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 - 97 . (canceled) 
     
     
         98 . A conjugate comprising a protein that has at least one lysine residue and is conjugated to a compound of the formula R 1 -(Leu) x -Gln-(Gly) y -(A-W—B—R 2 ) z  via a reaction of the at least one lysine residue with the Gln residue of the compound, wherein
 x is 0 or 1; y is 0 or 1; z is 0 or 1; 
 R 1  is 
 
       
         
           
           
               
               
           
         
         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)-; and 
         R 2  is selected from a fatty acid, linear or branched C 1 -C 3  alkyl-N 3 , cyclooctynyl, polysaccharide, —CH(OCH 3 ) 2 , 
       
       
         
           
           
               
               
           
         
       
       wherein each n is an integer independently selected from 0 to 6p and each n is selected from H and —NO 2 . 
     
     
         99 . The conjugate of  claim 98 , wherein the conjugate protein is CRM 197 . 
     
     
         100 . The conjugate of  claim 98 , wherein the conjugate protein is GBS 80 . 
     
     
         101 . (canceled) 
     
     
         102 . A conjugate comprising a protein that has at least one lysine residue and is conjugated to a compound selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein m is an integer selected from 1 to 2, n is an integer independently selected from 1 to 3, and q is an integer selected from 15 to 20 and wherein the conjugation is via a reaction of the at least one lysine residue with the Gln residue of the compound. 
     
     
         103 . The conjugate of  claim 98 , wherein W is polyethylene glycol having a molecular weight of between about 40 and about 80,000 amu. 
     
     
         104 . The conjugate of  claim 98 , wherein R 2  is selected from cyclooctynyl and 
       
         
           
           
               
               
           
         
       
     
     
         105 . The conjugate of  claim 98 , wherein R 2  is a fatty acid. 
     
     
         106 . A conjugate comprising a protein that has at least one lysine residue and is conjugated to a compound of the formula R 1 -(Leu) x -Gln-(Gly) y -(A-W—B—R 2 ) z  via a reaction of the at least one lysine residue with the Gln residue of the compound, wherein
 x is 0 or 1; y is 0 or 1; z is or 1; 
 R 1  is 
 
       
         
           
           
               
               
           
         
         W is 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)-; and 
         R 2  is a fluorophore. 
       
     
     
         107 . The conjugate of  claim 106 , wherein the fluorophore is selected from a fluorescein, rhodamine, a Cy dye, an Alexa dye, and a coumarin.