Synthesis of 1′-phenyl-2′-OMe ribose analogues connecting the thymine base at the 1′ position through a flexible linker for the formation of a stable anti-parallel triplex DNA

Article abstract of DOI:10.1016/j.tet.2012.11.016

We have previously developed the innovative bicyclic nucleoside analogues (WNA) for the formation of the triplex DNA. The WNA analogue consists of an aromatic ring and a recognition base on the bicyclic skeleton, and the recognition of the CG or TA interrupting sites has been achieved by the WNA analogues. However, the stabilization ability of the WNA analogue is dependent on its neighboring nucleobases within the TFO. We hypothesized that the sequence dependency might arise from the fixed conformation of the bicyclic ring of the WNA. Thus, it was expected that an open-linker between the sugar part and the nucleobase might produce the flexibility and improve the stabilizing effect of the nucleobase analogues. We now report the design and synthesis of a new nucleoside analogue as an open-form of WNA-βT, the 1′-phenyl- 2′-OMe-ribose derivative, connecting the thymine base to the ribose part through a methylene linker (1) or an ethylene linker (2). TFO containing the 3′-dA-1-dG context recognized the CG interrupting site, and that with the 3′-dG-1-dG context recognized the GC site. In contrast, 2 displayed a stabilizing effect on all four base pairs with some preferences for the TFO containing 3′-dA-2-dG and 3′-dG-2-dG. These results suggested that a flexible linker between the nucleobase and the ribose part may improve the sequence dependency for the triplex formation.

Full text of DOI:10.1016/j.tet.2012.11.016

Tetrahedron 69 (2013) 600e606
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of 10-phenyl-20-OMe ribose analogues connecting the
thymine base at the 10 position through a flexible linker for the
formation of a stable anti-parallel triplex DNA
,
,b,
Yosuke Taniguchi a b, Hidenori Okamura a, Norihiko Fujino a, Shigeki Sasaki a
*
a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
b CREST (JST), 4-1-8 Motomachi, 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:
Received 9 October 2012
Received in revised form 1 November 2012
Accepted 2 November 2012
Available online 9 November 2012
We have previously developed the innovative bicyclic nucleoside analogues (WNA) for the formation of
the triplex DNA. The WNA analogue consists of an aromatic ring and a recognition base on the bicyclic
skeleton, and the recognition of the CG or TA interrupting sites has been achieved by the WNA analogues.
However, the stabilization ability of the WNA analogue is dependent on its neighboring nucleobases
within the TFO. We hypothesized that the sequence dependency might arise from the fixed conformation
of the bicyclic ring of the WNA. Thus, it was expected that an open-linker between the sugar part and the
nucleobase might produce the flexibility and improve the stabilizing effect of the nucleobase analogues.
Keywords:
Disubstituted nucleoside analogue
Anti-parallel triplex DNA
Interrupting site
Non-selective stabilization
Antigene
We now report the design and synthesis of a new nucleoside analogue as an open-form of WNA-bT, the
10-phenyl-20-OMe-ribose derivative, connecting the thymine base to the ribose part through a methylene
linker (1) or an ethylene linker (2). TFO containing the 30-dA-1-dG context recognized the CG inter-
rupting site, and that with the 30-dG-1-dG context recognized the GC site. In contrast, 2 displayed
a stabilizing effect on all four base pairs with some preferences for the TFO containing 30-dA-2-dG and 30-
dG-2-dG. These results suggested that a flexible linker between the nucleobase and the ribose part may
improve the sequence dependency for the triplex formation.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
interrupting sites. In order to expand the triplex recognition code,
we developed innovative bicyclic nucleoside analogues called the
The triplex forming oligonucleotide (TFO) is the one of the
binding molecules to the major groove of DNA, and has the po-
W-shaped nucleoside analogues (WNAs). The WNA analogue has
a benzene ring as the stacking part and a nucleobase as the rec-
ognition part as shown by WNA-bT in Fig. 1. We have already
tential to be a biological tool for gene targeting and regulation.1,2
A
parallel triplex DNA is formed with the pyrimidine-rich TFO and the
homopurineehomopyrimidine duplex based on the stabilization
by the two Hoogsteen hydrogen bonds between the protonated dC
(dCþ) and the GC base pair and those between the T and AT base
pair. Due to the contribution of dCþ, the parallel triplex DNA is
stable under acidic conditions. On the other hand, the anti-parallel
triplex is formed by the interaction between the purine-rich TFO
and purine bases in the homopurineehomopyrimidine duplex, in
which two reverse Hoogsteen hydrogen bonds of the dG to GC base
pair and those of the dA to AT base pair are responsible for its high
stability under neutral conditions. However, the pyrimidine nu-
cleotide insertions in the homopurine strand, dC and T, inhibit the
stable triplex formation.3,4 Thus, the CG and TA base pairs are called
Stacking part
Recognition part
* Corresponding author. Tel./fax: þ81 92 642 6615; e-mail address: sasaki@
Fig. 1. Basic structure of WNA analogues (WNA-
phenyl-20OMe derivatives, 1 (right-top) and 2 (right-down), in this study.
b
T, left), and the structures of 10-
phar.kyushu-u.ac.jp (S. Sasaki).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.11.016

Y. Taniguchi et al. / Tetrahedron 69 (2013) 600e606
601
revealed that TFOs containing the WNA analogues could recognize
the CG or TA interrupting sites with a high selectivity and high
stability in an anti-parallel triplex formation.5 Recently, we have
presence of TMSCl and AgNO3.10 During this reaction, the 50-OH
group of 6 was silylated with TMSCl for protection during the re-
action with the isocyanate 7 to convert the amino group to the
corresponding ureido derivative. The ureido compound was treated
in 1 M H2SO4 in H2O to affect cyclization together with the
deprotection of the acetonide group to afford the triol intermediate
8. The N3 position of the thymine base and the 50- and 30-hydroxyl
groups of the ribose part were protected using acrylonitrile and
TIPDSCl2, respectively. The two isomers of the resulting compounds
9 were separated by column chromatography. Their stereochem-
istries were determined by analysis of their 1H-COSY and NOESY
demonstrated that TFO containing WNA-bT inhibited the cell pro-
liferation and induced a caspase-dependent apoptosis by the in-
hibition of the oncogene expression.6 Therefore, these artificial
nucleoside analogues, which are able to stabilize the triplex DNA,
have the potential to become a gene targeting agent. Unfortunately,
the stabilization effect of the WNA-containing TFO is dependent on
its neighboring nucleobases within TFO.7 The sequence de-
pendency of the TFO has been partially solved by the use of a variety
of WNA analogues. Nevertheless, the development of the nucleo-
side analogues for the formation of a stable triplex DNA without
a sequence dependency is a challenging theme.
We speculated that the sequence dependency of the WNA ana-
logues might arise from the rigid conformation of the bicyclic ring of
the WNA skeleton, although it contributed to the specific recogni-
tion for the CG or TA interrupting sites. Previously we synthesized
the 10-phenyl substituted nucleoside analogues whose recognition
base was directly connected to the sugar part at the 10 position
without a bicyclic sugar ring.8 However, these 10-phenyl branched
nucleoside analogues were unstable under strong acid or basic so-
lution conditions, and thereby it was impossible toincorporate them
into oligonucleotides. Therefore, in order to clarify the role of the
bicyclic ring of the WNA skeleton for the formation of the triplex
DNA, we designed new 10,10-disubstituted nucleoside analogues
with a flexible linker between the recognition base and the sugar
part. Thus, the 10-phenyl-20-OMe-ribose derivatives connecting the
thymine base to the ribose part through a methylene linker (1) and
that with an ethylene linker (2) were designed (Fig. 1). In this paper,
we describe the synthesis and evaluation of the triplex forming
ability of the new nucleoside analogues (1 and 2).
spectra. Subsequently, the 20-hyrodoxyl group of the desired
b-
phenyl substituted analogue was methylated using Meerwein re-
agent11 to yield 10. The TIPDS group and the cyanoethyl group of 10
were then removed by a sequential treatment with TBAF followed
by t-BuOK to produce the diol compound 1 in a good yield.
The synthesis of 2 is shown in Scheme 2. Compound 11 was
converted to the corresponding triol by the reaction with TBAF and
followed by DOWEX (Hþ), which was protected with TIPDS to
produce compound 12. The 20-hydroxyl group of 12 was methyl-
ated by MeI and NaHMDS to form 13. The allyl group of 13 was
converted to the corresponding ethylamino group (15) by a se-
quence of reactions. The vinyl group of 13 was subjected to an
oxidative cleavage reaction using OsO4 and NaIO4 to form the al-
dehyde 14. The aldehyde 14 was then transformed into the corre-
sponding oxime, followed by reduction with NaBH4/NiCl2 to
produce the amino compound 15.12 The amino group of 15 was
reacted with the isocyanate 79 to form the corresponding ureido
compound 16, which was treated with TBAF for the deprotection of
TIPDS followed by cyclization in 1 M H2SO4 to form the thymine
base, then the desired diol compound 2 was obtained.
2. Results and discussion
The synthesis of the 10,10-disubstituted nucleoside analogues
was started with the intermediates (4 and 11), which were pre-
viously reported by our group.5 The synthesis of 1 is shown in
Scheme 1. Compound 4 was subjected to cyanidation by the
treatment with TMSCN and ZnBr2 to produce 5 in a good yield as
a mixture of stereoisomers. The cyano group of 5 was converted to
the corresponding amino group by reduction with LiAlH4, and then
the TBDPS group was removed to form 6. The amino group of 6 was
reacted with 3-methoxy-2-methylacryloylisocyanate 79 in the
Scheme 2. Reagents and conditions: (a) (i) TBAF, THF (87%), (ii) DOWEX (Hþ), MeOH
(92%), (iii) TIPDSCl2, pyridine (97%). (b) MeI, NaHMDS, THF, 0 ꢀC (71%). (c) OsO4, NaIO4,
pyridine, H2O (74%). (d) (i) NH2OHeHCl, MeOH (90%). (ii) NaBH4, NiCl2, MeOH, (74%).
(e) (i) 7, benzene (90%). (f) (i) TBAF, THF (90%) (ii) 1 M H2SO4 aq, 90 ꢀC (72%).
The 10-phenyl substituted compound (3), as a control com-
pound, was synthesized from 4. The 10-hydroxyl group of 4 was
reduced by Et3SiH and TMSOTf, and then the stereoisomer at the 10
position was separated to produce the undesired
a-phenyl
substituted compound (11%) and a desired -phenyl substituted
b
compound (66%) by column chromatography. These isomers were
determined by an analysis 1H-COSY and NOESY spectra. The pro-
Scheme 1. Reagents and conditions: (a) TMSCN, ZnBr2, CH3NO2, 0 ꢀC, (97%). (b) LiAlH4,
THF, reflux, (90%). (c) (i) TMSCl, AgNO3, Et3N, 3-methoxy-2-methylacryloylisocyanate
(7), benzene, (ii) 1 M H2SO4 aq, 90 ꢀC, (81% for two steps). (d) (i) acrylonitrile, TBAH,
tecting groups of the
b-phenyl compound were removed using
TBAF and DOWEX (Hþ) in a good yield. The resulting triol com-
pound 17 was transformed into 3 by sequential reactions as de-
scribed for the synthesis of 1 and 2 (Scheme 3).
pyridine, reflux, (ii) TIPDSCl2, pyridine, (
a-Ph 66%, b-Ph (9) 28% for two steps). (e)
Me3OBF4, CH2Cl2, (99%). (f) (i) TBAF, THF, (ii) t-BuOK, t-BuOH, (75% for two steps).

602
Y. Taniguchi et al. / Tetrahedron 69 (2013) 600e606
selective stabilizing effect of 2 that the ethylene linker is flexible
enough to avoid steric repulsive interactions with adjacent nucle-
oside, which are supposed to be a problem of the rigid conformed
WNA analogues as sequence dependency.
3. Conclusion
In conclusion, we synthesized a new nucleoside analogue as an
open-form of WNA-b
T, 10-phenyl-20-OMe-ribose derivative with
the thymine base through the flexible linker, i.e., the methylene-
linked 1 and ethylene-linked 2 in order to avoid steric repulsion
of bicyclic WNA structure. The results of the triplex forming ability
have shown that TFO1(1) and TFO2(1) stabilize the triplex DNA
having the CG site and GC site, respectively. Furthermore, it has
been shown that TFO1e4(2) has an ability to form the non-
selective triplexes with all duplex targets without sequence de-
pendency. Consequently, the adjustment of the linker flexibility
between the recognition base and sugar skeleton of the nucleoside
analogues has the potential to enhance the stability and selectivity
of triplex DNA formation as a general sequence. New nucleoside
analogues have been developed based on this result.
Scheme 3. Reagents and conditions: (a) (i) Et3SiH, BF3$Et2O, CH2Cl2, ꢁ78 ꢀC to ꢁ40 ꢀ
C
(77%, a-Ph 11%, b
-Ph 66%), (ii) TBAF, THF, (iii) DOWEX (Hþ), MeOH, (82% for two steps).
(b) TIPDSCl2, pyridine (92%). (c) MeI, NaHMDS, THF, 0 ꢀC, (77%). (d) TBAF, THF, (68%).
Finally, these diol compounds (1, 2, and 3) were converted into
the corresponding amidite precursors (20, 21, and 22, respectively)
by conventional methods (Scheme 4). They were incorporated into
TFO1e4 with four different sequences using an automated DNA
synthesizer. The TFO1 in the sequence 30-AZG-50 containing 1 at the
position of Z was called TFO1(1). After purification by HPLC, the
purity and the structure were identified by MALDI-TOF MS mea-
surements. These TFOs were then evaluated for their triplex
forming ability by the gel-shift assay, and the association constant,
Ks, was calculated from the intensity of the radioactive bands, as
previously described.5 The Ks values are summarized in Fig. 2 as
a bar graph.
4. Experimental
4.1. General
The 1H NMR (400 MHz), 13C (125 MHz), and 1H-COSY, NOESY
spectra were recorded by Varian UNITY-400 and INOVA-500
spectrometers. The 31P NMR (162 MHz) spectrum was recorded
using 10% phosphoric acid in D2O as the internal standard at 0 ppm.
The IR spectra were obtained using
a PerkineElmer FTIR-
spectrumOne. The high-resolution mass spectra were recorded by
an Applied Biosystems Mariner System 5299 spectrometer.
4.2. Synthesis of 1
4.2.1. (1R,5R,6RS,8R)-6-Cyano-3,3-dimethyl-6-phenyl-8-(t-butyldi-
phenylsilyloxymethyl)-2,4,7-trioxabicyclo[3.3.0]octane (5). To a sus-
pension of ZnBr2 (2.7 g, 11.9 mmol) in CH3NO2 (100 mL) was added
a solution of 4 (3.0 g, 5.9 mmol) in CH3NO2 (20 mL) and TMSCN
(19.8 mL, 0.15 mol) at 0 ꢀC. After stirring for 2 h, the reaction
mixture was diluted with Et2O. The organic layer was washed with
satd NaHCO3 solution, dried over Na2SO4, then evaporated under
reduced pressure. The residue was purified by flash column chro-
matography (Hexane:EtOAc¼10/1) to give compound 5 as a color-
Scheme 4. Reagents and conditions: (a) (i) DMTrCl, pyridine, (ii) i-Pr2NP(Cl)
OCH2CH2CN, DIPEA, CH2Cl2,
synthesizer.
0
ꢀC, (63e73% for two steps). (b) DNA automated
Regarding the stabilization ability of the methylene-linked nu-
cleoside 1, TFO1(1) displayed a selective stabilization to the CG-
having duplex (Fig. 2A), while TFO2(1) was selective to the GC-
having duplex (Fig. 2B), while no stable triplexes were formed
with TFO3(1) and TFO4(1) (Fig. 2C and D). In comparison to the
control compound 3, 1 exhibited a stabilization effect in the se-
quence of TFO1 and TFO2, while none or a destabilizing effect was
observed in the sequence of TFO3 and TFO4, respectively. These
results indicated that the phenyl group within TFO4(3) was stacked
with the neighboring nucleobase in the sequence of 30-GZA-50, and
1 hampered the triplex formation without the hydrogen bonding
formation. Therefore, the methylene linker may not produce
enough flexibility to overcome the rigidity of the WNA structure.
The ethylene-linked nucleoside 2 showed a different feature
during the triplex formation. TFO1(2) and TFO2(2) formed stable
triplexes with all four target duplexes without remarkable selec-
tivity. Interestingly, the stabilities of these triplexes were higher
than the natural triplexes containing the GeGC triplet (Fig. 2A and
B). Although the stability was relatively low, TFO3(2) and TFO4(2)
formed triplexes with all the duplex targets (Fig. 2C and D), dem-
onstrating the unique property of 2. It is postulated from the non-
less oil (3.0 g, 5.8 mmol, 97%). lmax (neat) 2932, 1738 cmꢁ1
; dH
(400 MHz, CDCl3) 7.71e7.29 (15H, m), 5.08 (1H, d J 5.6 Hz), 4.94 (1H,
t, J 4.4 Hz), 4.93 (1H, d, J 5.6 Hz), 4.59 (1H, d, J 6.4 Hz), 4.53 (1H, d, J
6.0,12.2 Hz), 4.04e3.89 (2H, m),1.10 (9H, s),1.07 (3H, s),1.03 (3H, s);
dC (125 MHz, CDCl3) 136.9, 135.8, 135.7, 135.6, 135.5, 134.6, 132.9,
132.8, 129.9, 129.2, 128.9, 128.8, 128.1, 127.8, 126.4, 125.0, 120.6,
117.8, 117.3, 114.0, 89.1, 87.1, 86.4, 84.8, 84.0, 83.1, 81.1, 63.2, 63.1,
28.0, 27.0, 26.9, 26.8, 26.0, 25.5, 25.2, 25.0, 19.2; HRMS (ESI-TOF):
MþNaþ, found 536.2219. C31H35NO4SiNa requires 536.2228.
4.2.2. (1R,5R,6RS,8R)-6-Aminomethyl-3,3-dimethyl-8-hydroxymethyl-
6-phenyl-2,4,7-trioxabicyclo[3.3.0]octane (6). To a solution of 5 (2.3 g,
4.5 mmol)inTHF(90 mL)wasadded a solutionof LiAlH4 inTHF(1.0M,
23 mL, 22.4 mmol) at 0 ꢀC. The reaction mixture was refluxed for 2 h.
After cooling to 0 ꢀC, water was continuously added to the reaction
along with an aqueous 10% sodium hydroxide solution. The mixture
was filtered through a Celite545 pad, and then the filtrate was
extracted with Et2O. The organic layer was washed with a satd NaCl
solution, dried overNa2SO4, then evaporatedunder reducedpressure.
The residue was purified by flash column chromatography
(CHCl3:MeOH¼10/1) to give compound 6 as a pale yellow oil (1.1 g,

Y. Taniguchi et al. / Tetrahedron 69 (2013) 600e606
603
Fig. 2. Bar graphs of the association constants (Ks) of the TFOs. These values were determined from the results of the gel-shift assay. Triplex formation was done for 12 h at 22 ꢀC in
the buffer containing 20 mM TriseHCl, 5 mM MgCl2, 2.5 mM spermidine, and 10% sucrose at pH 7.5. Electrophoresis was done at 10 ꢀC with a non-denatured polyacrylamide gel. Ten
nano molar TFO containing the 32P-labeled one as the tracer was used. The association constant was calculated using the concentration of each component and the following
equation: Ks¼[Triplex]/[Duplex][TFO]. (A) TFO1: 30AZG50, (B) TFO2: 30GZG50, (C) TFO3: 30AZA50, (D) TFO4: 30GZA50, Z¼dG, WNA analogue, or new 10-phenyl-20OMe ribose de-
rivatives (1, 2, 3) as shown at the bottom of the graph.
4.1 mmol, 90%). lmax (neat) 3366, 2923, 2854 cmꢁ1
;
dH (400 MHz,
4.9 Hz), 4.08e3.76 (3.8H, m), 3.68 (0.6H, ddd, J 4.6, 11.9 Hz),
3.52e3.42 (1.4H, m), 1.64 (1.8H, s), 1.62 (1.2H, s); dC (125 MHz,
CD3OD) 166.5, 153.5, 144.2, 144.0, 140.6, 129.0, 128.8, 128.5, 128.2,
126.8, 110.0, 89.9, 84.6, 84.2, 79.7, 75.8, 72.8, 72.3, 64.0, 62.4, 56.0,
52.1, 49.3, 49.1, 48.9, 48.8, 48.6, 12.1, 12.0; HRMS (ESI-TOF): MþHþ,
found 349.1400. C17H21N2O6 requires 349.1394.
CDCl3) 7.67e7.20 (5H, m), 4.92e4.85 (1.7H, m), 4.69 (0.3H, dd, J 4.0,
7.0 Hz), 4.47 (0.7H, dd, J 2.1, 2.4 Hz), 4.24e4.18 (0.3H, m), 4.00 (0.7H,
dd, J 2.4, 12.2 Hz), 3.80 (0.3H, dd, J 3.7, 11.9 Hz), 3.70 (0.7H, dd, J 2.4,
12.2 Hz), 3.65 (0.3H, dd, J 3.7, 11.9 Hz), 3.24e2.99 (2H, m), 1.59 (0.9H,
s),1.36 (0.9H, s),1.24 (2.1H, s),1.22 (2.1H, s); dC (125MHz, CDCl3) 142.4,
128.5, 128.4, 127.5, 125.4, 125.3, 114.9, 88.1, 87.6, 82.7, 82.0, 63.2, 50.6,
47.0, 26.1, 24.8; HRMS (ESI-TOF): MþHþ, found 280.1588. C15H22NO4
requires 280.1543.
4.2.4. (1R,2R,3S,4R)-1-{3-(2-Cyanoethyl)thymine-1-yl}-methyl-1-
phenyl-3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxyl)ribose (9). To a so-
lution of 8 (2.0 g, 5.7 mmol) in pyridine (72 mL) was added acry-
lonitrile (0.38 mL, 5.7 mmol) and tetrabutylammonium hydroxide
4.2.3. (1RS,2R,3R,4R)-1-Phenyl-1-(thymin-1-yl)methylribose (8). To
a solution of 6 (2.1 g, 7.5 mmol) in benzene (77 mL) and triethyl-
amine (11 mL) was added AgNO3 (6.8 g, 38.1 mmol) and TMSCl
(1.94 mL, 15.3 mmol). After stirring for 1 h, the solution of 79 in
benzene (1.0 M, 15 mL, 15 mmol) was added to a reaction mixture.
After stirring for 3 h, the reaction was quenched with a satd
NaHCO3 solution and filtered through a Celite545 pad, and then the
filtrate was extracted with EtOAc. The organic layer was washed
with a satd NaCl solution, dried over Na2SO4, then evaporated
under reduced pressure. The residue was suspended in a 1 M H2SO4
solution (75 mL) and heated at 90 ꢀC. After stirring for 1.5 h, the
reaction mixture was neutralized by a satd NaHCO3 solution at
room temperature, and then extracted with EtOAc. The organic
layer was dried over Na2SO4, and then evaporated under reduced
pressure. The residue was purified by flash column chromatogra-
phy (CHCl3:MeOH¼15/1 to 10/1) to give compound 8 as a colorless
oil (2.2 g, 6.2 mmol, 81% for two steps). lmax (neat) 3374, 2920,
in MeOH (1.0 M, 172 m
L, 0.17 mmol). After stirring for 22 h at 90 ꢀC,
the reaction mixture was diluted with CH2Cl2 at room temperature.
The organic layer was washed with a satd NaCl solution, dried over
Na2SO4, then evaporated under reduced pressure. The residue was
purified by flash column chromatography (CHCl3:MeOH¼50/1 to
10/1) to give a white solid (1.7 g, 4.3 mmol). The white solid was
then dissolved in pyridine (43 mL) and TIPDSCl2 (3.4 mL,
10.9 mmol) at 0 ꢀC. After stirring for 5 h at room temperature, the
reaction mixture was diluted with EtOAc. The organic layer was
continuously washed with water and a satd NaCl solution, dried
over Na2SO4, then evaporated under reduced pressure. The residue
was purified by flash column chromatography (Hexane:EtOAc¼5/1
to 1/1) to give compound 9 as a colorless foam (775 mg, 1.2 mmol,
28%) and
a
-Ph isomer (1.8 g, 2.8 mmol, 66%). lmax (neat) 2260,1690,
dH (400 MHz, CD3OD) 7.43 (2H, d, J 7.0 Hz),
1660, 1638 cmꢁ1
;
7.28e7.21 (4H, m), 4.62 (1H, d, J 14.7 Hz), 4.42 (1H, d, J 4.3 Hz), 4.21
(1H, dt, J 8.5, 3.1, 2.4 Hz), 4.13 (1H, d, J 14.7 Hz), 4.14e3.40 (5H, m),
3.95e3.89 (1H, m), 2.46 (2H, m), 1.77 (3H, s), 1.09e0.72 (28H, m); dC
(125 MHz, CDCl3) 163.0, 151.1, 140.3, 140.0, 128.1, 127.8, 125.9, 117.1,
108.5, 86.8, 81.0, 78.3, 71.7, 62.3, 51.5, 36.5, 14.4, 17.3, 17.1, 17.0, 16.9,
1673 cmꢁ1
; dH (400 MHz, DMSO) 11.09 (0.6H, s), 10.85 (0.4H, s),
7.43e7.13 (6H, m), 5.43 (0.4H, d, J 5.8 Hz), 5.02 (0.4H, d, J 5.5 Hz),
4.81 (0.6H, t, J 5.8 Hz), 4.72 (0.4H, t, J 5.2 Hz), 4.70 (0.6H, d, J 6.4 Hz),
4.65 (0.6H, d, J 6.4 Hz), 4.37 (0.6H, d, J 14.6 Hz), 4.20 (0.6H, t, J

604
Y. Taniguchi et al. / Tetrahedron 69 (2013) 600e606
15.7, 13.3, 13.1, 12.8, 12.6; HRMS (ESI-TOF): MþHþ, found 644.3192.
3 h at room temperature, the reaction was quenched by water and
extracted with EtOAc. The organic layer was continuously washed
with water and a satd NaCl solution, dried over Na2SO4, then
evaporated under reduced pressure. The residue was purified by
flash column chromatography (Hexane:EtOAc¼6/1) to give com-
pound 12 as a colorless oil (4.78 g, 9.7 mmol, 97%). lmax (neat) 3503,
C
32H50N3O7Si2 requires 644.3182.
4.2.5. (1R,2R,3R,4R)-1-{3-(2-Cyanoethyl)thymine-1-yl}methyl-2-O-
methyl-1-phenyl-3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxyl)ribose
(10). To a solution of 9 (340 mg, 0.53 mmol) in CH2Cl2 (5.2 mL) was
added a proton-sponge (1.7 g, 7.9 mmol) and Me3OBF4 (935 mg,
6.3 mmol) at 0 ꢀC. After stirring for 5 h at room temperature, the
reaction mixture was quenched by a 5% citrate solution and diluted
with EtOAc. The mixture was filtered through a Celite545 pad, and
then the filtrate was continuously washed with 5% citrate solution
and satd NaCl solution, dried over Na2SO4, then evaporated under
reduced pressure. The residue was purified by flash column chro-
matography (Hexane:EtOAc¼5/1 to 3/2) to give compound 10 as
a colorless foam (344 mg, 0.52 mmol, 99%). lmax (neat) 2946, 2249,
2945, 2868, 1464 cmꢁ1
; dH (400 MHz, CDCl3) 7.43 (3H, d, J 7.3 Hz),
7.17e7.30 (2H, m), 5.50e5.60 (1H, m), 4.91 (2H, t, J 8.9 Hz), 4.25 (1H,
t, J 6.1 Hz), 4.03e4.11 (3H, m), 3.93 (1H, dd J 6.0, 10.1 Hz), 3.00 (1H,
s), 2.81 (1H, dd, J 7.0, 14.5 Hz), 2.70 (1H, dd, J 7.0, 14.5 Hz), 0.88e1.07
(24H, m); dC (125 MHz, CDCl3) 144.0, 133.3, 127.9, 126.7, 125.4, 117.4,
86.7, 81.4, 78.2, 72.6, 63.6, 39.1, 17.5, 17.4, 17.3, 17.2, 17.1, 17.0, 16.9,
13.3, 13.2, 12.9, 12.7, 12.6; HRMS (ESI-TOF): MþHþ, found 493.2784.
C26H45O5Si2 requires 493.2800.
1707, 1662, 1651 cmꢁ1
;
dH (400 MHz, CDCl3) 7.39 (2H, d, J 7.3 Hz),
4.3.2. (10S,20R,30R,40R)-10-Allyl-20-methoxy-10-phenyl-30,50-O-tetrai-
sopropyldisiloxyribose (13). To a solution of 12 (4.78 g, 9.7 mmol) in
THF (100 mL) was added methyliodide (6.1 mL, 98 mmol) and
NaHMDS in a THF solution (1.9 M, 15 mL, 29 mmol). After stirring
for 2 h, the reaction mixture was quenched by a satd NH4Cl solution
and extracted with EtOAc. The organic layer was continuously
washed with water and a satd NaCl solution, dried over Na2SO4,
then evaporated under reduced pressure. The residue was purified
by flash column chromatography (Hexane:EtOAc¼15/1) to give
compound 13 as a colorless oil (3.5 g, 6.9 mmol, 71%). lmax (neat)
7.23e7.14 (4H, m), 4.63 (1H, d, J 14.6 Hz), 4.17e3.98 (8H, m), 3.69
(3H, s), 2.30 (2H, dd, J 6.4, 5.8 Hz), 1.80 (3H, s), 1.05e0.73 (28H, m);
dC (125 MHz, CDCl3) 170.2, 163.0, 150.9, 140.5, 139.8, 127.9, 127.5,
126.0, 117.1, 108.4, 88.1, 86.6, 80.6, 70.9, 60.7, 60.5, 51.8, 43.1, 36.5,
17.5, 17.3, 17.1, 17.0, 15.7, 13.4, 13.1, 12.9, 12.7, 12.6; HRMS (ESI-TOF):
MþHþ, found 658.3315. C33H52N3O7Si2 requires 658.3338.
4.2.6. (1R,2R,3R,4R)-2-O-Methyl-1-phenyl-1-(thymine-1-yl)methyl-
ribose (1). To a solution of 10 (220 mg, 0.34 mmol) in THF (3.4 mL)
was added a solution of TBAF in THF (1.0 M, 840
mL, 0.84 mmol). After
2945, 2868, 1465 cmꢁ1
; dH (400 MHz, CDCl3) 7.43 (2H, d, J 2.1 Hz),
stirring for 45 min, the reaction mixture was diluted with a satd
NaHCO3 solution and EtOAc. The organic layer was washed with
a satd NaCl solution, dried over Na2SO4, then evaporated under re-
duced pressure. The residue was purified by flash column chroma-
tography (CHCl3:MeOH¼100/1 to 25/1) to give the desired
compound as a colorless foam (127 mg, 0.31 mmol, 92%). This col-
orless foam (110 mg, 0.27 mmol) was dissolved in a solution of po-
tassium t-butoxide (89.1 mg, 0.79 mmol) in t-butanol (5.3 mL). After
stirring for 2 h, the reaction mixture was treated with ammonium
chloride, dried over Na2SO4, then evaporated under reduced pres-
sure. The residue was purified by flash column chromatography
(CHCl3:MeOH¼50/1) to give compound 1 as a colorless foam
7.22e7.28 (2H, m), 7.14e7.18 (1H, m), 5.46e5.53 (1H, m), 4.85 (1H,
d, J 8.2 Hz), 4.22 (1H, dd, J 4.1, 8.4 Hz), 4.02e4.11 (2H, m), 3.96 (1H,
dd, J 2.1, 12.5 Hz), 3.62 (3H, s), 2.78 (1H, dd, J 7.0, 14.5 Hz), 2.63 (1H,
dd, J 7.0, 14.5 Hz), 0.85e1.04 (24H, m); dC (125 MHz, CDCl3) 144.6,
133.8, 127.8, 126.5, 125.6, 117.3, 88.1, 86.6, 80.5, 71.1, 61.5, 40.6, 17.6,
17.4, 17.3, 17.2, 17.1, 17.0, 13.4, 13.1, 12.8, 12.7; HRMS (ESI-TOF):
MþNaþ, found 529.2803. C27H46NaO5Si2 requires 529.2776.
4.3.3. (10S,20R,30R,40R)-10-(2-Formylethyl)-20-methoxy-10-phenyl-
30,50-O-tetraisopropyldisiloxyribose (14). To a solution of 13 (3.5 g,
6.9 mmol) in pyridine (80 mL) was added a solution of OsO4 in
water (0.131 M, 12.7 mL, 1.7 mmol) and NaIO4 in water (0.6 M,
70 mL, 41.5 mmol). After stirring for 3 h, the reaction mixture was
diluted with EtOAc. The organic layer was continuously washed
with water and a satd NaCl solution, dried over Na2SO4, then
evaporated under reduced pressure. The residue was purified by
flash column chromatography (Hexane:EtOAc¼10/1) to give com-
pound 14 as a colorless oil (2.6 g, 5.5 mmol, 74%). lmax (neat) 2943,
(77.3 mg, 0.21 mmol, 81%). lmax (neat) 3385, 2923, 1674 cmꢁ1
; dH
(400 MHz, CD3OD) 77.48 (2H, d, J 7.0 Hz), 7.29e7.19 (3H, m), 7.08 (1H,
s), 4.43 (1H, d, J 14.6 Hz), 4.31 (1H, d, J 14.6 Hz), 4.24 (1H, dd, J 4.9,
4.6 Hz), 4.13 (1H, t, J 4.9 Hz), 3.86 (1H, d, J 5.2 Hz), 3.70e3.59 (5H, m),
1.69 (3H, s); dC (125 MHz, CD3OD) 166.6, 152.9, 144.0, 129.0, 128.5,
126.6, 110.0, 89.8, 87.3, 85.7, 71.8, 63.8, 60.0, 51.5, 12.1; HRMS (ESI-
TOF): MþHþ, found 363.1579. C18H23N2O6 requires 363.1551.
2868, 1723, 1465 cmꢁ1
; dH (400 MHz, CDCl3) 9.61 (1H, s, J 1.8 Hz),
7.48e7.51 (2H, m), 7.29e7.32 (1H, m), 7.19e7.24 (2H, m), 4.28 (1H,
dd, J 3.4, 9.2 Hz), 4.21 (1H, dd, J 1.2, 13.0 Hz), 4.08e4.13 (1H, m), 3.98
(1H, dd, J 2,6,13.0 Hz), 3.76 (1H, dd, J 4.0), 3.60 (1H, s), 2.95 (1H, dd, J
3.7, 16.8 Hz), 2.65 (1H, dd, J 1.7, 16.7 Hz), 0.77e1.06 (30H, m); dC
(125 MHz, CDCl3) 170.8, 142.5, 128.4, 128.2, 127.7, 125.1, 125.0, 88.2,
85.6, 81.0, 60.7, 51.2, 42.6, 17.5, 17.4, 17.3, 17.2, 17.1, 17.0, 13.4, 13.3,
12.8, 12.7; HRMS (ESI-TOF): MþNaþ, found 531.2545.
4.3. Synthesis of 2
4.3.1. (10S,20R,30R,40R)-10-Allyl-10-phenyl-30,50-O-tetraisopropyl-dis-
iloxyribose (12). To a solution of 11 (7.1 g, 13 mmol) in a THF
(200 mL) was added TBAF in THF solution (1 M, 20 mL, 20 mmol).
After stirring for 2 h, the reaction mixture was diluted with EtOAc.
The organic layer was continuously washed with water and a satd
NaCl solution, dried over Na2SO4, then evaporated under reduced
pressure. The residue was purified by flash column chromatogra-
phy (Hexane:EtOAc¼3/2) to give a colorless oil (3.24 g, 11 mmol,
87%). This oil was dissolved in MeOH (50 mL). After the addition of
DOWEXÔ 50WX8-100 (1.15 g), the mixture was stirred for 26 h.
The reaction mixture was then filtered off. The filtrate was then
evaporated under reduced pressure. The residue was purified by
flash column chromatography (Hexane:EtOAc¼2/3) to give the triol
compound as a white solid (2.55 g, 10 mmol, 92%). Subsequently,
this white solid was dissolved in pyridine (100 mL), then this so-
C26H44NaO6Si2 requires 531.2569.
4.3.4. (10S,20R,30R,40R)-10-(2-Aminoethyl)-20-methoxy-10-phenyl-
30,50-O-tetraisopropyldisiloxyribose (15). To a solution of 14 (2.6 g,
5.5 mmol) in ethanol (30 mL) was added a solution of NH2OHeHCl
in pyridine (5 mL, 656 mg, 9.44 mmol), then the reaction mixture
was heated at 70 ꢀC for 16 h. The reaction mixture was diluted with
EtOAc, continuously washed with water and a satd NaCl solution,
dried over Na2SO4, then evaporated under reduced pressure. The
residue was purified by flash column chromatography
(Hexane:EtOAc¼6/1) to give a colorless oil (2.6 g, 5.0 mmol, 90%).
This colorless oil (554 mg, 1.06 mmol) was dissolved in methanol
(13 mL). NiCl2 (503 mg, 2.12 mmol) and NaBH4 (400 mg,10.6 mmol)
were added to this mixture at ꢁ78 ꢀC. After stirring for 2 h at the
lution was cooled to
tetraisopropyldisiloxane (3.82 mL, 12 mmol) was dropwise added
to the reaction mixture at the same temperature. After stirring for
0
ꢀC. The 1,3-dichloro-1,1,3,3-

Y. Taniguchi et al. / Tetrahedron 69 (2013) 600e606
605
same temperature, the reaction mixture was warmed up to room
temperature. After stirring for 2 h, the reaction mixture was
quenched by a satd NaHCO3 solution and extracted with CHCl3. The
organic layer was continuously washed with water and a satd NaCl
solution, dried over Na2SO4, then evaporated under reduced pres-
sure. The residue was purified by flash column chromatography
(CHCl3:MeOH¼20/1) to give compound 15 as a colorless oil
24.1 mmol) was added BF3$Et2O (3.8 mL, 30.1 mmol) at ꢁ78 ꢀC.
After stirring for 40 min at ꢁ40 ꢀC, the reaction mixture was diluted
with EtOAc, washed with a satd NaCl solution, dried over Na2SO4,
then evaporated under reduced pressure. The residue was purified
by flash column chromatography (Hexane:EtOAc¼30/1) to give the
a
-phenyl compound as a colorless oil (1.08 g, 2.2 mmol, 11%), and
the
b
-phenyl compound as a colorless oil (6.48 g, 13.3 mmol, 66%).
-phenyl compound (4.93 mmol, 10.1 mmol) was dis-
(400 mg, 0.79 mmol, 74%). lmax (neat) 2944, 2868, 1465 cmꢁ1
;
dH
The desired b
(400 MHz, CDCl3) 7.19e7.32 (5H, m), 4.52e4.58 (1H, m), 4.12 (1H, d J
4.1 Hz), 3.68 (1H, d, J 4.0 Hz), 3.33 (3H, s), 3.18 (1H, s), 2.67e2.70
(1H, m), 2.56e2.60 (1H, m), 2.10 (1H, dd, J 7.0, 14.3 Hz), 1.88 (1H, dd,
J 7.0, 14.3 Hz), 0.86e1.24 (30H, m); dC (125 MHz, CDCl3) 144.3, 128.1,
126.8,125.4, 88.6, 87.1, 80.2, 70.4, 60.8, 60.4, 37.8, 37.1,17.5,17.4,17.3,
17.1, 17.0, 13.4, 13.1, 12.8, 12.7; HRMS (ESI-TOF): MþHþ, found
510.3033. C26H48NO5Si2 requires 510.3066.
solved in THF (40 mL), and then TBAF$3H2O (3.82 g, 12.1 mmol) was
added to the reaction mixture. After stirring for 2 h, the reaction
mixture was evaporated under reduced pressure. The residue was
dissolved in MeOH (37 mL), and then DOWEX-50WX8-100 (3.12 g)
was added to the reaction mixture. After stirring for 40 h, the re-
action mixture was filtered off, and the filtrate was evaporated
under reduced pressure. The residue was purified by flash column
chromatography (Hexane:EtOAc¼1/1) to give compound 17 as
a colorless foam (1.42 g, 6.76 mmol, 82% for two steps). lmax (neat)
4.3.5. (10S,20R,30R,40R)-10-[(N-3-Methoxy-2-methylacryoyl)-ureido]-
20-methoxy-10-phenyl-30,50-O-tetraisopropyldisiloxy-ribose (16). To
a solution of 15 (370 mg, 0.73 mmol) in benzene (15 mL) was added
a solution of 7 in benzene (excess amount). After stirring for 5 h, the
reaction mixture was quenched by a satd NH4Cl solution and
extracted with EtOAc. The organic layer was continuously washed
with water and a satd NaCl solution, dried over Na2SO4, then
evaporated under reduced pressure. The residue was purified by
flash column chromatography (CHCl3:MeOH¼99/1) to give com-
pound 16 as a colorless oil (463 mg, 0.71 mmol, 90%). lmax (neat)
3346, 3272, 3201 cmꢁ1
; dH (400 MHz, CD3OD) 7.44e7.25 (5H, m),
4.70 (1H, d, J 7.2 Hz), 4.03 (1H, d, J 4.0 Hz), 3.98 (1H, q, J 4.0 Hz), 3.87
(1H, t, J 6.0 Hz), 3.80 (1H, dd, J 3.6, 12.0), 3.74 (1H, dd, J 5.2, 12.0 Hz);
dC (125 MHz, CDCl3) 141.9, 129.2, 128.7, 127.4, 86.3, 85.6, 79.1, 72.9,
63.7; HRMS (ESI-TOF): MþNaþ, found 233.0804. C11H14O4Na re-
quires 233.0784.
4.4.2. (1S,2R,3S,4R)-1-Deoxy-3,5-O-(1,3-disiloxanediyl-1,1,3,3-
tetraisopropyl)-1-phenylribose (18). To a solution of 17 (1.0 g,
4.76 mmol) in pyridine (48 mL) was added TIPDSCl2 (1.8 mL,
5.72 mmol) at 0 ꢀC. After stirring for 1 h at room temperature, the
reaction mixture was diluted with EtOAc, washed with a satd NaCl
solution, dried over Na2SO4, then evaporated under reduced pres-
sure. The residue was purified by flash column chromatography
(Hexane:EtOAc¼15/1 to 10/1) to give compound 18 as a colorless oil
2943, 2865, 1684, 1543 cmꢁ1
; dH (400 MHz, CDCl3) 8.67 (1H, s), 8.06
(1H, s), 7.46 (1H, d, J 7.3 Hz), 7.24 (1H, t, J 7.1 Hz), 7.17 (1H, t, J 7.2 Hz),
4.58 (1H, dd, J 2.0, 13.0 Hz), 4.48 (1H, dd, J 2.0, 13.0 Hz), 4.10e4.15
(1H, m), 3.90e4.00 (1H, m), 3.78 (1H, d, J 4.0 Hz), 3.40 (3H, s), 3.00
(1H, dd, J 5.2,16.2 Hz), 2.81 (1H, dd, J 5.2, 16.2 Hz), 2.70 (1H, dd, J 3.7,
16.8 Hz), 2.90e3.30 (1H, m), 2.18e2.38 (1H, m), 0.85e1.25 (30H, m);
dC (125 MHz, CDCl3) 168.8, 157.9, 154.0, 144.2, 128.1, 126.7, 125.4,
107.4, 88.7, 86.5, 80.4, 70.5, 61.3, 61.1, 60.2, 35.4, 35.1, 17.6, 17.4, 17.3,
17.2, 17.1, 17.0, 13.4, 13.1, 12.8, 12.7, 8.8; HRMS (ESI-TOF): MþNaþ,
found 673.3286. C32H54N2NaO8Si2 requires 673.3311.
(1.99 g, 4.40 mmol, 92%). lmax (neat) 3518, 2945, 2868 cmꢁ1
; dH
(400 MHz, CDCl3) 7.40 (2H, d, J 7.3 Hz), 7.32 (2H, t, J 7.3 Hz), 7.25 (1H,
t, J 7.3 Hz), 4.82 (1H, d, J 3.7 Hz), 4.36 (1H, d, J 6.1 Hz), 4.13e3.92 (3H,
m), 2.93 (1H, t, J 3.7 Hz), 1.10e0.93 (28H, m); dC (125 MHz, CDCl3)
140.1, 128.4, 127.62, 125.8, 85.5, 82.4, 71.7, 62.6, 17.5, 17.4, 17.3, 17.1,
17.1, 17.0, 13.4, 13.2, 12.9, 12.7; HRMS (ESI-TOF): MþHþ, found
453.2531. C23H41O5Si2 requires 453.2487.
4.3.6. (10S,20R,30R,40R)-10-[(3,4-Dihydro-2,4-dioxo-5-methyl-1(2H)
pyrimidinyl)]-20-methoxy-10-phenylribose (2). To a solution of 16
(430 mg, 0.66 mmol) in THF (5 mL) was added a solution of TBAF in
THF (1.59 mL, 1.59 mmol). After stirring for 2 h, the reaction mixture
was diluted with EtOAc. The organic layer was continuously washed
with water and a satd NaCl solution, dried over Na2SO4, then evap-
orated under reduced pressure. The residue was purified by flash
column chromatography (CHCl3:MeOH¼99/1) to give a colorless oil
(244 mg, 0.60 mmol, 90%). This colorless oil (220 mg, 0.54 mmol) was
dissolved in a solution of H2SO4 (1 M, 8 mL), then the mixture was
stirred for 3 h at 90 ꢀC. The reaction mixture was neutralized bya satd
NaHCO3 solution and extracted with EtOAc. The organic layer was
continuously washed with water and a satd NaCl solution, dried over
Na2SO4, then evaporated under reduced pressure. The residue was
purified by flash column chromatography (CHCl3:MeOH¼99/1) to
give compound 2 as a colorless foam (127 mg, 0.34 mmol, 72%). lmax
4.4.3. (1S,2R,3S,4R)-1-Deoxy-3,5-O-(1,3-disiloxanediyl-1,1,3,3-
tetraisopropyl)-2-O-methyl-1-phenylribose (19). To a solution of 18
(1.98 g, 4.37 mmol) in THF (44 mL) was added MeI (2.7 mL,
43.7 mmol) and NaHMDS (2.07 M in THF, 6.4 mL,13.1 mmol) at 0 ꢀC.
After stirring for 1 h, the reaction was quenched by a satd NH4Cl
solution, extracted with EtOAc, and then the organic layer was
washed with a satd NaCl solution, dried over Na2SO4, then evapo-
rated under reduced pressure. The residue was purified by flash
column chromatography (Hexane:EtOAc¼20/1) to give compound
19 as a colorless oil (1.57 g, 3.36 mmol, 77%). lmax (neat) 2946,
2866 cmꢁ1
; dH (400 MHz, CDCl3) 7.42 (2H, d, J 7.3 Hz), 7.31 (2H, t, J
7.6 Hz), 7.23 (1H, t, J 7.3 Hz), 4.95 (1H, s), 4.36 (1H, dd, J 3.6, 4.9 Hz),
4.19 (1H, dd, J 13.1, 2.4 Hz), 3.57 (3H, s), 3.56 (1H, t, J 4.9 Hz),
1.10e0.88 (28H, m); dC (125 MHz, CDCl3) 141.1, 128.3, 127.4, 125.7,
87.2, 84.6, 80.9, 70.6, 60.7, 58.9, 17.5, 17.4, 17.4, 17.3, 17.2, 17.0, 13.5,
13.1, 12.9, 12.6; HRMS (ESI-TOF): MþHþ, found 467.2660.
(neat) 3414, 2931, 2836, 1673, 1471, 1447 cmꢁ1
; dH (400 MHz, CDCl3)
8.86 (1H, s), 7.43 (2H, d, J 7.3 Hz), 7.32 (2H, t, J 7.3 Hz), 7.23 (1H, t, J
7.5 Hz), 6.63 (1H, s), 4.12 (2H, d J 3.6 Hz), 3.87e3.93 (1H, m),
3.66e3.81 (3H, m), 3.61 (1H, d J 3.6 Hz), 3.56 (3H, s), 3.35e3.45 (1H,
m), 2.44e2.51 (1H, m), 2.32e2.39 (1H, m), 2.12 (2H, s), 1.75 (3H, m);
dC (125 MHz, CDCl3) 164.1,150.7, 143.5,141.0, 128.6,127.4,109.8, 89.1,
85.4, 83.8, 70.5, 63.4, 60.2, 44.9, 33.0, 12.1; HRMS (ESI-TOF): MþNaþ,
found 399.1522. C19H24N2NaO6 requires 399.1527.
C24H43O5Si2 requires 467.2644.
4.4.4. (1S,2R,3S,4R)-1-Deoxy-2-O-methyl-1-phenylribose (3). To a so-
lution of 19 (1.54 g, 3.30 mmol) in THF (33 mL) was added a solution
of TBAF in THF (8.25 mL, 8.25 mmol). After stirring for 1 h, the re-
action mixture was diluted with EtOAc. The organic layer was con-
tinuously washed with water and a satd NaCl solution, dried over
Na2SO4, then evaporated under reduced pressure. The residue was
purified by flash column chromatography (Hexane:EtOAc¼2/1) to
give the compound 3 as a colorless oil (505.6 mg, 2.25 mmol, 68%).
4.4. Synthesis of 3
4.4.1. (1S,2R,3S,4R)-1-Deoxy-1-phenylribose (17). To a mixture of 4
(10.1 g, 20.1 mmol) in CH2Cl2 (67 mL) and Et3SiH (3.9 mL,

606
Y. Taniguchi et al. / Tetrahedron 69 (2013) 600e606
lmax (neat) 3406, 2931 cmꢁ1
;
dH (400 MHz, CDCl3) 7.37e7.26 (5H,
5859.51. TFO4(1): calcd for 5844.07, found 5843.88. TFO1(2): calcd
for 5874.07, found 5873.64. TFO2(2): calcd for 5890.06, found
5889.00. TFO3(2): calcd for 5874.07, found 5873.49. TFO4(2): calcd
for 5858.08, found 5873.49. TFO1(3): calcd for 5722.01, found
5721.14. TFO2(3): calcd for 5738.00, found 5737.89. TFO3(3): calcd
for 5722.01, found 5721.04. TFO4(3): calcd for 5706.02, found
5705.13.
m), 4.83 (1H, d J 5.5 Hz), 4.18 (1H, t, J 5.5 Hz), 3.99 (1H, dd, J 4.3,
3.7 Hz), 3.91 (1H, dd, J 3.6, 12.2 Hz), 3.77 (1H, dd, J 3.6, 12.2 Hz), 3.62
(1H, dd, J 5.5 Hz), 3.41 (3H, s); dC (125 MHz, CDCl3) 139.82, 128.59,
128.01, 126.01, 86.49, 84.46, 82.87, 70.55, 62.85, 58.40; HRMS (ESI-
TOF): MþHþ, found 225.1128. C12H17O4 requires 225.1121.
4.5. General procedure for synthesis of amidite precursor
4.7. Gel-shift assay
To a solution of the corresponding diol compound (1, 2, and 3) in
pyridine was added DMTrCl (2 equiv). After stirring for 1 h, the
reaction mixture was diluted with EtOAc. The organic layer was
continuously washed with water and a satd NaCl solution, dried
over Na2SO4, then evaporated under reduced pressure. The residue
was purified by flash column chromatography (CHCl3:MeOH sol-
vent system) to give the DMTr-protected compound as the colorless
foam. The DMTr-protected compound was dissolved in CH2Cl2 and
diisopropylethylamine (6.0 equiv) at 0 ꢀC. After stirring for 15 min
at the same temperature, N,N-diisopropylchlorophosphoramidite
(3.0 equiv) was added to the reaction mixture, which was stirred for
1 h at room temperature. The reaction mixture was diluted with
EtOAc, and the organic layer was washed with a satd NaHCO3 so-
lution, dried over Na2SO4, then evaporated under reduced pressure.
The residue was purified by flash column chromatography (Hex-
ane:EtOAc solvent system) to give the colorless foam. This foam was
dissolved in CH2Cl2 and poured into hexane at ꢁ78 ꢀC. The resulting
white powder was collected and dried under vacuum to give the
corresponding amidite precursor.
Triplex formation was done for 12 h at 22 ꢀC in the buffer con-
taining 20 mM TriseHCl, 5 mM MgCl2, 2.5 mM spermidine, and 10%
sucrose at pH 7.5. Electrophoresis was done at 10 ꢀC with a non-
denatured polyacrylamide gel. 10 nM TFO containing the 32P-la-
beled one as the tracer was used. The association constant was
calculated using the concentration of each component and the
following equation; Ks¼[Triplex]/[Duplex][TFO].
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Re-
search (S) and a Grant-in-Aid for Young Scientists (A) from the Ja-
pan Society for Promotion Science (JSPS) and CREST from the Japan
Science and Technology Agency (JST). We are grateful for the sup-
port from the Takeda Science Foundation and Teijin Pharma Award
in Synthetic Organic Chemistry Japan.
References and notes
Compound 20: as a white powder (73% for two steps): dH
(400 MHz, CDCl3) 7.61e7.17 (14H, m), 6.84 (1H, s), 6.76e6.73 (4H,
m), 6.51e3.81 (5H, m), 3.76 (6H, s), 3.56 (3H, s), 3.44e3.08 (2H, m),
2.58e2.22 (4H, m), 1.73 (3H, s), 1.29e0.85 (14H, m); dP (162 MHz,
CDCl3) 150.7, 150.1; ESI-TOF-MS: MþNaþ, found 887.3707.
1. (a) Govan, J. M.; Uprety, R.; Hemphill, J.; Lively, M. O.; Deiters, A. ACS Chem. Biol.
2012, 7, 1247e1256; (b) Duca, M.; Vekhoff, P.; Oussedik, K.; Halby, L.; Arimondo,
P. B. Nucleic Acids Res. 2008, 36, 4680e4688; (c) Jain, A.; Magistri, M.; Napoli, S.;
Cabone, G. M.; Catapano, C. Biochimie 2010, 92, 317e320; (d) Jain, A.; Wang, G.;
Vasquez, K. M. Biochimie 2008, 90, 1117e1130.
2. (a) Patterson, A.; Caprio, F.; Valleee-Beelisle, A.; Moscone, D.; Plaxco, K. W.;
Palleschi, G.; Ricci, F. Anal. Chem. 2010, 82, 9109e9115; (b) Obika, S.; Tomizu, M.;
Negoro, Y.; Orita, A.; Nakagawa, O.; Imanishi, T. ChemBioChem 2007, 8,
1924e1928.
C
48H57N4O9PNa requires 887.3755.
ꢀ
ꢀ
Compound 21: as a white powder (64% for two steps): lmax
(neat) 2969, 2345,1509 cmꢁ1
; dH (400 MHz, CDCl3) 8.01 (1H, s), 7.58
(1H, d, J 7.3 Hz), 7.52 (1H, d, J 7.3 Hz), 7.17e7.39 (12H, m), 6.78 (4H,
s), 6.65 (1H, d, J 11.0 Hz), 4.83 (0.2H, s), 4.70 (0.2H, s), 4.43 (0.5H, s),
4.20e4.33 (1H, m), 3.76 (7H, s), 3.45 (6H, d, J 5.9 Hz), 3.17e3.24 (1H,
m), 2.64 (1H, t, J 6.3 Hz), 2.56 (1H, t, J 6.3 Hz), 2.20e2.54 (2H, m),
2.12e2.29 (1H, m), 1.72 (5H, d, J 8.9 Hz), 1.62 (1H, s), 1.19e1.37 (14H,
m), 1.12 (2H, d, J 7.7 Hz), 0.95 (4H, d, J 6.7 Hz), 0.82 (8H, t, J 6.6 Hz);
dP (162 MHz, CDCl3) 151.3, 151.1; HRMS (ESI-TOF): MþNaþ, found
901.3902. C49H59N4NaO9P requires 901.3912.
Compound 22: as a white powder (63% for two steps): dH
(400 MHz, CDCl3) 7.50e7.18 (14H, m), 6.80 (4H, t, J 8.5 Hz), 4.88 (1H,
dd, J 3.6, 7.2 Hz), 4.37e4.24 (2H, m), 3.94e3.21 (15H, m), 2.64 (2H, t,
J 6.7), 2.30 (2H, t, J 6.8 Hz), 1.16e0.98 (12H, m); dP (162 MHz, CDCl3)
150.6, 150.1; HRMS (ESI-TOF): MþHþ, found 727.3510. C42H52N2O7P
requires 727.3507.
3. Soyfer, V. N.; Potaman, V. N. Triple-helical Nucleic Acids; Springer: New York, NY,
1996.
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Maeda, M. J. Am. Chem. Soc. 2004, 126, 516e528; (b) Sasaki, S.; Yamauchi, H.;
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TFO1(1): calcd for 5860.06, found 5859.80. TFO2(1): calcd for
5876.05, found 5875.34. TFO3(1): calcd for 5860.06, found