Synthesis and properties of trans-3′,4′-bridged nucleic acids having typical S-type sugar conformation

Article abstract of DOI:10.1021/jo051187l

The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest. The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing a 4,7-dioxabicyclo[4.3.0] nonane skeleton and a methoxy group at the C2' position. Conformational analysis showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphoramidite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleotides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary strands was evaluated by melting temperature (T_m) measurements. As a result, the ODN derivatives hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.

Full text of DOI:10.1021/jo051187l

Synthesis and Properties of Trans-3′,4′-Bridged Nucleic Acids
Having Typical S-type Sugar Conformation
Mitsuaki Sekiguchi,† Satoshi Obika,†,‡ Yasuki Harada,† Tomohisa Osaki,†
Roongjang Somjing,† Yasunori Mitsuoka,† Nao Shibata,† Miyuki Masaki,† and
Takeshi Imanishi*,†
Graduate School of Pharmaceutical Sciences, Osaka UniVersity, 1-6 Yamadaoka, Suita, Osaka 565-0871,
Japan, and PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
imanishi@phs.osaka-u.ac.jp
ReceiVed June 13, 2005
The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest.
The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing
a 4,7-dioxabicyclo[4.3.0]nonane skeleton and a methoxy group at the C2′ position. Conformational analysis
showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was
difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphora-
midite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleo-
tides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary
strands was evaluated by melting temperature (Tm) measurements. As a result, the ODN derivatives
hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the
duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.
Introduction
Development of oligonucleotide (ON) analogues that tightly
bind with single-stranded (ss) and/or double-stranded (ds)
nucleic acid has attracted considerable attention in the fields of
medical and molecular biology. Generally, the sugar moiety of
nucleosides or ONs has relatively high flexibility and is in
equilibrium between several metastable conformations (e.g., N-
and S-types). However, the conformational flexibility is re-
stricted in the hybridization process. The sugar moiety in dsRNA
predominantly adopts N-type conformation, while that in
dsDNA is rather flexible but mainly has S-type conformation
(Figure 1a).1 Conformational restriction of the ONs in a suitable
form to hybridize with the target strands is one promising
FIGURE 1. (a) Typical sugar puckering of nucleosides, N- and S-type
conformation. (b) Structure of 2′,4′-BNA/LNA bearing a locked N-type
sugar conformation.
approach to enhance the binding affinity and selectivity of the
ONs by means of an entropic gain in the hybridization process.2,3
* To whom correspondence should be addressed. Phone: +81 6 6879 8200.
Fax: +81 6 6879 8204.
† Osaka University.
‡ PRESTO.
(1) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag:
New York, 1984.
(2) Herdewijn, P. Liebigs Ann. 1996, 1337-1348.
(3) Kool, E. T. Chem. ReV. 1997, 97, 1473-1488.
10.1021/jo051187l CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/24/2006
1306
J. Org. Chem. 2006, 71, 1306-1316

Synthesis and Properties of Trans-3′,4′-Bridged Nucleic Acids
On the basis of this concept, many kinds of ON derivatives
with a conformationally restricted sugar moiety have been
prepared.2-10 The 2′-O-substitution of RNA is one simple but
effective ON modification, where the sugar conformation is
induced to be in N-type by a gauche effect between O2′ and
O4′ atoms.6 Oligonucleotide N3′fP5′ phosphoramidates, in
which the 3′-oxygen atom is replaced by the less electron-
withdrawing nitrogen atom, also have an N-type sugar confor-
mation, due to the reduced gauche effect between N3′ and O4′
atoms.7 Both ON analogues increase the stability of duplexes
formed with the complementary strand, especially with the
complementary RNA. Recently, our group11,12 and Wengel’s
group13 independently achieved the synthesis of 2′,4′-BNA/
LNA, of which the sugar conformation is exactly locked in
N-type by an additional methylene bridge between O2′ and C4′
atoms (Figure 1b).14 The ON derivatives containing 2′,4′-BNA/
LNA monomers showed unprecedentedly high binding affinity
with the RNA complement5,8,10-13 and have been applied in an
antisense strategy.15
On the other hand, studies on the ON analogues with a DNA-
selective binding affinity are of great interest. Wengel and co-
workers demonstrated the DNA-selective hybridization of the
pyrene-conjugated ON analogue, where the pyrene unit was
supposed to recognize the minor-groove structure of dsDNA.16
Very recently, bipyridyl-functionalized 2′-amino-LNA was
employed for high-affinity DNA targeting.17 On the other hand,
restriction of the sugar conformation of ONs within an S-type
by an additional bridged structure would be another strategy
for DNA-selective hybridization. Several nucleoside analogues
having an S-type sugar moiety with a bridged structure have
been synthesized and incorporated into ONs (Figure 2a).18-23
These ON analogues with S-type sugar conformation, however,
showed only moderate increase or considerable decrease in the
FIGURE 2. (a) Structure of selected bicyclic and tricyclic nucleoside
analogues with S-type sugar conformation. (b) Structure of trans-3′,4′-
BNAs 1 and 2. B ) nucleobases, T ) thymin-1-yl.
duplex stability, probably due to inappropriate restriction of the
sugar conformation or steric repulsion between the additional
bridged structure and neighboring nucleotide residues. Recently,
our group24 and Nielsen’s group25 independently reported the
synthesis of a novel S-type nucleoside analogue 1 (Figure 2b).
Preliminary molecular modeling showed that the additional
bridged structure between C3′ and C4′ atoms of 1 was located
outside the B-type DNA duplex structure; therefore, it is
expected that the bridged structure has no adverse contact with
the neighboring residues.
In this paper, we describe in detail the synthesis of nucleoside
analogues 1 and 2 (trans-3′,4′-BNA-thymine monomers) which
have a conformationally restricted S-type sugar moiety with a
4,7-dioxabicyclo[4.3.0]nonane skeleton (Figure 2b). The hy-
bridization properties of the ONs containing 2 are also discussed.
(4) Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167-179.
(5) Imanishi, T.; Obika, S. J. Synth. Org. Chem., Jpn. 1999, 57, 969-
980.
(6) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117-130.
(7) Gryaznov, S. M. Biochim. Biophys. Acta 1999, 1489, 131-140.
(8) Meldgaard, M.; Wengel, J. J. Chem. Soc., Perkin Trans. 1 2000,
3539-3554.
Results and Discussion
(9) Leumann, C. J. Bioorg. Med. Chem. 2002, 10, 841-854.
(10) Imanishi, T.; Obika, S. Chem. Commun. 2002, 1653-1659.
(11) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735-8738.
For the synthesis of trans-3′,4′-BNA monomers 1 and 2, we
chose the known D-allofuranose derivative 326 as the starting
material. As shown in Scheme 1, the acetonide group of 3 was
removed by treatment with 80% acetic acid at 40 °C to give
the diol 4 in 90% yield. Oxidative cleavage of the diol 4 with
sodium periodate gave the corresponding aldehyde, which was
then employed for an aldol-Canizzaro reaction to afford 5 in
(12) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401-5404.
(13) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
Commun. 1998, 455-456. Koshkin, A. A.; Singh, S. K.; Nielsen, P.;
Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J.
Tetrahedron 1998, 54, 3607-3630.
(14) The similar bridged nucleic acid analogues with N-type sugar
conformation were reported by other groups. See: Wang, G.; Girardet, J.-
L.; Gunic, E. Tetrahedron 1999, 55, 7707-7724. Wang, G.; Gunic, E.;
Girardet, J.-L.; Stoisavljevic, V. Bioorg. Med. Chem. Lett. 1999, 9, 1147-
1150. Morita, K.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone, J.;
Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. Lett. 2002,
12, 73-76. Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi,
S.; Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem.
2003, 11, 2211-2226.
(15) Vester, B.; Wengel, J. Biochemistry 2004, 43, 13233-13241. Jepsen,
J. S.; Sørensen, M. D.; Wengel, J. Oligonucleotides 2004, 14, 130-146.
(16) Bryld, T.; Højland, T.; Wengel, J. Chem. Commun. 2004, 1064-
1065.
(19) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1997, 119, 11548-
11549. Steffens, R.; Leumann, C. J.; J. Am. Chem. Soc. 1999, 121, 3249-
3255. Renneberg, D.; Leumann, C. J.; J. Am. Chem. Soc. 2002, 124, 5993-
6002.
(20) Altmann, K.-H.; Imwinkelried, R.; Kesselring, R.; Rihs, G. Tetra-
hedron Lett. 1994, 35, 7625-7628. Maier, M. A.; Choi, Y.; Gaus, H.; Barchi
Jr, J. J.; Marquez, V. E.; Manoharan, M. Nucleic Acids Res. 2004, 32, 3642-
3650.
(21) Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J. J. Chem.
Soc., Perkin Trans. 1 1997, 3423-3433.
(22) Ravn, J.; Thorup, N.; Nielsen, P. J. Chem. Soc., Perkin Trans. 1
2001, 1855-1861.
(17) Babu, B. R.; Hrdlicka, P. J.; McKenzie, C. J.; Wengel, J. Chem.
Commun. 2005, 1705-1707.
(23) Kværnø, L.; Wightman, R. H.; Wengel, J. J. Org. Chem. 2001, 66,
5106-5112.
(18) Tarko¨y, M.; Bolli, M.; Schweizer, B.; Leumann, C. HelV. Chim.
Acta 1993, 76, 481-510. Bolli, M.; Trafelet, H. U.; Leumann, C. Nucleic
Acids Res. 1996, 24, 4660-4667. Bolli, M.; Leumann, C. Angew. Chem.,
Int. Ed. Engl., 1995, 34, 694-696. Bolli, M.; Litten, J. C.; Schu¨tz, R.;
Leumann, C. J. Chem. Biol. 1996, 3, 197-206.
(24) Obika, S.; Sekiguchi, M.; Osaki, T.; Shibata, N.; Masaki, M.; Hari,
Y,; Imanishi, T. Tetrahedron Lett. 2002, 43, 4365-4368.
(25) Thomasen, H.; Meldgaard, M.; Freitag, M.; Petersen, M.; Wengel,
J.; Nielsen, P. Chem. Commun. 2002, 1888-1889.
(26) Prashad, M.; Fraser-Reid, B. J. Org. Chem. 1985, 50, 1564-1566.
J. Org. Chem, Vol. 71, No. 4, 2006 1307

Sekiguchi et al.
SCHEME 1 a
SCHEME 2 a
a Reagents, conditions, and yields: (a) 80% AcOH aq, 40 °C, 3 h, 90%;
(b) NaIO4, THF-H2O (1:8), 0 °C, 1 h; (c) 37% HCHO aq, 1 M NaOH aq,
THF-H2O (1:1), rt, 18 h, 82% over two steps; (d) NaH, BnBr, DMF, 0
°C, 1 h, 54%; (e) p-TsCl, Et3N, DMAP, CH2Cl2, rt, 20 h, 92%; (f) see
Table 1.
a Reagents, conditions, and yields: (a) 80% AcOH aq, 40 °C, 3 h; (b)
NaIO4, THF-H2O (1:1), 0 °C, 45 min; (c) 37% HCHO aq, 1 M NaOH aq,
THF-H2O (1:1), rt, 18 h, 65% over three steps; (d) NaH, BnBr, DMF, 0
°C, 1.5 h, 65%; (e) p-TsCl, Et3N, DMAP, CH2Cl2, rt, 19 h, 93%; (f) OsO4,
NaIO4, THF-H2O (1:1), 0 °C, rt, 2 h; (g) NaBH4, THF-H2O (1:1), 0 °C,
1 h 63% over two steps. (h) NaHMDS, THF, reflux, 12 h, 86%; (i) concd
H2SO4 (cat.), AcOH, Ac2O, rt.
TABLE 1. Hydroboration of 7 with Several Borane Reagentsa
yield of
yield of
run
reagent
borane-THF
equiv
8b (%)
9b (%)
1
2
3
4
3.0
3.0
1.5
1.5
23
58
64
borane-1,4-oxathiane
thexylborane
25
33
56
thexylborane with 2.0 mol %
of Rh(PPh3)3Cl
16
14 in 86% yield. Next, we attempted conversion of 14 to the
diacetate 15, which would be a key intermediate for coupling
with several nucleobases. However, reaction of 14 with acetic
acid, acetic anhydride and a catalytic amount of sulfonic acid
failed to give the desired diacetate 15. From this result, it was
supposed that the additional trans-fused six-membered ring
severely restricted the conformational flexibility of the furanose
ring and prevented the formation of the oxonium intermediate
(Scheme 2).
5
9-BBN
1.5
no reaction
a Every reaction was carried out at 0 °C. b Isolated yield.
82% yield. The pro-R hydroxymethyl group in 5 was selectively
benzylated to give 6 in 54% yield, and tosylation of 6 gave 7
in 92% yield. Next, hydroboration of 7 was carried out with
several borane reagents (Table 1). Treatment of 7 with borane-
tetrahydrofuran or borane-1,4-oxathiane complex gave the
desired terminal alcohol 8 in 23% and 25% yield, respectively
(runs 1 and 2). However, unfortunately, the Markovnikov-type
adduct 9 was obtained as a major product, probably due to
undesired chelation between the borane reagents and oxygen
atoms in 7. To avoid formation of such chelate structure, more
bulky borane reagents were used for further evaluation of the
reaction (runs 3-5). Although the reaction with 9-borabicyclo-
[3.3.1]nonane (9-BBN) did not proceed at all (run 5), the
reaction with thexylborane27 gave the desired compound 8 in
moderate yield (run 3). Addition of a catalytic amount of
Wilkinson’s reagent28 improved the yield up to 56% (run 4).
However, the yield and reproducibility of this hydroboration/
oxidation approach were still insufficient.
To overcome this problem, we selected an approach for the
synthesis of compound 8 via oxidative cleavage of olefin and
subsequent reduction of the corresponding allyl derivative
(Scheme 2),24 instead of the hydroboration/oxidation of the vinyl
derivative 7 (Scheme 1). Consequently, the compound 8 was
effectively obtained from the known allyl derivative 1029 as
shown in Scheme 2. Ring-closure reaction of 8 under alkaline
conditions successfully proceeded to give the tricyclic compound
As an alternative approach, we decided to introduce a
nucleobase into the sugar moiety before formation of the trans-
fused bicyclic ring structure (Scheme 3). Acetolysis of 8 gave
the corresponding triacetate 16 in 98% yield, which was coupled
with thymine nucleobase to afford the â-isomer 17 in 95% yield
according to Vorbru¨ggen’s procedure. After removal of both
acetyl groups in 17, the obtained diol was treated under alkaline
conditions. As a result, the ring-closure reaction from the 2′-
hydroxyl group proceeded exclusively and the 2,5-dioxabicyclo-
[2.2.1]heptane structure (2′,4′-BNA skeleton) was formed,
instead of the desired trans-fused 4,7-dioxabicyclo[4.3.0]nonane
structure. Therefore, it is necessary to protect the 2′-hydroxyl
group before the ring-closure reaction. Since it is known that a
2′-O-acyl group in fully acylated ribonucelosides showed high
reactivity under controlled conditions,30 we attempted several
reaction conditions. Consequently, regioselective deprotection
of the 2′-O-acetyl group in 17 was successfully accomplished
by using aqueous methylamine to give 18 in 87% yield.
Treatment of 18 with 2-methoxypropene gave 19 in 85% yield,
and removal of the acetyl group of 19 under alkaline conditions
afforded 20 in 87% yield. Ring-closure reaction of 20 by using
(29) Bar, N. C.; Roy, A.; Achari, B.; Mandal, S. B. J. Org. Chem. 1997,
(27) Negishi, E.; Brown, H. C. Synthesis 1974, 77-89.
(28) Evans, D. A.; Fu, G. C.; Hoveyda, A. H. J. Am. Chem. Soc. 1992,
114, 6671-6679.
62, 8948-8951.
(30) Nishino, S.; Takamura, H.; Ishido, Y. Tetrahedron 1986, 42, 1995-
2004.
1308 J. Org. Chem., Vol. 71, No. 4, 2006

Synthesis and Properties of Trans-3′,4′-Bridged Nucleic Acids
SCHEME 3 a
a Reagents, conditions, and yields: (a) Ac2O, CH3COOH, concd H2SO4 (cat.), rt, 6 h, 98%; (b) thymine, BSA, TMSOTf, ClCH2CH2Cl, reflux, 19 h, 95%;
(c) 40% MeNH2 aq, THF, 0 °C, 87%; (d) 2-methoxypropene, p-TsOH‚H2O, CH2Cl2, 0 °C, 1.5 h, 85%; (e) 2 M NaOH aq, THF-MeOH (3:1), rt, 1.5 h, 87%;
(f) NaHMDS, THF, reflux, 6 h, 73%; (g) p-TsOH‚H2O, THF-MeOH (2:1), 0 °C, 1 h, 85%.
SCHEME 4 a
a Reagents, conditions, and yields: (a) BOMCl, DBU, DMF, 0 °C, 45 min, 90% for 23; 3 h, 68% for 27; (b) NaH, MeI, DMF, 0 °C, 30 min, 86% for
24; 30 min, 93% for 28; (c) 20% Pd(OH)2/C, cyclohexene, MeOH, reflux, 7 h, 39%; (d) Dess-Martin periodinane, CH2Cl2, rt, 30 min, 97%; (e) DIBAL-H,
THF, -78 °C, 5 h, 67%; (f) 20% Pd(OH)2/C, ammonium formate, MeOH, reflux, 7.5 h, 63%.
sodium hexamethyldisilazide (NaHMDS) successfully proceeded
to afford 21 in 73% yield. The methoxypropyl (MOP) group in
21 was removed by treatment with p-toluenesulfonic acid to
give 22 in 85% yield. Thus, we achieved the synthesis of the
desired trans-3′,4′-BNA skeleton. To incorporate the monomer
into ONs, the 2′-hydroxyl group of 22 has to be removed or
appropriately protected. At first, 2′-deoxygenation of 22 was
attempted by the procedures previously reported.31-33 However,
unfortunately, the desired 2′-deoxy derivative was not obtained,
probably due to steric hindrance around the 2′-hydroxyl group.
Next, we examined the protection of the 2′-hydroxyl group. As
a protecting group we selected a methyl group because of its
small size. As shown in Scheme 4, thymine nucleobase of 22
was protected with a benzyloxymethyl (BOM) group to give
23 in 90% yield. The obtained 23 was then treated with sodium
hydride and iodomethane in DMF to afford the 2′-O-methyl
derivative 23 in 86% yield. Deprotection of the BOM group
and both 3′- and 5′-O-benzyl groups by Pd-mediated hydro-
genolysis afforded trans-3′,4′-BNA monomer 1 in 39% yield
along with its 3′-O-benzyl derivative. The low reactivity of the
(31) Robins, M. J.; Wilson, J. S. J. Am. Chem. Soc. 1981, 103, 932-
933.
(32) Pankiewicz, K.; Matsuda, A.; Watanabe, K. A. J. Org. Chem. 1982,
47, 485-488.
(33) Wang, Z.; Prudhomme, D. R.; Buck, J. R.; Park, M.; Rizzo, C. J.
J. Org. Chem. 2000, 65, 5969-5985.
J. Org. Chem, Vol. 71, No. 4, 2006 1309

Sekiguchi et al.
SCHEME 5 a
TABLE 2. Melting Temperatures (°C) of the Duplexes between
ODN1-ODN4 and Their Complementary Strandsa
target strands
DNA
RNA
sequence of ODNs (5′f3′)
complement
complement
d(GCGTTTTTTGCT) (ODN1)
d(GCGTTTTTTGCT) (ODN2)
d(GCGTTTTTTGCT) (ODN3)
d(GCGTTTTTTGCT) (ODN4)
50
36
37
38
45
33
35
36
a The UV melting profiles were recorded in 10 mm sodium phosphate
buffer (pH 7.2) containing 100 mm NaCl at a scan rate of 0.5 °C/min with
detection at 260 nm. The final concentration of each ODN was 4.0 µM.
The trans-3′,4′-BNA arabino-type monomer is shown in bold.
TABLE 3. Melting Temperatures (°C) of the Duplexes between
ODN1 or ODN2 and Their Complementary DNA Strands with or
without One Base Mismatcha
a Reagents, conditions, and yields: (a) DMTrCl, pyridine, 50 °C, 2.5 h,
77%; (b) DMTrOTf, CH2Cl2-pyridine, rt, 5 h, 100%; (c) NC(CH2)2-
OP[N(Pri)2]2, dicyanoimidazole, MeCN-THF, 50 °C, 15 h, 55%.
target sequence: 3′-d(CGCAANAAACGA)-5′
ODN
N ) A
G
C
T
ODN1
ODN2
50
36
41 (-9)b
37 (-13)b
20 (-16)b
38 (-12)b
23 (-13)b
25 (-11)b
3′-benzyloxy group was probably due to the steric hindrance
around the 3′-position that involves 1,3-diaxial interaction with
the hydrogens on the trans-fused six-membered ring and gauche
a Conditions for melting temperature measurements are shown in footnote
of Table 2 and in the Experimental Section. b The values in parentheses
are differences in Tm values between mismatched and matched duplexes.
1
interaction with the 2′-methoxy group. H NMR and X-ray
crystallographic analysis revealed that 1 has a typical S-type
(C3′-exo) conformation and the torsion angle δ (C5′-C4′-
C3′-O3′) of 174.6°.24 The torsion angle ø was -125.8°,
indicating anti orientation of the nucleobase moiety.24 Next, the
arabino-type derivative 2 was prepared as shown in Scheme 4.
The 2′-hydroxyl group of 22 was oxidized with Dess-Martin
periodinane to give the 2′-oxo derivative 25 in 97% yield.
Reduction of the obtained ketone 25 with sodium borohy-
dride showed no significant stereoselectivety (data not shown).
After several attempts with various reducing reagents, it was
found that the reduction with diisobutyl aluminum hydride
(DIBAL-H) gave the desired arabino-type product 26 stereo-
selectively. The stereochemistry at the C2′ position was con-
firmed by comparison of the coupling constants between H1′
and H2′ (3JH1′-H2′), which was changed from 7 Hz (for 22) to
4 Hz (for 26). Protection of the thymine nucleobase with the
BOM group, methylation of the 2′-hydroxyl group with sodium
hydride and iodomethane and Pd-mediated hydrogenolysis
afforded the desired compund 2 in 40% yield over three steps.
Reactivity of the 3′-benzyloxy group of the arabino-type com-
pound 28 was improved compared with that of the ribo-type
compound 24.
To incorporate the trans-3′,4′-BNA monomers 1 and 2 into
oligodeoxynucleotides (ODNs), we attempted to synthesize the
corresponding phosphoramidite derivatives (Scheme 5). The 5′-
O-dimethoxytrityl derivative 29 was obtained in 77% yield by
treatment of 1 with dimethoxytrityl chloride in pyridine.
However, the following phosphitylation of 29 did not proceed
at all, even if the reaction temperature was elevated up to 60
°C. We tried some other phosphitylation conditions; however,
the desired phosphoramidite derivative was not obtained at all.
On the other hand, the trans-3′,4′-BNA arabino-type monomer
2 was successfully converted to the corresponding phosphora-
midite compound. The dimethoxytritylation at the 5′-C-hydroxyl
group of 2 was successfully accomplished by treating with 4,4′-
dimethoxytrityl triflate (DMT-OTf)34 to give 30 in quantitative
yield. Phosphitylation of 30 proceeded at 50 °C to give the
corresponding phosphoramidite 31 in 55% yield.
Incorporation of the phosphoramidite 31 into 12-mer ODNs
ODN2-ODN4 was attempted by the usual phosphoramidite
protocol on an automated DNA synthesizer. However, the
coupling efficiency of phosphoramidite 31 was quite low under
standard coupling conditions. After several examinations of the
coupling conditions, the ODNs containing the trans-3′,4′-BNA
arabino-type monomer were synthesized with prolonged cou-
pling time (24 h) and elevated coupling temperature (50 °C).
The coupling efficiency, estimated from the absorption of the
released trityl cation, was improved up to 40%. After deblocking
and cleavage from the solid support by using aqueous ammonia
at 55 °C, the resultant ODNs were purified by reversed-phase
HPLC. The composition of ODNs (ODN2-ODN4) was con-
firmed by MALDI-TOF MS analysis.
The hybridization properties of ODN2-ODN4 toward their
complementary strands were evaluated by UV melting experi-
ments (Table 2). The melting temperatures (Tm’s) of the
duplexes between natural ODN (ODN1) and its DNA and RNA
complements were 50 and 45 °C, respectively. All three
modified ODNs (ODN2-ODN4) exhibited a decrease in Tm
value by -11 to -14 °C when they form duplexes with the
DNA complement. A similar decrease in Tm value (∆Tm ) -9
to -12 °C) was observed when targeting the RNA complement.
Thus, incorporation of the trans-3′,4′-BNA arabino-type mono-
mer into ODNs decreases the duplex stability, regardless of the
position of the modification and the type of the target nucleic
acid (DNA or RNA). To investigate in detail the duplex-forming
properties of the trans-3′,4′-BNA-modified ODNs, we examined
the sequence selectivity of ODN1 and ODN2 by means of Tm
measurement with the target DNA sequences containing one
base mismatch (Table 3). The Tm values of the mismatched
duplexes containing natural ODN (ODN1) decreased by -9 °C
to -13 °C, compared to that of the full-matched duplex. In the
case of trans-3′,4′-BNA modified ODN (ODN2), the differences
in Tm values between matched and mismatched duplexes are
larger than those observed for the duplexes containing ODN1,
(34) Tarko¨y, M.; Bolli, M.; Leumann, C. HelV. Chim. Acta 1994, 77,
716-744.
1310 J. Org. Chem., Vol. 71, No. 4, 2006

Synthesis and Properties of Trans-3′,4′-Bridged Nucleic Acids
methyl group of 2 might be more significant than that of the
conformationally unrestricted 2′-O-methylarabinonucleoside.
Another possibility to explain the destabilization would be the
large δ values (δ ) 174.6°) of trans-3′,4′-BNA monomers,24
which deviated from the mean δ value of one typical B-type
DNA duplex, Dickerson’s dodecamer (δ ) 123 ( 21°).36 This
difference in δ value might cause an extension of the helical
pitch and destabilization of the duplex structure. Alternatively,
the pattern of hydration, stabilizing the DNA helical structure,37
might be affected by the additional hydrophobic six-membered
ring in the trans-3′,4′-BNA monomer. To investigate the origin
of the decreased thermal stability in detail and develop a
conformationlly restricted nucleic acids with enhanced binding
affinity toward DNA complements, further chemical modifica-
tion of the trans-3′,4′-BNA is now in progress in our laboratory.
Conclusion
FIGURE 3. CD spectra of the DNA duplexes between ODN1 and
full-matched strand (thin line), ODN1 and one base mismatched strand
(dashed line), and ODN2 and full-matched strand (thick line). The
spectra were recorded in 10 mm sodium phosphate buffer (pH 7.2)
containing 100 mm NaCl at 10 °C. Concentration of each strand was
4 µm.
In this study, we have developed the nucleoside analogues,
trans-3′,4′-BNAs 1 and 2, bearing a trans-fused additional six-
membered ring for restriction of the sugar puckering into S-type
conformation. Conformational analysis revealed that these
nucleoside analogues have a C3′-exo sugar pucker, a typical
S-type conformation. The arabino-type analogue 2 was suc-
cessfully incorporated into ODNs. The corresponding ODN
derivatives containing 2 showed sequence-selective binding with
the complementary strands, though the binding affinity was
lower than that of the natural ODNs. We believe that the present
results will be useful for further development of the nucleic
acid analogues with S-type sugar conformation.
indicating that the trans-3′,4′-BNA-T moiety in ODN2 recog-
nized the corresponding adenine nucleobase in the complemen-
tary strand. To investigate the helical structure of the duplex
containing trans-3′,4′-BNA, we measured the CD spectrum of
the DNA duplex containing ODN2 at 10 °C. The spectrum is
shown in Figure 3 along with those of the natural DNA duplexes
between ODN1 and its complementary sequences with or
without one base mismatch. The CD spectrum of the duplex
containing ODN2 was quite similar to those of the matched or
mismatched natural DNA duplex. All spectra showed a large
positive Cotton band around 280 nm and large negative Cotton
band around 250 nm, indicating a typical B-type helix formation.
This result means that the modification of ODNs with one trans-
3′,4′-BNA unit does not affect the overall helical structure of
the DNA duplex, though the thermal stability of the duplexes
decreased significantly compared with that of the natural DNA
duplexes. The conformational change of the trans-3′,4′-BNA-
modified duplex is limited to a local one as well as a mismatched
DNA duplex.
Experimental Section
3-O-Benzyl-1,2-O-isopropylidene-3-C-vinyl-R-D-allofura-
nose (4). A solution of compound 326 (990 mg, 2.63 mmol) in 80%
aqueous acetic acid (15 mL) was stirred at 40 °C for 3 h. After
neutralization with 10 M aqueous sodium hydroxide, the reaction
mixture was extracted with EtOAc. The organic layer was washed
with H2O and saturated aqueous NaCl, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
flash silica gel column chromatography (EtOAc/n-hexane ) 3:2)
to give compound 4 as a colorless oil (793 mg, 90%). [R]27D: +68.6
(c 0.76, CHCl3). IR νmax (KBr): 3432, 2934, 1638, 1455, 1380
1
cm-1. H NMR (CDCl3): δ 1.39 (3H, s), 1.63 (3H, s), 2.17 (1H,
The sugar puckering of the trans-3′,4′-BNA monomers was
locked in C3′-exo conformation, which is predominantly
observed in DNA duplexes with B-type helical structure, and
the nucleobase orientation of trans-3′,4′-BNA monomers was
found to be anti that is favorable to Watson-Crick base pair
formation.24 However, modification of ODNs with the trans-
3′,4′-BNA arabino-type monomer 2 failed to stabilize the duplex.
One possibility to account for the destabilization of duplexes
by the trans-3′,4′-BNA modification would be the effect of the
2′-O-methyl group. It was previously reported that the ODNs
containing 2′-O-methylarabinonucleoside exhibited a sharp
decrease in thermal stability of the duplexes with their DNA
complements (∆Tm/modification ) -4 °C to -6 °C).35 In the
case of trans-3′,4′-BNA modification, a larger decrease in the
Tm value was observed (∆Tm/modification ) -12 °C to -14
°C). The sugar puckering of 2 was restricted in the C3′-exo
conformation with the arabino-configured 2′-O-methyl group
in an axial position. Therefore, the steric effect of the 2′-O-
t, J ) 6 Hz), 2.67 (1H, t, J ) 2 Hz), 3.63-3.82 (3H, m), 4.15 (1H,
d, J ) 8 Hz), 4.61, 4.70 (2H, ABq, J ) 11 Hz), 4.66 (1H, d, J )
4 Hz), 5.38 (1H, d, J ) 18 Hz), 5.55 (1H, d, J ) 11 Hz), 5.82 (1H,
d, J ) 4 Hz), 5.94 (1H, dd, J ) 11, 18 Hz), 7.26-7.38 (5H, m).
13C NMR (CDCl3): δ 26.7, 26.9, 64.2, 67.9, 70.2, 79.6, 80.9, 104.5,
118.9, 127.5, 127.7, 128.3, 134.2, 137.5. Mass (FAB): m/z 337
(M + H)+. Anal. Calcd for C18H24O6‚1/4H2O: C, 63.42; H, 7.24.
Found: C, 63.35; H, 7.10.
3-O-Benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-3-C-
vinyl-R-D-ribofuranose (5). To a stirred solution of compound 4
(682 mg, 2.03 mmol) in THF-H2O (1:8, 9.0 mL) was added NaIO4
(564 mg, 2.64 mmol) at 0 °C. After the mixture was stirred for 45
min, ethylene glycol (0.2 mL) was added and stirred for more 15
min. The reaction mixture was filtered and extracted with EtOAc.
The organic layer was washed with H2O and saturated aqueous
NaCl, dried over Na2SO4, and concentrated under reduced pressure.
The residue (640 mg) was dissolved in THF-H2O (1:1, 12 mL),
and aqueous 37% formaldehyde (0.8 mL, 10.5 mmol) and 1 M
(36) Dickerson, R. E.; Drew, H. R. J. Mol. Biol. 1981, 149, 761-786.
Drew, H. R.; Wing, R. M.; Takano, T.; Broka, C.; Tanaka, S.; Itakura, K.;
Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 2179-2183.
(37) Drew, H. R.; Dickerson, R. E. J. Mol. Biol. 1981, 151, 535-556.
(35) Gotfredsen, C. H.; Jacobsen, J. P.; Wengel, J. Tetrahedron Lett.
1994, 35, 6941-6944.
J. Org. Chem, Vol. 71, No. 4, 2006 1311

Sekiguchi et al.
aqueous sodium hydroxide (4.2 mL, 4.2 mmol) were added. The
whole was stirred at room temperature for 18 h. After neutralization
with diluted hydrochloric solution, the reaction mixture was
extracted with EtOAc. The organic layer was washed with H2O
and saturated aqueous NaCl, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash silica
gel column chromatography (EtOAc/n-hexane ) 1:1) to give
was added dropwise thexylborane (0.42 M in THF, 3.6 mL, 1.52
mmol) at 0 °C, and the whole was stirred at room temperature for
2 h. A mixture of 1 M aqueous sodium hydroxide (1.6 mL) and
30% hydrogen peroxide (0.6 mL) was added, and the whole was
heated under reflux for 1.5 h. After filtration through Celite, the
mixture was extracted with EtOAc. The organic layer was washed
with H2O and saturated aqueous NaCl, dried over Na2SO4, and
concentrated under reduced pressure. The resultant residue was
purified by flash silica gel column chromatography (EtOAc/n-
hexane ) 1:1) to give compound 8 as a colorless oil (340 mg,
56%).
compound 5 as a colorless oil (562 mg, 82% over two steps). [R]27
:
D
+10.9 (c 0.92, CHCl3). IR νmax (KBr): 3420, 2985, 1637, 1457,
1
1381 cm-1. H NMR (CDCl3): δ 1.37 (3H, s), 1.67 (3H, s), 2.48
(1H, brs), 2.59 (1H, brs), 3.67, 3.75 (2H, ABq, J ) 12 Hz), 4.07,
4.33 (2H, ABq, J ) 12 Hz), 4.57, 4.71 (2H, ABq, J ) 11 Hz),
4.73 (1H, d, J ) 4 Hz), 5.32 (1H, d, J ) 18 Hz), 5.50 (1H, d, J )
11 Hz), 5.88 (1H, d, J ) 4 Hz), 5.93 (1H, dd, J ) 11, 18 Hz),
7.25-7.36 (5H, m). 13C NMR (CDCl3): δ 25.9, 26.4, 63.8, 65.5,
67.7, 82.22, 87.0, 87.6, 104.6, 113.1, 117.5, 127.3, 127.7, 128.4,
135.5, 137.8. Mass (FAB): m/z 337 (M + H)+. Anal. Calcd for
C18H24O6‚1/5H2O: C, 63.59; H, 7.23. Found: C, 63.59; H, 7.10.
3,5-Di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-3-
C-vinyl-R-D-ribofuranose (6). To a suspension of 60% sodium
hydride (75 mg, 1.89 mmol) in anhydrous DMF (3.0 mL) was added
compound 5 (455 mg, 1.35 mmol) in anhydrous DMF (7.0 mL) at
0 °C and the mixture stirred for 30 min. Benzyl bromide (0.17
mL, 1.42 mmol) was added and the mixture stirred at room
temperature for 1.5 h. Methanol was added to the mixture and
evaporated to dryness under reduced pressure. The residue was
dissolved in ether, washed with H2O and saturated aqueous NaCl,
dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by flash silica gel column chromatography
(EtOAc/n-hexane ) 1:3) to give compound 6 as a colorless oil
(314 mg, 54%). [R]26D: +26.4 (c 1.12, CHCl3). IR νmax (KBr):
Method B (via Oxidative Cleavage of Allyl Group). To a
stirred solution of compound 13 (1.2 g, 2.0 mmol) in THF-H2O
(1:1, 12 mL) were added NaIO4 (1.3 g, 6.0 mmol) and OsO4 (0.07
M in tert-butyl alcohol, 0.28 mL, 0.02 mmol), and the mixture was
stirred at room temperature for 2 h. After filtration, the filtrate was
extracted with EtOAc. The organic layer was washed with H2O
and saturated aqueous NaCl, dried over Na2SO4, and concentrated
under reduced pressure. The residue was dissolved in THF-H2O
(1:1, 12 mL) and sodium tetrahydroborate (75 mg, 2.0 mmol) was
added at 0 °C. After the mixture was stirred for 1 h at the same
temperature, acetone was added, and the mixture was extracted with
EtOAc. The organic layer was washed with H2O and saturated
aqueous NaCl, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by flash silica gel column
chromatography (EtOAc/n-hexane ) 1:1) to give compound 8 as
a colorless oil (754 mg, 63%). [R]21D: +15.4 (c 0.83, CHCl3). IR
1
νmax (KBr): 3490, 2967, 1599, 1354 cm-1. H NMR (CDCl3): δ
1.28 (3H, s), 1.41 (3H, s), 1.91 (1H, dt, J ) 14, 7 Hz), 2.22 (1H,
dt, J ) 15, 6 Hz), 2.31 (3H, s), 3.38, 3.66 (2H, ABq, J ) 10 Hz),
3.75 (1H, dd, J ) 7, 11 Hz), 3.79 (1H, dd, J ) 6, 11 Hz), 4.35,
4.79 (2H, ABq, J ) 10 Hz), 4.39, 4.42 (2H, ABq, J ) 11 Hz),
4.58, 4.69 (2H, ABq, J ) 12 Hz), 4.66 (1H, d, J ) 4 Hz), 5.71
(1H, d, J ) 4 Hz), 7.14-7.35 (12H, m), 7.69 (2H, d, J ) 8 Hz).
13C NMR (CDCl3): δ 21.6, 25.9, 25.9, 33.8, 57.8, 66.4, 67.3, 69.1,
73.6, 83.8, 85.9, 86.7, 103.3, 113.2, 126.5, 127.1, 127.5, 127.6,
127.9, 128.1, 128.2, 129.5, 132.7, 137.3, 138.1, 144.3. Mass
(FAB): m/z 621 (M + Na)+. Anal. Calcd for C32H38O9S‚1/4H2O:
C, 63.72; H, 6.43; S, 5.36. Found: C, 63.79; H, 6.43; S, 5.13.
3-C-Allyl-3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-
R-D-ribofuranose (11). A solution of compound 1029 (4.9 g, 13
mmol) in 80% aqueous acetic acid (85 mL) was stirred at 40 °C
for 3 h. After neutralization with 10 M aqueous sodium hydroxide,
the reaction mixture was extracted with EtOAc. The organic layer
was washed with H2O and saturated aqueous NaCl, dried over
Na2SO4, and concentrated under reduced pressure. The residue (4.5
g) was dissolved in THF-H2O (1:1, 60 mL), and sodium periodate
(3.6 g, 17 mmol) was added at 0 °C. The mixture was stirred at
the same temperature for 45 min. Ethylene glycol (1.0 mL) was
added and stirred for further 15 min. After being filtered, the
solution was extracted with EtOAc. The organic layer was washed
with H2O and saturated aqueous NaCl, dried over Na2SO4, and
concentrated under reduced pressure. The residue was dissolved
in THF-H2O (1:1, 60 mL), and aqueous 37% formaldehyde (4.7
mL, 63 mmol) and 1 M aqueous sodium hydroxide (25 mL, 25
mmol) were added. The whole was stirred at room temperature for
18 h. After neutralization with diluted hydrochloric solution, the
reaction mixture was extracted with EtOAc. The organic layer was
washed with H2O and saturated NaCl solution, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified
by flash silica gel column chromatography (EtOAc/n-hexane ) 1:1)
to give compound 11 as a colorless oil (2.9 g, 65% over three steps).
[R]26D: -6.4 (c 0.82, CHCl3). IR νmax (KBr): 3415, 2942, 1637,
1
3560, 2938, 1605, 1455, 1379 cm-1. H NMR (CDCl3): δ 1.36
(3H, s), 1.67 (3H, s), 3.54 (2H, s), 4.14, 4.23 (2H, ABq, J ) 12
Hz), 4.48, 4.57 (2H, ABq, J ) 12 Hz), 4.56, 4.71 (2H, ABq, J )
12 Hz), 4.70 (1H, d, J ) 4 Hz), 5.26 (1H, d, J ) 18 Hz), 5.40 (1H,
d, J ) 11 Hz), 5.84 (1H, dd, J ) 11, 18 Hz), 5.89 (1H, d, J ) 4
Hz), 7.22-7.36 (10H, m). 13C NMR (CDCl3): δ 26.1, 26.5, 62.1,
67.4, 71.2, 73.5, 82.5, 87.1, 88.5, 104.7, 113.1, 117.3, 127.0, 127.3,
127.3, 127.4, 128.1, 128.2, 135.8, 138.2, 138.3. Mass (FAB): m/z
427 (M + H)+. Anal. Calcd for C25H30O6‚1/5H2O: C, 69.81; H,
7.12. Found: C, 69.80; H, 7.10.
3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-(p-toluenesulfo-
nyloxymethyl)-3-C-vinyl-R-D-ribofuranose (7). To a solution of
compound 6 (510 mg, 1.2 mmol) in anhydrous CH2Cl2 (10 mL)
were added Et3N (5.0 mL, 36 mmol), p-TsCl (343 mg, 1.8 mmol),
and DMAP (73 mg, 0.60 mmol) at room temperature, and the
mixture was stirred for 12 h. Saturated aqueous NaHCO3 was added,
and the mixture was stirred for a further 30 min and then extracted
with EtOAc. The organic layer was washed with H2O and saturated
aqueous NaCl, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by flash silica gel column
chromatography (EtOAc/n-hexane ) 1:3) to give compound 7 as
a light yellow solid (639 mg, 92%). Mp: 82-84 °C. [R]28D: +8.0
(c 1.06, CHCl3). IR νmax (KBr): 2933, 1361, 1178 cm-1. 1H NMR
(CDCl3): δ 1.32 (3H, s), 1.46 (3H, s), 2.31 (3H, s), 3.32, 3.57
(2H, ABq, J ) 9 Hz), 4.32 (2H, s), 4.69 (1H, d, J ) 4 Hz), 4.60,
4.76 (2H, ABq, J ) 11 Hz), 4.52, 4.64 (2H, ABq, J ) 10 Hz),
5.25 (1H, d, J ) 18 Hz), 5.39 (1H, d, J ) 11 Hz), 5.81 (1H, d, J
) 4 Hz), 5.89 (1H, dd, J ) 11, 18 Hz), 7.14-7.30 (12H, m), 7.75
(2H, d, J ) 9 Hz). 13C NMR (CDCl3): δ 21.6, 26.1, 26.1, 67.0,
68.9, 69.1, 73.1, 82.3, 86.8, 86.9, 104.6, 113, 118.3, 125.6, 127.0,
127.0, 127.2, 128.0, 128.0, 129.4, 133.1, 135.7, 138.0, 138.5, 144.1.
Mass (FAB): m/z 603 (M + Na)+. Anal. Calcd for C30H36O8S: C,
66.19; H, 6,25; S, 5.52. Found: C, 66.10; H, 6.29; S, 5.29.
3,5-Di-O-benzyl-3-C-hydroxyethyl-1,2-O-isopropylidene-4-C-
(p-toluenesulfonyloxymethyl)-R-D-ribofuranose (8). Method A
(via Hydroboration/Oxidation Approach). To a stirred solution
of compound 7 (590 mg, 1.02 mmol) and chlorotris(triphenylphos-
phine)rhodium (I) (18 mg, 0.02 mmol) in anhydrous THF (15 mL)
1
1381, 1211, 1019 cm-1. H NMR (CDCl3): δ 1.33 (3H, s), 1.65
(3H, s), 2.45 (1H, d, J ) 7 Hz), 2.50 (1H, d, J ) 7 Hz), 2.65 (1H,
ddt, J ) 6, 15, 2 Hz), 2.76 (1H, dd, J ) 8, 15 Hz), 3.86, 3.90 (2H,
ABX, J ) 12, 6 Hz), 4.12, 4.17 (2H, ABX, J ) 12, 7 Hz), 4.54
(1H, d, J ) 4 Hz), 4.65, 4.72 (2H, ABq, J ) 10 Hz), 5.20-5.30
(2H, m), 5.72 (1H, d, J ) 4 Hz), 5.88-6.04 (1H, m), 7.28-7.39
1312 J. Org. Chem., Vol. 71, No. 4, 2006

Synthesis and Properties of Trans-3′,4′-Bridged Nucleic Acids
(5H, m). 13C NMR (CDCl3): δ 25.8, 26.3, 37.0, 63.6, 64.0, 67.3,
83.1, 86.0, 87.4, 104.1, 112.6, 119.6, 127.4, 127.7, 128.3, 131.8,
137.9. Mass (FAB): m/z 373 (M + Na)+. HRMS (FAB): calcd for
C19H26O6 (M + H)+ 373.1627, found 373.1613. Anal. Calcd for
C18H24O6‚1/10H2O: C, 64.79; H, 7.50. Found: C, 64.52; H, 7.48.
3-C-Allyl-3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopro-
pylidene-R-D-ribofuranose (12). To a suspension of 60% sodium
hydride (2.83 g, 70.8 mmol) in anhydrous DMF (250 mL) was
added dropwise compound 11 (17.8 g, 50.5 mmol) in anhydrous
DMF (250 mL) at 0 °C. After the solution was stirred for 2 h,
benzyl bromide (6.24 mL, 53.1 mmol) was added, and the mixture
was stirred at room temperature for 1.5 h. MeOH was added,
evaporated to dryness under reduced pressure, and the residue was
extracted with ether and H2O. The organic layer was washed with
H2O and saturated aqueous NaCl, dried over MgSO4, and concen-
trated under reduced pressure. The resultant residue was purified
by flash silica gel column chromatography (EtOAc/CHCl3 ) 1:30)
to give compound 12 as a colorless oil (14.5 g, 65%). [R]25D: +14.2
(c 0.82, CHCl3). IR νmax (KBr): 3564, 2936, 1637, 1380, 1106
J ) 5 Hz), 7.19-7.41 (10H, m). 13C NMR (CDCl3): δ 26.3, 27.2,
28.4, 62.4, 65.3, 66.4, 73.8, 74.7, 77.9, 85.7, 88.0, 105.2, 115.6,
126.3, 126.8, 127.5, 127.8, 128.1, 128.4, 137.6, 139.0. Mass (EI):
m/z 91 (Bn+, 100), 426 (M+, 0.2). Anal. Calcd for C25H30O6‚
1/3H2O: C, 69.43; H, 7.15. Found: C, 69.38; H, 7.02.
3-C-Acetoxyethyl-1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-(p-tolu-
enesulfonyloxymethyl)-D-ribofuranose (16). To a stirred solution
of compound 8 (140 mg, 0.23 mmol) in acetic acid (1.0 mL) were
added acetic anhydrate (0.14 mL, 1.5 mmol) and concd H2SO4 (1
drop) at room temperature. After being stirred for 6 h, the solution
was neutralized with NaHCO3, and the mixture was extracted with
EtOAc and H2O. The organic layer was washed with saturated
aqueous NaHCO3, H2O, and saturated aqueous NaCl, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by flash silica gel column chromatography (EtOAc/n-
hexane ) 1:2) to give anomeric mixture 16 (R:â ) ca. 1:3) as a
colorless oil (154 mg, 98%). 1H NMR (CDCl3): δ 1.91 (3/4H, s),
1.93 (9/4H, s), 1.99 (3/4H, s), 2.01 (9/4H, s), 2.04 (3/4H, s) 2.05
(9/4H, s), 2.22-2.28 (1H, m), 2.39 (3H, s), 2.42-2.47 (1H, m),
3.44 (3/4H, d, J ) 10 Hz), 3.49 (1/4H, d, J ) 11 Hz), 3.73 (1H,
m), 4.18-4.28 (4H, m), 4.44-4.65 (15/4H, m), 4.83 (1/4H, d,
J ) 11 Hz), 5.49 (1/4H, d, J ) 5 Hz), 5.53 (3/4H, d, J )
1 Hz), 5.89 (3/4H, d, J ) 1 Hz), 6.32 (1/4H, d, J ) 5 Hz), 7.07
(6/4H, d, J ) 8 Hz), 7.19-7.37 (42/4H, m), 7.69 (2H, d, J ) 8
Hz). Mass (FAB): m/z 707 (M + Na)+. HRMS (FAB): calcd for
C35H40O12SNa (M + Na)+ 707.2138, found 707.2140. Anal. Calcd
for C35H40O12S‚1/5H2O: C, 61.07; H, 5.92; S, 4.68. Found: C,
61.07; H, 5.94; S, 4.56.
1
cm-1. H NMR (CDCl3): δ 1.31 (3H, s), 1.63 (3H, s), 2.24 (1H,
dd, J ) 5, 9 Hz), 2.49 (1H, dd, J ) 6, 15 Hz), 2.79 (1H, dd, J )
7, 15 Hz), 3.57, 3.71 (2H, ABq, J ) 10 Hz), 3.91 (1H, dd, J ) 9,
12 Hz), 4.29 (1H, dd, J ) 5, 12 Hz), 4.53 (1H, d, J ) 4 Hz), 4.54,
4.60 (2H, ABq, J ) 12 Hz), 4.66, 4.71 (2H, ABq, J ) 11 Hz),
5.14-5.19 (2H, m), 5.71 (1H, d, J ) 4 Hz), 5.84-5.99 (1H, m),
7.28-7.33 (10H, m). 13C NMR (CDCl3): δ 25.9, 26.4, 36.4, 61.6,
67.8, 68.8, 73.7, 83.4, 85.5, 88.4, 103.7, 112.7, 119.2, 127.0, 127.2,
127.5, 127.6, 128.2, 132.2, 137.8, 138.3. Mass (FAB): m/z 447
(M+Li)+. HRMS (FAB): calcd for C26H32O6Na (M + Na)+
463.2097, found 463.2093.
3′-C-Acetoxyethyl-2′-O-acetyl-3′,5′-di-O-benzyl-5-methyl-4′-
C-(p-toluenesulfonyloxymethyl)uridine (17). To a solution of
compound 16 (5.05 g, 7.38 mmol) in anhydrous 1,2-dichloroethane
(80 mL) were added thymine (1.4 g, 11.1 mmol) and N,O-bis-
(trimethylsilyl)acetamide (7.18 mL, 29.5 mmol), and the mixture
was heated under reflux for 2 h. The solution was cooled to 0 °C,
and TMSOTf (2.27 mL, 12.5 mmol) was added dropwise. The
mixture was heated under reflux for further 7 h. Saturated aqueous
NaHCO3 was added, and the solution was extracted with EtOAc.
The organic layer was washed with H2O and saturated aqueous
NaCl, dried over Na2SO4, and concentrated under reduced pressure.
The resultant residue was purified by flash silica gel column
chromatography (EtOAc/n-hexane ) 1:1) to give compound 17 as
a white foam (5.24 g, 95%). Mp: 68-72 °C. [R]21D: -41.7 (c
3-C-Allyl-3,5-di-O-benzyl-1,2-O-isopropylidene-4-C-(p-tolu-
enesulfonyloxymethyl)-R-D-ribofuranose (13). To a solution of
compound 12 (14.5 g, 30.0 mmol) in anhydrous CH2Cl2 (280 mL)
were added Et3N (18.4 mL, 132 mmol), p-TsCl (9.42 g, 49.4 mmol),
and DMAP (2.02 g, 16.5 mmol) at room temperature, and the
mixture was stirred for 19 h. Saturated aqueous NaHCO3 was added,
and the reaction mixture was stirred for a further 30 min and
extracted with EtOAc. The organic layer was washed with H2O
and saturated aqueous NaCl, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash silica
gel column chromatography (EtOAc/n-hexane ) 1:4) to give
compound 13 as a white solid (18.3 g, 93%). Mp: 115-116 °C.
[R]25D: +17.2 (c 0.89, CHCl3). IR νmax (KBr): 2936, 1361, 1177
0.79, CHCl3). IR νmax (KBr): 3033, 1746, 1693, 1367, 1230 cm-1
.
1
cm-1. H NMR (CDCl3): δ 1.27 (3H, s), 1.40 (3H, s), 2.31 (3H,
1H NMR (CDCl3): δ 1.42 (3H, d, J ) 1 Hz), 2.03 (3H, s), 2.09
(3H, s) 2.42 (3H, s), 2.34-2.42 (2H, m), 3.85, 3.92 (2H, ABq, J )
11 Hz), 4.09, 4.23 (2H, ABq, J ) 11 Hz), 4.11-4.22 (2H, m),
4.63, 4.73 (2H, ABq, J ) 12 Hz), 4.64, 5.00 (2H, ABq, J ) 12
Hz), 5.79 (1H, d, J ) 9 Hz), 6.21 (1H, d, J ) 9 Hz), 7.20-7.56
s), 2.44 (1H, dd, J ) 7, 15 Hz), 2.76 (1H, dd, J ) 7, 15 Hz), 3.42,
3.65 (2H, ABq, J ) 10 Hz), 4.37, 4.76 (2H, ABq, J ) 10 Hz),
4.38, 4.41 (2H, ABq, J ) 12 Hz), 4.50 (1H, d, J ) 4 Hz), 4.59,
4.70 (2H, ABq, J ) 12 Hz), 5.10-5.16 (2H, m), 5.65 (1H, d, J )
4 Hz), 5.76-5.92 (1H, m), 7.12-7.31 (12H, m), 7.69 (2H, d, J )
8 Hz). 13C NMR (CDCl3): δ 21.6, 25.9, 26.0, 35.8, 66.3, 67.2,
69.2, 73.4, 83.5, 85.5, 86.7, 103.5, 113.0, 119.4, 126.6, 127.0, 127.4,
127.5, 128.0, 128.1, 128.2, 129.5, 131.7, 132.9, 137.5, 138.3, 144.2.
Mass (FAB): m/z 617 (M + Na)+. Anal. Calcd for C33H38O8S: C,
66.65; H, 6,44; S, 5.39. Found: C, 66.49; H, 6.43; S, 5.31.
(1R,6S,7R,8R)-6-Benzyloxy-1-benzyloxymethyl-7,8-O-isopro-
pyridene-3,9-dioxabicyclo[4.3.0]nonane (14). To a solution of
compound 8 (182 mg, 0.30 mmol) in anhydrous THF (8.0 mL)
was added NaHMDS (1.0 M in THF, 1.2 mL, 1.2 mmol), and the
mixture was heated under reflux for 12 h. Saturated aqueous
NaHCO3 was added, and the solution was extracted with EtOAc.
The organic layer was washed with H2O and saturated aqueous
NaCl, dried over Na2SO4, and concentrated under reduced pressure
to give compound 14 as a white solid (110 mg, 86%). Mp: 105-
106 °C. [R]22D: +4.9 (c 0.70, CHCl3). IR νmax (KBr): 2943, 1456,
1377, 1210 cm-1. 1H NMR (CDCl3): δ 1.32 (3H, s), 1.35 (3H, s),
1.83-1.97 (1H, m), 2.36-2.41 (1H, m), 3.53, 4.53 (2H, ABq, J )
10 Hz), 3.56, (1H, dt, J ) 12, 3 Hz), 3.70, 4.03 (2H, ABq, J ) 11
Hz), 3.66-3.74 (1H, m), 4.51, 4.57 (2H, ABq, J ) 12 Hz), 4.63,
5.07 (2H, ABq, J ) 12 Hz), 4.99 (1H, d, J ) 5 Hz), 5.99 (1H, d,
(12H, m), 7.67 (1H, s), 7.68 (2H, d, J ) 6 Hz), 7.95 (1H, brs). 13
C
NMR (CDCl3): δ 11.9, 20.8, 21.0, 21.8, 28.1, 59.4, 67.0, 70.0,
71.2, 73.9, 77.6, 83.1, 83.1, 87.3, 111.4, 126.8, 127.3, 127.6, 127.9,
128.2, 128.4, 128.7, 129.8, 131.6, 135.5, 136.2, 137.4, 145.1, 150.6,
163.1, 169.5, 170.6. Mass (FAB): m/z 751 (M + H)+. Anal. Calcd
for C38H42 N2O12S: C, 60.43; H, 5.67; N, 3.71; S, 4.27. Found: C,
60.43; H, 5.67; N, 3.77; S, 4.03.
3′-C-Acetoxyethyl-3′,5′-di-O-benzyl-5-methyl-4′-C-(p-toluene-
sulfonyloxymethyl)uridine (18). To a stirred solution of compound
17 (1.32 g, 1.76 mmol) in THF (25 mL) was added 40% aqueous
MeNH2 (1.5 mL, 18 mmol) at 0 °C and was stirred for 1 h. Further
40% aqueous MeNH2 (1.5 mL) was added and stirred for 1 h. The
solvent was evaporated under reduced pressure, and the residue
was extracted with EtOAc. The organic layer was washed with H2O
and saturated aqueous NaCl, dried over Na2SO4, and concentrated
under reduced pressure. The resultant residue was purified by flash
silica gel column chromatography (CHCl3/MeOH ) 40:1) to give
compound 18 as a white foam (1.08 g, 87%). Mp: 69-71 °C.
[R]21D: -10.6 (c 0.73, CHCl3). IR νmax (KBr): 3381, 3064, 1741,
1
1704, 1366, 1234 cm-1. H NMR (CDCl3): δ 1.49 (3H, s), 2.08
(3H, s), 2.40 (3H, s), 2.40-2.47 (2H, m), 3.72, 3.81 (2H, ABq, J
J. Org. Chem, Vol. 71, No. 4, 2006 1313

Sekiguchi et al.
) 11 Hz), 4.06, 4.20 (2H, ABq, J ) 10 Hz), 4.38 (1H, d, J ) 8
Hz), 4.29-4.40 (2H, m), 4.54 (1H, d, J ) 11 Hz), 4.61-4.65 (2H,
m), 4.88 (1H, d, J ) 11 Hz), 5.89 (1H, d, J ) 8 Hz), 7.16-7.36
mg, 73%). Mp: 172-175 °C. [R]26 -129.8 (c 0.81, CHCl3). IR
D
νmax (KBr): 3178, 2955, 1695, 1460, 1088 cm-1
.
1H NMR
(CDCl3): δ 1.29 (3H, s), 1.38 (3H, s), 1.56 (3H, s), 1.93-2.05
(1H, m), 2.30-2.35 (1H, m), 3.01 (3H, s), 3.25 (1H, d, J ) 10
Hz), 3.77-3.85 (3H, m), 4.29-4.36 (2H, m), 4.58, 5.33 (2H, ABq,
J ) 11 Hz), 4.67 (2H, s), 5.42 (1H, d, J ) 8 Hz), 6.56 (1H, d, 8
Hz), 7.26-7.47 (10H, m), 7.96 (1H, s). 13C NMR (CDCl3): δ 12.1,
24.4, 26.4, 27.8, 49.0, 62.1, 66.1, 66.4, 73.9, 75.5, 76.5, 79.1, 83.6,
86.6, 101.0, 110.6, 127.3, 127.5, 127.7, 128.1, 128.3, 128.6, 136.6,
137.5, 138.4, 151.1, 163.4. Mass (FAB) m/z 567 (M + H)+. Anal.
Calcd for C31H38N2O8‚1/3H2O: C, 65.02; H, 6.81; N, 4.89. Found:
C, 64.98; H, 6.81; N, 4.83.
(12H, m), 7.49 (1H, s), 7.69 (2H, d, J ) 8 Hz), 8.20 (1H, brs). 13
C
NMR (CDCl3): δ 12.0, 21.1, 21.8, 28.9, 60.0, 67.1, 69.5, 71.7,
74.0, 79.1, 83.6, 86.7, 87.6, 111.2, 127.0, 127.6, 127.7, 127.9, 128.3,
128.4, 128.7, 129.9, 131.5, 135.6, 136.3, 137.1, 145.3, 155.0, 163.2,
170.7. Mass (FAB): m/z 709 (M + H)+. HRMS (FAB) calcd for
C36H41N2O11S (M + H)+ 709.2432, found 709.2424.
3′-C-Acetoxyethyl-3′,5′-di-O-benzyl-2′-O-(2-methoxypro-
pyl)-5-methyl-4′-C-(p-toluenesulfonyloxymethyl)uridine (19). To
a solution of compound 18 (4.77 g, 6.74 mmol) in anhydrous
CH2Cl2 (67 mL) were added 2-methoxypropene (9.69 mL, 0.10
mol) and p-TsOH‚H2O (25.6 mg, 0.14 mmol) at 0 °C. After the
mixture was stirred for 3 h, saturated aqueous NaHCO3 was added,
and the mixture was extracted with EtOAc. The organic layer was
washed with H2O and saturated aqueous NaCl, dried over Na2-
SO4, and concentrated under reduced pressure. The resultant residue
was purified by flash silica gel column chromatography (EtOAc/
n-hexane ) 1:1) to give compound 19 as a white foam (4.72 g,
90%). Mp: 72-74 °C. [R]25D: -37.6 (c 0.71, CHCl3). IR νmax
(KBr): 2989, 1692, 1367, 1235, 1178 cm-1. 1H NMR (CDCl3): δ
1.24 (6H, s), 1.49 (3H, s), 2.07 (3H, s) 2.20-2.28 (1H, m), 2.42
(3H, s), 2.53-2.61 (1H,m), 3.00 (3H, s), 3.88, 3.94 (2H, ABq, J )
11 Hz), 4.10 (1H, d, J ) 11 Hz), 4.19-4.26 (3H, m), 4.55-4.62
(3H, m), 4.68 (1H, d, J ) 11 Hz), 5.18 (1H, d, J ) 11 Hz), 6.14
(1H, d, J ) 8 Hz), 7.21-7.36 (12H, m), 7.49 (1H, s), 7.69 (2H, d,
J ) 8 Hz), 8.06 (1H, s). 13C NMR (CDCl3): δ 11.9, 21.1, 21.8,
24.7, 25.3, 27.9, 50.1, 59.8, 67.0, 70.7, 71.9, 74.0, 78.5, 83.4, 84.0,
86.0, 101.9, 110.8, 126.8, 127.3, 127.7, 127.9, 128.3, 128.3, 128.7,
129.7, 131.9, 136.5, 136.6, 138.3, 145.0, 150.7, 163.2, 170.7. Mass
(FAB): m/z 781 (M + H)+. Anal. Calcd for C40H48O12N2S‚
1/5H2O: C, 61.24; H, 6.22; N, 3.57; S, 3.93. Found: C, 61.20; H,
6.15; N, 3.49; S, 4.11.
3′,5′-Di-O-benzyl-3′-C,4′-C-ethoxymethylene-5-methyluri-
dine (22). To a solution of compound 21 (466 mg, 0.84 mmol) in
THF-MeOH (9.0 mL, 2:1) was added p-TsOH‚H2O (64 mg, 0.34
mmol) at 0 °C, and the whole was stirred for 1 h. Saturated aqueous
NaHCO3 was added, and the solution was extracted with EtOAc.
The organic layer was washed with H2O and saturated aqueous
NaCl, dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by flash silica gel column chromatography
(EtOAc/n-hexane ) 1:1) to give compound 22 as a white foam
(355 mg, 85%). Mp: 87-89 °C. [R]27D: -45.5 (c 0.66, CHCl3).
1
IR νmax (KBr): 3429, 1687, 1489, 1271, 1071 cm-1. H NMR
(acetone-d6): δ 1.62 (3H, s), 1.93-2.01 (1H, m), 2.48-2.54 (1H,
m), 3.61, 4.21 (2H, ABq, J ) 9 Hz), 3.69-3.86 (2H, m), 3.95,
4.02 (2H, ABq, J ) 10 Hz), 4.60, 4.66 (2H, ABq, J ) 12 Hz),
4.72, 5.43 (2H, ABq, J ) 11 Hz), 5.03 (1H, dd, J ) 3, 7 Hz), 5.20
(1H, d, J ) 3 Hz), 6.10 (1H, d, J ) 7 Hz), 7.26-7.51 (10H, m),
7.91 (1H, s), 10.1 (1H, brs). 13C NMR (CDCl3): δ 12.5, 27.7, 62.4,
65.1, 66.7, 71.0, 73.7, 80.0, 83.0, 86.0, 94.5, 109.9, 127.1, 127.2,
127.9, 128.2, 128.4, 136.4, 137.0, 138.9, 152.1, 163.7. Mass
(FAB): m/z 495 (M + H)+. HRMS (FAB): calcd for C27H30O7N2
(M + H)+ 495.2132, found 495.2137.
3′,5′-Di-O-benzyl-3-N-benzyloxymethyl-3′-C,4′-C-ethoxymeth-
ylene-5-methyluridine (23). To a solution of compound 22 (106
mg, 0.21 mmol) in anhydrous DMF (2 mL) were added DBU (0.063
mL, 0.42 mmol) and benzyl chloromethyl ether (0.044 mL, 0.32
mmol) at 0 °C. After the mixture was stirred for 45 min, MeOH
was added, and the solution was extracted with EtOAc. The organic
layer was washed with H2O and saturated aqueous NaCl, dried over
Na2SO4, and concentrated under reduced pressure. The resultant
residue was purified by flash silica gel column chromatography
(EtOAc/n-hexane ) 1:2) to give compound 23 as a white foam
(116 mg, 90%). Mp: 48-49 °C. [R]26D: -32.9 (c 0.77, CHCl3).
3′,5′-Di-O-benzyl-3′-C-hydroxyethyl-2′-O-(2-methoxypropyl)-
5-methyl-4′-C-(p-toluenesulfonyloxymethyl)uridine (20). To a
solution of compound 19 (974 mg, 1.25 mmol) in THF-MeOH
(3:1, 18 mL) was added 2 M aqueous NaOH (1.3 mL, 2.6 mmol)
at 0 °C. After being stirred for 1.5 h, H2O was added, and the
solution was extracted with CH2Cl2. The organic layer was washed
with H2O and saturated aqueous NaCl, dried over Na2SO4, and
concentrated under reduced pressure. The resultant residue was
purified by flash silica gel column chromatography (EtOAc/n-
hexane ) 1:1) to give compound 20 as a white foam (806 mg,
87%). Mp: 90-92 °C. [R]25 -52.3 (c 0.90, CHCl3). IR νmax
IR νmax (KBr): 3430, 2954, 2874, 1698, 1657, 1458, 1071 cm-1
.
D
(KBr): 3417, 3190, 2990, 1692, 1364, 1176 cm-1
.
1H NMR
1H NMR (CDCl3): δ 1.77 (3H, s), 1.77-1.85 (1H, m), 2.47 (1H,
d, J ) 14 Hz), 3.41, 3.97 (2H, ABq, J ) 10 Hz), 3.65-3.73 (1H,
m), 3.84-3.88 (1H, m), 3.92, 4.31 (2H, ABq, J ) 10 Hz), 4.47
(2H, s), 4.54 (1H, d, J ) 5 Hz), 4.62, 5.39 (2H, ABq, J ) 11 Hz),
4.70 (2H, s), 5.11 (1H, s), 5.45, 5.49 (2H, ABq, J ) 9 Hz), 5.69
(1H, d, J ) 5 Hz), 7.25-7.44 (16H, m), 7.83 (1H, s). 13C NMR
(CDCl3): δ 13.3, 27.8, 62.4, 64.9, 66.8, 70.6, 70.7, 72.4, 73.6, 80.0,
83.2, 86.0, 94.8, 109.4, 127.1, 127.2, 127.3, 127.5, 127.6, 127.8,
128.2, 128.2, 128.4, 134.8, 137.1, 137.9, 138.9, 152.8, 163.0. Mass
(FAB): m/z 615 (M + H)+. Anal. Calcd for C35H38N2O8‚1/10H2O:
C, 68.19; H, 6.23; N, 4.54. Found: C, 68.01; H, 6.25; N, 4.49.
3′,5′-Di-O-benzyl-3-N-benzyloxymethyl-3′-C,4′-C-ethoxymeth-
ylene-2′-O-methyl-5-methyluridine (24). To a stirred suspension
of 60% sodium hydride (9.0 mg, 0.23 mmol) in anhydrous DMF
(0.6 mL) was added dropwise a solution of compound 23 (109 mg,
0.18 mmol) in anhydrous DMF (1.4 mL) at 0 °C. After the mixture
was stirred for 30 min, MeI (0.06 mL, 0.9 mmol) was added and
stirred for 1 h at room temperature. MeOH was added and
evaporated to dryness under reduced pressure, and the residue was
extracted with EtOAc and H2O. The organic layer was washed with
H2O and saturated aqueous NaCl, dried over Na2SO4, and concen-
trated under reduced pressure. The resultant residue was purified
by flash silica gel column chromatography (EtOAc/n-hexane ) 1:2)
to give compound 24 as a white foam (97 mg, 86%). Mp: 44-45
(CDCl3): δ 1.24 (3H, s), 1.27 (3H, s), 1.48 (3H, d, J ) 1 Hz),
1.68 (1H, brs), 2.04-2.18 (1H, m), 2.43 (3H, s), 2.51-2.57 (1H,
m), 3.00 (3H, s), 3.77-3.81 (2H, m), 3.84, 4.01 (2H, ABq, J ) 11
Hz), 4.14, 4.37 (2H, ABq, J ) 11 Hz), 4.54, 5.15 (2H, ABq, J )
12 Hz), 4.58, 4.65 (2H, ABq, J ) 11 Hz), 4.64 (1H, d, J ) 8 Hz),
6.12 (1H, d, J ) 8 Hz), 7.17-7.39 (12H, m), 7.50 (1H, d, J ) 1
Hz), 7.69 (2H, d, J ) 8 Hz), 8.06 (1H, brs). 13C NMR (CDCl3):
δ 11.9, 21.8, 24.7, 25.3, 31.2, 50.0, 57.7, 66.8, 71.2, 71.8, 74.0,
78.4, 84.0, 84.2, 86.1, 101.8, 110.8, 126.7, 127.2, 127.6, 127.9,
128.2, 128.6, 129.7, 132.0, 136.7, 136.7, 138.6, 144.9, 150.7, 163.3.
Mass (FAB): m/z 739 (M + H)+. Anal. Calcd for C38H46N2O11S‚
1/2H2O: C, 61.03; H, 6.33; N, 3.75; S, 4.34. Found: C, 61.06; H,
6.25; N, 3.62; S, 4.24.
3′,5′-Di-O-benzyl-3′-C,4′-C-ethoxymethylene-2′-O-(2-meth-
oxypropyl)-5-methyluridine (21). To a stirred solution of com-
pound 20 (728 mg, 0.99 mmol) in anhydrous THF (16 mL) was
added NaHMDS (1.0 M in THF, 3.0 mL, 3.0 mmol), and the
mixture was heated under reflux for 6 h. After the solution was
cooled to 0 °C, saturated aqueous NaHCO3 was added, and the
mixture was extracted with EtOAc. The organic layer was washed
with H2O and saturated aqueous NaCl, dried over Na2SO4, and
concentrated under reduced pressure. The residue was recrystallized
from EtOAc-n-hexane to give compound 21 as a white solid (400
1314 J. Org. Chem., Vol. 71, No. 4, 2006

Synthesis and Properties of Trans-3′,4′-Bridged Nucleic Acids
°C. [R]24D: -89.1 (c 0.80, CHCl3). IR νmax (KBr): 2953, 2885,
s), 7.21-7.40 (10H, m), 7.45 (1H, s), 8.52 (1H, brs). 13C NMR
(CDCl3): δ 12.0, 24.0, 61.4, 65,1, 65.4, 74.1, 74.1, 78.6, 82.0, 82.0,
127.2, 109.8, 127.8, 128.3, 128.3, 128.7, 128.7, 135.7, 135.8, 137.2,
151.2, 163.0, 196.5. Mass (FAB): m/z 493 (M + H) +. Anal. Calcd
for C27H28N2O7‚3/4H2O: C, 64.09; H, 5.88; N, 5.54. Found: C,
64.11; H, 6.12; N, 5.27.
1
1712, 1669, 1456, 1090 cm-1. H NMR (CDCl3): δ 1.65 (3H, s),
2.02-2.14 (1H, m), 2.45 (1H, brd, J ) 14 Hz), 3.31, 4.33 (2H,
ABq, J ) 10 Hz), 3.44 (3H, s), 3.78 (2H, d, J ) 8 Hz), 3.86, 4.28
(2H, ABq, J ) 11 Hz), 4.65-4.67 (5H, m), 4.87 (1H, d, J ) 8
Hz), 5.29 (1H, d, J ) 11 Hz), 5.47, 5.50 (2H, ABq, J ) 10 Hz),
6.55 (1H, d, J ) 8 Hz), 7.23-7.45 (15H, m), 7.80 (1H, s). 13C
NMR (CDCl3): δ 13.1, 27.6, 59.4, 61.8, 66.1, 66.2, 70.7, 72.1,
73.9, 74.9, 79.1, 83.5, 86.6, 87.7, 110.3, 127.3, 127.3, 127.4, 127.4,
127.6, 128.1, 128.2, 128.3, 128.6, 135.5, 136.8, 137.9, 138.4, 151.8,
163.3. Mass (FAB): m/z 629 (M + H)+. Anal. Calcd for
C36H40N2O8‚1/5H2O: C, 68.38; H, 6.44; N, 4.43. Found: C, 68.31;
H, 6.43; N, 4.45.
1-(3,5-Di-O-benzyl-3-C,4-C-ethoxymethylene-â-D-arabino-pento-
furanosyl)thymine (26). To a solution of compound 25 (65 mg,
0.13 mmol) in anhydrous THF (1.5 mL) was added DIBALH (0.53
mL, 0.53 mmol) at -78 °C, and the whole was stirred for 5 h at
the same temperature. The reaction mixture was partitioned with
saturated aqueous NH4Cl, filtered through Celite, and extracted with
EtOAc. The combined organic layer was washed with H2O and
brine. The organic layer was dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (CHCl3/MeOH ) 40:1) to give compound
26 (44 mg, 67%) as a white solid. Mp: 133-135 °C. [R]24D: -34.7
(c 0.87, CHCl3). IR νmax (KBr): 3342, 2956, 1695, 1478, 1392,
1279, 1173, 1065 cm-1. 1H NMR (acetone-d6): δ 1.70 (3H, d, J )
1 Hz), 1.96-2.06 (1H, m), 2.23-2.29 (1H, m), 3.68-3.77 (1H,
dt, J ) 11, 3 Hz), 3.82-3.87 (1H, dd, J ) 4, 11 Hz), 4.05, 4.09
(2H, ABq, J ) 9 Hz), 4.14, 4.25 (2H, ABq, J ) 10 Hz), 4.56, 4.70
(2H, ABq, J ) 11 Hz), 4.58-4.72 (2H, m), 4.88 (1H, brt, J ) 5
Hz), 5.25 (1H, d, J ) 6 Hz), 6.10 (1H, d, J ) 4 Hz), 7.26-7.45
(10H, m), 7.90 (1H, d, J ) 1 Hz) 10.05 (1H, brs). 13C NMR
(acetone-d6): δ 165.3, 151.7, 140.0, 139.7, 138.4, 129.8, 129.7,
129.0, 128.9, 128.8, 109.2, 92.2, 87.1, 84.3, 74.3, 73.1, 70.3, 65.8,
65.5, 62.6, 26.7, 13.4. Mass (EI): m/z 494 (M+). Anal. Calcd for
C27H30N2O7‚1/2H2O: C, 64.40; H, 6.21; N, 5.56. Found: C, 64.16;
H, 6.17; N, 5.41.
3′-C,4′-C-Ethoxymethylene-2′-O-methyl-5-methyluridine (1).
To a suspension of 20% palladium hydroxide over carbon (150
mg) in EtOH (1.0 mL) was added a solution of compound 24
(278 mg, 0.44 mmol) in EtOH (3.0 mL) and cyclohexene (2.2
mL, 22 mmol). The mixture was heated under reflux for 7 h and
filtered. The solution was evaporated under reduced pressure, and
the residue was recrystallized from EtOH to give compound 1 (57
mg, 39%) as a white solid. Mp: 256-259 °C. [R]27D: -49.7 (c
1
0.53, CH3OH). IR νmax (KBr): 3238, 2966, 1705, 1469 cm-1. H
NMR (CD3OD): δ 1.84 (1H, dd, J ) 3, 13 Hz), 1.90 (3H, d, J )
1 Hz), 2.07 (1H, dt, J ) 13, 6 Hz), 3.42 (3H, s), 3.47 (1H, d, J )
9 Hz), 3.75 (1H, dd, J ) 5, 11 Hz), 3.95-4.08 (4H, m), 4.63 (1H,
d, J ) 7 Hz), 6.17 (1H, d, J ) 7 Hz), 8.10 (1H, d, J ) 1 Hz). 13
C
NMR (CDCl3): δ 12.6, 33.5, 59.2, 63.0, 64.7, 65.9, 84.7, 85.7,
89.7, 111.8, 139.5, 153.0, 166.1. Mass (FAB): m/z 329 (M + H)+.
Anal. Calcd for C14H20N2O7: C, 51.22; H, 6.14; N, 8.53. Found:
C, 51.25; H, 6.12; N, 8.31.
5′-O-(4,4′-Dimethoxytrityl)-3′-C,4′-C-ethoxymethylene-2′-O-
methyl-5-methyluridine (29). To a solution of compound 1 (58
mg, 0.18 mmol) in anhydrous pyridine (0.8 mL) were added
dimethoxytrityl chloride (88 mg, 0.26 mmol) and DMAP (4 mg,
0.04 mmol) at 50 °C, and the mixture was stirred for 2.5 h. Saturated
aqueous NaHCO3 was added, and the solution was extracted with
EtOAc. The organic layer was washed with H2O and saturated
aqueous NaCl, dried over Na2SO4, and concentrated under reduced
pressure. The resultant residue was purified by flash silica gel
column chromatography (EtOAc/n-hexane ) 3:1) to give compound
29 as a white foam (87 mg, 77%). Mp: 127-129 °C. [R]24D: -49.4
(c 0.91, CHCl3). IR νmax (KBr): 3458, 3195, 3007, 2953, 2837,
1-(3,5-Di-O-benzyl-3-C,4-C-ethoxymethylene-â-D-arabino-pento-
furanosyl)-3-N-benzyloxymethylthymine (27). To a solution of
compound 26 (600 mg, 1.2 mmol) in anhydrous DMF (12 mL)
were added DBU (0.2 mL, 1.33 mmol) and benzyl chloromethyl
ether (0.25 mL, 1.82 mmol) at 0 °C, and the mixture was stirred
for 3 h. The reaction mixture was partitioned with MeOH,
evaporated under reduced pressure, and extracted with CH2Cl2. The
organic layer was washed with H2O and brine. The organic layer
was dried over Na2SO4 and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(EtOAc/n-hexane ) 2:3) to give compound 27 (89 mg, 68%) as a
white solid. Mp: 72-74 °C. [R]24D: -1.88 (c 0.89, CHCl3). IR
1
1691, 1508, 1464, 1254 cm-1. H NMR (CDCl3): δ 1.28 (3H, s),
νmax (KBr): 3407, 3072, 3030, 2956, 1700, 1657, 1460, 1369, 1278,
1
1.45-1.53 (2H, m), 2.94 (1H, s), 3.40-3.48 (2H, m), 3.51 (2H,
s), 3.80 (6H, s), 3.91 (1H, dt, J ) 10, 4 Hz), 4.09 (1H, d, J ) 9
Hz), 4.74 (1H, d, J ) 6 Hz), 6.10 (1H, d, J ) 6 Hz), 6.84 (4H, dd,
J ) 2, 8 Hz), 7.24-7.39 (9H, m), 7.98 (1H, d, J ) 1 Hz), 8.18
(1H, brs). 13C NMR (CDCl3): δ 11.4, 32.4, 55.3, 59.2, 61.8, 65.8,
65.9, 75.8, 83.8, 84.3, 88.3, 88.5, 111.3, 113.1, 113.1, 127.5, 127.9,
128.8, 130.7, 130.7, 134.7, 137.3, 142.2, 158.9. Mass (FAB): m/z
631 (M + H)+. Anal. Calcd for C35H38N2O9‚1/2H2O: C, 65.72; H,
6.14; N, 4.38. Found: C, 65.72; H, 6.15; N, 4.31.
1166, 1065 cm-1. H NMR (acetone-d6): δ 1.74 (3H, d, J ) 2
Hz), 2.00-2.07 (1H, m), 2.24-2.28 (1H, m), 3.74 (1H, dt, J )
11, 4 Hz), 3.85 (1H, dd, J ) 5, 11 Hz), 4.07, 4.10 (2H, ABq, J )
10 Hz), 4.12, 4.21 (2H, ABq, J ) 10 Hz), 4.60, 4.69 (2H, ABq, J
) 12 Hz), 4.61, 4.67 (2H, ABq, J ) 12 Hz), 4.65 (2H, s), 4.94
(1H, dd, J ) 5, 6 Hz), 5.19 (1H, d, J ) 6 Hz), 5.40, 5.46 (2H,
ABq, J ) 9 Hz), 6.11 (1H, d, J ) 5 Hz), 7.24-7.45 (15H, m),
7.95 (1H, d, J ) 2 Hz). 13C NMR (CDCl3): δ 13.3, 25.6, 61.4,
64.2, 65.2, 69.7, 70.3, 70.3, 72.3, 72.9, 73.4, 82.6, 86.2, 91.9, 108.6,-
127.1, 127.1, 127.7, 127.7, 127.8, 127.8, 128.2, 128.4, 128.5, 135.7,
137.2, 137.2, 137.7, 151.0, 163.5. Mass (EI): m/z 614 (M +). Anal.
Calcd for C35H38N2O8‚3/4H2O: C, 66.92; H, 6.33; N, 4.46. Found:
C, 66.90; H, 6.18; N, 4.38.
3′,5′-Di-O-benzyl-2′-deoxy-3′-C,4′-C-ethoxymethylene-5-meth-
yl-2′-oxouridine (25). To a suspension of Dess-Martin periodinane
(1.26 g, 2.97 mmol) in CH2Cl2 (10 mL) was added a solution of
compound 22 (979 mg, 1.98 mmol) in CH2Cl2 (10 mL), and the
mixture was stirred at room temperature for 30 min. The mixture
of saturated aqueous Na2S2O3 and NaHCO3 (2:1) was added, and
the solution was extracted with CH2Cl2. The combined organic layer
was washed with saturated aqueous Na2S2O3, H2O, and brine. The
organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (EtOAc/n-hexane ) 1:1) to give compound 25 (945
mg, 97%) as a white solid. Mp: 86-88 °C. [R]22D: -121.7 (c
1.00, CHCl3). IR νmax (KBr): 3032, 2879, 1785, 1691, 1458, 1389,
1-(3,5-Di-O-benzyl-3-C,4-C-ethoxymethylene-2-O-methyl-â-D-
arabino-pentofuranosyl)-3-N-benzyloxymethylthymine (28). To
a suspension of 60% NaH (85 mg, 2.2 mmol) in anhydrous DMF
(5.0 mL) was added dropwise a solution of compound 27 (675 mg,
1.1 mmol) in anhydrous DMF (5.0 mL) at 0 °C. After being stirred
for 30 min, MeI (0.34 mL, 5.5 mmol) was added and the mixture
stirred for 30 min. The reaction mixture was partitioned with MeOH,
evaporated under reduced pressure, and extracted with EtOAc. The
combined organic layer was washed with H2O and brine. The
organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
1
1281, 1211, 1090, 1014 cm-1. H NMR (CDCl3): δ 1.49 (3H, s),
1.93-2.05 (1H, m), 2.27-2.32 (1H, m), 3.50, 4.23 (2H, ABq, J )
11 Hz), 3.75-3.82 (1H, m), 3.82-3.91 (1H m), 3.83, 4.52 (2H,
ABq, J ) 11 Hz), 4.34 (2H, t, J ) 10 Hz), 4.52 (2H, s), 6.51 (1H,
chromatography (EtOAc/n-hexane ) 2:3) to give compound 28 (645
:
mg, 93%) as a white solid. Mp: 86-88 °C. [R]22 +12.2 (c 0.79,
D
J. Org. Chem, Vol. 71, No. 4, 2006 1315

Sekiguchi et al.
CHCl3). IR νmax (KBr): 3073, 2951, 1703, 1658, 1457, 1370, 1280,
reaction mixture was partitioned with saturated NaHCO3 and
extracted with EtOAc. The organic layer was washed with H2O
and brine. The organic layer was dried over Na2SO4 and evaporated
under reduced pressure. The residue was purified by silica gel
column chromatography (EtOAc/n-hexane ) 2:3) to give compound
31 (88 mg, 55%) as a white solid. Mp: 75-77 °C. 31P NMR
(CDCl3): δ 141.4, 142.7. Mass (FAB): m/z 831 (M + H)+. HRMS
(FAB): calcd for C44H56N4O10P (M + H)+ 831.3735, found
831.3715.
1
1241, 1204, 1164, 1093 cm-1. H NMR (CDCl3): δ 1.80 (3H, d,
J ) 1 Hz), 1.90-2.02 (1H, m), 2.13-2.19 (1H, m), 3.35 (3H, s),
3.71 (1H, m), 3.87 (1H, m), 3.82, 4.01 (2H, AB, J ) 10 Hz), 4.18
(2H, AB, J ) 9 Hz), 4.37 (1H, d, J ) 5 Hz), 4.53 (2H, s), 4.54,
4.65 (2H, AB, J ) 12 Hz), 4.74 (2H, s), 5.51 (2H, s), 6.06 (1H, d,
J ) 5 Hz), 7.24-7.39 (15H, m), 7.91 (1H, d, J ) 1 Hz). 13C NMR
(CDCl3): δ 13.3, 25.7, 60.5, 61.2, 64.3, 64.6, 68.2, 70.2, 72.0, 73.1,
81.5, 82.1, 86.0, 90.3, 108.3, 126.8, 127.1, 127.5, 127.8, 128.1,
128.3, 128.5, 135.5, 136.9, 137.9, 150.7, 163.7. Mass (EI): m/z
628 (M+). Anal. Calcd for C36H40N2O8‚1/3H2O: C, 68.12; H, 6.46;
N, 4.13. Found: C, 67.91; H, 6.35; N, 4.34.
1-(3-C,4-C-Ethoxymethylene-2-O-methyl-â-D-arabino-pento-
furanosyl)thymine (2). To a suspension of 20% palladium
hydroxide over carbon (48 mg) in MeOH (1.0 mL) was added
compound 28 (96 mg, 0.15 mmol) in MeOH (2.0 mL) and
ammonium formate (4.8 g, 7.6 mmol). The mixture was heated
under reflux for 7.5 h and filtered. The solution was evaporated
under reduced pressure and the residue was purified by silica gel
column chromatography (CHCl3/MeOH ) 20:1) to give compound
2 (32 mg, 63%) as a white solid. Mp: 241-243 °C. [R]22D: +19.9
(c 1.00, MeOH). IR νmax (KBr): 3441, 1651, 1479, 1285, 1034
cm-1.1H NMR (CD3OD): δ 1.74-1.88 (1H, m), 1.88 (3H, d, J )
1 Hz), 1.97-2.09 (1H, m), 3.32 (3H, s), 3.77-3.83 (1H, m), 3.86-
3.90 (1H, m), 3.93, 4.07 (2H, AB, J ) 11 Hz), 3.95 (1H, d, J ) 5
Hz), 3.98 (2H, s), 6.13 (1H, d, J ) 5 Hz), 8.07 (1H, d, J ) 1 Hz).
13C NMR (CDCl3): δ 12.6, 16.6, 31.7, 60.2, 60.8, 62.4, 65.2, 77.7,
86.8, 87.6, 91.0, 109.5, 139.5, 152.2, 166.8. Mass (EI): m/z 328
(M+). Anal. Calcd for C14H20N2O7‚1/2H2O: C, 49.80; H, 6.27; N,
8.30. Found: C, 49.62; H, 6.11; N, 8.20.
Oligonucleotide Synthesis. Synthesis of ODNs containing trans-
3′,4′-BNA monomer 2 (ODN2-ODN4) was performed in 0.2 µmol
scale on an automated DNA synthesizer using the phosphoramidite
approach. As an activator, 5-ethylthio-1H-tetrazole (ETT) was used
for every coupling step. The trans-3′,4′-BNA amidite 30 was
manually coupled as follows. Just before the coupling step for
30, the column reactor was detached from the DNA synthe-
sizer. The column was filled with 0.1 M solution of 30 in MeCN
(0.05 mL) and 0.25 M solution of ETT in MeCN (0.05 mL) and
vigorously shaken for 24 h at 50 °C. The column was then attached
to the DNA synthesizer, and the reaction cycle was continued after
the coupling step of 30. Every ODN synthesis was performed on
DMTr-ON mode. Cleavage from the CPG support and removal of
protecting groups were accomplished by using 28% aqueous
ammonia (55 °C for 12 h). The crude ODNs bearing a DMTr group
were detritylated and purified with a C18 solid-phase extraction
cartridge [washed with 10% MeCN aqueous solution, detritylated
with 2% TFA aqueous solution, and eluted with 35% MeOH
aqueous solution]. The obtained ODNs were again purified by
reversed-phase HPLC [buffer A, 0.1 M TEAA; buffer B, 0.1 M
TEAA/MeCN ) 1:1, B 12% to 22.6%/20 min, linear gradient; flow
rate, 3.0 mL/min]. The composition of the ODN2-ODN4 was
confirmed by MALDI-TOF-MS analysis: m/z ODN2 [M - H]-
calcd 3718.51, found 3719.35; ODN3 [M - H]- calcd 3718.51,
found 3718.14; ODN4 [M - H]- calcd 3718.51, found 3718.92.
UV Melting Experiments. The UV melting profiles were
recorded in 10 mM sodium phosphate buffer (pH 7.2) containing
100 mM NaCl at a scan rate of 0.5 °C/min with detection at 260
nm. The final concentration of each ODN was 4.0 µM. The melting
temperatures were obtained as the maxima of the first derivative
of the melting curves.
1-[5-O-(4,4′-Dimethoxytrityl)-3-C,4-C-ethoxymethylene-2-O-
methyl-â-D-arabino-pentofuranosyl]thymine (30). To a suspen-
sion of silver trifluoromethanesulfonate (1.55 g, 6.0 mmol) in
anhydrous CH2Cl2 (1.0 mL) was added dropwise 4,4′-dimethoxy-
trityl chloride (2.1 g, 6.0 mmol) in anhydrous CH2Cl2 (5.0 mL),
and then the mixture was stirred for 1 h at room temperature. The
supernatant fluid (2.0 mL, 3.4 equiv) was added to a solution of
compound 2 (198 mg, 0.60 mmol) in anhydrous CH2Cl2-pyridine
(1:1, 2.5 mL), and the mixture was stirred for 5 h at room
temperature. The reaction mixture was partitioned with saturated
aqueous NaHCO3 and extracted with EtOAc. The organic layer
was washed with H2O and brine. The organic layer was dried over
Na2SO4 and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (CHCl3/MeOH )
30:1) to give compound 30 (382 mg, 100%) as a white solid. Mp:
270-272 °C. [R]25D: -31.3 (c 0.60, CHCl3). IR νmax (KBr): 3397,
Circular Dichroism (CD) Spectroscopy. CD spectra were
recorded at 10 °C in the quartz cuvette of 1 cm optical path length.
The samples were prepared in the same manner as described in
the UV melting experiments. The molar ellipticity was calculated
from the equation [θ] ) θ/cl, where θ is the relative intensity, c is
the sample concentration, and l is the cell path length in centimeters.
2957, 1689, 1608, 1509, 1465, 1279, 1250, 1176, 1106,1038 cm-1
.
1H NMR (CDCl3): δ 1.44 (3H, d, J ) 1 Hz), 1.64-1.69 (1H, m),
1.70 (3H, s), 1.76-1.88 (1H, m), 2.81 (1H, brs), 3.14 (3H, s), 3.64
(1H, m), 3.70 (1H, m), 3.76, 4.09 (2H, ABq, J ) 14 Hz), 3.77
(6H, s), 3.84-3.94 (2H, m), 6.05 (1H, d, J ) 4 Hz), 6.77-6.82
(4H, m), 7.21-7.42 (9H, m), 7.94 (1H, d, J ) 1 Hz), 9.25 (1H,
brs). 13C NMR (CDCl3): δ 12.2, 31.0, 55.3, 55.3, 60.0, 60.8, 61.3,
64.8, 77.5, 85.0, 85.3, 86.7, 90.0, 109.2, 112.8, 112.9, 126.9, 127.6,
128.4, 130.1, 130.2, 136.1, 136.1, 137.5, 144.8, 150.3, 158.4, 158.4,
164.4. Mass (FAB): m/z 631 (M + H)+. HRMS (FAB): calcd for
C35H39N2O9 (M + H)+ 631.2656, found 631.2640.
1-[3-O-(N,N′-Diisopropylamino-â-cyanoethoxyphosphino)-5-
O-(4,4′-dimethoxytrityl)-3-C,4-C-ethoxymethylene-2-O-methyl-
â-D-arabino-pentofuranosyl]thymine (31). To a solution of com-
pound 30 (121 mg, 0.19 mmol) in anhydrous MeCN-THF (1:1,
2.0 mL) were added 2-cyanoethyl N,N,N′,N′-tetraisopropylphos-
phordiamidite (0.15 mL, 0.46 mmol) and 4,5-dicyanoimidazole (46
mg, 0.39 mmol), and the whole was stirred for 15 h at 50 °C. The
Acknowledgment. This work was financially supported in
part by PRESTO from Japan Science and Technology Agency
(JST), a SUNBOR Grant from the Suntory Institute for
Bioorganic Research, a Grant-in-Aid from Japan Society for
the Promotion of Science, and a Grant-in-Aid from the Ministry
of Education, Science, and Culture, Japan.
Supporting Information Available: General experimental
1
procedures, copies of H and 13C NMR of compounds 1, 2, 4-8,
11-14, 17-30, HPLC chromatograms, and MALDI-TOF-MS
spectra for ODN2-ODN4. UV melting profiles for the duplexes
between ODN1-ODN4 and DNA or RNA complement and the
duplexes between ODN2 and DNA complement with or without
one base mismatch. This material is available free of charge via
the Internet at http://pubs.acs.org.
JO051187L
1316 J. Org. Chem., Vol. 71, No. 4, 2006