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

Abstract

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.

Organocatalytic asymmetric Michael addition of aldehydes and ketones to nitroalkenes catalyzed by adamantoyl l-prolinamide

“…24 To a stirred solution of corresponding freshly distilled aldehyde or ketones (3 mmol, 3.0 equiv) in indicated solvent (2 mL) were added catalyst and benzoic acid. The mixture was stirred at the 70 indicated temperature for 30 min, then corresponding nitroolifen (1 mmol, 1.0 equiv) was added.…”

Peptide Synthesis through Cell‐Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release

“…The amino acid sequence is fully controlled by the translation of genetic material . Native and engineered biosynthetic machinery can be used to incorporate a wide range of amino acids, in addition to those normally found in proteins, thereby expanding the utility of this method …”

“…Cell‐free expression using the supernatant of the lysate of Escherichia coli BL21 star (DE3) after centrifugation at 30 000 g was used to produce 1 a attached to the His 6 ‐tagged carrier protein peptidyl‐prolyl cis ‐ trans isomerase B (His 6 PpiB). After metal‐ion affinity chromatography, the purified fusion protein was incubated with enterokinase at 24 °C for 18 h to release 1 a , which was isolated through HPLC (Scheme ).…”

“…Previously we reported incorporation of chlorides 14 – 18 as substitutes for the corresponding aliphatic amino acids 11 – 13 during cell‐free expression of His 6 PpiB . The earlier studies were performed with a mixture of 16 and 17 , but these compounds have now been studied separately, and each is incorporated into protein as a substitute for Leu ( 12 ).…”

Biosynthetic Incorporation of Fluorinated Amino Acids into Peptides and Proteins

“…Cellular extracts used for cell-free expression also contain enzymes that catalyse the removal of a wide range of the protecting groups employed in the synthesis of amino acids, so the deprotection of an amino acid to be incorporated can be carried out in situ to avoid separate processing steps. [25,26] This is particularly useful with unstable amino acids, such as fluoroleucine 12. It rapidly decomposes through lactone formation but this can be avoided 5 6 Fig.…”