Synthesis and thermodynamic studies of oligodeoxyribonucleotides containing tandem lesions of thymidine glycol and 8-oxo-2′-deoxyguanosine

Article abstract of DOI:10.1021/tx060032l

Thymidine glycol (Tg), which is also known as 5,6-dihydroxy-5,6- dihydrothymidine, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) are two major types of DNA damage products induced by reactive oxygen species (ROS). Here, we report the synthesis of olig

Full text of DOI:10.1021/tx060032l

Chem. Res. Toxicol. 2006, 19, 837-843
837
Synthesis and Thermodynamic Studies of Oligodeoxyribonucleotides
Containing Tandem Lesions of Thymidine Glycol and
8-Oxo-2′-deoxyguanosine
Yuesong Wang and Yinsheng Wang*
Department of Chemistry-027, UniVersity of California at RiVerside, RiVerside, California 92521-0403
ReceiVed February 13, 2006
Thymidine glycol (Tg), which is also known as 5,6-dihydroxy-5,6-dihydrothymidine, and 8-oxo-7,8-
dihydro-2′-deoxyguanosine (8-oxodG) are two major types of DNA damage products induced by reactive
oxygen species (ROS). Here, we report the synthesis of oligodeoxyribonucleotides (ODNs) containing
both Tg and 8-oxodG. The dual incorporation of the two single-base lesions was achieved by using a
phosphoramidite building block of 8-oxodG with ultramild base protecting group and a building block
of Tg whose nucleobase hydroxyl groups were protected with acetyl functionality. The availability of
ODNs carrying neighboring 8-oxodG and Tg provided authentic substrates for assessing the formation
and examining the replication and repair of this kind of tandem lesions. In addition, thermodynamic
parameters derived from melting temperature data revealed that tandem lesions destabilized the double
helix to a greater extent than either of the two single-base lesions alone. The thermodynamic results
could offer a basis for understanding the repair of the tandem base lesions.
purified repair enzymes and in mammalian cells (25-31). To
understand the biological significances of tandem lesions with
a Tg adjacent to an 8-oxodG, it is crucial to assess their
formation and to examine the replication and repair of this type
of lesions. The lack of procedures for preparing pure and
sufficient tandem lesion-bearing substrates, however, hampers
such studies. This motivates us to design synthetic approaches
for the incorporation of both types of lesions into oligodeox-
yribonucleotides (ODNs).
Solid-phase synthesis of ODNs by the use of phosphoramidite
building blocks is advantageous over the postsynthetic methods
for the preparation of modified or damaged ODNs, because the
chain length and the sequence are not restricted and large-scale
preparation is available (32). In this regard, Iwai (33, 34)
described the synthesis of a building block of thymidine glycol
where the nucleobase hydroxyl group(s) was protected with a
tert-butyldimethylsilyl (TBDMS) group. Very recently, Cadet
et al. (35) reported the synthesis of a Tg building block where
the nucleobase hydroxyl group(s) was protected with a base-
labile levulinyl functionality. On the other hand, the deprotection
of ODNs synthesized from the commercially available building
block of 8-oxodG requires prolonged treatment with concen-
trated ammonium hydroxide; during such treatment, thymidine
glycol, however, was shown to be significantly degraded (33).
Therefore, dual insertion of 8-oxodG and thymidine glycol
necessitates alternative synthetic strategies.
Introduction
Reactive oxygen species (ROS)1 can lead to a plethora of
modifications to DNA, which yield nucleobase and deoxyribose
lesions, strand breaks, and DNA-protein cross-links (1, 2).
These oxidative DNA lesions may have implications in a variety
of pathological conditions, including diseases associated with
aging and cancer (1, 3). Thymidine glycol (or 5,6-dihydroxy-
5,6-dihydrothymidine, Tg) and 8-oxo-7,8-dihydro-2′-deoxygua-
nosine (8-oxodG) are two common monomeric base lesions
induced by ROS. Thymidine glycol lacks mutagenicity under
most conditions (4-7). However, it effectively blocks DNA
replication (8, 9). In contrast, 8-oxodG is a major mutagenic
lesion and can result in G f T transversion mutation (10, 11).
In recent years, several types of tandem DNA lesions (12-
20), where damage involves two adjacent nucleosides, have been
isolated and characterized. A first example of tandem lesions,
with an 8-oxodG and a formamido (dâF) residue adjacent to
each other, was reported by Box et al. (12, 21). Recently,
Bourdat et al. (15) showed that the amount of these tandem
lesions, dâF-8-oxodG and its isomeric 8-oxodG-dâF, formed
from γ radiation cannot account for the total amount of tandem
lesions involving 8-oxodG. It was, therefore, postulated that
other tandem lesions involving 8-oxodG may exist. Since
thymidine glycol and 8-oxodG are major types of single-
nucleobase lesions induced by ROS (2), we reason that tandem
lesions with a 8-oxodG adjacent to a Tg might be induced by
ROS.
Here in this paper, we reported the successful preparation of
ODN substrates bearing both 8-oxodG and Tg, which provides
substrates for examining the formation of the tandem lesions
with neighboring 8-oxodG and Tg induced by ROS. The
availability of this type of substrates also facilitates the
investigation of the replication and repair of this type of lesions
both in vitro and in vivo.
Clustered DNA damages, in which two or more lesions within
one or two helical turns of the DNA helix, can be initiated from
a single ionizing radiation track (22-24). It has been shown
that such damaged sites were difficult to be repaired with
* To whom correspondence should be addressed. E-mail,
yinsheng.wang@ucr.edu; fax, (951)827-4713; tel., (951)827-2700.
1 Abbreviations: ROS, reactive oxygen species; ODN, oligodeoxynucleo-
tides; BER, base excision repair; NER, nucleotide excision repair; ESI,
electrospray ionization; MS, mass spectrometry; MS/MS, tandem mass
spectrometry.
Experimental Procedures
Materials. Common reagents for solid-phase DNA synthesis
were obtained from Glen Research Co. (Sterling, VA). Unmodified
10.1021/tx060032l CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/28/2006

838 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Scheme 1. Synthesis of Phosphoramidite Building Block of Tga
Wang and Wang
a Reagents: (a) DMTrCl/pyridine/70 °C; (b) OsO4/pyridine; (c) Ac2O/DMAP/pyridine; (d) 70% acetic acid; (e) DMTrCl/pyridine; (f) NC(CH2)2OP(Cl)N(iPr)2/
DIEA/CH2Cl2.
ODNs used in this study were purchased from Integrated DNA
Technologies (Coraville, IA). Silica gel and TLC plates were
obtained from EM Science (Gibbstown, NJ). All chemicals, unless
otherwise specified, were from Sigma-Aldrich (St. Louis, MO).
Mass Spectrometry. Electrospray ionization-mass spectrometry
(ESI-MS) and tandem MS (MS/MS) experiments were carried out
on an LCQ Deca XP ion-trap mass spectrometer (ThermoFinnigan,
San Jose, CA). An equal-volume solvent mixture of acetonitrile
and water was used as solvent for electrospray, and a 2-µL aliquot
of ∼5 µM sample solution was injected in each run. The spray
voltage was 3.4 kV, and the instrument was operated in the
negative-ion mode.
HPLC. A 4.6 mm × 250 mm Apollo C18 column (5 µm in
particle size, 300 Å in pore size, Alltech Associate Inc., Deerfield,
IL) was used for the separation of synthetic ODNs. A solution of
50 mM triethylammonium acetate (TEAA, solution A) and a
mixture of 50 mM TEAA and acetonitrile (70/30, v/v, solution B)
were used as mobile phases. The flow rate was 0.8 mL/min, and a
gradient of 5 min 0-20% B, 45 min 20-50% B, and 5 min 50-
100% B was employed.
rated twice with dry pyridine (10 mL), and the dried residue was
dissolved again in dry pyridine (2 mL). DMAP (4-(dimethylamino)-
pyridine, 7.38 mg, 0.06 mmol) and acetic anhydride (0.36 mL, 3.84
mmol) were added into the solution under argon atmosphere. After
15 h, the solution was dried under reduced pressure, and the dried
residue was redissolved in EtOAc (80 mL). The organic layer was
washed with water (twice, 40 mL) and brine (40 mL), dried over
Na2SO4, and concentrated. Glycol 3 (262 mg, 82%) was isolated
with a silica gel column using a solvent gradient of 0-15% EtOAc
in CH2Cl2. Rf: 0.58 (EtOAc-CH2Cl2, 15:85). 1H NMR (400 MHz,
CDCl3): δ 8.64 (s, 1H), 7.15-7.40 (m, 18H), 6.69-6.81 (m, 8H),
6.35 (s, 1H), 6.03 (dd, 1H, J ) 5.8, 8.7 Hz), 4.26 (m, 1H), 4.10
(m, 3H), 3.76 (s, 6H), 3.72 (d, J ) 2.3 Hz, 6H), 3.22 (m, 2H), 2.02
(s, 3H), 2.00 (s, 3H), 1.90 (s, 3H). MALDI-MS: m/z 987.4 [M +
Na]+.
4. (5R,6S)-5,6-O-Acetyl-thymidine Glycol (4). Compound 3
(262 mg, 0.27 mmol) was treated with 70% acetic acid at room
temperature for 6 h. The resulting aqueous solution was dried under
reduced pressure. The dried residue was dispersed in ether (50 mL)
and extracted twice with water (50 mL each). The aqueous solutions
1
Synthesis and Characterization of Compounds (Scheme 1).
1. 3′,5′-O-(4,4′-Dimethoxytrityl)thymidine (1). The title compound
was synthesized according to the procedures reported by Sekine et
al. (36). In this respect, it is worth mentioning that the incorporation
of the bulky 4,4′-dimethoxytrityl (DMTr) functionality to the 3′-
hydroxyl group required a reaction temperature of 70 °C (36),
though the traditional DMTr protection of the 5′-hydroxyl group
is typically carried out at room temperature. Yield: 6.79 g (97%).
were combined and dried. Yield: 93 mg (95%). H NMR (400
MHz, CDCl3): δ 8.86 (s, 1H), 6.91 (s, 1H), 6.09 (t, 1H, J ) 7.3
Hz), 4.48 (m, 1H), 3.96 (m, 1H), 3.81 (m, 2H), 2.142 (m, 2H),
2.10 (s, 3H), 2.08 (s, 3H), 1.86 (s, 3H). ESI-MS: m/z 383.1 [M +
Na]+.
5. (5R,6S)-5′-O-(4,4′-Dimethoxytrityl)-5,6-O-acetyl-thymidine
Glycol (5). Compound 4 (90 mg, 0.26 mmol) was coevaporated
twice with dry pyridine (5 mL). The dried residue was redissolved
in dry pyridine (2 mL), and to the resulting solution was added
4,4′-dimethoxytrityl chloride (105 mg, 0.31 mmol) under argon
atmosphere. After 4 h, the solvent was removed under reduced
pressure. The product was loaded onto a silica gel column and eluted
with 0-2% methanol in a solvent mixture of Et3N and CH2Cl2
(1:99, v/v). Yield: 102 mg (61%). Rf: 0.17 (CH2Cl2-CH3OH, 19:
1
Rf: 0.38 (EtOAc-hexane, 1:1). H NMR (400 MHz, DMSO-d6):
δ 11.30 (br, 1H), 6.75-7.37 (m, 26H), 6.15 (m, 1H), 4.21 (m, 1H),
3.94 (m, 1H), 3.70 (s, 6H), 3.68 (d, J ) 3.2 Hz, 6H), 3.00 (m, 1H),
2.92 (m, 1H), 1.77 (m, 1H), 1.61 (m, 1H), 1.36 (s, 3H). MALDI-
MS: m/z 869.1 [M + Na]+.
2. (5R,6S)-3′,5′-O-(4,4′-Dimethoxytrityl)thymidine Glycol (2).
Compound 1 (1.67 g, 1.97 mmol) and OsO4 (0.5 g, 1.97 mmol)
were dissolved in pyridine (7.5 mL), and the mixture was stirred
at room temperature for 2 h. Sodium hydrogen sulfite (1.8 g),
dissolved in a mixture of water (30 mL) and pyridine (20 mL),
was then added to the reaction mixture. The mixture was stirred
for another 2 h. The product was extracted twice with CHCl3 (150
mL), and the organic layer was dried with Na2SO4. The solvent
was removed under reduced pressure. The reaction mixture was
then loaded onto a silica gel column and eluted with a CH2Cl2
solution containing 0.5% MeOH and 0.1% Et3N. Yield: 1.17 g
1
1). H NMR (400 MHz, CDCl3): δ 8.30 (s, 1H), 7.10-7.46 (m,
9H), 6.81-6.84 (m, 4H), 6.56 (s, 1H), 6.08 (t, 1H, J ) 7.2 Hz),
5.27 (s, 1H), 4.35 (m, 1H), 3.42 (dd, 1H, J ) 5.5, 9.8 Hz), 3.29
(dd, 1H, J ) 5.5, 9.8 Hz), 2.32 (m, 1H), 2.06 (m, 1H), 2.05 (s,
3H), 2.03 (s, 3H), 1.78 (s, 3H). ESI-MS: m/z 685.1 [M + Na]+.
6. (5R,6S)-5′-O-(4,4′-Dimethoxytrityl)-5,6-O-acetyl-thymidine
Glycol-3′-O-[(2-cyanoethyl)-N,N-diisopropyl-phosphoramid-
ite] (6). To a flask, which was suspended in an ice bath and
contained a solution of compound 5 (150 mg, 226 µmol) in dry
CH2Cl2 (1.8 mL), was added N,N-diisopropylethylamine (DIEA,
100 µL, 573 µmol) followed by dropwise addition of 2-cyanoethyl-
N,N-diisopropylchlorophosphoramidite (78 µL, 348 µmol). The
mixture was stirred at room temperature for 15 min. A second
portion of DIEA (100 µL, 573 µmol) was added to the mixture,
and the solution was stirred for another 15 min. Workup was carried
out by cooling the mixture in an ice bath followed by addition of
1
(67%). Rf: 0.24 (CH3OH-CH2Cl2, 1:19). H NMR (300 MHz,
CDCl3): δ 6.77-7.50 (m, 26H), 6.29 (m, 1H), 5.09 (s, 1H), 4.44
(m, 1H), 3.92 (m, 1H), 3.79 (m, 12H), 3.02 (m, 2H), 2.05 (m, 1H),
1.77 (m, 1H), 1.28 (s, 3H). MALDI-MS: m/z 903.4 [M + Na]+.
3. (5R,6S)-3′,5′-O-(4,4′-Dimethoxytrityl)-5,6-O-acetyl-thymi-
dine Glycol (3). Compound 2 (281 mg, 0.32 mmol) was coevapo-

Oligonucleotides with Thymidine Glycol and 8-OxodG
Chem. Res. Toxicol., Vol. 19, No. 6, 2006 839
Table 1. Sequences of Synthesized Oligodeoxynucleotides
oxidatively damaged products into ODNs. In addition, the 5-
and 6-hydroxyl groups in the commercially available Tg building
block were protected with TBDMS. The nucleobase deprotection
of those ODNs that are synthesized with the Tg building block,
therefore, requires treatments in two steps, one with ammonium
hydroxide and the other with tetrabutylammonium fluoride
(TBAF).
observed
m/z
theoretical
ODNs
sequencea
m/z
ODN1
ODN2
ODN3
ODN4
ODN5
5′-ATG GCG* TGC TAT-3′
5′-ATG GCG TgGC TAT-3′
5′-ATG GCG* TgGC TAT-3′
5′-ATG GCTg G*GC TAT-3′
5′-ATG* GCG TgGC TAT-3′
3691.2
3709.2
3725.2
3724.9
3725.2
3691.6
3709.6
3725.6
3725.6
3725.6
With the above analysis in mind, we decided to prepare the
phosphoramidite building blocks of 8-oxo-dG, whose N2 was
protected with a phenoxyacetyl group (37-39), and thymidine
glycol, whose 5- and 6-hydroxyl groups were protected with
acetyl functionality. The latter protection of thymidine glycol
obviates the needs for the deprotection with TBAF.
a G*: 8-oxodG.
CH3OH (0.36 mL). The solution was quickly extracted with EtOAc
(10 mL), and the organic layer was washed with NaHCO3 (4 mL)
and brine (4 mL) and dried with anhydrous Na2SO4. The solvent
was evaporated off to give 6 in white foam. The product was eluted
with 0-5% methanol in a solvent mixture of Et3N and CH2Cl2
(0.1:99.9, v/v). Yield: 133 mg (68%). Rf: 0.64, 0.73 (CH2Cl2-
CH3OH, 19:1). 31P NMR (CDCl3): δ 149.5, 149.2. ESI-MS: m/z
863.1 [M + H]+.
7. 5′-O-(4,4′-Dimethoxytrityl)-2-N-(phenoxyacetyl)-8-oxo-7,8-
dihydro-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)-N,N-diisopro-
pyl-phosphoramidite] (7). Compound 7 was prepared following
the previously published procedures (details shown in the Support-
ing Information) (37-39).
ODN Synthesis. ODNs (sequences shown in Table 1) were
synthesized on a Beckman Oligo 1000S DNA synthesizer (Ful-
lerton, CA) at 1-µmol scale. The phosphoramidite building blocks
6 and 7 were dissolved in anhydrous acetonitrile at a concentration
of 0.06 M; ultramild phosphoramidite building blocks (Glen
Research Inc., Sterling, VA) of dG, dC, and dA were employed,
and the factory-installed ODN assembly protocol was used without
any modification. After synthesis, the products were cleaved from
the controlled-pore glass (CPG) support and deprotected with 30%
NH4OH at room temperature for 1.5 h. The ODNs were then
purified by reversed-phase HPLC.
Thermodynamic Studies. UV absorbance-versus-temperature
profiles were recorded on a Varian Cary 500 spectrophotometer
(Varian, Inc., Palo Alto, CA), and the ODNs were dissolved in a
1.2-mL solution containing 250 mM NaCl, 10 mM sodium
cacodylate, and 0.1 mM EDTA (pH 7.0) at a total ODN concentra-
tion (Ct) of 2.0, 3.4, 5.6, 9.5, or 16 µM. The absorbance was
recorded in the reverse and forward directions for a temperature
range of 80-10 °C at a rate of 1 °C/min, and the melting
temperature (Tm) value was obtained by the derivative method.
The thermodynamic parameters were obtained from the van’t
Hoff plot (40), in which the reciprocal of Tm was plotted against
ln Ct/4:
To synthesize the thymidine glycol phosphoramidite building
block, we first protected the 3′- and 5′-hydroxyl groups of
thymidine with DMTr. The DMTr protecting group was chosen
for its easy removal upon acid treatment and its strong UV
absorbance, which allows for the monitoring of the elution of
the poorly UV-absorptive Tg during flash column chromatog-
raphy separation. The DMTr-protected compound was then
oxidized with OsO4 to offer the desired thymidine glycol (33).
We then protected both hydroxyl groups in thymine glycol with
acetyl groups, which was achieved by using DMAP as a catalyst
(44). It is worth noting that the acetylation of the tertiary
hydroxyl group was not complete without the addition of
DMAP. This result is in line with what Gasparutto et al. (35)
observed, where they showed that the protection of the 5-hy-
droxyl group of thymidine glycol with levulinyl functionality
was incomplete. The resulting compound was treated with 70%
acetic acid to give the 5,6-O-acetyl-thymidine glycol, which was
converted to the phosphoramidite building block using published
procedures (45).
With these two building blocks, we synthesized several ODNs
(sequences listed in Table 1). We found that the acetyl protecting
group of thymidine glycol can be readily removed upon
treatment with 30% NH4OH at room temperature for 1.5 h, and
thymidine glycol remains intact after such treatment. The ODNs
were then purified by reversed-phase HPLC (HPLC traces are
shown in the Supporting Information). It is worth noting that
the C6 position of the thymidine glycol functionality undergoes
epimerization during the deprotection step (34, 46). Therefore,
we expect that both the (5R,6S) and the (5R,6R) isomers are
present in the ODN, though only the major isomer of the
thymidine glycol, that is, the (5R,6S) isomer, from the OsO4
oxidation was employed for phosphoramidite building block
synthesis.
Ct
1
Tm
R
∆H°
∆S°
∆H°
)
ln
+
(
)
4
and
ESI-MS and MS/MS Characterization of ODNs Contain-
ing Both 8-OxodG and Tg. We next characterized the above
ODNs by ESI-MS and MS/MS (Table 1, Figures 1-2 and
Supporting Information). Here, we begin our discussion with
the characterizations of d(ATG*GCGTgGCTAT) (ODN5, G*
represents 8-oxodG). In this regard, ESI-MS shows that the
deconvoluted mass of the ODN is 50 Da higher than the
calculated mass of the unmodified d(ATGGCGTGCTAT)
(Figure 1a), which is consistent with the presence of both Tg
and 8-oxo-dG in this substrate.
∆G° ) ∆H° - T∆S°
where R is the ideal gas constant () 1.987 cal mol-1 K-1). The
error limits for ∆G°, ∆H°, and ∆S° derived from fitted parameters
were calculated by using previously described equations (41-43).
Results
Synthesis of Phosphoramidite Building Blocks for the Dual
Incorporation of Tg and 8-OxodG into ODNs. Phosphor-
amidite building blocks of both Tg and 8-oxodG are com-
mercially available (Glen Research). The labile nature of
thymidine glycol, which was shown to be decomposed upon
treatment with concentrated ammonia at room temperature for
an extended period of time (33), prevents the use of the
commercially available building block of 8-oxodG, which
necessitates deprotection with concentrated ammonia at room
temperature for 24-48 h, for the dual insertion of the two
The product-ion spectrum of the [M - 3H]3- ion (m/z 1240.7,
Figure 1b) of the ODN showed the formation of wn ions, that
is, w3, w4, w52-, w62-, w72-, w82-, and w113-, and [an - Base]
ions, that is, [a4 - Gua], [a5 - Cyt], [a6 - Gua], [a8 - Gua]2-
,
[a9 - Cyt]2-, and [a11 - Ade]2- ions [nomenclature for fragment
ions follows that reported by McLuckey et al. (47); “Ade”,
“Cyt”, and “Gua” represent adenine, cytosine, and guanine,
respectively]. Whereas the measured masses for the w3, w4, and

840 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Wang and Wang
Figure 2.
ESI-MS and MS/MS characterizations of
d(ATGGCG*TgGCTAT): (a) negative-ion ESI-MS; (b) product-ion
spectrum of [M - 3H]3- ion (m/z 1241).
Figure 1.
ESI-MS and MS/MS characterizations of
d(ATG*GCGTgGCTAT): (a) negative-ion ESI-MS; (b) product-ion
spectrum of the [M - 3H]3- ion (m/z 1241).
Thermodynamic Studies. We next measured the thermo-
dynamic parameters for duplex formation with the synthesized
lesion-bearing substrates. The specific nucleotide sequences
were the following: strand 1, 5′-ATGGCXYGCTAT-3′; strand
2, 5′-ATAGCMNGCCAT-3′, where XY/MN represents GT/
AC, GT/AA, TG/CA, G*T/AC, G*T/AA, GTg/AC, G*Tg/AC,
G*Tg/AA, or TgG*/CA (G* represents 8-oxodG). We refer to
the duplexes by abbreviations of the form of XY/MN (Table
2). We determined the ∆H° and ∆S° by fitting Tm-1 versus ln-
(Ct/4) as shown in Figure 3 (data for unmodified and single-
base, lesion-bearing ODNs are shown in the Supporting
Information). The ∆H°, ∆S°, ∆G°25 °C, and the Tm values at Ct
) 16.0 µM are listed in Table 2.
Our data on thermodynamic parameters (Table 2) showed
that the free energy change associated with the formation of
duplex at 25 °C (∆G°25 °C) with 8-oxodG/dA base pair (-15.1
kcal/mol) is very similar to that for the formation of the duplex
with 8-oxodG/dC base pair (-15.3 kcal/mol). These results are
consistent with previous thermodynamic results as well as NMR
and X-ray crystal structure studies (50-52), which showed that
8-oxodG can form base pairs with both dC and dA, depending
on whether the damaged nucleoside adopts an anti or syn
configuration about the glycosidic bond (50, 51).
2-
w5 ions are the same as the calculated masses for the
2-
corresponding ions of the unmodified ODN, the w62-, w7
,
and w82- ions exhibited 34 Da higher, and the w113- ion showed
50 Da higher, in mass than the corresponding fragments formed
from the unmodified d(ATGGCGTGCTAT). These results are
consistent with the presence of 8-oxo-dG and Tg at the third
and seventh positions in the ODN, respectively. The above
conclusion is further substantiated by the observed masses for
the [an - Base] ions. In this respect, the measured masses for
the [a4 - Gua], [a5 - Cyt], and [a6 - Gua] ions are 16 Da
higher, whereas the measured masses of the [a8 - Gua]2-, [a9 -
Cyt]2-, and [a11 - Ade]2- ions are 50 Da higher, than the
calculated masses of the corresponding fragments from the
unmodified d(ATGGCGTGCTAT). It is worth noting that we
did not observe the [a3 - Base] and w9 ion (Figure 1b), which
is consistent with previous findings about the fragmentations
of ODNs containing a 8-oxodG (48).
The sequences of ODN3 and ODN4 were also confirmed by
the similar ESI-MS and MS/MS analysis (Figure 2 shows the
MS and MS/MS for ODN3, and the mass spectrometric results
for ODN4 are shown in the Supporting Information). First, the
measured masses of these two ODNs are consistent with their
calculated masses (Table 1). In addition, the product-ion
spectrum of the [M - 3H]3- ion of ODN3 (Figure 2b) showed
Consistent with previous thermodynamic studies (34), our
results showed that the presence of thymine glycol markedly
decreases the duplex stability and it preferentially forms base
pairing with adenine. In addition, duplex ODN with tandem
lesion is less stable than the corresponding duplex with either
of the two single-base lesions alone (Table 2). Moreover, there
is no obvious difference in thermal stability of duplex ODNs
with the two types of tandem lesions, that is, 5′-G*Tg-3′ and
5′-TgG*-3′ (duplexes 7 and 9, Table 2).
that the w72-, w82-, w93-, w113-, [a8 - Gua]2-, [a9 - Cyt]2-
,
and [a11 - Ade]2- ions are 50 Da higher in mass than the
calculated masses for the corresponding ions of the unmodified
ODN. The measured masses of w3, w4, w5, [a3 - Gua], [a4 -
Gua], and [a5 - Cyt] ions, however, are the same as the
calculated masses for the respective ions of the unmodified
ODN. The product-ion spectrum of the [M - 3H]3- ion of
ODN4 is very similar to that of ODN3 except that the [a6 -
143], a characteristic ion for short ODNs carrying a Tg (49),
and w62- ions are found in the product-ion spectrum of ODN4
(Figure S15b, Supporting Information), but undetectable in that
of ODN3 (Figure 2b). Furthermore, the w5 ion is observed for
ODN3 (Figure 2b), but not for ODN4 (Figure S15b, Supporting
Information).
Discussion
In the present work, we successfully synthesized phosphor-
amidite building blocks of thymidine glycol and 8-oxodG, which
facilitate the simultaneous insertion of the two lesions into
ODNs. Additionally, with the use of acetyl group for the
protection of the hydroxyl functionalities on the thymine glycol
moiety, postsynthesis deprotection can be achieved in one step.

Oligonucleotides with Thymidine Glycol and 8-OxodG
Chem. Res. Toxicol., Vol. 19, No. 6, 2006 841
Table 2. Thermodynamic Parameters for Duplex Formation
a
Tm
∆H°
(kcal/mol)
∆S°
∆G°25 °C
(kcal/mol)
∆∆G°25 °C
(kcal/mol)
duplex
(°C)
(cal mol-1 K-1
)
5′-ATGGCXYGCTAT-3′
3′-TACCGNMCGATA-5′
1 (XY/MN: GT/AC)
2 (XY/MN: GT/AA)
3 (XY/MN: TG/CA)
4 (XY/MN: G*T/AC)
5 (XY/MN: G*T/AA)
6 (XY/MN: GTg/AC)
7 (XY/MN: G*Tg/AC)
8 (XY/MN: G*Tg/AA)
9 (XY/MN: TgG*/CA)
60.1
43.1
60.1
56.4
52.4
44.7
40.9
37.5
37.4
-83 ( 4.8
-72 ( 4.5
-77 ( 1.3
-83 ( 2.4
-90 ( 7.2
-78 ( 1.5
-69 ( 2.0
-81 ( 5.7
-76 ( 5.0
-220 ( 13
-200 ( 13
-207 ( 4
-230 ( 7
-250 ( 20
-222 ( 4
-195 ( 6
-240 ( 17
-220 ( 15
-16.2 ( 0.48
-11.6 ( 0.21
-15.6 ( 0.13
-15.3 ( 0.21
-15.1 ( 0.58
-12.2 ( 0.08
-11.1 ( 0.08
-10.7 ( 0.21
-10.5 ( 0.15
4.6
0.6
0.9b
-3.5b
4.0b
5.1b
0.9b
5.1b
a Ct ) 16 µM. b ∆G°25 °C (modified ODN) - ∆G°25 °C (corresponding unmodified ODN).
Scheme 2. Deamination of 5-Methycytosine Glycol and the
Implications of Tandem Lesions in mCG f TT Mutation
Figure 3. Plots of 1/Tm vs ln(Ct/4) for the duplexes containing
thymidine glycol and 8-oxodG tandem lesions. The duplex is d(ATG-
GCXYGCTAT)/d(ATAGCMNGCCAT), where XY/MN represents
G*Tg/AC(9), G*Tg/AA(b), and TgG*/CA(2).
both in vitro and in vivo, which may offer important insights
into the roles of this type of lesions in CpG mutagenesis.
Recent repair studies on cluster lesions showed that the repair
of one lesion can be hampered by the presence of another lesion
nearby (25-28, 31). Our thermodynamic measurement results
clearly showed that tandem lesions, where an 8-oxodG is
adjacent to a Tg, can cause more destabilization to DNA
duplexes than either of the two lesions alone. In this regard,
the presence of neighboring Tg and 8-oxodG destabilizes the
duplex DNA by ∼5.1 kcal/mol (Table 2). Although either Tg
or 8-oxodG, when present by itself, can be efficiently repaired
by base excision repair (BER) enzymes (62), the elevated
structural destabilization caused by the vicinal single-base
lesions may result in the compromised repair of these two lesions
by the BER machinery. In addition, both Tg and 8-oxodG could
be excised from DNA by NER enzymes in vitro (63); the
formation of these two lesions in tandem, due to the increased
structural destabilization, may also render the tandem lesions
to be excised more efficiently by NER than if the two lesions
are not proximal to each other. The availability of authentic
ODN substrates will again enable us to examine such possibili-
ties.
The availability of authentic ODN substrates enables the
assessment of the formation of this type of tandem lesions under
various oxidative stress conditions. In this respect, the tandem
lesion-carrying ODN substrates will facilitate the design and
optimization of enzymatic digestion procedures for the suc-
cessful release of this type of tandem lesion from duplex DNA.
Methylation of cytosine at CpG sites plays an important role
in the epigenetic silencing of genes in mammalian cells (53,
54). Damage occurring at CpG dinucleotide and the resulting
mutation at these sites may perturb the binding of DNA to
methyl-CpG binding proteins (55). We reason that the type of
tandem lesion that we synthesized here may have implications
in CpG mutagenesis. In this context, under oxidative stress, mC
could be damaged to form 5-methylcytosine glycol (56), which
is susceptible to deamination to yield thymine glycol. On the
other hand, the dG at CpG site can be oxidized to give a
8-oxodG (Scheme 2). Thymidine glycol is mostly a blocking
lesion. It, however, can be bypassed by translesion synthesis
DNA polymerases including yeast DNA polymerases η and ú
as well as human DNA polymerase κ, and all three polymerases
preferentially insert a dA opposite the lesion (5, 57, 58). On
the other hand, 8-oxodG can be bypassed by various DNA
polymerases, and both dC and dA can be inserted opposite the
lesion (59, 60). Therefore, the tandem lesions with a neighboring
pyrimidine glycol to 8-oxodG may contribute to the previously
observed ROS-induced mCG f TT mutation (61). The avail-
ability of ODN substrates bearing structurely defined tandem
lesions will allow for the replication studies of these lesions
Taken together, we reported here for the first time the dual
incorporation of Tg and 8-oxodG into ODNs. The availability
of ODNs carrying neighboring 8-oxodG and Tg builds a solid
foundation for examining the replication and repair of the
tandem oxidative lesions.
Acknowledgment. The authors thank the National Institutes
of Health (CA 96906 and CA101864) for supporting this
research.
Supporting Information Available: NMR spectra of synthetic
compounds, data fitting for thermodynamic measurements, HPLC

842 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Wang and Wang
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