Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor

Abstract

Site-specific isotopically-labeled valine, leucine, and isoleucine and biosynthetic precursors for these amino acids are provided. The amino acids are labeled with 13 C or 14 C at the methyl group carbon atom(s) most remote from the carboxyl group. Also disclosed are the biochemical precursors of these labeled amino acids, 2-keto-4-( n C)butyric acid and 2-keto-3-( n C-methyl)-4-( n C)-butyric acid in which n, at each occurrence, is 13 or 14. Also disclosed are proteins, protein fragments, and polypeptides containing these site-specifically isotopically labeled amino acids, and methods for preparing the biochemical precursors, the amino acids, and the proteins, protein fragments, and polypeptides.

We claim:  
     
         1 . A compound of formula I  
       
         
           
           
               
               
           
         
       
       or a salt thereof, wherein 
 R 1  is oxygen or NH 2 ;  
 R 2  is selected from the group consisting of  
                     wherein R 3  is hydrogen or  n CH 3 ;    the dotted line bond represents a second valence bond;    m is zero or one;    and n, at each occurrence, is 13 or 14;    with the provisos that    i) when R 1  is NH 2 , the second valence bond represented by the dotted line bond to R 1  is absent and the hydrogen attached to the dotted line bond is present;    ii) when R 1  is oxygen, the second valence bond represented by the dotted line bond to R 1  is present and the hydrogen atom attached to the dotted line bond is absent;    iii) when R 1  is oxygen, R 2  is B and m is zero; and    iv) when R 1  is NH 2 , R 3  is hydrogen or  n CH 3 .    
 
     
     
         2 . A protein, protein fragment, or polypeptide containing an aminoacyl residue derived from a compound according to  claim 1  wherein R 1  is —NH 2 .  
     
     
         3 . A compound of formula Ia  
       
         
           
           
               
               
           
         
       
       or a salt thereof, wherein R 3  is hydrogen or  n CH 3  and n, at each occurrence is 13 or 14.  
     
     
         4 . A compound according to  claim 3  selected from the group consisting of: 
 2-keto-4-( 13 C)-butyric acid;  
 2-keto-3-( 13 C-methyl)-4-( 13 C)-butyric acid;  
 2-keto-4-( 14 C)-butyric acid; and  
 2-keto-3-( 14 C-methyl)-4-( 14 C)-butyric acid;  
 or salts thereof.  
 
     
     
         5 . A compound of formula Ib  
       
         
           
           
               
               
           
         
       
       wherein 
 R 2  is selected from the group consisting of  
                     
 where R 3  is hydrogen or  n CH 3 ; m is zero or one; and n, at each occurrence, is 13 or 14.  
 
     
     
         6 . A compound according to  claim 5  selected from the group consisting of: 
 L-2-amino-3-methyl-5-( 13 C)-pentanoic acid;  
 L-2-amino-3-methyl-5-( 14 C)-pentanoic acid;  
 L-2-amino-3-( 13 C-methyl)-5-( 13 C)-butanoic acid;  
 L-2-amino-3-( 14 C-methyl)-5-( 14 C)-butanoic acid;  
 L-2-amino-4-( 13 C-methyl-5-( 13 C)-pentanoic acid; and  
 L-2-amino-4-( 4 C-methyl-5-( 14 C)-pentanoic acid; or salts thereof.  
 
     
     
         7 . A protein, protein fragment, or polypeptide containing one or more aminoacyl residues, wherein said one or more amino acyl residues comprise a compound of formula IIb  
       
         
           
           
               
               
           
         
       
       wherein R 2  is selected from the group consisting of  
       
         
           
           
               
               
           
         
       
       wherein R 3  is hydrogen or  n CH 3 ; m is zero or one; and n, at each occurrence, is 13 or 14.  
     
     
         8 . A method of preparing a compound of formula Ia  
       
         
           
           
               
               
           
         
       
       or a salt thereof, wherein R 3  is selected from the group consisting of hydrogen and  n CH 3 and n at each occurrence is 13 or 14,  
       which comprises 
 a) reacting a compound of formula IV  
                     
 with isotopically-labeled methyl iodide (H 3   n CI) to produce a compound of formula V  
                     
 and  
 b) removing the tert-butyl ester and dimethylhydrazino groups to produce 2-keto-4-( n C)-butyric acid.  
 
     
     
         9 . A method according to  claim 8 , which further comprises salifying the reaction product of step b).  
     
     
         10 . A method according to  claim 8 , which further comprises 
 c) reacting the product of step b) with isotopically-labeled methyl iodide (H 3   n CI), where n is 13 or 14, to produce a compound of formula VI                          and    d) removing the tert-butyl ester and dimethylhydrazino groups to produce 2-keto-3-( n C-methyl)-4-( n C)-butyric acid.    
     
     
         11 . A method according to  claim 10 , which further comprises salifying the reaction product of step d).  
     
     
         12 . A method of preparing a protein, protein fragment, or polypeptide containing at least one amino acyl residue selected from the group consisting of 4-( n C-methyl)-5-( n C)-leucyl, 5-( n C)-isoleucyl, and 3-( n C-methyl)-4-( n C)-valyl, which comprises 
 a) genetically modifying a suitable microorganism to express said protein, protein fragment, or polypeptide;    b) culturing said genetically modified microorganism in a nutrient medium containing a compound of formula Ia                          or salt thereof, wherein n, at each occurrence is 13 or 14, and R 3  is hydrogen or  n CH 3 ; and    c) isolating said protein, protein fragment, or polypeptide.    
     
     
         13 . A method of preparing a protein, protein fragment, or polypeptide containing at least one 3-methyl-5-( n C)-isoleucyl aminoacyl residue, which comprises 
 a) genetically modifying a suitable microorganism to express said protein, protein fragment, or polypeptide;    b) culturing said genetically modified microorganism in a nutrient medium containing a compound of formula Ia″                         or a salt thereof, wherein n, at each occurrence is 13 or 14; and    c) isolating said protein, protein fragment, or polypeptide.    
     
     
         14 . A method of preparing 2-amino-3-(C-methyl)-4-( n C)-butyric acid which comprises 
 a) genetically modifying a suitable microorganism to express a homopolymer of valine;    b) culturing said genetically modified microorganism in a nutrient medium containing a compound of formula Ia′                         or a salt thereof, where n, at each occurrence, is 13 or 14;    c) isolating the homopolymer of valine expressed by said genetically modified microorganism; and    d) fragmenting said homopolymer to produce 2-amino-3-( n C-methyl)-4-( n C)-butyric acid.    
     
     
         15 . A method of preparing 2-amino-4-( n C-methyl)-5-( n C)-pentanoic acid which comprises 
 a) genetically modifying a suitable microorganism to express a homopolymer of leucine;    b) culturing said genetically modified microorganism in a nutrient medium containing a compound of formula Ia′                         or a salt thereof, where n, at each occurrence, is 13 or 14;    c) isolating the homopolymer of leucine expressed by said genetically modified microorganism; and    d) fragmenting said homopolymer to produce 2-amino-4-( n C-methyl)-5-( n C)-pentanoic acid.    
     
     
         16 . A method of preparing 2-amino-3-methyl-5-( n C)-pentanoic acid which comprises 
 a) genetically modifying a suitable microorganism to express a homopolymer of isoleucine;    b) culturing the genetically modified microorganism in a nutrient medium containing a compound of formula Ia″                         or a salt thereof, where n, at each occurrence, is 13 or 14;    c) isolating the homopolymer of isoleucine expressed by said genetically modified microorganism; and    d) fragmenting said homopolymer to produce 2-amino-3-methyl-5-( n C)-pentanoic acid.