Premium
In Situ Deprotection and Incorporation of Unnatural Amino Acids during Cell‐Free Protein Synthesis
Author(s) -
Arthur Isaac N.,
Hennessy James E.,
Padmakshan Dharshana,
Stigers Dan J.,
Lesturgez Stéphanie,
Fraser Samuel A.,
Liutkus Mantas,
Otting Gottfried,
Oakeshott John G.,
Easton Christopher J.
Publication year - 2013
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201203923
Subject(s) - hydrazide , amino acid , chemistry , side chain , in situ , cell free protein synthesis , leucine , combinatorial chemistry , protein biosynthesis , organic chemistry , biochemistry , polymer
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 (2 S ,3 R )‐4‐chlorovaline into a protein through the direct use of their respective precursors, namely, ( S )‐4‐fluoroleucine hydrazide, ( S )‐4,5‐dehydroleucine hydrazide, and (2 S ,3 R )‐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.