Ribosomal synthesis of dehydroalanine-containing peptides.

As another approach to achieve genetic code reprogramming by a more general tRNA-acylation method, Murakami et al. reported a highly flexible-ribozyme- (flexizyme-) based tRNA-acylation system []. In the first demonstration of genetic code reprogramming using flexizymes, three sense codons were reassigned to three nonproteinogenic amino acids charged onto orthogonal tRNAs (). According to the reprogrammed genetic code, a 17 mer non-standard peptide possessing six nonproteinogenic amino acids was synthesized in a genetically encoded manner.

Ribosomal synthesis of unnatural peptides.

Total chemical synthesis of a ribozyme derived from a group I intron.

Ribosomal Synthesis of N-Methyl Peptides J.

Another approach to generate genetically encoded nonstandard peptide libraries involves the engineering of a ribosomal translation system, that is, cotranslational incorporation of nonproteinogenic amino acids into ribosomally synthesized polypeptides (). This approach is further divided into two strategies: genetic code expansion and genetic code reprogramming. In this paper, we discuss both strategies and their application for the generation of genetically encoded nonstandard peptide libraries as well as the use of these libraries for the selection/evolution of functional nonstandard peptides.

"Ribosomal Synthesis of N-methylated peptides" by Sara …

Nonstandard peptides, also known as unnatural peptides or peptidomimetics, are peptide-based small molecules containing a moiety that does not exist in standard (i.e., natural) peptides composed of only 20 proteinogenic amino acids. The nonproteinogenic moiety in nonstandard peptides, such as a nonproteinogenic side chain, a modified backbone, or a macrocyclized backbone, often contributes to improving the peptide's cell permeability, stability against peptidases, and conformational rigidity, thereby affording specific high affinity toward its target molecule [–]. Naturally occurring nonribosomal peptides (e.g., immunosuppressant cyclosporine A) are representative of nonstandard peptides, and given the success of nonribosomal peptides as therapeutics, the development of methods to construct highly diverse drug-like nonstandard peptide libraries is important for the discovery of novel drug candidates. Chemical synthesis can generate highly modified drug-like nonstandard peptide libraries, but the size of these libraries is relatively small (with a diversity of up to 106 unique compounds). In contrast, by using genotype-phenotype linking technology, ribosomal synthesis can generate genetically encoded peptide libraries with extremely high diversity (up to 1013 compounds) [–]. However, ribosomally synthesized peptide libraries are typically composed of just the 20 proteinogenic amino acids. This had led to the development of various strategies to generate highly diverse nonstandard peptide libraries; these strategies integrate biology-based methods to construct highly diverse libraries and chemistry-based methods to construct highly modified drug-like libraries.

Ribosomal synthesis of nonstandard peptides

The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.

Ribosomal Synthesis of N-methylated peptides - CORE

In summary, we have demonstrated the production of genetically encoded dehydropeptides. Our approach employs unmodified components from the translation machinery of E. coli, and mild but robust conditions to install multiple electrophilic functions in linear or cyclic peptides. The simplicity of this methodology should allow wide applicability, making highly decorated peptides available independently of solid phase peptide synthesis. Furthermore, having introduced ΔAla to the toolbox of mRNA-templated peptide synthesis, we may now embark on ultrahigh throughput selections for specific protein-reactive or catalytically active compounds.

Ribosomal Synthesis of Peptidase-Resistant Peptides …

The first model study of the in vitro display selection of a non-standard peptide prepared with a reconstituted translation system was reported by Forster et al. []. Five kinds of biotinylated peptides bearing different lengths of polypeptide spacer were synthesized in a ribosome display format, and biotinylated peptides with a spacer long enough to exit from the ribosome tunnel were selectively pulled down using streptavidin beads. However, in this paper, a diverse non-standard peptide library was not constructed and a novel functional non-standard peptide was not obtained. Very recently, several papers have been published on the selection of functional non-standard peptides by combining genetic code reprogramming with mRNA display selection. In this section, we review the preparation of non-standard libraries and the results of the selection in these reports.