Base-pair recognition ability of hydroxyphenyl nucleobases in parallel triplex DNA

Article abstract of DOI:10.1016/j.tet.2013.05.107

Oligonucleotides containing 2′-deoxyribonucleotides bearing hydroxyphenyl nucleobases or their 2′,4′-BNA-modified analogs were synthesized, and the triplex-forming ability of their oligonucleotides with double-stranded DNA targets was evaluated by UV melt

Full text of DOI:10.1016/j.tet.2013.05.107

Tetrahedron 69 (2013) 6381e6391
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Base-pair recognition ability of hydroxyphenyl nucleobases in
parallel triplex DNA
*
Yoshiyuki Hari , Satoshi Kashima, Hiroyasu Inohara, Shin Ijitsu, Takeshi Imanishi,
*
Satoshi Obika
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Oligonucleotides containing 20-deoxyribonucleotides bearing hydroxyphenyl nucleobases or their 20,40-
BNA-modified analogs were synthesized, and the triplex-forming ability of their oligonucleotides with
double-stranded DNA targets was evaluated by UV melting experiments. Results showed that 20-de-
oxyribonucleotide bearing 20-hydroxyphenyl nucleobase could be recognized by a dUA base pair while
no affinity to a TA base pair was observed. The ,40-BNA modification led to a further increase in the
binding affinity to a dUA base pair. The ,40-BNA bearing 3-hydroxyphenyl nucleobase showed moderate
binding affinity to a TA base pair, but without selectivity.
Article history:
Received 30 April 2013
Received in revised form 21 May 2013
Accepted 23 May 2013
Available online 30 May 2013
Keywords:
Artificial nucleobases
20,40-BNA
Ó 2013 Elsevier Ltd. All rights reserved.
Parallel motif triplex
Triplex DNA
Triplex-forming oligonucleotides
1. Introduction
Therefore, many artificial nucleic acids have been developed for
recognizing TA or CG base pairs in triplex formation.2 However,
Triplex DNA is formed by interaction between double-stranded
DNA (dsDNA) and an oligonucleotide, called a triplex-forming oli-
gonucleotide (TFO). Triplex formation is useful for targeting dsDNA
and can contribute to the development of various nucleic acid
technologies.1 However, there is the sequence limitation of target
dsDNA as a problem of triplex formation. Although AT or GC base
pairs within dsDNA are recognized by T or C in TFO in a parallel-
oriented manner to form T-AT or protonated C-GC (CþH-GC) base
triplets, respectively (Fig. 1), it is difficult to target dsDNA con-
taining pyrimidineepurine base pairs (TA or CG base pairs).
most of them target CG base-pair recognition and there are not
many approaches for a TA base pair.
On the basis of the structure of the T-CG base triplet,3 a previous
report described the design of a 2-pyridone nucleobase (P) for CG
base-pair recognition and demonstrated that 2-pyridone bound to
a CG base pair and that 20,40-BNA with P (PB) effectively recognized
a CG base pair (Fig. 2).4 Hydrogen bond formation between the 2-
carbonyl group in P and 4-amino group in C was considered as the
recognition mode (Fig. 2). This finding suggests that a hydroxyphenyl
nucleobase might be a promising candidate for a TA base pair and
could hydrogen-bond with the 4-carbonyl group in T through the
hydroxyl group, despite possible steric hindrance by the 5-methyl
group in T (Fig. 2). Therefore, in this study, TFOs including 20-de-
oxyribonucleotides with 2-, 3-, and 4-hydroxyphenyl nucleobases
Fig. 1. Structures of T-AT and CþH-GC base triplets.
* Corresponding authors. Tel./fax: þ81 6 6879 8201 (Y.H.); tel.: þ81 6 6879 8200;
fax: þ81 6 6879 8204 (S.O.); e-mail addresses: hari@phs.osaka-u.ac.jp (Y. Hari),
obika@phs.osaka-u.ac.jp (S. Obika).
Fig. 2. Design of nucleobase for a TA base-pair recognition.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.05.107

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Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
that have hydroxyl groups located at different positions, and TFOs
including 20,40-BNA with 2- and 3-hydroxyphenyl nucleobases, were
synthesized and the triplex-forming ability of their TFOs with dsDNA
was evaluated using UV melting experiments.5
with DMTrCl in pyridine to produce 22 in 91% yield. The desired
phosphoramidite 3 was obtained via phosphitylation.
The 20,40-BNA-modified phosphoramidites 23e25 with 2- and
3-hydroxyphenyl nucleobase units were synthesized according to
the synthetic route shown in Scheme 2. A previous study reported
2. Results and discussion
2.1. Synthesis
The synthesis of phosphoramidites 1e3 composed of a 20-de-
oxyribose unit and hydroxyphenyl units is shown in Scheme 1.
Coupling of compound 46 and 2-allyloxyphenyllithium followed by
the Mitsunobu reaction7 using N,N,N0,N0-tetramethyl azodicarbox-
amide (TMAD) and n-Bu3P gave 5 (
which had its allyl group deprotected to afford the desired
6 and its -isomer 7. A benzoyl group was chosen as the protecting
b
-isomer/
a
-isomer¼ca. 1:1),
b
-isomer
a
group of the phenolic hydroxyl group in oligonucleotide synthesis.
Reaction of 6 with BzCl in the presence of Et3N gave 8 in 97% yield,
which was converted into diol
9 by hydrogenolysis using
Pd(OH)2eC and cyclohexene. Dimethoxytritylation of 9 followed by
phosphitylation afforded 2-hydroxyphenyl phosphoramidite 1,
a suitable building block for oligonucleotide synthesis. For the 3-
hydroxyphenyl derivative, the
yield in two steps via reaction of
b
-isomer 11 was isolated in 28%
with 3-
4
methoxymethoxyphenyllithium and ring-closure. The methox-
ymethyl group (11) was converted into a benzoyl group (14)
through a deprotectioneprotection process. Then, the phosphor-
amidite
2
was synthesized via three steps (debenzyla-
tionedimethoxytritylationephosphitylation). The synthesis of 4-
hydroxyphenyl phosphoramidite 3 was achieved by a synthetic
route similar to that of 2-hydroxyphenyl derivative 1. Reaction of 4
with 4-allyloxyphenyllithium and ring-closure reaction yielded 17
as a 1:1 anomeric mixture. Palladium-catalyzed deallylation of 17
proceeded to give 18, which was benzoylated to afford a separable
Scheme 2. Reagents and conditions: (i) 2-allyloxyphenylmagnesium iodide, THF,
ꢀ40 ꢁC; TMAD, n-Bu3P, CH2Cl2, rt, 57% over two steps; (ii) NaBH4, Pd(PPh3)4, THF, rt,
80% (29) and quant. (35); (iii) BzCl, Et3N, CH2Cl2, rt, 93% (30) and 98% (36); (iv) 20%
Pd(OH)2eC, cyclohexene, EtOH, reflux, 89% (31) and 95% (37); (v) DMTrCl, pyridine, rt,
94% (32) and 91% (38); (vi) (i-Pr2N)2POCH2CH2CN, DIHT, MeCN/THF, rt, 92% (23), 81%
(24), and 71% (25); (vii) K2CO3, MeOH, rt, 5 min, 96%; (viii) allyl bromide, K2CO3, ac-
etone, rt, 14 h, 87%; (ix) 3-allyloxyphenylmagnesium iodide, THF, rt; TMAD, n-Bu3P,
CH2Cl2, rt, 70% over two steps.
mixture of b-isomer 19 and a-isomer 20. Hydrogenolysis of the b-
isomer 19 produced 21 in 82% yield, which underwent reaction
α
α
α
Scheme 1. Reagents and conditions: (i) 2-allyloxyphenyllithium, THF, ꢀ78 ꢁC; TMAD, n-Bu3P, CH2Cl2, rt, 38% (
b-isomer/
a
-isomer¼ ca. 1:1) over two steps; (ii) NaBH4, Pd(PPh3)4,
THF, rt, 55% (6) and 45% (7), and 91% (18,
b-isomer/
a
-isomer¼ca. 1:1); (iii) BzCl, Et3N, CH2Cl2, rt, 97% (8), 98% (14), and 91% (19/20¼ca. 1:1); (iv) 20% Pd(OH)2eC, EtOH, 70 ꢁC, 94% (9),
96% (15), and 82% (21); (v) DMTrCl, pyridine, rt, 98% (10), 72% (16), and 91% (22); (vi) i-Pr2NP(Cl)OCH2CH2CN, i-Pr2NEt, CH2Cl2, rt, 58% (1), 58% (2), and 95% (3); (vii) 3-
methoxymethoxyphenyllithium, THF, ꢀ78 ꢁC; TMAD, n-Bu3P, CH2Cl2, rt, 28% (11) and 24% (12) over two steps; (viii) 10% HCl aq, THF, rt,
2 days, 91%; (ix) 4-
allyloxyphenyllithium, THF, ꢀ60 ꢁC; TMAD, n-Bu3P, CH2Cl2, rt; 48% (
b-isomer/
a-isomer¼ca. 1:1) over two steps.

Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
6383
that reaction of aldehyde 26 with arylmagnesium reagents pro-
ceeded with high diastereoselectivity and that Mitsunobu reaction
of the resulting products led predominantly to 20,40-BNA-modified
C-nucleosides with
excess 2- or 3-allyloxyphenylmagnesium bromide in THF to give
coupling compounds; ring-closure using TMAD afforded the
anomers 27 and 28 in 57% and 70% yield, respectively. Interestingly,
in both cases, only the -anomer was isolated; its -isomer was not
b
-configuration.8 Thus, 26 was treated with
b
-
b
a
observed. Palladium-catalyzed reaction of 2-hydroxyphenyl de-
rivative 27 with NaBH4 gave deallylated compound 29, which was
benzoylated by BzCl in the presence of Et3N to produce 30 in high
yield. After debenzylation of 30 to produce 31, compound 32 was
obtained in 94% yield by dimethoxytritylation. Phosphitylation of
32 using (i-Pr2N)2POCH2CH2CN and diisopropylammonium tetra-
zolide (DIHT) gave benzoyl-protected phosphoramidite 23 with
a 2-hydroxyphenyl unit and 20,40-BNA modification in 92% yield. In
addition, the allyl-protected congener 24 was prepared through
debenzoylation of 32, followed by allylation and phosphitylation.
Analogous to synthesis of 23, 20,40-BNA phosphoramidite 25 with
a 3-hydroxyphenyl unit was synthesized. After conversion of the
allyl group of 28 into a benzoyl group (36) in two steps, 37 was
produced by the hydrogenolysis. The desired phosphoramidite 25
was obtained from 37 via dimethoxytritylated 38.
Fig. 4. TFOs synthesized in this study.
Table 1
Tm values (ꢁC) of triplexes between TFOs and dsDNA targetsa,b
TFO (X)
YZ
TA
UA
CG
AT
GC
Next, introduction of the phosphoramidites synthesized into
oligonucleotides was investigated using an automated DNA syn-
thesizer. The use of benzoyl-protected phosphoramidites 1e3 and
25, with the exception of the 20,40-BNA-modified 2-hydroxy analog
23, successfully led to the desired TFOs according to standard
phosphoramidite chemistry. For 23, two undesirable oligonucleo-
tides having an approximately þ17 mass unit difference from mo-
lecular weight of the desired TFO were obtained. This result implies
that a structure constrained by the methylene linkage between the
20-oxygen and 40-carbon atoms might trigger a ring-opening re-
action by conventional ammonia treatment as shown in Fig. 3.
Eventually, the desired TFO 42 was obtained by oligonucleotide
synthesis using allyl-protected phosphoramidite 24 followed by
treatment with NaBH4 and Pd(PPh3)4 to remove an allyl group. The
sequence of TFOs prepared is listed in Fig. 4.
39 (2H)
40 (3H)
41 (4H)
42 (2HB)
43 (3HB)
23
26
24
28
34
28
27
25
37
35
27
27
25
29
34
27
28
30
36
33
31
25
27
39
33
a
Conditions: 10 mM sodium cacodylate buffer (pH 6.8), 140 mM KCl, and 50 mM
MgCl2. The final concentration of each oligonucleotide used was 1.89 mM. C indicates
20-deoxy-5-methylcytidine.
b
Triplexes containing T-AT and PB-CG base triplets as X-YZ had Tm values of 44 ꢁ
C
and 37 ꢁC, respectively. PB indicates 20,40-BNA bearing 2-pyridone nucleobase.
and no significant sequence-selectivity toward a certain base pair
was observed because the hydroxyl group could function not only
as a hydrogen donor but also as a hydrogen acceptor. However, the
Tm value of a triplex with dsDNA (YZ¼UA) by TFO 39 was greater
than that with dsDNA (YZ¼TA) by þ5 ꢁC. In addition, TFO 39 pro-
vided nearly the same stability to TFO 41 (X¼4H) in the triplex with
dsDNA (YZ¼TA), while TFO 39 formed a stable triplex with dsDNA
(YZ¼UA) compared to TFO 41. These results suggest that a 2-
hydroxyphenyl nucleobase forms a hydrogen bond with U of a UA
base pair without forming any hydrogen bond with a TA base pair
because the 5-methyl group in T prevents bond formation (Fig. 5).
In contrast, TFO 40 (X¼3H) significantly stabilized triplexes formed
Fig. 3. Plausible mechanism of ring-opening reaction.
2.2. Evaluation
Triplex-forming ability of the TFOs 39e43 with dsDNA was
evaluated through UV melting experiments at 10 mM sodium
cacodylate (pH 6.8) plus 140 mM KCl and 50 mM MgCl2. As targets,
hairpin dsDNAs containing five types of base pairs (i.e., TA, its
demethylated UA, CG, AT, and GC base pairs) at the same position
were used. Results are summarized in Table 1. In general, TFOs
39e41 containing 20-deoxy derivatives 2H, 3H, and 4H, did not
sequence-specifically form stable triplexes with dsDNA (YZ¼TA)
Fig. 5. Proposed structures of base triplets by 2H and 3H.

6384
Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
with dsDNAs (YZ¼TA and UA) compared to TFO 41. The Tm values of
triplexes (X-YZ¼3H-TA and 3H-UA) were comparable to that of
triplex (X-YZ¼2H-UA). This suggests that the 3-hydroxyphenyl
nucleobase recognized not only a UA base pair, but also a TA base
pair by effective avoidance of the bulky 5-methyl group in T, despite
the lack of stability. When TFOs 42 and 43 containing 20,40-BNA
with 2- or 3-hydroxyphenyl nucleobases were used, affinities to
any dsDNA targets increased without any sequence-selectivity. TFO
42 (X¼2HB) formed a stable triplex with dsDNA (YZ¼UA) rather
than with dsDNA (YZ¼TA), which was comparable to triplexes
formed with TFOs, including 20,40-BNA (PB) with a 2-pyridone
nucleobase and dsDNA (YZ¼CG).9 As mentioned, PB likely bonds
to a CG base pair via a single hydrogen bond. Therefore, 2-
hydroxyphenyl nucleobase in 2HB forms a single hydrogen bond
with an UA base pair and cannot recognize a TA base pair because of
the 5-methyl group. In contrast, TFO 43 (X¼3HB) had Tm values of
34 ꢁC and 35 ꢁC against dsDNAs (YZ¼UA and TA), respectively. This
stability was significantly higher than that of the triplex (X-
YZ¼2HB-TA), although it was slightly lower than that of triplex (X-
YZ¼2HB-UA). These results suggest that 3-hydroxyphenyl nucleo-
base in 3HB should recognize both UA and TA base pairs through
a single hydrogen bond (Fig. 5). Results also indicated that 3-
hydroxyphenyl nucleobase can recognize a TA base pair in a paral-
lel triplex formation, although no sequence-selectivity was
observed.
[prepared from 2-allyloxyphenyl bromide (3.98 g, 18.7 mmol)
and n-BuLi (2.67 M in hexane, 6.9 mL, 18.4 mmol) in anhydrous
THF (20 mL) at ꢀ78 ꢁC], and the mixture was stirred at ꢀ78 ꢁC for
16 h. After addition of satd NH4Cl aq, the mixture was extracted
with Et2O. The extracts were washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. The res-
idue was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼5:1) to give the appropriate compounds (Rf¼ca.
0.1), which were dissolved in CH2Cl2 (7 mL); TMAD (750 mg,
4.35 mmol) and n-Bu3P (1.1 mL, 4.35 mL) were added at room
temperature. After being stirred at room temperature for 6 h,
water was added and the mixture was extracted with Et2O. The
extracts were washed with water and brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was pu-
rified by flash silica gel column chromatography (n-hexane/
AcOEt¼15:1) to give a ca. 1:1 anomeric mixture 5 (773 mg, 38%
from 4) as a colorless oil. 1H NMR (CDCl3)
d
1.77 (0.5H, ddd, J¼7.4,
10.2, 13.2 Hz), 1.96 (0.5H, ddd, J¼5.8, 7.8, 12.7 Hz), 2.56 (0.5H, ddd,
J¼1.0, 5.4, 13.2 Hz), 2.73 (0.5H, td, J¼6.8, 12.7 Hz), 3.56e3.71 (2H,
m), 4.12e4.13 (0.5H, m), 4.20e4.24 (0.5H, m), 4.27e4.31 (0.5H, m),
4.38e4.63 (6.5H, m), 5.22e5.25 (1H, m), 5.34e5.51 (1H, m),
5.96e6.06 (1H, m), 6.78e6.81 (1H, m), 6.91e6.98 (1H, m),
7.16e7.34 (11H, m), 7.52 (0.5H, d, J¼7.4 Hz), 7.59 (0.5H, d,
J¼6.8 Hz). HRMS (FAB) m/z calcd for C28H30NaO4 [MþNa]þ:
453.2036; found, 453.2049.
3. Conclusion
4.3. 2-(2-Deoxy-3,5-di-O-benzyl-b-D-ribofuranosyl)-1-
hydroxybenzene (6) and 2-(2-deoxy-3,5-di-O-benzyl-a-D-ri-
bofuranosyl)-1-hydroxybenzene (7)
Syntheses of 20-deoxyribonucleotides bearing 2-, 3-, and 4-
hydroxyphenyl nucleobases and 20,40-BNA-modified nucleotides
containing 2- and 3-hydroxyphenyl nucleobases were developed.
Evaluation of triplex-forming ability of TFOs containing their
hydroxyphenyl nucleobases with dsDNA showed 3-hydroxyphenyl
nucleobase might recognize a TA base pair through a single hy-
drogen bond without any sequence selectivity. Recently, we suc-
ceeded in the facile synthesis of various triazole or pyrimidinone
nucleobases on oligonucleotides using post-elongation modifica-
tion (PEM) and found promising nucleobases for CG base-pair
recognition.10 Thus, the synthesis of various 3-hydroxyphenyl units
using PEM could be effective for screening of nucleobases for TA
base-pair recognition. Further investigation of nucleobases based
on the 3-hydroxyphenyl structure is underway.
Under a nitrogen atmosphere, Pd(PPh3)4 (290 mg, 0.253 mmol)
was added to a solution of compound 5 (545 mg, 1.27 mmol) in
anhydrous THF (10 mL) and the mixture was stirred until solids of
Pd(PPh3)4 dissolved completely. Then, NaBH4 (58 mg, 1.52 mmol)
was added and the mixture was stirred at room temperature for
24 h. After addition of 3% HCl aq, the mixture was filtered through
a pad of CeliteÒ. The filtrate was extracted with AcOEt. The extracts
were washed with water and brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼20:1 to
10:1) to give compound 6 (270 mg, 55%) as a colorless oil and
compound 7 (221 mg, 45%) as a colorless oil. Compound 6: [a]
22
D
4. Experimental
þ54.0 (c 1.01, CHCl3). IR nmax (KBr) 3338, 3028, 2867, 1495, 1455,
1247, 1095, 1077 cmꢀ1. 1H NMR (CDCl3)
2.20e2.33 (2H, m), 3.55
d
4.1. General methods
(1H, dd, J¼2.5, 10.0 Hz), 3.67 (1H, dd, J¼3.0, 10.0 Hz), 4.18e4.20 (1H,
m), 4.24e4.26 (1H, m), 4.47 (1H, d, J¼12.0 Hz), 4.51 (1H, d,
J¼12.0 Hz), 4.54 (1H, d, J¼12.0 Hz), 4.68 (1H, d, J¼12.0 Hz), 5.20 (1H,
dd, J¼6.0, 10.5 Hz), 6.80 (1H, ddd, J¼2.0, 8.0, 8.0 Hz), 6.89 (1H, dd,
J¼2.0, 8.0 Hz), 7.01 (1H, d, J¼2.0, 8.0 Hz), 7.18 (1H, ddd, J¼2.0, 8.0,
All chemicals were purchased from chemical suppliers. For
column chromatography, Fuji Silysia silica gel BW-300, PSQ-100B,
and FL-100D were used. All melting points were measured on
a Yanagimoto micro melting point apparatus and are uncorrected.
1H NMR, 13C NMR, and 31P NMR spectra were recorded on JEOL
EX270, JEOL ECS400, and JEOL GX-500 spectrometers. IR spectra
were recorded on JASCO FT/IR-200 and JASCO FT/IR-4200 spec-
trometers. Optical rotations were recorded on a JASCO DIP-370
instrument. Mass spectra were obtained using a JEOL JMS-600 or
JEOL JMS-700 mass spectrometer. MALDI-TOF mass spectra were
recorded on a Bruker Daltonics Autoflex II TOF/TOF or JEOL JMS-
S3000 mass spectrometer.
8.0 Hz), 7.26e7.36 (10H, m), 8.10 (1H, s). 13C NMR (CDCl3)
d 39.4,
69.5, 71.3, 73.5, 81.3, 82.6, 84.7, 117.4, 119.4, 123.8, 125.9, 127.7, 127.8,
127.9, 127.9, 128.1, 128.5, 129.3, 137.3, 137.8, 155.8. HRMS (FAB) m/z
calcd for C25H26NaO4 [MþNa]þ: 413.1723; found 413.1742. Com-
pound 7: [
a]
23 þ6.6 (c 0.86, CHCl3). IR nmax (KBr) 3337, 3033, 2925,
D
2862, 1494, 1455, 1246, 1105, 1074 cmꢀ1. 1H NMR (CDCl3)
d
2.23 (1H,
ddd, J¼4.0, 8.0, 12.5 Hz), 2.65 (1H, ddd, J¼7.0, 7.0, 12.5 Hz),
3.58e3.64 (2H, m), 4.27e4.32 (1H, m), 4.37 (1H, ddd, J¼4.5, 4.5,
4.5 Hz), 4.49 (1H,d, J¼12.0 Hz), 4.50 (1H, d, J¼12.0 Hz), 4.55 (1H, d,
J¼12.0 Hz), 4.57 (1H, d, J¼12.0 Hz), 5.23 (1H, dd, J¼7.0, 8.0 Hz), 6.81
(1H, ddd, J¼2.0, 8.0, 8.0 Hz), 6.88 (1H, dd, J¼2.0, 8.0 Hz), 6.96 (1H,
dd, J¼2.0, 8.0 Hz), 7.17 (1H, ddd, J¼2.0, 8.0, 8.0 Hz), 7.25e7.38 (10H,
4.2. 1-Allyloxy-2-(2-deoxy-3,5-di-O-benzyl-D-ribofuranosyl)
benzene (5)
m), 8.09 (1H, s). 13C NMR (CDCl3)
d 39.4, 70.2, 71.6, 73.5, 79.9, 81.2,
Under a nitrogen atmosphere, a solution of 2-deoxy-3,5-di-O-
benzyl-
-ribofuranose 46 (1.47 g, 4.69 mmol) in anhydrous THF
(6 mL) was added to a solution of 2-allyloxyphenyllithium
82.9, 117.3, 119.5, 124.6, 127.5, 127.6, 127.7, 127.7, 127.8, 128.4, 129.1,
137.5, 137.5, 137.9, 155.4. HRMS (FAB) m/z calcd for C25H27O4
[MþH]þ: 391.1904; found 391.1895.
D

Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
6385
4.4. 1-Benzoyloxy-2-(2-deoxy-3,5-di-O-benzyl-
anosyl)benzene (8)
b-D
-ribofur-
(FAB) m/z calcd for
639.2350.
C
39H36NaO7 [MþNa]þ: 639.2353; found
Under a nitrogen atmosphere, BzCl (88
added to a solution of compound 6 (270 mg, 0.627 mmol) and Et3N
(105 L, 0.753 mmol) in anhydrous CH2Cl2 (6 mL) at room tem-
m
L, 0.753 mmol) was
4.7. 1-Benzoyloxy-2-{3-O-[2-cyanoethoxy(diisopropylamino)
phosphino]-2-deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-ribofur-
m
anosyl}benzene (1)
perature, and the mixture was stirred for 2 h. After addition of
water, the mixture was extracted with Et2O. The extracts were
washed with water and brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash silica gel
column chromatography (n-hexane/AcOEt¼10:1) to give com-
Under a nitrogen atmosphere, i-Pr2NP(Cl)OCH2CH2CN (22
mL,
0.098 mmol) was added to a solution of compound 10 (40 mg,
0.065 mmol) and i-Pr2NEt (57 mL, 0.327 mmol) in anhydrous
CH2Cl2 (1 mL) at room temperature and the mixture was stirred for
1 h. After addition of satd NaHCO3 aq, the mixture was extracted
with Et2O. The extracts were washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼5:1) to give compound 1 (31 mg, 58%) as a white
pound 8 (332 mg, 97%) as a colorless oil. [
a
]
24 þ57.1 (c 0.90, CHCl3).
D
IR nmax (KBr) 3062, 3032, 2867, 1738, 1489, 1452, 1263, 1219, 1181,
1081 cmꢀ1
.
1H NMR (CDCl3)
d
1.86 (1H, ddd, J¼6.0, 10.5, 13.0 Hz),
2.38 (1H, ddd, J¼1.5, 5.0, 13.0 Hz), 3.56 (1H, dd, J¼5.0, 10.0 Hz), 3.64
(1H, dd, J¼5.0, 10.0 Hz), 4.09e4.11 (1H, m), 4.20e4.23 (1H, m), 4.40
(2H, s), 4.57 (2H, s), 5.35 (1H, dd, J¼5.0,10.5 Hz), 7.14e7.36 (13H, m),
7.48 (2H, dd, J¼8.0, 8.0 Hz), 7.63 (1H, dd, J¼8.0, 8.0 Hz), 7.65 (1H, dd,
solid. 1H NMR (CDCl3)
d
0.90 (3H, d, J¼6.8 Hz), 0.96 (3H, d, J¼6.4 Hz),
1.06e1.09 (6H, m), 1.89e2.01 (1H, m), 2.20e2.43 (3H, m), 3.23e3.35
(2H, m), 3.38e3.48 (3H, m), 3.53e3.58 (1H, m), 3.79 (3H, s), 3.79
(3H, s), 4.15e4.16 (1H, m), 4.39e4.47 (1H, m), 5.29e5.36 (1H, m),
6.80e6.84 (4H, m), 7.14e7.38 (10H, m), 7.46e7.56 (4H, m),
J¼8.0, 8.0 Hz), 8.20 (2H, d, J¼8.0 Hz). 13C NMR (CDCl3)
d 39.8, 70.9,
73.4, 75.0, 81.2, 83.5, 122.1, 126.3, 126.7, 127.5, 127.5, 127.6, 127.6,
128.2, 128.3, 128.3, 128.128.7, 129.3, 130.1, 133.7, 134.2, 137.9, 138.1,
147.7, 165.0. HRMS (FAB) m/z calcd for C32H31O5 [MþH]þ: 495.2166;
found 495.2165.
7.64e7.74 (2H, m), 8.18e8.23 (2H, m). 31P NMR (CDCl3)
d 147.3,
148.3. HRMS (FAB) m/z calcd for C48H53N2NaO8P [MþNa]þ:
839.3432; found 839.3421.
4.5. 1-Benzoyloxy-2-(2-deoxy-b-D-ribofuranosyl)benzene (9)
4.8. 3-(2-Deoxy-3,5-di-O-benzyl-
thoxymethoxy)benzene (11) and 3-(2-deoxy-3,5-di-O-benzyl-
-ribofuranosyl)-1-(methoxymethoxy)benzene (12)
b-D-ribofuranosyl)-1-(me-
A solution of compound 8 (191 mg, 0.386 mmol), 20% Pd(OH)2/C
(120 mg), and cyclohexene (3.9 mL, 38.6 mmol) in EtOH (8 mL) was
stirred at 70 ꢁC for 1.5 h. The mixture was filtered through a pad of
CeliteÒ and the filtrate was concentrated under reduced pressure.
The residue was purified by flash silica gel column chromatography
a-D
Under a nitrogen atmosphere, a solution of compound 4
(180 mg, 0.573 mmol) in anhydrous THF (5 mL) was added to
a solution of 3-methoxymethoxyphenyllithium [prepared from 3-
methoxymethoxyphenyl bromide (690 mg, 3.18 mmol) and n-
BuLi (1.65 M in hexane, 1.9 mL, 3.18 mmol) in anhydrous THF
(20 mL) at ꢀ78 ꢁC], and the mixture was stirred at ꢀ78 ꢁC for 2 h.
After addition of satd NH4Cl aq, the mixture was extracted with
AcOEt. The extracts were washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified
by flash silica gel column chromatography (n-hexane/AcOEt¼1:1)
to give appropriate compounds (Rf¼ca. 0.5), which were dissolved
(CHCl3/MeOH¼60:1) to give compound 9 (114 mg, 94%) as a color-
22
less oil. [
a]
þ35.0 (c 1.03, CHCl3). IR nmax (KBr) 3410, 2918, 1732,
D
1451, 1263, 1219, 1179, 1086 cmꢀ1. 1H NMR (CDCl3)
d 2.01 (1H, ddd,
J¼6.5, 9.0, 13.5 Hz), 2.19 (1H, ddd, J¼2.5, 6.0, 13.5 Hz), 2.33 (2H, s),
3.58e3.69 (2H, m), 3.84 (1H, ddd, J¼4.0, 4.0, 4.0 Hz), 4.25e4.28 (1H,
m), 5.31 (1H, dd, J¼6.0, 9.0 Hz), 7.14 (1H, dd, J¼1.0, 8.0 Hz), 7.28 (1H,
ddd, J¼1.0, 1.0, 8.0 Hz), 7.34 (1H, ddd, J¼1.0, 8.0, 8.0 Hz), 7.50 (1H,
ddd, J¼1.0, 8.0, 8.0 Hz), 7.53 (2H, ddd, J¼1.0, 8.0, 8.0 Hz), 7.64 (1H,
ddd, J¼1.0, 8.0, 8.0 Hz), 8.19 (2H, dd, J¼1.0, 8.0 Hz). 13C NMR (CDCl3)
in CH2Cl2 (6 mL); TMAD (131 mg, 0.763 mmol), and n-Bu3P (190 mL,
d
42.6, 63.1, 73.2, 74.9, 86.6, 122.5, 126.4, 126.5, 128.6, 128.7, 129.1,
0.763 mmol) were added at room temperature. After being stirred
at room temperature for 2 h, water was added and the mixture was
extracted with AcOEt. The extracts were washed with brine, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼20:1) to give compound 11 (78 mg, 28%) as a col-
orless oil and compound 12 (66 mg, 24%) as a colorless oil. Com-
130.1, 133.8, 148.0, 165.3. HRMS (MALDI-TOF) m/z calcd for
C18H18NaO5 [MþNa]þ: 337.1046; found 337.1052.
4.6. 1-Benzoyloxy-2-[2-deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-
ribofuranosyl]benzene (10)
Under a nitrogen atmosphere, DMTrCl (60 mg, 0.176 mmol) was
added to a solution of compound 9 (46.0 mg, 0.146 mmol) in an-
hydrous pyridine (2 mL) at room temperature and the mixture was
stirred for 2 h. After addition of water, the mixture was extracted
with Et2O. The extracts were washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼5:1 to 3:1) to give compound 10 (89 mg, 98%) as
pound 11: [
a
]
23 þ26.2 (c 1.03, CHCl3). IR nmax (KBr) 2892,1488,1454,
D
1147, 1076 cmꢀ1
.
1H NMR (CDCl3)
d
1.91 (1H, ddd, J¼6.0, 10.5,
13.0 Hz), 2.36 (1H, ddd, J¼1.0, 6.0, 13.0 Hz), 3.45 (3H, s), 3.58 (1H,
dd, J¼5.5, 10.5 Hz), 3.67 (1H, dd, J¼4.5, 10.5 Hz), 4.16 (1H, m), 4.30
(1H, ddd, J¼2.5, 4.5, 5.5 Hz), 4.56 (2H, d, J¼12.0 Hz), 4.60 (2H, d,
J¼12.0 Hz), 5.12 (1H, dd, J¼5.0, 10.5 Hz), 5.14 (2H, s), 6.94 (1H, ddd,
J¼1.0, 2.0, 8.0 Hz), 7.01 (1H, m), 7.06 (1H, m), 7.20e7.38 (11H, m). 13C
NMR (CDCl3) d 41.1, 55.9, 71.0, 71.1, 73.4, 80.2, 81.5, 83.8, 94.4, 114.0,
a white solid. [
a]
26 þ30.2 (c 1.02, CHCl3). IR nmax (KBr) 3456, 2933,
115.1, 119.6, 127.6, 127.6, 127.7, 128.3, 128.4, 129.3, 138.1, 138.2, 143.4,
D
1737, 1607, 1509, 1450, 1252, 1176, 1079 cmꢀ1. 1H NMR (CDCl3)
d
1.69
157.3. HRMS (FAB) m/z calcd for C27H30NaO5 [MþNa]þ: 457.1985;
23
(1H, d, J¼4.0 Hz), 2.03 (1H, ddd, J¼6.5, 9.0, 13.0 Hz), 2.22 (1H, ddd,
J¼3.0, 6.5, 13.0 Hz), 3.28 (1H, dd, J¼5.0, 10.0 Hz), 3.35 (1H, dd, J¼5.0,
10.0 Hz), 3.79 (6H, s), 3.98 (1H, ddd, J¼5.0, 5.0, 5.0 Hz), 4.32e4.35
(1H, m), 5.35 (1H, dd, J¼6.5, 9.0 Hz), 6.82 (4H, d, J¼9.0 Hz),
7.15e7.36 (10H, m), 7.44e7.53 (4H, m), 7.62e7.68 (2H, m), 8.19 (2H,
found 457.1985. Compound 12: [
a
]
D
þ18.3 (c 1.24, CHCl3). IR nmax
(KBr) 2894, 1487, 1454, 1151, 1077 cmꢀ1. 1H NMR (CDCl3)
d 2.06 (1H,
ddd, J¼6.5, 8.0, 13.0 Hz), 2.62 (1H, ddd, J¼6.5, 6.5, 13.0 Hz), 3.45 (3H,
s), 3.62 (2H, m), 4.26 (1H, ddd, J¼4.5, 6.6, 6.5 Hz), 4.36 (1H, ddd,
J¼4.5, 4.5, 4.5 Hz), 4.49 (2H, d, J¼12.0 Hz), 4.59 (2H, d, J¼12.0 Hz),
5.05 (1H, dd, J¼6.5, 8.0 Hz), 5.16 (2H, s), 6.93 (1H, ddd, J¼1.0, 2.0,
8.0 Hz), 7.03 (1H, m), 7.08 (1H, m), 7.22e7.37 (11H, m). 13C NMR
d, J¼8.0 Hz). 13C NMR (CDCl3)
d 42.8, 55.2, 64.3, 74.3, 74.6, 85.5, 86.2,
113.1, 122.2, 126.3, 126.7, 126.8, 127.8, 128.2, 128.2, 128.7, 129.2,
130.1, 130.2, 133.7, 134.6, 136.0, 144.8, 147.6, 158.5, 164.9. HRMS
(CDCl3)
d 40.9, 56.0, 70.8, 71.7, 73.5, 80.0, 80.6, 82.9, 94.4, 113.9,

6386
Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
115.1,119.5,127.6,127.6,128.3, 128.4, 129.4,138.1, 144.5. HRMS (FAB)
143.4, 151.0, 163.3. HRMS (MALDI-TOF) m/z calcd for C18H18NaO5
[MþNa]þ: 337.1046; found 337.1058.
m/z calcd for C27H30NaO5 [MþNa]þ: 457.1985; found 457.1988.
4.12. 1-Benzoyloxy-3-[2-deoxy-5-O-(4,40-dimethoxytrityl)-
-ribofuranosyl]benzene (16)
b-
4.9. 3-(2-Deoxy-3,5-di-O-benzyl-b-D-ribofuranosyl)-1-
hydroxybenzene (13)
D
Under a nitrogen atmosphere, DMTrCl (49 mg, 0.145 mmol) was
10% HCl aq (7 mL) was added to a solution of compound 11
(286 mg, 0.657 mmol) in THF (10 mL), and mixture was stirred at
room temperature for 2 days. The mixture was extracted with Et2O
and the extracts were washed with water and brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by flash silica gel column chromatography (n-hexane/
added to a solution of compound 15 (38 mg, 0.121 mmol) in an-
hydrous pyridine (1.5 mL) at room temperature and the mixture
was stirred for 2 h. After addition of water, the mixture was
extracted with Et2O. The extracts were washed with water, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼5:1 to 3:1) to give compound 16 (53 mg, 72%) as
AcOEt¼5:1) to give compound 13 (234 mg, 91%) as a colorless oil.
23
[
a]
þ20.5 (c 0.96, CHCl3). IR nmax (KBr) 3331, 3032, 2861, 1591,
D
23 þ23.2 (c 1.16, AcOEt). IR nmax (KBr) 3454, 2932,
1456, 1077 cmꢀ1
.
1H NMR (CDCl3)
d
1.89 (1H, ddd, J¼5.5, 10.0,
a white solid. [
a
]
D
1736, 1607, 1508, 1252, 1176, 1080 cmꢀ1. 1H NMR (CDCl3)
d
1.76 (1H,
13.0 Hz), 2.33 (1H, ddd, J¼1.5, 5.0, 13.0 Hz), 3.58 (1H, dd, J¼5.0,
10.0 Hz), 3.67 (1H, dd, J¼4.5, 10.0 Hz), 4.15 (1H, m), 4.30 (1H, ddd,
J¼2.5, 4.5, 5.0 Hz), 4.55 (2H, s), 4.58 (2H, s), 5.08 (1H, dd, J¼5.0,
10.0 Hz), 5.21 (1H, s), 6.67 (1H, ddd, J¼1.0, 2.0, 8.0 Hz), 6.80 (1H, m),
6.86 (1H, m), 7.13 (1H, dd, J¼8.0, 8.0 Hz), 7.24e7.36 (10H, m). 13C
d, J¼4.0 Hz), 2.08 (1H, ddd, J¼6.0, 10.0, 13.5 Hz), 2.28 (1H, ddd,
J¼2.0, 6.0, 13.5 Hz), 3.25 (1H, dd, J¼4.5, 10.0 Hz), 3.35 (1H, dd, J¼5.0,
10.0 Hz), 3.75 (6H, s), 4.06 (1H, ddd, J¼4.5, 5.0, 7.0 Hz), 4.46 (1H, m),
5.20 (1H, dd, J¼6.0, 10.0 Hz), 6.75e6.85 (4H, m), 7.10e7.40 (11H, m),
7.45 (2H, dd, J¼1.0, 8.0 Hz), 7.48 (2H, dd, J¼8.0, 8.0 Hz), 7.62 (1H,
ddd, J¼1.0, 8.0, 8.0 Hz), 8.20 (2H, dd, J¼1.5, 8.0 Hz). 13C NMR (CDCl3)
NMR (CDCl3)
d 41.1, 71.1, 73.4, 80.3, 81.4, 83.7, 112.9, 114.6, 118.4,
127.7, 127.7, 128.4, 128.4, 129.5, 138.0, 138.2, 143.4, 155.8. HRMS
(FAB) m/z calcd for C25H26NaO4 [MþNa]þ: 413.1723; found
413.1712.
d
44.0, 55.1, 64.5, 74.6, 79.4, 86.2, 86.4, 113.1, 119.2, 120.8, 123.4,
126.7,127.8,128.1,128.5,129.3,129.5, 130.0,130.1,130.1,133.5,135.9,
136.0, 143.9, 144.8, 151.0, 158.4, 158.4, 165.1. HRMS (FAB) m/z calcd
for C39H36NaO7 [MþNa]þ: 639.2353; found 639.2376.
4.10. 1-Benzoyloxy-3-(2-deoxy-3,5-di-O-benzyl-b-D-ribofur-
anosyl)benzene (14)
4.13. 1-Benzoyloxy-3-{3-O-[2-cyanoethoxy(diisopropylamino)
phosphino]-2-deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-ribofur-
Under a nitrogen atmosphere, BzCl (90
added to a solution of compound 13 (234 mg, 0.599 mmol) and
Et3N (109 L, 0.779 mmol) in anhydrous CH2Cl2 (6 mL) at room
mL, 0.779 mmol) was
anosyl}benzene (2)
m
Under a nitrogen atmosphere, i-Pr2NP(Cl)OCH2CH2CN (30
0.134 mmol) was added to a solution of compound 16 (41 mg,
0.067 mmol) and i-Pr2NEt (58 L, 0.335 mmol) in anhydrous CH2Cl2
mL,
temperature and the mixture was stirred for 30 min. After addition
of water, the mixture was extracted with Et2O. The extracts were
washed with water and brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash silica gel
m
(1 mL) at room temperature and the mixture was stirred for 30 min.
After addition of satd NaHCO3 aq, the mixture was extracted with
Et2O. The extracts were washed with water, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼5:1 to
4:1) to give compound 2 (32 mg, 58%) as a white solid. 1H NMR
column chromatography (n-hexane/AcOEt¼20:1 to 10:1) to give
23
compound 14 (290 mg, 98%) as a colorless oil. [
a
]
þ12.7 (c 1.03,
D
CHCl3). IR nmax (KBr) 2860, 1738, 1451, 1233, 1078 cmꢀ1
.
1H NMR
(CDCl3)
d
1.93 (1H, ddd, J¼6.5, 10.0, 13.0 Hz), 2.40 (1H, ddd, J¼1.0,
5.0, 13.0 Hz), 3.58 (1H, dd, J¼5.0, 10.5 Hz), 3.67 (1H, dd, J¼4.5,
10.5 Hz), 4.16 (1H, m), 4.31 (1H, ddd, J¼2.0, 4.5, 5.0 Hz), 4.56 (2H, s),
4.58 (2H, s), 5.17 (1H, dd, J¼5.0, 10.0 Hz), 7.12 (1H, ddd, J¼1.0, 2.0,
8.0 Hz), 7.22e7.39 (13H, m), 7.50 (2H, dd, J¼8.0, 8.0 Hz), 7.63 (1H,
(CDCl3)
d
1.08 (3H, d, J¼6.8 Hz), 1.15e1.19 (9H, m), 2.02e2.11 (1H,
m), 2.34e2.47 (2H, m), 2.61 (1H, dd, J¼6.3, 6.8 Hz), 3.23e3.34 (2H,
m), 3.53e3.87 (4H, m), 3.74 (3H, s), 3.74 (3H, s), 4.24e4.25 (1H, m),
4.49e4.53 (1H, m), 5.18e5.22 (1H, m), 6.76e6.82 (4H, m), 7.12e7.18
(2H, m), 7.22e7.27 (2H, m), 7.31e7.41 (7H, m), 7.45e7.52 (4H, m),
dd, J¼8.0, 8.0 Hz), 8.20 (2H, d, J¼8.0 Hz). 13C NMR (CDCl3)
d 41.2,
7.59e7.63 (1H, m), 8.12e8.15 (2H, m). 31P NMR (CDCl3)
d 147.8,
71.0, 73.4, 79.9, 81.5, 83.9, 119.2, 120.8, 123.5, 127.5, 127.6, 127.7,
128.3, 128.4, 128.5, 129.3, 129.6, 130.1, 133.5, 138.0, 138.1, 143.8,
151.0, 165.1. HRMS (FAB) m/z calcd for C32H30NaO5 [MþNa]þ:
517.1985; found 517.2000.
148.0. HRMS (FAB) m/z calcd for C48H53N2NaO8P [MþNa]þ:
839.3422; found 839.3444.
4.14. 1-Allyloxy-4-(2-deoxy-3,5-di-O-benzyl-D-ribofuranosyl)
4.11. 1-Benzoyloxy-3-(2-deoxy-b-D-ribofuranosyl)benzene (15)
benzene (17)
A solution of compound 14 (230 mg, 0.466 mmol), 20% Pd(OH)2/
C (150 mg), and cyclohexene (4.7 mL, 46.6 mmol) in EtOH (10 mL)
was stirred at 70 ꢁC for 1.5 h. The mixture was filtered through a pad
of CeliteÒ and the filtrate was concentrated under reduced pres-
sure. The residue was purified by flash silica gel column chroma-
tography (n-hexane/AcOEt¼2:1) to give compound 15 (140 mg,
Under a nitrogen atmosphere, a solution of compound 4 (1.00 g,
3.18 mmol) in anhydrous THF (30 mL) was added to a solution of 4-
allyloxyphenyllithium [prepared from 4-allyloxyphenyl bromide
(2.70 g, 12.7 mmol) and s-BuLi (1.05 M in hexane and cyclohex-
ane,12.1 mL, 12.7 mmol) in anhydrous THF (130 mL) at ꢀ78 ꢁC], and
the mixture was stirred at ꢀ60 ꢁC for 1 h. After addition of satd
NH4Cl aq, the mixture was extracted with CH2Cl2. The extracts were
dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼3:2) to give appropriate compounds (Rf¼ca. 0.5),
which were dissolved in CH2Cl2 (30 mL); TMAD (600 mg,
3.48 mmol) and n-Bu3P (0.87 mL, 3.48 mL) were added at room
temperature. After being stirred at room temperature for 19 h,
water was added and the mixture was extracted with Et2O. The
96%) as a colorless oil. [
a]
24 þ19.1 (c 1.88, CHCl3). IR nmax (KBr) 3372,
D
2929, 1736, 1263 cmꢀ1. 1H NMR (CDCl3)
d
1.95 (1H, ddd, J¼6.5, 10.0,
13.0 Hz), 2.22 (1H, ddd, J¼2.0, 5.0, 13.0 Hz), 2.94 (1H, br s), 3.13 (1H,
br s), 3.68 (2H, m), 3.96 (1H, m), 4.30 (1H, m), 5.16 (1H, dd, J¼5.0,
10.0 Hz), 7.11 (1H, ddd, J¼1.0, 2.0, 8.0 Hz), 7.21 (2H, m), 7.37 (1H, dd,
J¼8.0, 8.0 Hz), 7.49 (2H, dd, J¼8.0, 8.0 Hz), 7.62 (1H, dd, J¼8.0,
8.0 Hz), 8.17 (2H, dd, J¼2.0, 8.0 Hz). 13C NMR (CDCl3)
d 43.6, 63.2,
73.4, 79.4, 87.4, 119.2, 120.9, 123.6, 128.6, 129.3, 129.5, 130.1, 133.7,

Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
6387
extracts were washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼5:1) to
give a ca. 1:1 anomeric mixture 17 (660 mg, 48% from 4) as a col-
7.24e7.38 (10H, m), 7.46 (2H, d, J¼8.0 Hz), 7.51 (2H, dd, J¼8.0,
8.0 Hz), 7.64 (1H, dd, J¼8.0, 8.0 Hz), 8.20 (2H, d, J¼8.0 Hz). 13C NMR
(CDCl3)
d 41.5, 70.8, 71.7, 73.5, 79.8, 80.6, 82.9, 121.5, 127.2, 127.6,
127.6,127.7,128.4,128.5,130.1,133.5,138.0,138.2,140.3,150.1,165.2.
HRMS (FAB) m/z calcd for C32H30NaO5 [MþNa]þ: 517.1985; found
517.1974.
orless oil. 1H NMR (CDCl3)
d
1.92 (0.5H, ddd, J¼5.9, 11.0, 13.2 Hz),
2.05 (0.5H, ddd, J¼6.2, 8.4, 12.8 Hz), 2.31 (0.5H, dd, J¼5.1, 13.2 Hz),
2.59 (0.5H, td, J¼6.6, 12.8 Hz), 3.55e3.69 (2H, m), 4.15e4.17 (0.5H,
m), 4.23e4.36 (1.5H, m), 4.46e4.64 (6H, m), 5.00e5.09 (1H, m),
5.26e5.30 (1H, m), 5.37e5.43 (1H, m), 5.99e6.11 (1H, m),
6.85e6.89 (2H, m), 7.26e7.34 (12H, m). HRMS (FAB) m/z calcd for
C29H31O4 [MþH]þ: 431.2217; found 431.2224.
4.17. 1-Benzoyloxy-4-(2-deoxy-b-D-ribofuranosyl)benzene (21)
A solution of compound 19 (170 mg, 0.343 mmol), 20% Pd(OH)2/
C (100 mg), and cyclohexene (3.5 mL, 34.3 mmol) in EtOH (7 mL)
was stirred at 70 ꢁC for 2 h. The mixture was filtered through a pad
of CeliteÒ and the filtrate was concentrated under reduced pres-
sure. The residue was purified by flash silica gel column chroma-
tography (n-hexane/AcOEt¼2:1) to give compound 21 (89 mg, 82%)
4.15. 4-(2-Deoxy-3,5-di-O-benzyl-D-ribofuranosyl)-1-
hydroxybenzene (18)
Under a nitrogen atmosphere, Pd(PPh3)4 (207 mg, 0.179 mmol)
was added to a solution of compound 17 (385 mg, 0.895 mmol) in
anhydrous THF (10 mL) and the mixture was stirred until solids of
Pd(PPh3)4 dissolved completely. Then, NaBH4 (41 mg, 1.07 mmol)
was added and the mixture was stirred at room temperature for
17 h. After addition of 3% HCl aq, the mixture was filtered through
a pad of CeliteÒ. The filtrate was extracted with Et2O. The extracts
were washed with water and brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (CHCl3 to CHCl3/
MeOH¼60:1) to give a ca. 1:1 anomeric mixture 18 (318 mg, 91%) as
as a white solid. [
a
]
D
22 þ34.8 (c 1.05, CHCl3). IR nmax (KBr) 3390, 2932,
1737, 1508, 1270, 1200, 1079 cmꢀ1. 1H NMR (CDCl3)
d
2.05 (1H, ddd,
J¼6.0, 10.5, 13.0 Hz), 2.14 (1H, br s), 2.19 (1H, br s), 2.26 (1H, ddd,
J¼1.5, 5.5, 13.0 Hz), 3.76 (2H, m), 4.01 (1H, ddd, J¼4.5, 4.5, 4.5 Hz),
4.42 (1H, m), 5.20 (1H, dd, J¼5.5, 10.5 Hz), 7.20 (2H, ddd, J¼1.0, 1.0,
8.0 Hz), 7.41 (2H, ddd, J¼1.0, 1.0, 8.0 Hz), 7.51 (2H, ddd, J¼1.0, 8.0,
8.0 Hz), 7.65 (1H, dddd, J¼1.0, 1.0, 8.0, 8.0 Hz), 8.20 (2H, dd, J¼1.0,
8.0 Hz). 13C NMR (CDCl3)
d 44.0, 63.4, 73.7, 79.6, 87.3, 121.7, 127.2,
128.6, 129.4, 130.2, 133.7, 138.8, 150.4, 165.3. HRMS (FAB) m/z calcd
for C18H18NaO5 [MþNa]þ: 337.1046; found 337.1035.
a colorless oil. 1H NMR (CDCl3)
d
1.91 (0.5H, ddd, J¼5.9, 10.8,
4.18. 1-Benzoyloxy-4-[2-deoxy-5-O-(4,40-dimethoxytrityl)-
-ribofuranosyl]benzene (22)
b-
13.2 Hz), 2.04 (0.5H, ddd, J¼6.4, 8.8, 12.7 Hz), 2.31 (0.5H, ddd, J¼1.0,
4.9, 13.2 Hz), 2.59 (0.5H, td, J¼6.4, 12.7 Hz), 3.56e3.68 (2H, m),
4.15e4.17 (0.5H, m), 4.23e4.30 (1H, m), 4.33 (0.5H, td, J¼4.9,
9.3 Hz), 4.47e4.62 (4H, m), 5.01 (0.5H, dd, J¼6.4, 8.8 Hz), 5.07 (0.5H,
dd, J¼4.9, 10.8 Hz), 5.12 (0.5H, br s), 5.14 (0.5H, br s), 6.71e6.75 (2H,
m), 7.20e7.35 (12H, m). HRMS (FAB) m/z calcd for C25H26NaO4
[MþNa]þ: 413.1723; found 413.1742.
D
Under a nitrogen atmosphere, DMTrCl (106 mg, 0.313 mmol)
was added to a solution of compound 21 (82 mg, 0.260 mmol) in
anhydrous pyridine (3 mL) at room temperature and the mixture
was stirred for 1 h. After addition of water, the mixture was
extracted with Et2O. The extracts were washed with water, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by flash silica gel column chromatography (n-
4.16. 1-Benzoyloxy-4-(2-deoxy-3,5-di-O-benzyl-
anosyl)benzene (19) and 1-benzoyloxy-4-(2-deoxy-3,5-di-O-
benzyl- -ribofuranosyl)benzene (20)
b-D-ribofur-
hexane/AcOEt¼3:1) to give compound 22 (145 mg, 91%) as a white
23
a
-D
solid. [
a
]
þ13.8 (c 1.06, CHCl3). IR nmax (KBr) 3454, 3004, 2933,
D
2837, 1739, 1607, 1508, 1263, 1079 cmꢀ1. 1H NMR (CDCl3)
d 1.87 (1H,
Under a nitrogen atmosphere, BzCl (129
added to a solution of compound 18 (318 mg, 0.923 mmol) and Et3N
(154 L, 1.11 mmol) in anhydrous CH2Cl2 (10 mL) at room temper-
ature and the mixture was stirred for 1.5 h. After addition of water,
the mixture was extracted with Et2O. The extracts were washed
with water and brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash silica gel col-
umn chromatography (n-hexane/AcOEt¼20:1) to give a separable
mixture of 19 and 20 (365 mg, 91%, 19/20¼ca. 1:1) as a colorless oil.
m
L, 1.11 mmol) was
br s), 2.05 (1H, ddd, J¼5.5, 10.0, 13.0 Hz), 2.25 (1H, J¼1.5, 5.5,
13.0 Hz), 3.28 (1H, dd, J¼4.5, 10.0 Hz), 3.36 (1H, dd, J¼5.0, 10.0 Hz),
3.78 (6H, s), 4.07 (1H, m), 4.30 (1H, m), 5.20 (1H, dd, J¼5.5, 10.0 Hz),
6.82 (4H, d, J¼9.0 Hz), 7.15e7.55 (15 H, m), 7.63 (1H, dd, J¼7.5,
m
7.5 Hz), 8.20 (1H, d, J¼7.5 Hz). 13C NMR (CDCl3)
d 43.9, 55.2, 64.4,
74.6, 79.5, 86.2, 86.3, 113.1, 121.5, 126.8, 127.1, 127.8, 128.2, 128.6,
129.5, 130.1, 130.1, 133.6, 136.0, 139.5, 144.8, 150.2, 158.4, 165.2.
HRMS (FAB) m/z calcd for C39H36NaO7 [MþNa]þ: 639.2353; found
639.2359.
Compound 19: [
a
]
.
22 þ14.7 (c 1.06, CHCl3). IR nmax (KBr) 2860, 1739,
D
1263, 1078 cmꢀ1
1H NMR (CDCl3)
d
1.94 (1H, ddd, J¼6.0, 10.5,
4.19. 1-Benzoyloxy-4-{3-O-[2-cyanoethoxy(diisopropylamino)
13.0 Hz), 2.38 (1H, ddd, J¼1.5, 5.5, 13.0 Hz), 3.60 (1H, dd, J¼5.0,
10.0 Hz), 3.67 (1H, dd, J¼5.0, 10.0 Hz), 4.18 (1H, m), 4.28 (1H, ddd,
J¼2.5, 5.0, 5.0 Hz), 4.58 (2H, d, J¼14.0 Hz), 4.60 (2H, d, J¼14.0 Hz),
5.18 (1H, dd, J¼5.5, 10.5 Hz), 7.20 (2H, ddd, J¼1.0, 1.0, 8.0 Hz),
7.25e7.38 (10H, m), 7.42 (2H, ddd, J¼1.0, 1.0, 8.0 Hz), 7.51 (2H, ddd,
J¼1.0, 8.0, 8.0 Hz), 7.63 (1H, ddd, J¼1.0, 1.0, 8.0 Hz), 8.20 (2H, ddd,
phosphino]-2-deoxy-5-O-(4,40-dimethoxytrityl)-
b-D-ribofur-
anosyl}benzene (3)
Under a nitrogen atmosphere, i-Pr2NP(Cl)OCH2CH2CN (36
0.161 mmol) was added to a solution of compound 22 (66 mg,
0.108 mmol) and i-Pr2NEt (96 L, 0.554 mmol) in anhydrous CH2Cl2
mL,
m
J¼1.0, 1.0, 8.0 Hz). 13C NMR (CDCl3)
d
41.3, 71.1, 71.1, 73.5, 80.0, 81.5,
(1 mL) at room temperature and the mixture was stirred for 25 min.
After addition of satd NaHCO3 aq, the mixture was extracted with
Et2O. The extracts were washed with water, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼5:1) to
give compound 3 (84 mg, 95%) as a white solid. 1H NMR (CDCl3)
83.9, 121.5, 127.2, 127.6, 127.7, 128.4, 128.4, 128.5, 129.5, 130.2, 133.6,
138.1, 138.2, 139.4, 150.2, 165.2. HRMS (FAB) m/z calcd for
C32H30NaO5 [MþNa]þ: 517.1985; found 517.2005. Compound 20:
[
a]
24 þ7.4 (c 0.87, CHCl3). IR nmax (KBr) 2864, 1739, 1264, 1078 cmꢀ1
.
D
1H NMR (CDCl3)
d
2.08 (1H, ddd, J¼6.0, 7.5, 13.5 Hz), 2.65 (1H, ddd,
J¼7.0, 7.0, 13.5 Hz), 3.59e3.67 (2H, m), 4.28 (1H, ddd, J¼4.5, 6.5,
6.5 Hz), 4.38 (1H, ddd, J¼4.5, 4.5, 4.5 Hz), 4.49 (1H, d, J¼12.0 Hz),
4.51 (1H, d, J¼12.0 Hz), 4.59 (1H, d, J¼12.0 Hz), 4.61 (1H, d,
J¼12.0 Hz), 5.12 (1H, dd, J¼7.0, 7.5 Hz), 7.18 (2H, d, J¼8.0 Hz),
d
1.09 (3H, d, J¼6.8 Hz), 1.16e1.20 (9H, m), 2.00e2.09 (1H, m),
2.32e2.49 (2H, m), 2.63 (1H, dd, J¼6.3, 6.9 Hz), 3.23e3.36 (2H, m),
3.53e3.89 (4H, m), 3.79 (3H, s), 3.79 (3H, s), 4.24e4.25 (1H, m),
4.51e4.55 (1H, m), 5.17e5.22 (1H, m), 6.80e6.84 (4H, m), 7.17e7.30

6388
Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
(5H, m), 7.35e7.38 (4H, m), 7.47e7.53 (6H, m), 7.62e7.66 (1H, m),
concentrated under reduced pressure, the mixture was extracted
with AcOEt. The extracts were washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼7:1) to give compound 30 (130 mg, 93%) as a col-
8.19e8.21 (2H, m). 31P NMR (CDCl3)
d 147.8, 148.0. HRMS (FAB) m/z
calcd for C48H53N2NaO8P [MþNa]þ: 839.3422; found 839.3443.
4.20. 1-Allyloxy-2-(3,5-di-O-benzyl-2-O,4-C-methylene-b-D-ri-
bofuranosyl)benzene (27)
orless oil. [
a
]
D
21 þ52.1 (c 1.14, CHCl3). IR nmax (KBr) 3063, 3032, 2877,
1737, 1489, 1452, 1259, 1203, 1029 cmꢀ1. 1H NMR (CDCl3)
d
3.86 (2H,
Under a nitrogen atmosphere, a solution of compound 268
(1.95 g, 5.69 mmol) in anhydrous THF (50 mL) was added to a so-
lution of 2-allyloxyphenylmagnesium iodide [prepared from 2-
allyloxyphenyl iodide (5.92 g, 22.8 mmol) and i-PrMgCl$LiCl
(1.3 M in THF, 17.5 mL, 22.8 mmol) in anhydrous THF (180 mL) at
ꢀ40 ꢁC], and the mixture was stirred at ꢀ40 ꢁC for 11 h. After ad-
dition of satd NH4Cl aq, the mixture was extracted with AcOEt. The
extracts were washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼3:1 to
1:1) to give an appropriate compound (Rf¼ca. 0.5, n-hexane/
AcOEt¼1:1), which was dissolved in anhydrous CH2Cl2 (50 mL);
TMAD (1.11 g, 6.47 mmol) and n-Bu3P (1.61 mL, 6.47 mmol) were
added at room temperature and the mixture was stirred for 2 h. The
mixture was concentrated under reduced pressure and the residue
was purified by flash silica gel column chromatography (n-hexane/
AcOEt¼5:1) to give compound 27 (1.48 g, 57% from 26) as a white
s), 4.01, 4.07 (2H, AB, J¼8 Hz), 4.09 (1H, s), 4.34 (1H, s), 4.44, 4.53
(2H, AB, J¼12 Hz), 4.66, 4.67 (2H, AB, J¼12 Hz), 5.29 (1H, s),
7.18e7.39 (13H, m), 7.48e7.54 (2H, m), 7.61e7.67 (2H, m), 8.14e8.20
(2H, m). 13C NMR (CDCl3)
d 66.3, 72.1, 73.4, 73.6, 77.7, 79.5, 80.1, 85.7,
122.1,126.1,127.3,127.4,127.5,127.6,128.2,128.3,128.4, 128.7, 128.9,
133.8, 137.4, 138.0, 147.0, 164.7. Mass (EI) m/z 522 (Mþ, 1.3). Anal.
Calcd for C33H30O6: C, 75.84; H, 5.79; found: C, 75.49; H, 5.88.
4.23. 1-Benzoyloxy-2-(2-O,4-C-methylene-b-D-ribofuranosyl)
benzene (31)
A solution of compound 30 (263 mg, 0.503 mmol), 20% Pd(OH)2/
C (100 mg), and cyclohexene (5.0 mL, 50.3 mmol) in EtOH (5 mL)
was refluxed for 1 h. The mixture was filtered through a pad of
CeliteÒ and the filtrate was concentrated under reduced pressure.
The residue was purified by flash silica gel column chromatography
(n-hexane/AcOEt¼1:2 to 1:5) to give compound 31 (153 mg, 89%)
powder. [
a]
21 þ60.3 (c 1.36, CHCl3). IR nmax (KBr) 3063, 3031, 2876,
as a white solid. [
a
]
D
22 þ4.2 (c 0.94, CH3OH). IR nmax (KBr) 3379, 2947,
D
1596,1490,1454,1239,1102, 1030 cmꢀ1. 1H NMR (CDCl3)
d
3.87, 3.88
1736, 1452, 1264, 1200, 1173, 1026 cmꢀ1
.
1H NMR (CD3OD)
d
3.87,
(2H, AB, J¼11 Hz), 4.02 (1H, s), 4.08, 4.12 (2H, AB, J¼8 Hz), 4.41 (2H,
s), 4.40, 4.50 (2H, AB, J¼12 Hz), 4.67 (2H, s), 5.26 (1H, dd, J¼1,11 Hz),
5.40 (1H, dd, J¼1,17 Hz), 5.35 (1H, s), 5.94e6.08 (1H, m), 6.80 (1H, d,
J¼8 Hz), 6.94 (1H, t, J¼8 Hz), 7.18e7.40 (11H, m), 7.54 (1H, d,
3.98 (2H, AB, J¼8 Hz), 3.90, 3.93 (2H, AB, J¼11 Hz), 4.13 (1H, s), 4.13
(1H, s), 5.11 (1H, s), 7.19e7.23 (1H, m), 7.31e7.38 (2H, m), 7.54e7.60
(2H, m), 7.68e7.73 (2H, m), 8.17e8.21 (2H, m). 13C NMR (CD3OD)
d
59.3, 71.5, 73.6, 80.9, 83.7, 88.3, 123.4, 127.2, 128.3, 129.6, 129.9,
J¼8 Hz). 13C NMR (CDCl3)
d
66.5, 68.3, 71.8, 73.3, 73.5, 78.0, 79.0,
130.2, 131.0, 132.7, 135.1, 148.5, 166.1. Mass (EI) m/z 342 (Mþ, 0.3).
80.5, 85.4, 110.7, 117.0, 120.5, 126.7, 127.3, 127.4, 127.4, 127.5, 127.5,
128.1, 128.2, 128.2, 132.8, 137.5, 138.0, 154.2. Mass (EI) m/z 458 (Mþ,
2.5). Anal. Calcd for C29H30O5: C, 75.96; H, 6.59; found: C, 75.94; H,
6.63.
Anal. Calcd for C19H18O6: C, 66.66; H, 5.30; found: C, 66.34; H, 5.33.
4.24. 1-Benzoyloxy-2-[5-O-(4,40-dimethoxytrityl)-2-O,4-C-
methylene-b-D-ribofuranosyl]benzene (32)
4.21. 2-(3,5-Di-O-benzyl-2-O,4-C-methylene-
anosyl)-1-hydroxybenzene (29)
b
-
D
-ribofur-
Under a nitrogen atmosphere, DMTrCl (85 mg, 0.25 mmol) was
added to a solution of compound 31 (58 mg, 0.17 mmol) in anhy-
drous pyridine (1 mL) at room temperature and the mixture was
stirred for 3 h. After addition of satd NaHCO3 aq, the mixture was
extracted with AcOEt. The extracts were washed with water and
brine, dried over Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by flash silica gel column chroma-
tography (n-hexane/AcOEt¼2:1) to give compound 32 (121 mg,
Under a nitrogen atmosphere, Pd(PPh3)4 (370 mg, 0.32 mmol)
was added to a solution of compound 27 (1.46 g, 3.18 mmol) in
anhydrous THF (30 mL) and the mixture was stirred until solids of
Pd(PPh3)4 dissolved completely. Then, NaBH4 (361 mg, 9.55 mmol)
was added and the mixture was stirred at room temperature for
19 h. After addition of 3% HCl aq, the mixture was filtered through
a pad of CeliteÒ. The filtrate was extracted with AcOEt. The extracts
were washed with water and brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was purified by
94%) as a white solid. [
a
]
D
22 ꢀ13.5 (c 1.00, CHCl3). IR nmax (KBr) 3458,
3007, 2949, 2836, 1737, 1606, 1508, 1451, 1254, 1175, 1068,
1030 cmꢀ1. 1H NMR (acetone-d6)
d
3.46, 3.54 (2H, AB, J¼11 Hz), 3.79
(6H, s), 3.91 (2H, s), 4.16 (1H, s), 4.35 (1H, s), 5.19 (1H, s), 6.89e6.92
(1H, m), 7.22e7.46 (10H, m), 7.57e7.66 (4H, m), 7.74e7.79 (1H, m),
flash silica gel column chromatography (n-hexane/AcOEt¼5:2) to
21
give compound 29 (1.07 g, 80%) as a white powder. [
a]
þ14.0 (c
7.91e7.94 (1H, m), 8.23e8.26 (2H, m). 13C NMR (acetone-d6)
d 55.6,
D
1.19, CHCl3). IR nmax (KBr) 3336, 3031, 2877, 1496, 1456, 1100,
61.0, 71.8, 73.6, 80.8, 82.9, 86.9, 86.9, 113.9, 123.5, 127.0, 127.6, 128.4,
128.6, 129.0, 129.4, 129.8, 130.1,130.8, 131.0,131.0,133.1,134.9, 136.7,
136.9, 146.2, 148.3, 159.5, 165.3. HRMS (FAB) m/z calcd for
C40H36NaO8 [MþNa]þ: 667.2308; found, 667.2304.
1029 cmꢀ1 1H NMR (CDCl3)
.
d
3.72 (2H, s), 3.97, 4.04 (2H, AB,
J¼8 Hz), 4.31 (2H, d, J¼2 Hz), 4.50, 4.59 (2H, AB, J¼11 Hz), 4.66 (2H,
d, J¼2 Hz), 5.42 (1H, s), 6.80e6.91 (3H, m), 7.13e7.37 (11H, m), 8.55
(1H, s). 13C NMR (CDCl3)
d 64.1, 72.3, 72.8, 73.6, 77.8, 82.1, 86.5, 118.0,
119.5,120.5,126.2,127.5,127.7,127.8,128.3,128.4, 128.9, 137.2,137.3,
155.3. HRMS (MALDI-TOF) m/z calcd for C26H26NaO5 [MþNa]þ:
441.1678; found, 441.1676.
4.25. 1-Benzoyloxy-2-{3-O-[2-
cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,40-dime-
thoxytrityl)-2-O,4-C-methylene-b-D-ribofuranosyl}benzene
(23)
4.22. 1-Benzoyloxy-2-(3,5-di-O-benzyl-2-O,4-C-methylene-b-
D
-ribofuranosyl)benzene (30)
Under a nitrogen atmosphere, i-Pr2NP(Cl)OCH2CH2CN (46
0.206 mmol) was added to a solution of compound 32 (33 mg,
0.051 mmol) and i-Pr2NEt (71 L, 0.408 mmol) in anhydrous
mL,
Under a nitrogen atmosphere, BzCl (42
mL, 0.36 mmol) was
m
added to a solution of compound 29 (112 mg, 0.28 mmol) and Et3N
(50 L, 0.36 mmol) in anhydrous CH2Cl2 (2 mL) at room tempera-
ture and the mixture was stirred for 3 h. After addition of water and
CH2Cl2 (1 mL) at room temperature and the mixture was stirred
for 1 h. After addition of satd NaHCO3 aq, the mixture was
extracted with AcOEt. The extracts were washed with satd
m

Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
6389
NaHCO3 aq, water, and brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash silica
gel column chromatography (n-hexane/AcOEt¼4:1) to give com-
mixture was concentrated under reduced pressure. The residue
was purified by flash silica gel column chromatography (n-hexane/
AcOEt¼2:1) to give compound 24 (75 mg, 81%) as a white solid. 1H
pound 23 (40 mg, 92%) as a white solid. 1H NMR (CDCl3)
d
0.87
NMR (CDCl3)
d
0.82 (3H, d, J¼6.6 Hz), 0.90 (3H, d, J¼6.6 Hz),
(3H, d, J¼6.4 Hz), 0.94 (3H, d, J¼6.9 Hz), 1.07e1.10 (6H, m),
2.29e2.32 (1H, m), 2.43e2.47 (1H, m), 3.39e3.56 (5H, m),
3.65e3.71 (1H, m), 3.81 (3H, s), 3.82 (3H, s), 3.86e3.90 (1H, m),
4.01e4.07 (1H, m), 4.31e4.45 (2H, m), 5.33 (0.5H, s), 5.36 (0.5H, s),
6.84e6.88 (4H, m), 7.22e7.46 (10H, m), 7.52e7.58 (4H, m),
7.65e7.69 (1H, m), 7.88e7.96 (1H, m), 8.19e8.24 (2H, m). 31P NMR
1.04e1.08 (6H, m), 2.26e2.31 (1H, m), 2.37e2.42 (1H, m),
3.36e3.55 (5H, m), 3.64e3.71 (1H, m), 3.80 (3H, s), 3.80 (3H, s),
3.88e3.94 (1H, m), 4.05e4.11 (1H, m), 4.21 (0.5H, d, J¼7.4 Hz), 4.34
(0.5H, d, J¼9.9 Hz), 4.42 (0.5H, s), 4.50 (0.5H, s), 4.56e4.59 (2H, m),
5.24e5.31 (1H, m), 5.37e5.40 (1.5H, m), 5.43e5.47 (0.5H, m),
5.97e6.12 (1H, m), 6.81e6.99 (6H, m), 7.21e7.34 (4H, m), 7.41e7.46
(4H, m), 7.54e7.57 (2H, m), 7.71e7.73 (0.5H, m), 7.77e7.79 (0.5H,
(acetone-d6)
d 146.9, 147.7. HRMS (FAB) m/z calcd for C49H54N2O9P
[MþH]þ: 845.3567; found, 845.3565.
m). 31P NMR (acetone-d6)
d 146.9, 147.7. HRMS (FAB) m/z calcd for
C45H54N2O8P [MþH]þ: 781.3618; found, 781.3638.
4.26. 2-[5-O-(4,40-Dimethoxytrityl)-2-O,4-C-methylene-
b-D-ri-
bofuranosyl]-1-hydroxybenzene (33)
4.29. 1-Allyloxy-3-(3,5-di-O-benzyl-2-O,4-C-methylene-b-D-ri-
bofuranosyl)benzene (28)
Under a nitrogen atmosphere, K2CO3 (103 mg, 0.75 mmol) was
added to a solution of compound 32 (120 mg, 0.19 mmol) in MeOH
(5 mL) at room temperature and the mixture was stirred for 5 min.
After removal of MeOH under reduced pressure and the mixture
was extracted with AcOEt. The extracts were washed with water
and brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by flash silica gel column
Under a nitrogen atmosphere, a solution of compound 26
(1.38 g, 4.03 mmol) in anhydrous THF (20 mL) was added to a so-
lution of 3-allyloxyphenylmagnesium iodide [prepared from 2-
allyloxyphenyl iodide (4.19 g, 16.1 mmol) and i-PrMgCl$LiCl
(1.3 M in THF, 12.4 mL, 16.1 mmol) in anhydrous THF (50 mL) at
room temperature], and the mixture was stirred for 15 h. After
addition of satd NH4Cl aq, the mixture was extracted with AcOEt.
The extracts were washed with water and brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified
by flash silica gel column chromatography (n-hexane/AcOEt¼3:1 to
1:1) to give an appropriate compound (Rf¼ca. 0.5, n-hexane/
AcOEt¼1:1), which was dissolved in anhydrous CH2Cl2 (30 mL);
TMAD (616 mg, 3.58 mmol) and n-Bu3P (0.89 mL, 3.58 mmol) were
added at room temperature and the mixture was stirred for 2 h.
After addition of water, the mixture was extracted with Et2O. The
extracts were washed with water, brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼5:1) to
chromatography (n-hexane/AcOEt¼3:1) to give compound 33
22
(96 mg, 96%) as a white solid. [
a
]
ꢀ2.3 (c 0.57, CHCl3). IR nmax
D
(KBr) 3338, 3006, 2949, 1606, 1507, 1457, 1296, 1250, 1030 cmꢀ1. 1H
NMR (acetone-d6)
d
2.76 (1H, br s), 3.32, 3.39 (2H, AB, J¼10 Hz),
3.66 (6H, s), 3.82 (2H, s), 4.09 (1H, s), 5.10 (1H, s), 6.70e6.80 (6H,
m), 6.97e7.03 (1H, m), 7.08e7.23 (3H, m), 7.28e7.33 (4H, m),
7.44e7.54 (3H, m). 13C NMR (acetone-d6)
d 55.6, 61.0, 71.8, 73.6,
80.8, 82.9, 86.9, 86.9, 113.9, 123.5, 127.0, 127.6, 128.4, 128.6, 129.0,
129.4, 129.8, 130.1, 130.8, 131.0, 131.0, 133.1, 134.9, 136.7, 136.9,
146.2, 148.3, 159.5, 165.3. HRMS (FAB) m/z calcd for C33H32NaO7
[MþNa]þ: 563.2046; found, 563.2050.
4.27. 1-Allyloxy-2-[5-O-(4,40-dimethoxytrityl)-2-O,4-C-methy-
give compound 28 (1.30 g, 70% from 26) as a colorless oil. [
a
]
26 þ7.1
D
lene-
b-D
-ribofuranosyl]benzene (34)
(c 1.45, CHCl3). IR nmax (KBr) 3030, 2878, 1600, 1449, 1281, 1105,
1031 cmꢀ1. 1H NMR (CDCl3)
d 3.86 (2H, s), 4.06 (1H, s), 4.07, 4.10 (2H,
Under a nitrogen atmosphere, K2CO3 (27 mg, 0.20 mmol) and
allyl bromide (17 L, 0.20 mmol) were added to a solution of
AB, J¼8 Hz), 4.22 (1H, s), 4.43, 4.49 (2H, AB, J¼12 Hz), 4.62 (2H, s),
4.65 (2H, s), 5.15 (1H, s), 5.25 (1H, d, J¼11 Hz), 5.36 (1H, d, J¼17 Hz),
5.92e6.06 (1H, m), 6.79 (1H, d, J¼8 Hz), 6.85 (1H, d, J¼8 Hz), 6.92
m
compound 33 (96 mg, 0.18 mmol) in anhydrous acetone (16 mL) at
room temperature and the mixture was stirred for 14 h. After ad-
dition of water, acetone was removed under reduced pressure. The
mixture was extracted with AcOEt. The extracts were washed with
water and brine, dried over Na2SO4, and concentrated under re-
duced pressure. The residue was purified by flash silica gel column
chromatography (n-hexane/AcOEt¼3:1) to give compound 34
(1H, s), 7.19e7.35 (11H, m). 13C NMR (CDCl3)
d 66.5, 68.6, 72.0, 73.5,
73.6, 77.5, 80.9, 83.7, 85.7, 111.5, 113.7, 117.5, 127.4, 127.5, 127.5, 127.6,
128.2, 128.2, 129.3, 133.1, 137.3, 137.9, 140.7, 158.5. Mass (EI) m/z 458
(Mþ, 12.5). Anal. Calcd for C29H30O5: C, 75.96; H, 6.59; found: C,
75.66; H, 6.62.
(90 mg, 87%) as a white solid. [
a
]
22 þ5.1 (c 0.93, CHCl3). IR nmax (KBr)
4.30. 3-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-D-ribofur-
anosyl)-1-hydroxybenzene (35)
D
3431, 3007, 2943, 2836, 1605, 1508, 1453, 1298, 1249, 1178, 1078,
1031 cmꢀ1. 1H NMR (acetone-d6)
d
3.47, 3.53 (2H, AB, J¼10 Hz), 3.77
(6H, s), 3.96 (2H, s), 4.21 (1H, s), 4.64 (2H, s), 5.25 (1H, s), 5.26 (1H, d,
J¼10 Hz), 5.47 (1H, d, J¼17 Hz), 6.05e6.15 (1H, m), 6.88e7.02 (6H,
m), 7.20e7.35 (4H, m), 7.44e7.47 (4H, m), 7.59e7.62 (2H, m),
Under a nitrogen atmosphere, Pd(PPh3)4 (162 mg, 0.14 mmol)
was added to a solution of compound 28 (640 mg, 1.40 mmol) in
anhydrous THF (150 mL) and the mixture was stirred until solids of
Pd(PPh3)4 dissolved completely. Then, NaBH4 (159 mg, 4.19 mmol)
was added and the mixture was stirred at room temperature for
17 h. After addition of 3% HCl aq, the mixture was filtered through
a pad of CeliteÒ. The filtrate was extracted with AcOEt. The extracts
were washed with water and brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼5:2) to
7.75e7.78 (1H, m). 13C NMR (acetone-d6)
d 55.6, 61.3, 69.1, 72.1, 73.5,
79.3, 81.1, 82.5, 86.7, 86.9, 112.2,113.9,117.1,121.4,127.6,127.9,128.6,
129.1, 129.3, 129.3, 131.0, 131.1, 134.5, 136.8, 137.0. HRMS (FAB) m/z
calcd for C36H36NaO7 [MþNa]þ: 603.2359; found 603.2357.
4.28. 1-Allyloxy-2-{3-O-[2-cyanoethoxy(diisopropylamino)
phosphino]-5-O-(4,40-dimethoxytrityl)-2-O,4-C-methylene-
b-
D
-ribofuranosyl}benzene (24)
give compound 35 (594 mg, quant.) as a colorless oil. [
a
]
25 þ11.7 (c
D
1.36, CHCl3). IR nmax (KBr): 3311, 3030, 2879, 1595, 1453, 1362, 1206,
Under a nitrogen atmosphere, (i-Pr2N)2POCH2CH2CN (64
mL,
1105,1032 cmꢀ1. 1H NMR (CDCl3)
d
3.85, 3.88 (2H, AB, J¼11 Hz), 4.08
0.20 mmol) was added to a solution of compound 34 (69 mg,
0.12 mmol) and DIHT (24 mg, 0.12 mmol) in anhydrous MeCN/THF
(2 mL, 3:1) at room temperature. After being stirred for 4 h, the
(1H, s), 4.07, 4.11 (2H, AB, J¼8 Hz), 4.22 (1H, s), 4.38, 4.49 (2H, AB,
J¼12 Hz), 4.66 (2H, s), 5.12 (1H, s), 5.59 (1H, s), 6.70 (1H, d, J¼8 Hz),
6.80 (1H, d, J¼8 Hz), 6.78 (1H, s), 7.13e7.34 (11H, m). 13C NMR

6390
Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
(CDCl3)
d
66.3, 72.1, 73.5, 73.7, 77.4, 80.9, 83.6, 85.8, 112.2, 114.4,
135.6, 141.0, 144.5, 151.0, 158.3, 165.0. HRMS (EI) m/z calcd for
C
117.2, 127.5, 127.6, 127.6, 127.7, 128.2, 128.3, 129.6, 137.3, 137.9, 140.8,
155.9. HRMS (MALDI-TOF) m/z calcd for C26H26NaO5 [MþNa]þ:
441.1678; found, 441.1677.
40H36O8 [M]þ: 644.2410; found: 644.2410.
4.34. 1-Benzoyloxy-3-{3-O-[2-
cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,40-dime-
thoxytrityl)-2-O,4-C-methylene-b-D-ribofuranosyl}benzene
(25)
4.31. 1-Benzoyloxy-3-(3,5-di-O-benzyl-2-O,4-C-methylene-b-
D-ribofuranosyl)benzene (36)
Under a nitrogen atmosphere, i-Pr2NP(Cl)OCH2CH2CN (45
0.20 mmol) was added to a solution of compound 38 (32 mg,
0.05 mmol) and i-Pr2NEt (87 L, 0.50 mmol) in anhydrous CH2Cl2
mL,
Under a nitrogen atmosphere, BzCl (200
mL, 1.70 mmol) was
added to a solution of compound 35 (594 mg, 1.42 mmol) and Et3N
(240 L, 1.70 mmol) in anhydrous CH2Cl2 (15 mL) at room tem-
m
m
(1 mL) at 0 ꢁC and the mixture was stirred at room temperature for
1 h. After addition of satd NaHCO3 aq, the mixture was extracted
with AcOEt. The extracts were washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. The residue
was purified by flash silica gel column chromatography (n-hexane/
AcOEt¼3:1) to give compound 25 (30 mg, 71%) as a white solid. 1H
perature and the mixture was stirred for 17 h. After addition of satd
NaHCO3 aq, the mixture was extracted with AcOEt. The extracts
were washed with water and brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (n-hexane/AcOEt¼3:1) to
25
give compound 36 (730 mg, 95%) as a colorless oil. [
a]
þ28.3 (c
D
NMR (400 MHz, CDCl3)
d
0.88 (3H, d, J¼6.9 Hz), 0.96 (3H, d, J¼6.8 Hz),
1.49, CHCl3). IR nmax (KBr) 3423, 3030, 2867, 1599, 1490, 1450, 1260,
1097 cmꢀ1. 1H NMR (CDCl3)
d
3.85, 3.87 (2H, AB, J¼11 Hz), 4.07 (1H,
1.06e1.09 (6H, m), 2.30e2.33 (1H, m), 2.42e2.45 (1H, m), 3.40e3.49
(5H, m), 3.66e3.69 (1H, m), 3.75e3.76 (6H, m), 3.92e3.95 (1H, m),
4.10e4.15 (1H, m), 4.21 (0.5H, d, J¼7.4 Hz), 4.28 (0.5H, s), 4.35 (0.5H,
d, J¼9.3 Hz), 4.39 (0.5H, s), 5.26 (1H, s), 6.80e6.83 (4H, m), 7.14e7.17
(2H, m), 7.24e7.27 (3H, m), 7.39e7.53 (10H, m), 7.60e7.64 (1H, m),
s), 4.09, 4.12 (2H, AB, J¼8 Hz), 4.25 (1H, s), 4.46, 4.52 (2H, AB,
J¼11 Hz), 4.64 (2H, s), 5.20 (1H, s), 7.11e7.42 (14H, m), 7.48e7.54
(2H, m), 7.62e7.67 (1H, m), 8.19 (2H, d, J¼7 Hz). 13C NMR (CDCl3)
d
68.7, 69.5, 71.9, 73.7, 74.5, 75.2, 81.7, 94.8, 89.1, 113.2, 114.3, 117.5,
8.12e8.16 (2H, m). 31P NMR (CDCl3)
d 148.2, 148.4. HRMS (FAB) m/z
119.5,127.6,127.8, 128.2, 128.4, 129.3, 133.1,137.3, 137.4,142.0, 158.5.
HRMS (MALDI-TOF) m/z calcd for C33H30NaO6 [MþNa]þ: 545.1940;
found, 545.1936.
calcd for C49H54N2O9P [MþH]þ: 845.3567; found, 845.3573.
4.35. Synthesis of TFOs
4.32. 1-Benzoyloxy-3-(2-O,4-C-methylene-b-D-ribofuranosyl)
benzene (37)
The 0.2 mmol scale synthesis of TFOs 39e43 was performed on
an automated DNA synthesizer (Applied Biosystems ExpediteÔ
8909 or Genedesign nS-8) using a standard phosphoramidite pro-
tocol. By treatment with 28% NH3 aq at room temperature for 4 h,
TFOs synthesized on DMTr-ON mode were cleaved from the CPG
resin and all protecting groups on TFOs were removed. The crude
TFOs 39e41 and 43 obtained were purified with NENSORBÔ PREP
or Sep-PakÒ Plus C18 cartridges followed by reversed-phase HPLC
(ChemcoPakÒ CHEMCOSORB 300-5C18, 4.6 mmꢂ250 mm or Wa-
A solution of compound 36 (730 mg, 1.40 mmol), 20% Pd(OH)2/C
(200 mg), and cyclohexene (14 mL, 140 mmol) in EtOH (14 mL) was
refluxed for 1 h. The mixture was filtered through a pad of CeliteÒ
and the filtrate was concentrated under reduced pressure. The
residue was purified by flash silica gel column chromatography (n-
hexane/AcOEt¼1:2 to 1:5) to give compound 37 (452 mg, 95%) as
26
ters XBridgeÒ MS C18 2.5
m
m, 10 mmꢂ50 mm). For TFO 42, before
reversed-phase HPLC purification, treatment with NaBH4 (2.2 mg,
58.2 mol) and Pd(PPh3)4 (2.1 mg,1.9 mol) in H2O (200 L) at room
a white solid. [
a
]
ꢀ17.4 (c 0.97, CHCl3). IR nmax (KBr) 3381, 2947,
D
1733,1591, 1487,1447,1264,1146, 1030 cmꢀ1. 1H NMR (CDCl3)
d 2.69
(1H, br s), 3.05 (1H, br s), 3.95 (2H, s), 3.97, 4.04 (2H, AB, J¼8 Hz),
4.16 (1H, s), 4.23 (1H, s), 5.13 (1H, s), 7.08e7.18 (3H, m), 7.26 (1H, s),
7.35e7.41 (1H, m), 7.47e7.53 (2H, m), 7.61e7.66 (1H, m), 8.17 (2H, d,
m
m
m
temperature for 3 h was performed to remove an allyl group. The
composition of the TFOs was confirmed by MALDI-TOF-MS analysis.
MALDI-TOF-MS data ([MꢀH]ꢀ) for TFOs 39e43; 39, found 4464.44
(calcd 4463.98); 40, found 4463.89 (calcd 4463.98); 41, found
4464.43 (calcd 4463.98); 42, found 4491.60 (calcd 4492.00); 43,
found 4492.34 (calcd 4492.00).
J¼7 Hz). 13C NMR (CDCl3)
d 59.0, 71.0, 72.3, 82.9, 83.6, 86.7, 118.6,
120.9, 122.6, 128.5, 129.1, 129.6, 130.1, 133.7, 140.5, 151.0, 165.2. Mass
(FAB) m/z 343 (MþHþ). Anal. Calcd for C19H18O6: C, 66.66; H, 5.30;
found: C, 66.55; H, 5.33.
4.33. 1-Benzoyloxy-3-[5-O-(4,40-dimethoxytrityl)-2-O,4-C-
4.36. UV melting experiments
methylene-b-D-ribofuranosyl]benzene (38)
UV melting experiments were performed on SHIMADZU UV-
1650 and SHIMADZU UV-1800 spectrophotometers equipped
with Tm analysis accessory. The TFOs 39e43 and hairpin dsDNA
targets were dissolved in 10 mM sodium cacodylate buffer (pH 6.8)
containing 140 mM KCl and 50 mM MgCl2 to give a final concen-
Under a nitrogen atmosphere, DMTrCl (175 mg, 0.515 mmol) was
added to a solution of compound 37 (147 mg, 0.429 mmol) in an-
hydrous pyridine (4 mL) at room temperature and the mixture was
stirred for 12 h. After addition of satd aq NaHCO3 solution, the
mixture was extracted with AcOEt. The extracts were washed with
water, dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by flash silica gel column chromatography
(n-hexane/AcOEt¼3:1 to 1:1) to give compound 38 (252 mg, 91%) as
tration of each strand of 1.89 mM. The samples were annealed in
boiling water followed by slow cooling to 5 ꢁC. The melting profiles
were recorded at 260 nm from 5 ꢁC to 90 ꢁC at a scan rate of 0.5 ꢁC/
min. The two-point average method was used to obtain the Tm
values and the final values were determined by averaging three
independent measurements, which were accurate to within 1 ꢁC.
a white solid. [
a
]
22 ꢀ12.8 (c 0.85, CHCl3). IR nmax (KBr): 3478, 3006,
D
2950, 1736, 1606, 1508, 1447, 1254, 1178, 1072, 1032 cmꢀ1. 1H NMR
(acetone-d6)
d
2.85 (1H, s), 3.41, 3.55 (2H, AB, J¼11 Hz), 3.74 (3H, s),
3.75 (3H, s), 3.98, 4.00 (2H, AB, J¼8 Hz), 4.18 (1H, s), 4.27, 4.41 (2H, AB,
J¼5 Hz), 5.15 (1H, s), 6.84e6.89 (4H, m), 7.11e7.31 (4H, m), 7.38e7.61
(11H, m), 7.68e7.75 (1H, m), 8.13e8.16 (2H, m). 13C NMR (CDCl3)
Acknowledgements
This work was supported in part by the Program to Disseminate
Tenure Tracking System of the Ministry of Education, Culture,
Sports, Science and Technology, Japan (MEXT), a Grant-in-Aid for
d
55.2, 60.2, 71.7, 72.9, 82,8, 83.3, 85.9, 86.3, 113.1, 118.6, 120.8, 122.7,
126.7,127.8,128.0,128.4,129.3,129.4,129.9,130.0,130.1,133.5,135.6,

Y. Hari et al. / Tetrahedron 69 (2013) 6381e6391
6391
Org. Biomol. Chem. 2011, 9, 326e336; (i) Hari, Y.; Obika, S.; Imanishi, T. Eur. J.
Org. Chem. 2012, 2875e2887.
3. (a) Yoon, K.; Hobbs, C. A.; Koch, J.; Sardaro, M.; Kutny, R.; Weis, A. L. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 3840e3844; (b) Radhakrishnan, I.; Patel, D. J. J. Mol.
Biol. 1994, 241, 600e619.
Scientific Research on Innovative Areas (No. 22136006) from MEXT,
and the Mochida Memorial Foundation for Medical and Pharma-
ceutical Research.
4. Obika, S.; Hari, Y.; Sekiguchi, M.; Imanishi, T. Angew. Chem., Int. Ed. 2001, 40,
2079e2081.
5. A part of the results was reported at the International Symposium on Nucleic
Acids Chemistry: (a) Obika, S.; Hari, Y.; Inohara, H.; Imanishi, T. Nucleic Acids
Res. Suppl. 2001, 171e172; (b) Inohara, H.; Obika, S.; Imanishi, T. Nucleic Acids
Symp. Ser. 2004, 63e64.
6. Wierenga, W.; Skulnick, H. I. Carbohydr. Res. 1981, 90, 41e52.
7. Tsunoda, T.; Otsuka, J.; Yamamiya, Y.; Ito, S. Chem. Lett. 1994, 539e542.
8. (a) Obika, S.; Hari, Y.; Morio, K.; Imanishi, T. Tetrahedron Lett. 2000, 41, 215e219;
(b) Hari, Y.; Obika, S.; Sakaki, M.; Morio, K.; Yamagata, Y.; Imanishi, T. Tetra-
hedron 2002, 58, 3051e3063.
9. A significant increase in the Tm value of the triplex with dsDNA (YZ¼CG) was
not observed by replacement of 2H with 2HB. This may imply that the 2-hy-
droxyphenyl nucleobase in 2HB worked only as a hydrogen-donor by the 20,40-
BNA modification though the reason is unclear.
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