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' m(7)GpppNNN...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 degrees C, pH 7.4). Product analysis of the Eu(THED)(3+) reaction upon the capped monoribonucleotide, m(7)GpppG, showed nucleotide diphosphates, GDP and m(7)GDP, and nucleotide monophosphate THED adducts, GMP-TEED and m(7)GMP-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 (k(0)) were 5.3 x 10(-5) and 4.5 x 10(-5) s(-1) far cleavage of GpppG and m(7)GpppG, respectively, at a complex concentration of 0.31 mM and substrate concentration of 0.031 mM (pH 7.4, 37 degrees 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 k(1) 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 be a nucleophilic displacement reaction, where the nucleophile is the metal activated hydroxyethyl group of the THED ligand.