —OC(O)—, —S—, ═NH—O—, ═NH—NH— and ═NH—N(C 1 -C 20 alkyl)-; 
         R 2  is selected from the group consisting of: a fatty acid, linear or branched C 1 -C 3  alkyl-N 3 , cyclooctynyl, fluorophore, polysaccharide, —CH(OCH 3 ) 2 , 
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         each n is an integer independently selected from 0 to 6; 
         each Q is selected from H and NO 2 . 
       
     
     
         2 - 97 . (canceled) 
     
     
         98 . A conjugate of a compound of the formula R 1 -(Leu) x -Gln-(Gly) y -A-W—B—R 2  wherein x is 0 or 1 is 0 or 1 z is 0 or 1;
 R 1  is selected from the group consisting of: acetyl, 
 
       
         
           
           
               
               
           
         
         
           wherein R 4  is selected from —H, —N 3 , and 
         
       
       
         
           
           
               
               
           
         
         W is selected from: C 1 -C 6  linear or branched alkyl or polyethylene glycol having a molecular weight of between about 40 and about 80,000 amu; 
         A is absent or selected from —O—, —NH— and —S—; 
         B is absent or selected from —O—, —C(O)—, —NH—, —C(O)NH—, —NHC(O)—, —NHC(O)O—, —OC(O)NH—, —OC(O)O—, —C═N(OH)—, —S(O2)-, —NHS(O 2 )—, —S(O 2 )NH—, —S(O)—, —NHS(O)—, —S(O)NH—; —C(O)O—, —OC(O)—, —S—, ═NH—O—, ═NH—NH— and ═NH—N(C 1 -C 20 alkyl); 
         R 2  is selected from the group consisting of: a fatty acid, linear or branched C 1 -C 3  alkyl-N 3 , cyclooctynyl, fluorophore, polysaccharide, —CH(OCH 3 ) 2 , 
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         each n is an integer independently selected from 0 to 6; 
         and each Q is selected from H and —NO 2 . 
       
     
     
         99 . The conjugate  claim 98 , wherein the conjugate protein is CRM 197 . 
     
     
         100 . The conjugate of  claim 98 , wherein the conjugate protein is GBS 80 . 
     
     
         101 . A vaccine comprising a conjugate of  claim 98 . 
     
     
         102 . A conjugate of a compound of claim  97 . 
     
     
         103 . A vaccine comprising a conjugate of  claim 99 . 
     
     
         104 . A vaccine comprising a conjugate of  claim 100 . 
     
     
         105 . A therapeutic protein comprising a compound of claim  96 . 
     
     
         106 . A therapeutic protein comprising a compound of claim  97 . 
     
     
         107 . An imaging agent comprising a compound of claim  96 . 
     
     
         108 . An imaging agent comprising a compound of claim  97 . 
     
     
         109 . A labeling tool comprising a compound of claim  96 . 
     
     
         110 . A labeling tool comprising a compound of claim  97 . 
     
     
         111 . The method of  claim 1 , wherein R 1  is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         112 . The conjugate of claim  97 , wherein R 1  is selected from the group consisting of: