Native Chemical Ligation of Hydrolysis-Resistant 3′-Peptidyl–tRNA Mimics

Abstract

Hydrolysis-resistant 3'-peptidyl-RNA conjugates that mimic tRNA termini represent a remarkable synthetic challenge, particularly if they contain amino acids with complex side-chain functionalities, such as arginines. Here we demonstrate a novel approach that combines solid-phase synthesis and bioconjugation to obtain these derivatives with high efficiency and purity. The key step is native chemical ligation of 3'-cysteinyl-RNA fragments to highly soluble peptide thioesters. The so-prepared 3'-peptidyl-RNA conjugates relate to resistance peptides that can render the ribosome resistant to macrolide antibiotics by a yet unknown ribosomal translation mechanism.

Selective Desulfurization Significantly Expands Sequence Variety of 3′‐Peptidyl–tRNA Mimics Obtained by Native Chemical Ligation

“…Desulfurization procedure: After native chemical ligation of a 3′-peptidyl-RNA conjugate, which was carried out as described previously,7 the reaction mixture (∼12 μL) was diluted with nanofiltered water (500 μL), transferred into a centrifugal concentrator with an ultrafiltration membrane (Vivaspin 500, 3000 MWCO PES, product number VS0191), and centrifuged for 20 min (Eppendorf MiniSpin, 13 400 rpm). The solution in the lower reservoir was discarded.…”

“…NCL was originally developed to link unprotected peptide fragments under mild conditions, and this approach eventually emerged as a major advance in chemical protein synthesis 8. Our work has shown that NCL can also work efficiently in the context of RNA7 and therefore might serve as a launching point for further investigations in the field of RNA bioconjugation.…”

“…We have recently elaborated a convergent strategy that involves native chemical ligation (NCL) of 3′-cysteinylamino-3′-deoxy-RNA and peptide thioesters 7. NCL was originally developed to link unprotected peptide fragments under mild conditions, and this approach eventually emerged as a major advance in chemical protein synthesis 8.…”

“…18 NCL was conducted under the optimized conditions based on our previously elaborated protocol,7 involving concentrations of 0.25 m m cysteinyl-RNA and 8 m m peptide thioester in Tris buffer (1 m ) at pH 8.0, in the presence of urea (7 m ) and tris(carboxyethyl)phosphine (TCEP; 0.1 m ) for S - t Bu removal in situ,19 and thiophenol (2 % ( v / v )) for formation of more reactive thioesters 20. After a typical reaction time of 20 h at 25 °C, the reaction mixture was applied to centrifugal concentrators with ultrafiltration membranes for desalting and separation from excess peptide thioesters; finally, the crude products were lyophilized.…”

The Synthesis of Methylated, Phosphorylated, and Phosphonated 3′‐Aminoacyl‐tRNASec Mimics

“…The structural complexity of the amino acid-RNA and peptide-RNA conjugates, in particular the 3'-peptidyl-tRNA mimics, present a series of challenges for de novo synthesis. [10][11][12][13][14][15][16][17][18][19][20][21] The importance of tRNA conjugates in probing fundamental biological enzymatic mechanisms argues that the development of efficient synthetic protocols for such ligands is warranted.…”