10.1002/chem.201902340
Chemistry - A European Journal
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amounts of stock solutions of 5mC and d3-5hmC. The overall volume was then
adjusted to 200 µl, for more details, see SI).
experimentally, that it is most likely the binding and orientation of
substrate in the active site pocket in TET enzymes (not the
strength of the C-H bond that is to be activated) that leads to this
unusual selectivity, highlighting the absolute requirement of a
second coordination sphere. In future investigations, nucleosides
and nucleotides should be used as substrates, this would slowly
increase the level of complexity in the substrate system in order
to resemble the natural substrate, DNA, more closely, as well as
a step wise incorporation of a second coordination sphere on the
side of the model complex.
These experiments show a large KIE which suggests a rate
determining C-H bond cleavage for the reaction of methylated
cytosine derivatives with 1 (step I in Scheme 5) opposed to a rate-
limiting rebound step II. Specifically, primary KIEs >1 (~9 and 29)
are observed for 5mC and 5hmC and these are well within the
range of commonly observed KIEs of Fe/α-KG enzyme
substrates.[6] It is important to highlight that the rate of formation
of the FeIV=O species is, here, not relevant for the C-H activation,
as substrates are added to an already formed active species 1.
The FeIV=O species in enzymes is of a fleeting nature and
impossible to trap at room temperature. This uncoupling of
essential steps in enzyme catalytic cycles is one major advantage
of biomimetic complexes.
Experimental Section
All experimental procedures such as optimized complex and substrate
syntheses as well as reaction workup procedure, GC-MS as well as UV vis
kinetic protocols and NMR spectra can be found in the supporting
information.
Acknowledgements
NSWJ and LJD are supported by the SFB1309 “Chemical Biology
of Epigenetic Modifications” Project C5. We would like to thank
Maren Haas of the research group of Prof. Oliver Trapp for her
help in finding a suitable GC-MS method. Additionally, we would
like to thank Eva Korytiakova of the research group of Prof.
Thomas Carell for her help with HPLC separations of 5hmC and
d2-5hmC and Johann de Graaff for his preliminary investigations
into the synthesis of d3-5mC.
Scheme 5. Proposed reaction mechanism of 1 with 5mC/d3-5mC consisting of
a hydrogen atom transfer from the substrate to the oxo-moiety (step I) and a
rebound step in which the product is formed via a transfer of the hydroxyl group
(step II).
Keywords: 5-methyl cytosine • DNA methylation • enzyme
models • TET enzymes • iron(IV)-oxo • bioinorganic chemistry
Conclusions
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