Selective Formation of Stable Triplexes Including a TA or a CG Interrupting Site with New Bicyclic Nucleoside Analogues (WNA)

Article abstract of DOI:10.1021/ja037211z

Triplex-forming oligonucleotides (TFOs) are potential DNA-targeting molecules and would become powerful tools for genomic research. As the stabilization of the TFO is partially provided by hydrogen bonds to purine bases, the most stable triplexes form wit

Full text of DOI:10.1021/ja037211z

Published on Web 12/23/2003
Selective Formation of Stable Triplexes Including a TA or a
CG Interrupting Site with New Bicyclic Nucleoside Analogues
(WNA)
Shigeki Sasaki,*,† Yosuke Taniguchi, Ryo Takahashi, Yusuke Senko,
Keiichi Kodama, Fumi Nagatsugi, and Minoru Maeda
Contribution from the Graduate School of Pharmaceutical Sciences, Kyushu UniVersity,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, and CREST, Japan Science and
Technology Agency
Received July 11, 2003; E-mail: sasaki@phar.kyushu-u.ac.jp
Abstract: Triplex-forming oligonucleotides (TFOs) are potential DNA-targeting molecules and would become
powerful tools for genomic research. As the stabilization of the TFO is partially provided by hydrogen bonds
to purine bases, the most stable triplexes form with homopurine/homopyrimidine sequences, and a pyrimidine
base in the purine strand of the duplex interrupts triplex formation. If a TFO can recognize sequences
including such an interrupting site, the target regions in the genome would be expanded to a greater extent.
However, this problem has not been generally solved despite extensive studies. We have previously reported
a new base analogue (WNA) constructed of three parts, a benzene ring, a heterocyclic ring, and a bicyclic
skeleton to hold these two parts. In this study, we have further investigated modification of WNA
systematically and determined two useful WNA analogues, WNA-âT and WNA-âC, for selective stabilization
of triplexes at a TA and a CG interrupting site, respectively. The triplexes with WNA analogues have exhibited
an interesting property in that they are more stable than natural-type triplexes even at low Mg2+ concentration.
From comparison of the results with H-WNA-âT lacking benzene and those with WNA-H without thymine,
it has been suggested that benzene is a major contributor for triplex stability and thymine provides selectivity.
Thus, it has been successfully demonstrated that WNA-âT/TA and WNA-âC/CG combinations may expand
triplex recognition codes in addition to the natural A/AT and G/GC base triplet codes. The results of this
study will provide useful information for the design of new WNA analogues to overcome inherent problems
for further expansion of triplex recognition codes.
Introduction
major groove with hydrogen bonds between the bases in the
third strand and those in the purine strand of the duplex, the
DNA-recognizing molecules would become powerful tools
for genomic research through gene inhibition, activation, gene
conversion and/or recombination, and gene therapy, etc. One
potential candidate of great interest has been triplex-forming
oligonucleotides (TFOs) and related molecules.1-7 Recent
successful illustrations of the application include the achieve-
ment of specific mutations by TFOs in mice, inhibition of cell
growth by PEG-TFO conjugates, and so on. There is, however,
an intrinsic limitation in the sequences that can be targeted by
TFOs.1,8 As a TFO recognizes DNA sequence by binding the
most stable triplexes form on homopurine/homopyrimidine
sequences. Even a single pyrimidine base in the purine strand
of the duplex interrupts triplex formation to a great extent. If
TFOs can recognize sequences including such an interrupting
site, their target regions within the genome would be expanded
to a greater extent. Despite numerous studies for more than a
decade, this limitation has remained an unsolved problem.9-13
In triplexes, pyrimidine-TFOs adopt parallel orientation, and
purine-TFOs tend toward an antiparallel orientation to the purine
strand of the duplex. Recently, interesting nucleoside analogues
have been reported from Leumann’s and Imanishi’s groups,
which stabilize parallel triplexes by forming one hydrogen bond
to the cytosine base at a C-G interrupting site.14-17 The bicyclic
† CREST, Japan Science and Technology Agency.
(1) Soyfer, V. N.; Potaman, V. N. Triple-Helical Nucleic Acids, Springer-
Verlag: New York, 1996.
(2) Rapozzi, V.; Cogoi, S.; Spessotto, P.; Risso, A.; Bonora, G.; Quadrifoglio,
F.; Xodo, L. Biochemistry 2002, 41, 502-510.
(3) Cogoi, S.; Rapozzi, V.; Quadrifoglio, F.; Xodo, L. Biochemistry 2001, 40,
1135-1143.
(9) Griffin, L. C.; Kiessling, L. L.; Beal, P. A.; Gillespie, P.; Dervan, P. B. J.
Am. Chem. Soc. 1992, 114, 7979-7982.
(10) Huang, C. Y.; Cushman, C. D.; Miller, P. S. J. Org. Chem. 1993, 58, 5048-
5049.
(4) Casey, B. P.; Glazer, P. M. Prog. Nucleic Acid Res. Mol. Biol. 2001, 67,
163-192.
(5) Vasquez, K. M.; Narayanan, L.; Glazer, P. M. Science 2000, 209, 530-
533.
(11) Sasaki, S.; Nakashima, S.; Nagatsugi, F.; Tanaka, Y.; Hisatome, M.; Maeda,
M. Tetrahedron Lett. 1995, 36, 9521-9524.
(6) Kuznetsova, S.; Ait-Si-Ali, S.; Nagibneva, I.; Troalen, F.; Le Villain, J.-
P.; Harel-Bellan, A.; Svinarchuk, F. Nucleic Acids Res. 1999, 27, 3995-
4000.
(12) Rao, T. S.; Hogan, M. E.; Revankar, G. R. Nucleotides Nucleosides 1994,
13, 95-107.
(7) Stanojevic, D.; Young, R. A. Biochemistry 2002, 41, 7209-7216.
(8) Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-650.
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9
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J. AM. CHEM. SOC. 2004, 126, 516-528
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A R T I C L E S
sugar structure of Imanishi’s nucleoside analogues (BNA or
LNA) is thought to bring an additional stabilizing effect.
Unfortunately, the validity of their design concept has not been
proved in a search for nucleoside analogues for a TA interrupting
site.
Compared to studies for parallel triplexes, fewer studies have
been made for antiparallel triplexes with interrupting sites. An
advantage of antiparallel triplexes is that they are formed under
neutral conditions with high stability, whereas a disadvantageous
point is that their triplex formation is inhibited by physiological
ionic conditions, especially in the presence of K+. We recently
developed a new general structure of nucleoside analogues
(WNA) with a bicyclo[3.3.0]octane skeleton bearing an aromatic
part for stacking and a nucleobase for Hoogesteen hydrogen
bonds.18-20 Our initial success encouraged us to undertake
further systematic investigations, and we have determined two
useful analogues for selective stabilization of triplexes at a TA
or a CG interrupting site. An interesting property of WNA
analogues is that the triplex stability with the WNA analogue
is higher than that of natural-type triplexes even at low Mg2+
concentration. Here, we describe in detail the design, synthesis,
and evaluation of the triplex-forming ability of the TFOs
incorporating the new base analogues (WNA).
Figure 1. Schematic illustration of difference in backbone geometry of
parallel and antiparallel triplex. Circles represent the position of C1′. (A)
T-AT triplet, (B) A-AT triplet, (C) an anticipated triplet of C1′ RA-TA,
(D) an anticipated triplet of WNA-7âG-TA.
Results
tion of a nucleobase unit might become restricted by construction
of a bicyclic ring (Figure 2B). Finally, we considered that a
benzene ring at the C1′-â position might occupy a space where
a base unit of a natural nucleoside is located and provide
stacking and/or van der Waals interactions (Figure 2C). A
simulated structure of the triplex including a WNA-7âG and a
TA interrupting site suggested that a benzene ring of WNA-
7âG might be stacked between adjacent purine bases of the TFO
(Figure 3A). It was also predicted that a benzene ring together
with a nucleobase might provide a shape complementary to a
TA base pair (Figure 3B). Thus, we proposed an original
approach of separating the hydrogen-bonding and base-stacking
contributions into triplex formation at the interrupting sites. As
a benzene ring, a five-membered ring and a nucleobase seem
to be aligned in a W-shape; we named the new molecule
W-shape nucleoside analogue (WNA) with the number (as
necessary) and R or â to represent the alkylation position of
the nucleobase and the stereochemistry of its glycosidic bond,
respectively. In the preliminary study, the validity of this
molecular design was demonstrated by the fact that the TFO
incorporating WNA-7âG (2b) forms the triplex toward the
duplex with a TA interrupting site.18
Design. Parallel and antiparallel triplexes differ remarkably
from each other in the geometry of the sugar-phosphate
backbone. It is anticipated that the backbone of the purine-TFO
in the antiparallel triplexes is located at approximately the central
position in the major groove, whereas the backbone of the
pyrimidine-TFO is much closer to the purine strand of the
duplex (Figure 1, A vs B). Taking such a difference into account,
nucleoside analogues with C1′-R-glycosidic bond were inves-
tigated, but selective formation of triplexes including interrupting
sites has not been clearly shown.13,21 Triplex stability is provided
by synchronization of interactions of hydrogen bonds, stacking
interactions, shape complimentarity, electrostatic interactions
with metal cations, and so on. It may be speculated that a
nucleobase with C1′-R stereochemistry might not adopt the
appropriate position for hydrogen bonds to the target base pair
(Figure 1C). It seemed to us that an appropriate spacer is needed
to bring a nucleobase so as to interact with a distant purine
base in the pyrimidine strand. Therefore, we designed new
nucleoside analogues with an appropriate spacer. Our basic
concept for the design of the new nucleoside analogue is
schematically illustrated in Figure 2 using WNA-7âG as an
example.
Encouraged by the initial success with WNA-7âG (2b),18 we
undertook further systematic investigation with a variety of
WNA analogues by altering the nucleobase unit. The compounds
used in this study are shown in Figure 4.
Based on molecular modeling, an ethylene spacer with C1′-R
stereochemistry was designed to connect a nucleobase unit for
hydrogen bonds to a distant purine base (Figure 2A). Conforma-
Synthesis. The WNA derivatives were synthesized with
D-ribose by a modification of the previously reported method18
(Scheme 1). The intermediate having a benzene ring at the
1-position (10) was prepared by a multistep synthesis including
protection of the 2,3-dihydroxyl group with acetonide, selective
protection of the 5-hydroxyl group with the TBDMS group,
oxidation of the 1-hydroxyl group to carbonyl,22 and addition
of phenyllithium. Allylation at the 1-position gave 11 in a ratio
(15) Prevot-Halter, I.; Leumann, C. J. Bioorg. Med. Chem. Lett. 1999, 9, 2657-
2660.
(16) Hari, Y.; Obika, S.; Sekiguchi, M.; Imanishi, T. Tetrahedron 2003, 59,
5123-5128.
(17) Obika, S.; Hari, Y.; Sekiguchi, M.; Imanishi, T. Angew. Chem., Int. Ed.
2001, 40, 2079-2081.
(18) Sasaki, S.; Yamauchi, H.; Nagatsugi, F.; Takahashi, R.; Taniguchi, Y.;
Maeda, M. Tetrahedron Lett. 2001, 42, 6915-6918.
(19) Sasaki, S.; Yamauchi, H.; Takahashi, R.; Taniguchi, Y.; Maeda, M. Nucleic
Acids Res. Supp. 2001, 1, 23-24.
(20) Taniguchi, Y.; Takahashi, R.; Kodama, K.; Senko, Y.; Maeda, M.; Sasaki,
S. Nucleic Acids Res. Supp. 2002, 2, 35-36.
(21) Thuong, N. T.; Helene, C. Angew. Chem., Intl. Ed. Engl. 1993, 32, 666-
690.
(22) Ogura, H.; Takahashi, H.; Itoh, T. J. Org. Chem. 1972, 37, 72-75.
9
J. AM. CHEM. SOC. VOL. 126, NO. 2, 2004 517

A R T I C L E S
Sasaki et al.
Figure 2. Design of a bicyclic structure having a base and a benzene ring.
Figure 3. Expected structure of triplex with a WNA-7âG/TA interrupting site. The square indicates the area of WNA-7âG/TA (A), the triplet structure of
which is shown in part B.
Figure 4. Structures of the WNA (W-shape nucleoside analogue). The number, R, or â represent the position of the nucleobase for glycosidic bond and its
stereochemistry, respectively.
of R:â ) 7:6 for the stereochemistry of the allyl group, which
was used for the following reaction without separation. Sub-
sequently, oxidative cleavage of the vinyl group and subsequent
deprotection of 2,3-O-acetonide spontaneously provided the
corresponding bicyclo[3.3.0]octane derivative, then the formed
hydroxyl groups were acetylated to afford the key intermediate
12. The cyclic compound 12 was easily separated from the
noncyclic compound which was derived from 11 with undesired
â-allyl stereochemistry. A similar hemiketal intermediate (14)
lacking a benzene ring was synthesized by a similar sequence
of reactions from 13.23
N-Glycosidation to 12 with the corresponding base derivatives
was done under different conditions to produce a mixture of
7-N and 9-N alkylated isomers in the synthesis of 16 and 17
(23) Mukaiyama, T.; Kobayashi, S. Carbohydr. Res. 1987, 171, 81-87.
9
518 J. AM. CHEM. SOC. VOL. 126, NO. 2, 2004

A R T I C L E S
Scheme 1 a
the TFO (10 nM) and different concentrations of the target
duplex (0-100 nM), and the triplex was identified as the slower
migrating band relative to the single-stranded TFO. Equilibrium
association constants were obtained by quantification of the
bands (Table 1).
Figure 5 illustrates the results of the electrophoresis with the
use of the TFO incorporating WNA-7RG and -9RG. In the
combinations of WNA-7RG/CG and 7RG/GC, triplex bands
were observed at high concentrations of the target duplex (Figure
5A), indicating that WNA-7RG displayed lower binding con-
stants with relative selectivity to a CG and a GC pair. In contrast,
the TFO with WNA-9RG produced triplexes toward a TA pair
selectively (Figure 5B). Compared with WNA-7âG,18 it is
interesting that the WNA-9RG exhibited higher potency in both
selectivity and stability to a TA site. The WNA-A derivatives
did not form stable triplexes, although some selectivity was
observed in the combinations of WNA-âA/TA and WNA-RA/
CG. As the triplexes with the WNA analogues connecting
guanine or adenine were not satisfactorily stable compared with
the natural triplexes (Z ) dG, dA), we next investigated the
WNA analogues bearing a pyrimidine base. To our surprise,
WNA-âT exhibited a high stabilizing effect with selectivity to
a TA base pair. It is also remarkable that selective stabilization
of the triplex at a CG interrupting site has been achieved with
WNA-âC. The WNA-pyrimidine analogues with R-stereochem-
istry did not produce a significant stabilizing effect. Triplex
stability with WNA-âT analogues as well as with natural bases
(dG and dA) could not be evaluated precisely, because the TFO
was converted to the triplex even at low concentration of the
target duplex. Therefore, we next examined triplex formation
in a buffer containing 5 mM MgCl2 instead of 20 mM MgCl2
in the previous assay. The results of the gel-shift assay are shown
in Figure 6, and the equilibrium association constants obtained
from them are summarized in Table 2. Under the conditions
with 5 mM MgCl2, the TFO with WNA-âT yielded almost
complete formation of a triplex with a TA interrupting site at a
10 nM duplex concentration (TFO/duplex ) 1:1), whereas the
other duplexes needed much higher concentrations for triplex
formation (Figure 6A). Interestingly, the triplex with the WNA-
âT/TA combination showed higher stability than the natural-
type triplexes (Table 2). Furthermore, the association constant
of the triplex with the WNA-âC/CG combination has been
shown to be higher than that of the natural-type triplexes in the
assay with 5 mM MgCl2, although it was estimated to be less
stable than the natural-type triplexes at 20 mM MgCl2 (Table
1). The stability of the triplexes formed with WNA analogues
is less affected by MgCl2 concentration than that of natural-
type triplexes. Consequently, it has been demonstrated that
WNA-âT is a selective and high-affinity recognition motif to a
TA interrupting site and that WNA-âC is a potential base
analogue for a CG interrupting site.
a (a) (1) acetone, H+, (2) TBDPSCl, TEA, DMAP, CH2Cl2, (3) PCC,
CH2Cl2, (b) PhLi, THF, (c) allyltrimethylsilane, ZnBr2, CH3NO2, (R:â )
7:6), (d) (1) OsO4, NaIO4, pyridine, (2) 5% H2SO4, THF, (3) Ac2O, pyridine,
(e) (1) OsO4, NaIO4, pyridine, (2) 2 M HCl-THF, (3) Ac2O, pyridine.
and R- and â-isomers in all syntheses, which were separated
by flash chromatography (Scheme 2).24,25 Regioisomers of 7-N
and 9-N alkylation were determined based on a comparison of
1
the chemical shift values of the H and 13C NMR spectra.26,27
The stereochemistry of the isolated isomers was determined by
1H-COSY and NOESY spectra.28 The abasic-type WNA-H was
prepared from 12 via reduction with Et3SiH and TMSOTf.
H-WNA-âT was similarly prepared from 14.
Each isomer of the new nucleoside analogues was transformed
to the corresponding amidite precursor with a 5′-O-dimethoxy-
trity (DMTr) protecting group and incorporated into the TFOs
by an automated DNA synthesizer. DNA synthesis was carried
out by the conventional method using DCI as the activator, TCA
for deprotection of the DMTr group, and iodine as the oxidizer.
Cleavage of the DMTr-bearing TFOs from the resin and removal
of the protecting groups were performed by heating in 28% NH4-
OH, followed by purification by HPLC with an ODS column.
Subsequently, deprotection of DMTr was done in 10% acetic
acid, and the structure and purity of the obtained TFO were
confirmed by MALDI-TOF MASS measurements. The target
duplexes were formed by annealing and purified by HPLC.
Evaluation of Triplex Formation. Triplexes were formed
by incubating the TFO, the 32P-labeled TFO as a tracer and the
duplex for 12 h at 22 °C in a buffer containing 20 mM Tris-
HCl, 20 mM MgCl2, 2.5 mM spermidine and 10% sucrose at
pH 7.5. The triplex formed was evaluated by gel shift assay
with 15% nondenatured polyacrylamide gel at 10 °C.29 As self-
aggregation of purine-rich TFO sometimes competes with the
triplex formation, TFO was used in this assay at low concentra-
tion such that no aggregation was observed. The triplex
formation was performed by using a constant concentration of
To clarify the role of the benzene and thymine parts of WNA-
âT, we evaluated the stability of the triplexes with the use of
WNA-H (7) lacking a heterocyclic part and H-WNA-âT without
a benzene ring (8) at a 5 mM MgCl2 concentration. The TFO
incorporating of WNA-H showed nonselective binding behavior
with relatively higher affinity to CG and GC base pairs. It is
apparent from these results that the bicyclic skeleton itself has
a stabilizing effect for triplex formation. Surprisingly, the
stability of the triplex containing WNA-H is higher than that
(24) Vorbru¨ggen H.; Krolikiewicz, K.; Bennua B. Chem. Ber. 1981, 114, 1234-
1255.
(25) Vorbru¨ggen H.; Ho¨fle, G. Chem. Ber. 1981, 114, 1256-1268.
(26) Garner, P.; Ramakanth, S. J. Org. Chem. 1988, 53, 1294-1298.
(27) Masko¨y, M.; Bolli, M.; Schweizer, B.; Leumann, C. J. HelV. Chim. Acta
1993, 76, 481-510.
(28) See the Supporting Information.
(29) Arimondo, P. B.; Barcelo, F.; Sun, J. S.; Maurizot, J. C.; Garestier, T.;
Helene, C. Biochemistry 1998, 37, 16627-16635.
9
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A R T I C L E S
Sasaki et al.
Scheme 2 a
a (a) Et3SiH, TMSOTf, CH2Cl2; (b) BSA, TMSOTf, CH3CN; (c) HMDS, TMSCl, SnCl4, CH3CN; (d) BSA, SnCl4,CH3CN; (e) (i) nBu4NF, THF, (ii)
aqueous NaOH, MeOH, (iii) DMTrCl, pyridine, (vi) iPr2NP(Cl)OCH2CH2CN, iPr2NEt, CH2Cl2; (f) (i) aqueous NaOH, MeOH, (ii) DMTrCl, pyridine, (iii)
iPr2NP(Cl)OCH2CH2CN, iPr2NEt, CH2Cl2.
Table 1. Equilibrium Association Constants (Ks, 109 M-1) of the
Triplexes at 20 mM MgCl2 Concentrationsa
X,Y
Z
T,A
A,T
C,G
G,C
dG
dA
0.16
0.069
0.08
>1.0
0.09
0.057
>1.0
0.40
WNA-7âG
WNA-9âG
WNA-7RG
WNA-9RG
0.02
<0.001
<0.001
0.10
0.01
0.01
0.008
0.013
<0.001
<0.001
0.023
<0.001
<0.001
0.021
0.017
0.017
WNA-âA
WNA-RA
0.26
0.007
0.015
0.009
0.063
0.067
0.029
0.036
WNA-âT
WNA-RT
>1.0
0.009
0.042
<0.001
0.091
<0.001
0.045
<0.001
WNA-âmC
0.42
<0.001
0.018
<0.001
0.03
<0.001
0.029
<0.001
WNA-RmC
WNA-âC
WNA-RC
0.057
0.014
0.032
<0.001
0.152
0.017
0.055
0.023
a Triplex formation and gel-shift assay were performed as described for
Figure 5, and the bands corresponding to the unbound TFO and the triplex
were quantified. The equilibrium association constant was calculated by
the equation Ks ) [triplex]/([TFO][duplex]). Concentrations of each
component represented as [triplex], [TFO], and [duplex] were estimated
from the relative ratio of the bands.
Figure 5. Gel-shift assay for determination of triplex formation with WNA-
7RG and -9RG. Triplex formation was done for 12 h at 22 °C in a buffer
containing 20 mM Tris-HCl, 20 mM MgCl2, 2.5 mM spermidine, and 10%
sucrose at pH 7.5. Electrophoresis was done at 10 °C with 20% nondenatured
polyacrylamide gel. 10 nM TFO containing the 32P-labeled one as the tracer
was used. The concentration of the duplex was increased from lane 1 to 5
(10, 20, 40, 60, 80 nM).
of the corresponding natural triplex including the G/GC triplet.
It has been revealed from comparison of association constants
between WNA-âT and WNA-H that the thymine unit of WNA-
âT causes a high stabilizing effect selectively to the TA site,
whereas it leads to large destabilization in the other three
combinations. On the other hand, in the case of WNA-âC,
destabilization to the CG pair is less than that to the TA and
the GC base pairs, resulting in selectivity to the CG pair. The
fact that H-WNA-âT did not produce stable triplexes at any
combination clearly suggests that the benzene ring brings about
a major stabilizing effect with the WNA analogues.
We also examined the triplex-forming ability of WNA-âT
to see whether it can stabilize a triplex containing the sequential
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A R T I C L E S
Figure 8. Gel-shift assay of the TFOs. TFOs were incubated under the
conditions described in Figure 5 in the presence or absence of KCl.
Electrophoresis was done at 10 °C with 15% nondenatured polyacrylamide
gel. 10 nM TFO was used in lanes 1, 2, and 4, and 3 µM TFO was used in
lanes 3 and 5.
A shortcoming of homopurine-TFOs in the formation of
triplexes is that they tend to form self-aggregation in the
presence of alkali metal cations to diminish the ability for triplex
formation.30 In the assay using 3 µM TFO concentration in the
presence of 150 mM KCl, the natural-type TFO (23, Z ) G)
was observed to form higher molecular-weight bands that are
suggestive of aggregation (Figure 8, lane 3). In a remarkable
contrast, the TFO incorporating WNA-âT did not indicate
aggregate formation under the same conditions (Figure 8, lane
5). These results have suggested another advantageous point of
the WNA analogues in that they may be useful in application
under physiological conditions.
Figure 6. Gel-shift assay for determination of triplex formation. The
conditions for triplex formation and gel-shift assay are the same as those
for Figure 5 except for the use of the buffer containing 5 mM MgCl2.
Table 2. Equilibrium Association Constants (Ks, 109 M-1) of the
Triplexes at 5 mM MgCl2 Concentrationsa
X,Y
Z
T,A
A,T
C,G
G,C
dG
dA
WNA-âT
WNA-âC
WNA-H
0.004
<0.001
0.30
<0.001
0.067
0.008
0.074
<0.001
0.025
0.008
<0.001
0.015
0.115
0.18
0.086
0.047
0.082
0.047
0.28
Discussion
The new nucleoside analogues (WNA) have been designed
based on our original concept of separating the hydrogen
bonding and base-stacking contributors into two parts, and
potential new compounds, WNA-âT (4b) for a TA and WNA-
âC (5b) for a CG interrupting site, have been developed for
selective stabilization of triplexes. As a matter of great interest,
the triplex stability with WNA-H without a base unit is higher
than that of the natural triplex (Table 2). It is apparent from
comparison of the stabilizing effect of H-WNA-âT without
benzene that benzene of WNA-H is a major contributor to triplex
stability. As a benzene nucleotide has been shown to stack with
slightly greater affinity than thymine in a duplex,31,32 the
stabilizing effect of a benzene of WNA-H might be also
attributable to stacking interactions in the triplex. The five-
membered ring between the 1′- and 2′-position might increase
hydrophobicity of the WNA-H, resulting in additional stabiliza-
tion of the triplex.33 Based on the stabilizing effect of WNA-
H, it is clear that the high selectivity of WNA-âT is brought
about by the high stabilizing effect of thymine to a TA site
together with its large destabilizing effect to the other base pairs
(Table 2). In a similar context, selectivity of WNA-âC to a CG
site may be explained in terms of differences in the destabilizing
effect of cytosine to the base pairs. Factors for stabilization
might include hydrogen bonds or van der Waals interactions to
a favorable base pair, and destabilizing effects might be due to
steric repulsions with unfavorable base pairs.
0.047
H-WNA-âT
0.002
0.003
0.009
0.042
a Triplex formation and gel-shift assay were performed as described for
Figure 6, and the bands corresponding to the unbound TFO and the triplex
were quantified. The equilibrium association constant was calculated by
the equation Ks ) [triplex]/([TFO][duplex]). Concentrations of each
component represented as [triplex], [TFO], and [duplex] were estimated
from the relative ratio of the bands.
Figure 7. Triplex formation with the TFO incorporating two continuous
WNA-âT analogues. The conditions for the triplex formation and gel-shit
assay are the same as described for Figure 5. Concentrations of the duplex
in lanes 1 to 6: 10, 20, 40, 60, 80, and 100 nM.
two TA interrupting sites or not (Figure 7). The gel-shift assay
showed that the TFO (25) with two continuous WNA-âT units
formed the triplex selectively with the target duplex with the
sequential two TA interrupting sites. Although stability of the
triplex formed with the TFO (25) is lower than that containing
a single WNA-âT/TA combination (23), achievement of triplex
formation with selectivity to two continuous TA interrupting
sites is of potential significance in future development of new
WNA analogues for further expansion of the target sequence.
(30) Olivas, W. M.; Maher, L. J., III. Nucleic Acids Res. 1995, 23, 1936-1941.
(31) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X.-F.; Sheils, C. J.; Paris, P.
L.; Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 8182-
8183.
(32) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X.-F.; Sheils, C. J.; Paris, P.
L.; Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 2213-
2222.
(33) See p 217 in ref 1 for a discussion of contribution of hydrophobicity in
triplex stabilization.
9
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A R T I C L E S
Sasaki et al.
Figure 9. Speculated complex structures of thymine to a TA and cytosine to a CG base pair. These complex structures were optimized with PM3. Electric
potentials were displayed by 3D mapped isosurface.
In the initial design of the WNA analogue, selective hydrogen
bonds between the guanine and the distant adenine of the TA
interrupting site were anticipated (Figure 1D). However, all the
WNA isomers with guanine and adenine did not produce stable
triplexes. In comparison with the stability of natural triplexes
or that with WNA-H, purine bases of the WNA analogues act
as destabilizing factors, probably because they might be
repulsive to the backbone of the pyrimidine strand of the duplex.
Molecular modeling has suggested that the thymine of the
WNA-âT might form hydrogen bonds to both bases of a TA
base pair rather than Hoogsteen-type hydrogen bonds with the
distant adenine. Similarly, hydrogen bonds of cytosine to both
bases of a CG base pair might be also plausible. Figure 9 depicts
such model triplets of T/TA and C/CG together with calculated
3D mapped isosurface of electric potential, presenting possible
hydrogen bondings at the junction region. Lower stability of
the triplex with WNA-âmC for a CG site might be rationalized
by assuming protonation at 3-N of 5-methycytosine. This
assumption is supported in principle by the observation that the
association constant of the triplex with a WNA-âmC/CG is
increased as the pH of buffer increased (relative ratio of the
association constant at 5 mM MgCl2 is 0.6 at pH 7.0, 1.0 at pH
7.5, and 1.9 at pH 8.0). In a complex structure determined for
T/CG mismatched base triplet in the antiparallel triplex,
hydrogen bonds of a water molecule between the guanine and
thymine at the junction were postulated to produce stabilizing
effect.34,35 Detailed study is needed for understanding of the
recognition mechanism of the WNA analogues.
Compared to the previous studies with limited success by
the use of nucleoside analogues with 1′-R stereochemistry, the
remarkable success of the WNA analogue with a bicyclo[3.3.0]-
octane skeleton may be attributable to cooperative action of a
benzene ring and a nucleobase unit. An advantageous property
of WNA analogues showing resistance to self-aggregation may
be due to steric disorder of the TFO caused by incorporating a
WNA analogue. In the case of triplex formation by the TFO
with two continuous WNA analogues, as triplex stability is
diminished to a large extent, its steric factor might become a
disadvantage. Detailed information on the structures of the
triplexes with the WNA analogues, together with elucidation
of the roles of each component, will be useful for the design of
new WNA analogues to expand triplex recognition codes.
Conclusion
In this study, we have determined two new nucleoside
analogues, WNA-âT (4b) and WNA-âC (5b), for the formation
of stable triplexes with selectivity to a TA and a CG interrupting
site, respectively. These useful properties are provided by
cooperative work of a benzene ring and a nucleobase attached
to the bicyclo[3.3.0]octane skeleton and are characteristic in that
the stability of the triplexes with them is higher than that of
natural-type triplexes in the presence of low Mg2+ concentration.
Thus, the new WNA analogues might have a high potential for
DNA recognition with TFOs in application to living systems.
A future challenge is the development of new WNA analogues
for expansion of triplex recognition codes to a general duplex
sequence having continuous or random interrupting sites, and
studies are now in progress along this line.
Experimental Section
General. 1H NMR (270, 400, 500, or 600 MHz) and 13C NMR (100,
125 MHz) spectra were recorded on a JEOL GX-270, Varian UNITY-
400, INOVA-500, or INOVA-600 spectrometer. Infrared (IR) spectra
were obtained using a SHIMADZU FTIR-8400 spectrometer. High-
resolution mass spectra analyses were recorded on an Applied Bio-
systems Mariner System 5299 spectrometer using bradykinin, neuro-
tensin, and angiotensin as an internal standard.
Synthesis. 5-O-tert-Butyldiphenylsilyl-2,3-O-isopropylidene-D-ri-
bono-1,4-lactone 9. A suspension of D-ribose (42.4 g, 0.28 mol) and
p-toluenesulfonic acid monohydrate (1.8 g, 9.6 mmol) in acetone (415
mL, 5.6 mol) was stirred for 20 h at room temperature. The reaction
mixture was quenched with NaHCO3 (1.2 g, 14.4 mmol), the solids
were filtered off, and then the filtrate was evaporated. To a solution of
the residue in CH2Cl2 (420 mL) at 0 °C were added Et3N (39 mL, 0.28
mol), tert-butylchlorodiphenylsilane (72.8 mL, 0.28 mol), and DMAP
(3.4 g, 0.028 mol). After stirring at room temperature for 1 h, the
reaction mixture was diluted with EtOAc, and the organic layers were
successively washed with sat. NH4Cl solution, water, and brine, dried
over Na2SO4, and evaporated. The residue was purified by flash
chromatography (silica gel, hexane/EtOAc ) 5:1) to give a colorless
oil in 83%. A solution of this colorless oil (99 g, 0.23 mol) in CH2Cl2
(190 mL) was added to a suspension of Celite545 (230 g) and PCC
(148.5 g, 0.69 mol) in CH2Cl2 (1.6 L). After stirring for 16 h at room
temperature, the reaction mixture was diluted with EtO2 and filtered
through a Celite545 pad, and then the filtrate was evaporated. The
residue was purified by flash chromatography (silica gel, hexane/EtOAc
(34) Dittrich, K.; Gu, J.; Tinder, R.; Hogan, M.; Gao, X. Biochemistry 1994,
33, 4111-4120.
(35) Floris, R.; Scaggiante, B.; Manzini1, G.; Quadrifoglio, F.; Xodo, L. E. Eur.
J. Biochem. 1999, 260, 801-809.
9
522 J. AM. CHEM. SOC. VOL. 126, NO. 2, 2004

A R T I C L E S
) 1:1) to give 9 as white solids (75.8 g, 0.18 mol, 77%). Mp 96-97
°C. H NMR (400 MHz, CDCl3/TMS) δ 1.04 (s, 9H), 1.39 (s, 3H),
was stirred for 3 h at room temperature, diluted with 10 mM phosphate
buffer (pH7), and extracted with ether. The organic layer was washed
with brine, dried over MgSO4, then evaporated. The residue was purified
by flash chromatography (silica gel, hexane/EtOAc ) 5:1) to give 13
as a colorless oil (9.18 g, 79%). 1H NMR (400 MHz, CDCl3): δ 1.34
(s, 3H), 1.54 (s, 3H), 2.10 (s, 3H), 2.34-2.38 (m, 2H), 4.00 (dd, 1H,
J ) 6.18, 10.9 Hz), 4.08-4.14 (m, 2H), 4.25-4.30 (m, 1H), 4.39 (dd,
1H, J ) 4.30, 6.74 Hz), 4.49 (dd, 1H, J ) 4.30, 6.79 Hz), 5.12-5.18
(m, 2H), 5.77-5.87 (m, 1H). FTIR (CHCl3) 1740 cm-1. HRMS
(ESIMS) m/z calcd for C13H20O5Na (M + Na)+ 279.1203, found
279.1172.
1
1.48 (s, 3H), 3.76 (d 1H, J ) 11.42 Hz), 3.91 (d, 1H, J ) 11.42 Hz),
4.57 (s, 1H), 4.73 (d, 1H, J ) 5.61 Hz), 4.89 (d, 1H, J ) 5.61 Hz),
7.39-7.48 (m, 6H), 7.62 (d, 2H, J ) 6.36 Hz), 7.63 (d, 2H, J ) 6.18
Hz). FTIR (film) 1790 cm-1. HRMS (ESIMS) m/z calcd for C24H30O5-
SiNa (M+Na)+ 449.1755, found 449.1747. Anal. Calcd for C24H30O5-
Si: C, 67.57; H, 7.09. Found: C, 67.87; H, 7.19.
(1R,5R,6RS,8R)-3,3-Dimethyl-6-phenyl-6-(2′-propenyl)-8-(tert-bu-
tyldiphenylsilyloxymethyl)-2,4,7-trioxabicyclo[3.3.0]octane 11. A
solution of phenyllithium in cyclohexane-diethyl ether solution (1.04
M, 13.9 mL, 14.4 mmol) was added to a solution of 9 (4.1 g, 9.60
mmol) in THF (60 mL) at -78 °C in portions. After stirring for 2 h at
-78 °C, the reaction mixture was allowed to warm to room temperature,
quenched with sat. NH4Cl solution, and extracted with EtOAc. The
organic layer was successively washed with water and brine, dried over
Na2SO4, and evaporated. The residue was purified by flash chroma-
tography (silica gel, hexane/EtOAc ) 7:1) to give 10 as a yellow oil
(3.83 g, 79%). The solution of this colorless oil (3.83 g, 7.59 mmol) in
CH3NO2 (10 mL) and allyltrimethylsilane (1.6 mL, 10.1 mmol) was
added to a suspension of zinc bromide (4.91 g, 21.8 mmol) at 0 °C.
After stirring for 2 h at room temperature, the reaction mixture was
quenched with sat. NaHCO3 and extracted with EtOAc. The organic
layers were successively washed with water and brine, dried over Na2-
SO4, and then evaporated. The residue was purified by flash chroma-
tography (silica gel, hexane/EtOAc ) 19:1) to give 11 as a colorless
oil (R-isomer/â-isomer ) 1.8 g/1.5 g, 82%). R-isomer: 1H NMR (270
MHz, CDCl3/TMS) δ 1.02 (s, 9H), 1.37 (s, 3H), 1.63 (s, 3H), 2.59 (dd
1H, J ) 6.9, 14.5 Hz), 2.79 (dd, 1H, J ) 7.3, 14.5 Hz), 3.73 (dd, 1H,
J ) 5.0, 10.9 Hz), 3.80 (dd, 1H, J ) 4.6, 10.9 Hz), 4.25 (dt, 1H, J )
3.3, 4.9 Hz), 4.67-4.75 (m, 2H), 4.86-4.89 (m, 1H), 4.92 (d, 1H, J )
1.7 Hz), 5.49-5.64 (m, 1H), 7.17-7.46 (m, 11H), 7.63-7.69 (m, 4H).
FTIR (film) 1640, 1420 cm-1. HRMS (ESIMS) m/z calcd for C33H40O4-
SiNa (M + Na)+ 551.2588, found 551.2602.
(1R,3R,4R,5R,7RS)-1-Phenyl-3-(tert-butyldiphenylsilyloxymethyl)-
4,7-diacetoxy-2,6-dioxabicyclo[3.3.0]octane 12. Aqueous solutions of
OsO4 (0.131 M, 2.0 mL, 0.262 mmol) and NaIO4 (0.6 M, 10 mL, 6.0
mmol) were added to a solution of 11 (1.03 g, 1.59 mmol) in pyridine
(13 mL), and the reaction mixture was stirred for 30 h at room
temperature. The reaction mixture was diluted with EtOAc and
successively washed with water and brine, dried over Na2SO4, and
evaporated. A solution of the residue in THF (20 mL)/5% H2SO4 (4
mL) was stirred for 6 h at 60 °C, quenched by the addition of sat.
NaHCO3 solution, and extracted with EtOAc. The organic layer was
successively washed with water and brine, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography (silica
gel, CHCl3/EtOAc ) 2:1) to give a colorless foam (269 mg, 28%, for
two steps). Acetic anhydride (0.45 mL, 4.76 mmol) was added to a
solution of the colorless foam (585 mg, 1.19 mmol) in pyridine (6 mL)
at 0 °C and stirred for 39 h at room temperature. The reaction mixture
was diluted with EtOAc and successively washed with water and brine,
dried over Na2SO4, and evaporated. The residue was purified by flash
chromatography (silica gel, hexane/EtOAc ) 5:1) to give 12 as a
colorless foam (613 mg, 90%). 1H NMR (500 MHz, CDCl3) δ 1.05 (s,
9H), 2.03 (s, 3H), 2.14 (s, 3H), 2.65 (dd 1H, J ) 1.83, 15.1 Hz), 2.86
(dd, 1H, J ) 5.95, 15.1 Hz), 3.76 (dd, 1H, J ) 3.44, 11.68 Hz), 4.06
(dd, 1H, J ) 2.75, 11.68 Hz), 4.22 (dt, 1H, J ) 2.98, 9.38 Hz), 4.83
(d, 1H, J ) 4.12 Hz), 5.05 (dd, 1H, J ) 4.12, 9.38 Hz), 6.62 (dd, 1H,
J ) 1.83, 5.95 Hz), 7.27-7.45 (m, 9H), 7.63-7.71 (m, 6H). FTIR
(film) 1740, 1370, 1230 cm-1. HRMS (ESIMS) m/z calcd for C33H38O7-
SiNa (M + Na)+ 597.2279, found 597.2256.
(1R,3R,4R,5S,7RS)-3-Acetoxymethyl-4,7-diacetoxy-2,6-dioxabicyclo-
[3.3.0]octane 14. Aqueous solutions of OsO4 (0.131 M, 83.8 mL, 11
mmol) and NaIO4 (0.6 M, 343 mL, 206 mmol) were added to a solution
of 13 (9.1 g, 34.3 mmol) in pyridine (168 mL), and the mixture was
stirred for 2 h at room temperature. The reaction mixture was diluted
with EtOAc and successively washed with water and brine, dried over
Na2SO4, and evaporated. The residue was purified by flash chroma-
tography (silica gel, hexane/EtOAc ) 3:1) to give a pale yellow oil
(5.96 g, 65%). A solution of this oil in 2 M HCl solution (36.5 mL)
and THF (183 mL) was stirred for 1 h at 40 °C. The reaction mixture
was neutralized with Et3N (10 mL), diluted with EtOAc, successively
washed with sat. NaHCO3 solution, water, and brine, and dried over
Na2SO4, then evaporated. The residue was purified by flash chroma-
tography (silica gel, CHCl3/MeOH ) 30:1) to give a bicyclic compound
as a pale yellow oil (3.47 g, 69%). A solution of the above product
(3.0 g, 13.7 mmol) and acetic anhydride (5.1 mL, 55.2 mmol) in
pyridine (120 mL) was stirred for 30 min at 0 °C and for 2 h at room
temperature. The reaction mixture was diluted with EtOAc and
successively washed with 10% HCl solution, brine, sat. NaHCO3
solution, and brine. The organic layer was dried over Na2SO4 and
evaporated. The residue was purified by flash chromatography (silica
gel, hexane/EtOAc ) 5:1) to give 14 as a colorless oil (2.4 g, 58%).
1H NMR (400 MHz, CDCl3): δ 2.01 (s, 3H), 2.07 (s, 3H), 2.11 (s,
3H), 2.35 (t, 2H, J ) 3.93 Hz), 4.05-4.13 (m, 2H), 4.30-4.33 (m,
1H), 4.81-4.87 (m, 2H), 4.93 (dd, 1H, J ) 4.68, 9.36 Hz), 6.46 (t,
1H, J ) 4.11 Hz). FTIR (CHCl3) 1743 cm-1. ESIMS (m/z): 325 (M
+ Na)+.
(1S,3R,4R,5R)-4-Acetoxy-1-phenyl-3-(tert-butyldiphenylsilyloxy-
methyl)-2,6-dioxabicyclo[3.3.0]octane, WNA-H, 15(1). Et3SiH (810
mg, 7.7 mmol) was added to a solution of 12 (400 mg, 0.7 mmol) in
CH2Cl2 (7 mL) at room temperature under argon, and the reaction
mixture was stirred at room temperature for 15 min, then cooled to 0
°C, followed by the addition of CF3SO3SiMe3 (0.317 mL, 1.75 mmol).
After stirring for 1 h at 0 °C, the mixture was diluted with sat. NaHCO3
solution and extracted with AcOEt. The organic layer was successively
washed with water and brine, dried over Na2SO4, and evaporated. The
residue was purified by flash chromatography (silica gel, hexane/AcOEt
) 10:1) to give WNA-H (15(1)) as a colorless oil (294 mg, 81%). 1H
NMR (270 MHz, CDCl3/TMS) δ 1.02 (s, 9H), 2.06 (s, 3H), 2.40-
2.57 (m, 2H), 3.73 (dd, 1H, J ) 3.63, 11.54 Hz), 4.01 (dd, 1H, J )
3.30, 11.55 Hz), 4.09 (dt, 1H, J ) 6.27, 8.91 Hz), 4.15-4.26 (m, 2H),
4.66 (d, 1H, J ) 4.29 Hz), 5.08 (dd, 1H, J ) 4.61, 9.23 Hz), 7.21-
7.70 (m, 15H). FTIR (CHCl3) 1740 cm-1. HRMS (ESIMS) m/z calcd
for C31H36O5SiNa (M + Na)+ 539.2224, found 539.2240.
Glycosidation. The synthesis of 16(1) and 17(1) was described as
a general procedure. The compounds (19-22) were synthesized in a
similar manner except for the reagents used.
(1′S,3′R,4′R,5′R,7′S and 7′R)-N2-Isobutyryl-9-and-7-{4′-acetoxy-
1′-phenyl-3′-(tert-butyldiphenylsilyloxymethyl)-2′,6′-dioxabicyclo-
[3.3.0]oct-7′-yl}guanine, WNA-9r,âG and -7r,âG, 16a,b(1) and
17a,b(1). N,O-Bis(trimethylsilyl)acetamide (BSA; 500 mL, 1.97 mmol)
was added to a suspension of N2-isobutyrylguanine (71 mg, 0.321
mmol) in CH3CN (25 mL) at 50 °C. A solution of TMSOTf (30 mL,
0.166 mmol) and 12 (166 mg, 0.289 mmol) in CH3CN (1.0 mL) was
added to the above mixture. The reaction mixture was stirred at 50 °C
(1R,5R,6R,8R)-8-Acetoxymethyl-3,3-dimethyl-6-(2′-propenyl)-
2,4,7-trioxabicyclo[3.3.0]octane 13. Trityl perchlorate (1.8 g, 5.3
mmol) was added to a solution of 1,5-O-diacetyl-2,3-O-isopropylidene-
D-ribose (14.6 g, 53.2 mmol) and allyltrimethylsilane (22.9 mL, 144
mmol) in 1,2-dimethoxyethane (292 mL) at 0 °C. The reaction mixture
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A R T I C L E S
Sasaki et al.
for 4 h and diluted with MeOH and evaporated. A solution of the above
residue in EtOAc was successively washed with sat. NaHCO3, water,
and brine, dried over Na2SO4, and evaporated. The isomers were
separated by flash chromatography (silica gel, acetone/hexane ) 1:2)
to give each isomer in total 88% yield. WNA-9RG (16a(1)): a white
powder (80 mg, 38%). Mp 122-125 °C. 1H NMR (500 MHz, CDCl3)
δ 0.99 (s, 9H), 1.26 (d, 6H, J ) 6.9 Hz), 1.94 (s, 3H), 2.55
(pseudoquintet, 1H, J ) 6.9 Hz), 3.04 (dd, 1H, J ) 7.9, 15.2 Hz), 3.16
(dd, 1H, J ) 4.3, 15.0 Hz), 3.67 (dd, 1H, J ) 3.6, 11.8 Hz), 3.93 (dd,
1H, J ) 3.0, 11.5 Hz), 4.10 (ddd, 1H, J ) 3.0, 3.6, 8.2 Hz), 4.97 (d,
1H, J ) 4.0 Hz), 5.07 (dd, 1H, J ) 4.0, 8.6 Hz), 6.42 (dd, 1H, J )
4.0, 7.9 Hz), 7.30-7.44 (m, 9H), 7.55-7.64 (m, 6H), 8.15 (bs, 1H),
8.20 (s, 1H), 12.0 (bs, 1H). FTIR (CHCl3) 1740, 1690 cm-1. FABMS
(m/z) 736 (M + H)+. WNA-9âG (16b(1)): a white powder (32 mg,
mg, 0.22 mmol), Me3SiCl (23.9 mg, 0.22 mmol), SnCl4 (84.4 mg, 0.324
mmol), thymine (37.4 mg, 0.275 mmol), 12 (155 mg, 0.27 mmol), CH3-
CN (2 mL). WNA-RT (19a(1)): a colorless foam (64.9 mg, 37%). 1H
NMR (500 MHz, CDCl3/TMS) δ 1.01 (s, 9H), 1.95 (s, 3H), 2.02 (s,
3H), 2.61 (dd, 1H, J ) 5.72, 15.33 Hz), 2.86 (dd, 1H, J ) 8.01, 15.33
Hz), 3.77 (dd, 1H, J ) 4.81, 11.44 Hz), 3.91 (dd, 1H, J ) 3.66, 11.44
Hz), 4.33 (dd, 1H, J ) 4.35, 8.01 Hz), 4.80 (dt, 1H, J ) 3.89 Hz),
4.99 (dd, 1H, J ) 8.70, 4.12 Hz), 6.53 (dd, 1H, J ) 5.72, 8.00 Hz),
7.25-7.46 (m, 10H), 7.60-7.65 (m, 6H), 8.04 (bs, 1H). FTIR (film):
3018, 1691 cm-1. HRMS (ESIMS) m/z calcd for C36H40N2O7SiNa (M
+ Na)+ 663.2497, found 663.2544. WNA-âT (19b(1)): a colorless
1
foam (72.3 mg, 42%). H NMR (500 MHz, CDCl3/TMS) δ 1.00 (s,
9H), 1.96 (s, 3H), 2.03 (s, 3H), 2.52 (dd, 1H, J ) 8.69, 13.95 Hz),
2.88 (dd, 1H, J ) 5.95, 13.95 Hz), 3.73 (dd, 1H, J ) 3.43, 11.67 Hz),
3.98 (dd, 1H, J ) 2.97, 11.67 Hz), 4.25 (dd, 1H, J ) 3.43, 8.92 Hz),
5.06 (dd, 1H, J ) 4.12, 8.92 Hz), 5.11 (d, 1H, J ) 4.12 Hz), 6.31 (dd,
1H, J ) 5.95, 8.69 Hz), 7.19-7.44 (m, 10H), 7.54-7.65 (m, 6H), 8.29
(bs, 1H). FTIR (film) cm-1 3018, 1691. HRMS (ESIMS) m/z calcd for
C36H40N2O7SiNa (M + Na)+ 663.2497, found 663.2456.
1
15%). Mp 120-125 °C. H NMR (500 MHz, CDCl3) δ 1.01 (s, 9H),
1.29 (d, 6H, J ) 6.9 Hz), 2.02 (s, 3H), 2.59 (pseudoquintet, 1H, J )
6.9 Hz), 3.01 (dd, 1H, J ) 6.6, 14.3 Hz), 3.08 (dd, 1H, J ) 7.7, 14.3
Hz), 3.75 (dd, 1H, J ) 3.6, 11.5 Hz), 4.01 (dd, 1H, J ) 3.0, 11.5 Hz),
4.31 (dt, 1H, J ) 3.3, 9.1 Hz), 5.14 (dd, 1H, J ) 3.8, 9.1 Hz), 5.22 (d,
1H, J ) 3.6 Hz), 6.30 (dd, 1H, J ) 6.9, 7.4 Hz), 7.27-7.45 (m, 9H),
7.60-7.66 (m, 6H), 7.82 (s, 1H), 7.88 (bs, 1H), 11.9 (bs, 1H). FTIR
(CHCl3) 1740, 1690 cm-1. FABMS (m/z): 736 (M + H)+. HRMS
(FABMS) m/z calcd for C40H46N5O7Si (M + H)+ 736.3175, found
736.3166. WNA-7RG (17a(1)): a white powder (54 mg, 25%). Mp
122-124 °C. 1H NMR (500 MHz, CDCl3) δ 0.98 (s, 9H), 1.26 (d, 6H,
J ) 7.1 Hz), 1.93 (s, 3H), 2.55 (pseudoquintet, 1H, J ) 7.1 Hz), 3.02
(dd, 1H, J ) 8.0, 15.1 Hz), 3.02 (dd, 1H, J ) 8.0, 15.1 Hz), 3.67 (dd,
1H, J ) 3.9, 11.5 Hz), 3.92 (dd, 1H, J ) 3.3, 11.5 Hz), 4.10 (ddd, 1H,
J ) 3.3, 3.6, 8.2 Hz), 4.96 (d, 1H, J ) 3.9 Hz), 5.05 (dd, 1H, J ) 3.9,
8.5 Hz), 6.40 (dd, 1H, J ) 3.6, 8.0 Hz), 7.27-7.45 (m, 9H), 7.05-
7.61 (m, 6H), 8.43 (s, 1H), 8.79 (bs, 1H), 12.1 (bs, 1H). FTIR
(CHCl3): 1740, 1690 cm-1. FABMS (m/z): 736 (M + H)+. WNA-
(1′S,3′R,4′R,5′R,7′S and 7′R)-N4-Benzoyl-1-{4′-acetoxy-1′-phenyl-
3′-(tert-butyldiphenylsilyloxymethyl)-2′,6′-dioxabicyclo[3.3.0]oct-7′-
yl}-5-methylcytosine, WNA-r and -âmC, 20a,b(1). Reagents: N4-
Benzoyl-cytosine (222.7 mg, 0.975 mmol), BSA (0.488 mL, 2.03
mmol), SnCl4 (0.303 mL, 2.63 mmol), 12 (465 mg, 0.81 mmol). WNA-
RmC (20a(1)): a colorless foam (85.7 mg, 14%). 1H NMR (270 MHz,
CDCl3/TMS) δ 1.03 (s, 9H), 2.06 (s, 3H), 2.19 (s, 3H), 2.68 (dd, 1H,
J ) 4.95, 15.18 Hz), 2.94 (dd, 1H, J ) 7.92, 15.18 Hz), 3.77 (dd, 1H,
J ) 2.97, 11.55 Hz), 3.90 (dd, 1H, J ) 3.63, 11.55 Hz), 4.34 (dd, 1H,
J ) 3.96, 8.24 Hz), 4.88 (d, 1H, J ) 4.29 Hz), 5.03 (dd, 1H, J ) 4.29,
8.25 Hz), 6.55 (dd, 1H, J ) 5.29, 7.29 Hz), 7.28-7.73 (m, 18H), 7.85
(s, 1H), 8.33 (d, 2H, J ) 6.93 Hz). FTIR (CHCl3) 1707, 1572 cm-1
.
HRMS (ESIMS) m/z calcd for C43H46N3O7Si (M + H)+ 744.3100, found
744.3112. WNA-âmC (20b(1)): a pale yellow foam (317.7 mg, 53%).
1H NMR (270 MHz, CDCl3/TMS) δ 1.01 (s, 9H), 2.05 (s, 3H), 2.16
(s, 3H), 2.54 (dd, 1H, J ) 8.58, 13.86 Hz), 2.97 (dd, 1H, J ) 5.61,
13.86 Hz), 3.75 (dd, 1H, J ) 3.63, 11.55 Hz), 4.01 (dd, 1H, J ) 2.97,
11.55 Hz), 4.26 (dd, 1H, J ) 5.61, 8.91 Hz), 5.08 (dd, 1H, J ) 3.96,
8.91 Hz), 5.15 (d, 1H, J ) 3.96 Hz), 6.36 (dd, 1H, J ) 5.6, 8.58 Hz),
7.18-7.72 (m, 18H), 8.32 (d, 2H, J ) 6.93 Hz). FTIR (CHCl3) 1707,
1570 cm-1. HRMS (ESIMS) m/z calcd for C43H46N3O7Si (M + H)+
744.3100, found 744.3128.
1
7âG (17b(1)): a white powder (22 mg, 10%). Mp 122-125 °C. H
NMR (500 MHz, CDCl3) δ 1.01 (s, 9H), 1.25 (d, 6H, J ) 6.9 Hz),
1.99 (s, 3H), 2.69 (pseudoquintet, 1H, J ) 6.9 Hz), 2.94 (dd, 1H, J )
5.8, 13.7 Hz), 3.16 (dd, 1H, J ) 8.5, 13.7 Hz), 3.78 (dd, 1H, J ) 4.1,
11.5 Hz), 4.00 (dd, 1H, J ) 3.3, 11.5 Hz), 4.29 (ddd, 1H, J ) 3.6, 3.9,
8.8 Hz), 5.11 (dd, 1H, J ) 3.6, 9.1 Hz), 5.34 (d, 1H, J ) 3.6 Hz), 6.39
(dd, 1H, J ) 5.8, 8.2 Hz), 7.26-7.45 (m, 9H), 7.63-7.73 (m, 6H),
8.01 (s, 1H), 9.25 (bs, 1H), 12.2 (bs, 1H). FTIR (CHCl3) 1740, 1690
cm-1. FABMS (m/z) 736 (M + H)+.
(1′S,3′R,4′R,5′R,7′S and 7′R)-N6-Benzoyl-9-{4′-acetoxy-1′-phenyl-
3′-(tert-butyldiphenylsilyloxymethyl)-2′,6′-dioxabicyclo[3.3.0]oct-7′-
yl}adenine, WNA-r and -âA, 18a,b(1). Reagents: N6-Benzoyl-
adenine (540 mg, 2.26 mmol), BSA (1.1 mL, 4.52 mmol), TMSOTf
(29 µL, 0.16 mmol), 12 (650 mg, 1.13 mmol). WNA-RA (18a(1)): a
white powder (129 mg, 15%). Mp 104-106 °C. 1H NMR (500 MHz,
CDCl3): δ 0.98 (s, 9H), 2.02 (s, 3H), 3.12 (dd, 1H, J ) 8.01, 15.11
Hz), 3.27 (dd, 1H, J ) 3.66, 15.11 Hz), 3.68 (dd, 1H, J ) 4.12, 11.67
Hz), 3.90 (dd, 1H, J ) 3.66, 11.67 Hz), 4.13-4.15 (m, 1H), 5.03 (d,
1H, J ) 4.12 Hz), 5.07 (dd, 1H, J ) 4.12, 8.47 Hz), 6.74 (dd, 1H, J
) 3.67, 8.01 Hz), 7.29-7.40 (m, 9H), 7.50-7.62 (m, 9H), 8.01 (d,
2H, J ) 7.09 Hz), 8.55 (s, 1H), 8.82 (s, 1H), 8.92 (bs, 1H). FTIR
(KBr): 1747, 1699, 1609, 1582 cm-1. ESIMS (m/z): 776 (M + Na)+.
WNA-âA (18b(1)): a white powder (237 mg, 28%). Mp 102-105
°C. 1H NMR (500 MHz, CDCl3) δ 1.07 (s, 9H), 2.05 (s, 3H), 3.09 (dd,
1H, J ) 6.18, 14.19 Hz), 3.47 (dd, 1H, J ) 8.24, 14.19 Hz), 3.84 (dd,
1H, J ) 4.12, 11.45 Hz), 4.07 (dd, 1H, J ) 3.2, 11.45 Hz), 4.41 (dt,
1H, J ) 3.66, 8.92 Hz), 5.16 (dd, 1H, J ) 3.89, 8.92 Hz), 5.16 (dd,
1H, J ) 3.66, 8.92 Hz), 5.34 (d, 1H, J ) 3.89 Hz), 6.56 (dd, 1H, J )
6.18, 8.02 Hz), 7.28-7.79 (m, 18H), 8.06 (d, 2H, J ) 7.09 Hz), 8.19
(s, 1H), 8.91 (s, 1H), 9.00 (bs, 1H). FTIR (KBr) 1747, 1699, 1609,
1582 cm-1. ESIMS (m/z) 754 (M + H)+.
(1′S,3′R,4′R,5′R,7′S and 7′R)-N4-Benzoyl-1-{4′-Acetoxy-1′-phenyl-
3′-(tert-butyldiphenylsilyloxymethyl)-2′,6′-dioxabicyclo[3.3.0]oct-7′-
yl}cytosine, WNA-r and -âC, 21a,b(1). Reagents: BSA (0.95 mL,
3.92 mmol), 12 (900 mg, 1.57 mmol) in 8 mL of CH3CN, N4-
benzoylcytosine (421 mg, 1.96 mmol) in 10 mL of CH3CN, SnCl4 (0.36
mL, 5.49 mmol). WNA-RC (21a(1)): a colorless foam (458.7 mg,
40%). 1H NMR (270 MHz, CDCl3/TMS) δ 1.02 (s, 9H), 2.06 (s, 3H),
2.71 (dd, 1H, J ) 4.95, 15.34 Hz), 3.10 (dd, 1H, J ) 7.26, 15.34 Hz),
3.75 (dd, 1H, J ) 4.29, 11.31 Hz), 3.82 (dd, 1H, J ) 4.29, 11.31 Hz),
4.24 (dt, 1H, J ) 4.29, 8.08 Hz), 5.00 (d, 1H, J ) 4.62 Hz), 5.08 (dd,
1H, J ) 4.62, 8.08 Hz), 6.41 (dd, 1H, J ) 4.95, 7.26 Hz), 7.27-7.70
(m, 19H), 7.93 (d, 2H, J ) 7.26 Hz), 8.27 (d, 1H, J ) 7.26 Hz). FTIR
(KBr) 3071, 2930, 1746, 1666, 1485 cm-1. HRMS (ESIMS) m/z calcd
for C42H44N3O7Si (M + H)+ 730.2943, found 730.2977. WNA-âC (21b-
1
(1)): a colorless foam (602.5 mg, 53%). H NMR (270 MHz, CDCl3/
TMS) δ 1.02 (s, 9H), 2.06 (s, 3H), 2.52 (dd, 1H, J ) 7.43, 14.35 Hz),
3.24 (dd, 1H, J ) 6.10, 14.35 Hz), 3.76 (dd, 1H, J ) 3.47, 11.71 Hz),
4.03 (dd, 1H, J ) 2.81, 11.71 Hz), 4.29 (ddd, 1H, J ) 2.81, 3.47, 9.07
Hz), 5.09 (dd, 1H, J ) 4.29, 9.07 Hz), 5.18 (d, 1H, J ) 4.29 Hz), 6.38
(dd, 1H, J ) 6.10, 7.43 Hz), 7.27-7.71 (m, 19H), 7.94 (d, 2H, J )
7.26 Hz), 8.01 (d, 1H, J ) 7.59 Hz). FTIR (KBr) 3069, 2930, 1747,
1665, 1485 cm-1. HRMS (ESIMS): m/z calcd for C42H44N3O7Si (M
+ H)+ 730.2943, found 730.2919.
(1′S,3′R,4′R,5′R,7′S and 7′R)-{4′-Acetoxy-1′-phenyl-3′-(tert-bu-
tyldiphenylsilyloxymethyl)-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}-
thymine, WNA-r and -âT, 19a, b(1). Reagents: (Me3Si)2NH (35.5
(1′R,3′R,4′R,5′S,7′S and 7′R)-{4′-Acetoxy-3′-acetoxymethyl-2′,6′-
dioxabicyclo[3.3.0]oct-7′-yl}thymine, H-WNA-rT and WNA-âT,
9
524 J. AM. CHEM. SOC. VOL. 126, NO. 2, 2004

A R T I C L E S
22a,b(1). Reagents: (Me3Si)2NH (192 mg, 1.19 mmol), Me3SiCl (129
mg, 1.19 mmol), SnCl4 (466 mg, 1.79 mmol), thymine (207 mg, 1.64
mmol), 14 (450 mg, 1.49 mmol), CH3CN (40 mL). H-WNA-RT (22a-
(1)): a colorless oil (29%): 1H NMR (500 MHz, CDCl3) δ 1.91 (s,
3H), 2.08 (s, 3H), 2.12 (s, 3H), 2.14-2.22 (m, 1H), 2.60 (dd, 1H, J )
6.18, 14.2 Hz), 4.08 (dd, 1H, J ) 5.27, 11.9 Hz), 2.16 (ddd, 1H, J )
2.51, 5.27, 8.02 Hz), 4.28-4.32 (m, 1H), 4.87 (dd, 1H, J ) 4.80, 7.79
Hz), 4.94 (t, 1H, J ) 5.04 Hz), 5.04 (t, 1H, J ) 4.58 Hz), 6.18 (dd,
1H, J ) 6.41, 7.56 Hz), 7.01 (d, 1H, J ) 1.14 Hz), 8.53 (bs, 1H).
FTIR (CHCl3) 3390, 1744, 1693 cm-1. HRMS (ESIMS) m/z calcd for
C16H21N2O8 (M + H)+ 369.1292, found 369.1327. H-WNA-âT (22b-
) 6.9 Hz), 2.72 (pseudoquintet, 1H, J ) 6.9 Hz), 3.09-3.22 (m, 2H),
3.62 (dd, 1H, J ) 5.6, 11.8 Hz), 3.78-3.87 (m, 2H), 3.90 (d, 1H, J )
3.3 Hz), 4.70 (d, 1H, J ) 3.3 Hz), 6.92 (dd, 1H, J ) 3.3, 6.9 Hz),
7.25-7.41 (m, 3H), 7.63-7.66 (m, 2H), 8.68 (s, 1H). 13C NMR (125
MHz, CD3OD) δ 19.35, 29.54, 50.23, 63.68, 73.38, 84.06, 89.07, 91.88,
93.42, 121.61, 126.64, 128.73, 129.51, 142.29, 143.53, 149.21, 154.89,
159.26, 181.83. FTIR (CHCl3) 3400, 1690 cm-1. FABMS (m/z) 456
(M + H)+. WNA-7âG (17b(2)): a white powder (88%). Mp 164-
1
169 °C. H NMR (270 MHz, CD3OD) δ 1.17-1.19 (m, 6H), 2.86 (t,
1H, J ) 6.3 Hz), 2.81 (dd, 3H, J ) 5.6, 13.1 Hz), 3.15-3.27 (m, 2H),
2.94 (dd, 1H, J ) 5.8, 13.7 Hz), 3.67 (t, 1H, J ) 5.9 Hz), 3.83 (dd,
1H, J ) 3.6, 9.2 Hz), 4.05 (ddd, 1H, J ) 2.6, 5.9, 8.9 Hz), 5.02 (d,
1H, J ) 4.0 Hz), 6.45 (dd, 1H, J ) 5.9, 8.8 Hz), 7.63-7.73 (m, 3H),
7.79-7.82 (m, 2H), 8.34 (bs, 1H). 13C NMR (125 MHz, CD3OD) δ
19.35, 36.93, 51.19, 63.78, 73.80, 84.77, 90.72, 90.75, 93.41, 112.00,
126.77, 128.57, 129.31, 141.54, 145.33, 149.57, 154.56, 160.65, 181.86.
FTIR (CHCl3) 3400, 1690 cm-1. FABMS (m/z) 456 (M + H)+.
1
(1)): a colorless oil (46%). H NMR (500 MHz, CDCl3) δ 1.86 (s,
3H), 2.08 (s, 3H), 2.09 (s, 3H), 2.44 (dd, 1H, J ) 8.70, 14.2 Hz), 2.77-
2.82 (m, 1H), 4.08 (dd, 1H, J ) 5.50, 11.7 Hz), 4.10-4.13 (m, 1H),
4.32 (dd, 1H, J ) 2.29, 11.7 Hz), 4.82 (dd, 1H, J ) 4.58, 8.47 Hz),
5.10 (d, 2H, J ) 5.49 Hz), 6.91 (d, 1H, J ) 4.81 Hz), 6.92 (d, 1H, J
) 5.27 Hz), 8.71 (bs, 1H). FTIR (CHCl3) 3427, 1740, 1720, 1659 cm-1
.
HRMS (ESIMS) m/z calcd for C16H20N2O8Na (M + Na)+ 391.1112,
(1′S,3′R,4′R,5′R,7′S and 7′R)-N6-Benzoyl-9-{4′-hydroxy-3′-hy-
droxymethyl-1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}adenine 18a,b-
found 391.1071.
1
Deprotection: General Procedure. A THF solution of the above
compound and TBAF (1.0 M THF solution, 2 equiv) was stirred for
1-2 h at room temperature, and the reaction mixture was diluted with
AcOEt. The organic layer was successively washed with water and
brine, dried over Na2SO4, and evaporated. The residue was purified by
flash chromatography (silica gel, CHCl3-CH3OH) to give the corre-
sponding compound, which was further deacetylated in a solution of
THF/methanol ) 9:1 containing 0.2 M NaOH (2 equiv) at 0 °C. After
stirring for 45-60 min at 0 °C, the reaction mixture was quenched
with acetic acid and diluted with MeOH, and then the solvent was
evaporated. The residue was purified by flash chromatography (silica
gel, CHCl3-CH3OH).
(1S,3R,4R,5R)-4-Hydroxy-3-hydroxymethyl-1-phenyl-2,6-
dioxabicyclo[3.3.0]octane, WNA-H, 15(2). A white powder (70%).
mp 122-125 °C. 1H NMR (400 MHz, CD3OD) δ 2.36 (ddd, 1H, J )
3.18, 5.99, 13.1 Hz), 2.50 (ddd, 1H, J ) 8.24, 9.73, 13.1 Hz), 3.83-
3.94 (m, 3H), 4.09 (ddd, 1H, J ) 5.99, 8.62, 9.73 Hz), 4.23 (dt, 1H, J
) 3.0, 8.24 Hz), 4.29 (d, 1H, J ) 4.11 Hz), 7.23 (t, 1H, J ) 7.49 Hz),
7.33 (t, 2H, J ) 7.49 Hz), 7.55 (d, 2H, J ) 7.49 Hz). 13C NMR (100
MHz, CD3OD): δ 45.43, 63.65, 71.32, 73.67, 84.82, 90.25, 94.07,
126.23, 128.14, 129.22, 143.46. FTIR (KBr) 3400 cm-1. HRMS
(ESIMS): m/z calcd for C13H17O4 (M + H)+ 237.1121, C13H16O4Na
(M + Na)+ 259.0941, found 237.1157, 259.0923.
(2). WNA-RA (18a(2)): a white powder (79%). Mp 125-128 °C. H
NMR (400 MHz, CD3OD) δ 3.21 (dd, 1H, J ) 6.37, 13.66 Hz), 3.29
(dd, 1H, J ) 8.42, 13.85 Hz), 3.67 (dd, 1H, J ) 5.99, 11.98 Hz), 3.83-
3.89 (m, 2H), 3.95-4.53 (m, 1H), 4.73 (d, 1H, J ) 3.74 Hz), 6.89 (dd,
1H, J ) 6.18, 8.42 Hz), 7.31 (t, 1H, J ) 7.30 Hz), 7.41 (t, 2H, J )
7.68 Hz), 7.57 (t, 2H, J ) 7.30 Hz), 7.65 (t, 2H, J ) 7.48 Hz), 7.70 (d,
2H, J ) 7.30 Hz), 8.09 (d, 2H, J ) 7.67 Hz), 8.75 (s, 1H), 8.86 (s,
1H). 13C NMR (125 MHz, CD3OD) δ 48.96, 63.71, 73.36, 83.95, 86.25,
91.41, 93.42, 109.52, 126.42, 128.74, 129.41, 129.53, 129.55, 134.18,
136.54, 141.10, 142.39, 151.32, 153.99, 164.61. FTIR (KBr) 3400,
1701, 1614, 1582 cm-1. HRMS (ESIMS) m/z calcd for C25H24N5O5
(M + H)+ 474.1772, found 472.1798. WNA-âA (18b(2)): a white
powder (59%). Mp 126-128 °C. 1H NMR (400 MHz, CD3OD) δ 3.00
(dd, 1H, J ) 6.37, 13.66 Hz), 3.51 (dd, 1H, J ) 8.42, 13.85 Hz), 3.73
(dd, 1H, J ) 5.99, 11.98 Hz), 3.91-3.95 (m, 2H), 4.13-4.17 (m, 1H),
5.02 (d, 1H, J ) 3.74 Hz), 6.67 (dd, 1H, J ) 6.18, 8.42 Hz), 7.29 (t,
1H, J ) 7.30 Hz), 7.40 (t, 2H, J ) 7.30 Hz), 7.56 (t, 2H, J ) 7.30
Hz), 7.65 (t, 1H, J ) 7.30 Hz), 8.06 (d, 2H, J ) 7.30 Hz), 8.09 (d, 2H,
J ) 7.30 Hz), 8.63 (s, 1H), 8.81 (s, 1H). 13C NMR (125 MHz, CD3-
OD) δ 49.21, 63.66, 73.75, 84.63, 88.82, 91.33, 93.56, 106.37, 126.65,
128.71, 129.43, 129.77, 133.90, 141.76, 144.99, 151.29, 153.24, 168.54.
FTIR (KBr) 3400, 1697, 1614, 1582 cm-1. HRMS (ESIMS) m/z calcd
for C25H24N5O5 (M + H)+ 474.1772, found 472.1814.
(1′S,3′R,4′R,5′R,7′S and 7′R)-N2-Isobutyryl-9-and-7-{4′-hydroxy-
3′-hydroxymethyl-1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}-
guanine 16a,b(2) and 17a,b(2). WNA-9RG (16a(2)): a white powder
(78%). Mp 156-159 °C. 1H NMR (270 MHz, CD3OD) δ 1.23 (d, 6H,
J ) 6.9 Hz), 2.72 (pseudoquintet, 1H, J ) 6.9 Hz), 3.09-3.29 (m,
2H), 3.65 (dd, 1H, J ) 6.6, 12.2 Hz), 3.80-3.89 (m, 2H), 4.02 (dd,
1H, J ) 2.6, 6.5 Hz), 4.63 (d, 1H, J ) 4.3 Hz), 6.60 (dd, 1H, J ) 4.3,
7.8 Hz), 7.28-7.42 (m, 3H), 7.64-7.67 (m, 2H), 8.40 (s, 1H). 13C
NMR (125 MHz, CD3OD) δ 19.32, 36.98, 51.19, 63.70, 73.33, 83.82,
86.46, 91.56, 93.34, 121.35, 126.41, 128.74, 129.52, 140.01, 142.37,
(1′S,3′R,4′R,5′R,7′S and 7′R)-(4′-Hydroxy-3′-hydroxymethyl-1′-
phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl)thymine 19a,b(2). WNA-RT
(19a(2)): a colorless foam (47%). 1H NMR (270 MHz, CD3OD) δ 2.16
(s, 3H), 2.74 (dd, 1H, J ) 3.63, 14.84 Hz), 2.92-3.04 (m, 1H), 3.67
(dd, 1H, J ) 6.92, 12.21 Hz), 3.80-3.92 (m, 2H), 4.11-4.16 (m, 1H),
4.57-4.62 (m, 1H), 6.61 (dd, 1H, J ) 3.96, 6.60 Hz), 7.26-7.59 (m,
6H), 7.71 (d, 2H, J ) 7.25 Hz), 8.17-8.24 (m, 1H), 8.27-8.31 (m,
2H). 13C NMR (100 MHz, CD3OD) δ 12.58, 48.69, 63.90, 73.44, 84.05,
87.10, 90.37, 93.41, 112.21, 126.30, 128.67, 129.50, 138.37, 142.70,
152.69, 166.31. FTIR (film) 3400, 1690 cm-1. FABMS (m/z): 361
[M + H]+. WNA-âT (19b(2)): a colorless foam (71%). 1H NMR (600
MHz, CD3OD) δ 1.91 (s, 3H), 2.71 (dd, 1H, J ) 8.42, 13.76 Hz), 2.75
(dd, 1H, J ) 6.03, 13.76 Hz), 3.68 (dd, 1H, J ) 6.04, 12.07 Hz), 3.86-
3.89 (m, 2H), 4.00-4.03 (m, 1H), 4.84 (d, 1H, J ) 3.65 Hz), 6.29 (dd,
1H, J ) 6.03, 8.43 Hz), 7.26 (t, 1H, J ) 7.44 Hz), 7.35 (t, 2H, J )
7.44 Hz), 7.69 (t, 3H, J ) 7.45 Hz). 13C NMR (100 MHz, CD3OD) δ
12.33, 49.21, 63.53, 73.69, 84.52, 90.08, 90.55, 93.18, 111.83, 126.51,
128.62, 129.35, 138.83, 141.54, 152.23, 166.40. FTIR (film) 3400, 1690
cm-1. HRMS (ESIMS) m/z calcd for C18H21N2O6 (M + H)+ 361.1394,
found 361.1412.
145.33, 150.88, 157.55, 181.72. FTIR (CHCl3) 3400, 1690 cm-1
.
FABMS (m/z) 456 (M + H)+. WNA-9âG (16b(2)): a white powder
(91%). Mp 160-163 °C. 1H NMR (270 MHz, CD3OD) δ 1.22 (d, 3H,
J ) 4.6 Hz), 1.24 (d, 3H, J ) 4.6 Hz), 2.73 (pseudoquintet, 1H, J )
6.9 Hz), 2.91 (dd, 1H, J ) 5.6, 13.5 Hz), 3.06 (dd, 1H, J ) 8.6, 13.5
Hz), 3.71 (dd, 1H, J ) 5.6, 11.9 Hz), 3.91 (dd, 1H, J ) 6.6, 12.2 Hz),
3.92 (dd, 1H, J ) 4.0, 9.2 Hz), 4.06 (ddd, 1H, J ) 2.3, 5.9, 8.6 Hz),
4.96 (d, 1H, J ) 3.6 Hz), 6.48 (dd, 1H, J ) 5.6, 8.6 Hz), 7.25-7.40
(m, 3H), 7.71-7.74 (m, 2H), 8.30 (s, 1H). 13C NMR (125 MHz, CD3-
OD) δ 19.33, 36.95, 50.18, 64.34, 73.72, 84.98, 87.69, 90.48, 93.22,
121.75, 126.64, 128.73, 129.44, 139.63, 141.50, 149.73, 150.26, 157.49,
181.73. FTIR (CHCl3) 3400, 1690 cm-1. FABMS (m/z) 456 (M + H)+.
WNA-7RG (17a(2)): a white powder (94%). Mp 157-160 °C. 1H
NMR (270 MHz, CD3OD) δ 1.20 (d, 3H, J ) 6.9 Hz), 1.22 (d, 3H, J
(1′S,3′R,4′R,5′R,7′S and 7′R)-N4-Benzoyl-1-(4′-hydroxy-3′-hy-
droxymethyl-1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl)-5-methyl-
1
cytosine 20a,b(2). WNA-RmC (20a(2)): a colorless foam (44%). H
NMR (270 MHz, CD3OD) δ 2.16 (dd, 1H, J ) 3.63, 14.84 Hz), 2.92-
9
J. AM. CHEM. SOC. VOL. 126, NO. 2, 2004 525

A R T I C L E S
Sasaki et al.
3.04 (m, 1H), 3.67 (dd, 1H, J ) 6.92, 12.21 Hz), 3.80-3.92 (m, 2H),
4.11-4.16 (m, 1H), 4.57-4.62 (m, 1H), 6.61 (dd, 1H, J ) 3.96, 6.60
Hz), 7.26-7.59 (m, 6H), 7.71 (d, 2H, J ) 7.25 Hz), 8.17-8.24 (m,
1H), 8.27-8.31 (m, 2H). 13C NMR (125 MHz, CD3OD): δ 23.51,
48.83, 56.67, 63.96, 73.42, 79.04, 82.46, 89.91, 107.21, 126.33, 127.60,
129.54, 133.51, 137.00, 140.45, 142.66, 149.59, 163.91, 172.42. FTIR
(KBr) 3400, 1701, 1560 cm-1. HRMS (ESIMS) m/z calcd for
C25H26N3O6 (M + H)+ 464.1816, found 464.1827. WNA-âmC (20b-
(2)): a colorless foam (72%). 1H NMR (270 MHz, CD3OD) δ 2.16 (s,
3H), 2.77-2.80 (m, 2H), 3.67 (dd, 1H, J ) 5.61, 11.88 Hz), 3.88 (dd,
1H, J ) 3.63, 8.25 Hz), 4.02 (dd, 1H, J ) 2.31, 8.25 Hz), 4.88-4.91
(m, 1H), 6.31 (dd, 1H, J ) 6.60, 7.26 Hz), 7.24-7.52 (m, 7H), 7.71
(d, 2H, J ) 7.26 Hz), 7.91-7.93 (m, 1H), 8.26-8.30 (m, 2H). 13C
NMR (125 MHz, CD3OD) δ 25.14, 49.30, 56.00, 63.52, 73.74, 84.59,
90.87, 93.30, 106.37, 126.58, 128.67, 129.40, 133.68, 136.96, 141.33,
with sat. NaHCO3 solution and extracted with AcOEt. The organic layer
was separated, dried over Na2SO4, and evaporated. The residue was
purified by flash chromatography (silica gel, hexane/AcOEt) to give
the purified material, which was crystallized in hexane at -78 °C. The
hexane was removed by decantation, and the solid material was dried
in a vacuum for several hours.
(1S,3R,4R,5R)-3-Dimethoxytrithyloxymethyl-4-O-(N,N-diisopro-
pyl-â-cyanoethylphosphoramidyl)-1-phenyl-2,6-dioxabicyclo[3.3.0]-
1
octane WNA-H, 15(3): a white powder (77%). H NMR (270 MHz,
CDCl3/TMS) δ 1.07 (d, 6H, J ) 6.6 Hz), 1.08 (d, 2H, J ) 6.6 Hz),
2.32-2.54 (m, 2H), 2.62 (t, 2H, J ) 6.27 Hz), 3.36-3.59 (m, 4H),
3.80 (s, 6H), 3.81-4.05 (m, 2H), 4.11-4.33 (m, 4H), 4.40 (d, 0.5H, J
) 3.96 Hz), 4.50 (d, 0.5H, J ) 4.29 Hz), 6.82-6.85 (m, 4H), 7.16-
7.39 (m, 10H), 7.46-7.51 (m, 2H), 7.57-7.64 (m, 2H). FTIR (CHCl3)
2341.4 cm-1. HRMS (ESIMS): m/z, calcd for C43H52N2O7P (M + H)+
739.3507, found 739.3554.
141.61, 156.55, 165.91, 169.48. FTIR (KBr) 3400, 1703, 1568 cm-1
.
HRMS (ESIMS) m/z calcd for C25H26N3O6 (M + H)+ 464.1816, found
(1′S,3′R,4′R,5′R,7′S and 7′R)-N2-Isobutyryl-9-and-7-{3′-dimethoxy-
trithyloxymethyl-4′-O-(N,N-diisopropyl-â-cyanoethylphosphora-
midyl)-1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}guanine 16a,b(3)
464.1812.
(1′S,3′R,4′R,5′R,7′S and 7′R)-N4-Benzoyl-1-(4′-hydroxy-3′-hy-
droxymethyl-1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl)cytosine 21a,b-
(2). WNA-RC (21a(2)): a colorless foam (34%). 1H NMR (270 MHz,
CD3OD) δ 2.72 (dd, 1H, J ) 4.5, 15.0 Hz), 3.07 (dd, 1H, J ) 7.6,
15.0 Hz), 3.64 (dd, 1H, J ) 6.6, 11.9 Hz), 3.79 (dd, 1H, J ) 2.6, 11.9
Hz), 3.93 (dd, 1H, J ) 4.2, 8.9 Hz), 4.05-4.12 (m, 1H), 4.77 (dd, 1H,
J ) 1.3, 2.0 Hz), 6.49 (dd, 1H, J ) 5.0, 7.6 Hz), 7.25-7.72 (m, 9H),
7.96-8.00 (m, 2H), 8.68 (d, 1H, J ) 7.6 Hz). 13C NMR (100 MHz,
d-DMSO) δ 48.84, 61.59, 71.47, 82.60, 87.42, 89.63, 91.19, 96.25,
125.26, 127.21, 128.11, 128.39, 128.42, 132.68, 133.13, 141.84, 145.13,
1
and 17a,b(3). WNA-9âG (16b(3)): a white powder (91%). H NMR
(270 MHz, CDCl3) δ 1.17 (d, 6H, J ) 6.6 Hz), 1.20 (d, 6H, J ) 6.9
Hz), 1.25 (d, 3H, J ) 3.3 Hz), 1.28 (d, 3H, J ) 3.3 Hz), 2.38-2.51
(m, 2H), 2.63 (pseudoquintet, 0.5H, J ) 6.9 Hz), 2.67 (pseudoquintet,
0.5H, J ) 6.6 Hz), 2.95-3.12 (m, 1H), 3.22-3.28 (m, 1H), 3.40-
3.73 (m, 4H), 3.79 (s, 3H), 3.80 (s, 3H), 4.07-4.35 (m, 3H), 5.22 (d,
0.5H, J ) 2.6 Hz), 5.31 (d, 0.5H, J ) 3.3 Hz), 6.32 (dd, 0.5H, J )
6.9, 7.6 Hz), 6.40 (dd, 0.5H, J ) 6.6, 7.6 Hz), 6.82 (dd, 4H, J ) 4.6,
8.6 Hz), 7.20-7.49 (m, 11H), 7.69-7.80 (m, 3H), 8.13 (s, 1H), 8.90
(bs, 1H), 12.0 (bs, 1H). WNA-9RG (16a(3)): a white powder (74%).
1H NMR (270 MHz, CDCl3) δ 1.17 (d, 6H, J ) 6.6 Hz), 1.20 (d, 6H,
J ) 6.9 Hz), 1.25 (d, 3H, J ) 3.3 Hz), 1.28 (d, 3H, J ) 3.3 Hz), 2.31
(dd, 2H, J ) 2.9, 6.9 Hz), 2.48 (t, 1H, J ) 6.6 Hz), 2.93-3.28 (m,
2H), 3.40-3.57 (m, 4H), 3.73 (s, 3H), 3.74 (s, 3H), 4.00-4.34 (m,
3H), 5.20 (d, 1H, J ) 2.6 Hz), 6.32 (dd, 0.5H, J ) 6.9, 7.6 Hz), 6.40
(dd, 0.5H, J ) 6.6, 7.6 Hz), 6.75-6.80 (m, 4H), 7.16-7.49 (m, 12H),
7.72-7.78 (m, 2H), 8.08 (s, 1H), 9.80 (bs, 1H), 12.2 (bs, 1H). WNA-
7âG (17b(3)): a white powder (92%). 1H NMR (270 MHz, CDCl3) δ
1.17 (d, 6H, J ) 6.6 Hz), 1.20 (d, 6H, J ) 6.9 Hz), 1.25 (d, 3H, J )
3.3 Hz), 1.28 (d, 3H, J ) 3.3 Hz), 2.31 (dd, 2H, J ) 2.9, 6.9 Hz), 2.48
(t, 1H, J ) 6.6 Hz), 2.93-3.28 (m, 2H), 3.40-3.57 (m, 4H), 3.76 (s,
3H), 3.77 (s, 3H), 4.00-4.34 (m, 3H), 5.20 (d, 1H, J ) 2.6 Hz), 6.36
(dd, 0.5H, J ) 6.9, 7.6 Hz), 6.44 (dd, 0.5H, J ) 6.6, 7.6 Hz), 6.77-
6.86 (m, 4H), 7.16-7.49 (m, 12H), 7.72-7.78 (m, 2H), 7.91 (s, 0.5H),
8.00 (s, 0.5H), 10.5 (bs, 1H), 12.3 (bs, 1H). WNA-7RG (17a(3)): a
152.95, 163.11, 167.27. FTIR (KBr) 3321, 1684, 1659, 1485 cm-1
.
ESIMS (m/z) 450 [M + H]+. WNA-âC (21b(2)): a colorless foam
1
(76% for tow steps). H NMR (270 MHz, CD3OD) δ 2.70 (dd, 1H, J
) 7.92, 14.02 Hz), 3.01 (dd, 1H, J ) 5.94, 14.02 Hz), 3.73-3.94 (m,
2H), 4.04-4.09 (m, 1H,), 4.94 (d, 1H, J ) 3.63 Hz), 6.34 (dd, 2H, J
) 5.94, 7.92 Hz), 7.23-7.70 (m, 9H), 7.96-8.00 (m, 2H), 8.40 (d,
1H, J ) 7.30 Hz). 13C NMR (100 MHz, CD3OD) δ 50.39, 63.43, 73.66,
84.38, 91.42, 92.26, 93.36, 98.71, 126.52, 128.66, 129.16, 129.40,
129.82, 134.10, 134.67, 141.63, 147.03, 157.68, 165.00, 169.14. FTIR
(KBr) 3381, 1653, 1560, 1487 cm-1. HRMS (ESIMS) m/z calcd for
C24H24N3O6 (M + H)+ 450.1660, found 450.1683.
(1′R,3′R,4′R,5′S,7′S)-{4′-Hydroxy-3′-hydroxymethyl-2′,6′-
dioxabicyclo[3.3.0]oct-7′-yl}thymine 22b(2). 0.2 M NaOH (2.3 mL,
0.468 mmol) was added to a solution of 22b(1) (48 mg, 0.13 mmol) in
THF/methanol ) 9:1 (0.98 mL) at 0 °C. After stirring for 1 h at 0 °C,
the reaction mixture was quenched with acetic acid and diluted with
MeOH, and then the solvent was evaporated. The residue was purified
by flash chromatography (silica gel, CHCl3/CH3OH ) 5:1) to give the
diol compound as a colorless oil (37 mg, 99%): 1H NMR (400 MHz,
CD3OD) δ 1.83 (s, 3H), 2.36 (ddd, 1H, J ) 1.68, 8.61, 14.22 Hz),
2.80 (ddd, 1H, J ) 4.87, 7.11, 14.22 Hz), 3.57 (dd, 1H, J ) 4.87,
11.98 Hz), 3.70 (ddd, 1H, J ) 2.25, 4.87, 8.98 Hz), 3.80 (dd, 1H, J )
2.25, 11.98 Hz), 3.88 (d, 1H, J ) 4.68, 8.98 Hz), 4.82 (d, 1H, J )
4.68 Hz), 4.97 (dt, 1H, J ) 1.68, 6.93 Hz), 6.90 (dd, 1H, J ) 4.87,
8.61 Hz), 7.17 (s, 1H). 13C NMR (125 MHz, CD3OD) δ 14.45, 40.03,
62.54, 73.89, 82.18, 84.23, 85.71, 87.04, 110.32, 137.77, 152.99, 168.65.
FTIR (film) 3081 cm-1. ESIMS (m/z) 307 (M + Na)+.
1
colorless oil (82%). H NMR (270 MHz, CDCl3) δ 1.07 (d, 6H, J )
6.9 Hz), 1.12 (d, 6H, J ) 5.6 Hz), 1.25 (d, 3H, J ) 4.3 Hz), 1.28 (d,
3H, J ) 4.3 Hz), 3.00-3.24 (m, 2H), 3.33-3.62 (m, 4H), 3.76 (s,
3H), 3.77 (s, 3H), 4.04-4.30 (m, 3H), 5.77 (d, 1H, J ) 2.6 Hz), 6.76-
6.81 (m, 4H), 6.88 (dd, 1H, J ) 2.9, 6.8 Hz), 6.91 (dd, 1H, J ) 3.6,
7.2 Hz), 7.15-7.40 (m, 12H), 7.51-7.58 (m, 2H), 8.14 (s, 1H), 9.98
(bs, 1H), 12.3 (bs, 1H).
(1′S,3′R,4′R,5′R,7′S and 7′R)-N6-Benzoyl-9-{3′-dimethoxytrithyl-
oxymethyl-4′-O-(N,N-diisopropyl-â-cyanoethylphosphoramidyl)-
1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}adenine 18a,b(3). WNA-
1
RA (18a(3)): a white powder (79%). H NMR (400 MHz, CDCl3/
General Procedure of the Synthesis of the â-Cyanoethylphos-
phoramidite Precursors of WNA. DMTrCl (1.5 equiv) was added to
a solution of the dihydroxyl derivative of WNA in pyridine, and the
mixture was stirred for 1 h. The mixture was diluted with EtOAc and
successively washed with water and brine. The organic layer was dried
over Na2SO4 and evaporated, and then the residue was purified by flash
chromatography (silica gel, CHCl3-CH3OH containing 0.5% pyridine)
to produce the corresponding DMTr-protected WNA. iPr2NP(Cl)OC2H4-
CN (6 equiv) was added to a solution of the above DMTr derivative
of WNA and iPr2NEt (3 equiv) in dry CH2Cl2 at 0 °C. After stirring
for 60 min at the same temperature, the reaction mixture was quenched
TMS): δ 0.87 (d, 2H, J ) 6.74 Hz), 1.06 (dd, 5H, J ) 3.56, 6.74 Hz),
1.11 (d, 2H, J ) 6.92 Hz), 1.26 (d, 3H, J ) 5.81, 6.78 Hz), 3.26-3.33
(m, 1H), 3.40-3.60 (m, 5H), 3.69-3.73 (m, 1H), 3.77 (s, 3H), 3.79
(s, 3H), 4.09-4.27 (m, 4H), 4.48 (d, 0.5H, J ) 3.93 Hz), 4.95 (d,
0.5H, J ) 3.74 Hz), 6.15 (s, 1H), 6.79-6.85 (m, 4H), 7.12-7.37 (m,
9H), 7.39-7.42 (m, 2H), 7.51-7.54 (m, 2H), 7.59-7.63 (m, 3H), 7.75
(s, 1H), 8.03 (d, 2H, J ) 7.49 Hz), 8.69 (d, 1H, J ) 4.49 Hz), 8.83 (d,
1H, J ) 5.43 Hz), 9.01 (bs, 1H). FTIR (KBr) 2252, 1701, 1609 cm-1
.
WNA-âA (18b(3)): a white powder (79%). 1H NMR (400 MHz,
CDCl3/TMS) δ 0.87 (d, 2H, J ) 6.74 Hz), 0.94 (d, 2H, J ) 6.74 Hz),
1.07 (dd, 5H, J ) 6.74, 9.73 Hz), 1.21-1.35 (m, 3H), 3.12-3.20 (m,
9
526 J. AM. CHEM. SOC. VOL. 126, NO. 2, 2004

A R T I C L E S
Table 3. Selected Chemical Shifts of 1H and 13C NMR of Diol
1H), 3.25-3.55 (m, 5H), 3.62-3.74 (m, 1H), 3.79 (s, 3H), 3.80 (s,
3H), 4.09-4.15 (m, 2H), 4.32-4.46 (m, 2H), 5.16 (d, 0.5H, J ) 2.81
Hz), 5.17 (d, 0.5H, J ) 3.56 Hz), 6.55 (dd, 0.5H, J ) 6.74, 7.49 Hz),
6.63 (dd, 0.5H, J ) 6.74, 7.67 Hz), 6.82 (d, 2H, J ) 8.79 Hz), 6.83 (d,
2H, J ) 8.98 Hz), 7.20-7.39 (m, 10H), 7.46-7.55 (m, 4H), 7.61 (t,
1H, J ) 7.49 Hz), 7.76 (d, 1H, J ) 6.93 Hz), 7.82 (d, 1H, J ) 6.93
Hz), 8.03 (d, 2H, J ) 7.68 Hz), 8.15 (s, 0.5H), 8.23 (s, 0.5H), 8.87 (d,
Derivatives of WNA (16(2) and 17(2))a
7-âG
9-âG
7-RG
9-RG
H-8b
8.39
90.72
160.65
112.00
145.33
8.28
87.69
150.26
121.75
139.63
8.68
89.07
159.26
112.61
143.53
8.40
86.46
145.33
121.35
140.01
C-anomericc
C-4c
C-5c
C-8b
1H, J ) 4.31 Hz), 9.00 (bs, 1H). FTIR (KBr) 2252, 1701, 1609 cm-1
.
(1′S,3′R,4′R,5′R,7′S and 7′R)-{3′-Dimethoxytrithyloxymethyl-4′-
O-(N,N-diisopropyl-â-cyanoethylphosphoramidyl)-1′-phenyl-2′,6′-
dioxabicyclo[3.3.0]oct-7′-yl}thymine 19a,b(3). WNA-RT (19a(3)): a
b
c
a ppm from TMS in CD3OD. 1H NMR measured at 500 MHz. 13C
NMR measured at 125 MHz.
1
Table 4. MALDI-TOF MS (negative mode) of the TFO Containing
white powder (37%). H NMR (270 MHz, CDCl3/TMS) δ 0.88 (d,
WNA Analogue (M-1 m/z)
2H, J ) 6.6 Hz), 0.99-1.13 (m, 6H), 1.16-1.38 (m, 4H), 2.04 (s,
3H), 2.34 (dd, 1H, J ) 6.2, 7.8 Hz), 2.55 (t, 1H, J ) 6.3 Hz), 2.62-
2.73 (m, 1H), 2.92 (ddd, 1H, J ) 2.6, 8.1, 14.9 Hz), 3.24-3.30 (m,
2H), 3.35-3.75 (m, 4H), 3.78 (s, 3H), 3.79 (s, 3H), 4.12 (dd, 2H, J )
6.9, 15.1 Hz), 4.23-4.36 (m, 2H), 4.55 (dd, 0.5H, J ) 3.3 Hz), 4.66
(d, 0.5H, J ) 3.9 Hz), 6.57 (dd, 1H, J ) 4.9, 7.5 Hz), 6.79-6.83 (m,
4H), 7.20-7.37 (m, 10H), 7.43-7.49 (m, 4H), 7.76 (s, 1H), 7.80 (s,
1H). FTIR (KBr) 1690 cm-1. WNA-âT (19b(3)): a white powder
(76%). 1H NMR (270 MHz, CDCl3/TMS) δ 0.99-1.13 (m, 6H), 1.16-
1.38 (m, 4H), 2.04 (s, 3H), 2.35 (dd, 1H, J ) 6.2, 11.1 Hz), 2.62 (dd,
2H, J ) 5.9, 14.1 Hz), 2.97-3.02 (m, 1H), 3.17-3.24 (m, 1H), 3.35-
3.75 (m, 4H), 3.78 (s, 3H), 3.79 (s, 3H), 4.12 (dd, 2H, J ) 6.9, 14.1
Hz), 4.21-4.37 (m, 4H), 4.91 (d, 0.5H, J ) 3.6 Hz), 5.03 (d, 0.5H, J
) 3.6 Hz), 6.21 (dd, 0.5H, J ) 6.6, 8.5 Hz), 6.40 (dd, 0.5H, J ) 6.6,
8.5 Hz), 6.80 (dd, 4H, J ) 1.9, 8.8 Hz), 7.20-7.35 (m, 10H), 7.42-
7.48 (m, 2H), 7.60 (dd, 2H J ) 1.9, 9.2 Hz), 7.65 (dd, 1H, J ) 1.9, 6.1
TFO
WNA
calcd
found
23
23
23
23
23
23
23
23
25
23
23
23
23
23
23
WNA-9RG
5883.06
5883.06
5883.06
5883.06
5867.05
5867.05
5858.29
5858.29
5951.56
5857.06
5857.06
5845.06
5845.06
5734.10
5782.01
5886.0
5882.64,
5883.3
WNA-7RG
WNA-9âG
WNA-7âG
WNA-RA
5880.68
5866.79
5866.06
5858.3,
5857.91
5955.46
5855.3
5859.97
5843.11
5843.44
5730.50
5780.66
WNA-âA
WNA-RT
WNA-âT
WNA-âT, WNA-âT
WNA-RmC
WNA-âmC
WNA-RC
WNA-âC
WNA-H
H-WNA-âT
Hz), 8.20 (bs, 1H). FTIR (CHCl3) 1690 cm-1
.
(1′S,3′R,4′R,5′R,7′S and 7′R)-N4-Benzoyl-1-{3′-dimethoxytrithyl-
oxymethyl-4′-O-(N,N-diisopropyl-â-cyanoethylphosphoramidyl)-
1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}-5-methylcytosine 20a,b-
(3). WNA- RmC (20a(3)): a white powder (99%). 1H NMR (270 MHz,
CDCl3/TMS) δ 0.90 (d, 2H, J ) 6.6 Hz), 1.05-1.42 (m, 6H), 1.48-
1.56 (m, 4H), 2.17 (s, 3H), 2.63-2.96 (m, 2H), 3.33-3.74 (m, 6H),
3.79 (s, 3H), 3.80 (s, 3H), 4.09-4.22 (m, 1H), 4.21-4.56 (m, 3H),
5.00-5.10 (m, 1H), 6.55-6.74 (m, 1H), 6.83 (dd, 4H, J ) 2.31, 7.58
Hz), 7.18-7.77 (m, 18H), 8.33 (d, 2H, J ) 7.59 Hz). FT-IR (CHCl3):
2341, 1707, 1572 cm-1. WNA- â mC (20b(3)) a white powder (67%).
1H NMR (270 MHz, CDCl3/TMS) δ 0.90 (d, 2H, J ) 6.6 Hz), 1.05-
1.42 (m, 6H), 1.48-1.56 (m, 4H), 2.19 (s, 3H), 2.48-2.89 (m, 2H),
3.00-3.19 (m, 2H), 3.33-3.74 (m, 4H), 3.79 (s, 3H), 3.80 (s, 3H),
4.01-4.09 (m, 1H), 4.21-4.38 (m, 3H), 4.95-5.08 (m, 1H), 6.23-
6.41 (m, 1H), 6.83 (dd, 4H, J ) 2.31, 7.58 Hz), 7.16-7.75 (m, 18H),
1.5H), 1.87 (s, 1.5H), 2.34-2.45 (m, 1H), 2.52 (dd, 1H, J ) 8.61,
13.1 Hz), 2.64 (t, 1H, J ) 7.11 Hz), 2.72-2.85 (m, 1H), 3.05 (dd, 1H,
J ) 5.61, 10.3 Hz), 3.28-3.63 (m, 4H), 3.78 (s, 3H), 3.79 (s, 3H),
3.83-3.89 (m, 1H), 3.93-3.98 (m, 1H), 4.05-4.08 (m, 1H), 4.16-
4.22 (m, 1H), 4.94 (t, 0.5H, J ) 4.12 Hz), 5.02 (t, 0.5H, J ) 4.50 Hz),
5.08-5.10 (m, 0.5H), 5.16-5.17 (m, 0.5H), 6.81 (dd, 4H, J ) 1.31,
7.30 Hz), 6.95 (dd, 1H, J ) 4.87, 8.42 Hz), 7.19 (t, 1H, J ) 6.92 Hz),
7.22-7.30 (m, 2H), 7.33-7.38 (m, 4H), 7.46 (t, 2H, J ) 7.11 Hz).
FTIR (film): 2253, 1717, 1651 cm-1. HRMS (ESIMS): m/z calcd for
C42H52N4O9P (M + H)+ 787.3466, found 787.3469.
Determination of the Regio- and Stereochemistry of 16(2) and
17(2). N7 or N9 alkylation was determined based on different chemical
shifts of guanine, in which signals of H-8, anomeric carbon, C-4, and
C-8 of N7-isomer appear more downfield than that of the N9-isomer
both in 1H and 13C NMR spectra. On the other hand, a signal of C-5 of
the N7-isomer appeared more upfield than that of the N9-isomer.27,28
Table 3 summarizes selected chemical shifts of each isomer.
Synthesis of the TFO Containing the WNA Analogue. The triplex-
forming oligodeoxynucleotides incorporating the WNA analogue were
synthesized by using an automated DNA synthesizer (Applied Bio-
systems 394 DNA/RNA Synthesizer) according to the standard protocol
except for the use of DCI as the activator. Cleavage and deprotection
of the synthesized oligomer were done in 28% NH4OH at 55 °C for 5
h. HPLC conditions: column, Nacalai Tesque COSMOSIL5C18-AR-
II; buffer A, 0.1 M TEAA; buffer B, CH3CN. B, 10% to 40%/20 min,
40% to 100%/30 min, linear gradient; flow rate, 4 mL/min. A peak
appeared at around tR ) 20 min was collected and freeze-dried. The
DMTr protecting group was cleaved in 10% aqueous acetic acid at
room temperature for 30 min, the resulting DMTr-OH was removed
by washing with ether, and the solvents were lyophilized. Structure
and purity of the synthesized TFO were confirmed by MALDI-TOF
MS measurements (Table 4).
8.33 (d, 2H, J ) 7.92 Hz). FTIR (CHCl3) 2341, 1709, 1572 cm-1
.
(1′S,3′R,4′R,5′R,7′S and 7′R)-N4-Benzoyl-1-{3′-dimethoxytrithyl-
oxymethyl-4′-O-(N,N-diisopropyl-â-cyanoethylphosphoramidyl)-
1′-phenyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}cytosine 21a,b(3). WNA-
1
RC (21a(3)): a white powder (94%). H NMR (270 MHz, CDCl3/
TMS) δ 1.01 (d, 2H, J ) 6.81 Hz), 1.14-1.65 (m, 12H), 2.04-2.17
(m, 1H), 2.33-2.35 (m, 1H), 2.57-2.70 (m, 1H), 3.27-3.57 (m, 4H),
3.78 (s, 3H), 3.79 (s, 3H), 4.23-3.92 (m, 4H), 6.24-6.31 (m, 1H),
6.80 (d, 2H, J ) 6.87 Hz), 6.81 (d, 2H, J ) 6.76 Hz), 7.20-7.61 (m,
20H), 7.91 (d, 1H, J ) 6.4 Hz). WNA-âC (21b(3)): a white powder
(83%). 1H NMR (270 MHz, CDCl3/TMS) δ 0.86-0.90 (m, 2H), 0.93-
1.32 (m, 12H), 2.02-2.08 (m, 1H), 2.34 (dd, 1H, J ) 6.10, 10.88 Hz),
2.45-2.63 (m, 1H), 3.18-3.76 (m, 4H), 3.79 (s, 6H), 4.08-4.16 (m,
1H), 4.29-4.39 (m, 1H), 4.95-5.06 (m, 1H), 6.31-6.43 (m, 1H), 6.82
(dd, 4H, J ) 1.65, 8.91 Hz), 7.20-7.64 (m, 18H), 7.29 (d, 1H, J )
7.92 Hz), 7.99 (d, 1H, J ) 7.26 Hz), 8.19 (d, 1H, J ) 7.26 Hz). ESIMS
(m/z) 952.4 (M + H)+.
(1′S,3′R,4′R,5′R,7′S)-{3′-Dimethoxytrithyloxymethyl-4′-O-(N,N-
diisopropyl-â-cyanoethylphosphoramidyl)-2′,6′-dioxabicyclo[3.3.0]-
oct-7′-yl}thymine 22b(3). H-WNA-âT (22b(3)): a white powder
(66%). H NMR (400 MHz, CDCl3/TMS) δ 0.91 (d, 2H, J ) 6.74
Hz), 1.09 (d, 6H, J ) 6.74 Hz), 1.14 (d, 4H, J ) 6.74 Hz), 1.85 (s,
Purification of the Target Duplex. A mixture containing equal
amounts of the complementary oligodeoxynucleotides was heated at
95 °C for 10 min, 55 °C for 30 min, 40 °C for 30 min, and 25 °C for
30 min. The duplex was purified by HPLC (Column: ZORBAX Oligo
Column (6.2 mm ID × 80 mm, 5µm); buffer A, 20% CH3CN, 80%
1
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A R T I C L E S
Sasaki et al.
0.02 M sodium phosphate (pH ) 7.0); buffer B, A + 1.0 M NaCl. B:
40% to 80%/15 min, 80% to 100%/20 min, linear gradient; flow rate,
1.0 mL/min) and by ethanol precipitation for desalination.
the following equation, Ks ) [Triplex]/([duplex][TFO]), and averaged
data from those obtained by multiple experiments are shown in Tables
1 and 2.
Gel-Shift Assay. TFOs were 5′ end-labeled by using [γ-32P]ATP
(4000 Ci/mmol, ICN Biomedicals, Inc.) and T4 polynucleotide kinase
(500 U, TAKARA Bio, Inc.) in T4 Kinase buffer according to the
standard protocol. After incubating for 45 min at 37 °C, 250 mM EDTA
and TEN 100 buffer were added to the mixture, and then the mixture
was purified with DE52 and DOWEX50. The purity of labeled TFO
was checked by 15% denatured polyacrylamide gel in the presence of
10 M urea. The mixture of the TFO (10 nM) containing the
corresponding 32P-labeled TFO (40000 cpm) and target duplex (0-
100 nM) was incubated in a buffer containing 20 mM Tris-HCl (pH )
7.5), 20 mM (or 5 mM) MgCl2, 2.5 mM spermidine, and 10% sucrose
for 12-15 h at 22 °C. The mixture was analyzed by electrophoresis
with 15% nondenatured polyacrylamide gel at 10 °C for 6-7 h at 110
V. Gels were visualized by BAS2500, and each band was quantified.
The stability constants (Ks) for each TFO were then calculated using
Acknowledgment. This work was supported by Grant-in-
Aid for Scientific Research (B) from Japan Society for the
Promotion of Science (JSPS), CREST from Japan Science and
Technology Corporation, and Advanced and Innovational
Research Program in Life Sciences from the Ministry of
Education, Culture, Sports, Science and Technology, the
Japanese Government and Kyushu University P&P “Green
Chemistry” Program.
Supporting Information Available: COSY and NOESY
spectra for determination of stereochemistry of glycosidic bond
of all WNA derivatives. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA037211Z
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