Cleavage of the 5‘ Cap Structure of mRNA by a Europium(III) Macrocyclic Complex with Pendant Alcohol Groups

Cellular and viral RNAs, synthesized by RNA polymerase II, have a unique chemical structure at their 5‘ termini, 5‘ m7GpppNNN... 3‘, referred to as the 5‘ cap. This structure plays a pivotal role in several processes of mRNA metabolism, including translation and turnover. Consequently, the 5‘ cap is a potential target for antisense directed chemistries. In this study the cyclen based lanthanide complexes, Eu(THED)3+, Eu(s-THP)3+, and La(TCMC)3+, were evaluated for their reactivity upon the 5‘ cap structure. Only Eu(THED)3+ showed significant reactivity upon a 5‘ capped RNA:antisense substrate (86% decapitation after 4 h in the presence of a 0.10 mM complex at 37 °C, pH 7.4). Product analysis of the Eu(THED)3+ reaction upon the capped monoribonucleotide, m7GpppG, showed nucleotide diphosphates, GDP and m7GDP, and nucleotide monophosphate THED adducts, GMP-THED and m7GMP-THED, as major products. The isolated adducts were characterized as phosphodiesters comprising a hydroxyethyl pendant group attached to the phosphate group. Pseudo-first-order rate constants (k0) were 5.3 × 10-5 and 4.5 × 10-5 s-1 for cleavage of GpppG and m7GpppG, respectively, at a complex concentration of 0.31 mM and substrate concentration of 0.031 mM (pH 7.4, 37 °C). Cleavage of GpppG was first order in Eu(THED)3+ for complex concentrations ranging from 0.158 to 1.26 mM. A plot of k1 versus concentration gave a second-order rate constant of 0.17 M-1 s-1. Cleavage of the 5‘ cap structure by Eu(THED)3+ is proposed to occur by a nucleophilic displacement reaction, where the nucleophile is the metal activated hydroxyethyl group of the THED ligand.