Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA

Article abstract of DOI:10.1016/j.bmc.2009.08.040

We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG interrupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural triplex-forming oligonucleotide (TFO). However, the

Full text of DOI:10.1016/j.bmc.2009.08.040

Bioorganic & Medicinal Chemistry 17 (2009) 6803–6810
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA)
having the pyrazole ring in an anti-parallel triplex DNA
Yosuke Taniguchi a,b, Yuko Uchida a, Tomoko Takaki a, Eriko Aoki a,b, Shigeki Sasaki a,b,
*
a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
b CREST, Japan Science and Technology Agency, Center Building, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG inter-
rupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural
triplex-forming oligonucleotide (TFO). However, the stabilization effect of WNA is dependent on the
neighboring nucleobases at both sides of the WNA analogs within the TFO. Considering that the base
is located at the hindered site constructed of three bases of the target duplex and the TFO, it was expected
that replacement of the pyrimidine base of the WNA analog with a smaller pyrazole ring might avoid ste-
ric repulsion to produce a greater stability for the triplex. In this study, the new WNA analogs bearing the
pyrazole ring, 3-aminopyrazole (AP), and 4-methyl-3-pyrazole-5-on (MP) were synthesized, incorporated
into the TFOs, then their stabilizing effects on the triplexes were evaluated. A remarkable success was
illustrated by the fact that the TFO containing WNA-bAP in the 30G-WNA-G-50 sequence formed a stable
triplex with selectivity to the CG interrupting site where the previous WNA-bC did not induce the triplex
formation.
Received 13 July 2009
Revised 18 August 2009
Accepted 19 August 2009
Available online 22 August 2009
Keywords:
W-shaped nucleoside analog (WNA)
Anti-parallel triplex
Antigene
5-Membered heterocycle
Nucleoside analog
Triplex DNA
CG interrupting site
Synthesis
Ó 2009 Elsevier Ltd. All rights reserved.
Evaluation
1. Introduction
small structural change to overcome this problem. A number of
modified WNA-bT derivatives were constructed with the o-,
Triplex-forming oligonucleotides (TFOs) associate with the ma-
jor groove of a duplex DNA and has attracted interest as potential
tools for genomic therapy, research, and technology.1–7 In the
anti-parallel triplex, the most stable triplex DNA is formed by the
interaction between the purine-rich TFO and homopurine/homo-
pyrimidine regions of the duplex DNA in a sequence specific
manner (dG/GC, dA/AT).8 Therefore, the stable triplex DNA is ham-
pered by one pyrimidine base insertion in the homopurine strand
(TA and CG pairs are called interrupting sites). Despite numerous
studies, this problem has not yet been fully solved.9–13
m-, p-bromo-, p-cyano, p-amino substituted benzene, or 3-pyridine
unit. The individual WNA derivative was determined to recognize
the TA interrupting site of each sequence, for example, the original
WNA-bT in the 30-AZG-50 and the 30-GZG-50 TFO, o-bromo-WNA-bT
O
O
O
O
O
O
WNA-βC
WNA-βT
O
O
We previously showed that W-shaped nucleoside analogs
(WNAs), WNA-bT and WNA-bC, recognized the TA and CG inter-
rupting site with a high selectivity, respectively (Fig. 1).14–16 Subse-
quently, it turned out that the recognition abilities of the WNA
analogs were affected by the neighboring nucleobases on the both
sides of the WNA. As the origin of a neighboring bases effect on
triplex stability, especially in the case of triplexes containing
non-natural base analogs, is not well understood,11,12 we initiated
a systematic investigation using a variety of WNA analogs with a
N
H
N
O
H
O
N
N
H
N
H
O
H
H
N
H
O
N
O
N
N
H
N
N
N
N
N
O
H
N
dR
dR
N
N
N
N
H
N
H
dR
O
dR
C
G
T
A
* Corresponding author. Tel./fax: +81 92 642 6651.
E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki).
Figure 1. Speculated recognition structure of WNA-bC/CG and WNA-bT/TA com-
binations. WNA (W-shaped nucleoside analogs).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.040

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Y. Taniguchi et al. / Bioorg. Med. Chem. 17 (2009) 6803–6810
in the 30-AZA-50 TFO, m-bromo-WNA-bT and p-cyano-WNA-bT in
the 30-GZA-50 TFO (Z = WNA analog) have been shown to be suit-
able for stabilization of the triplex having the corresponding TA
interrupting site.17–19 Thus, the combined use of the aromatic-
modified WNA analogs has provided a partial solution of the se-
quence-dependency problem of the WNA-bT analog. Nevertheless,
a general solution for the TA and CG interrupting site of the triplex
is still a challenging theme. In addition to the important role of the
benzene part, the effects of the recognition unit of the WNA analog
are somewhat complicated as shown by the facts that the order of
the stabilization effect on the triplexes is WNA-H lacking the rec-
ognition base with non-selectivity > WNA-bT with high selectiv-
ity > WNA-7bG having N7-guanine base with a low stabilization
effect.15 These results have indicated that the size of the recogni-
tion base significantly affects the triplex stability. These consider-
ations encouraged us to study replacement of the pyrimidine
base of the WNA analog with a smaller pyrazole ring to avoid steric
repulsion and to enhance hydrogen bond formation. In this paper,
we describe in detail the synthesis and evaluation of the triplex
On the other hand, the WNA analogs with the 4-methyl-3-pyr-
azole-5-on unit (WNA-MP) were synthesized as shown in Scheme
2. 4-Methyl-3-pyrazole-5-on (MP)21 was treated with benzoyl
chloride to afford 6 in good yield. As the benzoyl group of 6 mi-
grated to the other position, we used MP-Bz as the mixture of
regioisomers for the following coupling reaction without separa-
tion. The coupling of 6 with the bicyclic sugar intermediate 2 gave
four isomers 7a (13%), 7b (10%), 8a (20%), and 8b (11%). Fortu-
nately, these isomers could be completely separated by silica gel
column chromatography, and the regiochemistry and the stereo-
chemistry of each isomer were determined by its 1H-COSY and
NOESY spectra after removal of the TBDPS, acetyl and benzoyl
groups; WNA-2aMP (9a) in 78%, WNA-2bMP (9b) in 72%, WNA-
1aMP (10a) in 42%, WNA-1bMP 10b in 44% (Fig. 4). The coupling
position of the 4-methyl-3-pyrazole-5-on unit (MP) was unambig-
uously determined by the NOE cross peaks with 3-H; the 2-coupled
products (9a and 9b) did not produce cross peaks with the protons
of the sugar part, whereas the 1-coupled products (10a and 10b)
showed cross peaks with the protons of the sugar part as illus-
trated in Figure 4. At the same time, the stereochemistry of each
isomer was determined. The desired diol compounds (9a and 9b)
were converted to the corresponding phosphoramidite precursor;
forming ability of the new WNA analogs, WNA-bAP and -
aAP con-
taining 3-aminopyrazole and WNA-bMP and - MP containing 4-
a
methyl-3-pyrazole-5-on, whose hydrogen-bonding motifs are sim-
ilar to cytosine and thymine, respectively (Fig. 2).
WNA-2aMP (11a) in 51% yield, WNA-2bMP (11b) in 39% yield
(Scheme 3). Unfortunately, the diol compounds (10b and 10a)
led to multiple compounds using the same protection process.
2. Results and discussion
Therefore, we adopted WNA-2bMP and WNA-2aMP for incorpora-
tion into the TFO. The TFOs (TFO 1, 2, 3, and 4) having the WNA-2
a
Synthesis of the phosphoramidite derivatives of the WNA ana-
log with the 3-aminopyrazole unit (WNA-AP) was accomplished
as shown in Scheme 1. The amino group of 3-aminopyrazole (AP)
was protected by N,N-dimethylformamidedimethylacetal to give
1 in good yield.20 The protected aminopyrazole (1) was coupled
with the bicyclic sugar intermediate 214,15 to produce regioselec-
tively the N1-glycosidated products 3a (20%) and 3b (41%) after
separation by silica gel column chromatography, whose stereo-
chemistry was determined by their 1H-COSY and -NOESY spectra
(Fig. 3). Each isomer was subjected to deprotection of the TBDPS
and the acetyl groups to afford the corresponding diol compounds
4a (93%) and 4b (97%). These diol compounds were converted to
the corresponding phosphoramidite precursor by the conventional
or bMP derivative were obtained in good yields using an auto-
mated DNA synthesizer. The cleavage of the synthesized TFO from
the CPG resin was performed in a 28% NH4OH solution at room
temperature to prevent the removal of the pyrazole recognition
base. After separation by HPLC, the purity and structure of the TFOs
were confirmed by MALDI-TOF MS measurements.
The triplex forming abilities of the TFOs containing the WNA
analogs were evaluated by the gel-shift assay using the 32P-labeled
TFO as a tracer. The intensities of the radioactive bands were mea-
sured from which the association constants (Ks values) of the tri-
plex formations were calculated as previously described.15 The
examples of the electrophoresis of TFO containing WNA-bAP in
the sequence of 30-dG and 50-dG (TFO2(bAP)) are shown in Figure
5. The high mobility bands showed the single stranded TFO, on the
other hand, the low mobility bands showed the triplex DNA. The
triplex bands were observed at low TFO concentrations in Figure
5A, clearly indicating that TFO2(bAP) stabilized the triplex DNA
having the CG interrupting site with a high selectivity. The associ-
ation constant (Ks value) of all the TFOs are summarized in Figure
6. We previously reported that TFO1(bC) having WNA-bC in the
TFO sequence of 30-AZG-50 could stabilize the CG interrupting site
(Fig. 6A, green bars). TFO1(bAP) incorporating the aminopyrazole
unit with the b-stereochemistry formed a stable triplex with the
method (WNA-aAP; 5a in 60%, WNA-bAP; 5b in 66%). The TFOs
(TFO 1, 2, 3, and 4) containing the WNA-AP derivative were
obtained in good yield using an automated DNA synthesizer.
TFO1(bAP) represents the TFO1 containing WNA-bAP at position
Z in the sequence of 30-AZG-50. Cleavage of the synthesized TFO
from the CPG column and the deprotection of the aminopyrazole
unit were done in a solution of 28% NH4OH at 55 °C for 5 h. After
purification by reversed-phase HPLC, the synthesized TFOs were
identified by MALDI-TOF MS measurements.
GC pair at the complementary site (Fig. 6A, blue bars). TFO1(
aAP),
TFO1(2bMP), and TFO1(2 AP) indicated relatively higher stabil-
a
ization effects for the CG pair, although these stabilities and selec-
tivities were insufficient (Fig. 6A, pink and orange bars). Based on
these results, the recognition behavior of the pyrazole ring were
not clear in the sequence of TFO1 (30AZG). Remarkable results were
obtained with TFO2 in the sequence of 30-GZG-50 (Fig. 6B), where
the original WNA-bC was also not able to form stable triplexes
(Fig. 6B, green bars). TFO2(bAP) exhibited a high affinity for the
target DNA with the CG interrupting site (Fig. 6B, blue bars). It is
noteworthy that the CG recognition affinity of WNA-bAP is higher
than the natural dG/GC triplet. In this TFO sequence, a shortcoming
of the original WNA-bC was overcome with WNA-bAP. Unfortu-
NH
2
O
HO
HO
N
O
N
HO
HO
N
O
NH
O
O
HO
WNA-βAP
HO
WNA-2βMP
O
O
HO
O
N
N
O
N
HN
HO
NH
2
O
WNA-αAP
WNA-2αMP
nately, TFO2 with other WNA analogs (aAP, 2bMP, 2aMP) did
not show interesting stabilizing effects (Fig. 6B). The results of
Figure 2. Design of new WNA analogs, WNA-AP and WNA-2MP (AP: 3-aminopyr-
azole, MP: 4-methyl-3-pyrazole-5-on).
TFO3 in the sequence of 30-GZA-50 are summarized in Figure 6C.

Y. Taniguchi et al. / Bioorg. Med. Chem. 17 (2009) 6803–6810
6805
N
N
N
NH2
N
N
a
N
N
N
O
α
R'O
O
R'O
N
N
β
O
H
H
O
N
R''O
N
R''O
1
N
3-5b
3-5a
N
b
3a,3b: R' = TBDPS, R" = Ac
4a,4b: R', R" = H
5a,5b: R' = DMTr, R" = iPr2NPOC2H4CN
c
O
R'O
d
OAc
O
R''O
DNA synthesis
and purification
2
R' = TBDPS
R" = Ac
TFO1(α,βAP) 3'GGAAGGAZGGAGGAGGGA5'
TFO2(α,βAP) 3'GGAAGGGZGGAGGAGGGA5'
TFO3(α,βAP) 3'GGAAGGGZAGAGGAGGGA5'
TFO4(α,βAP) 3'GGAAGGAZAGAGGAGGGA5'
Z = WNA-αAP or -βAP
Scheme 1. Reagents and conditions: (a) N,N-dimethylformamidedimethylacetal, 100 °C, (92%); (b) 1, BSA, TMSOTf, CH3CN, rt (3b: WNA-bAP; 41%, 3a: WNA-
a
AP; 20%); (e) (1)
TBAF, THF, rt, (2) 0.2 M NaOHaq, MeOH, THF, 0 °C (4b: WNA-bAP; 97%, 4b: WNA-
a
AP; 93% for two steps); (f) (1) DMTrCl, pyridine, (2) iPr2NP(Cl)OCH2CH2CN, DIPEA, CH2Cl2,
0 °C (5b: WNA-bAP; 66%, 5a: WNA-
a
AP; 60% in two steps).
of the triplex stability with the WNA analogs. In the case of TFO4 in
the sequence of 30-AZA-50 where TFO4(bC) did not form the triplex
DNA at any target base pair, TFO4(bAP) formed triplexes for the GC
and AT pairs in the target duplexes, suggesting that the small rec-
ognition base may have a structural benefit for recognition in this
sequence (Fig. 6D, blue bars). The TFO4 sequences having other
N
NOE
N
TBDPSO
N
TBDPSO
N
β
H
H
H
O
O
O
O
H
α
N
H
OAc
H
OAc
N
NOE
N
WNAs (aAP, 2bMP, 2aMP) have a similar tendency for triplex sta-
bility although their complex stability constants were not suffi-
cient (Fig. 6D).
4a: WNA-βAP
4b: WNA-βAP
N
Figure 3. NOESY result for the determination of the stereochemistry at the
anomeric position of the WNA-AP derivatives.
3. Conclusion
In this study, by expecting that replacement of the pyrimidine
base of the WNA analog with a smaller pyrazole ring might avoid
steric repulsion with the base pair of the target duplex to produce
a greater stability for the triplex, the new WNA analogs with 3-
aminopyrazole or 4-methyl-3-pyrazole-5-on were designed, incor-
porated into the TFOs, and evaluated for their stabilizing effect of
the triplexes. A remarkable success has been illustrated by the for-
mation of a stable triplex with WNA-bAP in the TFO sequence of 30-
GZG-50 toward the target duplex having the CG interrupting site.
From the fact that the smaller pyrazole ring was useful for
improvement of the triplex formation, this study has provided use-
In contrast to the original WNA-bC that did not produce any triplex
stability in the TFO3 sequence, interesting binding profiles were
observed for all the new WNA analogs; WNA-bAP for the AT site,
WNA-aAP for all four base pairs, WNA-bMP for the AT and GC
pairs, and WNA-2aMP for the GC pair. In particular, the triplex sta-
bility formed with WNA-MP was similar to those formed with the
natural dG/GC triplet (Fig. 6C, yellow bars vs pink and orange bars).
As the WNA analog lacking a heterocyclic unit in the TFO3 se-
quence showed no stabilizing effect to any base pairs,18 it is appar-
ent that the pyrazole recognition base has a benefit for improving
O
O
O
a
NH
NH
N Bz
N
N
H
N
H
Bz
6
O
R
R
N
N
N
b
N
O
O
R'O
O
R'O
R'O
OAc
O
β
O
β
O
O
α
R''O
R''O
R''O
α
R
N
N
N
R' = TBDPS
R" = Ac
N
7,9b
8,10b
2
O
R
O
7,9a
8,10a
7a,7b,8a,8b: R' = TBDPS, R" = Ac, R = Bz
9a,9b,10a,10b: R', R", R = H
c
Scheme 2. Reagents and conditions: (a) BzCl, pyridine, (86%); (b) 6, BSA, TMSOTf, CH3CN, rt (7b: WNA-2bMP; 10%, 7a: WNA-2
MP; 20%); (c) (1) TBAF, THF, rt, (2) 0.2 M NaOHaq, MeOH, THF, 0 °C (9b: WNA-2bMP; 72%, 9a: WNA-2 MP; 78%, 10b: WNA-1bMP; 44%, 10a: WNA-1
steps).
a
MP; 13%, 8b: WNA-1bMP; 11%, 8a: WNA-
1a
a
aMP; 42% in two

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Y. Taniguchi et al. / Bioorg. Med. Chem. 17 (2009) 6803–6810
mer FTIR-SpectrumOne. The high-resolution mass spectra were re-
H
corded by an Applied Biosystems Mariner System 5299
spectrometer using nicotinic acid, bradykinin and neurotensin as
the internal standard. The MALDI-TOF/MS spectra were recorded
by a Bruker Microflex using 3-hydroxypicolinic acid matrix.
H
H
HN
H
O
HO
HO
H
H
N
N
H
H
β
β
NH
O
O
H
H
O
O
O
H
H
H
H
OH
OH
4.1.1. N3-Dimethylaminomethylidine-3-aminopyrazole (1)
N,N-Dimethylformamidedimethylacetal (0.53 mL, 4.14 mmol)
was added to a solution of 3-aminopyrazole (207 mg, 2.44 mmol)
in methanol (8.0 mL). The reaction mixture was refluxed at 100 °C
for 1 h, and then the solvent was evaporated under reduced pres-
sure. The residue was treated with hexane and the precipitates were
collected, whichwere recrystallizedfrom ether/ethanol = 9:1 togive
a pale green solid (312 mg, 2.26 mmol, 92%). 1H NMR (400 MHz,
CDCl3): d 7.85 (1H, s), 7.37 (1H, d, J = 2.0 Hz), 5.82 (1H, d,
J = 2.0 Hz), 3.01 (6H, s). 13C NMR (125 MHz, CDCl3): 155.0, 135.8,
91.8, 40.5, 34.7. FTIR (neat): 3151, 1645, 1541, 1403 cmꢀ1. HRMS
(ESI) m/z: calc for C6H11N4 (M+H)+ 139.0978, found 139.0978.
NOE
NOE
10b: WNA-1βMP
9b: WNA-2βMP
NOE
H
NOE
H
HO
HO
H
H
H
H
H
O
O
H
H
N
H
O
O
α
α
N
N
H
H
OH
OH
H
HN
O
O
9a: WNA-2αMP
10a: WNA-1αMP
4.1.2. N1- and N2-Benzoyl-4-methyl-3-pyrazolin-5-one (6)
Benzoyl chloride (0.3 mL, 2.56 mmol) was added to a solution of
4-methyl-3-pyrazolin-5-one (251 mg, 2.56 mmol) in pyridine
(15 mL). After stirring for 2.5 h, the reaction was quenched by a
10% aqueous HCl solution and extracted with EtOAc. The organic
layer was washed with water and an aqueous saturated NaCl solu-
tion, dried over Na2SO4, and evaporated. The residue was purified
by flash silica gel column chromatography (CHCl3/MeOH = 10:1)
to give a mixture of two regioisomers as a colorless oil (448 mg,
2.21 mmol, 86%). 1H NMR (400 MHz, CDCl3): d 8.19 (1H, dd,
J = 1.22, 8.39 Hz), 7.75 (1H, d, J = 7.02 Hz), 7.62 (0.5H, t,
J = 7.63 Hz), 7.60 (0.5H, s), 7.57 (0.5H, t, J = 7.33 Hz), 7.48 (1H, t,
J = 7.63 Hz), 7.46 (1H, t, J = 7.33 Hz), 7.33 (0.5H, s), 1.96 (1.5H, s),
1.95 (1.5H, s). 13C NMR (125 MHz, CDCl3): 171.3, 165.6, 164.3,
133.4, 132.3, 131.9, 130.8, 130.1, 129.6, 128.4, 128.3, 110.7, 7.0.
FTIR (neat): 3205, 2925, 1743, 1697, 1623, 1537, 1261 cmꢀ1. HRMS
(ESI) m/z: calcd for C11H11N2O2 (M+H)+ 203.0815, found 203.0841.
Figure 4. NOESY result for the determination of the stereochemistry and regio-
chemistry of WNA-MP.
ful information for further optimization of the WNA analog for tri-
plex stabilization.
4. Experiments
4.1. General
The 1H NMR (400 MHz), 13C NMR (125 MHz) and HH-COSY,
NOESY spectra were recorded using Varian UNITY-400 and INO-
VA-500 spectrometers. The 31P NMR (161 MHz) spectrum was re-
corded using 10% phosphoric acid in D2O for the internal
standard at 0 ppm. The IR spectra were obtained using a Perkin El-
O
DNA synthesis
TFO1(α,βMP) 3'GGAAGGAZGGAGGAGGGA5'
TFO2(α,βMP) 3'GGAAGGGZGGAGGAGGGA5'
TFO3(α,βMP) 3'GGAAGGGZAGAGGAGGGA5'
TFO4(α,βMP) 3'GGAAGGAZAGAGGAGGGA5'
Z = WNA-2αMP or -2βMP
N
and purification
N
R'
O
RO
β
O
α
"RO
N
N
9,11b
O
R'
9,11a
9a,9b: R, R', R" = H
11a,11b: R = DMTr, R' = Ac, R" = iPr NPOC H CN
a
2
2 4
Scheme 3. Reagents and conditions: (a) (1) DMTrCl, pyridine, (2) AcO2, pyridine, (3) (iPr2N)2POCH2CH2CN, DIPEA, CH2Cl2, 0 °C (11b: WNA-2bMP; 39%, 11a: WNA-2
aMP; 51%
in three steps).
(B) XY=TA
10 40 100
(C) XY=GC
10 40 100
(D) XY= AT
10 40 100 (nM)
A) XY = CG
10 40 100
Duplex
Concentration
0
4
0
4
0
4
0
4
Triplex
32P-TFO
TFO2 (βAP)
Target
Duplex
Figure 5. Gel-shift assay for determination of triplex formation of the TFO2 (bAP) containing WNA-bAP in the sequence of 30- and 50-dG. The triplex formation was done for
12 h at 22 °C in a buffer containing 20 mM Tris–HCl, 5 mM MgCl2, 2.5 mM spermidine and 10% sucrose at pH 7.5. Electrophoresis was done at 10 °C using 20% non-denatured
polyacrylamide gel. Ten nM TFO containing the 32P-labeled one as the tracer was used. The concentration of the duplex was increased; 0, 4, 10, 40, 100 nM.

Y. Taniguchi et al. / Bioorg. Med. Chem. 17 (2009) 6803–6810
6807
Figure 6. Bar graphs of the association constants (Ks) of the TFOs. These values were calculated from the result of the gel-shift assay the conditions of which were described in
Figure 5. Ks = [Triplex]/[ssTFO][Duplex]. (A) TFO1; 30AZG50, (B) TFO2; 30GZG50, (C) TFO3; 30GZA50 and (D) TFO4; 30AZA50, Z = WNA analogues or dG.
4.2. General procedure for glycosidation reaction
4.02 (1H, dd, J = 2.8, 11.6 Hz), 3.75 (1H, dd, J = 3.7, 11.1 Hz), 3.38
(1H, dd, J = 7.6, 14.2 Hz), 3.02 (6H, s), 2.81 (1H, dd, J = 6.7,
14.2 Hz), 1.96 (3H, s), 1.01 (9H, s). 13C NMR (125 MHz, CDCl3):
169.7, 154.8, 140.8, 140.0, 135.7, 135.6, 133.3, 133.1, 129.7,
129.6, 128.2, 127.7,127.6, 127.3, 125.8, 93.1, 86.4, 86.0, 80.2,
73.0, 63.0, 47.9, 26.8, 20.8, 19.2. FTIR (neat): 1745, 1635, 1533,
N,O-Bis(trimethylsilyl)acetamide (2.0 equiv) was added to a
suspension of the protected pyrazole 1 or 6 (1.0 equiv) in CH3CN.
A solution of TMSOTf (1.0 equiv) and 2 (0.7 equiv) in CH3CN was
added to the above mixture. After stirring for 3 h, the reaction mix-
ture was quenched with an aqueous saturated NaHCO3 solution,
and extracted with EtOAc. The organic layer was washed with
water and brine, dried over Na2SO4, and evaporated. The isomers
were separated by flash silica gel column chromatography (hex-
ane/CHCl3/acetone = 2:2:1 to 1:1:1) to give each isomer.
1237 cmꢀ1 HRMS (ESI) m/z: calcd for C37H45N4O5Si (M+H)+
.
653.3154, found 653.3126.
4.2.3. (10S,30R,40R,50R,70R)-N1-Benzoyl-2-{40-acetoxy-10-phenyl-
30-(t-butyldiphenylsilyloxymethyl)-20,60-dioxabicyclo[3.3.0]oct-
70-yl}-4-methyl-3-pyrazolin-5-one (7a)
4.2.1. (10S,30R,40R,50R,70R)-N3-Dimethylaminomethylidine-1-{40-
acetoxy-10-phenyl-30-(t-butyldiphenylsilyloxymethyl)-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-3-aminopyrazole (3a)
(WNA-2a
MP) 1H NMR (400 MHz, CDCl3): d 8.18 (2H, dd, J = 1.2,
7.9 Hz), 7.66–7.24 (19H, m), 6.17 (1H, dd, J = 4.5, 7.6 Hz), 5.00 (1H,
dd, J = 4.2, 8.8 Hz), 4.85 (1H, d, J = 3.9 Hz), 4.21 (1H, dt, J = 3.3,
8.8 Hz), 3.98 (1H, dd, J = 3.0, 11.5 Hz), 3.71 (1H, dd, J = 3.9,
11.5 Hz), 3.23 (1H, dd, J = 4.5, 15.1 Hz), 2.94 (1H, dd, J = 7.6,
15.1 Hz), 1.99 (3H, s), 1.94 (3H, s), 0.99 (9H, s). 13C NMR
(125 MHz, CDCl3): 170.1, 164.1, 154.2, 141.1, 135.7, 135.6, 133.7,
133.2, 133.1, 131.0, 130.4, 129.7, 127.6, 125.2, 111.5, 108.2, 92.5,
91.9, 87.4, 79.7, 72.5, 62.7, 47.8, 26.8, 20.7, 19.2, 7.6. FTIR (neat):
1745, 1601, 1448 cmꢀ1. HRMS (ESI) m/z: calcd for C42H45N2O7Si
(M+H)+ 717.2991, found 717.3039.
(WNA-a
AP) 1H NMR (400 MHz, CDCl3): d 7.71–7.29 (17H, m),
6.59 (1H, t, J = 7.6 Hz), 5.67 (1H, d, J = 1.8 Hz), 5.09 (1H, dd,
J = 4.6, 9.3 Hz), 4.68 (1H, dt, J = 9.2 Hz), 4.66 (1H, d, J = 4.3 Hz),
4.15 (1H, dd, J = 2.4, 11.6 Hz), 3.74 (1H, dd, J = 2.4, 11.9 Hz), 3.53
(1H, dd, J = 7.6, 14.3 Hz), 3.03 (6H, s), 2.83 (1H, dd, J = 7.3,
14.3 Hz), 1.92 (3H, s), 1.03 (9H, s). 13C NMR (125 MHz, CDCl3):
170.2, 154.8, 143.1, 139.9, 135.6, 133.3, 133.2, 129.6, 128.3,
127.7, 127.6, 127.2, 125.3, 91.9, 86.2, 85.9, 78.6, 71.8, 61.8, 46.5,
26.8, 20.7, 19.2. FTIR (neat): 1743, 1635, 1534, 1239 cmꢀ1. HRMS
(ESI) m/z: calcd for C37H45N4O5Si (M+H)+ 653.3154, found
653.3132.
4.2.4. (10S,30R,40R,50R,70S)-N1-Benzoyl-2-{40-acetoxy-10-phenyl-
30-(t-butyldiphenylsilyloxymethyl)-20,60-dioxabicyclo[3.3.0]oct-
70-yl}-4-methyl-3-pyrazolin-5-one (7b)
4.2.2. (10S,30R,40R,50R,70S)-N3-Dimethylaminomethylidine-1-{40-
acetoxy-10-phenyl-30-(t-butyldiphenylsilyloxymethyl)-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-3-aminopyrazole (3b)
(WNA-2bMP) 1H NMR (400 MHz, CDCl3): d 8.21 (2H, dd, J = 1.2,
7.0 Hz), 7.68–7.20 (19H, m), 6.09 (1H, t, J = 6.7 Hz), 5.08 (1H, dd,
J = 3.9, 9.1 Hz), 4.99 (1H, d, J = 3.9 Hz), 4.28 (1H, dt, J = 3.6,
9.1 Hz), 4.01 (1H, dd, J = 3.0, 11.6 Hz), 3.75 (1H, dd, J = 3.6,
11.6 Hz), 3.30 (1H, dd, J = 6.7, 14.4 Hz), 2.89 (1H, dd, J = 6.1,
14.4 Hz), 2.01 (3H, s), 1.94 (3H, s), 1.01 (9H, s). 13C NMR
(WNA-bAP) Colorless foam. 1H NMR (400 MHz, CDCl3): d 7.81–
7.26 (17H, m), 6.67 (1H, t, J = 7.0 Hz), 5.68 (1H, s), 5.14 (1H, dd,
J = 3.8, 9.3 Hz), 5.04 (1H, d, J = 3.7 Hz), 4.35 (1H, dt, J = 9.2 Hz),

6808
Y. Taniguchi et al. / Bioorg. Med. Chem. 17 (2009) 6803–6810
(125 MHz, CDCl3): 169.8, 164.0, 154.9, 140.1, 135.7, 135.6, 133.8,
133.2, 133.0, 130.5, 129.7, 129.5, 128.8, 128.6, 128.4, 127.7,
127.6, 125.6, 107.7, 92.7, 91.7, 86.6, 80.3, 72.9, 62.9, 48.5, 26.8,
20.7, 19.2, 7.4. FTIR (neat): 1746, 1600 cmꢀ1. HRMS (ESI) m/z: calcd
for C42H45N2O7Si (M+H)+ 717.2991, found 717.3032.
34.6. FTIR (neat): 3368, 1633 cmꢀ1. HRMS (ESI) m/z: calcd for
C19H25N4O4 (M+H)+ 373.1870, found 373.1898.
4.3.3. (10S,30R,40R,50R,70R)-2-(40-Hydroxy-30-hydroxymethyl-10-
phenyl-20,60-dioxabicyclo[3.3.0]oct-70-yl)-4-methyl-3-pyrazolin-
5-one (9a)
4.2.5. (10S,30R,40R,50R,70R)-N2-Benzoyl-1-{40-acetoxy-10-phenyl-
30-(t-butyldiphenylsilyloxymethyl)-20,60-dioxabicyclo[3.3.0]oct-
70-yl}-4-methyl-3-pyrazolin-5-one (8a)
(WNA-2a
MP) 1H NMR (400 MHz, CD3OD): d 7.60 (2H, d,
J = 8.2 Hz), 7.47 (1H, s), 7.37 (2H, t, J = 7.3 Hz), 7.27 (1H, t,
J = 7.3 Hz), 6.38 (1H, t, J = 6.6 Hz), 4.45 (1H, d, J = 4.3 Hz), 4.15–4.12
(1H, m), 3.87–3.84 (2H, m), 3.69 (1H, dd, J = 6.4, 11.9 Hz), 2.87 (1H,
d, J = 6.6 Hz), 1.86 (3H, s). 13C NMR (125 MHz, CD3OD): 162.5,
143.6, 131.0, 129.4, 128.5, 126.2, 103.6, 93.0, 90.5, 83.2, 73.1, 63.4,
48.3, 7.0. FTIR (neat): 3354, 1708, 1603, 1528 cmꢀ1. HRMS (ESI) m/
z: calcd for C17H21N2O5 (M+H)+ 333.1445, found 333.1473.
(WNA-1a
MP) 1H NMR (400 MHz, CDCl3): d 8.19 (2H, dd, J = 1.2,
7.9 Hz), 7.67–7.21 (19H, m), 6.17 (1H, t, J = 6.5 Hz), 4.98 (1H, dd,
J = 4.5, 9.1 Hz), 4.71 (1H, d, J = 4.2 Hz), 4.21 (1H, d, J = 9.1 Hz),
4.00 (1H, dd, J = 2.4, 11.8 Hz), 3.65 (1H, dd, J = 5.5 Hz, 14.9 Hz),
3.59 (1H, dd, J = 2.4, 11.8 Hz), 2.88 (1H, dd, J = 7.9, 14.9 Hz), 1.92
(3H, s), 1.90 (3H, s), 0.97 (9H, s). FTIR (neat): 1750, 1600,
1239 cmꢀ1
.
HRMS (ESI) m/z: calcd for C42H45N2O7Si (M+H)+
4.3.4. (10S,30R,40R,50R,70S)-2-(40-Hydroxy-30-hydroxymethyl-10-
phenyl-20,60-dioxabicyclo[3.3.0]oct-70-yl)-4-methyl-3-pyrazolin-
5-one (9b)
717.2991, found 717.3028.
4.2.6. (10S,30R,40R,50R,70S)-N2-Benzoyl-1-{40-acetoxy-10-phenyl-
30-(t-butyldiphenylsilyloxymethyl)-20,60-dioxabicyclo[3.3.0]oct-
70-yl}-4-methyl-3-pyrazolin-5-one (8b)
(WNA-2bMP) 1H NMR (400 MHz, CD3OD): d 7.73 (2H, d,
J = 7.6 Hz), 7.38 (1H, s), 7.34 (2H, t, J = 7.3 Hz), 7.24 (1H, t,
J = 7.3 Hz), 6.00 (1H, t, J = 6.8 Hz), 4.66 (1H, d, J = 3.6 Hz), 4.05–
4.00 (1H, m), 3.88 (1H, dd, J = 2.1, 11.9 Hz), 3.81 (1H, dd, J = 3.6,
10.7 Hz), 3.68 (1H, dd, J = 6.1, 10.7 Hz), 3.12 (1H, dd, J = 7.9,
14.0 Hz), 2.69 (1H, dd, J = 6.1, 14.0 Hz), 1.92 (3H, s). 13C NMR
(125 MHz, CD3OD): 162.7, 142.5, 131.9, 129.3, 128.4, 126.6,
102.9, 93.2, 90.0, 84.5, 73.6, 63.7, 49.1, 6.9. FTIR (neat): 3386,
1609, 1530 cmꢀ1. HRMS (ESI) m/z: calcd for C17H21N2O5 (M+H)+
333.1445, found 333.1420.
(WNA-1bMP) 1H NMR (400 MHz, CDCl3): d 8.21 (2H, dd, J = 1.2,
7.9 Hz), 7.70–7.21 (19H, m), 6.14 (1H, t, J = 6.7 Hz), 5.04 (1H, dd,
J = 3.6, 9.2 Hz), 4.98 (1H, d, J = 3.9 Hz), 4.20 (1H, dt, J = 3.3,
9.4 Hz), 3.93 (1H, dd, J = 3.0, 11.6 Hz), 3.68 (1H, dd, J = 3.6,
11.6 Hz), 3.39 (1H, dd, J = 7.0, 14.3 Hz), 2.87 (1H, dd, J = 6.7,
14.3 Hz), 1.92 (3H, s), 1.90 (3H, s), 0.97 (9H, s). FTIR (neat): 1751,
1600, 1240 cmꢀ1. HRMS (ESI) m/z: calcd for C42H45N2O7Si (M+H)+
717.2991, found 717.2979.
4.4. General procedure of synthesis for the WNA-AP
phosphoramidite
4.3. General procedure for deprotection reaction
A THF solution of the above compound and TBAF (1.0 M THF
solution, 2 equiv) was stirred for 3.5 h at room temperature. MeOH
and a 0.2 M aqueous NaOH solution (2 equiv) were then added to
this mixture at 0 °C. After stirring for 2.5 h at 0 °C, the reaction
was quenched with acetic acid and diluted with MeOH. The sol-
vents were removed under reduced pressure. The residue was
purified by flash silica gel column chromatography (CHCl3/
CH3OH = 10:1) to give the corresponding diol compound.
DMTrCl (3 equiv) was added to a solution of the diol compound
in pyridine. After stirring for 1 h, the reaction mixture was diluted
with EtOAc, and then washed with water and brine. The organic
layer was washed with water and brine, dried over Na2SO4, evap-
orated, and then the residue was purified by flash silica gel column
chromatography (CHCl3/CH3OH = 99:1 containing 0.5% pyridine) to
give the corresponding DMTr-protected WNA. iP2NEt (6 equiv) was
added to
a solution of the above DMTr-WNA and iPr2NP(-
Cl)OC2H4CN (3 equiv) in CH2Cl2 at 0 °C. After stirring for 1 h, the
reaction mixture was diluted with an aqueous saturated NaHCO3
solution, and extracted with EtOAc. The organic layer was dried
over Na2SO4, evaporated, then the residue was purified by flash sil-
ica gel column chromatography (hexane/EtOAc = 1:1) to give the
purified materials.
4.3.1. (10S,30R,40R,50R,70R)-N3-Dimethylaminomethylidine-1-(40-
hydroxy-30-hydroxymethyl-10-phenyl-20,60-
dioxabicyclo[3.3.0]oct-70-yl)-3-aminopyrazole (4a)
(WNA-a
AP) 1H NMR (400 MHz, CDCl3): d 7.73 (1H, s), 7.50 (2H,
d, J = 7.3 Hz), 7.44 (1H, s), 7.38 (2H, t, J = 7.6 Hz), 7.28 (1H, t,
J = 7.3 Hz), 6.63 (1H, t, J = 7.1 Hz), 5.71 (1H, s), 4.53 (1H, d,
J = 6.7 Hz), 4.34–4.32 (1H, m), 4.04 (1H, d, J = 7.3 Hz), 4.00 (1H,
dd, J = 1.8, 10.8 Hz), 3.80 (1H, dd, J = 4.6, 10.8 Hz), 3.23 (1H, dd,
J = 6.7, 14.6 Hz), 3.03 (6H, s), 2.79 (1H, dd, J = 7.3, 14.6 Hz). 13C
NMR (125 MHz, CDCl3): 154.9, 151.7, 144.0, 140.1, 128.6, 127.4,
127.6, 124.3, 92.6, 89.9, 87.9, 86.8, 83.2, 72.1, 62.7, 46.0, 40.4,
34.6. FTIR (neat): 3351, 1633 cmꢀ1. HRMS (ESI) m/z: calcd for
C19H25N4O4 (M+H)+ 373.1870, found 373.1908.
4.4.1. (10S,30R,40R,50R,70R)-N3-Dimethylaminomethylidine-1-{30-
dimethoxytrithyloxymethyl-40-O-(N,N-diisopropyl-b-
cyanoethylphosphoramidyl)-10-phenyl-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-3-aminopyrazole (5a)
(WNA-a
AP) 1H NMR (400 MHz, CDCl3): d 7.74 (1H, s), 7.67 (1H,
d, J = 7.3 Hz), 7.50–7.15 (12H, m), 6.78 (4H, dd, J = 1.5, 8.8 Hz), 6.60
(1H, t, J = 7.0 Hz), 5.67 (1H, s), 4.69–4.65 (1H, m), 4.50 (1H, d,
J = 4.8 Hz), 4.38–4.37 (1H, m), 3.99–3.87 (1H, m), 3.76 (6H, s),
3.77 (6H, s), 3.76–3.17 (7H, m), 3.05–3.01 (6H, m), 2.88–2.82
(1H, m), 1.06–0.78 (9H, m). 31P NMR (162 MHz, CDCl3): 149.32,
146.96. FTIR (neat): 2836, 1635, 1508, 1251 cmꢀ1. ESI-MS (m/z)
876.56 [M+H]+.
4.3.2. (10S,30R,40R,50R,70S)-N3-Dimethylaminomethylidine-1-(40-
hydroxy-30-hydroxymethyl-10-phenyl-20,60-dioxabicyclo[3.3.0]-
oct-70-yl)-3-aminopyrazole (4b)
(WNA-bAP) 1H NMR (400 MHz, CDCl3): d 7.75 (1H, s), 7.65 (2H,
d, J = 7.6 Hz), 7.46 (1H, s), 7.36 (2H, t, J = 7.6 Hz), 7.26 (1H, t,
J = 7.3 Hz), 6.66 (1H, t, J = 7.0 Hz), 5.73 (1H, s), 4.86 (1H, d,
J = 3.6 Hz), 4.07 (1H, d, J = 2.4 Hz), 4.01 (1H, d, J = 3.9 Hz), 3.99
(1H, dd, J = 3.3, 12.0 Hz), 3.80 (1H, dd, J = 4.9, 12.0 Hz), 3.45 (1H,
dd, J = 7.9, 13.9 Hz), 3.06 (6H, s), 2.82 (1H, dd, J = 6.1, 13.9 Hz).
13C NMR (125 MHz, CDCl3): 154.9, 151.9, 140.8, 140.3, 128.5,
127.6, 125.3, 92.4, 89.9, 87.9, 85.5, 84.0, 73.1, 63.2, 47.5, 40.4,
4.4.2. (10S,30R,40R,50R,70S)-N3-Dimethylaminomethylidine-1-{30-
dimethoxytrithyloxymethyl-40-O-(N,N-diisopropyl-b-
cyanoethylphosphoramidyl)-10-phenyl-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-3-aminopyrazole (5b)
(WNA-bAP) 1H NMR (400 MHz, CDCl3): d 7.89 (1H, d, J = 7.0 Hz),
7.82 (1H, d, J = 7.0 Hz), 7.75 (0.5H, s), 7.68 (0.5H, s), 7.50–7.44

Y. Taniguchi et al. / Bioorg. Med. Chem. 17 (2009) 6803–6810
6809
(2.5H, m), 7.41–7.34 (4.5H, m), 7.29–7.17 (4H, m), 6.79 (4H, d,
4.6. Synthesis of triplex-forming oligonucleotides
J = 8.8 Hz), 6.73–6.69 (1H, m), 5.69 (0.5H, d, J = 1.8 Hz), 5.64
(0.5H, d, J = 2.1 Hz), 4.85 (1H, dd, J = 3.6, 14.0 Hz), 4.38–4.37 (1H,
m), 4.24–4.18 (0.5H, m), 4.00–3.95 (0.5H, m), 3.77 (6H, s), 3.74–
3.68 (0.5H, m), 3.50–3.20 (6H, m), 3.02 (6H, s), 2.97–2.90 (1H,
m), 2.56–2.48 (0.5H, m), 2.36–2.25 (1.5H, m), 2.14 (0.5H, s),
1.05–1.00 (6H, m), 0.88–0.84 (6H, m), 0.78 (2H, d, J = 6.7 Hz). 31P
NMR (162 MHz, CDCl3): 149.13, 148.41. FTIR (neat): 2836, 1635,
1508, 1250 cmꢀ1. ESI-MS (m/z) 876.56 [M+H]+.
The triplex-forming oligonucleotides (TFOs) containing WNA
analogs having the pyrazole ring as a recognition base were syn-
thesized using an automated DNA synthesizer (Applied Biosystems
394 DNA/RNA Synthesizer). After cleavage from CPG, they were
purified by HPLC (HPLC conditions: COSMOSIL 5C18-MS-II
(10 ꢁ 250 mm), Buffer; A: 0.1 M TEAA, B: CH3CN, 10–40%/20 min,
40–100%/30 min linear gradient, Flow rate; 3.0 mL/min, UV-detec-
tor; 254 nm). All the synthesized TFOs were identified by the MAL-
DI-TOF MS (negative mode) measurements. MALDI-TOF MASS
4.5. General procedure of synthesis for WNA-MP
phosphoramidite
results: TFO1(bAP): calcd for 5815.05, found 5814.78, TFO1(
calcd for 5815.05, found 5811.49, TFO1(2bMP): calcd for 5830.27,
found 5832.73, TFO1(2 MP): calcd for 5830.27 found 5829.18,
TFO2(bAP): calcd for 5831.04, found 5831.01, TFO2( AP): calcd
for 5831.04, found 5831.38, TFO2(2bMP): calcd for 5846.26, found
5846.64, TFO2(2 MP): calcd for 5846.26 found 5846.49, TFO3-
(bAP): calcd for 5815.05, found 5814.04, TFO3( AP): calcd for
5810.05, found 5810.33, TFO3(2bMP): calcd for 5830.27, found
5828.69, TFO3(2 MP): calcd for 5830.27 found 5829.46, TFO4-
(bAP): calcd for 5799.06, found 5799.73, TFO4( AP): calcd for
5799.06, found 5797.73, TFO4(2bMP): calcd for 5814.28, found
5810.75, TFO4(2 MP): calcd for 5814.28 found 5812.68.
aAP):
a
DMTrCl (3 equiv) was added to a solution of the diol compound
in pyridine. After stirring for 1 h, the reaction mixture was diluted
with EtOAc, and then washed with water and brine. The organic
layer was washed with water and brine, dried over Na2SO4, evap-
orated, and then the residue was purified by flash silica gel column
chromatography (CHCl3/CH3OH = 99:1 containing 0.5% pyridine) to
give the corresponding DMTr-protected WNA. Acetic anhydride
(1.2 equiv) was added to the solution of DMTr-WNA in pyridine.
After stirring for 2 h, the reaction mixture was diluted with EtOAc
and washed with an aqueous saturated NaCl solution. The organic
layer was dried over Na2SO4, evaporated, then the residue was
purified by flash silica gel column chromatography (CHCl3/
CH3OH = 99:1 containing 0.5% pyridine) to give the purified
DMTr-Ac-protected WNA. iP2NEt (6 equiv) was added to a solution
of the above DMTr-Ac-WNA and (iPr2N)2POC2H4CN (3 equiv) in
CH2Cl2 at 0 °C. After stirring for 1 h, the reaction mixture was di-
luted with a satd NaHCO3 solution, and extracted with EtOAc.
The organic layer was dried over Na2SO4, and evaporated, and then
the residue was purified by flash silica gel column chromatography
(hexane/EtOAc = 1:1) to give the purified materials.
a
a
a
a
a
a
4.7. Evaluation of triplex formation by the gel-shift assay
The triplex formation was performed for 12 h at 22 °C in a buf-
fer containing 20 mM Tris–HCl, 5 mM MgCl2, 2.5 mM spermidine
and 10% sucrose at pH 7.5, and analyzed by electrophoresis at
10 °C with a 20% non-denatured polyacrylamide gel. Ten nM TFO
containing the 32P-labeled one as the tracers was used. The TFO
and the triplex bands were quantified to produce the relative con-
centrations, and the association constant was calculated using the
concentration of each component and the following equation,
Ks = [Triplex]/[Duplex][TFO].
4.5.1. (10S,30R,40R,50R,70R)-N1-Acethyl-2-{30-
dimethoxytrithyloxymethyl-40-O-(N,N-diisopropyl-b-
cyanoethylphosphoramidyl)-10-phenyl-20,60-
Acknowledgments
dioxabicyclo[3.3.0]oct-70-yl}-4-methyl-3-pyrazolin-5-one (11a)
(WNA-2a
MP) 1H NMR (400 MHz, CDCl3): d 7.75 (0.3H, s), 7.72
S.S. and Y.T. are grateful for the support by a Grant-in-Aid for
Scientific Research (A) and Young Scientists (B) from the Japan
Society for the Promotion of Science (JSPS), and CREST from the Ja-
pan Science and Technology Agency.
(0.7H, s), 7.57 (1.4H, d, J = 7.0 Hz), 7.55 (0.6H, d, J = 7.0 Hz), 7.42
(2H, d, J = 7.0 Hz), 7.34–7.16 (10H, m), 6.78 (4H, d, J = 8.8 Hz),
6.20–6.15 (1H, m), 4.72 (0.3H, d, J = 3.9 Hz), 4.62 (0.7H, d,
J = 3.9 Hz), 4.28 (0.7H, t, J = 3.9, 9.3 Hz), 4.18–4.16 (0.3H, m), 4.09
(0.7H, m), 3.96–3.95 (0.3H, m), 3.77 (6H, s), 3.73–3.70 (1H, m),
3.55–3.46 (2H, m), 3.44–3.38 (2H, m), 2.34–3.15 (2H, m), 2.96–
2.89 (1H, m), 2.29 (0.9H, s), 2.28 (2.1H, s), 1.95 (0.9H, s), 1.92
(2.1H, s), 1.33–1.18 (3H, m), 1.09 (4H, d, J = 6.7 Hz), 1.04 (1.8H, d,
J = 6.7 Hz), 1.03 (4.2H, d, J = 6.7 Hz), 0.83 (2H, d, J = 6.7 Hz). 31P
NMR (162 MHz, CDCl3): 150.21, 148.85. FTIR (neat): 2252, 1766,
1607, 1583, 1508 cmꢀ1. ESI-MS (m/z) 878.43 [M+H]+.
References and notes
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2008, 36, 5123.
2. Rusling, D. A.; Broughton-Head, V. J.; Brown, T.; Fox, K. R. Curr. Chem. Biol. 2008,
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3. Simon, P.; Cannata, F.; Concordet, J.-P.; Giovannangeli, C. Biochimie 2008, 90,
1109.
4. Lange, S. S.; Reddy, M. C.; Vasquez, K. M. DNA Repair 2009, 8, 865.
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4.5.2. (10S,30R,40R,50R,70S)-N1-Acethyl-2-{30-
dimethoxytrithyloxymethyl-40-O-(N,N-diisopropyl-b-
cyanoethylphosphoramidyl)-10-phenyl-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-4-methyl-3-pyrazolin-5-one (11b)
(WNA-2bMP) 1H NMR (400 MHz, CDCl3):
d 7.65 (2H, d,
J = 6.7 Hz), 7.45 (2H, t, J = 7.6 Hz), 7.38–7.18 (11H, m), 6.79 (4H,
d, J = 8.8 Hz), 6.12–6.06 (1H, m), 4.79 (0.5H, d, J = 3.6 Hz), 4.71
(0.5H, d, J = 2.7 Hz), 4.33–4.22 (1.5H, m), 4.03–3.98 (0.5H, m),
3.78 (3H, s), 3.77 (3H, s), 3.72 (1H, dd, J = 6.7, 14.8 Hz), 3.50–3.19
(6H, m), 3.00–2.92 (1H, m), 2.47 (1H, t, J = 6.4 Hz), 2.32 (2H, s),
2.31 (2H, s), 1.91 (1.5H, s), 1.89 (1.5H, s), 1.07 (3H, d, J = 6.7 Hz),
1.04 (3H, d, J = 6.7 Hz), 0.95 (3H, d, J = 6.7 Hz), 0.82 (3H, d,
J = 6.7 Hz). 31P NMR (162 MHz, CDCl3): 150.52, 149.27. FTIR (neat):
2252, 1766, 1607, 1583, 1508 cmꢀ1. ESI-MS (m/z) 878.66 [M+H]+.
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