Analysis of an Active Deformylation Mechanism of 5-Formyl-deoxycytidine (fdC) in Stem Cells

Article abstract of DOI:10.1002/anie.202000414

The removal of 5-methyl-deoxycytidine (mdC) from promoter elements is associated with reactivation of the silenced corresponding genes. It takes place through an active demethylation process involving the oxidation of mdC to 5-hydroxymethyl-deoxycytidine

Full text of DOI:10.1002/anie.202000414

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Accepted Article
Title: Analysis of an active deformylation mechanism of 5-formyl-
deoxycytidine (fdC) in stem cells
Authors: Alexander Schön, Ewelina Kaminska, Florian Schelter,
Eveliina Ponkkonen, Eva Korytiaková, Sarah Schiffers, and
Thomas Carell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202000414
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10.1002/anie.202000414
Angewandte Chemie International Edition
RESEARCH ARTICLE
Analysis of an active deformylation mechanism of 5-formyl-
deoxycytidine (fdC) in stem cells
Alexander Schön,# Ewelina Kaminska,# Florian Schelter,# Eveliina Ponkkonen, Eva Korytiaková, Sarah
Schiffers and Thomas Carell*
Abstract: 5-Methyl-deoxycytidine (mdC) is considered the fifth base
of the genetic system. Its presence in promoter elements takes part
in silencing the corresponding gene. Its removal, associated with the
reactivation of silenced genes, is possible via an enigmatic active
demethylation process that requires the oxidation of mdC to
5-hydroxymethyl-deoxycytidine (hmdC) and further on to 5-formyl-
deoxycytidine (fdC) and 5-carboxy-deoxycytidine (cadC) with the help
of α-ketoglutarate dependent Tet-oxygenases. The removal of mdC
occurs at the level of the fdC and cadC oxidation state. One pathway
involves the action of a dedicated glycosylase (TDG), which cleaves
fdC out of the genome, allowing its replacement by dC. A second is
proposed to involve C-C bond cleavage that converts fdC directly into
dC. While the TDG-induced repair-based pathway is well
characterized, information about the C-C bond cleaving process is
very limited. Here, we report the synthesis of a novel 6-aza-5-formyl-
deoxycytidine (a-fdC) probe molecule, which was fed to various
somatic cell lines and induced mESCs, together with a 2’-fluorinated
fdC analog (F-fdC). While deformylation of F-fdC was clearly
observed in vivo, it did not occur with a-fdC, suggesting that the C-C
bond cleavage.[15, 16] Evidence for the existence of such a direct
deformylation process was recently reported.[17] Model studies
showed that direct deformylation of fdC and potentially also
decarboxylation of 5-carboxy-deoxycytidine (cadC) is indeed
possible.[15] Nevertheless, it requires activation of the
nucleobases by a nucleophilic addition to the C6 position. For fdC,
an additional hydrate formation step of the formyl group seems to
be necessary as depicted in Fig. 1A. Although the activation with
a helper nucleophile is well known as the central mechanistic
process during methylation of dC to mdC by DNA-
methyltransferases (Dnmts),[18, 19] it remains yet to be confirmed if
such activation occurs in vivo as well. Here, we investigated this
hypothesis with the two probe molecules, 2’-fluorinated-fdC 2 (F-
fdC) and 6-aza-fdC
3
(a-fdC). Both compounds were
simultaneously fed to different cell types, including primed stem
cells. This led to random incorporation of these bases at the “C”
sites into the respective genomes. Furthermore, it led to the
presence of F-fdC and a-fdC not only at CpG sites. Ultrasensitive
UHPLC-QQQ-MS2 was subsequently used to interrogate the
chemical processes that occur at F-fdC and a-fdC in the genomes.
The data show that while F-fdC is efficiently deformylated, this
does not occur for a-fdC. The only difference between both
nucleobases is the presence of an in-ring nitrogen atom (6-aza
atom), which features a lone pair that prohibits a nucleophilic
addition. The experiment provides consequently strong evidence
that the nucleophilic activation in vivo is the central governing
mechanistic event that is required for C-C bond cleavage.
bond cleaving deformylation reaction must be initiated by
nucleophilic activation.
a
5-Formyl-deoxycytidine (fdC) 1, is a dC-derived nucleobase that
is found in stem cells during early development and in brain.[1-5]
These tissues are particularly rich in 5-hydroxymethyl-
deoxycytidine (hmdC) from which fdC
1
is produced.[6, 7]
Formation of hmdC and fdC requires oxidation reactions that are
performed by α-ketoglutarate-dependent Tet enzymes with
5-methyl-deoxycytidine (mdC) being the initial starting
molecule.[8-10] This cascade of oxidation reactions is a part of an
active demethylation process, in which mdC as a silencer of
transcription is replaced by unmodified dC.[11] The central
molecule that is removed seems to be fdC.[12, 13] It can be cleaved
out of the genome by a dedicated DNA glycosylase, which creates
an abasic site, that is further processed leading to the insertion of
an unmodified dC.[14] Because abasic sites are harmful DNA
repair intermediate can cause genome instability. It was early on
suggested that fdC might be directly deformylated to dC by C-C
[a]
M. Sc. A. Schön, B. Sc. E. Kaminska, M. Sc. F. Schelter, M. Sc. E.
Ponkkonen, M. Sc. E. Korytiaková, Dr. S. Schiffers and Prof. Dr. T.
Carell
Department of Chemistry, Ludwig-Maximilians Universität München
Butenandtstr. 5-13, 81377 München
Fax: (+) 49 2180 77756
E-mail: thomas.carell@lmu.de
Homepage: www.carellgroup.de
Figure 1. (A) Depiction of the mdC removal pathways that involve oxidation to
hmdC, fdC and cadC followed by either base excision repair (magenta) or C-C
bond cleavage (blue). (B) Representation of fdC 1 and the two probe molecules
2 and 3 used for this study.
# these authors contributed equally.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.202000414
Angewandte Chemie International Edition
RESEARCH ARTICLE
The fluorinated nucleoside F-fdC 2 was recently introduced by us
as a deformylation probe.[17] 2 is an antimetabolite that is
effectively incorporated into the genomes of growing cells. The
2’-Fluoro group is required to block all types of glycosylases, so
that base excision repair is efficiently inhibited. This ensures high
levels of F-fdC 2 in the genome, as required to observe potential
deformylation processes.
The synthesis of the novel nucleoside a-fdC 3 is depicted in
Scheme 1. The synthesis was started with bromo pyruvic acid 4,
which we first converted into the semicarbazone 5 followed by the
conversion into its acid chloride, subsequent cyclization and
hydrolysis to give hydroxymethylated 6-aza-uracil 6.[20]
Vorbrüggen nucleosidation with Hoffers’ chlorosugar provided
subsequently the nucleoside 7 as a mixture of the α- and β-
anomers, which could be separated by recrystallization. Next, we
acetyl-protected the hydroxymethylgroup to 8 and then used a
standard procedure to convert the U-base 8 into the C-derived
base 9 by amination of the 4-triazole intermediate with ammonium
hydroxide. This led to the concomitant cleavage of the acetyl-
protecting group. Dess-Martin oxidation of 9 to 10 and final
deprotection of the toluoyl-groups furnished the 6-aza-5-formyl-
deoxycytidine nucleoside (a-fdC) 3 in a good total yield of 22% in
respect to 6 (SI).
dividing cells. Initial studies, in which we fed the nucleosides
individually allowed us to determine that both compounds do not
decrease cell viability until a concentration of 400 µM, thus the
experiments were conducted below the toxicity level. In addition,
we tested 2 and 3 at 350 µM on E14 TDG +/- and -/- mouse
embryonic stem cells (mESC) under a three-day priming process
with C/R media. This system allowed us to exclude the BER
pathway, leading to a detectable and quantifiable accumulation of
natural fdC (SI). After three days, the cells were harvested and
lysed, the genomic DNA was extracted using an optimised
protocol (SI). This was followed by an enzymatic digestion of
gDNA to single nucleosides and analysed according to a protocol
that we reported recently in detail.[21] The obtained nucleoside
mixture containing mostly the canonical nucleosides dA, dC, dG
and dT, plus the non-canonical nucleosides mdC, hmdC and fdC,
as well as the incorporated molecules F-fdC and a-fdC and their
potential follow-up products (F-dC, F-mdC, a-dC, a-mdC).
Nucleosides were separated by ultra-HPL-chromatography and
characterized by coupling of the UHPLC system to a triple
quadrupole mass spectrometer. For exact quantification of the
nucleosides by isotope dilution, isotopically labelled standards of
F-fdC and of the product F-dC were spiked into the analysis
mixture as internal standards (SI). To enable exact quantification,
calibration curves using these standards were determined (SI).
Quantification was performed in the linear region.
During the analysis, we noted an unusually low amount of
detected a-fdC 3 because it showed a broad elution profile with
very low intensity (Fig. 2B). All attempts to sharpen the elution
profile in order to gain sensitivity failed. NMR analysis of
compound 3 showed the reason for broad elution profile (SI). Due
to the additional electron withdrawing in-ring nitrogen atom,
compound 3 exists partially as its hydrate in aqueous solution
(20%, see SI). Although the ease of hydrate formation may foster
deformylation, the hydrate/carbonyl equilibrium makes efficient
detection of compound 3 basically impossible. In order to
circumvent the problem, we started to derivatize a-fdC 3 before
the analysis with methoxyamine. Addition of CH3ONH2 (150 mM)
to the digestion solution provided indeed the methoxyoxime of
a-fdC in quantitative yield already after 15 min at 25 °C and pH 10.
The naturally present fdC 1 and the probe molecule F-fdC 2,
however, react as well, but unfortunately not quantitatively. To
reduce impurities during MS measurements we decided against
using a catalyst for oxime formation. We therefore decided to
analyse the digested DNA in two batches. The first one contained
the digested untreated DNA to quantify all bases other than a-fdC.
In the second batch, we treated the digested DNA with
methoxyamine for a-fdC quantification. For quantification
purposes of derivatized a-fdC we performed an external
calibration curve (SI).
Compound 3 features a nitrogen atom instead of a carbon atom
at C6, which possesses a lone pair that blocks any nucleophilic
addition to this position. Compound 3 is consequently a perfect
model system to investigate, if such a nucleophilic activation is
required for the deformylation as mechanistically postulated
(Fig. 1A).
Scheme 1. Synthesis of the probe molecule a-fdC 3. a. semicarbazide∙HCl,
NaOAc, HOAc, H2O, 0 °C to r.t., 2.5 h, 49%. b. pyridine, SOCl2, 80 °C, 75 min.
c. H2O, 110 °C., 17 h, 74% over 2 steps. d. TMSCl, HMDS, 135 °C, 75 min,
then e. Hoffer´s chlorosugar, CHCl3, r.t.,17 h, 56% over 2 steps. f. Ac2O,
pyridine, r.t., 22 h, 96%. g. 1,2,4-triazole, POCl3, NEt3, MeCN 0 °C to r.t., 18 h,
then h. NH4OH, 1,4-dioxane, 40 °C, 5 h, 84%. i. Dess-Martin periodinane,
CH2Cl2, -15 °C to r.t., 1 h, 89%. j. NaOMe, MeOH, benzene, r.t., 1.5 h, then k.
reversed-phase HPLC, 54%.
The nucleosides 2 and 3 were subsequently added at a
concentration of 350 µM to the media of Neuro-2a, RBL-2H3,
CHO-K1 cells for 72 hours (see SI). During this time, the
nucleosides are converted in vivo into the corresponding
triphosphates and then incorporated into the genome of the
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10.1002/anie.202000414
Angewandte Chemie International Edition
RESEARCH ARTICLE
can certainly exclude that deformylation in vivo is occurring only
with F-fdC. It is unfortunate that we are unable to measure the
direct deformylation of fdC because of the presence of efficient
BER processes. A TDG -/- cell line showed a huge increase of
fdC compared to the TDG +/-, whereas a-fdC and F-fdC stay
constant showing that these compounds are indeed not repaired
by the TDG protein (see SI). The bisulfite studies however show
that the F-fdC compound is not a perfect but sufficient reporter of
this C-C bond cleavage reaction. Treatment of the a-fdC
compound 3 with bisulfite provided under no circumstances the
deformylated product a-dC, showing that the inability to react with
a nucleophile at position C6 totally blocks the C-C bond cleavage
reaction. We can therefore conclude that the deformylation of fdC
during active demethylation requires oxidation of mdC to fdC. fdC
can undergo a direct C-C bond cleavage to dC, but this reaction
requires a helper nucleophile to attack the C6-position, which is
blocked in the case of a-fdC by the lone pair of the C6 carbon to
nitrogen exchange. While the chemistry that allows the
transformation of fdC to dC is now elucidated, we next need to
find the nucleophiles that perform the reaction in vivo.
Figure 2. (A) Overview of the experimental steps with the feeding and analysis.
(B) Analysis scheme and depiction of the reaction of a-fdC with methoxyamine
to block hydrate formation and of a typical UHPL-chromatogram before (C-8
column) and after derivatization (C-18 column) for exact quantification. Peak
splitting is due isomerization (blue peaks: a-fdC, red peaks: fdC and purple
peaks: F-fdC).
With this method in hand, we next quantified all nucleosides
present in the genome of the cells treated with a mixture of (2+3).
Fig. 3A shows that we indeed detected the fluorinated F-dC
compound 2, confirming very efficient deformylation activities. We
tested different cell types and found different levels of
deformylation activities. But in all cases, the conversion of F-fdC
into F-dC was clearly detectable. Most interesting is that we
observed the largest deformylation activites in cells associated
with neuronal properties. It goes in line with neurons featuring the
highest levels of hmdC and fdC. In contrast, Fig. 3B shows for the
a-fdC compound 3 that we were unable to detect any formation of
the deformylated compound a-dC despite the high propensity of
3 to exist in the hydrated form which is one prerequisite for
efficient C-C bond cleavage. This result suggests that the ability
to react with a nucleophile at position C6 is also in vivo required
for efficient deformylation.
In order to substantiate this result, we next performed in vitro
studies with bisulfite. Bisulfite is a strong nucleophile that was
reported to cause deformylation of fdC by first attacking the C6
position, followed by conversion of the C5-C6 saturated fdC
adduct into the bisulfite adduct that then undergoes
deformylation.[22] The deformylated product dC is then further
converted into dU by the well-known bisulfite induced
deamination reaction of dC (see SI). Indeed, when we reacted fdC
with bisulfite, we observed efficient deformylation and
deamination to dU. We then studied to which extent the reaction
is influenced by the C2’-F atom present in F-fdC, in order to
estimate if the in vivo deformylation could be just the result of the
present 2’-F atom. Treatment of F-fdC with bisulfite also led to
deformylation and deamination to F-dU and indeed the reaction is
a little faster compared to fdC (see Fig. 3C). Although the
difference is measurable, it is in total rather small. With these data
in hand, we can conclude that we may overestimate the amount
of deformylation that can occur with fdC lacking the 2’-F atom. We
Figure 3. (A) Depiction of the deformylation data of F-fdC in different cell types
showing that F-fdC is deformylated in very different cells. Deformylation rate
was calculated by the F-dC + F-mdC/dN per F-fdC/dN, then the values were
normalized to the cell line with the lowest deformylation level (CHO-K1=1). (B)
Depiction of the deformylation of F-fdC/dG and a-fdC/dG showing the induced
differences within the C6 carbon to nitrogen exchange. (C) Depiction of the
bisulfite data showing that the deformylation of fdC and F-fdC is comparable
proving that the 2’-F substitution has only a small accelerating effect. Whereas
the reaction of a-fdC could not be detected.
Acknowledgements
Funded by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) GRK2338 (Project ID 321812289),
SFB1309 (PID 325871075), SFB1361 (PID 393547839) and
SPP1784 (PID 255344185). This project has received additional
This article is protected by copyright. All rights reserved.

10.1002/anie.202000414
Angewandte Chemie International Edition
RESEARCH ARTICLE
funding from the European Research Council (ERC) under the
European Union's Horizon 2020 research and innovation
programme (grant agreement n° EPiR 741912) and through a
H2020 Marie Skłodowska-Curie Action (LightDyNAmics, 765866).
Keywords: epigenetics • formylcytidine • deformylation • C-C
bond cleavage • active demethylation
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10.1002/anie.202000414
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RESEARCH ARTICLE
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
In the course of active
demethylation in stem cells,
mdC is oxidized to fdC which is
deformylated potentially after
addition of a nucleophile at the
C6-position.
Alexander Schön, Ewelina
Kaminska, Florian Schelter,
Eveliina Ponkkonen, Eva
Korytiaková, Sarah Schiffers,
Thomas Carell*
Page No. – Page No.
Analysis of an active
deformylation mechanism of
5-formyl-deoxycytidine (fdC)
in stem cells
This article is protected by copyright. All rights reserved.