The pH effect on the naphthoquinone-photosensitized oxidation of 5-methylcytosine

Article abstract of DOI:10.1002/chem.200800840

The pH effect on the one-electron photooxidation of 5-methyl-2′- deoxycytidine (d^mC) by sensitization with 2-methyl-1,4-naphthoquinone (NQ) was investigated. Photoirradiation of an aqueous solution containing d^mC and NQ under slightly acidic conditions of pH 5.0 efficiently produced 5-formyl-2′-deoxycytidine, whereas similar NQ-photosensitized oxidation of d^mC proceeded to a lesser extent under more acidic or basic conditions. Fluorescence-quenching experiments re vealed that the less-efficient photooxidation at pH values below 4.5 is attributed to the decreased rate of one-electron oxidation of d^mC owing to protonation at the N(3)-position. The NQ-photosensitized oxidation of an N(4)-dimethyl-substituted d^mC derivative under various pH conditions also sug gests that the pH change in the range of 5.0 to 8.0 may be responsible for a reversible deprotonation-protonation equilibrium at the N(4)-exocyclic amino group of the d^mC radical cation. In accord with the photochemical reactivity of monomeric d^mC, the 5-methylcytosine residue in oligodeoxynucleotides was oxidized efficiently by photoexcited NQ-tethered oligodeoxynucleotides under slightly acidic conditions to form an alkali-labile 5-formylcytosine residue.

Full text of DOI:10.1002/chem.200800840

FULL PAPER
DOI: 10.1002/chem.200800840
The pH Effect on the Naphthoquinone-Photosensitized Oxidation of
5-Methylcytosine
[
a, b]
[a]
[a]
[a]
Hisatsugu Yamada,
Kazuhito Tanabe,* Takeo Ito, and Sei-ichi Nishimoto*
Abstract: The pH effect on the one-
electron photooxidation of 5-methyl-2’-
deoxycytidine (d C) by sensitization
vealed that the less-efficient photooxi-
dation at pH values below 4.5 is attrib-
uted to the decreased rate of one-elec-
gests that the pH change in the range
of 5.0 to 8.0 may be responsible for a
reversible deprotonation–protonation
equilibrium at the N(4)-exocyclic
m
m
with
2-methyl-1,4-naphthoquinone
tron oxidation of d C owing to proto-
m
(
NQ) was investigated. Photoirradia-
nation at the N(3)-position. The NQ-
photosensitized oxidation of an N(4)-
amino group of the d C radical cation.
tion of an aqueous solution containing
In accord with the photochemical reac-
m
m
m
d C and NQ under slightly acidic con-
dimethyl-substituted d C derivative
tivity of monomeric d C, the 5-methyl-
ditions of pH 5.0 efficiently produced
under various pH conditions also sug-
cytosine residue in oligodeoxynucleoti-
des was oxidized efficiently by photo-
excited NQ-tethered oligodeoxynucleo-
tides under slightly acidic conditions to
form an alkali-labile 5-formylcytosine
residue.
5-formyl-2’-deoxycytidine, whereas sim-
ilar NQ-photosensitized oxidation of
Keywords: methylcytosine · DNA
methylation · epigenetic analysis ·
nucleic acids · photooxidation
m
d C proceeded to a lesser extent under
more acidic or basic conditions. Fluo-
rescence-quenching experiments re-
[
3–7]
Introduction
tion have been reported in which differences in chemical,
[8]
[9]
biological, or photochemical reactivity between cytosine
m
Cytosine methylation is a physiological modification that
takes place after DNA replication. In the course of such a
modification, a methyl group is transferred from (S)-adeno-
sylmethionine to the C5 position of cytosine by DNA meth-
Cytosine methylation is believed to play a
number of important biological roles, typically the epigenet-
ic repression of genetic information, although it is not fully
(C) and 5-methylcytosine ( C) are evaluated.
[4–5]
At present, the bisulfite method
is most commonly
used among various different protocols for the detection of
cytosine methylation. This protocol is based on the sodium
bisulfite modification of normal C but not C and allows the
clear discrimination between C and C in genomic DNA
with high selectivity. There are, however, practical draw-
backs to the method including complicated procedures and
long reaction periods for chemical modification. To replace
the bisulfite method, several chemical protocols based on
[
1,2]
m
yltransferases.
m
[1,2]
elucidated.
For a better understanding of the biological
effects of cytosine methylation, there is increasing interest
in developing an effective method to assess the methylation
status of specific cytosine residues in genomes. Various
methods for identification and analysis of cytosine methyla-
m
the direct modification of C but not C have been reported.
m
Okamoto et al. showed that treatment of a single C bulge
m
of duplex DNA with osmium tetroxide produces an C–
[6]
osmium adduct in a sequence-selective manner. Bareyt
and Carell reported that the C in duplex DNA is oxidized
selectively by pentavalent vanadium species or sodium per-
AHCTUNGTRENNUNGi odate in the presence of lithium bromide. These reactions
[
a] Dr. H. Yamada, Dr. K. Tanabe, Dr. T. Ito, Dr. S.-i. Nishimoto
Department of Energy and Hydrocarbon Chemistry
Graduate School of Engineering, Kyoto University
Katsura Campus, Nishikyo-ku, Kyoto 615-8510 (Japan)
Fax : (+81)75–383–2501
m
[7]
have been applied to sequence-specific DNA-methylation
E-mail: tanabeka@scl.kyoto-u.ac.jp
analysis.
nishimot@scl.kyoto-u.ac.jp
m
Recently, we proposed a protocol for discriminating
C
[
b] Dr. H. Yamada
m
based on the one-electron photooxidation of C in a given
oligodeoxynucleotide (ODN) sensitized by 2-methyl-1,4-
naphthoquinone (NQ)-tethered ODN. Photoirradiation of
an NQ-tethered duplex in an aqueous solution induced the
Nano-Medicine Merger Education Unit, Kyoto University
Katsura Campus, Nishikyo-ku, Kyoto 615-8530 (Japan)
[9]
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800840.
Chem. Eur. J. 2008, 14, 10453 – 10461
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10453

m
efficient one-electron oxidation of the C residue to form 5-
formylcytosine when the NQ chromophore was arranged so
m
[9a,b]
as to be in close contact with the target C.
Subsequent
treatment of the photoirradiated aqueous solution with hot
m
piperidine led to exclusive strand cleavage at the original C
m
site. In contrast, an ODN analogue, which replaced C with
normal C, thymine (T), adenine (A), or guanine (G) under-
went less oxidative strand cleavage at the alternative target
site probably because of charge-transfer and charge-recom-
bination processes between the base radical cation and the
NQ radical anion. Suppressed strand cleavages at C and T
were also explained by less-efficient NQ-sensitized photoox-
idation to form the corresponding radical cations owing to
the considerably smaller free-energy change of charge sepa-
m
Figure 1. HPLC profiles of the photooxidation of d C (200 mm) sensitized
by NQ (200 mm) upon 312-nm irradiation in 2 mm sodium cacodylate
buffer solution containing 20 mm NaCl (pH 6.0) and 10% acetonitrile.
[9b]
ration for photooxidation of C and T by excited NQ. In
addition, well-designed incorporation of the NQ chromo-
phore into the interior of ODN suppressed the competitive
strand cleavage exclusively at consecutive G bases, which
occurred as a result of positive charge transfer. Thus, such a
striking photooxidative reactivity leading to strand cleavage
course of HPLC profiles observed in the NQ-photosensi-
tized oxidation of d C at pH 6.0. Consistent with previous
reports, the photoirradiation produced d C along with the
degradation of d C and the corresponding HPLC peaks
m
[10]
[11]
f
m
m
allowed us to detect the target C as a positive band on the
sequencing gel.
Although the NQ-photosensitized oxidation accompanied
by selective strand cleavage at C in DNA is an attractive
were assigned by reference to the respective authentic sam-
ples. In addition to the formation of d C, a few minor prod-
uct peaks, which may have included 5-(hydroperoxymethyl)-
f
m
method for identification of the methylation site, relatively
lower sensitivity of the detection method owing to reduced
efficiency in the photooxidation of C still remains as a
2’-deoxycytidine as a reaction intermediate and 5-hydroxy-
hm
[12]
methyl-2’-deoxycytidine (d C), were observed.
Figure 2
m
major issue that must be improved to establish a status of
general availability. Herein, we characterized the pH effect
on the NQ-photosensitized one-electron oxidation of 5-
m
methyl-2’-deoxycytidine (d C) to obtain insight into the
m
design of photochemical systems involving the C-selective
strand cleavage with high sensitivity. A pH change is among
the potential external triggers for regulation of various pho-
m
toreactions. Upon photoirradiation of monomeric d C in
the presence of NQ under various pH conditions, a signifi-
cant pH-dependent formation of 5-formyl-2’-deoxycytidine
f
f
(
d C) was observed. We confirmed that the formation of d C
occurs with maximum efficiency at pH 5.0, whereas the effi-
ciency of photooxidation was lower under more acidic or
basic conditions. In addition, we investigated the NQ-photo-
f
i
Figure 2. pH-dependent variation of the initial rate (k) of d C formation,
as observed in the NQ-sensitized photooxidation of d C.
m
m
sensitized oxidative cleavage reaction of C in a duplex in
which the NQ chromophore was incorporated into the
strand for immobilization at a specific position, thus obtain-
ing evidence that optimization of the pH environment in the
photosensitized oxidation results in much larger amount of
f
shows variation of the initial rate of d C formation as a func-
f
tion of the pH value. The initial rate of d C formation,
which increased upon photoirradiation with decreasing pH
values from 8.0 to 5.0, attained its maximum at pH 5.0 and
then decreased dramatically at pH values below 5.0. Similar
m
strand cleavage at C in DNA.
m
pH dependency was also observed for the rate of d C degra-
Results and Discussion
dation during the photosensitization. These results indicate
that pH value of the sample solution affected the NQ-pho-
m
m
f
We initially performed the photooxidation of d C by sensiti-
zation with NQ at various pH values. Aerobic solutions of
d C (200 mm) and NQ (200 mm) in 2 mm sodium cacodylate
buffer solution containing 20 mm NaCl and 10% acetonitrile
at a pH range of 4.0 to 8.0 were photoirradiated by using
12-nm UV light. Figure 1 shows a representative time
tosensitized oxidation of d C into d C.
m
The photooxidation of d C by an NQ sensitizer has been
m
studied extensively by using flash photolysis and detailed
[
11,13–14]
product analysis.
Scheme 1 shows a mechanistic out-
m
f
line of the photochemical conversion of d C to d C based
on the results of previous studies. The initial step of oxida-
3
10454
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Chem. Eur. J. 2008, 14, 10453 – 10461

Naphthoquinone-Photosensitized Oxidation of AHCUTNGRENGUN5 -Methylcytosine
FULL PAPER
m
Scheme 1. A plausible reaction mechanism for the NQ-photosensitized oxidation of d C.
m
tion involves electron transfer from d C to the triplet-excit-
the initial oxidation and/or the reversible deprotonation–
protonation process at the amino group, but not the irrever-
sible oxygenation of methyl-centered d C radical, may be
m
ed state NQ, thereby producing the corresponding d C base
radical cations and NQ radical anions.
d C radical cation undergoes deprotonation at the methyl
group to form the corresponding methyl-centered radical in-
termediate as shown by electron paramagnetic resonance
EPR) study, followed by reaction with molecular oxygen
to produce hydroperoxide that is converted to the final pho-
tooxidation products, such as d C and d C. In competition
with the deprotonation process at the methyl group, there is
a protonation–deprotonation equilibrium at the exocyclic
amino group on the d C radical cation, which is associated
with regeneration of the original d C by reduction and sub-
sequent protonation. Both the deprotonation at the methyl
group and the protonation–deprotonation equilibrium at the
exocyclic amino group of the d C radical cation intermedi-
[13,15]
m
The resulting
m
sensitive to the pH change ranging from 4.0 to 8.0.
To obtain a mechanistic insight into the pH effect on the
m
initial one-electron oxidation step of d C, an attempt was
[16]
(
made to measure the triplet lifetime of NQ in aqueous solu-
tion and thereby determine whether varying pH values can
alter the one-electron oxidizing ability of photoexcited NQ.
Upon laser flash excitation at 355 nm of NQ (50 mm) in de-
oxygenated phosphate buffer solution (pH 5.0–8.0), a transi-
f
hm
m
3
*
ent absorption band assigned to the triplet NQ ( NQ ) at
m
[13,15b,19–20]
around 350 nm was observed,
showing exponential
[20]
decay by a self-quenching mechanism (see the Supporting
Information). As shown in Table 1, the triplet lifetimes of
m
ate are substantially affected by the pH change.
[
a]
T
Table 1. Triplet lifetimes (t ) of NQ at various pH values.
In a previous paper, we predicted that deprotonation of
m
pH
(ms)
a] t
ance at 345 nm by using 355-nm laser flash photolysis in argon-saturated
0 mm phosphate buffer solution containing 50 mm NQ.
5.0
6.0
7.0
8.0
the C radical cation may occur on a time-scale region com-
t
T
1.6Æ0.1
1.9Æ0.2
1.4Æ0.2
1.4Æ0.1
parable with the positive-charge transfer though DNA bases
by reference to a proton-coupled electron-transfer pro-
[
T
was determined by the first-order kinetics for the decay of absorb-
[
17–18]
cess
in which electron-transfer and proton-transfer pro-
1
cesses occur concertedly upon the one-electron redox reac-
tion of nucleobases. This also suggests that the d C radical
m
cation might favor deprotonation at the methyl group into a
methyl-centered d C radical in the present pH range (4.0–
NQ measured at various pH values were substantially simi-
m
3
*
lar, indicating that the chemical properties of transient NQ
are pH independent. This result is also supported by a previ-
8
.0), probably owing to a much lower pK value (! 4.0) for
a
3
*
the corresponding protonation–deprotonation equilibrium.
ous report that the quenching of NQ by thymidine was of
m
[13]
In view of the evidence that both the degradation of d C
the same rate constants at pH 3.0, 5.0, and 8.0. Thus, it
f
and formation of d C proceed in a pH-dependent manner,
may be concluded that the pH change does not affect the
Chem. Eur. J. 2008, 14, 10453 – 10461
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10455

K. Tanabe, S.-I. Nishimoto et al.
+
[22]
one-electron oxidizing ability of NQ sensitizer in the triplet
excited state.
phate by AcrH . Thus, these results clearly indicate that
m
lower efficiencies for the degradation of d C and the forma-
tion of d C at pH values below 4.5 in the NQ-photosensi-
tized oxidation of d C are attributable to the decreased rate
of one-electron oxidation of d C that is protonated at N(3)
f
To further clarify the mechanistic details of the initial
one-electron oxidation step, we also evaluated the pH de-
pendency of the photoinduced electron-transfer reaction of
m
m
m
d C by fluorescence-quenching experiments. The rate con-
accompanied by the increased oxidation potential.
stant of electron-transfer fluorescence quenching of photo-
excited organic sensitizer by d C was conveniently estimat-
To evaluate the effect of the reversible protonation–de-
m
m
protonation equilibrium at the N(4) amino group of the d C
m
ed by Stern–Volmer analysis. Alternative photooxidizing flu-
radical cation on the photochemical conversion of d C to
+
f
orophores of the 10-methylacridinium ion (AcrH ) and
d C, we also performed the NQ-photosensitized oxidation of
9
,10-dicyanoanthracene (DCA) were employed to perform
N(4)-dimethyl-substituted 5-methyl-2’-deoxycytidine (M-
[21]
m
appropriate fluorescence-quenching measurements.
Both
d C) with a lack of deprotonation at the N(4) position. M-
m
fluorophores have sufficient reduction potentials (2.43 and
.95 V vs. NHE (NHE=normal hydrogen electrode) in the
d C was prepared from 3’,5’-O-bis(tert-butyldimethylsilyl)-
1
thymidine 1, as outlined in Scheme 2. Before investigating
[22]
m
singlet excited state, respectively) to oxidize the 5-methyl-
cytosine base (1.73 V vs. NHE).
AcrH or DCA (25 mm) at 358 or 390 nm, respectively, in
deoxygenated phosphate buffer solution under various pH
conditions (pH 3.0–8.0), the intensity of each fluorescence
emission decreased with increasing concentration of d C.
The fluorescence-quenching rate constants (k ) of AcrH
and DCA were determined by the Stern–Volmer plots of
the photooxidation of M-d C, we measured the fluorescence
[15]
*
m
m
Upon excitation of
quenching of DCA by M-d C to confirm that M-d C has
+
m
an oxidation potential similar to that of the original d C.
The k values of DCA at pH 7.0 in the presence of d C and
M-d C were determined to be 3.4ꢁ10 and 4.2ꢁ10 m s ,
respectively, indicating that incorporation of the methyl
group at the N4 position did not significantly decrease the
m
q
m
9
9
À1 À1
m
+
*
q
*
m
oxidation potential of d C (see the Supporting Information).
the data. The kq values evaluated at the respective pH
This result was also supported by the similar energy levels
of the highest-occupied molecular orbitals of d C
+
*
m
values are plotted in Figure 3. Both k values of AcrH and
q
m
(
À5.85 eV) and M-d C (À5.80 eV), as estimated by ab initio
[24]
calculations at the B3LYP/6–31G* level.
An aerobic aqueous solution of M-d C (200 mm) was pho-
toirradiated by using 365-nm UV light in the presence of
NQ (200 mm) in 2 mm sodium cacodylate buffer solution
containing 20 mm NaCl in the pH range between 5.0 and
.0. The pH dependency of the NQ-photosensitized deg-
radation of M-d C was evaluated by HPLC (Figure 4). In
contrast with the remarkable pH dependence of d C degra-
m
[25]
[26]
8
m
m
dation, small changes were observed in the photosensitized
m
degradation profile of M-d C with varying pH values, thus
indicating that the reversible deprotonation at the N4 amino
m
group of the d C radical cation seems to be responsible for
Figure 3. pH-dependent variation of the fluorescence quenching rate con-
the present pH-dependent NQ-photosensitized oxidation of
+
stant (k
q
) for AcrH
(
) or DCA (~) photosensitizing one-electron oxi-
&
m
f
m
d C to produce d C in the pH range between 5.0 and 8.0. In
a separate experiment, we also examined the competitive
dation of d C in anoxic phosphate buffer solution (5 mm; pH 3.0–8.0).
m
m
photosensitized reaction of d C and M-d C. Similar photoir-
radiation at 365 nm for 10 min of an aerobic aqueous solu-
*
m
m
DCA were fairly constant in the pH region between 5.0
tion (pH 7.0) of d C and M-d C in the presence of NQ re-
and 8.0, indicating that one-
m
electron oxidation of d C was
not affected by the pH change
in this range. In contrast, kq
values decreased drastically at
pH values below 4.5. Such a de-
crease in the kq value may be
attributed to the increase in ox-
m
idation potential of d C owing
to protonation at the N(3) posi-
[23]
tion (pK =4.4). A similar ob-
a
servation was also reported for
photoinduced, one-electron oxi-
m
Scheme 2. Synthesis of N(4)-dimethyl-substituted 5-methyl-2’-deoxycytidine (M-d C). Conditions: a) 1,2,4-tri-
3 3 3 3 2
AHCTUNGTRENNGNUa zole, POCl , Et N, CH CN, 95%; b) 50% Dimethylamine, CH CN, quantitative; c) TBAF 3H O, THF, 87%.
dation of cytosine 5’-monophos- TBAF=tetra-n-butylammonium fluoride, TBDMS=tert-butyldimethylsilyl.
10456
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Chem. Eur. J. 2008, 14, 10453 – 10461

Naphthoquinone-Photosensitized Oxidation of AHCUTNGRENGUN5 -Methylcytosine
FULL PAPER
m
librium between the d C radical cation and its deprotonated
form at the amino group may favor a shift to an increased
m
amount of d C radical cations at pH 5.0, leading to the effi-
cient deprotonation at the C(5) methyl group. With increas-
ing pH values from 5.0 up to more basic conditions such as
m
pH 8.0, the d C radical cation intermediate undergoes com-
petitive deprotonation at the N(4)-exocyclic amino group to
[
28]
form an oxidizing aminyl radical
radical intermediate,
or its tautomer iminyl
[29]
À
which may be reduced by O2
C
.
These deprotonation and one-electron reduction processes
m
may regenerate the original d C, thus resulting in a de-
creased yield of the photooxidation product d C. Although
f
Figure 4. The pH effect on the NQ-photosensitized (200 mm) oxidative
degradation of M-d C (200 mm) in aerated 2 mm sodium cacodylate
buffer solution containing 20 mm NaCl and 10% acetonitrile (pH 5.0, *;
pH 6.0, &; pH 7.0, ~; pH 8.0, *).
m
the pK value for the protonation–deprotonation equilibri-
a
m
um at the N(4)-exocyclic amino group of the d C radical
cation has not been determined at present, it seems that the
protonation–deprotonation equilibrium presumably occurs
under weakly acidic conditions such as pH<7.0 in light of
the remarkable pH-dependence of d C degradation and d C
formation (see Figure 2).
sulted in their degradation with a conversion ratio of 9:50,
despite similar oxidation potentials of these analogues (see
the Supporting Information). It is therefore most likely that
m
f
m
the d C radical cation intermediate may undergo deproto-
To characterize the photooxidative strand cleavage at the
C site in DNA, we performed the NQ-photosensitized one-
m
nation at the exocyclic N(4) amino group into an N(4)-
amino-centered radical under neutral and even weakly
acidic conditions, which suppresses an alternative deproto-
nation at the C(5) methyl group into a C(5)-methyl-centered
electron oxidation at various pH values by using a modified
ODN with the NQ chromophore in the interior of a strand
(ODN ACHNUTGTNERN(GU NQ )). The modified strand of ODN ACHTUNGETRNN(GNU NQ ) was pre-
1 1
f
[9a]
radical followed by oxygenation to produce d C (see also
pared by coupling of N-hydroxysuccinimidyl ester 4 with
Scheme 1). In contrast, the efficient oxygenation of the M-
ODNs with various base sequences and a common amino-
hexyl linker (Scheme 3). In this study, we targeted the C in
m
m
d C radical cation intermediate may proceed in the absence
m
of deprotonation at the N(4)-amino group of M-d C.
a partial sequence of the human p53 gene corresponding to
[30]
Scheme 1 shows a plausible mechanism for the NQ-photo-
codons 280–285 of exon 8. The sequences of ODNs used
in this study are summarized in Figure 5a. Photoirradiation
m
sensitized oxidation of d C to form the final oxidation prod-
[
31]
32
ucts. The pH effects on this oxidation can be summarized as
at 312 nm of the duplex comprising ODN ACHTUNGERTNNUNG( NQ ) and P-
1
m
m
follows: 1) In the pH range below 4.5, d C is predominantly
5’-end-labeled ODN( C) was carried out in sodium cacody-
late buffer solution (pH 4.0–8.0) containing 20 mm NaCl at
08C in air, and the reaction was analyzed by polyacrylamide
gel electrophoresis after treatment with hot piperidine. As
in an N(3)-protonated form with a higher oxidation poten-
tial, therefore difficulties are encountered in undergoing
one-electron oxidation to produce the corresponding radical
m
m
cation intermediate. This unfavorable property of d C for
shown in Figure 5b, strand cleavage at C was observed in
one-electron oxidation accounts for decreased efficiency of
the duplex after photoirradiation for 2 h in the pH range of
5.0–8.0, whereas only a background level of strand cleavage
was observed at pH values below 4.5. As expected, the effi-
f
the d C formation at relatively lower pH values below 4.5.
m
2
) In the pH range between 5.0 and 8.0, d C is free from
m
protonation at the N(3) position and so can readily undergo
ciency of strand cleavage at C increased with decreasing
m
one-electron oxidization into the d C radical cation inter-
pH, attaining a maximum efficiency at pH 5.0. These results
clearly indicate that NQ-photosensitized one-electron oxida-
mediate by NQ in the excited state, regardless of the pH
conditions. Under slightly acidic conditions such as pH 5.0,
m
tion at the C site in DNA and subsequent formation of a
m
the resulting d C radical cation undergoes irreversible de-
methyl-centered radical occur efficiently at pH 5.0, as in the
protonation at the C(5) methyl
group to form a methyl-cen-
tered
radical
intermediate,
which leads to a higher yield of
f
d C via addition of molecular
oxygen to the C(5) position.
Steenken reported that the rad-
ical cation of 2’-deoxycytidine is
in equilibrium with its depro-
tonated form at the amino
group even under slightly acidic
[27]
conditions. Thus, it is reason-
able to presume that the equi- Scheme 3. Synthesis of oligodeoxynucleotides with an NQ chromophore.
Chem. Eur. J. 2008, 14, 10453 – 10461
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10457

K. Tanabe, S.-I. Nishimoto et al.
Conclusion
In summary, we studied the pH
effect on the NQ-photosensi-
tized one-electron oxidation of
m
d C. The efficiency of oxidative
f
m
formation of d C from d C in-
creased with decreasing pH
from 8.0 to 5.0, attained its
maximum value at pH 5.0, and
then decreased dramatically
upon a further decrease in pH
values below 4.5. Fluorescence
quenching experiments suggest
that the lower efficiency for the
f
formation of d C at pH 4.5 is at-
tributable to the decreased rate
of one-electron oxidation of
m
d C owing to protonation at
the N(3) position. The pH-inde-
m
pendent degradation of M-d C
by NQ-photosensitized oxida-
tion indicates that the deproto-
nation–protonation equilibrium
at the N(4)-exocyclic amino
Figure 5. a) Sequences of the oligodeoxynucleotides used in this study; b) Representative autoradiogram of de-
naturing gel electrophoresis for ODN (NQ ) and P-5’-end-labeled ODN ( C) upon 312 nm photoirradiation
1
for 2 h in 2 mm sodium cacodylate buffer solution containing 20 mm NaCl (pH 4.0–8.0) at 08C. After treatment
with hot piperidine (908C, 20 min), the samples were purified through electrophoresis through a denaturing
3
2
m
m
group of the d C radical cation
1
5% polyacrylamide/7m urea: lane 1, pH 4.0; lanes 2, pH 4.5; lane 3, pH 5.0; lane 4, pH 6.0; lane 5, pH 7.0; may be a key factor in the for-
f
lane 6, pH 8.0; lane 7, Maxam-Gilbert G+A sequencing lanes; c) pH-dependent efficiency of strand cleavage
mation of d C in the pH range
between 5.0 and 8.0. We also
confirmed that photosensitiza-
tion of the NQ-tethered duplex
at pH 5.0 allows enhanced oxi-
m
at the C site estimated by densitometoric analysis of the gel image. Each error bar represents the standard
deviation calculated from three sets of experimental data.
m
m
similar photooxidation of monomeric d C. Thus, the NQ-
dation of C to produce 5-formylcytosine.
photosensitized oxidation under slightly acidic conditions is
Although optimization of the pH environment in the pho-
tosensitized oxidation was revealed to give an efficient
strand cleavage at C in DNA, further improvements are
m
essential for strong strand cleavage at the C site in DNA.
m
m
Along with the cleavage at C, competitive cleavage at adja-
cent G residues was also observed at pH 5.0 but not at
needed to establish a standard method to analyze cytosine
[32]
m
pH>6.0. Douki and Cadet reported that dG is the most
degradable nucleoside among four DNA bases owing to the
positive-charge transfer in the NQ-sensitized photooxida-
methylation. First, the further sensitivity for detection of
C
is required. One of the key strategies for the highly sensitive
m
detection of C is the employment of fluorescence emission,
[
33]
tion.
Our previous results also implied that the positive
which provides a distinct signal. We have started to develop
m
m
charge of the C radical cation generated by NQ photosen-
sitization migrates to the adjacent G base. The correspond-
ing G radical cation is then produced in competition with
the formation of a methyl-centered C radical intermediate
via deprotonation at the C(5)-methyl group. In this con-
text, the present result suggests that the equilibrium be-
a protocol for the fluorometric detection of C by using a
combination of photooxidative strand cleavage and invasive
[9c]
cleavage reaction. Second, high selectivity for the DNA
m
m
cleavage at C is indispensable. To overcome this problem,
[9b]
one must suppress the competitive oxidative cleavage at G
caused by the positive-charge transfer in the NQ-sensitized
photooxidation. Our current study focuses on the improve-
m
tween the C radical cation and its deprotonated form at
m
the amino group may favor a shift to an increased amount
ment of the photosensitized oxidation to give C-specific
m
of C radical cation under slightly acidic conditions such as
cleavage by using other photosensitizers.
pH 5.0, thus leading to the positive-charge transfer to a
neighboring G base as a well-known positive-charge trap-
ping site.
Even though the methylcytosine-selective reaction is im-
m
perative for correct detection of C, there are few protocols
[34,35]
m
for detecting C by using methylcyotsine-positive and cyto-
sine-negative reactions. Conventional reactions are based on
cytosine-selective but not methylcytosine-selective reactions.
m
Given these contexts, our system to directly oxidize C but
10458
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 10453 – 10461

Naphthoquinone-Photosensitized Oxidation of AHCUTNGRENGUN5 -Methylcytosine
FULL PAPER
1
not C could be promising for the efficient analysis of the
status of cytosine methylation at a specific site in a gene.
1838C; H NMR (D
6
4
2
O, 400 MHz): d=7.41 (s, 1H), 6.13 (t, 1H, J=
.6 Hz), 4.32 (m, 1H), 3.90 (m, 1H), 3.68 (ddd, 2H, J=31.5, 12.3,
.9 Hz), 3.07 (s, 6H), 2.27–2.12 (2H), 2.09 ppm (s, 3H); C NMR
]DMSO, 100 MHz): d=164.9, 153.7, 141.0, 101.8, 87.2, 84.6, 70.3,
1
3
(
[D
6
6
2
2
1.2, 40.2, 39.8, 18.1 ppm; FABMS (matrix: 3-nitrobenzyl alcohol): m/z
+
+
20 3 4
70 [M+H] ; HRMS: calcd for C12H N O : 270.1453 [M+H] ; found:
Experimental Section
70.1454.
Photosensitized oxidation by 2-methyl-1,4-naphthoquinone: Solutions of
-methyl-2’-deoxycytidine (200 mm) or N(4)-dimethyl-substituted 5-
methyl-2’-deoxycytidine (200 mm) in 2 mm sodium cacodylate buffer solu-
tion (various pH values) containing 20 mm NaCl were added to an aceto-
nitrile solution of 2-methyl-1,4-naphthoquione (200 mm). The solutions
Materials: 2-Methyl-1,4-naphthoquinone was purchased from Wako Pure
Chemical Industry, Japan, and was further purified by recrystallization
from methanol before use. 5-Methyl-2’-deoxycytidine and 9,10-dicya-
noanthracene were obtained commercially from MP Biomedicals and
Tokyo Chemical Industry, respectively, and were used without further pu-
rifications. 5-Formyl-2’-deoxycytidine was prepared as described previ-
5
(
100 mL) in a 1.5-mL Eppendorf tube were exposed to 312 nm UV light
with a Lourmat TFX-20m transilluminator (Vilber Lourmat, France) or
65 nm UV light with a TFL-40X transilluminator (UVP, USA) at 08C.
[
36]
+
À
ously. 10-Methylacridinium iodide (AcrH I ) was prepared by the re-
3
[
37]
actions of acridine with methyl iodide in acetonitrile, and the resulting
iodide salt was purified by recrystallization from 25% methanol/acetoni-
trile. The reagents for the DNA synthesizer were purchased from Glen
Research. BD Uni-Link AminoModifier was purchased from BD Biosci-
ences Clontech. Calf intestinal alkaline phosphatase (AP), nuclease P1
Analytical HPLC was performed with a Shimadzu 6 A HPLC system.
Sample solutions (10 mL) were injected onto a reversed-phase column
(
Inertsil ODS-3, GL Sciences Inc., f 4.6 mm ꢁ 150 mm). The solvent
mixture of triethylamine acetate (0.1m, pH 7.0) containing various con-
centrations of acetonitrile (5 or 15 vol%) was delivered as the mobile
phase. The column eluents were monitored by the UV absorbance at
(
P1), and phosphodiesterase I were purchased from PROMEGA,
3
2
À1
YAMASA, and ICN, respectively. [g- P]ATP (6000 Cimmol ) and T4
polynucleotide kinase (10 unitsmL ) were obtained from Amersham
2
60 nm.
À1
Nanosecond laser flash photolysis: The laser-flash-photolysis experiments
were carried out with a Unisoku TSP-601 flash spectrometer. A Continu-
um Surelite-I Nd:YAG (Q-swiched) laser with the third harmonic at
Bioscience and Nippon Gene, respectively. All aqueous solutions were
prepared by using purified water (YAMATO, WR600 A).
4
(
(
-(N-1-Triazoyl)-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxythymidine
2): 1,2,4-Triazole (6.4 g, 93 mmol) was suspended in acetonitrile
150 mL), which was cooled to 08C and POCl (2 mL, 21.9 mmol) was
3
55 nm (approximately 50 mJ per 6-ns pulse) was employed for the flash
photoirradiation. Further details of the laser flash system have been de-
3
[
38]
scribed previously. Solutions of NQ (50 mm) in 10 mm phosphate buffer
solution (pH 5–8) were de-aerated by passing argon through the solution
prior to the laser flash photolysis experiments.
then slowly added. Triethylamine was then added dropwise and the sus-
pension was stirred for 30 min. 3’,5’-O-Bis(tert-butyldimethylsilyl)thymi-
dine (1; 2.0 g, 4.25 mmol) was dissolved in acetonitrile (30 mL) and
added to the solution, which was then continuously stirred for another
Fluorescence quenching: Quenching experiments of the fluorescence of
photosensitizers were carried out on a Shimadzu RF-5300PC spectropho-
tometer. The excitation wavelengths were 358 and 390 nm for 10-methyl-
acridinium ion (25 mm) and 9,10-dicyanoanthracene (25 mm), respectively.
The monitoring wavelengths were those corresponding to the respective
emission bands at 453 and 487 nm, respectively. The dynamic quenching
3
0 min. The reaction was quenched by water and extracted with ethyl
acetate. The organic layer was washed with brine, dried over MgSO , fil-
tered, and concentrated. The crude product was purified by column chro-
matography (SiO , 33% ethyl acetate–hexane) to give 2 (2.1 g, 95%) as a
colorless oil. H NMR (400 MHz, CDCl ): d=9.24 (s, 1H), 8.21 (s, 1H),
.07 (s, 1H), 6.26 (t, 1H, J=6.3 Hz), 4.38–4.35 (1H), 4.03 (1H), 3.93 (dd,
H, J=11.5, 2.7 Hz), 3.77 (dd, 1H, J=11.5, 2.4 Hz), 2.61 (ddd, 1H, J=
3.4, 6.1, 3.7 Hz), 2.41 (s, 1H), 2.04 (td, J=12.0, 5.0 Hz), 0.88 (s, 9H),
.86 (s, 9H), 0.09 (d, 6H, J=5.1 Hz), 0.05 ppm (d, 6H, J=4.9 Hz);
4
2
1
3
rate constant k
+k [5-methyl-2’-deoxycytidine], where I
intensities in the absence and presence of 5-methyl-2’-deoxycytidine and
is the lifetime of the singlet excited state of 10-methylacridinium ion
q
was determined by the Stern–Volmer equation: I
0
/I=
8
1
1
0
1
q
t
0
0
/I is the ratio of the emission
t
0
[
37]
[39]
1
3
(35 ns)
quencher.
or 9,10-dicyanoanthracene (15.1 ns)
in the absence of
3
C NMR (CDCl , 100 MHz): d=158.0, 153.7, 153.3, 146.6, 145.0, 105.2,
8
8.7, 87.8, 62.5, 42.6, 25.9, 25.7, 18.4, 18.0, 17.2, À4.9, À5.4 ppm; FABMS
+
(
matrix: 3-nitrobenzil alcohol): m/z 522 [M+H] ; HRMS: m/z calcd for
Synthesis of ODNs: Synthesis of oligodeoxynucleotides was performed
on an Applied Biosystems Model 392 DNA/RNA synthesizer by using
standard phosphoroamidite chemistry techniques. We used BD Uni-Link
AminoModifier for DNA synthesis to introduce an aminohexyl group
into oligomers. After automated DNA synthesis, the oligomer was puri-
fied by reversed-phase HPLC with a 0–30% linear gradient (over
60 min) of acetonitrile/0.1m TEAA buffer solution (TEAA=triethylam-
monium acetate) at pH 7.0. The purity and concentration of the oligomer
were determined by complete digestion with AP, P1, and phosphodiester-
ase I at 378C for 4 h. The synthesized oligomers were identified by
MALDI-TOF mass spectrometry (negative mode: calcd. 5312.47, found
+
C
24
H
44
N
5
O
4
Si
-(Dimethylamino)-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxythymidine
3): 50% Dimethylamine in aqueous solution (8 mL) was added to a so-
2
: 522.2932 [M+H] ; found: 522.2942.
4
(
lution of 2 (1.0 g, 1.92 mmol) in acetonitrile (20 mL) and the mixture was
stirred at 08C for 5 min. The reaction mixture was diluted with water and
extracted with ethyl acetate. The organic layer was washed with brine,
dried over MgSO
rified by column chromatography (SiO
give 3 (1.0 g, quantitative) as a colorless oil. H NMR (300 MHz, CDCl
d=7.40 (s, 1H), 6.27 (t, 1H, J=6.5 Hz), 4.31–4.27 (1H), 3.84–3.66 (3H),
.06 (s, 6H), 2.31 (ddd, 1H, J=13.3, 6.1, 3.7 Hz), 2.06 (s, 3H), 1.96–1.87
1H), 0.85 (s, 9H), 0.81 (s, 9H), 0.03 (d, 6H, J=1.3 Hz), À0.02 ppm (d,
4
, filtered, and concentrated. The crude product was pu-
2
, 33% hexane–ethyl acetate) to
1
3
):
3
5312.05).
A solution of 3-(N-hydroxysuccinimidylethyl)-2-methyl-1,4-
[
9a]
(
6
1
naphthoquinone (68 mg, 0.2 mmol) and saturated NaHCO (20 mL) was
3
1
3
H, J=1.5 Hz); C NMR (75 MHz, CDCl
3
): d=165.9, 155.0, 140.2,
added to a solution (total volume 50 mL) of oligomer with an amino
linker in the interior of the strand and subsequently incubated at 258C
for 4 h. The reaction mixture was purified by reversed-phase HPLC with
a 0–30% linear gradient (over 60 min) of acetonitrile/0.1m TEAA buffer
solution at pH 7.0. The purity and concentration of NQ-modified ODN
were determined by complete digestion by AP, P1, and phosphodiestera-
02.1, 87.3, 85.4, 71.5, 62.6, 41.8, 40.1, 25.8, 25.6, 18.3, 17.9, À4.7,-5.5 ppm;
+
FABMS (matrix: 3-nitrobenzyl alcohol): m/z 498 [M+H] ; HRMS:
calcd for C24
+
H
48
N
3
O
4
Si
2
: 498.3183 [M+H] ; found: 498.3192.
m
4
-(Dimethylamino)-2’-deoxythymidine (M-d C): Tetrabutylammonium
fluoride trihydrate (0.94 g, 3.0 mmol) was added to a solution of 3 (0.5 g,
.0 mmol) in dry THF (10 mL) and the mixture was stirred at room tem-
se I at 378C for 4 h. The synthesized ODNs were identified by MALDI-
1
À
TOF mass spectrometry (ODN (NQ
found: 5539.59).
1
); m/z: calcd for 5538.69 [MÀH] ;
perature for 20 min. The reaction mixture was then concentrated and the
crude residue was resuspended with water and then washed with chloro-
form. The aqueous layer was concentrated in vacuo. The crude product
3
2
Preparation of 5’- P-end labeled ODNs: ODNs (400 pmol strand con-
3
2
was roughly purified by column chromatography (SiO
2
, 0–10% metha-
centration) were labeled by phosphorylation with 4 mL of [g- P]ATP and
[
40–41]
nol-chloroform) and then purified by reversed-phase HPLC (15% aceto-
46 mL of T4 polynucleotide kinase by using standard procedures.
The
m
nitrile–water) to give M-d C (234 mg, 87%) as a white solid. mp. 182–
5’-end-labeled ODNs were recovered by ethanol precipitation and fur-
Chem. Eur. J. 2008, 14, 10453 – 10461
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10459

K. Tanabe, S.-I. Nishimoto et al.
f
ther purified by 15% nondenaturing gel electrophoresis and isolated by
the crush-and-soak method.
[10] We also confirmed that the yield of d C slightly decreased after
[
42]
f
60 min of irradiation, indicating that d C was degraded by prolonged
3
2
NQ-photosensitized oxidation (see the Supporting information).
Photooxidative cleavage of ODNs: P-5’-end labeled ODNs (<400 nm
strand concentration) were hybridized by their complementary ODNs
with NQ chromophore (500 nm) in 2 mm sodium cacodylate buffer solu-
tion (pH 7.0) containing 20 mm NaCl. Hybridization was achieved by
heating the sample at 908C for 5 min and slowly cooling to room temper-
[
11]
These results are also supported by the previous reports.
11] C. Bienvenu, J. R. Wagner, J. Cadet, J. Am. Chem. Soc. 1996, 118,
1406–11411.
[
[
1
12] 5-(Hydroperoxymethyl)-2’-deoxycytidine, 5-(hydroxymethyl)-2’-de-
oxycytidine and 5-formyl-2’-deoxycytidine have been identified as
the photooxidation products by previous spectroscopic experiments
3
2
ature. The P-5’-end labeled duplex was irradiated at 312 nm with a
transilluminator at 08C. After irradiation, all reaction mixtures were pre-
cipitated with the addition of 10 mL of herring-sperm DNA or salmon-
1
13
[11]
( H and C NMR, UV spectroscopy, and mass spectroscopy).
À1
sperm DNA (1 mgmL ), 10 mL of 3m sodium acetate, and 800 mL of eth-
[13] J. R. Wagner, J. E. van Lier, L. J. Johnson, Photochem. Photobiol.
1990, 52, 333–343.
[14] T. Douki, J. L. Ravanat, D. Angelov, J. R. Wagner, J. Cadet, in
Long-Range Charge Transfer in DNA I, Vol. 236 (Ed.: G. B. Schus-
ter), Springer, New York, 2004, pp.1–25.
anol. The precipitated DNA was washed with 100 mL of 80% cold etha-
nol and then dried in vacuo. The precipitated DNA was disolved in 50 mL
of 10% piperidine (v/v), heated at 908C for 20 min and concentrated.
The resulting samples were then analyzed by polyacrylamide gel electro-
[
9]
phoresis, of which the experimental details were described previously.
[15] The free-energy change of charge separation (DGCS) for photooxida-
m
3
*
tion of C by NQ was estimated to be À0.58 eV by using Wellerꢃs
equation (1):
(E
is the NQ triplet energy
[
15a]
DGCS =À
A
T
N
T
N
U
G
T
+Erdn)+Eox
(1)
[
15b]
where E
T
(2.52 eV), Erdn is its reduction
(À0.21 V vs. NHE), and Eox is the oxidation potential
[
15c]
potential
Acknowledgement
of 5-methylcytosine (1.73 V vs. NHE) as calculated from the ioniza-
tion potential
[
15d]
[15e]
(8.39 eV).
See: a) D. Rehm, A. Weller, Isr. J.
H.Y. acknowledges a fellowship from Japan Society for the Promotion of
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1
4511–14517.
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a
value for M-d C was determined to be 3.8 from the pH-re-
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m
N(3) protonation of M-d C should be negligible in the pH range
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10460
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 10453 – 10461

Naphthoquinone-Photosensitized Oxidation of AHCUTNGRENGUN5 -Methylcytosine
FULL PAPER
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m
[
[
30] C in codon 282 of exon 8 in human p53 gene is marked as a meth-
[
2c]
ylation hot spot.
31] It is well known that direct absorption of UV-B in the 290–320 nm
wavelength region by DNA results in formation of dimeric photo-
[
31a,b]
products between adjacent pyrimidine bases.
In the present se-
quence, there are no consecutive T bases and therefore we did not
confirm the formation of T dimers. Although the T dimers may
form upon UV-B irradiation of DNA bearing consecutive T bases,
the effect of this undesirable photoreaction will be negligible for the
m
clear detection of C by using PAGE. According to the previous re-
[
31c]
port,
the strand cleavage at consecutive T bases did not occur
upon similar photoirradiation at 312 nm by using a transilluminator.
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[
32] Small amounts of unselective strand cleavage was also observed
under more acidic conditions (pH<4.5). In view of the evidence
m
that NQ-photosensitized oxidation of C was difficult to proceed
under more acidic condition such as pH values less than 4.5, we as-
sumed that the unselective strand cleavage observed in this study
may be attributed to acid damage of DNA bases.
Received: May 3, 2008
Revised: August 4, 2008
[
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Published online: October 1, 2008
Chem. Eur. J. 2008, 14, 10453 – 10461
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