Synthesis and properties of DNA oligomers containing 2′- deoxynucleoside N-oxide derivatives

Article abstract of DOI:10.1021/jo7021845

(Chemical Equation Presented) Cytosine and adenine N-oxide derivatives have long been known as products resulting from the oxidative damage of DNA by peroxides such as hydrogen peroxide. Although the synthesis and properties of 2′-deoxynucleoside N-oxide

Full text of DOI:10.1021/jo7021845

Synthesis and Properties of DNA Oligomers Containing
2′-Deoxynucleoside N-Oxide Derivatives
Hirosuke Tsunoda, Akihiro Ohkubo, Haruhiko Taguchi, Kohji Seio, and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, CREST, JST (Japan Science and Technology
Agency), Yokohama, 226-8501, Japan
msekine@bio.titech.ac.jp
ReceiVed August 7, 2007
Cytosine and adenine N-oxide derivatives have long been known as products resulting from the oxidative
damage of DNA by peroxides such as hydrogen peroxide. Although the synthesis and properties of 2′-
deoxynucleoside N-oxide derivatives have been well described, little has been reported about the chemical
and biochemical behavior of initially formed DNA oligomers containing these N-oxide bases. In this
study, we established a convenient method for the solid-phase synthesis of oligodeoxynucleotides
incorporating 2′-deoxycytidine N-oxide (dCO) or 2′-deoxyadenosine N-oxide (dAO) by using the
postsynthetic oxidation of N-protected DNA oligomers except for the target dC or dA site with m-CPBA
in MeOH in a highly selective manner. In this strategy, the benzoyl, phthaloyl, and (4-isopropylphenoxy)-
acetyl groups proved to serve as base protecting groups to avoid oxidation of adenine, cytosine, and
guanine, respectively, at the unmodified sites.
Introduction
block the chain elongation by DNA polymerases or to lead
mutations.8-11
DNA base oxidation has been recognized as a major cause
of genetic mutation. Oxidative DNA damage is associated with
cancer, aging, and genetic diseases.1,2 Reactive oxygen species
such as hydroxyl radical, peroxyl radical, and hydrogen peroxide
have been known to oxidize DNA bases. Free radicals such as
hydroxyl radical reacts with pyrimidine and purine bases to give
many oxidation products.3-7 For example, oxidatively modified
structures such as thymine glycol, uracil glycol, and 8-oxogua-
nine have been found. These modifications have been found to
On the other hand, nonradical processes are also important
in understanding the mechanism of oxidative DNA damage.
Hydrogen peroxide is a major nonradical species in living cells
produced by a series of metabolic processes of a nonreactive
superoxide anion.12,13 Adenine N1-oxide is a specific product
derived from the adenine residue of DNA upon treatment with
H2O2 under nonradical conditions,14 and it has been detected
in cellular DNA after exposure to nonlethal concentrations of
H2O2 (Figure 1).15,16 Brown et al. previously reported strong
growth inhibitory activity of adenosine N1-oxide and AMP N1-
(1) Cooke, M. S.; Evans, M. D.; Dizdaroglu, M. FASEB J. 2003, 17,
1195-1214.
(8) Simandan, T.; Sun, J.; Dix, T. A. Biochem. J. 1998, 335, 233-240.
(9) Kusumoto, R.; Masutani, C.; Iwai, S.; Hanaoka, F. Biochemistry 2002,
41, 6090-6099.
(10) Purmal, A. A.; Lampman, G. W.; Bond, P. J.; Hatahet, Z.; Wallace,
S. S. J. Biol. Chem. 1998, 273, 10026-10035.
(11) Shibutani, S.; Bodepudi, V.; Johnson, F.; Grollman, A. P. Biochem-
istry 1993, 32, 4615-4621.
(12) Boveris, A. AdV. Exp. Med. Biol. 1977, 78, 67-82.
(13) Badwey, A.; Kanovsky, M. L. Annu. ReV. Biochem. 1980, 49, 695-
726.
(14) Rhaese, H. J. Biochim. Biophys. Acta 1968, 166, 311-336.
(15) Mouret, J. F.; Odin, F.; Polverelli, M.; Cadet, J. Chem. Res. Toxcol.
1990, 3, 102-110.
(2) Ames, B. N.; shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 7915-7822.
(3) Bjelland, S.; Seeberg, E. Mutat. Res. 2003, 531, 37-80.
(4) Slupphaug, G.; Kavli, B.; Krokan, H. E. Mutat. Res. 2003, 531, 231-
251.
(5) Cadet, J.; Douki, T.; Gasparutto, D.; Ravanat, J. L. Mutat. Res. 2003,
531, 5-23.
(6) Gasparutto, D.; Bourdat, A. G.; D’ Ham, C.; Duarte, V.; Romieu,
A.; Cadet, J. Biochimie 2000, 82, 19-24.
(7) Cadet, J.; Delatour, T.; Douki, T.; Gasparutto, D.; Pouget, J. P.;
Ravanat, J. L.; Sauvaigo, S. Mutat. Res. 1999, 424, 9-21.
10.1021/jo7021845 CCC: $40.75 © 2008 American Chemical Society
Published on Web 01/18/2008
J. Org. Chem. 2008, 73, 1217-1224
1217

Tsunoda et al.
TABLE 1. Synthesis of 2′-Deoxycytidine N-Oxide Derivative 2 by
Treatment of 1 with Several Peroxidesa
FIGURE 1. Adenine and cytosine N-oxide derivatives.
oxide in mouse sarcoma 180 cell.17 2′-Deoxycytidine N3-
oxide18-21 was also considered as a principle component of the
oxidation products of DNA upon treatment with hydrogen
peroxide, although this modified species has not been detected
from modified 2′-deoxynucleosides induced by oxidative DNA
damage. There is a possibility that these 2′-deoxynucleoside
N-oxides are mutagenic in DNA replication like the well-known
8-oxoguanine.22
The chemical synthesis of cytosine and adenine N-oxides have
been reported previously in a number of laboratories. Adenine
underwent preferential N1-oxidation with 30% aqueous hydrogen
peroxide in acetic acid.23 Monoperoxyphthalic acid has been
used to convert cytosine and cytidine into the corresponding
N3-oxides.18 m-Chloroperoxybenzoic acid (m-CPBA), a com-
mercially available stable peroxide, has also been used for the
synthesis of adenosine and cytidine N-oxide derivatives.19-21
Although many studies concerning the formation and properties
of nucleoside N-oxide derivatives have been conducted until
date, little attention has been paid to oligodeoxynucleotides
containing cytosine or adenine N-oxide. If oligomers incorporat-
ing a nucleoside N-oxide at an appropriate position could be
synthesized, it is possible to clarify its biological behavior. With
this background in mind, we studied the synthesis of DNA
oligomers containing a 2′-deoxynucleoside N-oxide.
entry
reagent (equiv)
t-BuOOH (1)
solvent
time (h)
yield (%)
1
2
3
4
5
6
7
8
9
CH3CN
CH2Cl2
CH3CN
CH2Cl2
THF
CH3CN
CH2Cl2
MeOH
CH3CN
CH2Cl2
MeOH
MeOH
24
24
24
24
4
no reaction
no reaction
t-BuOOH (1)
30% H2O2 (2), TFAA (2)
30% H2O2 (2), TFAA (2)
UHP (2), TFAA (2)
UHP (2), TFAA (2)
UHP (2), TFAA (2)
UHP (2), TFAA (2)
m-CPBA (1)
m-CPBA (1)
m-CPBA (1)
m-CPBA (3)
31
19
39
33
14
10
48
55
71
89
4
12
24
24
24
24
90 min
10
11
12
a UHP ) ureahydrogenperoxide; TFAA ) trifluoroacetic anhydride.
SCHEME 1. Synthesis of 2′-Deoxyadenosine N-Oxide
Derivative 4 by Treatment of 3 with m-CPBA
formed in a low yield of 31% (entry 3). The combined use of
urea hydrogen peroxide (UHP) and trifluoroacetic anhydride
(TFAA) was effective for the conversion of pyridine derivatives
into their N-oxide derivatives.27 However, the N-oxide 2 could
only be obtained in moderate yields (entries 5-8). In contrast,
when mCPBA was used in MeOH, the N-oxidation proceeded
more smoothly (entries 9-12). The use of 3 equiv of m-CPBA
in MeOH gave the best result. Under similar conditions, 3′,5′-
O-bis(tert-butyldimethylsilyl)-2′-deoxyadenosine 3 could be
oxidized to give the N-oxide derivative 4 in 92% yield, as shown
in Scheme 1.
Results and Discussion
For the N-oxidation of deoxynucleosides, several methods
using peroxides have been reported. To find the most favorable
conditions for the N-oxidation, reactions of 3′,5′-bis(tert-
butyldimethylsilyl)-2′-deoxycytidine 1 with several peroxides
were examined in aqueous and nonaqueous solvents, as shown
in Table 1. t-BuOOH is known as a widely used reagent for
the oxidation of phosphite triester intermediates in DNA
synthesis.24-26 It turned out that this reagent was completely
inactive toward the cytosine base for 24 h (entries 1 and 2).
When compound 1 was treated with 30% hydrogen peroxide
and trifluoroacetic anhydride,27 the N-oxide derivative 2 was
We attempted to synthesize the phosphoramidite unit 8 of
2′-deoxycytidine N-oxide for the synthesis of oligodeoxynucle-
otides containing dCO in the usual manner (Scheme 2). Reaction
of 2 with dibutylformamide dimethyl acetal in MeOH gave the
4-N-protected product 5 in 99% yield. During this reaction, the
N-oxide function was stable. The usual tritylation of 5 with
DMTrCl gave the 5′-masked product 7 in 71% yield. However,
the last phosphitylation of 7 with chloro(2-cyanoethoxy)-
(diisopropylamino)phosphine resulted in a complex mixture.
Bis(diisopropylamino)(2-cyanoethoxy)phosphine when used as
a phosphitylating agent gave a similar result.
(16) Mouret, J. F.; Polverelli, M.; Sarrazini, F.; Cadet, J. Chem. Biol.
Interact. 1991, 77, 187-201.
(17) Brown, G. B.; Clarke, D. A.; Biesele, C. J.; Kaplan, L.; Stevens,
M. A. J. Biol. Chem. 1958, 233, 1509-1512.
(18) Cramer, F.; Seidel, H. Biochim. Biophys. Acta 1963, 72, 157-161.
(19) Sako, M.; Kawada, H. J. Org. Chem. 2004, 69, 8148-8150.
(20) Subbaraman, L. R.; Subbaraman, J.; Behrman, E. J. Biochemistry
1969, 8, 3059-3066.
(21) Delia, T. J.; Olsen, M. J.; Brown, G. B. J. Org. Chem. 1965, 30,
2766-2768.
To investigate this result, we checked the stability of
compound 5 with various tervalent phosphorus reagents using
1H and 31P NMR. It was found that compound 5 was rapidly
deoxygenated within 10 min by chloro(2-cyanoethoxy)(diiso-
propylamino)phosphine to give 9 (Scheme 3). Deoxygenation
could not be suppressed when bis(diisopropylamino)(2-cyano-
ethoxy)phosphine was employed. A slower but similar deoxy-
genation was observed when diethyl chlorophosphite was used
as the phosphorus reagent.
(22) Shibutani, S.; Takeshita, M.; Grollman, A. P. Nature 1991, 349,
431-434.
(23) Stevens, M. A.; Magrath, D. I.; Smith, H. W. J. Am. Chem. Soc.
1958, 80, 2755-2758.
(24) Engels, J.; Ja¨ger, A. Angew. Chem., Int. Ed. Engl. 1982, 21, 912.
(25) Ja¨ger, A.; Engels, J. Tetrahedron Lett. 1984, 25, 1437-1440.
(26) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett. 1986,
35, 4191-4194.
(27) Caron, S.; Do, N. M.; Sieser, J. E. Tetrahedron Lett. 2000, 41, 2299-
2302.
1218 J. Org. Chem., Vol. 73, No. 4, 2008

DNA Oligomers Containing 2′-Deoxynucleoside N-Oxide DeriVatiVes
SCHEME 2. Synthesis of the 2′-Deoxycytidine N3-Oxide 3′-Phosphoramidite Building Block 8
SCHEME 3. Deoxygenation of 5 with Phosphorus(III)
Compounds
for suppressing the generation of dT; however, the N-oxidation
was not completed under these conditions. To improve the yield
of the d[COpT] dimer, the concentration of mCPBA in MeOH
was changed from 0.04 to 0.1 M. Under these conditions, the
d[COpT] dimer was successfully obtained without an increase
of dT (Figure 2b). Therefore, we selected a 0.1 M solution of
m-CPBA in MeOH for 1 h as the best conditions for oxidation
of the cytosine base.
Under the above-mentioned conditions, the oxidation of d[T6-
CT7] proceeded similarly to give the desired oligomer d[T6COT7]
in 36% yield (Figure 3a). It should be noted that the cleavage
of the oligodeoxynucleotide 14mer did not occur. Similarly, a
DNA 14mer d[T6AOT7] containing 2′-deoxyadenosine N-oxide
was synthesized by a similar postsynthetic oxidation in 27%
yield (Figure 3b). These oligomers were characterized by
MALDI-TOF mass spectroscopy and enzymatic hydrolysis (see
the Supporting Information).
The N-oxide function was found to be very stable toward
triethyl phosphite for at least 24 h. Interestingly, the reaction
of 5 with diphenyl phosphite led to the deoxygenation of the
N-oxide moiety (t1/2 ) 4 h). In connection with our study,
Hammer has recently reported the facile reduction of a 2′-
deoxynucleoside analog using a thiazole N-oxide nucleobase
with phosphitylating reagents.28
There is a possibility that 2′-deoxycytidine N-oxide was
converted to N4-hydroxy-2′-deoxycytidine in the DNA oligomer.
To check this possibility, N4-hydroxy-2′-deoxycytidine was
synthesized by the previous method.30 A new peak obtained by
the enzymatic digestion of the modified oligomer d[T6COT7]
was not consistent with that of 4-N-hydroxy-2′-deoxycytidine,
as evidenced by reversed-phase HPLC analysis (Figure 4).
Therefore, it seemed that 2′-deoxycytidine N-oxide remained
intact in the DNA oligomer.
Screening of Suitable Protecting Groups Resistant to
N-Oxidation of Nucleobases. To study the biological property
of DNA incorporating a 2′-deoxynucleoside N-oxide, we needed
DNA oligomers with appropriate sequences containing dA, dG,
dC, and dT along with a damaged 2′-deoxynucleoside dCO or
dAO. It is necessary to selectively oxidize a cytosine or adenine
base at an appropriate position in the sequence and avoid side
reactions on other bases by protecting them with suitable
protecting groups. Acyl-type protecting groups were chosen as
the protecting groups for the amino groups of dA, dG, and dC
because they significantly decrease the nucleophilicity of the
amino groups of the adenine and cytosine bases to prevent
N-oxide formation. In the case of the adenine base, the acetyl
Based on these results, it was concluded that the synthesis
of the phosphoramide building block 8 was difficult, and it
seemed that an alternative H-phosphonate method could not be
used as suggested by its mechanism that involves activated
tervalent phosphorus intermediates. Therefore, we considered
that the desired oligomer should be synthesized by the selective
oxidation of oligodeoxynucleotide containing N-free 2′-deoxy-
cytidine or 2′-deoxyadenosine.
The synthesis of DNA dimer d[COT] containing dCO was
carried out, as shown in Scheme 4. A dimer with the sequence
of CT was constructed on a T-loaded highly cross-linked
polystyrene (HCP) according to the N-unprotected phosphora-
midite method.29 After removal of the 5′-terminal DMTr group,
the HCP resin was treated with m-CPBA in MeOH. When a
0.04 M solution of m-CPBA in MeOH was used as the oxidizing
agent at room temperature for 8 h, the cytosine residue was
found to be almost quantitatively oxidized in the solid-phase
(Figure 2a); however, dT was observed along with the desired
dimer d[COpT]. It was likely that the phosphotriester bond of
the d[CpT] dimer was cleaved under acidic conditions for long
time periods. Therefore, the reaction time was shortened to 1 h
(28) Miller, T. J.; Farquar, H. D.; Sheybani, A.; Tallini, C. E.; Saurage,
A. S.; Fronczek, F. R.; Hammer, R. P. Org. Lett. 2002, 4, 877-880.
(29) Ohkubo, A.; Ezawa, Y.; Seio, K.; Sekine, M. J. Am. Chem. Soc.
2004, 126, 10884-10896.
(30) Felczak, K.; Miazga, A.; Poznanski, J.; Bretner, M.; Kulikowski,
T.; Dzik, J. M.; Golos, B.; Zielinski, Z.; Ciesla, J.; Rode, W. J. Med. Chem.
2000, 43, 4647-4656.
J. Org. Chem, Vol. 73, No. 4, 2008 1219

Tsunoda et al.
SCHEME 4. Solid-phase Synthesis of a Dimer Containing 2′-Deoxycytidine N3-Oxide
tested, as shown in Table 2. It was found that 4-N-benzoyl-2′-
deoxyadenosine derivative 10b was resistant to m-CPBA in
MeOH and was almost recovered without N-oxidation of the
adenine base (entry 2). In contrast, the N-acetylated and
N-benzoylated 2′-deoxycytidine derivatives 11a and 11b formed
considerable N-oxidation products and the recovery of these
compounds was not very high (entries 3 and 4). When
compound 11b was treated with m-CPBA in MeOH, a complex
mixture was formed. The use of the acetyl group gave a better
result but the recovery of 11a was low. Interestingly, it was
found that the 4-N-phthaloyl-2′-deoxycytidine derivative 11c
remained completely intact as judged by TLC under these
conditions and was recovered in 89% yield (entry 5).
FIGURE 2. Reversed-phase HPLC profiles of the crude mixtures
obtained by oxidation of the dimer d[CpT]: (a) 0.04 M mCPBA/MeOH,
8 h; (b) 0.1 M mCPBA/MeOH, 1 h.
and benzoyl groups used for the usual DNA synthesis were
FIGURE 3. Anion-exchange HPLC profiles of the crude mixtures obtained by using a postsynthetic oxidative method (left) and the
oligodeoxynucleotide obtained by HPLC purification (right): (a) d[T6COT7]; (b) d[T6AOT7].
FIGURE 4. Reversed-phase HPLC profiles of (a) the 2′-deoxynucleoside obtained by enzymatic hydrolysis and (b) 4-N-hydroxy-2′-deoxycytidine.
1220 J. Org. Chem., Vol. 73, No. 4, 2008

DNA Oligomers Containing 2′-Deoxynucleoside N-Oxide DeriVatiVes
TABLE 2. Stability of the Protecting Groups of the Amino Groups for the Adenine and Cytosine Bases under the Conditions for N-Oxidation
entry
compd
base
prot
recovery (%)
1
2
3
4
5
10a
10b
11a
11b
11c
Ade
Ade
Cyt
Cyt
Cyt
ac
bz
ac
bz
77
91
74
63
89
phth
SCHEME 5. Synthesis of a 2′-Deoxycytidine 3′-Phosphoramidite Building Block 14 Protected with a Phthaloyl Group
SCHEME 6. Solid-Phase Synthesis of DNA Oligomers 16 and 18 Containing dCO and dAO
Synthesis of Appropriately Protected 2′-Deoxynucleoside
3′-Phosphoramidite Building Blocks. We synthesized the 2′-
deoxycytosine 3′-phosphoramidite building block 14 protected
with a phthaloyl group to synthesize the desired DNA oligomers
according to a previously reported method (Scheme 5).31-33
Reaction of 2′-deoxycytidine with phthaloyl chloride in pyridine
gave the 4-N-protected product 12 in 60% yield. The usual
tritylation of 12 with DMTrCl gave the 5′-protected product 13
in 70% yield. Phosphitylation of 13 with bis(diisopropylamino)-
(2-cyanoethoxy)phosphine gave the phosphoramidite unit 14,
which was isolated in 70% yield by silica gel column chroma-
tography with 1% pyridine.
Synthesis of Oligodeoxynucleotides Incorporating dA, dG,
dC, and dT Along with dCO or dAO. For the synthesis of
DNA oligomers with appropriate sequences containing dA, dG,
dC, and dT along with a damaged 2′-deoxynucleoside dCO or
dAO, we synthesized appropriately protected DNA oligomers
15 and 17 having N-unprotected dA and dC, respectively, at
one point in their base sequences on HCP resins. These DNA
oligomers were oxidized by mCPBA in MeOH as described
for N-oxidation. As expected, N-oxidation of the other bases
was efficiently suppressed, and the desired DNA oligomers 16
and 18 were obtained in 13% and 11% yields, respectively, by
HPLC purification (Scheme 6 and Figure 5). These oligomers
were characterized by MALDI-TOF mass spectroscopy and
enzymatic hydrolysis (see the Supporting Information).
(31) Kume, A.; Sekine, M.; Hata, T. Chem. Lett. 1983, 12, 1597-1600.
(32) Kume, A.; Iwase, R.; Sekine, M.; Hata, T. Nucleic Acids Res. 1984,
12, 8525-8538.
(33) Beier, M.; Pfleiderer, W. HelV. Chim. Acta 1999, 82, 633-644.
J. Org. Chem, Vol. 73, No. 4, 2008 1221

Tsunoda et al.
FIGURE 5. Anion-exchange HPLC profiles of the crude mixtures obtained by using a postsynthetic oxidative method (left) and the desired
oligodeoxynucleotide obtained by HPLC purification (right): (a) oligodeoxynucleotide 16; (b) oligodeoxynucleotide 18.
-5.6, -5.5, -4.9, -4.6, 17.9, 18.3, 25.7, 25.9, 42.0, 61.9, 70.3,
86.6, 87.7, 131.8, 149.2, 155.4; HRMS (ESI) m/z (M + H) calcd
Conclusion
In this study, we have synthesized oligodeoxynucleotides
containing 2′-deoxycytidine or 2′-deoxyadenosine N-oxide. It
was difficult to prepare the 2′-deoxynucleoside N-oxide 3′-
phosphoramidite derivatives required for the usual protocol in
the phosphoramidite approach. This is because the N-oxide
function was very sensitive to tervalent phosphorus species. To
overcome this inherent problem, we tried to use the postsynthetic
approach using our recently reported N-unprotected phosphora-
midite method.29 Thus, we synthesized the desired oligodeoxy-
nucleotides with modified bases. Using established methods,
we were unable to synthesize oligomers having nucleoside
N-oxide, while the synthesis based on the postsynthetic modi-
fication method was successful without deoxygenation of
N-oxide. In addition, we were able to synthesize oxidized
oligomers having various sequences using appropriate base-
protecting groups. Further biological studies on the properties
of modified oligodeoxynucleotides with dCO or dAO are now
under way.
+
for C21H42N3O5Si2 472.2678, found 472.2650.
Oxidation of 3 to 4 by Use of m-CPBA. The conditions used
for these experiments are shown in Table 2. Typical procedure:
Compound 3 (192.0 mg, 0.40 mmol) was dissolved in MeOH
(4 mL), and this solution was treated with m-CPBA for an
appropriate time. The reaction was monitored by TLC analysis.
The reaction mixture was diluted with CHCl3 (40 mL) and washed
with aqueous NaHCO3. The organic phase was collected, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with
CHCl3-MeOH (100:0-95:5, v/v) to give the fractions containing
4. The fractions were collected and evaporated under reduced
pressure to give 4. The yields of 4 are listed in Table 2. Compound
1
4: UV (MeOH) λmax 262 nm, λmax 234 nm; H NMR (CDCl3) δ
0.09-0.11 (m, 12H), 0.91-0.92 (m, 18H), 2.41-2.50 (m, 1H),
2.57-2.67 (m, 1H), 3.77 (dd, 1H, J ) 3.1 Hz, J ) 11.2 Hz), 3.88
(dd, 1H, J ) 4.0 Hz, J ) 11.2 Hz), 4.01-4.05 (m, 1H), 4.58-4.63
(m, 1H), 6.40 (t, 1H, J ) 6.3 Hz), 7.35 (br s, 2H), 8.25 (s, 1H),
8.70 (s, 1H); 13C NMR (CDCl3) δ -5.5, -5.4, -4.8, -4.7, 17.9,
18.26, 25.7, 25.92, 41.3, 62.6, 71.7, 84.6, 88.0, 119.4, 141.5, 142.1,
143.9, 148.6; HRMS (ESI) m/z (M + H) calcd for C22H42N5O4-
Experimental Section
+
Si2 496.2770, found 496.2774.
Oxidation of 1 to 2 by Use of Various Oxidizing Agents. The
conditions used for these experiments are shown in Table 1. Typical
procedure: Compound 1 (45.6 mg, 0.10 mmol) was dissolved in a
solvent (1 mL), and the solution was treated with an oxidizing agent
for an appropriate time. The reaction was monitored by TLC
analysis. The reaction mixture was diluted with CHCl3 (10 mL)
and washed with aqueous NaHCO3. The organic phase was
collected, dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of silica
gel with CHCl3-MeOH (100:0-95:5, v/v) to give the fractions
containing 2. The fractions were collected and evaporated under
reduced pressure to give 2. The yields of 2 are listed in Table 1:
UV (MeOH) λmax 274 nm, λmax 227 nm; 1H NMR (CDCl3) δ 0.00-
0.05 (m, 12H), 0.82-0.86 (m, 18H), 2.00-2.09 (m, 1H), 2.30-
2.39 (m, 1H), 3.71 (dd, 1H, J ) 2.9 Hz, J ) 12.0 Hz), 3.83-3.85
(m, 2H), 4.31-4.37 (m, 1H), 6.22 (t, 1H, J ) 5.4 Hz), 6.46 (d,
1H, J ) 7.4 Hz), 7.73 (d, 1H, J ) 7.7 Hz); 13C NMR (CDCl3) δ
Synthesis of Compound 5. Compound 2 (472 mg, 1.0 mmol)
was rendered anhydrous by repeated coevaporation with dry
pyridine (1 mL × 3), dry toluene (1 mL × 1), and dry CH3CN (1
mL ×1) and dissolved in dry MeOH (10 mL). To the solution was
added N,N-dibutylformamide dimethyl acetal (610 mg, 3.0 mmol).
After being stirred at room temperature for 1 h, the solution was
evaporated under reduced pressure. The residue was chromato-
graphed on a column of silica gel with hexane-CHCl3 (25:75-
0:100, v/v) and then CHCl3-MeOH (100:0-95:5, v/v) to give the
fractions containing 5. The fractions were collected and evaporated
under reduced pressure to give 5 (603 mg, 99%): 1H NMR (CDCl3)
δ 0.00-0.06 (m, 12H), 0.84-0.91 (m, 24H) 1.26-1.37 (m, 2H),
1.51-1.62 (m, 2H), 2.05-2.14 (m, 1H), 2.37-2.46 (m, 1H), 3.39
(br s, 2H), 3.72 (d, 1H, J ) 9.6 Hz), 3.87-3.91 (m, 2H), 4.30-
4.36 (m, 1H), 5.92 (d, 1H, J ) 7.6 Hz), 6.24 (t, 1H, J ) 5.4 Hz),
7.69 (d, 1H, J ) 7.6 Hz), 10.58 (s, 1H); 13C NMR (CDCl3) δ -5.6,
-5.5, -5.0, -4.6, 13.6, 13.7, 17.9, 18.3, 19.6, 20.1, 25.7, 25.9,
1222 J. Org. Chem., Vol. 73, No. 4, 2008

DNA Oligomers Containing 2′-Deoxynucleoside N-Oxide DeriVatiVes
29.3, 30.7, 41.8, 42.0, 47.1, 61.7, 69.8, 87.1, 87.7, 102.7, 128.5,
1H, J ) 7.3 Hz), 7.79-7.83 (m, 2H), 7.95-7.98 (m, 2H), 8.63 (d,
1H, J ) 7.3 Hz); 13C NMR (CDCl3) δ -5.5, -5.4, -4.9, -4.5,
18.0, 18.4, 25.7, 25.7, 25.90, 25.94, 42.1, 61.5, 69.4, 87.5, 87.9,
100.4, 124.4, 131.5, 135.1, 145.4, 154.7, 159.1, 165.1; HRMS (ESI)
m/z (M + H) calcd for C29H44N3O6Si2+ 586.2763, found 586.2762.
Stability of 10a,b and 11a-c under Conditions for N-
Oxidation. The conditions used for these experiments are shown
in Table 2. Typical procedure: The compound (0.10 mmol) was
dissolved in MeOH (1 mL), and m-CPBA (22.4 mg, 0.10 mmol;
77% purity) was added. After being stirred at room temperature
for 24 h, the reaction mixture was diluted with CHCl3 (10 mL)
and washed with aqueous NaHCO3. The organic phase was
collected, dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of silica
gel with CHCl3-MeOH (100:0-95:5, v/v) to give the fractions
containing starting material. The fractions were collected and
evaporated under reduced pressure to give starting material. The
recovery yields of 10a,b and 11a-c are listed in Table 2.
151.5, 154.4, 159.6; HRMS (ESI) m/z (M + H) calcd for
+
C30H59N4O5Si2 611.4019, found 611.4010.
Synthesis of Compound 7. To a solution of compound 5
(1.22 g, 2.0 mmol) in dry THF (10 mL) were added triethylamine
trihydrofluoride (977 µL, 6.0 mmol) and triethylamine (836 µL).
After being stirred at room temperature for 2 h, the reaction was
quenched by addition of H2O. The mixture was partitioned between
CHCl3/i-PrOH (3:1, v/v) and H2O. The organic phase was collected,
dried over Na2SO4, filtered, and evaporated under reduced pressure.
Subsequently, the residue was rendered anhydrous by repeated
coevaporation with dry pyridine (1 mL × 1) and dissolved in dry
pyridine (20 mL). To the solution was added DMTrCl (813 mg,
2.4 mmol), and the mixture was stirred at room temperature for
2 h. The reaction was quenched by addition of saturated aqueous
NaHCO3. The mixture was partitioned between CHCl3 and H2O.
The organic phase was collected, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chromato-
graphed on a column of silica gel with CHCl3-MeOH (100:0-
95:5, v/v) containing 0.5% Et3N to give the fractions containing 7.
The fractions were collected and evaporated under reduced pressure.
The residue was evaporated by repeated coevaporation three times
each with toluene and CHCl3 to remove the last traces of Et3N to
give 7 (979 mg, 71%): 1H NMR (CDCl3) δ 0.93 (t, 6H, J ) 7.2
Hz), 1.26-1.36 (m, 2H), 1.54-1.65 (m, 2H), 2.21-2.30 (m, 1H),
2.62-2.71 (m, 1H), 3.36-3.50 (m, 4H), 3.73-3.78 (m, 8H), 4.18-
4.20 (m, 1H), 4.49-4.55 (m, 1H), 5.75 (d, 1H, J ) 7.7 Hz), 6.33
(t, 1H, J ) 5.5 Hz), 6.83 (d, 4H, J ) 8.7 Hz), 7.26-7.42 (m, 9H),
7.66 (d, 1H, J ) 7.7 Hz), 10.43 (s, 1H); 13C NMR (CDCl3) δ 13.7,
19.8, 29.1, 30.6, 41.9, 44.5, 52.1, 55.1, 62.9, 70.4, 86.4, 86.5, 87.8,
102.5, 113.1, 126.8, 127.8, 128.1, 129.4, 130.0, 130.1, 130.3, 130.5,
144.5, 151.4, 154.7, 158.4, 159.5; HRMS (ESI) m/z (M + H) calcd
Synthesis of Compound 12. 2′-Deoxycytidine monohydride
(2.27 g, 10.0 mmol) was rendered anhydrous by repeated coevapo-
ration with dry pyridine (2 mL × 1) and dissolved in dry pyridine
(36 mL). To the solution was added chlorotrimethylsilane (3.16
mL, 25.0 mmol), and the mixture was stirred for 1 h. Then DMAP
(10 mg) was added, and phthaloyl chloride (2.16 mL, 15.0 mmol)
in dioxane (4 mL) was added dropwise at 0 °C for 15 min. After
being stirred at room temperature for 1 h, the mixture was
hydrolyzed with ice-water, and stirring was continued for 15 min.
The mixture was partitioned between 10% pyridine in CHCl3 and
H2O. The organic phase was extracted with H2O (50 mL), and the
combined aqueous phases were washed twice with 10% pyridine
in CHCl3 (50 mL). The organic phase was collected, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was evaporated by repeated coevaporation with toluene and
CH3CN to remove pyridine. The residue was washed with CH2Cl2
to give 12 (2.13 g, 60%): 1H NMR (DMSO) δ 2.10-2.16 (m,
1H), 2.36-2.43 (m, 1H), 3.57-3.68 (m, 2H), 3.90-3.94 (m, 1H),
4.21-4.28 (m, 1H), 5.11 (t, 1H, J ) 5.2 Hz), 5.30 (d, 1H, J ) 4.3
Hz), 6.11 (t, 1H, J ) 6.2 Hz), 6.71 (d, 1H, J ) 7.1 Hz), 7.92-8.03
(m, 4H), 8.63 (d, 1H, J ) 7.3 Hz); 13C NMR (DMSO) δ 40.9,
60.7, 69.7, 87.1, 88.3, 101.1, 124.0, 131.2, 135.4, 146.0, 154.2,
+
for C39H49N4O7 685.3596, found 685.3593.
Deoxygenation of 5 by Use of Various Phosphorus Reagents.
The conditions used for these experiments are shown in Scheme
3. Typical procedure: Compound 5 (61.1 mg, 0.10 mmol) was
dissolved in CH3CN (1 mL), and this solution was treated with a
phosphorus agent for an appropriate time. The reaction was
monitored by TLC analysis. The reaction mixture was diluted with
CHCl3 (10 mL) and washed once with aqueous NaHCO3. The
organic phase was collected, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chromato-
graphed on a column of silica gel with CHCl3-MeOH (100:0-
95:5, v/v) to give the fractions containing 9. The fractions were
+
158.9, 165.1; HRMS (ESI) m/z (M + H) calcd for C17H16N3O6
358.1034, found 358.1038.
Synthesis of Compound 13. Compound 12 (715 mg, 2.0 mmol)
was rendered anhydrous by repeated coevaporation with dry
pyridine (1 mL × 1) and dissolved in dry pyridine (20 mL). To
the solution was added DMTrCl (813 mg, 2.4 mmol), and the
mixture was stirred at room temperature for 3 h. The reaction was
quenched by addition of saturated aqueous NaHCO3. The mixture
was partitioned between CHCl3 and H2O. The organic phase was
collected, dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of silica
gel with hexane-ethyl acetate (25:75, v/v) containing 1% pyridine
to give the fractions containing 13. The fractions were collected
and evaporated under reduced pressure. The residue was finally
evaporated by repeated coevaporation three times each with toluene
and CHCl3 to remove the last traces of pyridine to give 13 (920
mg, 70%): 1H NMR (CDCl3) δ 2.36-2.40 (m, 1H), 2.67-2.77
(m, 1H), 3.42 (dd, 1H, J ) 3.4 Hz, J ) 11.3 Hz), 3.52 (dd, 1H, J
) 2.7 Hz, J ) 11.1 Hz), 3.75 (s, 6H), 4.12-4.13 (m, 1H), 4.54-
4.60 (m, 1H), 6.21 (t, 1H, J ) 5.4 Hz), 6.37 (d, 1H, J ) 7.1 Hz),
6.81 (d, 4H, J ) 8.7 Hz), 7.11-7.37 (m, 9H), 7.75-7.79 (m, 2H),
7.90-7.93 (m, 2H), 8.54 (d, 1H, J ) 7.1 Hz); 13C NMR (CDCl3)
δ 41.7, 55.1, 62.1, 69.6, 86.3, 86.6, 87.7, 100.5, 113.2, 124.2, 126.9,
127.9, 128.1, 129.9, 131.1, 135.0, 135.3, 135.4, 144.1, 145.5, 155.0,
158.4, 159.0, 164.8; HRMS (ESI) m/z (M + H) calcd for
1
collected and evaporated under reduced pressure to give 9: H NMR
(CDCl3) δ 0.00-0.07 (m, 12H), 0.83-0.91 (m, 24H), 1.26-1.34
(m, 2H), 1.49-1.60 (m, 2H), 2.06-2.12 (m, 1H), 2.36-2.46 (m,
1H), 3.21-3.32 (m, 2H), 3.43-3.54 (m, 2H), 3.73 (dd, 1H, J )
2.3 Hz, J ) 11.5 Hz), 3.82-3.93 (m, 2H), 4.31-4.38 (m, 1H),
5.95 (d, 1H, J ) 7.3 Hz), 6.27 (t, 1H, J ) 5.5 Hz), 8.06 (d, 1H, J
) 7.3 Hz), 8.78 (s, 1H); 13C NMR (CDCl3) δ -5.59, -5.55, -5.0,
-4.6, 13.6, 13,7, 17.9, 18.3, 19.7, 20.0, 25.7, 25.9, 29.0, 30.9, 42.2,
45.2, 52.0, 61.7, 69.7, 86.0, 87.1, 102.4, 141.3, 156.3, 158.1, 171.8;
+
HRMS (ESI) m/z (M + H) calcd for C30H59N4O4Si2 595.4069,
found 595.4069.
Synthesis of Compound 11c. Compound 2 (228 mg, 0.50 mmol)
was rendered anhydrous by repeated coevaporation with dry
pyridine (1 mL × 1) and dissolved in dry pyridine (5 mL). To the
solution was added phthaloyl chloride (86.4 mg, 0.6 mmol), and
the mixture was stirred at room temperature for 30 min. The reaction
mixture was diluted with CHCl3 (10 mL) and washed with aqueous
NaHCO3. The organic phase was collected, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel with CHCl3-MeOH
(100:0-98:2, v/v) to give the fractions containing 11c. The fractions
were collected and evaporated under reduced pressure to give 11c
(246 mg, 84%): 1H NMR (CDCl3) δ 0.05-0.11 (m, 12H), 0.86-
0.91 (m, 18H), 2.20-2.23 (m, 1H), 2.53-2.63 (m, 1H), 3.76-
4.01 (m, 2H), 4.34-4.42 (m, 1H), 6.19-6.23 (m, 1H), 6.61 (d,
+
C38H34N3O8 660.2340, found 660.2349.
Synthesis of Compound 14. Compound 13 (990 mg, 1.5 mmol)
was rendered anhydrous by repeated coevaporation with dry
pyridine (1 mL × 3), dry toluene (1 mL × 1), and dry CH3CN (1
J. Org. Chem, Vol. 73, No. 4, 2008 1223

Tsunoda et al.
mL × 1) and dissolved in dry CH2Cl2 (15 mL). To the solution
were added bis(diisopropylamino)(2-cyanoethoxy)phosphine
(524 µL, 1.65 mmol), 1H-tetrazole (63.2 mg, 0.90 mmol), and
diisopropylamine (127 µL, 0.90 mmol), and the mixture was stirred
at room temperature for 2 h. The mixture was partitioned between
CHCl3 and aqueous NaHCO3. The organic phase was collected,
dried over Na2SO4, filtered, and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel with
hexane-ethyl acetate (40:60-30:70, v/v) containing 1% pyridine
to give the fractions containing 13. The fractions were collected
and evaporated under reduced pressure. The residue was finally
evaporated by repeated coevaporation three times each with toluene
and CHCl3 to remove the last traces of pyridine to give 13 (900
mg, 70%): 1H NMR (CDCl3) δ 1.04-1.17 (m, 12H), 2.40-2.45
(m, 2H), 2.62 (t, 1H, J ) 6.3 Hz), 2.72-2.82 (m, 1H), 3.40-3.61
(m, 6H), 3.78 (s, 6H), 4.20-4.21 (m, 1H), 4.59-4.69 (m, 1H),
6.22-6.32 (m, 2H), 6.81-6.86 (m, 4H), 7.26-7.39 (m, 9H), 7.79-
7.82 (m, 2H), 7.94-7.97 (m, 2H), 8.51 and 8.60 (2d, 1H, J ) 7.3
Hz); 13C NMR (CDCl3) δ 20.1, 20.2, 20.4, 24.4, 24.5, 24.6, 40.7,
41.1, 43.1, 43.2, 43.3, 53.4, 55.2, 58.1, 58.4, 61.5, 61.9, 70.9, 71.2,
71.8, 72.1, 85.8, 86.9, 87.5, 87.6, 100.5, 113.2, 117.3, 117.5, 124.3,
127.1, 128.0, 128.1, 128.2, 130.07, 130.13, 131.4, 135.1, 135.3,
144.0, 145.2, 154.6, 158.6, 159.1, 164.9; 31P NMR (CDCl3) δ 149.6,
150.2 (2s); HRMS (ESI) m/z (M + H) calcd for C47H51N5O9P+
860.3419, found 860.3411.
Synthesis of the Dimer d[COpT]. A thymidine-loaded HCP (1.0
µmol, 27 µmol/g, succinate linker) was used. Each cycle of chain
elongation consisted of the following steps: (1) detritylation (3%
trichloroacetic acid in CH2Cl2, 2 mL, 1 min); (2) washing [CH2Cl2
(1 mL × 3), CH3CN (1 mL × 3)]; (3) the first coupling [an
appropriate phosphoramidite unit (20 µmol) in CH3CN (200 µL),
HOBt (5.4 mg, 40 µmol) in CH3CN (200 µL), 1 min]; (4) washing
[CH3CN (1 mL × 3)]; (5) the second coupling [an appropriate
phosphoramidite unit (20 µmol) in CH3CN (200 µL), HOBt (5.4
mg, 40 µmol) in CH3CN (200 µL), 1 min]; (6) washing [CH3CN
(1 mL × 3)]; (7) oxidation [0.1 M I2, pyridine-H2O (9/1, v/v), 2
min]; and (8) washing [pyridine (1 mL × 3), CH3CN (1 mL × 3),
CH2Cl2 (1 mL × 3)]. Generally, the average yield per cycle was
estimated to be 98-99% by the DMTr cation assay. After chain
elongation was finished, the DMTr group was removed by treatment
with 3% trichloroacetic acid in CH2Cl2 (2 mL) for 1 min, and the
resin was washed with CH2Cl2 (1 mL × 3), CH3CN containing
1% Et3N (1 mL × 3), and CH3CN (1 mL × 3). Subsequently, the
synthesized dimer d[CpT]-loaded HCP (50 nmol, 27 µmol/g,
succinate linker) was used. General procedure for N-oxidation
reaction: (1) Oxidation [0.02-0.1 M m-CPBA in MeOH, 1-8 h];
(2) washing [MeOH (1 mL × 3), CH3CN (1 mL × 3)]. The dimer
was deprotected and released from the polymer support by treatment
with concentrated NH3 aq (500 µL) for 1 h. The polymer support
was removed by filtration and washed with CH3CN (1 mL × 3).
The filtrate was evaporated and purified by reversed-phase HPLC:
HRMS (ESI) m/z (M + H) calcd for C19H27N5O12P+ 548.1388,
found 548.1388.
The mass spectral data of oligodeoxynucleotides are as follows.
Oligodeoxynucleotide d[T6COT7]: MALDI-TOF mass (M + H)
+
calcd for C140H185N29O95P13 4194.71, found 4198.69. Oligode-
oxynucleotide d[T6AOT7]: MALDI-TOF mass (M + H) calcd for
+
C
141H185N31O94P13 4218.72, found 4222.71.
Synthesis of Oligodeoxynucleotides 16 and 18. The synthesis
of oligodeoxyribonucleotides by use of an ABI 392 DNA synthe-
sizer was carried out.21 After chain elongation was finished, the
DMTr group was removed by treatment with 3% trichloroacetic
acid in CH2Cl2 (2 mL) for 1 min, and the resin was washed with
CH2Cl2 (1 mL × 3), CH3CN containing 1% pyridine (1 mL × 3),
and CH3CN (1 mL × 3). In the synthesis of a DNA 14mer
containing a 2′-deoxynucleoside N-oxide, the unmodified oligomer-
loading HCP (250 nmol, 27 µmol/g, succinate linker) was oxidized
by treatment of a solution of 0.1 M m-CPBA in MeOH (150 µL),
and washed with MeOH (1 mL × 3) and CH3CN (1 mL × 3). The
oligomer was deprotected and released from the polymer support
by treatment with concentrated NH3 aq (500 µL) for 12 h. The
polymer support was removed by filtration and washed with distilled
water (1 mL × 3). The filtrate was evaporated and purified by
reversed-phase HPLC or anion-exchange HPLC. The yields of these
modified oligodeoxynucleotides were calculated using the ꢀ value
of this oligonucleotide, which was estimated by the sum of the ꢀ
values at 260 nm of all nucleotides involving the modified
nucleoside in consideration of a 15% hyperchromic effect: dAO, ꢀ
) 7.93 × 103 at 260 nm; dCO, ꢀ ) 3.91 × 103 at 260 nm. For the
modifiedoligodeoxynucleotides,thefollowingꢀwasused: d[T6COT7],
ꢀ ) 9.95 × 104 at 260 nm; d[T6AOT7], ꢀ ) 1.03 × 105 at 260 nm;
d[GACTGACOTGACT] 16, ꢀ ) 1.09 × 105 at 260 nm; d[GACT-
GAOCTGACT] 18, ꢀ ) 1.05 × 105 at 260 nm.
The mass spectral data of oligodeoxynucleotides are as follows.
Oligodeoxynucleotide 16: MALDI-TOF Mass (M + H) calcd for
C
117H149N45O71P11+ 3660.65, found 3660,08. Oligodeoxynucleotide
+
18: MALDI-TOF mass (M + H) calcd for C117H149N45O71P11
3660.65, found 3664.44.
Enzyme Assay of Oligodeoxynucleotides. The enzymatic
digestion was performed by using an appropriate oligodeoxynucle-
otide (0.2 OD), snake venom phosphodiesterase (10 µg), nuclease
P1 (10 µg), and calf intestine alkaline phosphatase (0.4 units) in
20 µL of alkaline phosphatase buffer [500 mM Tris-HCl (pH 9.0),
10 mM MgCl2] at 37 °C for 4 h. After the enzymes were deactivated
by heating at 80 °C for 5 min, the solution was diluted and filtered
by a 0.45 µm filter (Millex-HV, Millipore). The mixture was
analyzed by reversed-phase HPLC. For d[T6COT7], dCO:dT ) 1.0:
13.1. For d[T6AOT7], dAO:dT ) 1.0:13.0. For oligodeoxynucleotide
16, dCO:dG:dA:dC:dT ) 1.0:3.2:2.9:2.0:3.0. For oligodeoxynucle-
otide 18, dAO:dG:dA:dC:dT ) 1.0:3.2:2.0:3.0:3.1.
Synthesis of Oligodeoxynucleotides d[T6COT7] and d[T6AOT7].
The synthesis of oligodeoxyribonucleotides by use of an ABI 392
DNA synthesizer was carried out.21 After chain elongation was
finished, the DMTr group was removed by treatment with 3%
trichloroacetic acid in CH2Cl2 (2 mL) for 1 min, and the resin was
washed with CH2Cl2 (1 mL × 3), CH3CN containing 1% Et3N (1
mL × 3), and CH3CN (1 mL × 3). In the synthesis of a DNA
14mer containing a 2′-deoxynucleoside N-oxide, the unmodified
oligomer-loading HCP (250 nmol, 27 µmol/g, succinate linker) was
oxidized by treatment of a solution of 0.1 M m-CPBA in MeOH
(150 µL) and washed with MeOH (1 mL × 3) and CH3CN (1 mL
× 3). The oligomer was deprotected and released from the polymer
support by treatment with concentrated NH3 aq (500 µL) for 1 h.
The polymer support was removed by filtration and washed with
distilled water (1 mL × 3). The filtrate was evaporated and purified
by reversed-phase HPLC or anion-exchange HPLC.
Acknowledgment. This work was supported by a Grant from
CREST of JST (Japan Science and Technology Agency) and a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan. This
work was supported in part by a grant of the Genome Network
Project from the Ministry of Education, Culture, Sports, Science
and Technology, Japan, and by the COE21 project.
Supporting Information Available: The 1H, 13C, and 31P NMR
data of all new products. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO7021845
1224 J. Org. Chem., Vol. 73, No. 4, 2008