Insight into the recognition mechanism of DNA cytosine-5 methyltransferases (DNMTs) by incorporation of acyclic 5-fluorocytosine (FC) nucleosides into DNA

Article abstract of DOI:10.1016/j.bmcl.2018.05.008

DNA cytosine-5 methyltransferase (DNMT) catalyzes methylation at the C5 position of cytosine in the CpG sequence in double stranded DNA to give 5-methylCpG (mCpG) in the epigenetic regulation step in human cells. The entire reaction mechanism of DNMT is d

Full text of DOI:10.1016/j.bmcl.2018.05.008

Accepted Manuscript
Insight into the Recognition Mechanism of DNA Cytosine-5 Methyltransferases
(DNMTs) by Incorporation of Acyclic 5-Fluorocytosine (FC) Nucleosides into
DNA
Shohei Utsumi, Kousuke Sato, Satoshi Ichikawa
PII:
S0960-894X(18)30397-4
https://doi.org/10.1016/j.bmcl.2018.05.008
BMCL 25825
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 March 2018
27 April 2018
3 May 2018
Please cite this article as: Utsumi, S., Sato, K., Ichikawa, S., Insight into the Recognition Mechanism of DNA
Cytosine-5 Methyltransferases (DNMTs) by Incorporation of Acyclic 5-Fluorocytosine (FC) Nucleosides into DNA,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.008
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Bioorganic & Medicinal Chemistry Letters
Insight into the Recognition Mechanism of DNA Cytosine-5 Methyltransferases
(DNMTs) by Incorporation of Acyclic 5-Fluorocytosine (FC) Nucleosides into
DNA
Shohei Utsumia, Kousuke Satob* and Satoshi Ichikawaa,c*
a
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Kanazawa 1757, Tobetsu, Ishikari-gun 061-0293, Japan
Center of Research and Education on Drug Discovery, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
b
c
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
DNA cytosine-5 methyltransferase (DNMT) catalyzes methylation at the C5 position of cytosine
in the CpG sequence in double stranded DNA to give 5-methylCpG (mCpG) in the epigenetic
regulation step in human cells. The entire reaction mechanism of DNMT is divided into six
steps, which are scanning, recognition, flipping, loop locking, methylation, and releasing. The
methylation and releasing mechanism are well-investigated; however, few reports are known
about other reaction steps. To obtain insight into the reaction mechanism, we planned the
incorporation of acyclic nucleosides, which make it easy to flip out the target nucleobase, into
oligodeoxynucleotides (ODNs) and investigated the interaction between the ODN and DNMT.
Here, we describe the design and synthesis of ODNs containing new acyclic 5-fluorocytosine
nucleosides and their physiological and biological properties, including their interactions with
DNMT. We found that the ODNs containing the acyclic 5-fluorocytosine nucleoside showed
higher flexibility than those that contain 5-fluoro-2′-deoxycytidine. The observed flexibility of
ODNs is expected to influence the scanning and recognition steps due to the decrease in helicity
of the B-form.
Keyword_5:
Epigenetic
Acyclic nucleosides
5-fluoro-2′-deoxycytidine
Covalent bond
DNA methyltransferase
DNA methylation has a profound effect on gene expression,
particularly in gene promoter regions.1,2 There are a number of
repetitive DNA sequences, and some genes are hypermethylated,
leading to silencing of the gene.3-5 Unusual DNA methylation
patterns are found in tumor cells, and their inhibition could be a
promising target in anticancer drug development. Methylation at
the C5 position of cytosine in a CpG sequence in double stranded
DNA is catalyzed by DNA cytosine-5 methyltransferase
(DNMT) to give 5-methylCpG (mCpG), using S-adenosyl-L-
methionine (SAM) as a methyl group donor. The whole reaction
of DNMT is shown in Figure 1A.6,7 Genome DNA is associated
with DNMT in a sequence independent manner and scans its
recognition site (a: scanning). After finding its specific
recognition site, DNMT forms a complex (b: recognition) and
then makes a transition to flip out the complex (c: flipping). The
resulting complex causes an induced fit to the loop locking
conformation for the methylation reaction (d: loop locking).
Then, the methylation reaction and releasing of the methylated
DNA occur (e and f: methylation and releasing). The methylation
and releasing mechanism have been investigated well, as shown
in Figure 1B. First, the Glu residue in the catalytic pocket of
DNMT protonates the nitrogen in the cytosine, and a thiol of the
Cys residue in the active site acts as a nucleophile and attacks the
C6 position of the cytosine to form an enzyme-linked
intermediate. Then, the C5 position of the cytosine is methylated
by SAM. Upon deprotonation of the C5 position, -elimination
of the added Cys residue occurs to complete the reaction.
Contrary to the methylation and releasing steps, little is known
about other reaction steps, which are scanning, recognition,
flipping and loop locking (Figure 1A(a) to (d)).8 Ring-opening of
the ribose of the nucleoside and its incorporation into an
oligodeoxynucleotide (ODN) increases the flexibility at the
modification site and decreases the thermal stability of duplex.9-11
The flexibility at the acyclic nucleotide site would easily flip out
its nucleotide by DNMT (Figure 1A(c)), and the decrease in
thermal stability of the modified ODN would influence the
interaction of ODN and DNMT by the conformational change of
ODN (Figure 1A(a) and (b)). To eliminate the discussion of the
releasing step (Figure 1A(f)), which could cause concern with
thermal stability of the duplex, 5-fluorocytosine (FC) is used as a
nucleobase in our study; 5-fluorocytosine works as one of the
mechanism-based inhibitors of DNMT,12,13 and the proposed
mechanism is illustrated in Figure 1C. Methylation will occur in
the same manner as natural cytosine methylation steps, the
releasing step is blocked, and the corresponding intermediate is
trapped. It is easy to discuss the complicated reaction steps by its
simplification and detect the covalent-bonding ODN-DNMT
complex using the difference of the molecular weight by

polyacrylamide gel electrophoresis (PAGE) and/or mass
spectrum. Therefore, we planned the incorporation of new
acyclic nucleosides, which make it easy to flip out from double
stranded DNA, into ODNs and investigate the interaction
between the ODN and DNMT. Here, we describe the synthesis of
new acyclic nucleosides containing 5-fluorocytosine, the
interaction between ODNs containing acyclic nucleosides and
DNMT, and the physiological properties of modified ODNs. We
also discuss the scanning, recognition and flipping mechanism
nor-2′-deoxynucleoside15, which has a short internucleotide
linkage and a longer linker from the backbone to FC. These
compounds were synthesized as presented in Schemes 1 and 2.
The hydroxyl group of the protected glycerol derivative 116 was
chloromethylated, and then 5-fluorouracil derivative 2 was
synthesized by Herdewijn’s method.17 The benzyl group of 2 was
removed by Pd black and 1,4-cyclohexadiene under microwave
irradiation. After protection of the resulting hydroxy group by the
TBS group, the 5-fluorouracil moiety was converted to 5-
fluorocytosine by the conventional method18 to give 4. The N4
amino group of 4 was protected by the benzoyl group, and then
selective removal of the benzoyl protection of hydroxyl group
was carried out by NaOH in THF-H2O to yield 5. The primary
alcohol of 5 was protected by the 4,4′-dimethoxytrityl (DMTr)
group to produce 6, and then
6 was converted to a
phosphoramidite unit 7. The phosphoramidite 14 was prepared as
shown in Scheme 2 in a manner similar to the synthesis of 7.
Namely, the hydroxyl group of dibenzyloxy glycerol derivative
819 was chloromethylated, and then 5-fluorouracil derivative 9
was synthesized. The 5-fluorouracil moiety was converted to 5-
fluorocytosine to give 10, and the N4 amino group of 10 was
protected by the benzoyl group to give 11, and the benzyl groups
were removed under the same conditions in Scheme 1 to yield
12. The primary alcohol of 12 was protected by the 4,4′-
dimethoxytrityl (DMTr) group to produce 13, and then 13 was
converted to phosphoramidite unit 14. The control compound 5-
fluoro-2′-deoxycytidine (dFC) phosphoramidite unit was also
synthesized using a previous method.20
F
F
The ODNs containing C-ac1 and C-ac 2 (ODN-FC-ac1 and
ODN-FC-ac2) were prepared using DNA synthesizer (Figure 1D).
Figure 1. A) Models for the interactions between DNA and DNMT during
methylation reaction. B) Proposed mechanism of cytosine methylation by
DNMTs. C) Proposed mechanism of inhibition of DNMTs by 5-
fluorocytosine (FC). D) Sequences of synthesized ODNs containing FC-ac1,
FC-ac2, and FC.
Scheme 2. Synthesis of phosphoramidite unit of FC-ac2 (14). Reaction
conditions: a) i) (CH2O)n,
4
M
HCl/dioxane,
0
°C ii) N,O-
bis(trimethylsilyl)acetamide, SnCl4, MeCN, 2 steps 56%; b) TPSCl, Et3N,
DMAP, MeCN, then aq. NH4OH, 82%; c) BzCl, Et3N, DMAP, CH2Cl2,
quant; d) 1,4-cyclohexadiene, Pd black, MeCN, microwave 120 °C, 37%; e)
(Figure 1A(a), (b) and (c)) of DNMT.
DMTrCl,
pyridine,
60%;
f)
2-cyanoethyl
N,N-
Results and Discussion
diisopropylchlorophosphoramidite, iPr2NEt, DMAP, CH2Cl2, 80%.
We designed FC-ac1 and 2 (Figure 1D) to investigate the
recognition by DNMT based on the difference of the glycerol
skeleton. FC-ac1 is a ganciclovir-type 2′-deficient acyclic
nucleoside14, which has the same number of bonds between 5′
ODN-FC was used for control experiments, and its complement
ODN-G was also synthesized. To ascertain whether the
methylation steps of ODN-FC-ac1 and ODN-FC-ac2 were the
same as that of ODN-FC, we first studied the interaction between
ODN-FC-ac1 or ODN-FC-ac2 and a bacterial DNMT, M.HhaI,
from Haemophilus haemolyticus. M.HhaI typically methylates
the internal cytosine in the target sequence 5′-GCGC-3′; in our
F
and 3′, and a glycerol skeleton containing C as a nucleobase for
F
trapping DNMT. On the other hand, C-ac2 is an analogue of 2′-
F
studies, FC-ac1 and C-ac2 were substituted in place of the
cytosine. Each double stranded ODN (ODN-FC-ac1:G, ODN-FC-
ac2:G, and ODN-FC:G) was incubated with 4 equiv. of M.HhaI at
37 °C for 120 min in the presence or absence of SAM. It was
F
reported that ODNs containing C instead of cytosine in a CpG
sequence had a covalent complex formation with M.HhaI in the
presence of SAM (Figure 1C).21 ODN-FC-ac1 and ODN-FC-ac2
formed a stable complex with M.HhaI, mainly in the presence of
SAM in denaturing polyacrylamide gel (Figure 2; lanes 3, 6 and
9), as well as ODN-FC. Moreover, to investigate the mechanism
of ODN-FC-ac1 and ODN-FC-ac2, the reversibility assay of the
ODN-M.HhaI complex was carried out in Figure S1. The
complexes were not disrupted by the addition of 100 equiv. of
Scheme 1. Synthesis of phosphoramidite unit of FC-ac1 (7). Reaction
conditions: a) i) (CH2O)n,
4
M
HCl/dioxane,
0
°C, ii) N,O-
bis(trimethylsilyl)acetamide, SnCl4, MeCN, 2 steps 58%; b) i) 1,4-
cyclohexadiene, Pd black, MeOH, microwave 120 °C, ii) TBSCl,
imidazole, CH2Cl2, 2 steps 88%; c) TPSCl, Et3N, DMAP, MeCN, then aq.
NH4OH, 96%, d) i) BzCl, Et3N, HOAt, CH2Cl2, ii) NaOH, THF, H2O, 2
steps 35%; e) i) DMTrCl, pyridine, ii) TBAF, THF, 2 steps 60%; f) 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite, iPr2NEt, DMAP,
CH2Cl2, 44%.

ODN-M.HhaI complex, giving greater than 90% yield after 120
min reaction time (Figure 3A, lane 5). On the other hand, a band
corresponding to a complex with M.HhaI was observed in 15%
yield after 120 min for ODN-FC- ac1:G (Figure 3A, lane 10),
and in 79% yield after 120 min for ODN-FC-ac2:G (Figure 3A,
lane 15). The relative rate constant (krel) values of complex
formation are shown in Figure 3B. ODN-FC:G revealed the
fastest complex formation, and ODN-FC-ac1:G had a 37.0 times
lower krel value (0.027). On the other hand, the krel value of ODN-
FC-ac2:G (0.30) was 11.1 times greater than that of ODN-FC-
ac1:G, and the value was still 3.3 times lower than ODN-FC:G.
To confirm what the difference of krel values depends on, we
investigated the flexibility and the structure of ODN-FC-ac1:G
and ODN-FC-ac2:G.
lane
1
2
3
4
5
6
7
8
9
ODN
FC
+
FC-ac1
FC-ac2
M.HhaI
－
－
+
－
－
+
+
－
－
+
+
SAM
SAM
SAM
cofactor
－
－
－
ODN/M.HhaI
complex
free ODN
Figure 2. Electrophoretic mobility shift assay of ODN-FC:G, ODN-FC-
ac1:G, and ODN-FC-ac2:G with or without SAM: The reactions were
performed with a 25 nM ODN and 240 M SAM by incubating at 37 °C for
2 h with 100 nM M.HhaI in 50 mM Tris-HCl (pH 7.5), 10 mM EDTA and 5
mM -mercaptoethanol, and analyzed by denaturing PAGE (8%, 140 V, 40
min). Lanes 1, 4 and 7; free ODN (control), lanes 2, 5 and 8; ODN without
SAM, lanes 3, 6 and 9; ODN with SAM.
Table 1. Tm values and thermodynamic parameters of ODN-FC:G,
ODN-FC-ac1:G and ODN-FC-ac2:G.
5′-TCTGATGXGCTGACAT-3′
3′-AGACTACGCGACTGTA-5′
ODN
Tm (ºC) DTm (ºC) –DHº(kJ/mol) –TDSº(kJ/mol) –DGº(kJ/mol)
ODN-FC:G
66.1
61.4
62.1
—
82.9
69.5
75.5
414
340
379
331
271
303
A
ODN-FC-ac1:G
ODN-FC-ac2:G
– 4.7
– 4.0
lane
1
2
3
4
5
6
7
8
9
10
11
12
13 14 15
FC
+
FC-ac1
+
FC-ac2
+
ODN
M.HhaI
―
―
―
―
―
―
10
30
60 120
10
30
60 120
time (min)
10
30
60 120
ODN/M.HhaI
complex
F
To evaluate the flexibility of C-ac1 and 2 in ODNs, Tm was
measured (Table 1). ODN-FC-ac1:G and ODN-FC-ac2:G showed
61.4 and 62.1 °C as the Tm value, respectively. These values are
4.7 and 4.0 °C lower than those of ODN-FC:G (66.1 °C). These
results suggested that the flexibility of an acyclic nucleoside
modification influenced the thermal stability of ODN duplexes
entirely, and ODN-FC-ac1 was suggested to be more flexible than
ODN-FC-ac2. The –H°, –TS°, and –G° values of the
unmodified ODN-FC:G (414, 331 and 82.9 kJ/mol, respectively)
were the highest of all. On the other hand, the –TS° values of
the ODN-FC-ac1:G and ODN-FC-ac2:G were 271 and 303 kJ/mol,
respectively, which were lower than those of the unmodified one.
The –H° and –G° values were also lower, and the results
implied that the lower –TS° was due to the flexibility of ODN
compensating the unfavorable disadvantage in –H°. When
comparing ODN-FC-ac1:G with ODN-FC-ac2:G, ODN-FC-ac1:G
had the lower –H°, –TS°, and –G°. The thermodynamic
parameters indicated that the flexibility of ODN-FC-ac1:G is
higher than that of ODN-FC-ac2:G.
free ODN
krel
B
100
80
60
40
20
1
0.30
ODN-FC:G
ODN-FC-ac1:G
ODN-FC-ac2:G
0.027
0
30
60
90
120
time (min)
Figure 3. Electrophoretic mobility shift assay of ODN-FC:G, ODN-FC-
ac1:G, and ODN-FC-ac2:G: A) The reactions were performed with 25 nM
ODN and 240 M SAM by incubating at 37 °C for 0-120 min with 100 nM
M.HhaI in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM NaCl, 5 mM -
mercaptoethanol, 5% glycerol, and analyzed by denaturing PAGE (8%, 140
V, 1 h). Lanes 1, 6 and 11; 0 min, lanes 2, 7 and 12; 10 min, lanes 3, 8 and
13; 30 min, lanes 4, 9 and 14; 60 min, lanes 5 10 and 15; 120 min. B) Time
course of binding rate between ODN and M.HhaI and relative rate constants
(krel); ODN-FC:G (solid), ODN-FC-ac1:G (dashed), ODN-FC-ac2:G (dot).
To study the influences of the flexibility on the duplex
structure, we measured CD spectra of the duplex of ODN-FC-
ac1:G, ODN-FC-ac2:G, and ODN-FC:G at 25 °C (Figure 4). The
observed CD spectra of ODN-FC-ac1:G, ODN-FC-ac2:G, and
ODN-FC:G consisted of a positive cotton effect (an increase at
275 nm and a decrease at 245 nm) due to the nucleobase stacking
and the helicity of the duplex similar to a conventional B-form
duplex.22 ODN-FC-ac2:G has a lower increase at 275 nm, and
ODN-FC-ac1:G has a lower increase and decrease showing not
only at 275 nm but also at 245 nm compared with ODN-FC:G.
These results suggested that both of the ODNs containing acyclic
nucleosides revealed a weaker nucleobase stacking effect due to
the increase in flexibility at the acyclic modification site, and
ODN-FC-ac1:G had a decrease of helicity of the B-form duplex.
The differences in the CD spectra corresponded to the difference
of thermodynamic parameters, especially a lower –TS°.
non-labeled ODN after 24 h of incubation. These results showed
that ODN-FC-ac1 and ODN-FC-ac2 formed a covalent complex
with M.HhaI, which demonstrated the same finding as previously
known for ODN-FC. Therefore, ODN-FC-ac1 and ODN-FC-ac2
containing an acyclic nucleoside would form the covalent
complex by the same reaction mechanism as ODN-FC. From
these results, ODN-FC, ODN-FC-ac1, and ODN-FC-ac2 would
share the same mechanism in methylation step (Figure 1A (e) and
Figure 1C) and eliminate the discussion of releasing step (Figure
1A (f) and Figure 1C). The reactivities of ODN-FC-ac1 and
ODN-FC-ac2 were lower than ODN-FC. To assess the difference
of their reactivities, the detailed time course of the ODN-M.HhaI
complex formation was investigated in Figure 3. The reaction
with ODN-FC:G showed rapid and efficient formation of an

clarify the recognition mechanism of DNMT and to develop
other modified ODNs, which make minor structural changes in
the helicity of the B-form.
5′-TCTGATGXGCTGACAT-3′
3′-AGACTACGCGACTGTA-5′
6
4
2
0
ODN-FC:G
ODN-FC-ac1:G
ODN-FC-ac2:G
Acknowledgments
This work was supported in part by Grant-in-Aid for Scientific
Research (C) (Grant No. 17K05919) from Japan Society for the
Promotion of Science (JSPS), Hokkaido University, Global
Facility Center (GFC), Pharma Science Open Unit (PSOU),
funded by MEXT under "Support Program for Implementation of
New Equipment Sharing System", the Platform Project for
Supporting Drug Discovery and Life Science Research (Basis for
Supporting Innovative Drug Discovery and Life Science
Research; BINDS) from the Japan Agency for Medical Research
and Development (AMED).
200
250
300
350
–2
–4
–6
wavelength (nm)
Figure 4. CD spectra of ODNs containing FC-ac1, FC-ac2, and FC; ODN-
FC:G (solid), ODN-FC-ac1:G (dashed), ODN-FC-ac2:G (dot). The duplex
(1.5 M) at 25 °C in a buffer of 10 mM Na cacodylate (pH 7.0) with 150
mM NaCl was used for each measurement.
References and notes
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ODN-FC, ODN-FC-ac1, and ODN-FC-ac2 would share the
same mechanism in the methylation step (Figure 1A (e) and
Figure 1C); however, the krel values of ODN-FC-ac1:G and ODN-
FC-ac2:G are lower than ODN-FC:G, and a negative correlation
was observed between the flexibility and ODN-DNMT complex
formation. These results suggested that not only the methylation
reaction step but also the scanning, recognition, and flipping
steps (Figure 1A (a), (b) and (c)) are very important for the entire
reaction of DNMT. X-ray structure of the cocrystal between
ODN containing FC, DNMT, and S-adenosyl-L-homocysteine
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In summary, to obtain insight into the recognition mechanism
of DNMT, we designed and synthesized acyclic nucleosides
containing a FC nucleobase, FC-ac1, and FC-ac2. The ODNs
containing ODN-FC-ac1 and ODN-FC-ac2 showed a higher
flexibility than that of ODN FC, and ODN-FC-ac1 was more
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thermodynamic parameters and CD spectra.
A negative
correlation was observed between the flexibility and ODN-
DNMT complex formation. A weaker nucleobase stacking effect
due to the increase in flexibility at the acyclic modification site
may provide a higher flip out effect (Figure 1A(c)); however, the
local flexibility would influence the scanning and recognition
steps (Figure 1A(a) and (b)) due to the decrease in helicity of the
entire shape of the B-form duplex shown in our CD spectra
(Figure 4). Further investigations are necessary in our lab to
Supplementary Material
Supplementary data associated with this article can be found
in the online version.

Design and synthesis of new acyclic nucleosides
into ODNs and investigate the interaction between
the ODN and DNMT.
The synthesized ODNs showed a higher flexibility
from the data obtained by the thermodynamic
parameters and CD spectra.
A negative correlation was observed between the
flexibility and ODN-DNMT complex formation.