A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

Article abstract of DOI:10.1016/j.bmc.2012.10.035

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4, 5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN ^N nucleoside unit possessing tribenzoyl gr

Full text of DOI:10.1016/j.bmc.2012.10.035

Bioorganic & Medicinal Chemistry 20 (2012) 7095–7100
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A practical post-modification synthesis of oligodeoxynucleotides containing
4,7-diaminoimidazo[50,40:4,5]pyrido[2,3-d]pyrimidine nucleoside
Noriko Tarashima a, Yosuke Higuchi a, Yasuo Komatsu b, Noriaki Minakawa a,
⇑
a Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1-78-1, Tokushima 770-8505, Japan
b Institute for Biological Resources and Functions and Fellow Research Group, National Institute of Advanced Industrial Science and Technology (AIST, Hokkaido),
2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing
4,7-diaminoimidazo[50,40:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImNN). Since the ImNN nucleoside unit
possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable
under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the
conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification
synthesis of the desired ODNs containing the ImNN unit. Starting with protected 4-amino-7-chloro-1-
Received 11 September 2012
Revised 30 September 2012
Accepted 1 October 2012
Available online 29 October 2012
Keywords:
Nucleoside
(2-deoxy-b-D
-ribofuranosyl)imidazo[50,40:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the
corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best
protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification syn-
thesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support
was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form
the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs
containing ImNN were obtained in good yield.
Imidazopyridopyrimidine
Oligodeoxynucleotide
Post-modification synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
base pair was selectively recognized as a complementary base by
DNA polymerases due to the four H-bonds and the shape comple-
Thus far, a large number of chemically modified nucleoside deriv-
atives have been incorporated into oligodeoxynucleotides (ODNs)
with the aim of biological, bioengineering, and therapeutic applica-
tions. However, the desired ODN synthesis has sometimes failed be-
cause of instability of modified nucleoside units in ODN throughout
the conditions of solid-phase synthesis. If this is the case, a post-
modification synthesis would be an effective alternative approach
to prepare the desired ODNs.1–3 In this method, a labile structure
is substituted to the stable form, which is converted into the desired
structure in the last stage of ODN synthesis.4
Among our studies, we have been investigating the design and
synthesis of artificial base-pairing motifs beyond the Watson–Crick
base pairs.5–10 We had previously reported the synthesis of a series
of nucleoside derivatives possessing an imidazo[50,40:4,5]pyr-
ido[2,3-d]pyrimidine (Im) and a 1,8-naphthyridine (Na) moieties,
which were expected to form base pairs (a series of Im:Na pairs)
with four hydrogen bonds (H-bonds) when they were introduced
into ODNs. In fact, an ImON:NaNO pair (Fig. 1A) in an ODN, for exam-
ple, markedly stabilized DNA duplexes thermally and thermody-
namically.6 Furthermore, we demonstrated that the ImON:NaNO
mentarity of the pair with the purine:pyrimidine base pair.11,12
Quite recently, we designed a new Im:Na pair, that is, the
ImNN:NaOO pair (Fig. 1B) consisting of a DAAD:ADDA H-bonding
pattern (D = donor and A = acceptor), and demonstrated the
ImNN:NaOO pair was more thermally and thermodynamically sta-
ble because of a favorable secondary interaction due to the arrange-
ment of H-bonding pattern.10 In order to utilize this artificial base
pair for a variety of applications, including a study of enzymatic rec-
ognition by DNA polymarases and structural analysis by NMR and
X-ray, preparation of more ODNs containing ImNN and NaOO was
required. However, the overall yield of ODN synthesis containing
A
B
H
H
OA D H N
N HD A O
N A DH N
N A DH N
N HD A O
N
N
A N
N HD
N
N
N A DH N
N HD A O
N
N
H
H
ImON:NaNO pair
ImNN:NaOO pair
⇑
Corresponding author. Tel./fax: +81 88 633 7288.
E-mail address: minakawa@tokushima-u.ac.jp (N. Minakawa).
Figure 1. Structures of Im:Na base pairs.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.035

7096
N. Tarashima et al. / Bioorg. Med. Chem. 20 (2012) 7095–7100
ImNN was quite low, unlike that of ODN synthesis containing other
Im and Na nucleoside derivatives including NaOO.
In this work, we investigated why incorporation of ImNN unit
into ODN resulted in low yield, and found that multiple electron-
withdrawing groups such as benzoyl group on the nucleobase
accelerate cleavage of the glycosidic linkage. Accordingly, we
developed an efficient post-modification synthesis of ODN contain-
ing ImNN. Herein, we present the experimental details of the re-
sults of this research.
2. Results and discussion
As described in our previous papers, we prepared ODNs con-
taining ImNN using a phosphoramidite unit 5 (Scheme 1). In accor-
dance with our previous method,5 we planned to prepare a new
30-mer ODN containing an ImNN unit (50-GTGGGCAAGImNNGTGC-
GCTGACCATCCAGAAC-30). After carrying out the usual procedure
for ODN synthesis, followed by C-18 cartridge column purification,
the resulting solution was analyzed by reversed-phase high-per-
formance liquid chromatography (HPLC) using a C-18 column. As
a result, this analysis afforded a complex HPLC profile (Fig. 2A),
distinct from the case of ODN synthesis containing NaOO, and iso-
lation yield of the desired ODN was quite low (the peak corre-
sponding to the desired 30-mer ODN is marked by asterisk). In
the solid-phase synthesis of ODN using DNA synthesizer, the elon-
gated ODN on controlled pore glass (CPG) support is exposed by
acidic (3% trichloroacetic acid (TCA) in CH2Cl2), coupling (1H-tetra-
zole in CH3CN), capping (Ac2O in THF/pyridine), and oxidation
(0.02 M I2 in THF/H2O/pyridine) conditions until the desired length
is reached. Among these conditions, it is known that the acidic con-
ditions often cause a cleavage of the glycosidic linkage, especially
in purine nucleoside units.13 Since the phosphoramidite unit 5
has three benzoyl groups on its nucleobase moiety, the cleavage
of the glycosidic linkage under the acidic conditions was suspected.
To confirm this assumption, UV spectra of 4 in CH2Cl2 (neutral) and
3% TCA in CH2Cl2 (acidic) solutions were compared. Consequently,
the resulting two UV spectra had no inflection point (compare the
spectra 1 and 2), which suggested a structural change other than
protonation on its nucleobases, that is, the cleavage of the
glycosidic linkage of 4 had occurred. Since the spectrum under
acidic conditions did not change even after 1 h (compare the
spectra 2 and 3), this undesired cleavage was presumed to occur
Figure 2. HPLC profiles of ODN synthesis (A) using phosphoramidite 5; (B) using 9,
after C-18 cartridge column purification. The peak marked with asterisk is the
desired 30-mer.
immediately under the acidic conditions (Fig. 3A).14 On the other
hand, UV spectra of unprotected ImNN derivative 3 under the neu-
tral and acidic conditions had an inflection point around 340 nm
(compare the spectra 1 and 2), suggesting the structural change
from neutral to protonation form. The spectrum under the acidic
conditions then gradually changed (compare the spectra 2 and
3), arising from the cleavage of the glycosidic linkage. (Fig. 3B,
the inflection point of this alteration was observed at 360 nm).14
These results suggested that the ImNN derivative possessing tribe-
nzoyl groups on its exocyclic amino groups was labile, giving aba-
sic site under the acidic conditions arising from a protonation of
the N3 position (Scheme 2). To stabilize its glycosidic linkage, we
examined preparation of other protected ImNN units, such as
dibenzoate and dibutylaminomethylene derivatives. However,
none of the attempts afforded the desired compounds in good yield
(data not shown).
As described in the Introduction, the post-modification synthe-
sis is an effective alternative approach for ODN synthesis. Accord-
ingly, we planned to adopt this approach for ODN synthesis
containing the ImNN. As shown in Scheme 1, ImNN derivative 3
was prepared via 4-amino-7-chloro derivative 15 by treatment
with a mixture of ammonium hydroxide–1,4 dioxane (1:1) at
NBz2
NBz2
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
a
N
a
a
N
DMTrO
NC
N
N
N
TIPSO
TIPSO
RO
O
P
O
4
O
3
O
N
NHBz
N
NHBz
N
NH2
N
Cl
O
O
TIPSO
TIPSO
RO
NiPr2
1: R = TIPS
2: R = H
b
5
c
NH(pBrBz)
NH(pBrBz)
NH(pBrBz)
NH(pBrBz)
N
N
N
N
N
N
N
N
N
N
N
N
N
N
e
f
DMTrO
DMTrO
NC
RO
O
8
O
P
O
N
H
Cl–
N
N
N
Cl
Cl
N
Cl
N
N
HO
O
RO
NiPr2
6: R = TIPS
10
d
O
7: R = H
9
Scheme 1. Reagents: (a) Ref. 5; (b) NH4F in MeOH, reflux; (c) p-BrBzCl, Hunig’s base in CH2Cl2, then NaOEt in EtOH–THF (1:1); (d) TBAF in THF; (e) DMTrCl, Et3N in DMF; (f) 2-
cyanoetyl N,N-diisopropylchlorophosphoramidite, iPr2NEt, DMAP in CH2Cl2.

N. Tarashima et al. / Bioorg. Med. Chem. 20 (2012) 7095–7100
7097
Figure 3. UV spectra of (A) compound 4; (B) compound 3; (C) compound 6, in CH2Cl2 (neutral) and 3% TCA in CH2Cl2 (acidic). Spectra 1 were UV absorption under neutral
conditions, and 2 (0 sec) and 3 (after 1800 or 3600 s) were those under acidic conditions. The inflection points were marked with asterisk.
Bz
N
Bz
Bz
Bz
5'
N
N
H
5'
N
3% TCA
CH2Cl2
5'
N
N
O
O
P
OH
3'
N
N
decomposition
N
N
O
O
O
P
O
P
Bz
Bz
O
O
N
N
H
O
O
N
N
H
Bz
N
Bz
N
NC
O
O
O
O
O
O
O
O
H
N
N
NC
NC
N
3'
3'
Bz
N
N
H
Scheme 2. Proposed mechanism of the cleavage of glycosidic linkage.
100 °C. If this nucleophilic substitution occurs under milder condi-
tions, a 4-amino-7-chloroimidazopyridopyrimidine skeleton like 1
would be a suitable structure for the post-modification synthesis.
To confirm this strategy, compound 2 prepared form 1 was treated
with ammonium hydroxide at 55 °C for 17 hr, which are the usual
conditions for ODN synthesis. As a result, complete conversion of 2
into the corresponding ImNN derivative was confirmed by HPLC
analysis (see the Supplementary data,Fig. S2). Since the alternative

7098
N. Tarashima et al. / Bioorg. Med. Chem. 20 (2012) 7095–7100
route has been suggested, protection of the excyclic amino group
on 4-position of 1 with a benzoyl group was examined. However,
the resulting monobenzoate was a rather polar compound com-
pared with the corresponding tribenzoate derivative, and thus this
protecting group was unsuitable for further conversion, especially
in the chromatographic purification of the corresponding phospho-
ramidite unit. In order to decrease the polarity and to ease the
chromatographic purification of the resulting compounds, a p-bro-
mobenzoyl (p-BrBz) group was chosen as the protecting group. The
desired protection functioned well when 1 was treated with p-bro-
mobenzoyl chloride in CH2Cl2 in the presence of Hunig’s base,15
followed by NaOEt in a mixture of EtOH and THF (1:1) to give
monoacylated product 6 in 74% yield by two steps. The resulting
6 was then converted into the free nucleoside 7 by treatment with
tetrabutylammonium fluoride in THF. When 7 was treated with
dimethoxytrityl chloride (DMTrCl) in pyridine,15 a polar spot was
observed in TLC analysis and no desired 8 was obtained. The result-
ing polar compound had no signal corresponding to the sugar por-
tion, and the 1H NMR analysis indicated this could be 7-pyridinium
derivative 10 (see the Supplementary data, Fig. S3). Since pyridine
was found to be unsuitable as reaction solvent, dimethoxytrityla-
tion of 7 was carried out in DMF in the presence of triethylamine16
to give 8 in 78% yield. Treatment of 8 with N,N-diisopropylchloro-
phosphoramidite in CH2Cl2 in the presence of Hunig’s base affor-
ded the phosphoramidite unit 9.
Prior to the ODN synthesis, stability of the glycosidic linkage was
evaluated using 6 by monitoring of UV absorption. As can be seen in
Figure 3C, the UV spectra of 6 under the neutral and the acidic con-
ditions had the inflection point around 320 nm (compare the spec-
tra 1 and 2), and the spectrum under the acidic conditions gradually
changed (compare the spectra 2 and 3), which suggested the cleav-
age of the glycosidic linkage (the inflection point of this alteration
was observed at 340 nm).14 The velocity of the cleavage reaction
was rather slow, and the t1/2 of 6 was estimated to be 350 sec, which
was longer than not only that of 4 (t1/2 = <30 s) but also that of sily-
lated N-benzoyladenine (t1/2 = 300 s). This result indicates that the
4-amino-7-chloroimidazopyridopyrimidine nucleoside unit pos-
sessing a p-BrBz group such as 9 in ODN can tolerate ODN synthesis.
Once the conditions had been fixed, synthesis of the desired 30
mer was tried again. After carrying out the usual procedure for
ODN synthesis using the phosphoramidite 9, the ODN supported
on CPG was treated with ammonium hydroxide at 55 °C for 17 h,
and the reaction mixture after C-18 cartridge column purification
was analyzed by HPLC using a C-18 column. As can be seen in Fig-
ure 2B, the HPLC profile of the mixture was quite simple, distinct
from the previous experiment ( Fig. 2A), giving the desired se-
quence as a major product. Further purification of the peak corre-
sponding to the desired sequence was carried out to give pure
ODN. To confirm the nucleoside composition, the resulting ODN
was treated with a mixture of nuclease P1, snake venom phospho-
diesterase, and alkaline phosphatase, and the reaction mixture was
analyzed by HPLC. The peak corresponding to the ImNN, confirmed
by co-elution with an authentic sample, was clearly observed, and
the nucleoside composition calculated from the areas of the peaks
supported the sequence of ODN (Fig. 4). Since no peak correspond-
ing to 2 was observed in the HPLC profile, this post-modification
synthesis via nucleophilic substitution of 7-chloro group into 7-
amino group worked well. Using this synthetic method, we tried
the synthesis of another sequence (50-CGAAImNNAACC-30) for
structural analysis by NMR study. This attempt also afforded the
desired 9-mer in good yield.
In conclusion, we investigated why incorporation of ImNN unit
in ODN does not result in high yield, and revealed that the cause
arises from the instability of the glycosidic linkage of ImNN deriv-
ative possessing tribenzoyl groups on its exocyclic amino groups
under acidic conditions. Accordingly, post-modification synthesis
of the desired ODN was examined as an alternative approach.
Starting with 4-amino-7-chloro derivative 1, which is a precursor
of 4,7-diamino derivative, conversion into the corresponding phos-
phoramidite unit was examined. The p-BrBz group was the best
protecting group of 4-amino group of 1 for further chemical con-
version. Since dimethoxytritylation of 7 in pyridine afforded unex-
pected 10, compound 8 was prepared by treatment of 7 with
DMTrCl in DMF in the presence of triethylamine. The resulting 8
was then converted into the phosphoramidite unit 9 for the post-
modification synthesis. After carrying out the ODN synthesis linked
to the CPG support, the support was treated with ammonium
hydroxide at 55 °C for 17 hr to remove the protecting groups, de-
tach the ODN form the CPG support, and convert 7-chloro group
into the amino group. As a result, the desired ODNs containing
ImNN were obtained in good yield. Using these ODNs, a study of
enzymatic recognition by DNA polymarases and structural analysis
of the ImNN:NaOO pair is now under investigation. The results will
be reported in due course.
3. Experimental section
3.1. General methods
Physical data were measured as follows: Melting points are
uncorrected. 1H and 13C NMR spectra were recorded at 400 or
500 MHz and 100 or 125 MHz instruments (Bruker FT-NMR
AV400 or AV500) in CDCl3 or DMSO-d6 as the solvent with tetra-
methylsilane as an internal standard. Chemical shifts are reported
in parts per million (d), and signals are expressed as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), or br (broad). All
exchangeable protons were detected by addition of D2O. TLC
was done on Merck Kieselgel F254 precoated plates. Silica gel
used for column chromatography was KANTO CHEMICAL Silica
Gel 60 (spherical) 63–210 or Silica Gel 60 N (spherical, neutral)
63–210.
3.2. 4-Amino-7-chloro-1-(2-deoxy-b-
D-ribofuranosyl)imidazo-
[50,40:4,5]pyrido[2,3-d]pyrimidine (2)
A solution of 13 (97 mg, 0.15 mmol) in MeOH (5 mL) containing
ammonium fluoride (56 mg, 1.5 mmol) was heated under reflux for
50 h. The reaction mixture was cooled and concentrated in vacuo.
The residue was purified by a short silica gel column, eluted with
MeOH in CHCl3 (10–40%), to give 2 (36 mg, 72%) as a white solid:
1H NMR (DMSO-d6) d 9.25 and 8.64 (each s, each 1H), 8.02 (br s,
2H, exchangeable with D2O), 6.64 (dd, 1H, J = 5.5 Hz), 5.43 (m,
1H, exchangeable with D2O), 4.98 (m, 1H, exchangeable with
D2O), 4.40 (m, 1H), 3.96 (m, 1H), 3.51 (dd, 1H, J = 4.0, 12.0 Hz),
3.43 (dd, 1H, J = 4.5, 12.0 Hz), 2.79 (m, 1H), 2.52 (m, 1H); 13C
NMR (DMSO-d6) d 159.34, 157.78, 157.41, 154.37, 141.04, 131.05,
128.42, 107.03, 88.15, 85.89, 69.82, 60.90; ESIMS-LR m/z = 359
Figure 4. HPLC profile of the 30-mer ODN after complete hydrolysis.

N. Tarashima et al. / Bioorg. Med. Chem. 20 (2012) 7095–7100
7099
(MNa+); ESIMS-HR Calcd for C13H13ClN6O3Na 359.0635, found
359.0660.
(704 mg, 78%) as a white solid: 1H NMR (DMSO-d6) d 11.46 (s,
1H, exchangeable with D2O), 9.97 and 8.87 (each s, each 1H),
7.92 and 7.73 (each d, each 2H, J = 8.3 Hz), 7.11–6.58 (m, 13 H),
6.56 (m, 1 H), 5.50 (d, 1H, J = 5.8 Hz, exchangeable with D2O),
4.54 (m, 1H), 4.08 (m, 1H), 3.65 (s, 6H), 3.25 (m, 1H), 3.13 (dd,
1H, J = 2.5, 10.6 Hz), 2.78 (dd, 1H, J = 4.7, 10.6 Hz), 2.63 (m, 1H);
13C NMR (DMSO-d6) d 165.31, 158.48, 157.84, 157.81, 157.48,
156.18, 152.88, 144.50, 141.95, 135.28, 135.20, 133.89, 132.71,
132.31, 131.50, 130.61, 129.32, 129.27, 129.18, 127.56, 127.50,
127.31, 126.45, 112.83, 108.52, 85.80, 85.25, 84.92, 79.15, 68.90,
62.06, 54.89, 37.88; ESIMS-LR m/z = 844 (MNa+); ESIMS-HR Calcd
for C41H34BrClN6O6 843.1278, found 843.1309.
3.3. N4-p-Bromobenzoylamino-7-chloro-1-(2-deoxy-3,5-di-O-
triisopropylsilyl-b-
d]pyrimidine (6)
D
-ribofuranosyl)imidazo[50,40:4,5]pyrido[2,3-
To a solution of 1 (1.07 g, 1.6 mmol) in CH2Cl2 (16 mL) including
N,N-diisopropylethylamine (1.66 mL, 9.8 mmol) was added p-
BrBzCl (1.07 g, 4.9 mmol), and the reaction mixture was stirred
for 12 h at room temperature. The reaction was quenched by addi-
tion of ice and the reaction mixture was partitioned between CHCl3
and H2O. The separated organic layer was washed with saturated
aqueous NaHCO3 and brine. The organic layer was dried (Na2SO4)
and concentrated in vacuo. Then, the residue was dissolved in a
mixture of EtOH–THF (32 mL each) and NaOEt (2.66 mL of 20%
EtOH solution) was added at 0 °C. After being stirred for 10 min,
the reaction mixture was neutralized by 1 N HCl. The solvent
was removed in vacuo, and the residue was partitioned between
CHCl3 and H2O. The separated organic layer was washed with sat-
urated aqueous NaHCO3 and brine. The organic layer was dried
(Na2SO4) and concentrated in vacuo, and the residue was purified
by a silica gel column (neutralized), eluted with MeOH in CHCl3
(0–5%), to give 6 (1.07 g, 80%) as a white solid: 1H NMR (DMSO-
d6) d 11.45 (s, 1H, exchangeable with D2O), 9.77 and 8.70 (each s,
each 1H), 7.98 and 7.77 (each d, each 2H, J = 8.5 Hz), 6.87 (dd,
1H, J = 5.3 Hz), 4.76 (m, 1H), 4.09 (m, 1H), 3.78 (dd, 1H, J = 3.8,
11.4 Hz), 3.65 (dd, 1H, J = 3.9, 11.4 Hz), 3.15 (m, 1H), 2.69 (ddd,
1H, J = 5.9, 13.3 Hz), 1.12–0.97 (m, 42H); 13C NMR (DMSO-d6) d
165.19, 158.08, 157.35, 156.07, 152.86, 141.65, 133.83, 132.68,
132.37, 131.55, 130.62, 126.47, 108.53, 87.87, 85.56, 70.71, 62.07,
17.83, 17.51, 11.56, 11.16; ESIMS-LR m/z = 853 (MNa+); ESIMS-HR
Calcd for C38H56BrClN6O4Si2Na 853.2671, found 853.2660.
Physical data for N4-p-Bromobenzoylamino-7-pyridiniumimi-
dazo[50,40:4,5]pyrido[2,3-d]pyrimidine chiloride (10): 1H NMR
(DMSO-d6) d 12.16 (br s, 1H, exchangeable with D2O), 10.28 and
8.78 (each s, each 1H), 10.18 (d, 2H, J = 5.7 Hz), 8.96 (t, 1H,
J = 7.5 Hz), 8.45 (m, 2 H), 8.10 and 8.82 (each d, each 2H).
3.6. N4-p-Bromobenzoylamino-7-chloro-1-[2-deoxy-5-O-(4,40-
dimethoxytrityl)-b-D
-ribofuranosyl]imidazo[50,40:4,5]pyrido-
[2,3-d]pyrimidine 2-cyanoethyl N,N0-diisopropylphospho-
ramidite (9)
To a mixture of 8 (205 mg, 0.25 mmol), N,N-diisopropylethyl-
amine (174
CH2Cl2 (5 mL) was added 2-cyanoethyl N,N-diisopropylchlorophos-
phoramidite (61 L, 0.28 mmol) at 0 °C. After the mixture was stir-
lL, 1.0 mmol), and dimethylaminopyridine (5 mg) in
l
red for 1 h at room temperature, the mixture was diluted with CHCl3.
The mixture was washed with saturated aqueous NaHCO3, followed
by brine. The separated organic layer was dried (Na2SO4), and con-
centrated in vacuo. The residue was purified by a silica gel column
(neutralized), eluted with hezane/AcOEt (4:1 to 1:2), to give 9
(148 mg, 58%) as a white foam: ESIMS-LR m/z 1044 (MNa+);
ESIMS-HR Calcd. for
C50H51BrClN8O7PNa 1045.2367, found
3.4. N4-p-Bromobenzoylamino-7-chloro-1-(2-deoxy-b-
D-
1045.2405; 31P NMR (CDCl3) d: 149.63, 149.07.
ribofuranosyl)imidazo[50,40:4,5]pyrido[2,3-d]pyrimidine (7)
3.7. Synthesis and characterization of ODNs containing ImNN
using the post-modification method
To a solution of 6 (400 mg, 0.48 mmol) in THF was added TBAF
(1 M in THF, 1.0 mL, 1 mmol) at 0 °C. After being stirred for 40 min
at room temperature, the the reaction was quenched by addition of
ODNs were synthesized on a DNA synthesizer (NTS H-6, Nihon
Techno Service) using the phosphoramidite unit 9, and commer-
cially available deoxyribonucoside phosphoramidite units (benzoyl
protected dA, acetyl protected dC, and isobutyryl protected dG) at
acetic acid (60 lL, 1.0 mmol). The solvent was removed in vacuo,
and the residue was purified by a silica gel column, eluted with
MeOH in CHCl3 (0–15%), to give 7 (250 mg, quant) as a white solid:
1H NMR (DMSO-d6) d 11.47 (br s, 1H, exchangeable with D2O), 9.75
and 8.82 (each s, each 1H), 7.99 and 7.77 (each d, each 2H,
J = 8.5 Hz), 6.80 (m, 1H), 5.46 (d, 1H, J = 5.0 Hz, exchangeable with
D2O), 4.88 (t, 1H, J = 5.2 Hz, exchangeable with D2O), 4.46 (m, 1H),
3.99 (m, 1H), 3.53 (ddd, 1 H, J = 4.3, 4.9, 11.8 Hz), 3.45 (m, 1 H), 2.90
(m, 1H), 2.58 (m, 1H); 13C NMR (DMSO-d6) d 157.34, 142.23,
131.55, 130.68, 126.46, 88.20, 85.98, 69.56, 60.66; ESIMS-LR m/
z = 540 (MH+); ESIMS-HR Calcd for C20H16BrClN6O4 541.0003,
found 540.9855.
0.2 lmol scale following the standard procedure. For the incorpo-
ration of 9 into the ODNs, a 0.11 M solution in dry CH3CN, and cou-
pling time of 10 min was used. After completion of synthesis, the
CPG support was then treated with concentrated NH4OH (2 mL)
at 55 °C for 17 h, and 0.2 M TEAA buffer (2 mL, pH 7.0) was added
to the reaction mixture. The whole mixture was pored onto a C-18
cartrige column (YMC dispoSPE, f 0.6 ꢀ 1.6 cm), and the column
was washed with 10% CH3CN in 0.1 M TEAA buffer (pH 7.0)
(2 mL), followed by H2O (3 mL). Then, a solution of 3% aq TFA
(4 mL) was eluted to remove the DMTr group. After being washed
with H2O (3 mL), 0.1 M TEAA buffer (2 mL), and H2O (3 mL), the
ODN was eluted with 20% and 50% aq CH3CN (3 mL each). An ali-
quot of eluate was analyzed by reverse-phase HPLC, using a
J’sphere ODN M80 column (4.6 ꢀ 150 mm, YMC) with linear gradi-
ent of acetonitrile (from 5 to 20% over 25 min) in 0.1 M TEAA buffer
(pH 7.0). The resulting ODN was further purified by reverse-phase
HPLC under the same conditions to give a highly purified ODNs.
The structure of ODNs containing ImNN were confirmed by MAL-
DI–TOF/MS: 50-GTGGGCAAGImNNGTGCGCTGACCATCCAGAAC-30,
calculated mass: C295H367N124O174P29 9331.0 (MHꢁ), observed
mass: 9336.2; 50-CGAAImNNAACC-30, calculated mass: C90H110N41
O47P8 2764.54 (MHꢁ), observed mass: 2766.52.
3.5. N4-p-Bromobenzoylamino-7-chloro-1-[2-deoxy-5-O-(4,40-
dimethoxytrityl)-b-
[2,3-d]pyrimidine (8)
D
-ribofuranosyl]imidazo[50,40:4,5]pyrido-
To a solution of 7 (571 mg, 1.1 mmol) in DMF (10 mL) contain-
ing triethylamine (0.91 mL, 6.6 mmol) was added DMTrCl (1.11 g,
3.3 mmol), and the reaction mixture was stirred for 22 h at room
temperuture. The reaction was quenched by addition of ice and
the reaction mixture was partitioned between CHCl3 and H2O.
The separated organic layer was washed with saturated aqueous
NaHCO3 and brine. The organic layer was dried (Na2SO4) and con-
centrated in vacuo, and the residue was purified by a silica gel col-
umn (neutralized), eluted with MeOH in CHCl3 (0–13%), to give 8

7100
N. Tarashima et al. / Bioorg. Med. Chem. 20 (2012) 7095–7100
The former sequence was further confirmed by complete hydro-
Supplementary data
lysis to give nucleoside units. The purified 30-mer ODN (0.25 OD
units at 260 nm) was incubated with snake venom phosphodies-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.035.
terase (6
lL, 68 Unit/mL), nuclease P1 (10
l
L, 0.5 U/mL), and
alkaline phosphatase (5
l
L, 0.1 U/mL) in
a buffer containing
100 mM Tris-HCl (pH 7.6) and 2 mM MgCl2 (total 500 lL) at
References and notes
37 °C for 18 h. Hyperchromicity of each ODN was determined by
comparing UV absorbencies at 260 nm of the solutions before
and after hydrolysis. The extinction coefficients (at 260 nm) of each
ODN were determined using the following equation: 2ODN = the
sum of 2nucleoside/hyperchromicity. The extinction coefficients (at
260 nm) of nucleosides used for calculations were as follows: dA,
15,400; dG, 11,700; dT, 8800; dC, 7300; ImNN, 26,200. After the
reaction mixture was heated in boiling water for 5 min, the en-
zymes were removed from the reaction mixture by filtration with
MicropureÒ-EZ device (MILLIPORE), and the filtrate was concen-
trated. Nucleoside composition was determined by analysis of
the residue with reverse-phase HPLC, using a J’sphere ODN 80 col-
umn (4.6 ꢀ 250 mm, YMC) with a linear gradient of acetonitrile
(from 5% to 25% over 30 min) in 0.1 M TEAA buffer (pH 7.0). The re-
sults were shown in Fig. 4.
1. MacMillan, A. M.; Verdine, G. L. J. Org. Chem. 1990, 55, 5931.
2. Kim, S. J.; Stone, M. P.; Harris, C. M.; Harris, T. M. J. Am. Chem. Soc. 1992, 114,
5480.
3. Ono, A.; Haginoya, M.; Kiyokawa, M.; Minakawa, N.; Matsuda, A. Bioorg. Med.
Chem. Lett. 1994, 4, 361.
4. Nakane, M.; Ichikawa, S.; Matsuda, A. J. Org. Chem. 2008, 73, 1842.
5. Minakawa, N.; Kojima, N.; Hikishima, S.; Sasaki, T.; Kiyosue, A.; Atsumi, N.;
Ueno, Y.; Matsuda, A. J. Am. Chem. Soc. 2003, 125, 9970.
6. Hikishima, S.; Minakawa, N.; Kuramoto, K.; Fujisawa, Y.; Ogawa, M.; Matsuda,
A. Angew. Chem., Int. Ed. 2005, 44, 596.
7. Hikishima, S.; Minakawa, N.; Kuramoto, K.; Ogata, S.; Matsuda, A. ChemBioChem
2006, 7, 1970.
8. Hirama, Y.; Abe, H.; Minakawa, N.; Matsuda, A. Tetrahedron 2010, 66, 8402.
9. Hirama, Y.; Minakawa, N.; Matsuda, A. Bioorg. Med. Chem. 2011, 19, 352.
10. Kuramoro, K.; Tarashima, N.; Hirama, Y.; Kikuchi, Y.; Minakawa, N.; Matsuda,
A. Chem. Commun. 2011, 47, 10818.
11. Minakawa, N.; Ogata, S.; Takahashi, M.; Matsuda, A. J. Am. Chem. Soc. 2009, 131,
1644.
12. Ogata, S.; Takahashi, M.; Minakawa, N.; Matsuda, A. Nucleic Acids Res. 2009, 37,
5602.
Acknowledgments
13. Septak, M. Nucleic Acids Res. 1996, 24, 3053.
14. Full data of UV spectrum monitoring were shown in the Supplementary data.
15. When the reaction with p-bromobenzoyl chloride was carried out in pyridine,
unexpected cleavege of the glycosidic linkage occurred to give a product like
compound 10. Since no decomposition has occurred when 1 was stirred in
pyridine, acylation of the exocyclic amino group seemed to be necessary for
such cleavage reaction.
This investigation was supported in part by a Grant-in-Aid for
Scientific Research from the Japan Society for Promotion of Science
(No. 24390027). We would like to thank Mr. H. Kitaike (Center
for Instrumental Analysis, The University of Tokushima) for
measurement of analytical data.
16. Craynest, N. V.; Guianvarc’h, D.; Peyron, C.; Benhida, R. Tetrahedron Lett. 2004,
45, 6243.