Reprogramming the Translation Initiation for the Synthesis of Physiologically Stable Cyclic Peptides

Abstract

The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report here a new technology to reprogram the initiation event, where various amino acids and those bearing N (alpha)-acyl groups can be used as an initiator for peptide synthesis. The technology is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technology to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiologically stable bond, and it is also extended to the custom synthesis of its analogues with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodology, we also prepared a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technology offers a new means to prepare a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.

Initiating translation with D-amino acids

“…Because initiated peptide synthesis without formyl modification on their N terminus, giving the corresponding H-peptides only. This was in sharp contrast to our previously reported result that L aa-initiated peptides were generally formylated at their N terminus (Goto et al 2008). This unmistakable difference in occurrence of the N-terminal formylation of peptide initiated with did not occur during the translation in these cases; even if it occurred, its degree should be far less than 1%, which would be negligible.…”

“…All oligonucleotides were purchased from Operon Biotechnologies. Flexizyme and tRNA molecules were synthesized using the same procedure as previously described (Murakami et al 2006;Ohta et al 2007;Goto et al 2008). …”

“…The flexizyme system consists of artificially evolved ribozymes, enabling us to charge a wide variety of amino acids onto any desired tRNAs, including the initiator tRNA fMet CAU (Murakami et al 2003a(Murakami et al ,b, 2006. Indeed, we have previously reported that various L aa-tRNA fMet CAU molecules prepared by the flexizyme system can function as an initiator in the Met-withdrawn wPURE system (Goto et al 2008). Importantly, the flexizyme system tolerates not only L aa with nonproteinogenic side chains but also D aa with a variety of side chains (Murakami et al 2006).…”

“…Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1020708. initiated (Goto et al 2008). This suggests that the initiation governance can be overridden by such a genetic code reprogramming strategy.…”

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

“…Several attempts to recode the N-terminus of proteins with ncAAs have been pursued, mostly using in vitro approaches. [64][65][66][67][68] Interestingly, several Met analogues are known to work in protein initiation in vivo, indicating that they are formylated by methionyl-tRNA fMet formyltransferase (FMT) and recognized by the initiation factors (IFs). [69] In addition, their N-terminal processing is attenuated [69] and can be regulated by changing the identity of the second amino acid in the protein sequence.…”