A new water soluble copper N-heterocyclic carbene complex delivers mild O⁶G-selective RNA alkylation

Abstract

We show here that copper carbenes generated from diazo acetamides alkylate single RNAs, mRNAs, or pools of total transcriptome RNA, delivering exclusively alkylation at the O6 position in guanine (O6G). Although the reaction is effective with free copper some RNA fragmentation occurs, a problem we resolve by developing a novel water-stable copper N-heterocyclic carbene complex. Carboxymethyl adducts at O6G are known mutagenic lesions in DNA but their relevance in RNA biochemistry is unknown. As a case-in-point we re-examine an old controversy regarding whether O6G damage in RNA is susceptible to direct RNA repair.

Synthesis of Various Water Soluble and Stable Copper Complexes Bearing N‐Heterocyclic Carbene Ligands and Their Activity in DNA Alkylation

“…e., 5, 6b, and 6c) are the best-performing catalysts. The complex is easily stored as a solid (X-ray data on 6 is reported) [8] or can be kept as a frozen DMSO stock solution for months without losing catalytic activity. The ability to perform click reactions directly on the copper complex is especially practical and means that complex 6 can serve as a starting point for producing Cu-NHC complexes customized for any particular application.…”

“…The O 6 G selectivity was further confirmed by complete digestion and comparison to authentic standards as previously described. [8] Helv. Chim.…”

“…Bi-or tridentate N-donor ligands can reduce the redox problem, [6] but our interest in aqueous copper catalysis stems from the unique ability of copper(I) carbenes to alkylate guanine in various nucleic acids with high chemoselectivity (see Scheme 1). [7,8] This reaction is completely suppressed by the N-donor ligands in common usage for the copper-catalyzed azide alkyne cycloaddition (CuAAC), but proceeds smoothly with Cu-NHC complexes. [7,8] This promising result has been difficult for us to build on because there are so few water-soluble copper-NHC (Cu-NHC) complexes available.…”

“…[7,8] This reaction is completely suppressed by the N-donor ligands in common usage for the copper-catalyzed azide alkyne cycloaddition (CuAAC), but proceeds smoothly with Cu-NHC complexes. [7,8] This promising result has been difficult for us to build on because there are so few water-soluble copper-NHC (Cu-NHC) complexes available. Herein, we describe our results on trying to build a stable, modular, and water-soluble family of Cu-NHC complexes.…”

Transition‐Metal‐Mediated Modification of Biomolecules

“…In contrast to donor‐acceptor copper carbenes, which reacted mainly through N−H alkylation of the exocyclic amine groups (Section 3), unstabilized copper carbenes were shown by Gillingham's group to result in a completely different reactivity: selective O 6 guanine alkylation in ss DNA and RNA (Figure ). Mechanistic studies pointed to a substrate directed reaction, where copper coordinates to N 7 of guanine, directing the position of alkylation towards O 6 and resulting in cleaner and quicker reactions (up to 92 % in only 0.5 h).…”