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 compound, or salt thereof, comprising Formula (I): 
       
         
           
           
               
               
           
         
       
       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; 
 J is 
 
       
         
           
           
               
               
           
         
         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; at least one protected carbonyl group, including a protected dicarbonyl group; at least one masked carbonyl group, including a masked dicarbonyl group; or combinations thereof; 
       
       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; 
       or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof. 
     
     
         2 . The compound of  claim 1 , wherein A is substituted or unsubstituted lower alkylene, or an unsubstituted or substituted arylene selected from the group consisting of a phenylene, pyridinylene, pyrimidinylene or thiophenylene. 
     
     
         3 . The compound of  claim 1 , corresponding to 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). 
       
     
     
         4 . The compound of  claim 3 , corresponding to Formula (XXXIII-A): 
       
         
           
           
               
               
           
         
       
     
     
         5 . The compound of  claim 3 , corresponding to Formula (XXXIII-B): 
       
         
           
           
               
               
           
         
       
     
     
         6 . The compound of  claim 1 , corresponding to Formula (XXXIV): 
       
         
           
           
               
               
           
         
         wherein X 1  is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R 8  and R 9  on each CR 8 R 9  group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl aryl, or any R 8  and R 9  can together form ═O or a cycloalkyl, or any to adjacent R 8  groups can together form a cycloalkyl. 
       
     
     
         7 . The compound of  claim 6 , corresponding to Formula (XXXIV-A): 
       
         
           
           
               
               
           
         
       
     
     
         7 . The compound of  claim 6 , corresponding to Formula (XXXIV-B): 
       
         
           
           
               
               
           
         
       
     
     
         8 . The compound of  claim 1 , corresponding to Formula (XXXV): 
       
         
           
           
               
               
           
         
         wherein X 1  is C, S, or S(O); and L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene). 
       
     
     
         9 . The compound of  claim 8 , corresponding to Formula (XXXV-A): 
       
         
           
           
               
               
           
         
       
     
     
         10 . The compound of  claim 8 , corresponding to Formula (XXXV-B): 
       
         
           
           
               
               
           
         
       
     
     
         11 . The compound of  claim 1 , corresponding to Formula (XXXX): 
       
         
           
           
               
               
           
         
       
       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; 
 
     
     
         12 . The compound of  claim 11 , corresponding to Formula (XXXXIII): 
       
         
           
           
               
               
           
         
       
     
     
         13 . The compound of  claim 12 , selected from: 
       
         
           
           
               
               
           
         
       
     
     
         14 . The compound of  claim 1 , 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. 
       
     
     
         15 . The compound of  claim 14 , selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         16 . The compound of  claim 1 , 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. 
       
     
     
         17 . The compound of  claim 16 , selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         18 . The compound of  claim 1 , 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. 
       
     
     
         19 . The compound of  claim 18 , selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         20 . The compound of  claim 1 , wherein the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, protected carbonyl group or masked carbonyl group. 
     
     
         21 . The compound of  claim 20  selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         22 . The compound of  claim 1 , wherein the -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, protected carbonyl group or masked carbonyl group. 
     
     
         23 . The compound of  claim 22  having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         24 . A polypeptide incorporating at least one compound of  claim 1 . 
     
     
         25 . A compound, or salt thereof, comprising Formula (XI): 
       
         
           
           
               
               
           
         
       
       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; 
 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; 
 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, -(alkylene or substituted alkylene)-ON(R″) 2 , -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —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 selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; and any combination of the above; 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—, -(alkylene or substituted alkylene)-O—N═CR′—, -(alkylene or substituted alkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O) k -(alkylene or substituted alkylene)-S—, -(alkylene or substituted alkylene)-S—S—, —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; 
 or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof. 
 
     
     
         26 . The compound of  claim 25 , wherein A is phenylene or substituted phenylene. 
     
     
         27 . The compound of  claim 25 , corresponding to Formula (XII): 
       
         
           
           
               
               
           
         
       
     
     
         28 . A polypeptide incorporating at least one compound of  claim 25 . 
     
     
         29 . The compound of  claim 25 , wherein X is a biologically active agent selected from the group consisting of a peptide, protein, enzyme, antibody, drug, dye, lipid, nucleosides, oligonucleotide, cell, virus, liposome, microparticle, and micelle. 
     
     
         30 . The compound of  claim 29 , wherein X is a drug selected from the group consisting of an antibiotic, fungicide, anti-viral agent, anti-inflammatory agent, anti-tumor agent, cardiovascular agent, anti-anxiety agent, hormone, growth factor, and steroidal agent. 
     
     
         31 . The compound of  claim 29 , wherein X is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase. 
     
     
         32 . The compound of  claim 25 , wherein X is a detectable label selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating, electron dense, magnetic, intercalating, radioactive, chromophoric, and energy transfer moiety.