Synthesis of RNA-peptide conjugates - acylated tRNA mimics

The translation of short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. These antibiotics bind to the ribosomal peptidyl transferase center (PTC) next to the place of peptide bond formation and closed the entrance of the ribosomal exit tunnel. It is known that the amino acid sequence and size of peptides are critical for their activity. Moreover, a significant correlation between different peptide consensus sequences and structurally different macrolide antibiotics to which resistance is conferred, suggest a direct interaction between the peptide, the drug, and the ribosome. Since the peptide alone does not cause resistance, a likely scenario is that translation of these peptides may result in expulsion of the macrolide antibiotics. In this context, the two major goals of this proposal on synthetic and bioorganic chemistry are 1) the development of a synthetic approach for the efficient access to non-hydrolizable RNA-peptide conjugates that mimic aminoacylated transfer-RNA at the ribosomal peptidyl-transferase center (to date, their limited availability represents a major hurdle for studies on the aforedescribed resistance phenomenon). 2) to unravel the molecular mechanism of macrolide antibiotic resistance and associated ribosomal stalling by chemical and biochemical methods and - as a long-term aim - by X-ray crystallography (based on the conjugates synthesized). Ad 1) We will elaborate a strategy to synthesize oligoribonucleotides that are linked via a terminal 3`-amino-3`- deoxyadenosine to the C-terminus of a peptide of variable sequence by a non-hydrolizable amide bond. Importantly, our design should allow synthesis of both the oligonucleotide and the peptide on the same solid support via a specifically functionalized nucleoside/amino acid building block. The synthetic challenge is to explore protecting group concepts that are compatible with RNA and peptide solid-phase synthesis and - at the same time - retain high flexibility with respect to the chemical diversity of amino acid side chains and their chemical modification. We will select the sequences of our target conjugates based on recently discovered, short peptides that when translated at the ribosome cause macrolide antibiotic resistance and/or ribosome stalling. The nucleotide sequences of our target conjugates will contain the CCA terminus of tRNA. Another key feature of our strategy faces tailoring of conjugates for enzymatic ligation of RNA as well as native chemical ligation of peptides to enable access to non-hydrolizable full-length tRNAs with large peptide chains. Ad 2) Once the chemical synthesis of these conjugates has been established we first aim at verification of their binding to the ribosomal peptidyl-transferase center (by probing experiments) and then proceed with biochemical and structural characterization of macrolide antibiotic resistance to reveal the underlying molecular mechanism. This part of the project will be extended to a collaborative effort together with the laboratories of Norbert Polacek (Medical University Innsbruck) and Daniel Wilson (Gene Center, LMU, Munich).

The overall scientific goal of the project was to establish robust approaches for the synthesis of non-hydrolizable RNA-peptide conjugates that mimic aminoacylated transfer-RNA at the ribosomal peptidyl-transferase center. These derivatives are frequently needed for structural and molecular biology approaches to elucidate ribosomal complexes and their function. When the project started in 2010, efficient access to synthetic peptidyl-tRNA mimics was almost completely lacking. Only a single study was reported in the literature. The obvious need and requests for these compounds, mainly from structural and molecular biologists who were working in the ribosome field, prompted us to put serious efforts into the creation of efficient synthetic routes towards these derivatives. To achieve this goal, the following hurdles were addressed and successfully solved: i) We increased the spectrum of appropriate solid-phase supports for the synthesis of peptidyl-RNA conjugates. ii) We developed a flexible synthetic route to hydrolysis-resistant tRNA mimics, allowing the implementation of modern bioconjugation reactions such as Click and Staudinger ligations. iii) We elaborated a semisynthetic path for tRNAs that contain their naturally occurring modifications together with a hydrolysis-resistant linkage to the peptidyl chain. iv) We created a synthetic approach based on the concept of native chemical ligation to guarantee utmost flexibility of amino acid side chains with respect to the synthesis of hydrolysis-resistant peptidyl-tRNA mimics. v) We extended the above synthesis strategies towards implementation of posttranslational amino acid modifications such as methylations and phosphorylations for the synthesis of hydrolysis-resistant peptidyl-tRNA mimics.This project therefore contributed significantly to make peptidyl-tRNA mimics available for comprehensive investigations of ribosome function. In particular, structural biology approaches for the investigation of ribosome complexes will benefit from the facile access. Currently, these RNA-peptide conjugates are key compounds in an ongoing international collaboration (Prof. Alexander Mankin, University of Illinois at Chicago) to unravel the molecular mechanism of macrolide antibiotic resistance and associated ribosomal stalling. Importantly, a new collaboration with Prof. Marat Yusupov (IGBMC Strasbourg) resulted in crystal structures of the eukaryotic ribosome bound to diprolyl-tRNA mimic and to the translation factor eIF5A provide mechanistic insights into how prolines induce translational stalling and how this stalling is alleviated by hypusine an unusual amino acid present on a single cellular protein eIF5Aa. This work has just been submitted for publication in a high IF journal.