Functionalized polystyrene supports for solid-phase synthesis of glycyl-, alanyl-, and isoleucyl-RNA conjugates as hydrolysis-resistant mimics of peptidyl-tRNAs

Abstract

RNA-peptide conjugates that mimic amino acid-charged tRNAs and peptidyl-tRNAs are of high importance for structural and functional investigations of ribosomal complexes. Here, we present the synthesis of glycyl-, alanyl-, and isoleucyladenosine modified solid supports that are eligible for the synthesis of stable 3'-aminoacyl- and 3'-peptidyl-tRNA termini with an amide instead of the natural ester linkage. The present work significantly expands the range of accessible peptidyl-tRNA mimics for ribosomal studies.

Graphical abstract

Figure 1

Constitution and sequence annotation of an exemplary target RNA-peptide conjugate that mimics 3’-peptidyl-tRNA and comprises a hydrolysis-resistant 3’-ribose amide linkage.

Scheme 1

Synthesis of the modified solid supports 8, 9, and 10. Reaction conditions: (a) 1.3 equiv Fmoc-Ala-OBt, Fmoc-Gly-OBt or Fmoc-Ile-OBt, 2.2 equiv Me₃P in tetrahydrofuran, 0 °C to rt, 16 h, 74–81%; (b) 5 equiv adipic acid bis(pentafluorophenyl)ester, 1 equiv DMAP in N,N-dimethylformamide/pyridine (1/1, v/v), rt, 1 h, 66–79%; (c) 3 equiv (w/w) amino-functionalized polystyrene support (GE Healthcare, Custom Primer Support™ 200 Amino), 2 equiv pyridine in N,N-dimethylformamide, rt, 22 h, loading: 45–63 μmol/g. Me₃P = trimethylphosphine, DMAP = 4-(N,N-dimethylamino)pyridine, HOBt = N-hydroxybenzotriazole, Fmoc = N-(9-fluorenyl)methoxycarbonyl.

Figure 2

Synthesis of RNA-peptide conjugates based on the modified solid supports 9 and 10. Anion-exchange HPLC profiles of crude and purified (insets) conjugates S7, S4, and S6 (A–C, upper panels) and LC-ESI mass spectra of purified products (A–C, lower panels). Anion-exchange chromatography conditions: Dionex DNAPac®PA-100 (4 × 250 mm) column; temperature: 60 °C; flow rate: 1 mL/min; eluant A: 25 mM Tris–HCl (pH 8.0), 6 M urea; eluant B: 25 mM Tris–HCl (pH 8.0), 6 M urea, 500 mM NaClO₄; gradient: 0–35% B in A within 30 min; UV detection at 260 nm.