Synthesis and triplex-forming ability of oligonucleotides bearing 1-substituted 1H-1,2,3-triazole nucleobases

Article abstract of DOI:10.1016/j.bmc.2010.12.049

Using the copper(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition, a post-elongation modification of 1-ethynyl substituted nucleobases has been employed to construct 18 variations of oligonucleotides from a common oligonucleotide precursor. The triplex

Full text of DOI:10.1016/j.bmc.2010.12.049

Bioorganic & Medicinal Chemistry 19 (2011) 1162–1166
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and triplex-forming ability of oligonucleotides bearing 1-substituted
1H-1,2,3-triazole nucleobases
⇑
⇑
Yoshiyuki Hari , Motoi Nakahara, Juanjuan Pang, Masaaki Akabane, Takeshi Kuboyama, Satoshi Obika
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Using the copper(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition, a post-elongation modification of
1-ethynyl substituted nucleobases has been employed to construct 18 variations of oligonucleotides from
a common oligonucleotide precursor. The triplex-forming ability of each oligonucleotide with dsDNA was
evaluated by the UV melting experiment. It was found that triazole nucleobases generally tend to exhibit
binding affinities in the following order: CG > TA > AT, GC base pairs. Among the triazole nucleobases
examined, a 1-(4-ureidophenyl)triazole provided the best result with regard to affinity and selectivity
for the CG base pair.
Received 22 November 2010
Revised 22 December 2010
Accepted 23 December 2010
Available online 30 December 2010
Keywords:
Oligonucleotide
Synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
Click chemistry
Triple helix
CG recognition
1. Introduction
that a hydrogen-acceptor at the a-position of glycosidic bond could
be vital for CG base pair recognition as depicted in Figure 1.4
In addition, we have recently reported the facile synthesis and
the respective duplex-forming abilities of several oligonucleotides,
including 20-deoxyribonucleotides, having 1-substituted 1,2,3-tria-
zol-4-yl nucleobases. The chemical syntheses of these compounds
were effected by implementation of the post-elongation modifica-
tion (PEM) using the copper(I)-catalyzed alkyne-azide 1,3-dipolar
cycloaddition (CuAAC) reaction.7,8 The PEM method facilitates the
preparation of oligonucleotides bearing various 1-substituted
1,2,3-triazol-4-yl nucleobases from a common oligonucleotide pre-
cursor in only one synthetic step (i.e., CuAAC reaction). Moreover,
Triplex formation of double-stranded DNA (dsDNA) and an oli-
gonucleotide is a foundation of nucleic acid-based technologies
such as gene therapy and gene diagnosis.1 However, natural
oligonucleotides bind only to the homopurine strand of the homop-
urine–homopyrimidine tract within dsDNA, forming triplex DNA
through either the Hoogsteen hydrogen bonds or the reverse-
Hoogsteen interactions. In other words, no natural nucleobase can
form a base triplet with a pyrimidine–purine (CG or TA) interrup-
tion within a homopurine–homopyrimidine tract of dsDNA. The
development of artificial nucleic acids, particularly artificial nucle-
obases capable of recognizing CG or TA base pairs, has been
explored to address this issue.2,3 The development of a more
efficiently recognizable nucleobase remains a challenge and is
currently in great demand.
the triazole nucleobases have a nitrogen atom at the a-position of
the glycosidic bond, which may be necessary for CG base pair rec-
ognition. Thus, we set out to explore the affinities and selectivities
of nucleobases bearing a triazole core with the aim of finding
a nucleobase that effectively recognizes CG base pairs. Here we
We have also engaged in the development of a nucleic acid
combined with 20,40-BNA (20,40-bridged nucleic acid) modification
and nucleobases which sequence-selective and strongly recognize
CG or TA base pairs.4–6 As previously demonstrated by our group,
20,40-BNA bearing an oxazol-4-yl nucleobase (OB) recognizes the
N
N
N
α
α
O
N
CG base pair with
a slight sequence-selectivity. However,
BNA
OB
BNA
IB
the BNA–imidazol-1-yl analog (IB) exhibited the least affinity for
the CG base pairs among all of the natural base pairs. This indicates
H
H
H
N
N
O
H
O
N
N
N
N
O
H
O
N
N
N
N
C
C
N
N
H
H
H
H
DNA
DNA
N
N
N
G
G
⇑
Corresponding authors. Tel./fax: +81 6 6879 8201 (Y.H.); tel.: +81 6 6879 8200;
fax: +81 6 6879 8204 (S.O.).
DNA
DNA
H
H
E-mail addresses: hari@phs.osaka-u.ac.jp (Y. Hari), obika@phs.osaka-u.ac.jp (S.
Obika).
Figure 1. Plausible structures of OB-CG and IB-CG base triplets.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.049

Y. Hari et al. / Bioorg. Med. Chem. 19 (2011) 1162–1166
1163
(i)
report our successful studies toward this goal culminating in the
synthesis of oligonucleotides bearing a wider variety of 1-substi-
tuted 1,2,3-triazole nucleobases and their subsequent evaluation
of base pair recognition in the formation of triplex DNA. The details
of these results are described in this work.9
R
6
5'-TTTTTCTXTCTCTCT-3'
N
N
7a-r
N
O
O
X
=
O
P
O
O
2. Results and discussion
R =
The phosphoramidite 1 was synthesized as depicted in Scheme 1.
H
N
Methanolysis of the anomeric mixture of toluoyl esters 2 (
a:b = ca.
OEt
Me
3:1) afforded the diol 3, which was treated with DMTrCl in pyridine
to afford 4 and 5 chemoselectively. At this point, the anomeric mix-
ture could be separated by silica gel column chromatography to pro-
O
O
7a
7b
7c
7h
7d
7e
vide the desired b-isomer 4 (18% yield) and the
a-isomer 5 (56%
N
OH
7j
yield). Phosphitylation of 4 provided the desired phosphoramidite
1, which was subsequently employed in an oligonucleotide synthe-
sis using an automated DNA synthesizer to give the oligonucleotide
6. Next, the construction of triazole nucleobases was examined
according to our previously optimized CuAAC conditions.9 The tri-
plex-forming oligonucleotides (TFOs) that were prepared (7a–r)
are depicted in Scheme 2 (see experimental for details).10
7g
7i
7f
OH
OH
O
OH
Me
O
OH
OH
N
NH2
H
O
7k
7l
OH
7p
7m
7n
O
NH2
H
N
NH2
The triplex-forming ability of 7a–r bearing triazole nucleobases
with dsDNA targets (XY = CG, TA, GC or AT), was evaluated by the
UV melting experiment under neutral conditions [7 mM phosphate
buffer (pH 7.0), 140 mM KCl and 10 mM MgCl2]. These results are
summarized in Table 1. When substituents at the 1-position of the
triazole nucleobases were aliphatic groups, such as benzyl (7a),
cyclohexyl (7b), adamantyl (7c), (ethoxycarbonyl)methyl (7d) or
2-(acetamido)ethyl (7e), the Tm values with dsDNA (XY = CG) ran-
ged from 17 °C to 20 °C. Athough this interaction occurred with
selectivity for the CG base pair, its magnitude is less than that of
natural TFO 8 (25 °C) with dsDNA (XY = CG).11 However, even
TFO 7c, containing a bulky adamantyl group, was observed to form
the triplexes. The installation of an additional functional group
(e.g., 7d and 7e) allowed for the formation of hydrogen bonds,
resulting in the loss of selectivity for the CG base pair caused by
an increase in affinity for the TA base pair. The TFOs 7f–r contain
the aryl-substituted triazole nucleobases. Interestingly, the phe-
nyltriazole nucleobase 7f led to a significant increase in the affinity
to CG and TA base pairs as compared to that observed with the ali-
phatic-substituted triazoles 7a–e. This is presumably due to the
reduction of entropic penalty for base pair-recognition by the con-
formational rigidity of the phenyl substituent and an increase in
OH
7q
O
7o
7r
Scheme 2. Reagents and conditions: (i) R–N3 (10 equiv), CuSO4 (2 equiv), sodium
ascorbate (4–20 equiv), tris[(1-benzyl-1,2,3-triazol-4-yl)methyl]amine (TBTA)
(4 equiv), DMSO-phosphate buffer (10 mM, pH 7.0), rt, 1–24 h.
Table 1
Tm values (°C) of triplexes between TFOs 7 and four dsDNA targetsa
TFO
XY
CG
TA
17
GC
AT
T (8)b
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
25
19
20
17
20
20
25
25
24
17
21
23
22
22
21
23
24
21
24
20
44
11
<10
<10
<10
12
<10
<10
<10
15
<10
<10
<10
<10
14
<10
17
12
<10
<10
<10
<10
13
<10
12
<10
<10
12
12
11
14
14
17
15
15
15
14
14
15
17
13
18
16
<10
13
12
<10
<10
<10
<10
<10
<10
<10
<10
13
RO
(i)
DMTrO
(iii)
DMTrO
(ii)
O
O
O
(β)
(α)
+
RO
HO
HO
a
Conditions: 7 mM sodium phosphate buffer (pH 7.0), 140 mM KCl and 10 mM
4
5
2
)
R = Tol (
MgCl2. The final concentration of each oligonucleotide used was 1.5 lM. The
sequence of dsDNA targets is as follows: 50-GCTAAAAAGAXAGAGAGATCG-30/50-
CGATCTCTCTYTCTTTTTAGC-30 (XY = CG, TA, GC or AT).
3
R = H ( )
b
The sequence of TFO 8 is 50-TTTTTCTTTCTCTCT-30.
DMTrO
O
X
6
5'-TTTTTCT TCTCTCT-3'
(iv)
O
P
the stability effected by additional
Tm value observed with the triplex dsDNA complex (XY = CG) and
the Tm difference ( Tm), were 25 °C and 8 °C, respectively. The for-
p-stacking interactions. The
O
O
i-Pr2N
OCH2CH2CN
X =
D
1
mer was analogous to that of a triplex containing a T-CG base trip-
let formed by TFO 8 and dsDNA (XY = CG). Previously, it was
reported by Dervan’s group that the binding affinity of the 4-phen-
ylimidazole nucleobase (D2) to all four base pairs was very weak
(Fig. 2a).12 Therefore, it was suggested that a nitrogen atom at
O
P O
O
Scheme 1. Reagents and conditions: (i) NaOMe, MeOH, rt, 3 h, 92%; (ii) DMTrCl
(1.5 equiv), pyridine, rt, 2 h, 18% for 4 and 56% for 5; (iii) (i-Pr2N)2POCH2CH2CN
(1.5 equiv), diisopropylammonium tetrazolide (1.0 equiv), MeCN–THF (3:1), rt, 4 h,
76%; (iv) DNA synthesis (C = 20-deoxy-5-methylcytidine).
the
a-position (e.g., triazole nucleobases) of the glycosidic bond
could form a hydrogen bond with the 4-amino group of C. The

1164
Y. Hari et al. / Bioorg. Med. Chem. 19 (2011) 1162–1166
(b)
40
(a)
DNA
N
O
P
30
DNA
N
N
D2
H
N
N
O
H
O
N
N
20
10
0
N
C
N
H
H
DNA
N
N
G
DNA
H
(c)
O
O
5
25
45
65
85
Bun
Prn
N
Temp. (°C)
N
H
N
H
H
DNA
N
DNA N
N
U2
N
D4
Figure 3. UV melting profiles for triplexes between TFO 7r and dsDNAs (XY = CG,
circle; XY = TA, triangle; XY = GC, cross; XY = AT, line) as shown in Table 2. The
presented melting curves were normalized.
Figure 2. (a) The structure of D2. (b) The structure of a P-CG base triplet. (c) The
structures of U2 and D4.
NHCONH2
formation of triplexes was further observed with dsDNA bearing
purine–pyrimidine base pairs (XY = GC or AT) with the TFOs 7g
and 7h possessing an o-hydroxy phenyl or a 2-hydroxypyridin-3-
yl group. On the contrary, a carboxyl group (e.g., 7i) at the o-posi-
tion led to a dramatic decrease in the Tm value with dsDNA
(XY = CG) as compared to that of the unsubstituted TFO 7f; this
is most likely due to the increase in steric bulk imposed by the
ortho substituent. Meta substitution on the phenyl group (e.g.,
7j–m) led to somewhat decreased affinities for CG and TA base
pairs. A para methyl substituent (i.e., 7n) decreased both affinities
to CG and TA base pairs by 4 °C as compared with those of the
unsubstituted phenyl TFO 7f. An additional effect of an amino
(i.e., 7o) or a hydroxymethyl (i.e., 7p) group on base pair recogni-
tion ability was not observed. Interestingly, the p-carboxyl substi-
tuent in 7q led to a greater decrease of the Tm value with dsDNA
(XY = TA) than that observed with dsDNA (XY = CG). Consequently,
N
N
DNA
N
7r
H
N
N
O
H
O
N
N
N
C
N
H
H
DNA
N
N
G
DNA
H
Figure 4. Plausible structure of a 7r-CG base triplet.
however, detailed explanation of this observation requires further
experimentation.
Among five-membered heteroaromatic-containing nucleobases
(e.g., U214 and D415 shown in Fig. 2c) developed for CG base pairs,
the 1-(4-ureidophenyl)triazole nucleobase could be a promising
candidate, although further improvements in stability and selectiv-
ity are required. Furthermore, our present work demonstrates that
distal substituents on the triazole core (i.e., ureido moiety) in 7r
can significantly affect the base pair recognition ability and/or se-
quence-selectivity.
the
DTm was greater than 11 °C, although the Tm value with dsDNA
(XY = CG) was not sufficiently large. Gratifyingly, the TFO 7r bear-
ing a p-ureido group was found to exhibit high sequence-selectiv-
ity without the loss of the affinity for dsDNA (XY = CG). The Tm and
D
Tm of the TFO 7r dsDNA complex (XY = CG) was 24 °C and 11 °C,
respectively. This result is comparable to that of the 2-pyridone
nucleobase CG base complex (Fig. 2b).13
The triplex-forming ability of 7r was evaluated and compared
to those of unsubstituted 7f and natural 8 (Table 2 and Figure 3)
under different conditions, that is, 10 mM sodium cacodylate buf-
fer (pH 6.8), 100 mM KCl and 50 mM MgCl2. Analogous to the
results obtained in phosphate buffer shown in Table 1, the TFO
7r exhibited high sequence-selectivity with dsDNA containing CG
base pairs (Fig. 4). The Tm value of 7r (29 °C) was comparable with
those observed with the dsDNA complexes (XY = CG) with 7f and 8.
The results provided from the TFOs 7f and 7r indicate that a
p-ureido group decreases only the affinity for TA base pairs;
3. Conclusion
An oligonucleotide including an ethynyl moiety was efficiently
synthesized and subsequently converted to a wide variety of
1-substituted triazole derivatives by employment of the CuAAC
reaction as a PEM method. The triazole nucleobases within oligo-
nucleotides exhibited a significant binding affinity for CG base
pairs in a sequence-selective fashion. Among the substrates tested,
it was found that the 1-(4-ureidophenyl)triazole nucleobase pro-
vided the best result with regard to the binding affinity and CG se-
quence-selectivity. Moreover, the relationship between the
structure and base-pair recognition obtained in this study could
provide additional insight into the design of nucleobases contain-
ing a five-membered heteroaromatic core to provide more effective
CG base pair ligands.
Table 2
Tm values (°C) of triplexes between TFOs 7f and 7r, and four dsDNA targetsa
TFO
XY
CG
TA
GC
AT
T (8)b
7f
7r
28
29
29
21
23
18
22
17
17
45
19
18
4. Experimental
4.1. General
a
Conditions: 10 mM sodium cacodylate buffer (pH 6.8), 100 mM KCl and 50 mM
MgCl2. The final concentration of each oligonucleotide used was 1.9 lM. The
sequence of dsDNA targets is as follows: 50-GCTAAAAAGAXAGAGAGATCG-30/50-
CGATCTCTCTYTCTTTTTAGC-30 (XY = CG, TA, GC or AT).
All chemicals were purchased from chemical suppliers. For col-
umn chromatography, Fuji Silysia silica gel PSQ-100B and FL-100D
b
The sequence of TFO 8 is 50-TTTTTCTTTCTCTCT-30.

Y. Hari et al. / Bioorg. Med. Chem. 19 (2011) 1162–1166
1165
were used. All melting points were measured on a Yanagimoto mi-
cro melting point apparatus and are uncorrected. 1H NMR, 13C NMR
and 31P NMR spectra were recorded on a JEOL ECS400 spectrome-
ter. IR spectra were recorded on JASCO FT/IR-200 and JASCO FT/IR-
4200 spectrometers. Optical rotations were recorded on a JASCO
DIP-370 instrument. Mass spectra were measured on a JEOL JMS-
600 or JEOL JMS-700 mass spectrometer. MALDI-TOF mass spectra
were recorded on a Bruker Daltonics Autoflex II TOF/TOF mass
spectrometer. EYELA Cute Mixer CM-1000 was used as a shaker.
resin by treatment with 28% aqueous NH3 solution at room tem-
perature for 1.5 h. Additional treatment with 28% aqueous NH3
solution at 55 °C for 15 h underwent the removal of all protecting
groups on TFOs. The obtained crude TFOs were purified with Sep-
PakÒ Plus C18 cartridges (Waters) followed by reversed-phase
HPLC (Waters XTerraÒ MS C18 2.5
lm, 10 ꢂ 50 mm). The composi-
tion of the TFOs was confirmed by MALDI-TOF-MS analysis.
MALDI-TOF-MS data ([MꢁH]ꢁ) for 6: found 4395.39 (calcd
4395.91).
4.1.1. 1-Ethynyl-2-deoxy-
D
-ribose (3)
4.3. Azide synthesis
NaOMe (521 mg, 9.64 mmol) was added to a solution of 216 in
MeOH (30 mL) at room temperature and the mixture was stirred
for 3 h. The solvent was removed under reduced pressure and
the residue was purified by silica gel column chromatography
Among azide reagents used for click chemistry, 1-(3-azidophe-
nyl)urea and 1-(4-azidophenyl)urea were new compounds which
were prepared according to the following procedure.
(CHCl3/MeOH = 20:1) to give compound 317
(a:b = 3:1).
4.3.1. 1-(3-Azidophenyl)urea
4.1.2. 1-(b)-Ethynyl-5-O-(4,40-dimethoxytrityl)-2-deoxy-
D
-ribose
NaN3 (10 mg, 15.3
phenyl)urea (20 mg, 76.6
7.66 mol), sodium ascorbate (3.5 mg, 17.7
N,N0-dimethylethylenediamine (1.0
L, 9.23 mol) in DMSO–H2O
l
mol) was added to a solution of 1-(3-iodo-
mol), copper(I) iodide (1.5 mg,
mol) and
(4) and 1-(
a
)-ethynyl-5-O-(4,40-dimethoxytrityl)-2-deoxy-
D
-
l
ribose (5)
l
l
Under a nitrogen atmosphere, DMTrCl (1.21 g, 2.95 mmol) was
added to a solution of compound 3 ( :b = 3:1, 280 mg, 1.97 mmol)
l
l
a
(5:1, 1.0 mL) at room temperature and the mixture was stirred
for 10 h. The mixture was extracted with AcOEt. The organic ex-
tracts were washed with water and brine and dried over Na2SO4.
The organic layer was evaporated and the residue was purified
by flash column chromatography (toluene/acetone = 3:1) to give
compound (12.6 mg, 93%) as yellow solids. mp 137–139 °C. IR
(KBr) 3508, 3376, 3318, 3201, 3141, 3086, 2116, 1659, 1592,
in anhydrous pyridine (60 mL) at room temperature and the mix-
ture was stirred for 2 h. After addition of saturated aqueous NaH-
CO3 solution, the mixture was extracted with AcOEt. The organic
extracts were washed with water, dried over Na2SO4 and concen-
trated under reduced pressure. The residue was purified by flash
silica gel column chromatography (n-hexane/AcOEt = 4:1) to give
compound 4 (159 mg, 18%) and 5 (461 mg, 56%). Compound 418
24.4 (c 1.03, CHCl3). 1H NMR (CDCl3)
1.80 (1H, d,
:
1552, 1487, 1439, 1349, 1317, 1307 cmꢁ1 1H NMR (CD3OD) d
.
½
a 2D6
ꢀ
d
6.67 (1H, ddd, J = 2.0, 8.0 and 10 Hz), 7.04 (1H, ddd, J = 2.0, 8.5
and 10 Hz), 7.24 (1H, dd, J = 8.0 and 8.5 Hz), 7.31 (1H, dd, J = 2.0
and 2.0 Hz). 13C NMR (CD3OD) d 110.3, 113.8, 116.4, 131.1, 141.9,
142.6, 159.1. FABMS: 178 (MH+). Anal. Calcd for C7H7N5O: C,
47.46; H, 3.98; N, 39.53. Found: C, 47.61; H, 4.10; N, 39.30.
J = 3.3 Hz), 2.15 (1H, ddd, J = 3.0, 6.6 and 17.2 Hz), 2.24 (1H, ddd,
J = 6.0, 8.7 and 17.2 Hz), 2.46 (1H, d, J = 2.1 Hz), 3.15 (1H, dd,
J = 6.0 and 12.8 Hz), 3.26 (1H, dd, J = 4.5 and 12.8 Hz), 3.78 (6H,
s), 3.86–3.91 (1H, m), 4.35–4.38 (1H, m), 4.76 (1H, m), 6.78–6.83
(4H, m), 7.16–7.69 (9H, m). Compound 5: ½a D25
ꢀ
0.926 (c 0.98,
CHCl3). IR mmax (KBr) 3280, 2934, 2044, 1611, 1509, 1251, 1221,
4.3.2. 1-(4-Azidophenyl)urea
1177, 1085, 1034 cmꢁ1
.
1H NMR (CDCl3) d 2.07 (1H, dt, J = 3.6
NaN3 (20 mg, 30.5
phenyl)urea (40 mg, 153
15.3 mol), sodium ascorbate (6.5 mg, 32.8
N,N0-dimethylethylenediamine (1.7
L, 15.6 mol) in DMSO-H2O
l
mol) was added to a solution of 1-(4-iodo-
mol), copper(I) iodide (2.9 mg,
mol) and
and 13.4 Hz), 2.11 (1H, d, J = 6.7 Hz), 2.45 (1H, ddd, 6.7, 7.9 and
13.4 Hz), 2.51 (1H, d, J = 1.8 Hz), 3.10 (1H, dd, J = 5.5 and 9.8 Hz),
3.16 (1H, dd, J = 4.3 and 9.8 Hz), 3.72 (6H, s), 4.09–4.12 (1H, m),
4.21–4.25 (1H, m), 4.80–4.85 (1H, m), 6.78–6.83 (4H, m),
7.12–7.40 (9H, m). 13C NMR (CDCl3) d 41.8, 55.2, 64.0, 67.3, 73.9,
74.6, 84.0, 86.2, 113.1, 126.8, 127.8, 128.0, 130.0, 135.8, 144.7,
158.4. HRMS (FAB) m/z calcd for C28H28NaO5 [M+Na]+: 467.1834;
found, 467.1840.
l
l
l
l
l
(5:1, 3.0 mL) at room temperature and the mixture was stirred
for 12 h. The mixture was extracted with AcOEt. The organic ex-
tracts were washed with water and brine and dried over Na2SO4.
The organic layer was evaporated and the residue was purified
by flash column chromatography (n-hexane/acetone = 4:1) to give
compound (23.8 mg, 88%) as yellow solids. mp 174–177 °C. IR
(KBr) 3418, 3351, 3318, 2575, 2491, 2449, 2141, 1613, 1581,
4.1.3. 1-(b)-Ethynyl-3-O-[2-
cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,40-
1534, 1512, 1485 cmꢁ1 1H NMR (CD3OD)
. d 6.96 (2H, d,
dimethoxytrityl)-2-deoxy-
D
-ribose (1)
J = 8.0 Hz), 7.38 (2H, d, J = 8.0 Hz). 13C NMR (CD3OD) d 120.3,
121.9, 135.5, 138.1, 159.3. FABMS: 178 (MH+). Anal. Calcd for
C7H7N5O: C, 47.46; H, 3.98; N, 39.53. Found: C, 47.44; H, 4.09; N,
39.13.
Under a nitrogen atmosphere, 2-cyanoethyl N,N,N0,N0-tetra-
isopropylphosphordiamidite (0.10 mL, 0.33 mmol) was added to
a solution of compound 4 (77 mg, 0.17 mmol) and diisopropylam-
monium tetrazolide (35 mg, 0.17 mmol) in anhydrous MeCN–THF
(3:1, 2.4 mL) at room temperature and the mixture was stirred
for 4 h. The solvent was removed under reduced pressure and
the residue was purified by flash silica gel column chromatography
(n-hexane/AcOEt = 5:1) to give compound 119 (80 mg, 69%) as a
colorless oil. 31P NMR (CDCl3) d 148.5, 149.1.
4.4. Click chemistry: Typical procedure
A solution of 1-(4-azidephenyl)urea (10 mM in DMSO, 3
was added to a mixture of CuSO4 (2 mM in H2O, 3 L), TBTA
(2 mM in DMSO, 6 L), sodium ascorbate (10 mM in H2O, 3 L), 6
(0.9 mM in H2O, 3.3 L) and H2O (8.7 L) in a 1.5-mL Eppendorf
lL)
l
l
l
l
l
4.2. Oligonucleotide synthesis
tube. The mixture was shaken at room temperature for 20 h using
a shaker (1000 rpm). The whole was purified by reversed-phase
The synthesis of 6 was performed on a 0.2-
l
mol scale on an
HPLC [column: Waters XTerraÒ MS C18 2.5
l
m, 4.6 ꢂ 50 mm; gra-
automated DNA synthesizer (Applied Biosystems Expedite™
8909) using the common phosphoramidite protocol except for a
prolonged coupling time of 5 min for unnatural phosphoramidites.
TFOs synthesized on DMTr-ON mode were cleaved from the CPG
dient: 8–20% acetonitrile in 0.1 M triethylammonium acetate buf-
fer (pH 7.0) for 30 min; flow rate: 1.0 mL/min] to give the desired
TFO 7r, the composition of which was confirmed by MALDI-TOF-
MS analysis.

1166
Y. Hari et al. / Bioorg. Med. Chem. 19 (2011) 1162–1166
MALDI-TOF-MS data ([MꢁH]ꢁ) for TFOs 7a–r: 7a, found
References and notes
4528.47 (calcd 4529.06); 7b, found 4522.96 (calcd 4521.08); 7c,
found 4573.22 (calcd 4573.16); 7d, found 4525.32 (calcd
4525.03); 7e, found 4523.65 (calcd 4524.04); 7f, found 4515.98
(calcd 4515.03); 7g, found 4530.16 (calcd 4531.03); 7h, found
4532.02 (calcd 4532.35); 7i, found 4559.93 (calcd 4559.04); 7j,
found 4531.39 (calcd 4531.03); 7k, found 4544.19 (calcd
4545.06); 7l, found 4558.90 (calcd 4559.04); 7m, found 4573.21
(calcd 4573.07); 7n, found 4531.03 (calcd 4529.06); 7o, found
4527.82 (calcd 4530.05); 7p, found 4545.18 (calcd 4545.06); 7q,
found 4559.13 (calcd 4559.04); 7r, found 4573.53 (calcd 4573.07).
1. For reviews. (a) Buchini, S.; Leumann, C. J. Curr. Opin. Chem. Biol. 2003, 7, 717;
(b) Duca, M.; Vekhoff, P.; Oussedik, K.; Halby, L.; Arimondo, A. B. Nucleic Acids
Res. 2008, 36, 5123.
2. For reviews (a) Luyten, I.; Herdewijn, P. Eur. J. Med. Chem. 1998, 33, 515; (b)
Purwanto, M. G. M.; Weisz, K. Curr. Org. Chem. 2003, 7, 427.
3. Recent papers selected. (a) Guianvarc’h, D.; Fourrey, J. L.; Maurisse, R.; Sun, J. S.;
Benhida, R. Bioorg. Med. Chem. 2003, 11, 2751; (b) Sasaki, S.; Taniguchi, Y.;
Takahashi, R.; Senko, Y.; Kodama, K.; Nagatsugi, F.; Maeda, M. J. Am. Chem. Soc.
2004, 126, 516; (c) Wang, W.; Purwanto, M. G. M.; Weisz, K. Org. Biomol. Chem.
2004, 2, 1194; (d) Craynest, N. V.; Guianvarc’h, D.; Peryon, C.; Benhida, R.
Tetrahedron Lett. 2004, 45, 6243; (e) Ranasinghe, R. T.; Rusling, D. A.; Powers, V.
E. C.; Fox, K. R.; Brown, T. Chem. Commun. 2005, 2555; (f) Rusling, D. A.; Powers,
V. E. C.; Ranasinghe, R. T.; Wang, Y.; Osborne, S. D.; Brown, T.; Fox, K. R. Nucleic
Acids Res. 2005, 33, 3025; (g) Taniguchi, Y.; Togo, M.; Aoki, E.; Uchida, Y.; Sasaki,
S. Tetrahedron 2008, 64, 7164.
4.5. UV melting experiments (Tm measurements)
4. Hari, Y.; Obika, S.; Inohara, H.; Ikejiri, M.; Une, D.; Iminishi, T. Chem. Pharm. Bull.
2005, 53, 843.
UV melting experiments were carried out on a Shimadzu
UV-1650PC spectrophotometer equipped with a Tm analysis acces-
sory. The UV melting profiles were recorded in 7 mM sodium phos-
phate buffer (pH 7.0), 140 mM KCl and 10 mM MgCl2 or in 10 mM
sodium cacodylate buffer (pH 6.8), 100 mM KCl and 50 mM MgCl2
from 5 °C to 85 °C at a scan rate of 0.5 °C/min at 260 nm. The final
concentration of each oligonucleotide used was 1.5 lM or 1.9 lM.
A Tm value was designated the maximum of the first derivative cal-
culated from the profile.
5. Obika, S.; Hari, Y.; Morio, K.; Iminishi, T. Tetrahedron Lett. 2000, 41, 221.
6. (a) Obika, S.; Hari, Y.; Sekiguchi, M.; Iminishi, T. Angew. Chem., Int. Ed. 2001, 40,
2079; (b) Obika, S.; Hari, Y.; Sekiguchi, M.; Iminishi, T. Chem. Eur. J. 2002, 8,
4796; (c) Obika, S.; Hari, Y.; Sekiguchi, M.; Iminishi, T. Tetrahedron 2003, 59,
5123; (d) Obika, S.; Inohara, H.; Hari, Y.; Imanishi, T. Bioorg. Med. Chem. 2008,
16, 2945; (e) Hari, Y.; Matsugu, S.; Inohara, H.; Hatanaka, Y.; Akabane, M.;
Imanishi, T.; Obika, S. Org. Biomol. Chem. 2010, 8, 4176.
7. Nakahara, M.; Kuboyama, T.; Izawa, A.; Hari, Y.; Imanishi, T.; Obika, S. Bioorg.
Med. Chem. Lett. 2009, 19, 3316.
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1128.
9. The synthesis of some oligonucleotides has already been reported as
a
Acknowledgments
communication.7
10. Unfortunately, the reaction of oligonucleotide 6 with 3-azido-2-nitro- or 3-
azido-pyridines did not proceed at all in this reaction system.
11. Yoon, K.; Hobbs, C. A.; Koch, J.; Sardaro, M.; Kutny, R.; Weis, A. L. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 3840.
12. Griffin, L. C.; Kiessling, L. L.; Beal, P. A.; Gillespie, P.; Dervan, P. B. J. Am. Chem.
Soc. 1992, 114, 7976.
This work was supported in part by Special Coordination Funds
for Promoting Science and Technology of the Ministry of Education,
Culture, Sports, Science and Technology, Japan (MEXT), by the
Molecular Imaging Research Program from MEXT and by a Grant-
in-Aid for Science Research (KAKENHI) from the Japan Society for
the Promotion of Science (JSPS).
13. Under same conditions, the Tm and
DTm of TFO bearing P to dsDNA (XY = CG)
were 24 °C and 8 °C, respectively.6a
14. Purwanto, M. G. M.; Weisz, K. Tetrahedron Lett. 2006, 47, 3849.
15. Wachowius, F.; Rettig, M.; Palm, G.; Weisz, K. Tetrahedron Lett. 2008, 49,
7264.
16. Novák, P.; Pohl, R.; Kotora, M.; Hocek, M. Org. Lett. 2006, 8, 2051.
17. Adamo, M. F. A.; Pergoli, R. Org. Lett. 2007, 9, 4443.
18. Chiba, J.; Takeshima, S.; Mishima, K.; Maeda, H.; Nanai, Y.; Mizuno, K.; Inouye,
M. Chem. Eur. J. 2007, 13, 8124.
Supplementary data
Supplementary data (1H and 13C spectra for compound 5) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2010.12.049.
19. Fujimoto, K.; Yamada, S.; Inouye, M. Chem. Commun. 2009, 7164.