Angewandte
Chemie
DOI: 10.1002/anie.201002033
Hydroxymethylcytosine
Quantification of the Sixth DNA Base Hydroxymethylcytosine in the
Brain**
Martin Mꢀnzel, Daniel Globisch, Tobias Brꢀckl, Mirko Wagner, Veronika Welzmiller,
Stylianos Michalakis, Markus Mꢀller, Martin Biel, and Thomas Carell*
The genetic code is established by the sequence of the four
canonical DNA nucleosides dA, dC, dG, and dT.[1] Of these
four bases, only the dC base is chemically modified inside cells
of higher organisms to control transcriptional activity.[2]
Special methyltransferases replace the H atom at position 5
by a methyl group to form methylcytosine (5-MedC).[3]
Methylation occurs only in CpG sequences and is mostly
responsible for the silencing of genes.[4] In two recent
publications 5-hydroxymethylcytosine (5-HOMedC) was estab-
lished as a new post-replicatively formed DNA nucleoside
(Scheme 1). Kriaucionis and Heintz detected 5-HOMedC in
cerebellar purkinje neurons.[5] Tahiliani et al. reported the
presence of traces of 5-HOMedC (ca. 0.032% of all nucleosides)
in mouse embryonic stem cells and observed the new base in
CpG sequences in human embryonic kidney (HEK) cells
when they overexpressed the hydroxylating 2-oxoglutarate
and FeII-dependent enzyme TET in these cells.[6] It was
additionally shown that these TET enzymes are able to
oxidize the 5-methyl group of 5-MedC to give the hydroxy-
methyl group in vitro. The new base was detected using thin-
layer chromatography after radioactive labeling of the
nucleotides. The function of 5-HOMedC is currently unclear,
but it is speculated that it may establish another level of
transcriptional control or that it is an intermediate of a
putative oxidative demethylation mechanism.[7]
We developed a quantitative LC-MS method to inves-
tigate the distribution of 5-HOMedC in mammal brains and to
determine the relative quantities of 5-HOMedC and 5-MedC. To
this end, we synthesized both nucleosides in natural and
isotope-labeled forms[8] (Scheme 2) and quantified their
amounts in different mouse brain tissues.
5-HOMedC and 5-MedC were labeled as 18O and CD3
derivatives, respectively. The synthesis of 5-HOMedC started
Scheme 1. Structure of the four canonical nucleosides and of the post-
replicatively formed bases 5-MedC and 5-HOMedC.
[*] Dipl.-Chem. M. Mꢀnzel,[+] Dipl.-Chem. D. Globisch,[+]
Dipl.-Chem. T. Brꢀckl, Dipl.-Chem. M. Wagner,
Dipl.-Chem. V. Welzmiller, Dr. M. Mꢀller, Prof. Dr. T. Carell
Center for Integrated Protein Science (CiPSM) at the Department of
Chemistry, Ludwig-Maximilians-University
Butenandtstrasse 5–13, 81377 Munich (Germany)
E-mail: thomas.carell@cup.uni-muenchen.de
Dr. S. Michalakis, Prof. Dr. M. Biel
Center for Integrated Protein Science (CiPSM) at the Department of
Pharmacy, Ludwig-Maximilians-University
Butenandtstrasse 5–13, 81377 Munich (Germany)
Scheme 2. Syntheses of the isotope-labeled nucleosides 5-HOMedC,
[18O]5-HOMedC, 5-MedC, and [D3]5-MedC. a) H2O, DIPEA, 72%, b) TBSCl,
imid., 46%, c) NaH, TPSCl, d) NH3/MeOH, 76% over two steps,
e) 3HF·NEt3, 52%, f) NaH, TPSCl, g) NH3/MeOH, 81% over two
steps, h) HF·pyr, 88%, i) CD3MgI, CuCl, [Pd(PPh3)4], 90% inseparable
mixture of 6 and TBSdC, j) HF·pyr, 84% (based on pure 6).[18O]5-
HOMedC was synthesized by the same route as 5-HOMedC. The asterisk
(*) indicates the 18O label. DIPEA=N,N-diisopropylethylamine, imi-
d.=imidazole, pyr=pyridine, TBS=tert-butyldimethylsilyl, TPS=2,4,6-
triisopropylbenzenesulfonyl.
[+] These authors contributed equally to this work.
[**] We thank the excellence cluster CiPSM, SFB 646, and SFB 749 for
generous support. M. Mꢀnzel is grateful to the Fonds of the
Chemical Industry for a Kekulꢁ fellowship. We thank V. Hammel-
mann for support during this project.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2010, 49, 5375 –5377
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5375