BODIPY-modified 2′-deoxyguanosine as a novel tool to detect DNA damages

Article abstract of DOI:10.1016/j.bmcl.2011.05.084

BODIPY-modified 2′-deoxyguanosine was synthesized for use as a detection reagent for genotoxic compounds. BODIPY-FL is a well known fluorescence reagent whose fluorescent light emission diminishes near a guanine base by a photo-induced electron transfer process. We attached BODIPY-Fl to the 5′ position of the deoxyribose moiety of 2′-deoxyguanosine. Although this compound has low fluorescence activity, when depurination by the action of alkylating reagents and dG oxidation by singlet oxygen occurred, the emission of strong fluorescence was observed. BODIPY-dG was found, therefore, to be a very useful tool for selectively detecting DNA damaging activity particularly in natural environmental extracts.

Full text of DOI:10.1016/j.bmcl.2011.05.084

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4206–4209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
BODIPY-modified 20-deoxyguanosine as a novel tool to detect DNA damages
⇑
Takeji Takamura-Enya , Ryoko Ishii
Department of Applied Chemistry, Kanagawa Institute of Technology, Shimo-Ogino 1030, Atsugi-shi 243-0292, Japan
a r t i c l e i n f o
a b s t r a c t
BODIPY-modified 20-deoxyguanosine was synthesized for use as a detection reagent for genotoxic
compounds. BODIPY-FL is a well known fluorescence reagent whose fluorescent light emission diminishes
near a guanine base by a photo-induced electron transfer process. We attached BODIPY-Fl to the 50 position
of the deoxyribose moiety of 20-deoxyguanosine. Although this compound has low fluorescence activity,
when depurination by the action of alkylating reagents and dG oxidation by singlet oxygen occurred, the
emission of strong fluorescence was observed. BODIPY-dG was found, therefore, to be a very useful tool
for selectively detecting DNA damaging activity particularly in natural environmental extracts.
Ó 2011 Elsevier Ltd. All rights reserved.
Article history:
Received 1 February 2011
Revised 16 May 2011
Accepted 23 May 2011
Available online 30 May 2011
Keywords:
BODIPY
Mutagen
Alkylating reagents
Oxidative damages
20-Deoxygunosine
Modifications of DNA with exogenous and/or endogenous
sources are now believed to be the initial step of carcinogene-
sis.1–3 Such modifications would trigger DNA mutations, which,
in some cases, could also be linked to several chronic diseases.
Numerous DNA modifications have now been identified. These in-
clude strand breaks, the covalent modification of nucleobases with
chemicals, oxidative damage, and the depurination of bases.4
Guanine bases in DNA are also known to be the most reactive sites
to genotoxic compounds.4 The oxidation of 20-deoxyguanosine
(dG) by X-rays or activated oxygen produces several oxidative
products, including 8-oxo-dG, 4,8-dihydro-4-hydroxy-8-oxo-
20-deoxyguanosine 1, and imidazolone derivatives (Fig. 1).5 Lipid
peroxidation products can also attack dG to form etheno-dG deriv-
atives.4 Amino/nitro-arenes, carcinogens in foods and the atmo-
sphere, react at the C8 position of dG in major amounts.6,7 PAH,
when metabolically activated, can react with the N2 amino group
of dG.8 Alkylating reagents can react with N7 of dG to form N7
alkylating compound 2, which undergoes a facile depurination
reaction, releasing N7-alkylated guanine base (Fig. 1).4
analysis, and for QSAR studies to predict the mutagenic activities of
compounds. Although these biological and chemical methods for
detecting genotoxic compounds are all useful and widely used, they
also have some drawbacks such as the need for special equipment
and/or low sensitivities to active compounds. The objective of our
study was to synthesis compounds that could detect mutagenic
compounds in a facile and rapid manner. To enable the detection
of mutagens, we focused on 4,4-difluoro-4-bora-3a,4a-diaza-s-
indacene (BODIPYÒ) dyes, which are generally known to be high
fluorescence compounds and are used in biological research.11,12
BODIPYÒ is a strong fluorescence dye used in the biological field.
This dye is also known to have its fluorescence quenched by proxim-
ity to a guanine base through a photo-induced electron transfer
mechanism.13 When an oligonucleotide was modified with a BODI-
PYÒ derivative, the fluorescence intensity of the BODIPYÒ dimin-
ished by up to 95% compared to that of the original compound.13
This property can be used to detect an oligonucleotide sequence
and GTPase activities in cells.14–16 Given the reactive nature of dG
To date, these DNA modifications have generally been identified
by biological methods using an Ames salmonella reverse mutation
assay to determine the mutagenicity of a compound and a salmonella
UMU test to determine its genotoxicity. In an alternative method, 4-
nitrobenzylpyridine can be used to detect DNA alkylating reagents.
9,10 Electrophilic compounds can attack the N atom of the pyridine
moiety. The resulting N-alkyl compound then turns blue under alka-
li conditions with the loss of a benzylic proton.9,10 The 4-NBP meth-
od can be widely used to detect alkylating compounds, includingTLC
⇑
Figure 1. 20-Deoxyguanosine (dG) and selected DNA damages, 4-OH-8-oxo-20-
deoxyguanosine 1, and N7 alkylation compound 2.
Corresponding author. Tel.: +81 46 291 3072; fax: +81 46 242 8760.
E-mail address: takamura@chem.kanagawa-it.ac.jp (T. Takamura-Enya).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.084

T. Takamura-Enya, R. Ishii / Bioorg. Med. Chem. Lett. 21 (2011) 4206–4209
4207
Figure 3. Synthetic procedure for BODIPY-dG 3.
The compound had a weak fluorescence nature and its emission
maximum was 524 nm, which was slightly red shifted compared to
that of BODIPY-FL (513 nm). In order to check our hypothesis, rep-
resentative alkylating compounds, methyl methanesulfonate 5 and
epichlorohydrin 6, were added to the solution containing the
BODIPY-dG derivative 3 under the slightly acidic condition of pH
5 (Fig. 4).21 After 12 h, the solutions with the added alkylating re-
agents were observed to show strong green fluorescence as ex-
pected (Fig. 5, the visualization of this fluorescence enhancement
with alkylating reagents is shown in the Supplementary data,
Fig. 1). The green fluorescence of the solutions could be easily de-
tected with the naked eye under a fluorescent light or a black light.
The fluorescence spectra produced by the addition of the two alkyl-
ating compounds were totally equal, and there was a quantitative
recovery of the fluorescence.
Alkylating reagents are generally known to attack N7 of dG,
with the resulting N7-modified dG 2 being readily depurinated
by a glycosidic bond cleavage. In the case of methyl methanesulfo-
nate, the methylation of N7 generally occurs. The modification of
the N7 of dG is relatively stable and the resulting methylated com-
pound can be present for several hours under an aqueous condi-
tion. We found that 90% of starting material 3 was consumed
after 4 h when it reacted with 5. The resulting methylated adduct
was depurinated within several hours at pH5 which is in accor-
dance with the half-life of 4.4 h at pH 4.2 for N7 alkylated deoxy-
guanosine.22 This fact well explains our experimental finding that
a reaction time of 12 h was needed to enhance the fluorescence
activity. We have also confirmed the final product was a ribofura-
nose attached with BODIPY-FL at the 50 position and the same
product had been efficiently obtained from acid treatment of BOD-
IPY-dG (Fig. 4, Supplementary data).
Figure 2. Concept of fluorescence detection of alkylating reagents by
BODIPY-modified dG 3.
to mutagenic compounds, we theorised that by attaching the
BODIPYÒ to dG at the nearest position, like 50-OH of dG, the expected
compound 3 would generally show only weak fluorescence emis-
sion. However, once some modifications occur at the guanine bases,
the fluorescence of BODIPYÒ will re-emerge (Fig. 2). This fluores-
cence recovery can efficiently occur when alkylating compounds at-
tack the BODIPYÒ-modified dG, after which the depurination of the
modified guanine bases occurs.
The target compound 3 that should be synthesized for this pur-
pose has a very simple but compromising chemical structure,
where 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-
3-propionic acid (known as BODIPYÒ-FL) is attached to the 50 posi-
tion of dG. The synthesis of BODIPYÒ-FL was performed based on
the published paper of Malan et al.17 Initially, BODIPYÒ-FL was re-
acted to dG directly, under the general conditions of dicyclohexyl-
carbodiimide (DCC) and pyridine with 4-dimethylaminopyridine
(4-DMAP), because the 50-OH of dG can be expected to easily react
prior to the hydroxy group at the 30 position. However, this ap-
proach failed and only a crude mixture was obtained, probably be-
cause of the instability of BODIPYÒ under the applied reaction
conditions. No direct modification of dG with BODIPYÒ-FL has yet
been reported. We did find one paper that described how a dG ad-
duct modified with
a mutagen can be converted into a
BODIPYÒ-FL-modified dG derivative in dichloromethane as a sol-
vent, although both 30-OH and 50-OH were reacted with BODIPYÒ-
Fl.18 This experimental evidence encouraged us to attempt to use
another protocol for synthesis. Because dG is insoluble in dichloro-
methane, it was necessary to protect it with appropriate protective
groups. In order to enhance the solubility and protect the reactive
sites, except for 50-OH, 30-OH was protected by TBDMS, and the
N2 of dG was protected by a phenoxyacetyl (PAC) group, which is
shown as compound 4 in Figure 3. This compound was obtained
by the sequence of 30,50-OH-bisTBDMS protection, N2 protection
with PAC anhydride,19 followed by the selective desilylation of 50-
OH by TBAF with acetic acid. Compound 4 was further reacted with
BODIPY-FL using DCC in dichloromethane with 4-DMAP. Protective
groups were further de-protected with diisopropylamine in metha-
nol and a trihydrogen fluoride–triethylamine complex in THF. After
the solvent was evaporated, the compound was resuspended in
acetonitrile, and the obtained precipitate was washed thoroughly,
giving the desired BODIPY-modified dG 3.20
In order to further elucidate the usefulness of BODIPY-dG 3, we
next attempted to check whether oxidative damages to dG could
Figure 4. DNA damaging reagents tested in this study.

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T. Takamura-Enya, R. Ishii / Bioorg. Med. Chem. Lett. 21 (2011) 4206–4209
In conclusion, a simple modification of dG with BODIPY-FL
could be used as a mutagen detector for oxidative DNA damages
and alkylating reagents. It would be possible to use reagent 3 in
an in vivo system such as for cell lines or in a test paper application
to test for mutagens in the environment similar to pH test paper.
Evaluation of these approaches of compound 3 and the detailed
mechanism of its on/off fluorescence are now being studied in
our laboratory.
Acknowledgments
This work was supported in part by Grants-in-Aid from the
Ministry of Education, Science, Sports and Culture and by the Japan
Society for the Promotion of Science and The Science Research
Promotion Fund.
Supplementary data
Figure 5. Fluorescence intensity of BODIPY-dG treated with alkylating reagents, 5
and 6.
Supplementary data (the visualization of the fluorescence of
BODIPY-dG with alkylating reagents, fluorescence recovery of
13
BODIPY-dG with DMBA under UVA light condition, 1H- and
C-NMR of compound 3) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.05.084.
be detected in the same system. Phenalenone 7 is a known con-
comitant present in the atmosphere and is also known to be a sin-
glet oxygen producer under black light irradiation (Fig. 4). Singlet
oxygen, once produced, can attack dG to form a highly oxidized
product, 4-OH-8-oxo-dG 1, with a yield of around 40%.22,23 Because
the electronic properties of the oxidized compound 1 are different
from those of dG, the fluorescence quenching by the electron trans-
fer pathway observed in the original compound 3 will not be ob-
served when the oxidation of dG occurs. Indeed, BODIPY-dG 3
with phenalenone 7 under UV light conditions had strong, dose-
dependent fluorescence emission (Fig. 6).24 Moreover, DMBA 8
can also recover the fluorescence of BODIPY-dG, probably because
of the oxidative damage to the dG moiety of compound 3 (Fig. 4,
Supplementary Fig. 2). Koutaka et al. showed that the on/off fluo-
rescence of the fluorophore can be explained by the HOMO level
of the donor (dG) and acceptor (BODIPY) compounds.25 A semi-
empirical chemical calculation showed that the HOMO energies
of BODIPY, dG, and 1 were ꢀ8.881, ꢀ8.811, and ꢀ9.985 eV, respec-
References and notes
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Y.; Kurane, R. Nucl. Acids Res. 2001, 29, e34.
26
tively. The lower HOMO value of 1 compared to that of BODIPY
could explain why the highly oxidized dG moiety in compound 3
reduced its quenching property.
15. McEwen, D. P.; Gee, K. R.; Kang, H. C.; Neubig, R. R. Anal. Biochem. 2001, 291,
109.
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20. Preparation of the compound 3: Into the solution of N2-phenoxyacetyl-30-
TBDMS-20-deoxyguanosine (0.2 mmol) and BODIPY-FL (0.4 mmol) in
dichloromethane was added DCC (2 equiv) with DMAP (2 equiv). After the
reaction mixture was left overnight, it was filtered and filtrate was evaporated
to dryness. Residual material was subjected to a column chromatography on
silica gel to give BODIPY-modified protected dG, which were further subjected
to diisopropylamine in methanol for 2 h. After purified by column
chromatography, resulting compounds were treated with triethylamine
trihydrogen fluoride complex in THF. After 24 h, solvent was evaporated and
the residual material was suspended in acetonitrile. Resulting precipitation
was obtained by centrifugation and washed twice with acetonitrile to give the
desired 50-BODIPY-FL-20-deoxyguanosine 3. 1H NMR (400 MHz, DMSO-
d6)™ = 10.50 (br s, 1H), 7.75 (s, 1H), 7.57(s, 1H), 6.93 (d, J = 4.1 Hz, 1H) 6.34
(br s, 1H), 6.23 (d, J = 3.7 Hz, 1H), 6.18(s, 1H) 6.01 (t, J = 6.9 Hz, 1H), 5.32 (br s,
1H), 4.25 (br s, 1H), 4.10 (ddd, J = 42.2, 11.8, 5.1 Hz, 2H), 3.85 (br s, 1H), 2.98 (t,
J = 7.5 Hz, 2H), 2.67–2.60 (m, 2H), 2.40–2.36 8 (m, 2H), 2.34 (s, 3H), 2.13 (t,
J = 7.1 Hz, 3H), 13C NMR (100 MHz, DMSO-d6)™ = 171.80, 159.77, 156.73,
156.17, 153.69, 150.99, 144.53, 135.24, 134.69, 132.99, 128.72, 125.53, 120.52,
116.78, 116.54, 83.98, 82.41, 70.65, 64.44, 32.15, 23.43, 14.62, 11.07, 10.83,
Figure 6. Fluorescence intensity of BODIPY-dG
phenalenone. Phenalenone is abbreviated as PhO.
3 treated with compound 7,

T. Takamura-Enya, R. Ishii / Bioorg. Med. Chem. Lett. 21 (2011) 4206–4209
4209
HR-HR-MS (ESI-TOF): m/z [M+H]+ calcd for C24H27BF2N7O5:542.2129; found
542.1989.
21. Typical procedure for detection of alkylating compounds: To the buffered solution
24.
A
96-well plate version of the fluorescence detection of singlet oxygen
generating compounds: to the buffered solution (PBS, pH 7, 100 L) containing
an appropriate amounts of phenalenone 7 or DMBA 8 was added 100 L of the
l
l
(pH5) were added 10
alkylating reagents (10
sample was diluted and the fluorescence was measured at an excitation of
490 nm. The emission spectra from 495 to 510 nm were recorded.
22. Gates, K. S.; Nooner, T.; Dutta, S. Chem. Res. Toxicol. 2004, 17, 839.
23. Stevenson, G. A. C.; Davies, R. J. Chem. Res. Toxicol. 1999, 12, 38.
l
L
of BODIPY-dG (1 mg/mL DMSO) followed by
BODIPY-dG solution (1 mg/mL in DMSO). After UVA irradiation for 20 min, the
reaction mixture was diluted by a factor of 1000 and em 485 nm/ex 535 nm
fluorescence was recorded by a fluorescence plate reader.
lL of compound 1 and 5 L of 2). After 12 h, the
l
25. Koutaka, K.; Kosuge, J.; Fukasaku, N.; Hirano, T.; Kikuchi, K.; Urano, Y.; Kojima,
H.; Nagano, T. Chem. Pharm. Bull. 2004, 52, 700.
26. MOPAC calculation (PM3) was performed by ChemBio 3D 12.0 with MOPAC.