Ribosomal Synthesis of Peptides with C‐Terminal Lactams, Thiolactones, and Alkylamides

Abstract

All change at the C terminus: We have established a novel methodology for the ribosomal synthesis of peptides featuring C-terminal cyclization and various modifications, including macrocyclization, by making use of genetic code reprogramming. The C-terminal amide modification of linear and cyclic peptides should enhance their physiological stabilities, and open up the possibility of developing new drug-like peptides. The C terminus of a peptide expressed by the translation apparatus generally ends in a carboxylate group. On the other hand, the C termini of some naturally occurring peptides have amide moieties instead of carboxylates, which are believed to give better biostability. Here, we describe a new strategy for the ribosomal synthesis of peptides featuring C-terminal lactam, thiolactone, and alkylamide units. The method was based on the concept of genetic code reprogramming involving the flexizymes (flexible tRNA acylation ribozymes) and the PURE (peptide synthesis using recombinant elements) system, in which vacant codons are reassigned to nonproteinogenic amino acids; this enabled us to convert the C termini of peptides into the above functionalities. We have also applied this method to the synthesis of a macrocyclic peptide closed by an amide bond formed between a lysine side chain and the peptide C terminus. This method thus offers us new opportunities to express various peptides with C-terminal modifications as well as macrocyclic peptides using the translation apparatus, and potentially to accelerate the discovery of peptidic drugs designed for various therapeutic targets.

In Vitro Biosynthesis of Peptides Containing Exotic Azoline Analogues

“…We further attempted to expand the repertoire of the ring size from five to six by using Hcy and Dab, which contained γ‐sulfhydryl and amino groups, respectively. The precursor peptides bearing these non‐proteinogenic amino acids were expressed in the reprogrammed FIT system by using the previously reported side‐chain protection strategy . It should be noted that, although cyclodehydration of Hcy by McbB/C, a split YcaO‐like cyclodehydratase involved in the biosynthesis of microcin B17, had previously been tested by using a synthetic peptide ligated to the cognate LP, the Hcy residue was not modified .…”

Engineering the Translation Apparatus to Incorporate Nonnatural Amino Acids

“…Impressively, this system is able to initiate translation with tRNA charged with peptides [40]. The incorporation of nonnatural amino acids by flexizymes and successive reactions between nonnatural amino acids can be further utilized as a novel way to generate cyclic peptides, which may have important physiological effects [38,[41][42][43][44][45]. In addition, the flexizyme has been further modified to increase its tRNA specificity by adding a sequence to its 3'-end [46].…”

Protein Based Drug Discovery

“…Novel research strategy for the ribosomal synthesis of peptides featuring Cterminal lactam, thiolactone, and alkylamide units is described. The method is based on the concept of genetic code reprogramming involving the flexizymes (flexible tRNA acylation ribozymes) and the PURE (peptide synthesis using recombinant elements) system, in which vacant codons are reassigned to nonproteinogenic amino acids; this enabled us to convert the C termini of peptides into the above functionalities [78]. CD-NP is a novel chimeric natriuretic peptide (NP) consisting of the 22-amino-acid (AA) human C-type natriuretic peptide (CNP), a venodilating peptide with limited renal actions and minimal effects on blood pressure, and the 15-AA C-terminus of Dendroaspis NP (DNP).…”