TET‐Like Oxidation in 5‐Methylcytosine and Derivatives: A Computational and Experimental Study

Abstract

The epigenetic marker 5-methylcytosine (5mC) is an important factor in DNA modification and epigenetics. It can be modified through a three-step oxidation performed by ten-eleven-translocation (TET) enzymes and we have previously reported that the iron(IV)-oxo complex [Fe(O)(Py 5 Me 2 H)] 2 + (1) can oxidize 5mC. Here, we report the reactivity of this iron(IV)-oxo complex towards a wider scope of methylated cytosine and uracil derivatives relevant for synthetic DNA applications, such as 1methylcytosine (1mC), 5-methyl-iso-cytosine (5miC) and thy-mine (T/5mU). The observed kinetic parameters are corroborated by calculation of the CÀ H bond energies at the reactive sites which was found to be an efficient tool for reaction rate prediction of 1 towards methylated DNA bases. We identified oxidation products of methylated cytosine derivatives using HPLC-MS and GC-MS. Thereby, we shed light on the impact of the methyl group position and resulting CÀ H bond dissociation energies on reactivity towards TET-like oxidation.

Spectroscopic and in vitro Investigations of Fe2+/α‐Ketoglutarate‐Dependent Enzymes Involved in Nucleic Acid Repair and Modification

“…[77] While HPLC(-MS) can be used to detect both nucleoside and nucleobase substrates, GC-MS requires the digestion of DNA to the nucleobases since (derivatized) nucleosides are not stable under the conditions of GC-MS. Derivatization of nucleobases with for example N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in acetonitrile and subsequent detection as well as quantification has been reported for a range of epigenetically relevant cytosine derivatives as well as for artificial DNA bases. [78] Not only single nucleosides or nucleobases can be processed with HPLC, DNA oligomers can also serve as analytes. In 2009, Yu and Hunt published their results on EcAlkB activity on trimeric (TmdCT or TmdAT or TɛdAT) or pentameric (3mdC or 1mdA in different combinations with C/T/A, as well as 1mdG, 3mT and ɛA) substrates.…”

Selective Functionalisation of 5‐Methylcytosine by Organic Photoredox Catalysis

“…A key requirement for DNA is selectivity for the C5methyl group of 5mC over the C5-methyl group of the canonical thymine (T) nucleobase (Figure 1A). The bond dissociation energies of the CÀ H bond in C5-methyl groups of 5mC and T differ by less than 5 kJ mol À 1 (387.3 kJ mol À 1 and 383.1 kJ mol À 1 respectively), [8] therefore it would be challenging to achieve selectivity via 5mC-methyl radical formation by direct hydrogen-atom abstraction. 5mC-methyl radical formation can also be achieved via deprotonation of the 5mC radical cation.…”

Selective Functionalisation of 5‐Methylcytosine by Organic Photoredox Catalysis

“…A key requirement for DNA is selectivity for the C5‐methyl group of 5mC over the C5‐methyl group of the canonical thymine (T) nucleobase (Figure 1A ). The bond dissociation energies of the C−H bond in C5‐methyl groups of 5mC and T differ by less than 5 kJ mol −1 (387.3 kJ mol −1 and 383.1 kJ mol −1 respectively), [8] therefore it would be challenging to achieve selectivity via 5mC‐methyl radical formation by direct hydrogen‐atom abstraction. 5mC‐methyl radical formation can also be achieved via deprotonation of the 5mC radical cation.…”