In Situ Deprotection and Incorporation of Unnatural Amino Acids during Cell-Free Protein Synthesis

The S30 extract from E. coli BL21 Star (DE3) used for cell‐free protein synthesis removes a wide range of α‐amino acid protecting groups by cleaving α‐carboxyl hydrazides; methyl, benzyl, tert‐butyl, and adamantyl esters; tert‐butyl and adamantyl carboxamides; α‐amino form‐, acet‐, trifluoroacet‐, and benzamides; and side‐chain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)‐4‐fluoroleucine, (S)‐4,5‐dehydroleucine, and (2S,3R)‐4‐chlorovaline into a protein through the direct use of their respective precursors, namely, (S)‐4‐fluoroleucine hydrazide, (S)‐4,5‐dehydroleucine hydrazide, and (2S,3R)‐4‐chlorovaline methyl ester. These results also show that the fluoro‐ and dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively. Well‐protected: An E. coli extract used in cell‐free protein synthesis removes a wide range of amino acid protecting groups for direct incorporation of the resulting free amino acids into proteins (see scheme). The synthesis of unnatural amino acids is simplified and problems associated with chemical instability of amino acids during their deprotection and protein synthesis are circumvented.