Tyrosine Bioconjugation through Aqueous Ene-Like Reactions

Abstract

A new and versatile class of cyclic diazodicarboxamides that reacts efficiently and selectively with phenols and the phenolic side chain of tyrosine through an Ene-like reaction is reported. This mild aqueous tyrosine ligation reaction works over a broad pH range and expands the repertoire of aqueous chemistries available for small molecule, peptide, and protein modification. The tyrosine ligation reactions are shown to be compatible with the labeling of native enzymes and antibodies in buffered aqueous solution. This reaction provides a novel synthetic approach to bispecific antibodies. This reaction will find broad utility in peptide and protein chemistry and in the chemistry of phenol-containing compounds.

1 . A compound having Formula I: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein: 
         R 1  and R 2  are each H when 
       
       
         
           
           
               
               
           
         
       
       is a single bond, and R 1  and R 2  are each absent when 
       
         
           
           
               
               
           
         
       
       is a double bond;
 W is independently a direct bond or is O; 
 R 3  is independently hydrogen, halogen, carboxyl, cyano, nitro, amino, substituted or unsubstituted alkyl, substituted or unsubstituted thioalkyl, perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryloxy; substituted or unsubstituted alkylaryl, or substituted or unsubstituted alkylheteroaryl, or two R 3's  form a cyclic or heterocyclic ring, wherein each R 3  is optionally independently substituted with 1 to 3 groups selected from halogen, carboxyl, cyano, nitro, amino, alkyl, alkenyl, alkynyl, perfluoroalkyl, thioalkyl, alkoxy, aryloxy, aryl, alkylaryl, heteroaryl, and alkylheteroaryl; 
 L is independently H, N 3 , CH 3 , C═CH, C═CHN 3 , CH═CHN 3 , CH 2 CH 2 N 3 , O(CH 2 )N 3 , C 6 H 5 , COCH 3 , OCH 2 C≡CH, OCH 2 COCH 3 , OCOCF 3 , or X—[CH 2 CH 2 —Y] n —(CH 2 ) q —N 3 ; 
 X and Y are each independently CH 2 , O, NH, S, NHCO or CONH; and 
 n and q are each independently an integer from 0 to 12. 
 
     
     
         2 . The compound of  claim 1 , wherein the compound of Formula I has Formula II, III, IV, or V: 
       
         
           
           
               
               
           
         
       
     
     
         3 . The compound of  claim 1 , wherein the compound of Formula I has Formula VI: 
       
         
           
           
               
               
           
         
       
     
     
         4 . The compound of  claim 3 , wherein the compound of Formula VI has Formula VII or Formula VIII: 
       
         
           
           
               
               
           
         
       
     
     
         5 . (canceled) 
     
     
         6 . A compound having Formula IX: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein: 
         W is independently a direct bond or is O; 
         R 3  is independently hydrogen, halogen, carboxyl, cyano, nitro, amino, substituted or unsubstituted alkyl, substituted or unsubstituted thioalkyl, perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryloxy; substituted or unsubstituted alkylaryl, or substituted or unsubstituted alkylheteroaryl, or two R 3's  form a cyclic or heterocyclic ring, wherein each R 3  is optionally independently substituted with 1 to 3 groups selected from halogen, carboxyl, cyano, nitro, amino, alkyl, alkenyl, alkynyl, perfluoroalkyl, thioalkyl, alkoxy, aryloxy, aryl, alkylaryl, heteroaryl, and alkylheteroaryl; 
         L is independently H, N 3 , CH 3 , C≡CH, C≡CHN 3 , CH═CHN 3 , CH 2 CH 2 N 3 , O(CH 2 )N 3 , C 6 H 5 , COCH 3 , OCH 2 C≡CH, OCH 2 COCH 3 , OCOCF 3 , or X—[CH 2 CH 2 —Y] n —(CH 2 ) q —N 3 ; 
         X and Y are each independently CH 2 , O, NH, S, NHCO or CONH; 
         n and q are each independently an integer from 0 to 12; and 
         R 6  is a tyrosine moiety or a tyrosine residue in a peptide or a protein. 
       
     
     
         7 . The compound of  claim 6 , wherein the tyrosine moiety is N-acyl tyrosine methylamide, or H-Gly-Gly-Tyr-OH; and the tyrosine residue in a peptide or a protein is in Chymotrypsinogen A, Myoglobin, Bovine Serum Albumin (BSA), or tocinoic acid. 
     
     
         8 - 19 . (canceled) 
     
     
         20 . A method of producing a multi-specific antibody with binding specificity for binding peptides, polypeptides, and organic compounds, comprising the steps of:
 a) cyclizing a compound of Formula XX with a peptide of Formula XXV to form a 1,4-triazole compound of Formula XXVI;   
       
         
           
           
               
               
           
         
         b) converting the 
       
       
         
           
           
               
               
           
         
       
       single bond in the compound of Formula XXVI to a 
       
         
           
           
               
               
           
         
       
       double bond to provide a compound of Formula XXVII: 
       
         
           
           
               
               
           
         
         c) conjugating the compound of Formula XXVII with Herceptin to provide the Herceptin antibody of Formula XXVIII: 
       
       
         
           
           
               
               
           
         
         wherein: 
         X and Y are each independently CH 2 , O, NH, S, NHCO or CONH; and 
         n and q are each independently an integer from 0 to 12; wherein the compound of Formula XIV has multi-specific antibody binding specificity for binding peptides, polypeptides, and organic compounds. 
       
     
     
         21 . A method of site-specific tyrosine labeling at a specific site on human IgG heavy chain proteins, the method comprising the step of exposing human IgG heavy chain proteins to o-benzaldehyde diazonium hexafluorophosphate. 
     
     
         22 . The method of  claim 21 , wherein the specific site on the human IgG heavy chain protein is at the CH 2  domain of the heavy chain tyrosine 319 residue according to Kabat numbering. 
     
     
         23 . The method of  claim 21 , wherein the human IgG heavy chain protein is an antibody. 
     
     
         24 . The method of  claim 21 , wherein the antibody is herceptin, rituxan or erbitux. 
     
     
         25 . A method of chemoselectively modifying a moiety containing the amino acid tyrosine, the method comprising the steps of:
 a) reacting a compound of Formula X with a compound of Formula XXIX to provide a compound of Formula XXX; and   
       
         
           
           
               
               
           
         
         b) reacting the compound of Formula XXX with a compound of Formula XXXI to provide a compound of Formula XXXII; 
       
       
         
           
           
               
               
           
         
         wherein: 
         X −  is independently F − , Cl − , Br − , I − , ClO 4   − , NO 3   − , HSO 3   − , PF 6   −  or BF 4   − ; 
         Y is independently NH or O; 
         R 7 , R 8  and R 9  are each independently hydrogen, hydroxyl, amino, substituted or unsubstituted alkyl, substituted or unsubstituted thioalkyl, perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryloxy, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, or R 7 , R 8  and R 9  are in a tyrosine residue of a peptide or a protein; 
         R 10  is independently hydrogen, halogen, nitro, cyano, trifluoromethyl, substituted or unsubstituted alkyl or alkyl, substituted or unsubstituted alkoxy or alkoxy, substituted or unsubstituted aryl or aryl substituted, substituted or unsubstituted aryloxy or aryloxy, substituted or unsubstituted heteroaryl or heteroaryl substituted, substituted or unsubstituted heteroaryloxy or heteroaryloxy; 
         R 11  is independently hydrogen, alkyl, alkoxy, phenoxy, or alkylaryloxy; 
         R 12  is independently small organic molecule, fluorescence unit, enzyme, peptide or antibody; 
         Linker is (CH 2 ) l -A-[CH 2 CH 2 —Z] m —(CH 2 ) n —B; 
         A and B are each independently bond, C(═O), CONH or NHCO; 
         Z is independently CH 2  or O; and 
         l, m, and n are each independently an integer from 0 to 12. 
       
     
     
         26 . The method of  claim 25 , wherein the compound of Formula XXIX has Formula XXXIII: 
       
         
           
           
               
               
           
         
       
     
     
         27 . The method of  claim 26 , wherein the compound of Formula XXXIII has Formula XXXIV: 
       
         
           
           
               
               
           
         
       
     
     
         28 . A compound having Formula XXXIII: 
       
         
           
           
               
               
           
         
       
     
     
         29 . The compound of  claim 28 , wherein the compound of Formula XXXIII has Formula XXXIV: 
       
         
           
           
               
               
           
         
       
     
     
         30 . A method of bioconjugating momomethyl auristatin E (MMAE) to a cancer targeting monoclonal antibody (mAb) to provide a bioconjugated mAb, the method comprising the step of reacting the compound of Formula XXXV with a monoclonal antibody (mAb) to provide the bioconjugated mAb: 
       
         
           
           
               
               
           
         
       
     
     
         31 . The method of  claim 30 , wherein the monoclonal antibody (mAb) is CD22, CD30, CD33, GPNMB or ErbB2. 
     
     
         32 . A method of bioconjugating momomethyl auristatin E (MMAE) to a cancer targeting monoclonal antibody (mAb) to provide a bioconjugated mAb, the method comprising the step of reacting the compound of Formula XXXVI with a monoclonal antibody (mAb) to provide the bioconjugated mAb: 
       
         
           
           
               
               
           
         
       
     
     
         33 . The method of  claim 32 , wherein the monoclonal antibody (mAb) is CD22, CD30, CD33, GPNMB or ErbB2. 
     
     
         34 . A method of chemoselectively modifying a glucaon-like protein receptor (GLP-1R) agonist, the method comprising the steps of reacting a GLP-1R agonist with a compound of Formula XI: 
       
         
           
           
               
               
           
         
         wherein: 
         W is independently a direct bond or is O; 
         R 3  is independently hydrogen, halogen, carboxyl, cyano, nitro, amino, substituted or unsubstituted alkyl, substituted or unsubstituted thioalkyl, perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryloxy; substituted or unsubstituted alkylaryl, or substituted or unsubstituted alkylheteroaryl, or two R 3's  form a cyclic or heterocyclic ring, wherein each R 3  is optionally independently substituted with 1 to 3 groups selected from halogen, carboxyl, cyano, nitro, amino, alkyl, alkenyl, alkynyl, perfluoroalkyl, thioalkyl, alkoxy, aryloxy, aryl, alkylaryl, heteroaryl, and alkylheteroaryl; 
         L is independently H, N 3 , CH 3 , C≡CH, C≡CHN 3 , CH═CHN 3 , CH 2 CH 2 N 3 , O(CH 2 )N 3 , C 6 H 5 , COCH 3 , OCH 2 C≡CH, OCH 2 COCH 3 , OCOCF 3 , or X—[CH 2 CH 2 —Y] n —(CH 2 ) q —N 3 ; 
         X and Y are each independently CH 2 , O, NH, S, NHCO or CONH; 
         n and q are each independently an integer from 0 to 12. 
       
     
     
         35 . (canceled) 
     
     
         36 . A method of chemoselectively modifying a glucaon-like protein receptor (GLP-1R) agonist, the method comprising the steps of reacting a GLP-1R agonist with a compound of Formula XXXVII: 
       
         
           
           
               
               
           
         
         wherein: 
         X −  is independently F − , Cl − , Br − , I − , ClO 4   − , NO 3   − , HSO 3   − , PF 6   −  or BF 4   − ; 
         R 10  is independently hydrogen, halogen, nitro, cyano, trifluoromethyl, substituted or unsubstituted alkyl or alkyl, substituted or unsubstituted alkoxy or alkoxy, substituted or unsubstituted aryl or aryl substituted, substituted or unsubstituted aryloxy or aryloxy, substituted or unsubstituted heteroaryl or heteroaryl substituted, substituted or unsubstituted heteroaryloxy or heteroaryloxy; and 
         R 11  is independently hydrogen, alkyl, alkoxy, phenoxy, or alkylaryloxy. 
       
     
     
         37 . The method of  claim 36 , further comprising reacting the modified GLP-1R agonist with a compound of Formula XXXI:
   NH 2 —Y-Linker-R 12   (XXXI),
   wherein:   Y is independently NH or O;   R 12  is independently small organic molecule, fluorescence unit, enzyme, peptide or antibody;   Linker is (CH 2 ) l -A-[CH 2 CH 2 —Z] m —(CH 2 ) n —B;   A and B are each independently bond, C(═O), CONH or NHCO;   Z is independently CH 2  or O; and   l, m, and n are each independently an integer from 0 to 12.   
     
     
         38 - 39 . (canceled)