# Aldehyde or Hydrate? Investigation into the Oxidation of 5‐Formylcytosine Derivatives Using a Computational and Experimental Approach

## TL;DR

This study explores how iron(IV)-oxido complexes oxidize 5-formyl nucleobases, revealing that hydrate forms play a key role in the reaction mechanism.

## Contribution

The study identifies hydrate-mediated oxidation as a novel pathway for 5-formyl nucleobase oxidation by iron(IV)-oxido complexes.

## Key findings

- Hydrate formation significantly influences the oxidation kinetics of 6-aza-derivatives.
- Rate constants correlate with C—H bond dissociation values for 5-hydroxymethyl nucleobase oxidation.
- Geminal diol intermediates accelerate hydrogen-atom transfer in the oxidation process.

## Abstract

This study investigates the oxidation of 5‐hydroxymethyl and 5‐formyl nucleobases using an iron(IV)‐oxido complex that mimics the function of TET enzymes. A central question in this context is whether the oxidation of formyl substrates proceeds via the aldehyde or the hydrate form. To investigate the possible different reaction kinetics of these two forms, nucleobases containing a 6‐aza‐moiety are employed, giving rise to significantly more aldehyde hydrate as compared to the unaltered nucleobase. The concentration changes of substrates and products during oxidation were followed with 1H NMR spectroscopy. To analyze the kinetics of the oxidation reactions, a detailed numerical simulation of the stepwise sequential oxidation process is applied. 5‐Hydroxymethyl nucleobases are first oxidized to the respective 5‐formyl derivatives, which exist in equilibrium with their hydrate forms, and then further oxidized to the final 5‐carboxyl nucleobases. The rate constants for 5‐hydroxymethyl nucleobase oxidation show a good correlation with C—H bond dissociation values. The influence of hydrate formation on sequential oxidation is most prominent in the 6‐aza‐derivatives. The results not only deepen our understanding of substrate oxidation by iron‐oxido species but also pave the way for future studies on related biological oxidation mechanisms.

Hydrate‐mediated oxidation of 5‐formyl nucleobases by the iron(IV)‐oxido complex proceeds via geminal diol intermediates, accelerating hydrogen‐atom transfer and revealing aza‐dependent kinetic and mechanistic pathways.© 2025 WILEY‐VCH GmbH