Ribosomal Incorporation of Consecutive β-Amino Acids

Abstract

Due to their unique characteristics, which are not shared by canonical α-peptides, peptides that contain stretches of consecutive β-amino acids are attractive scaffolds for novel peptide drugs and nanomaterials. Although ribosomal incorporation of single or nonconsecutive β-amino acids into peptides has previously been reported, the incorporation of consecutive β-amino acids has not yet been accomplished. This is primarily due to their incompatibility with the ribosomal translation system. Here, we took advantage of engineered β-aminoacyl-tRNAs bearing optimized T-stem and D-arm motifs for enhancing binding affinity to EF-Tu and EF-P, respectively. Combined with a reconstituted E. coli translation system and optimized translation factor concentrations, up to seven consecutive β-amino acids could be incorporated into a model peptide. Furthermore, the synthesis of macrocyclic β-peptides closed by a thioether bond between two d-α-amino acids is also demonstrated. This represents the first example of the ribosomal synthesis of peptides containing stretches of consecutive β-amino acids.

Ribosomal Incorporation of Aromatic Oligoamides as Peptide Sidechain Appendages

“…Amf(-Gly-(Q Dap ) 3 analogues, which is consistent with our recent studies on translation initiation. [13] The translated products were systematically accompanied by a truncated peptide, which depended on the template, shedding light on different aspects of elongation.…”

“…of non-proteinogenic amino acids into peptides and proteins, greatly expanding available structures, functions, and chemistries. Despite eons of evolution, the ribosome displays startling tolerance towards residues significantly deviating from the twenty canonical a-amino acids: d-, [1,2] b- [3,4] and Nmethyl- [5] amino acids, and a-amino acids equipped with bulky and exotic sidechains [6] have been charged on tRNA by means of flexizymes and, when combined with a reconstituted flexible in vitro translation (FIT) system, [7] have been ribosomally incorporated within a-peptides. Capitalizing on the even greater tolerance of the ribosome during translation initiation, exotic peptides, [8] terpenes [9] and, recently, nonamino acid substrates [10,11] and aromatic oligoamide foldamers, [12,13] have been incorporated as the first unit of ribosomal peptide synthesis to produce various types of hybrid molecules.…”

“…Amf(-Gly-foldamer) amino acids were activated as cyanomethyl esters and the ability of flexizyme to load these new substrates onto tRNA was assessed using an established protocol involving microhelix RNA (Supporting Information, Figures S1-S3). [7] The results of the aminoacylation reaction ( Table 1) indicated that for Amf(-Gly-Q-Ac) (1,2) and Amf(-Gly-Q 2 -Ac) (3,4), the reaction proceeded adequately both with negatively charged Q Asp and positively charged Q Dap . In contrast, for sequences including P, that is Amf(-Gly-QP-Ac) and Amf(-Gly-QPQ-Ac), the reaction only worked with Q Dap (5,7).…”

“…The size and rigidity of these sidechains is comparable to that of other large previously reported residues. [23] Amf bearing two foldamer units (3)(4)(5) was incorporated within the peptide sequence, but only the positively charged, 3 and 5, were incorporated at the C-terminus of the peptide. Overall, these results exceeded our expectations given their size and aromatic nature.…”

Exploring the Minimal RNA Substrate of Flexizymes

“…Further in vitro flexizyme selection resulted in dFx, optimized for an amino acid substrate with a DBE moiety. Since then, dFx has been used to synthesize acyl‐tRNAs with amino acids bearing nonaromatic side chains, including those with different side chains (e.g., l ‐Ala, l ‐Ser, and l ‐Arg), with the d ‐configuration (e.g., d ‐Ala, d ‐Ser, and d ‐Arg), with N‐modified backbones (e.g., N ‐acyl‐Lys, N ‐methyl‐Ala, and N ‐alkyl‐Gly), and also with methylene‐extended backbones [e.g., l ‐β‐homoIle ( l ‐βhIle), l ‐β‐homoPhg ( l ‐βhPhg), and l ‐β‐homoGlu ( l ‐βhGlu)] …”