Palladium-catalyzed direct N-arylation of nucleosides, nucleotides, and oligonucleotides for efficient preparation of dG-N2 adducts with carcinogenic amino-/nitroarenes

Article abstract of DOI:10.1021/jo0605243

A method for direct palladium-catalyzed N-arylation reaction of nucleobases was developed for the convenient synthesis of DNA adducts with carcinogenic compounds. Using xantphos as the phosphine ligand and tetraethylammonium fluoride as the base in DMSO,

Full text of DOI:10.1021/jo0605243

Palladium-Catalyzed Direct N-Arylation of Nucleosides, Nucleotides,
and Oligonucleotides for Efficient Preparation of dG-N2 Adducts
with Carcinogenic Amino-/Nitroarenes
Takeji Takamura-Enya,* Shigeki Enomoto, and Keiji Wakabayashi
Cancer PreVention Basic Research Project, National Cancer Centre Research Institute,
Tokyo 104-0045, Japan
tenya@gan2.res.ncc.go.jp
ReceiVed March 9, 2006
A method for direct palladium-catalyzed N-arylation reaction of nucleobases was developed for the
convenient synthesis of DNA adducts with carcinogenic compounds. Using xantphos as the phosphine
ligand and tetraethylammonium fluoride as the base in DMSO, several o-iodonitroarenes could be
efficiently coupled with 2′-deoxyguanosine, 2′-deoxyadenosine, and 2′-deoxycytidine. The presence of a
3′-phosphate group in the deoxyribose moiety was found to be compatible with this N-arylation reaction;
further, oligonucleotides could serve as substrates. The facile nitroreduction of the coupling compounds
(12) yielded 2′-deoxyguanosin-N2-ylarylamine adducts, which are known to be biologically important.
Compound 12 was easily converted to phosphoramidite derivatives, allowing the preparation of site-
specific modified oligonucleotides with arylamine after the nitroreduction.
Introduction
directly attack N7 of 2′-deoxyguanosine (dG); this results in
the subsequent transfer of the arylamine at the N7 position of
dG to the C8 position, forming N-(deoxyguanosin-8-yl)aryl-
amine, i.e., dG-C8 adduct 1. Carbocations that are resonance
stabilized from the arylnitrenium ions also attack dG to form
2′-deoxyguanosin-N2-yl-arylamine (dG-N2 adduct 3) wherein the
C atom located at the ortho position of the amino group often
attaches covalently to the N2 position of dG.1d-f Biologically,
the efficiency of amino-/nitroarene-derived dG-N2 adduct
formation is generally lower than that of the formation of the
corresponding dG-C8 adducts. However, the in vivo efficiency
of the DNA repair enzymes in the repair of dG-N2 adducts is
less than that in the repair of dG-C8 adducts. Thus, dG-N2
adducts have attracted significant interest in genotoxicity
studies.2 In addition, although it is generated to a far lesser
extent, 2′-deoxyadenosine (dA) serves as a target for the
arylnitrenium ions, forming 2′-adenosin-8-ylaminoarene (dA-
C8 adduct 2) and 2′-adenosin-6-ylaminoarene (dA-N6 adduct
4), which are analogous to the dG adducts (Scheme 1).1
Aromatic amino and nitro compounds, which are common
mutagens/carcinogens widely distributed in the environment,
covalently bind to DNA after metabolic activation; this is
recognized as the crucial first step in mutagenesis/carcinogen-
esis.1 In mammalian cells, the manner of DNA adduct formation
with these classes of carcinogens is very similar; aminoarenes
are oxidatively activated by the P450 species, whereas nitro-
arenes are reductively activated by coenzymes of P450 such as
NADPH.1 Both of these reactions yield hydroxyamines that are
esterified by acetyl- or sulfotransferases to form O-esterified
hydroxyaminoarenes. The spontaneous fission of the N-O bond
generates highly active arylnitrenium ions to attack the DNA
molecules. Generally, the arylnitrenium ions are believed to
* To whom correspondence should be addressed. Tel: +81-3-3547-5201,
Fax: +81-3-3543-9305. Present address: Department of Applied Chemistry,
Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi-shi 243-0292,
Japan. Tel: +81-46-291-3072. Fax: +81-46-242-8760. E-mail: takamura@
chem.kanagawa-it.ac.jp.
10.1021/jo0605243 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/20/2006
J. Org. Chem. 2006, 71, 5599-5606
5599

Takamura-Enya et al.
SCHEME 1. Metabolic Activation and DNA Adduct
Formation of Amino-/Nitroarenes
under conditions of Pd(0)/BINAP with lithium hexamethyl-
disilazide (LiHMDS), potassium tert-butoxide (tBuOK), or K3-
PO4, yielding dG-C8 adduct 1 derived from carcinogenic
heterocyclic amines by subsequent deprotection.10 Initially,
Schoffer et al.11a reported that the protection of the N6 atom of
adenosine derivatives in the arylamination reaction was not
required for the preparation of adenosine-C8-PAH adducts,
and Elmquist et al. later utilized the same technique for the
preparation of dG-C8 adducts 1.11b We have already demon-
strated that a Pd(0)/xantphos system works well in the arylami-
nation reaction for both suitably protected 8-aminonucleosides
with bromoarenes and for protected 8-bromonucleosides with
heterocyclic amines.12 Generally, the phosphoramidite deriva-
tives of these DNA adducts could be obtained on a large scale
by the Pd-catalyzed amination strategy for the production of
site-specific adducted oligonucleotides that are useful tools for
the physicochemical and biological studies investigating the
fundamental mechanisms underlying mutation as the initial step
in carcinogenesis.11b,13
In these arylamination studies, it was necessary to protect
the OH groups of the ribose moiety in all of the reactions in
order to enhance solubility of nucleosides in organic solvents;
in particular, the amide moiety at the N1 position of dG required
protection to remove the unfavorable acidic proton that may
have obstructed arylamination. This protection/deprotection
strategy leads to easy separation, but inevitably adds two steps
to synthetic procedures, reduces the overall yields, and some-
times requires expensive chemicals for protection.
While studying the synthesis of dG-C8 adducts using the
Pd(0)/xantphos system, we obtained evidence that activated
halogenated arenes might enable the direct preparation of DNA
adducts, i.e., coupling to nucleosides without protective groups
is feasible. Moreover, once this direct coupling reaction of
nucleosides has occurred, nucleotides and oligonucleotides could
also serve as substrates for the arylamination reaction. To our
knowledge, there have been no previous studies on the direct
arylamination of nucleotides and oligonucleotides without
protective groups. In this paper, we report the development of
a successful method for the direct arylamination of nucleosides,
nucleotides, and oligonucleotides, and its application in the
syntheses of dG-N2 adducts with amino-/nitroarenes and site-
specific modified oligonucleotides.
The DNA adducts of carcinogenic amino-/nitroarenes are
classically obtained by a simple electrophilic amination reaction3
and can now also be prepared using the Buchwald-Hartwig
N-arylation strategy.4 The classical conditions, however, have
certain limitations,3 whereas it has been demonstrated that the
Buchwald-Hartwig palladium (Pd)-catalyzed arylamination
reaction is widely applicable for the generation of a variety of
DNA adducts, including dG-C8 1, dG-N2 3, dA-C8 2, and
dA-N6 4 adducts.4 Johnson’s group demonstrated examples of
the preparation of some dG-N2-type 3 and dA-N6-type 4
adducts by the Buchwald-Hartwig arylamination reaction using
Pd(0)/2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) sys-
tems with Cs2CO3.5 This system was later applied to the
preparation of several N2-modified dG derivatives.6 The N2
adducts of dG 3 with nitropyrene and also with an imidazo-
quinoline-type mutagen could be synthesized in a very similar
manner.7 Some related PAH-derived dG and dA adducts were
initially prepared using Lakshman’s Pd(0) with a series of
biphenylphosphine ligands or BINAP system.8 Nitrous acid-
mediated cross-linking nucleosides have also been described.9
N,O-Protected 8-bromonucleosides were found to readily un-
dergo an arylamination reaction of carcinogenic arylamines
Results and Discussion
(1) (a) Purohit, V.; Basu, K. Chem. Res. Toxicol. 2000, 13, 673. (b)
Tokiwa, H.; Ohnishi, Y. CRC Crit. ReV. Toxicol. 1986, 17, 23. (c) IARC
Monographs on the EValuation of Carcinogenic Risk to Humans; Interna-
tional Agency for Research on Cancer (IARC): Lyon, 1989; Vol. 46. (d)
Beland, F. A.; Marques, M. M. DNA Adducts: Identification and Biological
Significance; IARC: Lyon, 1984; Vol. 125. (e) Fu, P. P. Drug Metabolism
ReV. 1990, 22, 209. (f) Nagao, M. Food Borne Carcinogens, Heterocyclic
Amines; John Wiley & Sons Ltd.: Chichester, UK, 2000.
(2) (a) Yasui, M.; Dong, H.; Bonala, R. R.; Suzuki, N.; Ohmori, H.;
Hanaoka, F.; Johnson, F.; Grollman, A. P.; Shibutani, S. Biochemistry 2004,
43, 15005. (b) Gupta, R. C.; Dighe, N. R.; Randerath, K.; Smith, H. C.
Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6605. (c) Beland, F. A.; Kadlubar,
F. F. EnViron. Health Perspect. 1985, 62, 19. (d) Kriek, E. Cancer Res.
1972, 29, 1799.
(3) (a) Sano, T.; Kaya, K. Chem. Res. Toxicol. 1995, 8, 699. (b) Bonala,
R.; Yu, P. L.; Johnson, F. Tetrahedron Lett. 1999, 40, 597. (c) Ve´liz, E.
A.; Beal, P. A. J. Org. Chem. 2001, 66, 8592. (d) Scheer, S.; Steinbrecher,
T.; Boche, G. EnViron. Health Perspect. 1994, 102, 151.
(4) For reviews, see: (a) Lakshman, M. K. J. Organomet. Chem. 2002,
653, 234. (b) Lakshman, M. K. Curr. Org. Synth. 2005, 2, 83.
(5) De Riccardis, F.; Bonala, R. R.; Johnson, F. J. Am. Chem. Soc. 1999,
121, 10453.
Arylamination of O-Protected dG. To select the most
effective catalytic system for the following direct arylamination
(8) (a) Lakshman, M. K.; Keeler, J. C.; Hilmer, J. H.; Martin, J. Q. J.
Am. Chem. Soc. 1999, 121, 6090. (b) Lakshman, M. K.; Hilmer, J. H.;
Martin, J. Q.; Keeler, J. C.; Dinh, Y. Q.; Ngassa, F. N.; Russon, L. M. J.
Am. Chem. Soc. 2001, 123, 7779. (c) Lakshman, M. K.; Ngassa, F. N.;
Bae, S.; Buchanan, D. G.; Hahn, H. G.; Mah, H. J. Org. Chem. 2003, 68,
6020. (d) Lakshman, M. K.; Gunda, P. Org. Lett. 2003, 5, 39.
(9) (a) Harwood, E. A.; Hopkins, P. B.; Sigurdsson, S. T. J. Org. Chem.
2000, 65, 2959. (b) Harwood, E. A.; Siguedsson, S. T.; Edfeldt, N. B. F.;
Reid, B. R.; Hopkins, P. B. J. Am. Chem. Soc. 1999, 121, 5081. (c)
Riccardis, F. D.; Johnson, F. Org. Lett. 2000, 2, 293.
(10) (a) Wang, Z.; Rizzo, C. J. Org. Lett. 2001, 3, 565. (b) Gillet, L. C.;
Scharer, O. D. Org. Lett. 2002, 4, 4205. (c) Meier, C.; Gra¨sl, S. Synthesis
Lett. 2002, 802.
(11) (a) Schoffer, E.; Olsen, P.; Means, J. C. Org. Lett. 2001, 3, 4221.
(b) Elmquist, C. E.; Stover, J. S.; Wang, Z.; Rizzo, C. J. J. Am. Chem. Soc.
2004, 126, 11189.
(12) (a) Takamura-Enya, T.; Ishikawa, S.; Mochizuki, M.; Wakabayashi,
K. Tetrahedron Lett. 2003, 44, 5969. (b) Takamura-Enya, T.; Ishikawa, S.;
Mochizuki, M.; Wakabayashi, K. Nucleic Acid Res. Suppl. 3 2003, 23.
(13) Radha, R.; Bonala, M.; Torres, C.; Attaluri, S., Iden, C. R.; Johnson,
F. Chem. Res. Toxicol. 2005, 18, 457.
(6) Bonala, R. R.; Shishkina, I. G.; Johnson, F. Tetrahedron Lett. 2000,
41, 7281.
(7) (a) Chakraborti, D.; Colis, L.; Schneider, R.; Basu, A. K. Org. Lett.
2003, 5, 2861. (b) Stover, J. S.; Rizzo, C. J. Org. Lett. 2004, 6, 4985.
5600 J. Org. Chem., Vol. 71, No. 15, 2006

Preparation of dG-N2 Adducts with Carcinogenic Amino-/Nitroarenes
SCHEME 2. Effect of Phosphine Ligand on the Synthesis
of 11aa
SCHEME 3. Arylamination Reaction of O-TBDMS-dG with
3-Iodobiphenyl
SCHEME 4. Direct Pd-Catalyzed Arylamination with DG
and Selected Iodoarenesa
a Key: (1) Conversion after 10 h reaction time; (2) isolation yield.
CHART 1. Ligands Tested in This Study
a Key: (1) isolated yield; (2) Cs2CO3 was used.
coupling reaction, 1-iodonaphthalene 9a and 3′,5′-O-TBDMS-
dG 10 were considered as the initial substrates, and this
combination was tested with several phosphine ligands and Pd2-
(dba)3 to determine the optimal conditions for catalytic N-
arylation (Scheme 2). Without O6-protection, the arylamination
reaction with bromoarenes was slow, as expected; however, this
sluggishness might be overcome by using iodoarenes since they
are generally recognized to be more reactive than the corre-
sponding bromoarenes. The ligands screened were BINAP,
xantphos, DPEphos, 2-(dicyclohexylphosphino)-2′-(N,N-dimeth-
ylamino)biphenyl 5, Popd2 6, butyldi-1-adamantylphosphine 7,
and 1,3-bis(2,6-diisopropylphenyl)imidazolium chloride 8, as
illustrated in Chart 1, that are commonly used or were newly
discovered in a Pd-catalyzed C-N bond forming process.8d,14,15
The screening reactions were carried out under standard
conditions of Pd2(dba)3 (10 mol %), the ligand (30 mol %),
and Cs2CO3 (2 equiv to the nucleoside derivatives) in dioxane
at 100 °C for 10 h (Scheme 2).
Dioxane was selected over other inert solvents such as toluene
because the protected dG solubilizes better in dioxane. No
reaction occurred in the control experiment without the Pd
catalyst/ligands. The reactions employing ligands 5-8 also
failed. BINAP and xantphos yielded moderate conversion as
did DPEphos to a lesser extent. Xantphos and BINAP demon-
strated a similar activity in the conversion of the starting
materials, although the former was somewhat superior to the
latter with regard to the isolation yields. The Pd(0)/xantphos
system also proved to be efficient for the coupling of 3-iodobi-
phenyl 9b at a higher reaction temperature of 120 °C, although
3-bromobiphenyl did not yield any catalyzed reaction products
under the same reaction conditions (Scheme 3).
Direct Arylamination of dG for the Preparation of dG-
N2 Adducts. The system that was optimized for O-protected
dG 10 and 9a was applied for the direct arylamination of dG
without any protective groups. However, only poor conversion
was achieved, probably due to the sparing solubility of dG in
dioxane. DMSO, the next choice as a more polar solvent with
greater solubilization activity, was found to produce N2-aryl
nucleoside 12a at low yields (Scheme 4).
(14) For an adamantylphosphine ligand, see: (a) Ehrentraut, A.; Zapf,
A.; Beller, M. J. Mol. Catal. 2002, 182/183, 515. For xantphos, see: (b)
Ali, M. H.; Buchwald, S. L. J. Org. Chem. 2001, 66, 2560. (c) Yin, J.;
Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 6043. For Popd2, see: (d)
Li, G. Y.; Zheng, G.; Noonan, A. F. J. Org. Chem. 2001, 66, 8677. For an
imidazol type ligand, see: (e) Grassa, G. A.; Viciu, M. S.; Huang, J.; Nolan,
S. P. J. Org. Chem. 2001, 66, 7729. For DPEphos, see: (f) Belfield, A. J.;
Brown, G. R.; Foubister, A. J. Tetrahedron 1999, 11399.
Changing the base to tetraethylammonium fluoride (TEAF)
also yielded 12a with a similar efficiency. It was encouraging,
(15) Wolfe, J. P.; Tomori, H.; Sadighi, J. P.; Yin J.; Buchwald, S. L. J.
Org. Chem. 2000, 65, 1158.
J. Org. Chem, Vol. 71, No. 15, 2006 5601

Takamura-Enya et al.
TABLE 1. Arylamination Reaction of Nucleosides with
o-Iodonitrobenzene
however, that the use of more activated o-iodonitrobenzene with
the Pd(0)/xantphos/TEAF system produced the corresponding
N2-nitrobenzene-dG 12c, which can be used as a precursor for
the generation of the desired dG-N2-aniline adduct 19c in a
moderate yield. Generally, Cs2CO3 has a coupling efficiency
similar to that of TEAF. However, due to its ease of handling
during the isolation steps, TEAF is superior. With TEAF in
DMSO, the modified nucleoside derivatives were highly soluble
in general organic solvents such as CHCl3/CH3OH making it
easy to apply the derivatives to silica gel column chromatog-
raphy, although some of the compounds were isolated as
tetraethylammonium (TEA) salts. To our knowledge, this is the
first demonstration that TEAF can act as the base in Pd-catalyzed
arylamination. It should be noted that this Pd(0)/xantphos/TEAF
system could not be used with other solvents such as THF. When
the same catalyst and base load were used in THF, no conversion
of the starting materials was observed. Another quaternary
ammonium salt, tetraethylammonium bromide, did not yield
coupling compound 12 in DMSO.
The direct coupling of dG and a series of iodoarenes 9d-g
was carried out to determine the utility of the reaction. Targets
were chosen so as to yield dG-N2 adducts of carcinogenic
amino-/nitroarenes or their precursors. Ortho-substituted iodo-
nitroarenes containing 3-iodo-4-nitrobiphenyl 9d and 1-iodo-
2-nitronaphthalene 9e could couple within 4 h at 90 °C at a
good yield. O-Acetylamide iodobenzene 9f gave no reaction
products even at 120 °C, whereas 6-acetylamide-1-iodopyrene
9g gave dG-N2-yl-6-acetylaminopyrene 12g at moderate yields;
this is a deacetylation derivative that is known to be generated
in the reaction of the ultimate mutagenic forms of 1-nitropyrene
with dG.7a,16 The other nucleosides dA and dC also gave the
corresponding adducts modified with nitrobenzene, i.e., 15c and
16c, respectively, at excellent yields in the presence of Pd(0)/
xantphos/TEAF (Table 1, entries c and d). It is noteworthy that
the coupling reaction with dC proceeded even at room temper-
ature. The Pd(0)/xantphos/Cs2CO3 system in dioxane worked
well for the N-arylation of dA as observed by TLC analyses;
however, due to the sparing solubility of the reaction products
in commonly used organic solvents, the isolation was tedious
and resulted in a low isolation yield.
a Ar ) O-nitrobenzene. Reactions were performed with nucleotide/
nucleoside (1 mmol), o-iodonitrobenzene (2 mmol), Pd2(dba)3 (0.1 mol),
xantphos (0.2 mol), and TEAF (2 mmol).
SCHEME 5. Arylamination of Oligonucleotide TTTGTTT
with o-Iodonitrobenzene
Given the success of the Pd(0)/xantphos/TEAF system for
the direct N-arylation of nucleosides, its potential application
with nucleotides was explored. The nucleotide, 2′-deoxygua-
nosine-3′-phosphate (2′-dGp), was coupled with o-iodonitroben-
zene to yield 2′-deoxyguanosin-N2-ylnitrobenzene 13c in 61%
yield (Table 1, entry a). In DNA samples, a DNA adduct with
a 3′-phosphate group is an essential substrate that is used for
quantitative determination by 32P-postlabeling analysis.17 Au-
thentic samples of this type of nucleotide have only been
obtained by a nitrenium ion-mediated reaction with 2′-dGp
derivatives or a post-phosphorylation reaction with the corre-
sponding modified nucleosides.18,19 The oligonucleotide TTT-
GTTT 17 could also be coupled efficiently by using the Pd-
(0)/xantphos/TEAF system, although 100 mol % of Pd(0) was
loaded in 100 µL of DMSO due to the technical difficulty of
weighing the correct amount for a small-scale reaction (300
µmol) and to prevent the cleavage of phosphate bonds during
the prolonged heating conditions at 90 °C (Scheme 5). The
reaction was completed within 1 h with 100% conversion, and
the isolated yield of the modified oligonucleotide 18c after
HPLC was estimated to be 68% using UV absorbance at 260
nm. Although the reaction with mixed sequence oligonucleotides
containing dA an dC has not been tested yet, dA and dC in
oligonucleotides are expected to couple with iodoarenes because
amino groups of dA and dC are known to be more nucleophilic
than those of dG. Regarding the Pd(0)-catalyzed coupling
(16) Herreno-Saenz, D.; Evans, F. E.; Beland, F. A.; Fu, P. P. Chem.
Res. Toxicol. 1995, 8, 269.
(17) Reddy, M. V.; Randerath, K. Carcinogenesis 1986, 7, 1543.
(18) (a) Prusiewicz, C. M.; Sangaiah, R.; Tomer, K. B.; Gold, A. J. Org.
Chem. 1999, 64, 7628. (b) Haack, T.; Boche, G.; Kliem, C.; Weissler, M.;
Albert, D.; Schmeiser, H. G. H. Chem. Res. Toxicol. 2004, 17, 776.
(19) (a) Godschalk, R.; Nair, J.; Kliem, H.-C.; Weissler, M.; Bouvier,
G.; Bartsch, H. Chem. Res. Toxicol. 2002, 15, 433. (b) Kawanishi, M.; Enya,
T.; Suzuki, H.; Takebe, H.; Matsui, S.; Yagi, T. Mutat. Res. 2000, 470,
133.
5602 J. Org. Chem., Vol. 71, No. 15, 2006

Preparation of dG-N2 Adducts with Carcinogenic Amino-/Nitroarenes
SCHEME 6. Nitroreduction of 12c and 12e
SCHEME 7. Synthesis of Oligonucleotide Containing dG-N2
Adduct 3c
reaction, the Sonogashira reaction of protected oligonucleotides
was previously performed by Khan and Grinstaff.20
Synthesis of dG-N2 Adduct and Its Application for the
Synthesis of Site-Specific Modified Oligonucleotides. Since
the Pd/xantphos system displayed high reactivity with o-
iodonitroarenes, we were interested in the synthesis of authentic
dG-N2-arylamine adduct 3 specimens that are found in
biological samples. The direct reduction of the nitro group of
12c and 12e would yield the N2-arylamine adducts of dG 3.
We tested several nitroreduction systems including Pd/C with
H2, cyclohexadiene with Pd black, Na2S2O4, and Zn dust.
Among them, an efficient reduction to the desired amine was
observed in the system of cyclohexadiene in DMF with Pd black
heated at 90 °C and with Zn dust in a 1:1 mixture of DMSO
and 500 mM CH3COONH4 at pH 4.5. Treatment of 12c with
aqueous Na2S2O4 in aqueous DMSO also resulted in rapid
reduction of a nitro group but always involved the sulfonylation
products of the resulting aromatic amine, as confirmed by liquid
chromatography-mass spectrometry (LC-MS) techniques.
Pd/C with H2 gas at atmospheric pressure did not cause the
conversion of the starting materials. The Zn/500 mM CH3-
COONH4 system at pH 4.5 proved superior to the cyclohexa-
diene/Pd black system because the former reaction proceeded
under mild conditions without heating and was completed within
1 h at room temperature. The desired 3c compound was purified
by passing the filtrate of the reaction mixture through a C18
RP-silica gel column and eluting with a water/MeOH gradient.
This reduction procedure could be applied to another nitro
derivative 3e (Scheme 6).
dG and iodonitroarene 9c under the conditions of Pd(OAc)2/
xantphos/Cs2CO3 in dioxane at 90 °C, resembling the conditions
for O-silyl protected dG (Table 1, entry b). In this case, Pd-
(OAc)2 was used instead of Pd2(dba)3. When Pd2(dba)3 was
employed as the Pd source, the reaction gave rise to some
colored impurities as well as the desired compounds that could
not be separated by silica gel chromatography. The dimethoxy-
trityrated N2-nitrophenyl-dG adduct 14c was phosphitylated by
using the 2-cyanoethyltetraisopropylphosphorodiamidite/tetra-
zole system in CH2Cl2. Careful workup with silica gel chro-
matography gave the desired 5′-DMTr-N2-2-nitrophenyl-2′-
deoxyguanosine phosphoramidite 19c. The amidite 19c was then
subjected to a DNA synthesizer with a slight modification where
the coupling time of the modified amidite was changed to 15
min. The coupling efficiency was generally higher than 80%
as determined by UV monitoring during tritanol elimination.
Oligonucleotide 13mer 20c modified with nitrobenzene was thus
obtained purified by HPLC. The UV spectrum of the oligo-
nucleotide showed absorbance at 400-500 nm, which was
derived from the nitrobenzene moiety at the N2 position. MS
analysis of this modified oligonucleotide showed a peak of m/z
4088. Enzymatic hydrolysates of the oligonucleotide clearly
indicated the presence of N2-nitrophenyl-dG 12c. The nitro
group in oligonucleotide 20c was effectively converted to an
amino group by using Zn dust in Na citrate at pH 5. The Zn/
CH3COONH4 system worked well for the nitro reduction;
however, some minor HPLC peaks were always observed,
The nitrophenyl-dG derivative12c could be used directly for
general oligonucleotide synthesis by the phosphoramidite ap-
proach (Scheme 7). Compound 12c could be converted to the
5′-dimethoxytriphenylmethyl (DMTr) derivative 14c in the
general manner of 4,4′-dimethoxytriphenylmethyl chloride
(DMTrCl) in pyridine. Compound 14c was also obtained
efficiently by the direct coupling reaction between 5′-DMTr-
(20) Khan, S. I.; Grinstaff, M. W. J. Am. Chem. Soc. 1999, 121, 4704.
J. Org. Chem, Vol. 71, No. 15, 2006 5603

Takamura-Enya et al.
monitored by TLC, and upon completion, the reaction mixtures
were dissolved in a CHCl3-MeOH solution, directly subjected to
column chromatography on silica gel, and eluted with a step
gradient of MeOH in CHCl3. The fractions containing the desired
products were combined, evaporated, and finally dried under high
vacuum to remove traces of the solvent.
N2-Naphthalen-1-yl-2′-deoxyguanosine (12a): 1H NMR (DMSO-
d6) δ 10.9 (s, 1H), 8.89 (s, 1H), 8.13-8.08 (m, 2H), 8.03 (s, 1H),
7.96 (dd, J ) 7.8 Hz, 1.0, 1H), 7.73 (d, J ) 8.1 Hz, 1H), 7.62-
7.51 (m, 3H), 6.10 (t, J ) 6.8 Hz, 1H), 5.24 (d, J ) 4.1 Hz, 1H),
4.84 (t, J ) 5.5 Hz, 1H), 4.23 (td, J ) 6.2 Hz, 3.2 Hz, 1H), 3.76
(dd, J ) 7.8 Hz, 4.6 Hz, 1H), 3.40 (tt, J ) 16.3 Hz, 5.7 Hz, 1H),
2.59-2.51 (m, 1H), 2.18 (dq, J ) 13.2 Hz, 3.2 Hz, 1H); 13C NMR
(DMSO-d6) 156.4, 150.1, 149.4, 136.5, 133.6, 133.2, 128.3, 126.5,
126.0, 125.7, 124.1, 121.3, 118.9, 118.3, 87.6, 82.8, 70.5, 61.6;
FAB-HRMS (nba) m/z [M + H+] calcd for C20H20N5O4 394.1515,
found 394.1492.
probably due to the formation of hydroxyamine derivatives from
insufficient nitro reduction, whereas overnight treatment with
Zn in Na citrate buffer resulted in the complete reduction of
the nitro group in oligonucleotide 20c. Liquid chromatography
electrospray ionization tandem mass spectrometry (LC-ESI-
MS) indicated that the nitrophenyl oligonucleotide 20c was
converted to aminophenyl oligonucleotide 21c whose enzymatic
hydrolysates also showed the presence of N2-aminophenyl-
dG 3c. The synthesis of site-specific modified oligonucleotides
with the dG-N2 adduct was recently reported by Johnson’s
group; they used the phosphoramidite derivatives of acetylami-
nophenyl-dG or trifluoroacetylaminophenyl-dG followed by the
alkali deprotection of the trifluoroacetyl moiety.14 The system
using nitro groups as the protective group of the dG-N2 adduct
is now an efficient alternative tool for the synthesis of site-
specific modified oligonucleotides.
N2-2-Nitrobenzen-1-yl-2′-deoxyguanosine (12c): 1H NMR (DM-
SO-d6) δ 11.8 (s, 1H), 9.60 (s, 1H), 8.26 (d, J ) 8.5 Hz, 1H),
8.11-8.07 (m, 2H), 7.74 (t, J ) 5.9 Hz, 1H), 7.29 (t, J ) 6.7 Hz,
1H), 6.09 (t, J ) 6.7 Hz, 1H), 5.25 (d, J ) 3.7 Hz, 1H), 4.86 (t,
J ) 5.2 Hz, 1H), 4.30 (br s, 1H), 3.78 (q, J ) 3.9 Hz, 1H), 3.46
(tt, J ) 16.2 Hz, 5.2 Hz, 1H), 2.57-2.51(m, 1H), 2.26-2.18 (m,
1H); 13C NMR (DMSO-d6) 156.4, 148.4, 148.3, 139.0, 136.9, 134.6,
133.2, 125.3, 123.9, 123.4, 119.2, 87.6, 83.0, 70.5, 61.5; FAB-
HRMS (nba) (m/z) [M + H+] calcd for C16H17N6O6 389.1210, found
389.1225.
N2-4-Nitrobiphenyl-3-yl-2′-deoxyguanosine (12d): 1H NMR
(DMSO-d6) δ 12.06 (br s, 1H), 9.88 (br s, 1H), 8.85 (s, 1H), 8.22
(dd, J ) 8.8 Hz, 1.0, 1H), 8.17 (d, J ) 1.0 Hz, 1H), 7.78 (d, J )
7.8 Hz, 2H), 7.60-7.53 (m, 3H), 7.46 (t, J ) 7.0 Hz, 1H), 6.24 (t,
J ) 6.7 Hz, 1H), 5.30 (d, J ) 3.2 Hz, 1H), 4.91 (t, J ) 4.9 Hz,
1H), 4.28 (s, 1H), 3.85 (s, 1H), 3.30 (s, 2H), 2.63-2.55 (m, 1H),
2.28-2.20 (m, 1H), -0.87 (m, 18H), 0.15-0.02 (s, 9H); 13C NMR
(DMSO-d6) 156.5, 148.5, 148.7 146.1, 137.6, 136.7, 136.6, 134.3,
129.3, 129.0, 127.0, 126.4, 121.1, 120.9, 119.3, 87.9, 82.7, 70.7,
61.6; FAB-HRMS (nba) m/z [M + H +] calcd for C22H21N6O6Si2-
Na 465.1522, found 465.1488.
N2-2-Nitronaphthalen-1-yl-2′-deoxyguanosine (12e): 1H NMR
(DMSO-d6) δ 8.30 (d, J ) 8.3 Hz, 1H), 8.11 (dd, J ) 7.9 Hz, 1.1
Hz, 1H), 8.02 (s, 2H), 7.95 (s, 1H), 7.78-7.69 (m, 2H), 5.79 (t,
J ) 6.8 Hz, 1H), 5.10 (d, J ) 3.2 Hz, 1H), 4.71 (br s, 1H), 4.00
(br s, 1H), 3.18 (br s, 2H), 2.31-2.22 (m, 1H), 1.98 (dq, J ) 13.1
Hz, 3.3 Hz, 1H), -0.87 (m, 18H), 0.15-0.02 (s, 9H); 13C NMR
(DMSO-d6) 150.6, 149.3, 141.6, 136.3, 135.1, 129.3, 128.7, 128.3,
127.9, 126.6, 124.8, 120.7, 118.2, 87.6, 82.8, 70.5, 61.6; FAB-
HRMS (nba) m/z [M + H+] calcd for C20H19N6O6 439.1366, found
439.1329.
Conclusion
Here, we described the efficient preparation of dG-N2
adducts from carcinogenic aromatic amino/nitro compounds via
the direct N-arylation reaction. This methodology will be applied
for a wide variety of dG-N2 adducts and also to dA-N6 DNA
adducts; in particular, it will be used for the preparation of site-
specific adducted oligonucleotides with dG-N2 adducts during
solid-phase oligonucleotide synthesis.
Experimental Section
Typical Procedure for the Coupling of Silyl-Protected 2′-
Deoxyguanosine 10 with Iodoarene 9. Compound 10 (1 mmol),
Pd2(dba)3 (0.1 mmol), phosphine ligand (0.3 mmol), iodoarene 9
(2 mmol), and Cs2CO3 (2 mmol) were dissolved in 3 mL of dioxane
and stirred at 100 °C for 10 h. The reaction mixtures were then
evaporated, and the residues were dissolved in CHCl3, subjected
to column chromatography on silica gel, and eluted using a step
gradient of MeOH in CHCl3. The fractions containing the desired
products were combined, evaporated, and finally dried under high
vacuum to remove traces of the solvent.
3′,5′-Di-tert-butyldimethylsilyl-N2-naphthalen-1-yl-2′-deoxy-
guanosine (11a): 1H NMR (DMSO-d6) δ 10.89 (s, 1H), 8.84 (s,
1H), 8.03 (d, J ) 6.6 Hz, 2H), 7.92-7.87 (m, 2H), 7.67 (d, J )
8.1 Hz, 1H), 7.56-7.47 (m, 2H), 7.41 (t, J ) 7.9 Hz, 1H), 6.06 (t,
J ) 6.7 Hz, 1H) (s, 1H), 4.28 (br s, 1H), 3.71 (br s, 1H), 3.47 (d,
J ) 5.4 Hz, 1H), 3.11 (d, J ) 5.1 Hz, 1H), 2.65-2.55 (m, 1H),
2.16-2.07 (s, 1H), 0.81 (m, 9H), 0.77 (m, 9H) 0.05 (s, 6H), -0.09
(m, 6H); 13C NMR (CDCl3) 156.3, 150.1, 149.3, 136.3, 133.6,
133.2, 128.3, 126.6, 125.9, 125.3, 124.1, 121.4, 118.8, 118.3, 87.1,
82.5, 72.2, 62.7, 25.7, 25.6, 17.9, 17.6, -4.7, -4.9, -5.4; FAB-
HRMS (nitrobenzyl alcohol/nba) m/z [M + Na+] calcd for
C61H74N9O6Si2Na 644.3064, found 644.3123.
N2-(8-Acetylamidopyren-1-yl)-2′-deoxyguanosine (12g): 1H
NMR (DMSO-d6) δ 10.35 (s, 1H), 8.59 (d, J ) 8.5 Hz, 1H), 8.40-
8.10 (m, 8H), 6.09 (t, J ) 7.0 Hz, 1H), 5.19 (br s, 1H), 4.82 (br s,
1H), 4.21 (m, 1H), 3.76 (m, 1H), 3.16 (tm, 2H), 2.60 (m, 1H),
2.49 (s, 3H), 2.18 (m, 1H); 13C NMR (DMSO-d6) 168.9, 156.6,
153.5, 150.7, 126.2, 135.1, 131.5, 128.2, 127.5, 126.3, 125.9, 125.1,
124.8, 124.5, 123.4, 123.2, 122.6, 122.1, 118.4, 116.5, 87.5, 82.7,
82.4, 70.6, 61.6, 14.1; FAB-HRMS (nba) m/z [M + Na+] calcd
for C28H24N6O5Na 547.1706, found 547.1590.
3′,5′-Di-tert-butyldimethylsilyl-N2-biphenyl-3-yl-2′-deoxygua-
nosine (11b): 1H NMR (CDCl3) δ 12.24 (br s, 1H), 9.96 (br s,
1H), 8.31 (br s, 1H), 8.15-7.97 (m, 1H), 7.87-7.72 (m, 3H), 7.42-
7.25 (m, 6H), 6.30-6.21 (m, 1H), 4.55-4.45 (m, 1H), 4.02-3.95
(m, 1H), 3.80-3.65 (m, 2H), 2.55-2.25 (m, 2H), 0.99-0.87 (m,
18H), 0.15-0.02 (s, 9H); 13C NMR (CDCl3) 158.7, 150.3, 149.5,
141.0, 140.8, 139.5, 139.1, 136.0, 128.6, 128.3, 128.2, 127.1, 126.9,
122.4, 120.9, 120.2, 118.9, 118.7, 118.4, 118.2, 87.6, 84.0, 72.0,
62.8, 40.7, 31.5, 25.9, 25.7, 22.6, 18.3, 17.9, 14.1, -4.59, -4.62,
-4.73, -4.75, -5.37, -5.47; FAB-HRMS (nba) m/z [M + Na+]
calcd for C34H49N5O4Si2Na 670.3220, found 670.3261.
Typical Procedure for the Direct N-Arylation of Nucleosides
with Iodoarene 9. Nucleoside (1 mmol), Pd2(dba)3 (0.1 mmol),
xantphos (0.3 mmol), iodoarene 9 (2 mmol), and TEAF (2 mmol)
were dissolved in 3 mL of DMSO and stirred at the temperature
indicated in the Results and Discussion. The reactions were
N6
-2-Nitrobenzen-1-yl-2′-deoxyadenosine (15c): 1H NMR
(DMSO-d6) δ 10.6 (s, 1H), 8.60 (s, 1H), 8.43 (d, J ) 8.3 Hz, 1H),
8.39 (d, J ) 0.5 Hz, 1H), 8.11 (d, J ) 8.3 Hz, 1H), 7.77 (t, J ) 7.4
Hz, 1H), 7.31 (t, J ) 7.7 Hz, 1H), 6.42 (t, J ) 6.8 Hz, 1H), 5.34
(d, J ) 4.1 Hz, 1H), 5.06 (t, J ) 5.6 Hz, 1H), 4.46-4.40 (m, 1H),
3.89 (dd, J ) 7.2 Hz, 4.3 Hz, 1H), 3.66-3.60 (m, 1H), 3.56-3.49
(m, 1H), 2.81-2.72 (m, 1H), 2.33 (dq, J ) 13.2 Hz, 3.2 Hz, 1H);
13C NMR (DMSO-d6) 151.4, 150.8, 149.4, 141.6, 139.7, 134.6,
133.6, 125.3, 124.3, 123.5, 120.8, 87.9, 83.9, 70.7, 61.6; FAB-
HRMS (nba) m/z [M + H+] calcd for C16H17N6O5 373.1260, found
373.1237.
5604 J. Org. Chem., Vol. 71, No. 15, 2006

Preparation of dG-N2 Adducts with Carcinogenic Amino-/Nitroarenes
N6-2-Nitrobenzen-1-yl-2′-deoxycytidine (16c): 1H NMR (DMSO-
d6) δ 9.90 (s, 1H), 7.92 (d, J ) 8.1 Hz, 2H), 7.64 (br s, 2H), 7.30
(br s, 1H), 6.07 (t, J ) 8.1 Hz, 1H), 5.98 (br s, 1H), 5.16 (t, J )
4.1 Hz, 1H), 4.93 (t, J ) 5.1 Hz, 1H), 4.15 (dt, J ) 8.5 Hz, 3.4
Hz, 1H), 3.73 (d, J ) 3.2 Hz, 1H), 3.50 (ddd, J ) 21.7 Hz, 12.0
Hz, 4.8 Hz, 2H), 2.15-2.05 (m, 1H), 1.98-1.87 (m, 1H); 13C NMR
(DMSO-d6) 153.9, 142.7, 141.9, 133.7, 131.5, 126.6, 124.8, 94.8,
87.4, 85.0, 70.2, 61.2, 48.5; FAB-HRMS (nba) m/z [M + Na+]
calcd for C15H16N4O6Na 371.0967, found 373.0968.
Typical Procedure for the Coupling of Nucleotide with 9c.
Nucleotide (0.04 mmol), Pd2(dba)3 (0.02 mmol), xantphos (0.03
mmol), 9c (0.08 mmol), and TEAF (0.08 mmol) were dissolved in
1 mL of DMSO and stirred at 90 °C for 4 h. After the reactions
were completed as judged from the TLC analyses, 5 mL of water
followed by 4 mL of CHCl3 were added to the reaction mixtures.
The aqueous layer was subjected to column chromatography on
octadecyl silica (ODS) and eluted using a step gradient of MeOH
in water. The fractions containing the desired products were
combined, evaporated, and finally dried under high vacuum to
remove traces of the solvent.
3′-O-[(N,N-Diisopropylamino)-2-cyanoethoxyphosphinyl]-5′-
O-(4,4-dimethoxytrityl)-N2-2-nitrobenzen-1-yl-2′-deoxyguano-
sine (19c). A premixed solution of N,N-diisopropylaminotetrazolide
(1.2 equiv) and 2-cyanoethyltetraisopropylphosphorodiamidite (1.2
equiv) was added to a solution of 14c (80 mg) in CH2Cl2 (5 mL).
The reaction was stirred for 2 h, and the solvent was then removed.
The residue was purified by column chromatography on a neutral
silica gel column and eluted with chloroform containing 2%
triethylamine to yield 19c (70%): 1H NMR (CDCl3) δ 10.6 (s,
1H), 8.06 (s, 1H), 7.86 (d, J ) 8.1 Hz, 1H), 7.72 (d, J ) 12.0 Hz,
1H), 7.31 (t, J ) 6.3 Hz, 2H), 7.22-7.07 (m, 7H), 6.88 (br s, 1H),
6.70 (t, J ) 6.3 Hz, 4H), 6.17 (t, J ) 5.4 Hz, 1H), 4.53 (br s, 1H),
4.16 (br s 1H), 3.87-3.44 (m, 10H), 3.30-3.20 (m, 2H), 2.75-
2.25 (m, 4H), 1.25-1.00 (m, 12H); 13C NMR (CDCl3) 158.3, 149.6,
144.3, 135.7, 135.5, 135.4, 134.8, 134.0, 129.9, 128.0, 127.9, 127.8,
127.4, 126.8, 125.2, 123.6, 122.2, 119.5, 117.5, 117.3, 113.0, 86.3,
85.5, 85.3, 83.7, 73.8, 63.6, 58.9, 55.2, 45.6, 43.3, 40.3, 31.6, 24.6,
22.6, 20.2, 14.1, 8.79; 31P NMR (CDCl3) 149.0, 148.6; FAB-HRMS
(nba) m/z [M + H+] calcd for C46H52N8O9P 891.3594, found
891.3452.
Solid-Phase Synthesis of Oligonucleotide 20c. Oligonucleotide
5′-d(GAACC-9c-GAGGCCC) 20c was prepared on a 1 µmol scale
by using general isobutyryl- or benzoyl-protected cyanoethylphos-
phoramidites and modified phosphoramidite 19c. The manufactur-
er’s standard synthesis protocol was followed, except for the
prolonged coupling reaction time of 15 min. The deprotection of
the oligonucleotides was achieved by treatment with concentrated
ammonia and 0.25 M mercaptoethanol at 50 °C for 17 h. The
purification was performed using a Sep-Pak C18 column followed
by HPLC (column, Unison US-C18 (3.0 × 150 mm); column
temperature, 50 °C; eluent, linear gradient of 3%-40% acetonitrile
over 30 min at a flow rate of 0.5 mL/min) to yield 20c (27%,
MALDI-MS analysis: [M - 1] calcd for 20c 4088.72, found
4088.94). Nucleotide digestion yielded 20c along with three normal
nucleotides in the correct stoichiometric ratio. HPLC conditions:
column, Cosmosil AR-II ODS column (4.6 × 250 mm); eluent, 50
mM ammonium acetate gradient of 2%-10% over 18 min, aceto-
nitrile gradient of 10%-100% over 15 min, and isocratic elution
for 10 min.
Typical Procedure of Nitroreduction of 20c. To an aqueous
solution (100 µL) of 21c (10 ng) in 200 mM Na citrate at pH 5.0
was added 3 mg of Zn dust. The reaction was allowed to proceed
for 24 h, and the product was subjected to HPLC under the
following conditions: column, Xterra MS C8 (4.6 × 50 mm);
eluent: linear gradient of 0%-34% CH3OH in 0.25% TEA adjusted
to pH 7.0 with CH3COOH at a flow rate of 0.5 mL/min to yield
21c (63%).
N2-2-Nitrobenzen-1-yl-2′-deoxyguanosine-3′-phosphate (13c):
1H NMR (DMSO-d6) δ 8.12 (d, J ) 8.1 Hz, 1H), 8.01 (s, 1H),
8.00 (d, J ) 6.8 Hz, 1H), 7.70 (t, J ) 7.6 Hz, 1H), 7.19 (t, J ) 7.8
Hz, 1H), 6.01 (t, J ) 6.7 Hz, 1H), 4.75 (br s, 1H), 3.99 (q, J ) 3.3
Hz, 1H), 3.55-3.43 (m, 1H), 3.18 (q, J ) 7.1 Hz, 8H), 2.60-2.52
(m, 1H), 2.45-2.37 (m, 1H), 1.13 (t, J ) 6.6 Hz, 12H); 13C NMR
(DMSO-d6) 158.54, 150.0, 148.7, 139.6, 135.9, 134.4, 133.7, 125.1,
123.9, 122.8, 119.0, 86.5, 82.6, 74.1, 61.8, 51.3, 7.10; 31P NMR
(DMSO-d6) 1.38; FAB-HRMS (nba) m/z [M + H+] calcd for
C16H18N6O9P 469.0872, found 469.0848.
Direct N-Arylation of Oligonucleotide 17 with 9c. In a screw-
capped vessel containing 0.3 µmol of oligonucleotide 17 was added
100 µL of a premixed solution of 9c (1.2 µmol), Pd2(dba)3 (0.3
µmol), xantphos (0.6 µmol), and TEAF (1.2 µmol) in DMSO. The
reaction was allowed to proceed at 90 °C for 1 h until the complete
conversion to 17 was confirmed by HPLC analysis. The reaction
was extracted with CHCl3, and the aqueous layer was subjected to
HPLC under the following conditions: column, Cosmosil ODS AR
II (4.6 × 250 mm); eluent, linear gradient of 0%-40% CH3CN in
0.25% TEA-AcOH over 20 min at a flow rate of 1.0 mL/min to
yield 18c: ESI-MS m/z [M - 2H]2-, 1106.2; [M - 3H]3-, 737.4;
[M - 4H]4-, 553.1.
5′-O-(4,4′-Dimethoxytrityl)-N2-2-nitrobenzen-1-yl-2′-deoxy-
guanosine (14c): Procedure A. Five milliliters of dioxane was
added to a reaction vessel containing 5′-O-(4,4′-dimethoxytrityl)-
2′-deoxyguanosine (0.5 mmol), Pd(OAc)2 (0.05 mmol), Cs2CO3
(1 mmol), xantphos (0.15 mmol), and 9c (1 mmol) The reaction
mixture was stirred for 4 h at 90 °C and evaporated; the residue
was then purified by column chromatography. The fractions
containing the desired products were combined, evaporated, and
finally dried under high vacuum to remove traces of the solvent.
Typical Procedure of Nitroreduction of 9. To a solution of 9
(0.1 mmol) in 2 mL of DMSO/500 mM NaCOOCH3 (1:1) was
added 50 mg of Zn dust. The color of the solution disappeared
immediately. After confirmation of completion of the reaction by
TLC (CHCl3/MeOH ) 5:1), the filtrate was directly subjected to
column chromatography on an ODS column and eluted using a
step gradient of MeOH/water.
Procedure B. To a solution of 12c (0.2 mmol) in pyridine was
added 0.3 mmol of 4,4′-dimethoxytrityl chloride After the reaction
was stirred at room temperature for 4 h, another aliquot of 4,4′-
dimethoxytrityl chloride (0.3 mmol) was added. After a 6 h reaction,
triethylamine (3 mL) and MeOH (3 mL) were added to the solution.
The mixture was then evaporated and applied to a chromatographic
N2-2-Aminobenzen-1-yl-2′-deoxyguanosine (3c): 1H NMR
(DMSO-d6) δ 10.6 (s, 1H), 7.96 (s, 1H), 7.93 (s, 1H), 7.38 (d, J )
7.3 Hz, 1H), 6.91 (t, J ) 7.2 Hz, 1H), 6.76 (d, J ) 7.3 Hz, 1H),
6.60 (t, J ) 7.2 Hz, 1H), 6.04 (t, J ) 7.0 Hz, 1H), 5.23 (d, J ) 3.4
Hz, 1H), 4.94 (s, 2H), 4.83 (t, J ) 4.8 Hz, 1H), 4.26 (br s, 1H),
3.76 (dd, J ) 7.1 Hz, 4.1 Hz, 1H), 3.43 (ddd, J ) 22.6 Hz, 12.0
Hz, 5.0 Hz, 1H), 2.58-2.51 (m, 1H), 2.17 (dq, J ) 13.2 Hz, 2.0,
1H); 13C NMR (DMSO-d6) 156.6, 150.7, 150.0, 142.0, 135.9, 125.5,
125.0, 123.1, 117.6, 116.4, 115.8, 87.6, 82.6, 70.7, 61.7; FAB-
HRMS (nba) m/z [M + H+] calcd for C16H19N6O4 359.1467, found
359.1447.
1
column, affording the desired compound (79%): H NMR (CDCl3)
δ 10.2 (s, 1H), 8.33 (s, 1H), 7.83 (s, 1H), 7.63 (br s, 1H), 7.32-
7.02 (m, 11H), 6.82 (br s, 1H), 6.70 (d, J ) 8.1 Hz, 4H), 6.21 (br
s, 1H), 4.52 (br s, 1H), 4.12 (br s 1H), 3.71 (br s, 1H), 3.64 (s,
6H), 3.31 (br s, 1H), 3.23 (br s, 1H), 2.72-2.45 (m, 2H); 13C NMR
(CDCl3) 158.2, 148.9, 147.9, 144.3, 136.9, 135.5, 135.4, 134.8,
133.8, 129.8, 129.0, 127.9, 127.6, 126.7, 125.4, 122.5, 119.0, 113.0,
86.3, 85.6, 83.8, 72.0, 64.1, 63.6, 40.6, 30.9, 29.2; FAB-HRMS
(nba) m/z [M + H+] calcd for C37H35N6O8 691.2516, found
691.2411.
N2-2-Aminonaphthalen-1-yl-2′-deoxyguanosine (3f): 1H NMR
(DMSO-d6) δ 8.30 (s, 1H), 7.89 (s, 1H), 7.69 (d, 1H), 7.61 (d,
1H), 7.54 (d, 1H), 7.32 (dt, J ) 10.5 Hz, 3.8, 1H), 7.15-7.12 (m,
1H), 7.09 (d, J ) 8.8 Hz, 1H), 5.84 (br s, 1H), 5.41 (s, 2H), 5.09
J. Org. Chem, Vol. 71, No. 15, 2006 5605

Takamura-Enya et al.
(s, 1H), 4.70 (s, 1H), 4.36 (t, J ) 5.0 Hz, 1H), 4.07 (s, 1H), 3.63
(s, 1H), 3.50-3.34 (m, 1H), 3.26 (s, 1H), 2.42-2.35 (m, 1H), 2.05-
1.95 (m, 1H); 13C NMR (DMSO-d6) 156.6, 152.0, 150.5, 143.3,
135.5, 132.3, 127.8, 126.7, 126.3, 121.0, 120.3, 118.8, 117.3, 87.5,
82.2, 70.7, 61.6, 55.9; FAB-HRMS (nba) m/z [M + H+] calcd for
C20H21N6O4 409.1624, found 409.1577.
Supporting Information Available: General experimental
information; 1H NMR and 13C NMR for compounds 11a,b, 12a,c-
e,g, 13c, 14c, 15c, 16c, 3c,e, and 19c; and 31P spectra for 13c and
19c; mass spectra of 17 and 18c; HPLC profiles of the coupling
reaction of 17 yielding 18c and enzymatic hydrolysates of 17 and
18c and nitro reduction of 21 yielding 22c and enzymatic hydroly-
sates of 21 and 22c. This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. This study was supported by a Grant-
in-Aid for Cancer Research from the Ministry of Health, Labour,
and Welfare, Japan.
JO0605243
5606 J. Org. Chem., Vol. 71, No. 15, 2006