Compositions Containing, Methods Involving, and Uses of Non-Natural Amino Acids and Polypeptides

Abstract

Disclosed herein are non-natural amino acids and polypeptides that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The non-natural amino acids, by themselves or as a part of a polypeptide, can include a wide range of possible functionalities, but typical have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such polypeptides. Typically, the modified non-natural amino acid polypeptides include at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, including therapeutic, diagnostic, and other biotechnology uses.

1 . A method of producing a polypeptide comprising at least one amino acid having a structure of Formula (I): 
       
         
           
           
               
               
           
         
       
       the method comprising incorporating the amino acid of Formula (I) into a terminal or internal position within the polypeptide, wherein:
 A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; 
 B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — where k is 1, 2, or 3, —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; 
 
       
         
           
           
               
               
           
         
         R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; 
         each R″ is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl; 
         R 1  is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and 
         R 2  is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; 
         each of R 3  and R 4  is independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3  and R 4  or two R 3  groups optionally form a cycloalkyl or a heterocycloalkyl; 
         or the A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; 
         or the J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; 
       
       with a proviso that when A is phenylene and each R 3  is H, B is present; and that when A is —(CH 2 ) 4 — and each R 3  is H, B is not —NHC(O)(CH 2 CH 2 )—; and that when A and B are absent and each R 3  is H, R is not methyl. 
     
     
         2 . The method of  claim 1 , wherein the amino acid is incorporated at a specific site into the polypeptide using a translation system comprising:
 (i) a polynucleotide encoding the polypeptide, wherein the polynucleotide comprises a selector codon corresponding to the pre-designated site of incorporation of the amino acid of Formula (I), and   (ii) a tRNA comprising the amino acid, wherein the tRNA is specific to the selector codon.   
     
     
         3 . The method of  claim 2 , wherein the translation system comprises a tRNA that is aminoacylated to the amino acid of Formula (I). 
     
     
         4 . The method of  claim 3 , wherein the translation system is an in vivo translation system comprising a cell selected from the group consisting of a bacterial cell, archeaebacterial cell, and eukaryotic cell. 
     
     
         5 . The method of  claim 4 , wherein the amino acid has a structure corresponding to Formula (III): 
       
         
           
           
               
               
           
         
         wherein each R a  is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′) 2 , —C(O) k R′ where k is 1, 2, or 3, —C(O)N(R′) 2 , —OR′, and —S(O) k R′, where each R′ is independently H, alkyl, or substituted alkyl. 
       
     
     
         6 . The method of  claim 5 , wherein the amino acid is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         7 . The method of  claim 4 , wherein the amino acid has a structure corresponding to Formula (VI): 
       
         
           
           
               
               
           
         
         wherein each R a  is independently selected from the group consisting of H, halogen, alkyl substituted alkyl —N(R′) 2 , —C(O) k R′ where k is 1, 2, or 3, —C(O)N(R′) 2 , —OR′, and —S(O) k R′, where each R′ is independently H, alkyl or substituted alkyl. 
       
     
     
         8 . The method of  claim 7 , wherein the amino acid is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The method of  claim 4 , wherein the amino acid has a structure corresponding to Formula (IX): 
       
         
           
           
               
               
           
         
         wherein each R a  is independently selected from the group consisting of H, halogen, alkyl substituted alkyl, —N(R′) 2 , —C(O) k R′ where k is 1, 2, or 3, —C(O)N(R′) 2 , —OR′, and —S(O) k R′, where each R′ is independently H, alkyl or substituted alkyl. 
       
     
     
         10 . The method of  claim 9 , wherein the amino acid is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         11 . The method of  claim 4 , wherein the A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group. 
     
     
         12 . The method of  claim 11  wherein the amino acid is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The method of  claim 4 , wherein the J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group. 
     
     
         14 . The method of  claim 13  wherein the amino acid is: 
       
         
           
           
               
               
           
         
       
     
     
         15 . The method of  claim 4 , wherein the amino acid of Formula (I) has the structure of Formula (XXX): 
       
         
           
           
               
               
           
         
         wherein X 1  is C, S, or S(O); and L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene). 
       
     
     
         16 . The method of  claim 4 , wherein the amino acid of Formula (I) has the structure of Formula (XXXIII): 
       
         
           
           
               
               
           
         
         wherein X 1  is C, S, or S(O); and L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene). 
       
     
     
         17 . The method of  claim 4 , corresponding to Formula (XXXX): 
       
         
           
           
               
               
           
         
       
       wherein: 
       
         
           
           
               
               
           
         
          where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups; and 
         T 3  is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl substituted alkyl cycloalkyl, or substituted cycloalkyl; 
       
     
     
         18 . The method of  claim 17 , corresponding to Formula (XXXXIII): 
       
         
           
           
               
               
           
         
       
     
     
         19 . A method for derivatizing a polypeptide comprising an amino acid of Formula (I), the method comprising contacting the polypeptide with a reagent of Formula (XIX), wherein Formula (I) corresponds to: 
       
         
           
           
               
               
           
         
       
       wherein:
 A is optional, and when present is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; 
 B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — where k is 1, 2, or 3, —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; 
 
       
         
           
           
               
               
           
         
         R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; 
         each R′ is independently H, alkyl, or substituted alkyl; 
         R 1  is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and 
         R 2  is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; 
         each R 3  and R 4  is independently H, halogen, lower alkyl, or substituted lower alkyl; 
       
       wherein Formula (XIX) corresponds to: 
       
         
           
           
               
               
           
         
       
       wherein:
 each X is independently a detectable label, biologically active agent, or polymer; 
 each L is a linker independently selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — where k is 1, 2, or 3, —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)NR′C(O)O-(alkylene or substituted alkylene)-, —O—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(O)O-(alkylene or substituted alkylene)-, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or substituted alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; 
 L 1  is optional, and when present, is —C(R′) p —NR′—C(O)O-(alkylene or substituted alkylene)- where p is 0, 1, or 2; 
 W is —ON(R 1 ) 2  or —C(═O)R 2 , where each R 1  is independently H or an amino protecting group, and R 2  is H or OR′; and 
 n is 1 to 3. 
 
     
     
         20 . The method of  claim 19 , wherein the amino acid corresponds to Formula (II): 
       
         
           
           
               
               
           
         
       
     
     
         21 . The method of  claim 19 , wherein the reagent corresponds to Formula (XXVII): 
       
         
           
           
               
               
           
         
       
     
     
         22 . The method of  claim 19 , wherein the derivatized polypeptide comprises at least one oxime containing amino acid having the structure of Formula (XI): 
       
         
           
           
               
               
           
         
       
       wherein:
 A is optional, and when present is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; 
 B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — where k is 1, 2, or 3, —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; 
 R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; 
 R 1  is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and 
 R 2  is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; 
 each R 3  and R 4  is independently H, halogen, lower alkyl, or substituted lower alkyl; 
 R 5  is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, —C(O)R″, —C(O) 2 R″, or —C(O)N(R″) 2 , wherein each R″ is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or R 5  is L-X, where
 X is a detectable label, biologically active agent, or polymer; and
 L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — where k is 1, 2, or 3, —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; 
 
 
 with a proviso that when A and B are absent, R is not methyl. 
 
     
     
         23 . The method of  claim 19 , wherein the polypeptide is contacted with the reagent of Formula (XIX) in aqueous solution under mildly acidic conditions. 
     
     
         24 . The method of  claim 23 , wherein the conditions are pH 2 to 8. 
     
     
         25 . The method of  claim 19 , wherein the polypeptide is contacted with the reagent of Formula (XIX) in the presence of an accelerant selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         26 . The method of  claim 19 , wherein the amino acid of Formula (I) has the structure of Formula (XXX): 
       
         
           
           
               
               
           
         
         wherein X 1  is C, S, or S(O); and L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene). 
       
     
     
         27 . The method of  claim 19 , wherein the amino acid of Formula (I) has the structure of Formula (XXXIII): 
       
         
           
           
               
               
           
         
         wherein X 1  is C, S, or S(O); and L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene). 
       
     
     
         28 . The method of  claim 19 , corresponding to Formula (XXXX): 
       
         
           
           
               
               
           
         
       
       wherein: 
       
         
           
           
               
               
           
         
          where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups; and 
         T 3  is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl substituted alkyl cycloalkyl, or substituted cycloalkyl; 
       
     
     
         29 . The method of  claim 28 , corresponding to Formula (XXXXIII):