Synthesis and properties of 2-azidodeoxyadenosine and its incorporation into oligodeoxynucleotides

Article abstract of DOI:10.1016/S0040-4039(01)02028-7

2-Azidodeoxyadenosine (7) was conveniently synthesized from deoxyguanosine (2) by use of a combined reagent of TMSN₃-BuONO. The structure of the tautomer of the azido derivative was determined by ¹H NMR. Reaction of 7 with iPr₂NP(OEt)₂ gave an intermediate 10 of the Staudinger reaction. Incorporation of 7 into a DNA 13mer resulted in a significant decrease of the T_m value of the DNA duplex upon hybridization with the complementary strand. The thermal stability was discussed based on the hydrogen bond energy and electrostatic repulsion.

Full text of DOI:10.1016/S0040-4039(01)02028-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 9215–9219
Synthesis and properties of 2-azidodeoxyadenosine and its
incorporation into oligodeoxynucleotides
Takeshi Wada, Akira Mochizuki, Seiichiro Higashiya, Hiroyuki Tsuruoka, Shun-ichi Kawahara,
Masahide Ishikawa and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Received 20 September 2001; revised 22 October 2001; accepted 26 October 2001
Abstract—2-Azidodeoxyadenosine (7) was conveniently synthesized from deoxyguanosine (2) by use of a combined reagent of
TMSN3–BuONO. The structure of the tautomer of the azido derivative was determined by 1H NMR. Reaction of 7 with
iPr2NP(OEt)2 gave an intermediate 10 of the Staudinger reaction. Incorporation of 7 into a DNA 13mer resulted in a significant
decrease of the Tm value of the DNA duplex upon hybridization with the complementary strand. The thermal stability was
discussed based on the hydrogen bond energy and electrostatic repulsion. © 2001 Elsevier Science Ltd. All rights reserved.
Oligonucleotides containing a nucleoside derivative
capable of photo-crosslinking have proved to be useful
for determination of the precise site of interaction
We found an effective method for the synthesis of 7
from 2%-deoxyguanosine (2). The reaction of 2 with
hexamethyldisilazane in the presence of a catalytic
between protein–DNR/RNA1 and RNA–RNA.2
A
amount of Me3SiCl in acetonitrile gave
a fully
trimethylsilylated species 3. It turned out that addition
of ethanol to the mixture resulted in 3%,5%-O-
variety of photo-crosslinking reactions using oligonu-
cleotides containing thionucleoside derivatives such as
4-thiouridine and 6-thioguanosine have been reported.3
On the other hand, azido-nucleoside derivatives, which
are expected to be photo-activated, have not been
utilized at the oligonucleotide level at all.4 Only exam-
ples of 6-azidopurine nucleoside 5%-di- and tri-phos-
phate derivatives have been studied in connection with
the mechanism of biological reactions associated with
kinases and ATPases.5
OR
O
N
N
N
N
N
NH
NH2
N
NH2
N
a
TMSO
HO
O
O
OH
OTMS
2
3: R = TMS
4: R = H
b
c
5: R = 2,4,6-trimethyl-
Recently, Higashiya et al. reported a new method for
the synthesis of 2-azidoadenosine (1) by treatment of a
2-amino-6-chloropurine riboside derivative with
TMSN3 in the presence of butyl nitrate.6 This effective
reaction provides a new possibility that 2-azido-2%-
deoxyadenosine could be obtained by a similar reaction
and used as a deoxynucleoside component that can be
incorporated into DNA and activated selectively by
photo-irradiation. Here, we report the synthesis of 2-
azidodeoxyadenosine (7) and its chemical and struc-
tural properties as well as its incorporation into
oligodeoxynucleotides.
benzenesulfonyl
O
NH2
N
N
S
O
O
N
N
d
N
e
5
N
N
N3
HO
O
N
N3
HO
O
OH
OH
6
7
Scheme 1. (a) HMDS (3.0 equiv.), TMSCl (0.05 equiv.),
CH3CN, rt, 1 h; (b) EtOH, rt, 1 h; (c) MsCl (1.3 equiv.), Et3N
(3.0 equiv.), DMAP (0.1 equiv.), CH2Cl2, rt, 48 h; (d) (1)
TMSN3 (10 equiv.), BuONO (10 equiv.), CH2Cl2, −20°C to rt,
24 h, (2) H2O–MeOH (1:1, v/v), rt, 1 h; (e) conc. NH3 (20
equiv.), dioxane, rt, 60 h.
* Corresponding author.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)02028-7

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T. Wada et al. / Tetrahedron Letters 42 (2001) 9215–9219
bis(trimethylsilyl)deoxyguanosine (4). In situ treatment
of 4 with mesitylenesulfonyl chloride gave the 6-O-
mesitylated species 5 in 83% yield (Scheme 1).
min and tcomp=10 min in 1% trifluoracetic acid in
CH2Cl2.
In the current chemical synthesis of DNA, two
approaches are available.10 One is the phosphoramidite
method.11 The other is the H-phosphonate approach.12
Since it is well known that azide compounds easily
react with tervalent phosphorus compounds to give
iminophosphorane derivatives,13 we examined the com-
patibility of 7 with phosphoramidite reagents which
have been used as deoxynucleotide building blocks in
the phosphoramidite approach. As a model compound,
diethyl N,N-diisopropylphosphoramidite (9) was cho-
sen for this study.
The reaction of 5 with 10 equiv. each of trimethylsilyl
azide and butyl nitrite in CH2Cl2 followed by hydrolysis
gave the product 6 in 45% yield. Further treatment of 6
with conc. NH3 gave 7 in 89% yield.
It was reported that 2-azidoadenosine 1a exists in equi-
librium with two tricyclic tautomers 1b and 1c having a
tetrazole ring (Fig. 1).7
1
However, the H NMR spectrum of the deoxy counter-
part 7 suggested that 7 exists in equilibrium between the
actual azide derivative 7a and a tricyclic tetrazole
derivative 7b or 7c. At 20°C the ratio of the two
tautomers was 6:4, as shown in Fig. 2. When the
temperature increased to 100°C, the ratio changed to
9:1. It has been generally recognized that, in the azide–
tetrazole equilibrium, higher temperatures favor the
azide species.8 Therefore, the enriched tautomer was
When compound 7 was allowed to react with 1.5 equiv.
of 9 in DMF–CD3CN (9:1, v/v), a new resonance signal
at 34.0 ppm in the 31P NMR spectrum appeared at the
initial stage, as shown in Fig. 3. After 6 h, this peak
disappeared and a new resonance signal at 13 ppm was
observed as the main peak. The FAB mass of the
mixture was analyzed. Consequently, it was found that
the initial product has a molecular weight of 513 and
the final product has a molecular weight of 485. From
these results, we concluded that the initial product is
the actual intermediate 10 of the Staudinger reaction, as
shown in Scheme 2.13 The final product is the
iminophosphorane type product 11 (Fig. 3).
1
determined to be the azido derivative 7a. The H NMR
spectrum of 7 showed that there is only a slight differ-
ence (0.14 ppm) in the chemical shift between the
anomeric protons of 7a and the teterazole form which
appeared at 6.26 and 6.40 ppm, respectively. This result
suggested that the tetrazole species is the one described
as 7b, since it is expected that, if 7c were present, the
chemical shift of the anomeric proton of 7c should be
significantly shifted to a low-magnetic field compared
with that of 7a because the tetrazole ring approaches
very close to the anomeric proton so that a consider-
able deshielding effect is expected.
NH2
NH2
NH2
N
N
N
N
N
N
R
N
N
X
N
N
N
N
and/or
N
N3
N
N
N
X
N
N
azido-form
tetrazole-form
1a: X = R
1b: X = R
1c: X = R
This conclusion was also supported by the following
theoretical calculation: the ab initio MO calculations of
the tautomers 8a–c of 9-methyl-2-azidoadenine were
carried out at the level of MP2/6-31G*.9 The tetra-
zolo[5,1a]-9-methyladenine derivative 8b is 0.46 kcal/
mol stable than the tetrazolo[5,1,b]-9-methyladenine
derivative 8c.
7a: X = dR
7b: X = dR
7c: X = dR
8a: X = CH3
8b: X = CH3
8c: X = CH3
Figure 1. Tautomerism of 2-azidoadenosine (1a), 2-azido-2%-
deoxyadenosine (7a), and 9-methyl-2-azidoadenine (8a).
The detailed analysis of the thermodynamic parameters
in the equilibrium between the azido form 7a and
1
tetrazole form 7b was carried out by H NMR. The
thermodynamic parameters DH, DS, and DG were cal-
culated to be 3.89, 1.44×10−2, and −0.44 kcal/mol
(27°C).
To test if 2-azidodeoxyadenosine can be incorporated
into DNA, several chemical properties of this com-
pound were studied. It was found that 7 was stable
under basic conditions such as conc. NH3–pyridine
(9:1, v/v) at room temperature for 48 h and conc.
NH3–EtOH (3:1, v/v) at room temperature for 48 h. On
the other hand, the glycosidic bond of 7 was found to
be considerably stable under acidic conditions: (1) t1/2
=
4 h, tcomp=24 h in 3% dichloroacetic acid in CH2Cl2;
(2) t1/2=1 h, tcomp=8 h in 1% trifluoroacetic acid in
CH2Cl2. Compared with these results, N-benzoyldeoxy-
Figure 2. 1H NMR spectra of a tautomeric mixture of 2-
azido-2%-deoxyadenosine 7a and its tetrazole derivative 7b at
various temperatures in DMSO-d6.
cytidine showed less stability of t1/2 <5 min and tcomp
=
30 min in 3% dichloroacetic acid in CH2Cl2 and t1/2 <5

T. Wada et al. / Tetrahedron Letters 42 (2001) 9215–9219
9217
Scheme 2. Reaction of 2-azidodeoxyadenosine (7) with
diethyl N,N-diisopropylphosphoramidite (9) in DMF–
CD3CN (9:1, v/v).
Figure 3. 31P NMR analysis of the reaction of 2-azido-
deoxyadenosine (7) with diethyl N,N-diisopropylphospho-
ramidite (9) in DMF–CD3CN (9:1, v/v).
From the viewpoint of the synthesis of oligodeoxynu-
cleotides incorporating 7, these results indicated that it
is impossible to synthesize 2%-azidodeoxyadenosine 3%-
phosphoramidite derivatives required for the standard
phosphoramidite approach. On the other hand, the
H-phosphonate method involves the use of pentavalent
H-phosphonate building blocks. Quite recently, we
have demonstrated that the 2-azidobenzoyl group can
be used as the base protecting group that is compatible
with the H-phosphonate approach.14 Therefore, we
tried to synthesize 2-azido-2%-deoxyadenosine 3%-H-
phosphonate building block 13 (Scheme 3).
NH2
NH2
N
N
N
N
N
N
N
N3
a
N
N3
DMTrO
HO
O
O
OH
12
OH
7
NH2
N
N
N
O
b
N
N3
DMTrO
Reaction of 7 with 4,4%-dimethoxytrityl chloride in pyr-
idine gave the 5%-O-tritylated product 12 in 84% yield.
Reaction of 12 with 7 equiv. of diphenyl phosphonate
in pyridine followed by hydrolysis gave the H-phospho-
nate building block 13 in 77% yield without damage to
the azide function.
O
H
O
P
O-TEAH+
13
Scheme 3. Synthesis of 2-azidodeoxyadenosine 3%-H-phos-
phonate unit. (a) DMTrCl (1.2 equiv.), pyridine, rt, 1 h; (b)
diphenyl phosphonate (7 equiv.), pyridine, rt, 1.5 h; (c) H2O–
Et3N (1:1, v/v), rt, 1 h.
Condensation of 13 with 3%-O-acetylthymidine (14) in
the presence of BOP-Cl15 (2 equiv.) in pyridine at room
temperature for 1.5 h gave the H-phosphonate dimer
15 in 88% yield, as shown in Scheme 4. This product
was oxidized by I2 treatment, and the protecting groups
were removed by successive treatments with 80% AcOH
and conc. NH3–pyridine. Thus, the fully deprotected
dimer 16 was obtained in an overall yield of 43%. This
product showed a characteristic peak (2155 cm−1) of the
azido group in its IR spectrum and a single resonance
peak at −0.38 ppm in its 31P NMR spectrum. The
1
structure of the dimer was also confirmed by H and
13C NMR (Fig. 4).
These results strongly suggested that it is possible to
synthesize oligodeoxynucleotides containing 7 by the
H-phosphonate method. Therefore, we synthesized
d(T6AN3T6) on a T-loaded CPG gel support by the
H-phosphonate method using a powerful condensing
reagent BOMP.16 This modified 13mer was isolated in
22% yield by anion exchange HPLC. Hybridization of
the 13mer with the complementary d(A6TA6) resulted
in a sharp drop (DTm=−7.4°C) of the Tm value down
Scheme 4. Synthesis of 2-azidodeoxyadenosine 3%-H-phos-
phonate unit 13 and 2-azido-2%-deoxyadenyl(5%-3%)thymidine
(16). (a) BOP-Cl (2.0 equiv.), pyridine, rt, 1.5; (b) (1) 2% I2,
pyridine–H2O (98:2, v/v), rt, 1 h, (2) conc. NH3–pyridine (9:1,
v/v), rt, 1 h.

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T. Wada et al. / Tetrahedron Letters 42 (2001) 9215–9219
tives would be generally possible by use of the H-phos-
phonate method. RNAs containing its ribonucleoside
counterpart would be more important in molecular
biology since much attention has been paid to clarifica-
tion of RNA–protein interaction.2 The chemical synthe-
sis of such modified RNAs would be realized in the
near future using the present method. During this
study, we were also able to detect the intermediate of
the Staudinger reaction. This observation is noteworthy
in phosphorus chemistry.
Acknowledgements
Figure 4. Melting temperature curve of d(T6AT6)/d(A6TA6)
duplex. Conditions: 10 mM NaHPO4, 1 M NaCl, 0.1 mM
EDTA (pH 7.9), conc. of an oligonucleotide 2.0 mM.
This work was supported by a Grant from ‘Research
for the Future’ Program of the Japan Society for the
Promotion of Science (JSPS-RFTF97I00301) and a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan.
to 33.7°C compared with the normal DNA duplex
d(T6AT6)/d(A6TA6), which had a Tm value of 41.1°C.
This destabilization reflects formation of an unfavor-
able base pair between T and AN3. Theoretically, we
also calculated the hydrogen bond energy DE of this
unfavored base pair at the level of MP2/6-31G*9 using
2-azido-9-methyladenine and 1-methylthymine. As a
result, the hydrogen bond energy was calculated to be
−10.63 kcal/mol. The hydrogen bond energy of 9-
methyladenine and 1-methylthymine was also calcu-
lated to be −12.50 kcal/mol. Therefore, the effect of the
2-azido group on the destabilization of the hydrogen
bonding is estimated to be 1.87 kcal/mol. Moreover,
the electron density of the nitrogen attached to C2 was
estimated to be −0.515e. Even if the sterically favorable
2-azidoadenine base can form a base pair with the
thymine base, this local charge is responsible for the
considerable destabilization of the modified base pair
since there must be electrostatic repulsion between this
nitrogen and the carbonyl oxygen of the 1-
methylthymine. If this is not the case, it is obvious that
the destabilization observed is essentially due to the
presence of the tricyclic tetrazole form like 7b that
cannot form a base pair with the thymine base (Fig. 5).
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H N
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