Triplex-forming ability of oligonucleotides containing 1-aryl-1,2,3- triazole nucleobases linked via a two atom-length spacer

Article abstract of DOI:10.1016/j.bmc.2013.05.034

Phosphoramidites containing 2-propynyloxy or 1-butyn-4-yl as nucleobase precursors were synthesized and introduced into oligonucleotides using an automated DNA synthesizer. Copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition of the oligonucleotides wi

Full text of DOI:10.1016/j.bmc.2013.05.034

Bioorganic & Medicinal Chemistry 21 (2013) 5583–5588
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Triplex-forming ability of oligonucleotides containing
1-aryl-1,2,3-triazole nucleobases linked via a two atom-length spacer
⇑
⇑
Yoshiyuki Hari , Motoi Nakahara, 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:
Phosphoramidites containing 2-propynyloxy or 1-butyn-4-yl as nucleobase precursors were synthesized
and introduced into oligonucleotides using an automated DNA synthesizer. Copper-catalyzed
alkyne-azide 1,3-dipolar cycloaddition of the oligonucleotides with various azides gave the correspond-
ing triazolylated oligonucleotides, triplex-forming ability of these synthetic oligonucleotides with
double-stranded DNA targets was evaluated by UV melting experiments. It was found that nucleobases
containing 2-(1-m-carbonylaminophenyl-1,2,3-triazol-4-yl)ethyl units likely interacted with A of a TA
base pair in a parallel triplex DNA.
Received 1 May 2013
Revised 22 May 2013
Accepted 23 May 2013
Available online 31 May 2013
Keywords:
Artificial nucleobases
1,3-Dipolar cycloaddition
Triazoles
Ó 2013 Elsevier Ltd. All rights reserved.
Triplex DNA
Triplex-forming oligonucleotides
1. Introduction
tal results and their evaluation can be implemented. Therefore, we
considered that the PEM method can be applied to investigate
A triplex-forming oligonucleotide (TFO) that can form a triplex
DNA with double-stranded DNA (dsDNA) would be a promising
tool for gene-targeting therapy and genetic diagnosis.1 In general,
triplex DNA can be sequence-selectively formed by recognition of
AT and GC base pairs in dsDNA by T and C in TFO via Hoogsteen
hydrogen bonds, respectively. The sequence of dsDNA targeted
by TFO is limited to these purine–pyrimidine base pairs. Therefore,
towards recognition of pyrimidine–purine base pairs such as CG
and TA, a number of synthetic nucleotides have been developed
to date.2 Concerning CG base pair recognition, several promising
nucleotides have already been synthesized. In contrast, there is al-
most no nucleotide recognizing a TA base pair with high selectivity
and stability. The difficulty to find nucleotides for TA base pair rec-
ognition is considered to be caused by disturbance of hydrogen
bonding interaction with the 4-carbonyl group due to the steric
bulkiness of the 5-methyl group in T.
nucleobases for TA base pair recognition and this could bring use-
ful information concerning molecular design for TA recognition. In
this study, nucleobases with two atom-length spacers such as
methyleneoxy and ethylene were designed to avoid the steric
repulsion from 5-methyl group of T and with the recognition unit
to bind to the opposite A. The recognition unit was synthesized by
using copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition as
PEM method to investigate the fine structure (Fig. 1). The details
of our results obtained are described.
2. Results and discussion
2.1. Synthesis
The synthesis of an oligonucleotide 1 bearing a 2-propynyloxy
group was carried out according to Scheme 1. Chlorosugar 2
Recently, we have tried to develop nucleotides capable of recog-
nizing a CG base pair by means of the post-elongation modification
(PEM) methods such as copper-catalyzed alkyne-azide 1,3-dipolar
cycloaddition3 and nucleophilic substitution reaction4 to construct
artificial nucleobases. Since this PEM method allows us to
efficiently synthesize various derivatives in a short period, the
detailed molecular design of nucleobases based on the experimen-
spacer
recognition unit
X
X
copper-catalyzed
N
1,3-diplar cycloaddition
N
N
+
H
N
N
(X = O or CH2)
H
O
N3
Me
N
N
H
N
N
⇑
T
Corresponding authors. Tel./fax: +81 6 6879 8201 (Y. Hari); tel.: +81 6 6879
N
A
O
8200; fax: +81 6 6879 8204 (S. Obika).
E-mail addresses: hari@phs.osaka-u.ac.jp (Y. Hari), obika@phs.osaka-u.ac.jp
(S. Obika).
Figure 1. Nucleobases designed in this study.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.034

5584
Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 5583–5588
CO2Et
underwent reaction with propargyl alcohol in MeCN to give de-
sired b-isomer 3 in 34% yield, the stereochemistry of which was
determined by NOESY correlations of deprotected 4 shown in
Scheme 1. The primary alcohol of 4 was protected by DMTrCl in
the presence of pyridine and phosphitylation of 5 gave phospho-
ramidite 6, a building block for oligonucleotide synthesis. The
oligonucleotide precursor 1 for copper-catalyzed alkyne-azide
1,3-dipolar cycloaddition was prepared on an automated DNA
synthesizer using standard phosphoramidite chemistry.
The synthesis of oligonucleotide 7 with the incorporation of
1-butyn-4-yl group was shown in Scheme 2. Reduction of com-
pound 8 prepared according to the report5 with LiAlH4, tosylation
followed by treatment with lithium acetylide–ethylenediamine
complex was carried out in three-steps; this gave the compound
9 in 53% yield. Phosphoramidite 10 was obtained by phosphityla-
tion of 9 and the yield was 97%.
DMTrO
DMTrO
O
O
(i)-(iii)
(iv)
OH
8
OH
9
DMTrO
O
(v)
X
5'-TTTTTCT TCTCTCT-3'
O
P
7
i-Pr2N
OCH2CH2CN
10
O
O
X
=
O
P
O
O
Copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition of
oligonucleotides 1 and 7 with various azides was carried out
according to the reaction conditions previously reported in our
earlier study.3a This was performed to convert the oligonucleotides
into TFOs (Scheme 3, and see experimental for the synthesis of
azides). In all cases, the corresponding TFOs 11a–h and 12a–j were
successfully obtained by reversed-phase HPLC purification after
the reaction.
Scheme 2. Reagents and conditions: (i) LiAlH4, THF, 0 °C, 4 h, (ii) TsCl, pyridine, rt,
12 h, (iii) lithium acetylide–ethylenediamine complex, THF, rt, 9 h, 53% for three-
steps, (iv) i-Pr2NP(Cl)OCH2CH2CN, i-Pr2NEt, CH2Cl2, rt, 5 h, 97%, (v) DNA synthesis
(C = 20-deoxy-5-methylcytidine).
o-position (Fig. 2). Among TFOs 11d–h including m- or p-substi-
tuted phenyl analogs, only TFO 11e with m-hydroxymethyl group
and TFO 11h with m-ureidophenyl one seem to stabilize the tri-
plexes with dsDNA (YZ = TA) compared to that observed with
TFO 11a including an unsubstitued phenyl group though without
stabilization selective to dsDNA (YZ = TA) (Table 1 and Fig. 2).
Among TFOs 12 in which an ethylene spacer was incorporated,
unsubstitued phenyl analog (12a) increased the Tm value by +2 °C
against dsDNA for either TA or CG base pair compared to the cor-
responding methyleneoxy spacer (11a). o-Substituents (12b and
12c) decreased the stability of triplexes formed with all dsDNA tar-
gets (Fig. 3). This is likely due to the steric repulsion between the
o-substituents and the target base pairs. No major changes were
observed in TFOs 12d–f. TFO 12g containing m-carbamyl group
2.2. Evaluation
In triplex DNA between TFO and dsDNA, base pair-recognition
ability of the nucleobases consisting of 1-aryl-1,2,3-triazol-4-yl
unit and spacer unit in TFO was evaluated by UV-melting experi-
ment. The results of TFOs 11 with methyleneoxy spacer and TFOs
12 with ethylene spacer are summarized in Tables 1 and 2, respec-
tively. The measurement was carried out under neutral conditions
and hairpin dsDNA targets containing four kinds of natural base
pairs, TA, CG, GC and AT base pairs for YZ were used (For details
see footnote of Table 1). Regarding TFOs 11 with methyleneoxy
spacer, when substituent at 1-position of triazole was a simple
phenyl group (11a) without any functional group for hydrogen
bond formation, the range of Tm values against all four hairpin
dsDNA targets was from 20 °C to 22 °C. o-Hydroxyphenyl deriva-
tive (11b) gave almost same Tm values as 11a, while o-carbamoyl
congener (11c) led to a decrease in Tm values against any base pairs
likely due to the existence of bulky carbamoyl group at the
stabilized the triplex with dsDNA (YZ = TA) and the
served was +3 °C. Significant improvement was observed in TFO
12h containing the m-ureido group. The Tm value of 12h against
dsDNA with TA base pair was +6 °C, which was significantly higher
DTm value ob-
D
than that observed (
DTm = +2 °C to +3 °C) against the other dsDNA
targets.
TolO
TolO
HO
O
O
H
O
O
O
(i)
(ii)
Cl
H
OTol
OTol
OH
4
2
3
NOESY
(v)
DMTrO
O
DMTrO
O
O
(iii)
(iv)
O
O
P
OH
5
i-Pr2N
OCH2CH2CN
6
O
O
O
O
X
5'-TTTTTCT TCTCTCT-3'
X
=
1
O
P
O
Scheme 1. Reagents and conditions: (i) propargyl alcohol, MeCN, rt, 4 h, 34%, (ii) NaOMe, MeOH, rt, 5 h, quant., (iii) DMTrCl, pyridine, rt, 6 h, 98%, (iv) i-Pr2NP(Cl)OCH2CH2CN,
i-Pr2NEt, CH2Cl2, rt, 15 h, 90%, (v) DNA synthesis (C = 20-deoxy-5-methylcytidine).

Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 5583–5588
5585
5'-TTTTTCTXTCTCTCT-3'
Table 2
Tm values (°C) of triplexes between TFOs 12 including a ethylene spacer and four
hairpin dsDNA targetsa,b
O
O
O
O
TFO
YZ
TA
R
N
O
O
(i)
N
N
X =
CG
GC
AT
12a
12b
12c
12d
12e
12f
12g
12h
12i
22
24
20
21
O
P
O
P
18 (ꢀ4)
18 (ꢀ4)
22 ( 0)
23 (+1)
22 ( 0)
25 (+3)
28 (+6)
27 (+5)
27 (+5)
22 (ꢀ2)
20 (ꢀ4)
22 (ꢀ2)
23 (ꢀ1)
23 (ꢀ1)
25 (+1)
27 (+3)
27 (+3)
26 (+2)
19 (ꢀ1)
19 (ꢀ1)
21 (+1)
20 ( 0)
19 (ꢀ1)
22 (+2)
23 (+3)
23 (+3)
21 (+1)
20 (ꢀ1)
16 (ꢀ5)
21 ( 0)
19 (ꢀ2)
19 (ꢀ2)
22 (+1)
23 (+2)
22 (+1)
21 ( 0)
O
O
O
O
1
11a-h
O
O
R
N
O
O
(i)
N
N
12j
O
P
O
P
a
Conditions are shown in the footnote of Table 1.
O
O
O
O
b
D
Tm: Difference in the Tm value from that of 12a is shown in parenthesis.
7
12a-j
11b
11c
11d
11e
11f
11g
11h
R =
3
2
1
OH
OH
NH2
O
0
11a,12a
11d,12d
11b,12b
11c,12c
-1
-2
-3
OH
OH
NH2
dsDNA targets: (YZ = TA), (YZ = CG), (YZ = GC), (YZ = AT).
11g,12g
11f,12f
11e,12e
O
O
Figure 2. The difference in the Tm values of TFOs 11b–h from that of TFO 11a.
HN
NH2
NH2
O
NH2
O
N
N
H
12b
12c
12d
12e
12f
12g
12h
12i
12j
12i
12j
6
5
N
H
11h,12h
O
Me
4
3
2
1
Scheme 3. Reagents and conditions: (i) R-N3, CuSO4, sodium ascorbate, tris[(1-
benzyl-1,2,3-triazol-4-yl)methyl]amine (TBTA), DMSO-phosphate buffer (10 mM,
pH 7.0), rt, 0.5–12 h, 67–91%.
0
-1
-2
-3
-4
-5
Table 1
Tm values (°C) of triplexes between TFOs 11 including a methyleneoxy spacer and four
hairpin dsDNA targetsa,b
TFO
YZ
TA
dsDNA targets: (YZ = TA), (YZ = CG), (YZ = GC), (YZ = AT).
CG
GC
AT
11a
11b
11c
11d
11e
11f
20
22
22
20
Figure 3. The difference in the Tm values of TFOs 12b–j from that of TFO 12a.
20 ( 0)
17 (ꢀ3)
21 (+1)
22 (+2)
19 (ꢀ1)
19 (ꢀ1)
23 (+3)
23 (+1)
21 (ꢀ1)
24 (+2)
25 (+3)
22 ( 0)
23 (+1)
25 (+3)
20 (ꢀ2)
19 (ꢀ3)
21 (ꢀ1)
23 (+1)
19 (ꢀ3)
20 (ꢀ2)
23 (+1)
20 ( 0)
18 (ꢀ2)
20 ( 0)
23 (+3)
19 (ꢀ1)
19 (ꢀ1)
21 (+1)
aryltriazole moiety attached to ethylene spacer lies in a suitable
space for TA base pair recognition.
C2-Symmetric 3,5-bisureidophenyl group (12i) designed with
consideration of free rotation of bond between aryl moiety and tri-
azole ring gave almost same result observed with the monoureido
11g
11h
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 1.89 lM. The
derivative (12h). Interestingly,
DTm value of N-acetyl-1,2,3,4-tetra-
sequence of hairpin dsDNA targets is as follows: 50-GGCAAAAAGAYAGAGAGACGC-
hydroquinoline (12j) to the TA base pair was +5 °C. The results of
TFOs 12h–j strongly suggest that the carbonyl oxygen of the com-
mon 2-(1-m-carbonylaminophenyl-1,2,3-triazol-4-yl)ethyl unit
forms hydrogen bond-mediated recognition with A (probably with
6-amino group in A) of the TA base pair, as shown in Figure 4. In
fact, under same conditions, the Tm value of triplex of TFO 12h
and dsDNA (YZ = TA) was comparable to that (Tm = 29 °C) of triplex
containing a T-CG base triplet forming a single hydrogen bond
though this was lower than that (Tm = 32 °C) of triplex containing
a G-TA base triplet.6 The above results do not imply that TFOs
12h–j formed a stable and selective triplex to only dsDNA (YZ = TA)
among all four dsDNA targets. However, this result would provide
hexaethyleneglycol-GCGTCTCTZTCTTTTTGCC-30 (YZ = TA, CG, GC or AT).
b
D
Tm: Difference in the Tm value from that of 11a is shown in parenthesis.
In the above analysis on substituent in the phenyl ring (Tables 1
and 2), TFO 12 with ethylene spacer showed higher Tm values
than TFO 11 with methyleneoxy spacer (Figs. 2 and 3). In addition,
a high Tm value of TFO 12h to dsDNA (YZ = TA) was observed
D
D
when compared with TFO 11h with the same m-ureidophenyl
group. These results may imply that the ethylene spacer is less
flexible than methyleneoxy spacer and consequently the

5586
Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 5583–5588
7.90–7.95 (4H, m). 13C NMR (101 MHz, CDCl3) d 21.6, 21.7, 39.2,
54.2, 64.1, 74.2, 74.4, 81.5, 102.2, 122.4, 126.9, 127.0, 129.1,
129.1, 129.6, 129.8, 143.8, 143.9, 166.2, 166.4. MS (EI) m/z 408
(M+, 100); HRMS (EI) m/z Calcd for C24H24O6: 408.1573. Found
408.1580.
NH
N
N
NH2
N
O
N
O
H
N
N
H
Me
4.3. Prop-2-ynyl 2-deoxy-b-D-ribofuranoside 4
N
H
N
N
T
N
A
Under a nitrogen atmosphere, NaOMe (240 mg, 4.41 mmol) was
added to a solution of 3 (600 mg, 1.47 mmol) in anhydrous MeOH
(5 mL) at room temperature and the mixture was stirred for 5 h.
The solvent was removed under reduced pressure and the residue
was purified by flash silica gel column chromatography (n-hexane/
AcOEt = 1:7) to give compound 4 (251 mg, quant) as a colorless oil.
O
Figure 4. Plausible recognition mode of a TA base pair by 2-[1-(m-ureidophenyl)-
1,2,3-triazol-4-yl]ethyl nucleobase in TFO 12h. The moiety displayed in black is
2-(1-m-carbonylaminophenyl-1,2,3-triazol-4-yl)ethyl unit.
½
a 2D6
ꢁ
+216.4 (c 1.00, CD3OD); IR mmax (KBr) 3284, 2928, 1086,
useful information on the design of nucleobase for TA base pair
recognition in the formation of triplex DNA.
1034 cmꢀ1 1H NMR (400 MHz, CD3OD) d 1.78 (1H, ddd, J = 1.5,
;
3.5, 14.0 Hz), 2.26 (1H, ddd, J = 5.0, 8.0, 14.0 Hz), 2.70 (1H, t,
J = 2.5 Hz), 3.48 (1H, dd, J = 5.0, 12.0 Hz), 3.57 (1H, dd, J = 3.5,
12.0 Hz), 3.80–3.84 (1H, m), 4.02–4.06 (1H, m), 5.25 (1H, dd,
J = 1.5, 5.5 Hz); 13C NMR (101 MHz, CD3OD) d 42.2, 54.7, 63.0,
72.2, 75.4, 80.5, 87.0, 103.1; MS (FAB) m/z 174 [M+H]+; HRMS
(FAB) m/z Calcd for C8H12O4 [M+H]+: 173.0814. Found 173.0809.
3. Conclusion
Oligonucleotides bearing two type of spacers attached to acety-
lene unit were synthesized and by the copper-catalyzed alkyne-
azide 1,3-dipolar cycloaddition of them with various azides the
preparation of oligonucleotides with the corresponding
substituted triazoles was achieved. The evaluation of their
triplex-forming ability with dsDNA demonstrated that 2-(1-m-
carbonylaminophenyl-1,2,3-triazol-4-yl)ethyl unit could make a
hydrogen bond to a TA base pair though the stability and se-
quence-selectivity of the triplex formed was not satisfactory as as-
sessed by UV-melting experiments. Our results obtained could
contribute considerably in finding of nucleobases to recognize a
TA base pair at a practical level.
4.4. Prop-2-ynyl 2-deoxy-5-O-(4,40-dimethoxytrityl)-b-
D-
ribofuranoside 5
Under a nitrogen atmosphere, 4,40-DMTrCl (283, 0.836 mmol)
was added to a solution of 4 (120 mg, 0.697 mmol) in anhydrous
pyridine (5 mL) at room temperature and the mixture was stirred
for 6 h. After addition of water, the mixture was extracted with
AcOEt. The organic extracts were washed with water and brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by flash silica gel column chromatography
(n-hexane/AcOEt = 2:1) to give compound 5 (324 mg, 98%) as a col-
4. Experimental
4.1. General
orless oil. ½a 2D3
ꢁ
+86.5 (c 1.03, CDCl3); IR
mmax (KBr) 1607, 1509, 1444,
1301, 1251, 1177, 1084, 1034 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d
2.07 (1H, d, J = 13.5 Hz), 2.25–2.31 (1H, m), 2.43 (1H, t,
J = 2.5 Hz), 3.16 (2H, d, J = 4.5 Hz), 3.77 (6H, s), 4.19–4.24 (2H, m),
4.28 (1H, dd, J = 1.0, 2.5 Hz), 5.46 (1H, d, J = 5.0 Hz), 6.80–6.83
(4H, m), 7.17–7.32 (7H, m), 7.40–7.42 (2H, m). 13C NMR
(101 MHz, CDCl3) d 14.1, 41.1, 53.9, 55.1, 63.9, 73.2, 74.3, 79.3,
86.0, 86.9, 102.7, 113.0, 126.7, 127.7, 128.0, 130.0, 135.8, 135.9,
144.7, 158.4; MS (FAB) m/z 697 [M+Na]+; HRMS (FAB) m/z Calcd
for C29H30NaO6 [M+Na]+: 497.1935. Found 497.1944.
All chemicals were purchased from chemical suppliers. For col-
umn chromatography, Fuji Silysia silica gel PSQ-100B and FL-100D
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 or JEOL
AL300 spectrometer. 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 mea-
sured on a JEOL JMS-600 or JEOL JMS-700 mass spectrometer. MAL-
DI-TOF mass spectra were recorded on a Bruker Daltonics Autoflex
II TOF/TOF or JEOL JMS-S3000 mass spectrometer. EYELA Cute Mix-
er CM-1000 was used as a shaker.
4.5. Prop-2-ynyl 3-O-[2-cyanoethoxy(diisopropylamino)-
phosphino]-2-deoxy-5-O-(4,40-dimethoxytrityl)-b-
D-
ribofuranoside 6
Under
sopropylchlorophosphoramidite (40
to a solution of compound 5 (70 mg, 0.148 mmol) and N,N-diiso-
propylethylamine (75 L, 0.443 mmol) in anhydrous CH2Cl2
(2 mL) at room temperature and the mixture was stirred for 15 h.
After addition of water, the solvent was removed under reduced
pressure and the residue was purified by flash silica gel column
a
nitrogen atmosphere, 2-cyanoethyl N,N-dii-
4.2. Prop-2-ynyl 2-deoxy-3,5-di-O-toluoyl-b-
D
-ribofuranoside 3
L,
lL, 0.177 mmol) was added
Under nitrogen atmosphere, propargyl alcohol (360
a
l
l
9.25 mmol) was added to a solution of 2 (3.0 g, 7.71 mmol) in
anhydrous CH3CN (75 mL) at 0 °C and the mixture was stirred for
6 h at room temperature. After addition of water, the mixture
was extracted with AcOEt. The organic extracts were washed with
saturated NaHCO3 aq, water and brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt = 5:1) to
give compound 3 (1.2 g, 39%) as a colorless oil. Compound 3:
chromatography (n-hexane/AcOEt = 4:1) to give compound
6
(90 mg, 90%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 1.00
(2H, d, J = 3.0 Hz), 1.11–1.16 (9H, m), 1.86–1.98 (3H, m), 2.52–
2.62 (1H, m), 3.08–3.14 (1H, m), 3.28–3.41 (1H, m), 3.51–3.60
(3H, m), 3.78 (6H, s), 4.12–4.34 (4H, m), 5.43 (1H, m), 6.80–6.84
(4H, m), 7.19–7.35 (7H, m), 7.42–7.46 (2H, m); 31P NMR
(101 MHz, CDCl3) d 147.3, 148.2; MS (FAB) m/z 697 [M+Na]+;
HRMS (FAB) m/z Calcd for C38H47N2NaO7P [M+Na]+: 697.3019.
Found 697.3049.
½
a 2D6
ꢁ
+138.0 (c 1.00, CHCl3). IR mmax (KBr) 1718, 1611, 1273, 1178,
1109, 1020 cmꢀ1 1H NMR (400 MHz, CDCl3) d 2.29 (1H, dd,
;
J = 2.0, 14.5 Hz), 2.40–2.41 (7H, m), 2.54–2.61 (1H, m), 4.28 (1H,
dd, J = 2.5, 15.5 Hz), 4.50–4.56 (2H, m), 4.62–4.66 (1H, m), 5.42–
5.46 (1H, m), 5.51 (1H, d, J = 5.5 Hz), 7.21–7.24 (4H, m),

Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 5583–5588
5587
4.6. 1-(1-Butyn-4-yl)-1,2-dideoxy-5-O-(4,40-dimethoxytrityl)-b-
nS-8) using the common phosphoramidite protocol. TFOs synthe-
sized on DMTr-ON mode were cleaved from the CPG resin and all
the protecting groups on TFOs were removed by treatment with
28% NH3 aq at room temperature for 3 h. The obtained crude TFOs
were purified on Sep-PakÒ Plus C18 cartridges (Waters) followed
D
-ribofuranose 9
Under a nitrogen atmosphere, LiAlH4 (360 mg, 9.47 mmol) was
added to a solution of 85 (1.2 g, 2.37 mmol) in anhydrous THF
(20 mL) at room temperature and the mixture was stirred for 9 h.
After addition of water, the mixture was extracted with AcOEt.
The organic extracts were washed with water and brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by flash silica gel column chromatography (n-hexane/
AcOEt = 5:1) to give compound 5 (1.1 g) as a colorless oil. This com-
pound was not subjected to further purification and a portion of
this was used in the next step. Under a nitrogen atmosphere, p-TsCl
(246 mg, 1.29 mmol) was added to a solution of alcohol (500 mg,
1.08 mmol) in anhydrous pyridine (20 mL) at room temperature
and the mixture was stirred for 12 h. After addition of water, the
mixture was extracted with AcOEt. The organic extracts were
washed with water and brine, dried over Na2SO4, and concentrated
under reduced pressure. Flash silica gel column chromatography
(n-hexane/AcOEt = 3:2) of the residue was performed to obtain
appropriate compound [539 mg, Rf = 0.3 (n-hexane/AcOEt = 1:1)]
as a colorless oil, 250 mg of this compound was dissolved in anhy-
drous THF (3 mL). Under a nitrogen atmosphere, lithium acetylide
ethylenediamine complex (82 mg, 0.889 mmol) was added to the
solution at room temperature and the mixture was stirred for
9 h. After addition of saturated NH4Cl aq, the mixture was ex-
tracted with AcOEt. The organic extracts were washed with water
and brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by flash silica gel column chro-
matography (n-hexane/AcOEt = 3:2) to give compound 9 (124 mg,
by reversed-phase HPLC (Waters XBridgeÒ OST C18 2.5
lm,
10 mm ꢂ 50 mm). The composition of the TFOs was confirmed by
MALDI-TOF-MS analysis. MALDI-TOF-MS data ([MꢀH]ꢀ) for 1 and
7:1, found 4423.01 (calcd 4423.94). 7, found 4424.31 (calcd
4423.96).
4.9. Azide synthesis
Among azide reagents used for click chemistry, 2-azidobenza-
mide, 3-azidobenzamide, 1,10-(5-azido-1,3-phenylene)diurea and
N-acetyl-7-azide-1,2,3,4-tetrahydroquinoline were new com-
pounds which were prepared according to the following
procedure.
4.9.1. 2-Azidobenzamide
Under a nitrogen atmosphere, SOCl2 (1 mL) was added to 2-
azidobenzoic acid (200 mg, 1.23 mmol) and the mixture was re-
fluxed for 2 h. The organic layer was evaporated. 10% aqueous
NH3 (3 mL) was added to the residue at 0 °C and the mixture
was stirred for 0.5 h. The mixture was extracted with CHCl3.
The organic extracts were washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. The res-
idue was purified by flash silica gel column chromatography
(CHCl3/MeOH = 10:1) to give desired compound (185 mg, 93%)
as yellow solids. Mp 130–131 °C. IR (KBr) 3368, 3168, 2130,
2103, 1655, 1620, 1599, 1574, 1483, 1452, 1403, 1230, 1163,
53% for three-steps) as a colorless oil. ½a D24
ꢁ
+3.0 (c 0.31, CHCl3);
IR mmax (KBr) 2933, 1607, 1509, 1445, 1301, 1251, 1177, 1074,
1127, 1084 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 7.22 (1H, dt,
.
1034 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d 1.73–1.80 (2H, m), 1.92–
J = 1.5, 7.5 Hz), 7.33 (1H, dd, J = 1.5, 7.5 Hz), 7.50 (1H, dt, J = 1,5
and 7.5 Hz), 7.56 (1H, br s), 7.57 (1H, dd, J = 1.5 and 7.5 Hz),
7.73 (1H, br s). 13C NMR (101 MHz, CDCl3) d 119.7, 124.8,
128.3, 129.7, 131.4, 136.6, 167.1. MS (EI) m/z 162 (M+, 100);
HRMS (EI) m/z Calcd for C7H6N4O: 162.0542. Found 162.0550.
1.99 (3H, m), 2.27–2.36 (2H, m), 3.06 (1H, dd, J = 6.0, 10.0 Hz),
3.20 (1H, dd, J = 5.0, 10.0 Hz), 3.77 (6H, s), 3.86–3.90 (1H, m),
4.26–4.33 (2H, m), 6.80–6.83 (4H, m), 7.17–7.44 (9H, m); 13C
NMR (101 MHz, CDCl3) d 15.3, 34.5, 40.4, 55.1, 64.4, 68.5, 74.6,
83.9, 85.6, 86.1, 113.1, 126.7, 127.6, 128.1, 130.0, 136.0, 144.8,
158.4; HRMS (MALDI-TOF) m/z Calcd for C30H32NaO5 [M+Na]+:
495.2142. Found 495.2141.
4.9.2. 3-Azidobenzamide
Under a nitrogen atmosphere, SOCl2 (2 mL) was added to 3-azi-
dobenzoic acid (120 mg, 0.88 mmol) and the mixture was refluxed
for 2 h. The organic layer was evaporated. 10% aqueous NH3 (3 mL)
was added to the residue at 0 °C and the mixture was stirred for
0.5 h. The mixture was extracted with CHCl3. The organic extracts
were washed with water and brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by flash
silica gel column chromatography (CHCl3/MeOH = 9:1) to give de-
sired compound (100 mg, 83%) as brown solids. Mp 135–136 °C. IR
mmax (KBr) 3358, 3171, 2198, 2113, 1658, 1483, 1444, 1395, 1314,
4.7. 1-(1-Butyn-4-yl)-3-O-[2-
cyanoethoxy(diisopropylamino)phosphino]-1,2-dideoxy-5-O-
(4,40-dimethoxytrityl)-b-
D-ribofuranose 10
Under
sopropylchlorophosphoramidite (51
to a solution of compound 9 (90 mg, 0.190 mmol) and N,N-diiso-
propylethylamine (97 L, 0.571 mmol) in anhydrous CH2Cl2
a
nitrogen atmosphere, 2-cyanoethyl N,N-dii-
l
L, 0.223 mmol) was added
l
(2 mL) at room temperature and the mixture was stirred for 5 h.
After addition of water, the solvent was removed under reduced
pressure and the residue was purified by flash silica gel column
chromatography (n-hexane/AcOEt = 1:1) to give compound 10
(124 mg, 97%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 1.17
(3H, d, J = 7.0 Hz), 1.13–1.18 (9H, m), 1.74–1.89 (3H, m), 1.97–
2.13 (2H, m), 2.30–2.46 (3H, m), 2,44 (1H, m), 3.07–3.18 (2H, m),
3.52–3.85 (10H, m), 4.05–4.11 (1H, m), 4.26–4.32 (1H, m), 4.40–
4.47 (1H, m), 6.80–6.84 (4H, m), 7.16–7.46 (9H, m); 31P NMR
(162 MHz, CDCl3) d 147.5, 147.7; MS (FAB) m/z 673 [M+H]+; HRMS
(FAB) m/z Calcd for C39H50N2O6P [M+H]+: 673.3407. Found
673.3434.
1288, 1164, 1129 cmꢀ1 1H NMR (300 MHz, DMSO-d6) d 7.25 (1H,
.
d, J = 7.5 Hz), 7.46 (1H, br s), 7.48 (1H, t, J = 7.5 Hz), 7.58 (1H, s),
7.68 (1H, d, J = 7.5 Hz), 8.06 (1H, br s). 13C NMR (101 MHz,
DMSO-d6) d 118.0, 121.9, 124.2, 130.0, 136.0, 139.6, 167.1. MS
(EI) m/z 162 (M+, 100); HRMS (EI) m/z Calcd for C7H6N4O:
162.0542. Found 162.0546.
4.9.3. 1,10-(5-Azido-1,3-phenylene)diurea
Under a nitrogen atmosphere, SnCl2 (3.2 g, 1.70 mmol) was
added to
a solution of 5-iodo-1,3-dinitrobenzene (500 mg,
1.70 mmol) in anhydrous EtOH (10 mL) at room temperature and
the mixture was stirred for 3 h at 70 °C. Ice (10 g) was added and
the pH of the solution was controlled to approximately 9 by 10%
NaOH aq. After the solids were removed by filtration through Cel-
iteÒ, the filtrate was extracted with AcOEt. The organic extracts
were washed with brine, dried over Na2SO4, and concentrated un-
der reduced pressure. Under a nitrogen atmosphere, 0.4 N HCl aq
4.8. Oligonucleotides 1 and 7
The synthesis of 1 and 7 was performed on a 0.2-lmol scale or
1.0- mol scale on an automated DNA synthesizer (Gene Design
l

5588
Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 5583–5588
(10 mL) and KNCO (340 mg, 4.08 mmol) were added to the residue
and the mixture was stirred for 16 h at room temperature. The sol-
ids obtained by filtration was purified by flash silica gel column
chromatography (AcOEt/MeOH = 30:1) to give 1,10-(5-iodo-1,3-
phenylene)diurea (105 mg, 19% from 5-iodo-1,3-dinitrobenzene)
as light brown solids. Mp 135–138 °C. IR mmax (KBr) 3292, 3195,
4575.09); 11g, 69%. Found 4587.89 (calcd 4588.09); 11h, 79%.
Found 4603.52 (calcd 4603.10); 12a, 80%. Found 4543.77 (calcd
4543.09); 12b, 83%. Found 4559.18 (calcd 4559.09); 12c, 82%.
Found 4586.26 (calcd 4586.11); 12d, 83%. Found 4559.51 (calcd
4559.09); 12e, 74%. Found 4573.72 (calcd 4573.11); 12f, 82%.
Found 4573.90 (calcd 4573.11); 12g, 71%. Found 4586.99 (calcd
4586.11); 12h, 77%. Found 4602.02 (calcd 4601.13); 12i, 71%.
Found 4659.83 (calcd 4659.17); 12j, 90%. Found 4640.51 (calcd
4640.20).
1672, 1585, 1544, 1442, 1347 cmꢀ1
.
1H NMR (400 MHz, DMSO-
d6)
d
5.92 (4H, br s), 7.78 (1H, t, J = 1.8 Hz), 7.48 (2H, d,
J = 1.8 Hz), 8.81 (2H, br s). 13C NMR (101 MHz, DMSO-d6) d 94.6,
105.6, 118.6, 142.2, 155.8. HRMS (MALDI) m/z Calcd for C8H9IN4-
NaO2: 342.9662. Found 342.9652.
4.11. UV melting experiments
According to the reaction conditions reported previously,7 NaN3
(26 mg, 0.41 mmol), CuI (7.7 mg, 41
l
mol), sodium ascorbate
UV melting experiments were performed on SHIMADZU UV-
1650 and SHIMADZU UV-1800 spectrophotometers equipped with
Tm analysis accessory. TFOs 11 and 12 and hairpin dsDNA targets
were dissolved in 10 mM sodium cacodylate buffer (pH 6.8) con-
taining 100 mM KCl and 50 mM MgCl2 to give a final concentration
(25 mg, 0.12 mmol) and N,N0-dimethylethylenediamine (4.4
lL,
0.81 mmol) were added to a solution of 1,10-(5-iodo-1,3-pheny-
lene)diurea (95 mg, 0.30 mmol) in DMSO-H2O (5:1, 10 mL) and
the mixture was stirred for 19 h at room temperature. After addi-
tion of brine, the mixture was extracted with AcOEt. The organic
extracts were washed with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by flash
silica gel column chromatography (AcOEt/MeOH = 15:1) to give
the desired compound (50 mg, 72%) as white powder. Mp
of each strand of 1.89 lM. The samples were annealed in boiling
water followed by slow cooling to 5 °C. The melting profiles were
recorded at 260 nm from 5 °C to 90 °C at a scan rate of 0.5 °C/
min. A two-point average method was used to obtain the Tm values
and the final values were determined by averaging three indepen-
dent measurements which were accurate to within 1 °C.
>300 °C. IR mmax (KBr) 3258, 2115, 1668, 1594, 1556 cmꢀ1 1H
.
NMR (300 MHz, CD3OD) d 6.92 (2H, d, J = 1 Hz), 7.10 (1H, t-like,
J = 1 Hz). 13C NMR (76 MHz, CD3OD) d 104.3, 106.6, 142.3, 142.8,
159.1. HRMS (MALDI) m/z Calcd for C8H9N7NaO2: 258.0710. Found
258.0707.
Acknowledgments
This work was supported in part by the Program to Disseminate
Tenure Tracking System of the Ministry of Education, Culture,
Sports, Science and Technology, Japan (MEXT), a Grant-in-Aid for
Scientific Research on Innovative Areas (No. 22136006) from
MEXT, a Grant-in-Aid for JSPS Fellows of Japan Society for the Pro-
motion of Science (JSPS) and Mochida Memorial Foundation for
Medical and Pharmaceutical Research.
4.9.4. N-Acetyl-7-azido-1,2,3,4-tetrahydroquinoline
Under a nitrogen atmosphere, t-BuONO (270
lL, 2.28 mmol)
and TMSN3 (240 L, 1.51 mmol) was added to a solution of N-acet-
l
yl-7-amino-1,2,3,4-tetrahydroquinoline8 (270 mg, 1.42 mmol) at
0 °C. The mixture was stirred for 9 h at room temperature. After
addition of water, the mixture was extracted with AcOEt. The or-
ganic extracts were washed with saturated NaHCO3 aq, water
and brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by flash silica gel column chro-
matography (n-hexane/AcOEt = 1:1) to give the compound
(280 mg, 91%) as light yellow solids. Mp 53–55 °C. IR mmax (KBr)
2944, 2109, 2050, 1656, 1606, 1576, 1498, 1454, 1406, 1352,
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.034.
References and notes
1300, 1263, 1231, 1209, 1136, 1019 cmꢀ1 1H NMR (400 MHz,
.
1. Reviews: (a) Praseuth, D.; Guieysse, A. L.; Hélène, C. Biochim. Biophys. Acta 1999,
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CDCl3) d 1.96 (2H, quint, J = 7.0 Hz), 2.25 (3H, s), 2.72 (2H, t.,
J = 7.0 Hz), 3.76 (2H, t, J = 7.0 Hz), 6.79 (1H, d, J = 7.0 Hz), 6.94
(1H, br s), 7.12 (1H, d, J = 7.0 Hz). 13C NMR (101 MHz, CDCl3) d
23.2, 23.6, 26.3, 43.2 (br s), 115.1, 115.2, 129.4, 137.5, 139.9 (br
s), 169.7. MS (FAB) m/z 217 [M+H]+; HRMS (FAB) m/z Calcd for
C
11H13N4O [M+H]+: 217.1084. Found 217.1087.
4.10. Click chemistry: general procedure
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A solution of azide compound (10 mM in DMSO, 3 lL) was
added to a mixture of CuSO4 (2 mM in H2O, 3
DMSO, 6 L), sodium ascorbate (10 mM in H2O, 3
[0.9 mM in phosphate buffer (pH 7.0), 3.3 L] and H2O (8.7
l
L), TBTA (2 mM in
L), 1 or 7
L) in
l
l
l
l
a 1.5 mL Eppendorf tube. The mixture was shaken at room temper-
ature using a shaker (1000 rpm) until the reaction was complete.
The entire product was purified by reversed-phase HPLC [column:
Waters XBridgeÒ OST C18 2.5
lm, 4.6 mm ꢂ 50 mm; eluent: gradi-
ent system of MeCN/0.1 M triethylammonium acetate buffer (pH
7.0); flow rate: 1.0 mL/min] to give the desired TFO 11 or 12, the
identification was confirmed by MALDI-TOF-MS analysis. Isolated
yield and MALDI-TOF-MS data ([MꢀH]ꢀ) for TFOs 11a–h and
12a–j: 11a, 67%. Found 4545.85 (calcd 4545.06); 11b, 74%. Found
4560.24 (calcd 4561.06); 11c, 91%. Found 4588.03 (calcd
4588.09); 11d, 85%. Found 4561.66 (calcd 4561.06); 11e, 86%.
Found 4575.27 (calcd 4575.09); 11f, 74%. Found 4574.77 (calcd
5. Hovinen, J.; Salo, H. J. Chem. Soc., Perkin Trans. 1 1997, 3013.
6. It is also known that G forms a single hydrogen bond with TA base pair: (a) Yoon,
K.; Hobbs, C. A.; Koch, J.; Sardaro, M.; Kutny, R.; Weis, A. L. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 3840; (b) Griffin, L. C.; Dervan, P. B. Science 1989, 245, 967.
7. Andersen, J.; Madsen, U.; Björkling, F.; Lianng, X. Synlett 2005, 2209.
8. Kolmakov, K.; Belov, V. N.; Wurm, C. A.; Harke, B.; Leutenegger, M.; Eggeling, C.;
Hell, S. W. Eur. J. Org. Chem. 2010, 3593.