Non-natural labeled amino acid and method for producing a conjugate of said amino acid and trna

Abstract

This invention relates to: a labeled amino acid that can be introduced into a protein with the aid of a protein synthesis system; a functional protein having functions derived from a label compound; a labeled amino acid comprising an aromatic ring bound to an amino acid side chain and a label compound bound thereto via the aromatic ring; a functional protein to which the labeled amino acid has been introduced; and a novel method for effectively obtaining a labeled amino acid-tRNA complex.

1 . A labeled amino acid comprising a label compound bound to an amino acid via an aromatic ring, wherein the aromatic ring is contained in a side chain of a naturally-occurring amino acid or is bound to an amino acid side chain. 
     
     
         2 . The labeled amino acid according to  claim 1 , wherein the amino acid is phenylalanine, tyrosine, tryptophan, or a derivative thereof. 
     
     
         3 . The labeled amino acid according to  claim 2 , wherein the amino acid is an aminophenylalanine or a derivative thereof. 
     
     
         4 . The labeled amino acid according to  claim 1 , wherein the label compound is bound to the aromatic ring at its meta or para position to an amino acid skeleton. 
     
     
         5 . The labeled amino acid according to  claim 4 , wherein the label compound is bound to the aromatic ring at its para position to the amino acid skeleton. 
     
     
         6 . The labeled amino acid according to  claim 1 , wherein the label compound is bound to the amino acid via a spacer. 
     
     
         7 . The labeled amino acid according to  claim 1 , wherein the spacer comprises 2 to 18 linearly bound C, O, N, or S atoms or a combination thereof. 
     
     
         8 . The labeled amino acid according to  claim 7 , wherein the spacer comprises 4 to 15 linearly bound C, O, N, or S atoms or a combination thereof. 
     
     
         9 . The labeled amino acid according to  claim 1 , wherein the label compound is selected from the group consisting of a dye compound, a fluorescent substance, a chemiluminescent substance, a bioluminescent substance, an enzyme substrate, a coenzyme, an antigenic substance, and a protein-binding substance. 
     
     
         10 . The labeled amino acid according to  claim 9 , wherein the label compound is a fluorescent substance. 
     
     
         11 . The labeled amino acid according to  claim 10 , wherein the fluorescent substance has an excitation wavelength and an emission wavelength in the visible light range. 
     
     
         12 . The labeled amino acid according to  claim 11 , wherein the fluorescent substance is a chemical compound comprising, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene as a basic skeleton, a salt thereof, or a derivative thereof. 
     
     
         13 . The labeled amino acid according to  claim 11 , wherein the fluorescent substance is 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt thereof. 
     
     
         14 . A labeled amino acid represented by formula I: 
       
         
           
           
               
               
           
         
       
     
     
         15 . A labeled amino acid represented by formula II: 
       
         
           
           
               
               
           
         
       
     
     
         16 . The labeled amino acid according to  claim 1 , wherein the labeled amino acid is bound to pdCpA. 
     
     
         17 . A functional protein comprising at least one labeled amino acid according to  claim 1 . 
     
     
         18 . The functional protein according to  claim 17 , wherein the labeled amino acid is labeled with a compound comprising 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt thereof. 
     
     
         19 . A functional protein comprising at least one labeled amino acid according to  claims 14  or  15  and being labeled with a compound comprising 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt thereof. 
     
     
         20 . The functional protein according to  claims 17  or  18 , wherein the functional protein is an antibody, streptavidin derivative, or green fluorescent protein derivative. 
     
     
         21 . The functional protein according to  claim 20 , wherein the functional protein is a derivative of an anti-lysozyme camel antibody. 
     
     
         22 . The derivative of an anti-lysozyme camel antibody according to  claim 21 , wherein fluorescence polarization increases upon binding to a lysozyme molecule, wherein the lysozyme molecule is an antigen. 
     
     
         23 . A method for synthesizing the functional protein comprising the labeled amino acid according to  claim 1 , wherein the label compound is introduced via a cell-free protein synthesis system or a protein synthesis system in living cells. 
     
     
         24 . The method for synthesizing the functional protein according to  claim 23 , further comprising utilization of one selected from the group consisting of a four-base codon, a termination codon, and an artificial base codon. 
     
     
         25 . The method for synthesizing the functional protein according to  claim 24 , wherein the method of protein synthesis utilizes a four-base codon. 
     
     
         26 . An aminoacyl-tRNA, wherein the labeled amino acid according to  claim 1  and tRNA having an anticodon complementary to a codon that is designed to encode the amino acid are bound to each other. 
     
     
         27 . A method for synthesizing the aminoacyl-tRNA according to  claim 26 , comprising:
 binding pdCpA to an amino acid to obtain a pdCpA-amino acid conjugate;   reacting the pdCpA-amino acid conjugate with a label compound to prepare a labeled amino acid-pdCpA conjugate; and   binding the labeled amino acid-pdCpA conjugate to tRNA(-CA).   
     
     
         28 . The functional protein according to  claim 19 , wherein the functional protein is an antibody, streptavidin derivative, or green fluorescent protein derivative. 
     
     
         29 . The functional protein according to  claim 28 , wherein the functional protein is a derivative of an anti-lysozyme camel antibody. 
     
     
         30 . The functional protein according to  claim 29 , wherein fluorescence polarization increases upon binding to a lysozyme molecule, wherein the lysozyme molecule is an antigen.