Design, synthesis, and evaluation of a novel bridged nucleic acid, 2′,5′-BNAON, with S-type sugar conformation fixed by N-O linkage

Article abstract of DOI:10.1016/j.tet.2008.12.073

We designed a novel 2′-O,5′-N bridged nucleic acid, 2′,5′-BNA^ON, whose sugar puckering was fixed to S-type conformation by an N-O linkage. A dimer unit formed from 2′,5′-BNA^ON-U and thymidine was synthesized via a coupling reaction b

Full text of DOI:10.1016/j.tet.2008.12.073

Tetrahedron 65 (2009) 2116–2123
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design, synthesis, and evaluation of a novel bridged nucleic acid, 20,50-BNAON,
with S-type sugar conformation fixed by N–O linkage
,
Tetsuya Kodama a, Chieko Matsuo a, Hidetsugu Ori a, Tetsuya Miyoshi a, Satoshi Obika a
,
*
,
a
Kazuyuki Miyashita b , Takeshi Imanishi
*
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
b Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan
a r t i c l e i n f o
a b s t r a c t
We designed a novel 20-O,50-N bridged nucleic acid, 20,50-BNAON, whose sugar puckering was fixed to
S-type conformation by an N–O linkage. A dimer unit formed from 20,50-BNAON-U and thymidine was
synthesized via a coupling reaction between a protected 20,50-BNAON-U monomer and a thymidine de-
rivative. Introduction of 20,50-BNAON-U into DNA was carried out using conventional phosphoramidite
chemistry with a DNA synthesizer. The hybridization abilities of 20,50-BNAON-U-modified oligonucleo-
tides against DNA or RNA complement were evaluated.
Article history:
Received 1 November 2008
Received in revised form 18 December 2008
Accepted 18 December 2008
Available online 30 December 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Oligonucleotide
Nucleic acid
BNA
Conformational restriction
Carbamate
S-Type
1. Introduction
III showed depressed hybridization ability (ꢀ7 to ꢀ12 ꢁC/modifi-
cation)3 even compared with II. These results naturally lead us to an
Artificial or modified oligonucleotides, which can specifically
interact with complementary oligonucleotides and which show
resistance to enzymatic degradation are potential tools for genome
technology. For this reason, numerous nucleic acid analogues have
been synthesized and characterized.
analogue having a carbamate linkage and a conformationally re-
stricted sugar moiety to gain high thermal stability.
Conformational restriction is a powerful strategy for designing
artificial nucleic acids possessing excellent hybridization abilities
and resistance to enzymatic digestion. We have developed the
One approach to oligonucleotide modification is replacement of
a phosphodiester moiety by another linkage. For example, re-
placement with a phosphorothioate1 linkage is known to be
a useful modification in several fields. Oligonucleotides in which
a phosphodiester linkage is replaced by a carbamate linkage (O30–
CO–N50) also have potential as materials for genome technology
because of their resistance to enzymatic digestion (Fig. 1).2,3 The
hybridization abilities of oligonucleotides containing carbamate
linkages have been reported previously, and showed interesting
tendencies. The simple DNA analogue II, which contains a carba-
mate linkage, showed slightly lower hybridization ability (ꢀ3 to
ꢀ5 ꢁC/modification),2 while the conformationally flexible analogue
O
O
O
Thy
Thy
Thy
O
O
O
O
O
O
O
O
O
O
α
NH
β
NH
O
1
NR
O
N
2
Thy
O
N
Thy
γ
O
O
χ
1'
O
P O
O
P O
O
P O
O
O
O
(R = H, alkyl)
I
III
Imanishi et al.3
II
Waldner et al.2
This study
* Corresponding authors. Tel.: þ81 6 6879 8200; fax: þ81 6 6879 8204. (S.O);
tel.:/fax: +81 721 24 9652 (K.M).
E-mail addresses: obika@phs.osaka-u.ac.jp (S. Obika), miyasik@osaka-ohtani.
ac.jp (K. Miyashita).
Figure 1. Structures of DNA analogues with carbamate moieties (O30–CO–N50) re-
placing the natural phosphodiester linkage. Thy; thymin-1-yl.
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.12.073

T. Kodama et al. / Tetrahedron 65 (2009) 2116–2123
2117
OH
various types of conformationally restricted nucleic acid ana-
logue,4–9 and found that 20,40-BNAs (bridged nucleic acids, often
represented as LNA10) whose sugar conformation is fixed to N-type
show high binding affinity with the RNA complement (Fig. 2A).5–7
In particular, the 20,40-BNANC-modified oligonucleotides, which
contain an N–O bond between the 20- and 40-positions, showed
extremely high tolerance to nuclease and high binding affinity with
RNA complement and double-stranded DNA (dsDNA).7
OH
A
Base
Base
O
O
4'
HO
4'
2'
2'
HO
2',4'-BNANC
NH
O
O
N
H
2',4'-BNA/LNA
OH
O
B
5'
Base
Base
O
Although the unique characteristics of nucleic acid analogues
with S-type sugar conformation have not yet been identified well,
BNA with S-type sugar conformation are believed to behave as DNA
analogues based on the good hybridizing abilities of tricyclo-DNA,11
50-amino-30,50-BNA,8 and 20-deoxy-trans-30,40-BNA.9 In these ana-
logues, the sugar pucker of the furanose part is restricted to S-type
(Fig. 2B). To comparewith the hybridization abilityof DNA analogues
3'
OH
OH
tricyclo-DNA
5'-amino-3',5'-BNA
OH
O
H
N
Base
O
Base
O
5'
II and III, we designed a novel bridged nucleic acid I, 20,50-BNAON
,
O
with S-type sugar pucker and a carbamate linkage (Figs.1 and 2B). A
preliminary molecular modeling study suggested that the sugar
puckering of this novel BNA would be rigidly restricted to S-type by
2'
OH
OH
2',5'-BNAON
2'-deoxy-trans-3',4'-BNA
an N–O bond between C20 and C50 and that its dihedral angle (N50–
g
C50–C40–C30)would beca.15ꢁ. Itis thoughtthattheseconformational
characteristics are suitable for DNA duplex formation because these
Figure 2. Examples of nucleic acid analogue possessing (A) N-type or (B) S-type sugar
conformation.
characteristics (e.g., S-type sugar pucker and moderate
g dihedral
angle) are found mainly in crystal structures of natural dsDNA.12
2. Result and discussion
O
N
O
N
2.1. Synthesis of 20,50-BNAON
NBOM
O
NBOM
O
To evaluate the characteristics of 20,50-BNAON, we synthesized
a uridine analogue 20,50-BNAON-U. Uridine derivative 1,13,14 which
was prepared from uridine according to a known procedure was
transformed to mesylated diol 2 over two steps (Scheme 1). The
primary hydroxyl group of 2 was substituted by N,O-di-Boc-
hydroxyamine under Mitsunobu reaction conditions15 to afford
N,O-di-Boc-50-deoxy-50-hydroxyaminouridine derivative 3a. Pro-
tection of the hydroxyl group at the 30-position with TBDPSCl and
imidazole followed by the removal of Boc group on the hydroxy-
amino group using TFA gave compound 4a. Next, the reaction to
obtain compound 5a, with a bridged structure between the 20- and
50-position of 4a, was investigated under various reaction condi-
tions (e.g., Et3N, LDA, KHMDS). However, the only compound that
could be identified was not the desired compound 5a but com-
pound 6 (Scheme 2). We attempted the bridging reaction using N-
Cbz-protected 50-deoxy-50-hydroxyaminouridine derivative 4b as
well, but the desired 20,50-BNAON derivative 5b was not obtained.
The characteristic tetracyclic structure of 6 was determined by
X-ray crystallographic analysis after derivatizing to compound 7,
which was provided by treating compound 6 with TBAF (Scheme 2
and Fig. 3).
HO
O
O
O
a,b
Si
O
HO OMs
Si
OH
O
1
2
c
O
N
O
Boc
NBOM
O
NBOM
HO
R
O
R
N
O
N
N
O
O
d,e
TBDPSO OMs
HO OMs
3a: R = Boc
3b: R = Cbz
4a: R = H
4b: R = Cbz
Scheme 1. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, 0 ꢁC; (b) TBAF, THF, 85%
over two steps; (c) Boc-NH-O-Boc, Ph3P, DBAD, THF 71% for 3a, Cbz-NH-O-Boc, Ph3P,
DBAD, THF, 65% for 3b; (d) TBDPSCl, imidazole, DMF; (e) TFA, CH2Cl2, 78% for 4a; 64%
for 4b over two steps.
In order to obtain 20,50-BNAON skeleton, we investigated the
possibility of carrying out N5–C6 bond cleavage of 6 by N-alkylative
b-elimination (Scheme 3. Table 1). First, a dichloromethane solu-
tion of 6 was refluxed in presence of benzyl bromide, but no
O
O
O
O
C5
NBOM
O
H
H
R
N
O
C6
N5'
NBOM
O
NBOM
O
HO
R
NBOM
O
N
N
N
N
N
N
N
C5'
O
O
O
O
b
a
O
O
C1'
C2'
TBDPSO OMs
OTBDPS
OH
OTBDPS
5a: R = H
5b: R = Cbz
6
4a: R = H
4b: R = Cbz
7
Scheme 2. Reagents and conditions: (a) KHMDS, THF, 0 ꢁC, 53%; (b) TBAF, THF, 98%.

2118
T. Kodama et al. / Tetrahedron 65 (2009) 2116–2123
Table 1
b
-Elimination reaction of 50-amino group from uracil moiety
Run
Conditionsa
Results
1
BnBr, CH2Cl2
BnBr, toluene
BnBr, o-xylene
MPMCl, o-xylene
No reaction
8a (18%)b
8a (46%)b
8b (50%)b
8c (11%)b
2
3b
4
5
p-Nitrophenyl chloroformate, o-xylene
a
Reactions were performed under reflux conditions until there was no change on
TLC profile.
b
Isolated yield.
reagent. The 20,50-BNAON-U dimer unit 17 was incorporated into the
center of an oligonucleotide 19 using an automated DNA synthesizer
and standard phosphoramidite protocols. The modified oligonu-
cleotide 19 was purified by HPLC, and its sequence composition was
verified by MALDI-TOF mass spectroscopy.
Figure 3. ORTEP plot of compound 7.
2.2. Duplex-forming ability
reaction occurred (run 1). Higher-temperature conditions were
effective for this reaction, and N50-benzyl derivative 8a was
obtained in 18% and 46% yields, respectively, by refluxing in toluene
(run 2) and o-xylene (run 3). It was found that benzyl bromide
could be replaced by other electrophiles to give methoxybenzyl-
and p-nitrophenoxycarbonyl derivatives 8b (run 4) and 8c (run 5).
Evidence for the occurrence of these b-elimination reactions was
provided by NMR spectra of 8 showing vinyl signals at the C5 and
C6 positions of the uracil base.
For synthesis of the dimer unit I conjugated with a carbamate
linker, we attempted to prepare the 50-amino derivative 12 bearing
a free secondary amino group as follows (Scheme 4). After the N3-
BOM group of 6 was removed via 9 by hydrogenolysis and
hydrolysis, the N50–C6 bond of 10 was cleaved attending on
methoxybenzylation to afford 11. Unfortunately, treatment of 11
with DDQ induced addition of 50-amino group to the C6 position,
reforming 10. These results suggested that the distance between
N50- and C6 position is too close to stand 5a and/or 12.
As an alternative method, synthesis of the dimer unit I with
a carbamate structure was performed via compound 8c, avoiding 12.
First, since the desired 8c was not obtained efficiently under the
conditions described in Table 1, the reaction conditions were opti-
mized. It was found that addition of tetra-n-butylammonium iodide
and 2,6-di-tert-butyl-4-methylpyridine to the reaction mixture
worked well, allowing compound 8c to be obtained in 55% yield
(Scheme 5). Treatment with NaH in DMF resulted in a coupling re-
action between 8c and 3-N-benzyloxymethyl-50-O-(4,40-dime-
thoxytrityl)thymidine16 to give the fully protected 20,50-BNAON-U
dimer unit 13 successfully. After deprotections and the additional
suitable protection, the phosphoramidite building block 17 was fi-
nally obtained by phosphitylation with a phosphorodiamidite
The duplex-forming ability of oligonucleotide 19, containing
20,50-BNAON, with complementary DNA (20Dna) and RNA (20Rna)
was evaluated by means of UV melting experiments. Single modi-
fication of the oligonucleotide led to significant destabilization of
its duplex DNA and RNA (ca. ꢀ30 ꢁC, Table 2), and the modified
oligonucleotides did not show any mismatch recognition abilities
(data not shown). These results suggest that the uracil base of 20,50-
BNAON-U could not pair with any bases on the opposite strand. One
reason for the loss of its hydrogen bonding ability due to 20,50-
BNAON modification might be steric and/or electrostatic repulsion
between the 6-H atom of the thymine base and the 20-O atom of the
bridged structure: the torsion angle around glycoside bond
c would
be fixed at an unsuitable direction for hybridizations. Nielsen and
co-workers17 reported that the torsion angle of a tricyclic nucleo-
side containing an arabino-type oxy group, deviated slightly from
the values of around typically found for nucleosides with S-type
conformation, and this resulted in strong destabilization of its du-
plexes. In addition, the replacement of a phosphate linkage with
a carbamate linkage is another possible cause of the destabilization.
20,50-BNAON must have had less flexibility than the expected around
the 50-position, and the linkage described by torsion angle
have been unfavorably restricted.
a may
O
O
H
H
NR
O
NH
O
N
N
N
N
O
O
O
O
b
OTBDPS
6: R = BOM
9: R = CH2OH
OTBDPS
10
a
O
O
c
5
H
R
N
O
6
O
O
NBOM
O
d
5'
NBOM
O
N
O
N
N
MPM
O
O
conditions
Table 1
H
NH
O
NH
O
N
O
N
O
N
N
O
O
OTBDPS
OTBDPS
6
8a: R = Bn
OTBDPS
OTBDPS
8b: R = MPM
NO2
11
O
12
8c: R =
Scheme 4. Reagents and conditions: (a) cyclohexene, Pd(OH)2, EtOH, reflux; (b) NaOH,
THF, H2O, THF, 75% over two steps; (c) MPMCl, o-xylene, reflux, 45%; (d) DDQ, CH2Cl2,
H2O, 0 ꢁC, 50%.
O
Scheme 3.
b-Elimination reaction.

T. Kodama et al. / Tetrahedron 65 (2009) 2116–2123
2119
O
O
N
O
N
NBOM
N
OH
NH
N
O
O
O
O
DMTrO
O
HO
O
HO
O
O2N
O
O
O
O
O
O
N
O
O
O
O
O
OH
O
NBOM
O
NBOM
O
NH
O
a
b
c
N
O
N
O
N
O
N
O
N
N
6
N
N
O
O
O
O
or
OTBDPS
OTBDPS
OTBDPS
OTBDPS
8c
13
14
O
O
O
NH
NH
NH
O
N
O
N
N
O
DMTrO
O
DMTrO
O
DMTrO
O
O
O
O
O
O
O
e
O
O
f
O
N
d
NH
O
NH
O
NH
O
N
O
N
O
N
N
N
O
O
O
O
O
P
O
OH
OTBDPS
NC
NiPr2
17
16
15
Scheme 5. Reagents and conditions: (a) p-nitrophenyl chloroformate, TBAI, 2,6-di-tert-butyl-4-methylpyridine, o-xylene, reflux, 55%; (b) 3-N-BOM-50-O-DMTr-thymidine, NaH,
THF, 0 ꢁC, 87%; (c) BCl3, CH2Cl2, ꢀ78 ꢁC, 84%; (d) DMTrCl, pyridine, 63%; (e) TBAF, THF, 97%; (f) (iPr2N)2POCH2CH2CN, diisopropylammonium tetrazolide, CH3CN, THF, 78%.
atmosphere. Melting points were measured on a Yanagimoto micro
melting points apparatus and are uncorrected. 1H, 13C, and 31P NMR
spectra were recorded on JEOL EX-270 (1H, 270 MHz; 13C,
67.8 MHz) and GX-500 (1H, 500 MHz; 31P 202 MHz) instruments.
Table 2
Tm Values of 20,50-BNAON-U oligonucleotides with complementary DNA and RNAa,b
Oligonucleotides
Tm Values (ꢁC)
20Dna
20Rna
Values for d are in parts per million relative to tetramethylsilane or
50-d(GCGTTTTTTGCT)-30 (18)
50-d(GCGTTTUTTGCT)-30 (19)
50
48
deuterated solvent as internal standard. 31P NMR spectrum was
recorded at 121.5 MHz with 85% H3PO4 as external standard. IR
spectra were recorded on a JASCO FT/IR-200 spectrometer. FAB-
Mass spectra were measured on a JEOL JMS-600 or JMS-700 mass
spectrometer. Column chromatography was carried out using Fuji
Silysia BW-127ZH, BW-300, and FL-100D. X-ray crystallography
was carried out on a Rigaku R-AXIS RAPID-S instrument. DNA
synthesis was performed using an Applied Biosystems ExpediteÔ
8909 synthesizer. MALDI-TOF-MS spectra were recorded in nega-
tive ion mode on Brucker Daltonics Autoflex II TOF/TOF instrument.
UV melting experiments were carried out using a SHIMADZU UV-
1650PC instrument.
17 (ꢀ33)c
18 (ꢀ30)c
a
Target sequence: 50-d(AGCAAAAAACGC)-30 (20Dna) and 50-r(AGCAAAAAACGC)-
30 (20Rna).
b
Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl
and 4 mM of each oligonucleotide strand.
c
Parentheses showed difference between Tnmatural and Tmmodified. TU: dimer unit I
(structure was illustrated in Fig. 1).
3. Conclusion
We synthesized a novel bridged nucleic acid containing a uracil
base, 20,50-BNAON-U, whose sugar puckering was fixed to S-type by
an N–O linkage, and successfully incorporated the resulting dimer
unit, linked by a carbamate moiety, into an oligonucleotide. The
hybridization ability of the 20,50-BNAON-modified oligonucleotide
with the complementary single strand DNA/RNA was found to be
lower than that of the natural oligonucleotide.18
4.2. 3-N-Benzyloxymethyl-20-O-methanesulfonyluridine (2)
To a mixture of 1 (1.65 g, 2.71 mmol) and Et3N (1.18 mL,
8.33 mmol) in CH2Cl2 (33 mL) was added MsCl (0.32 mL,
4.16 mmol) at 0 ꢁC, and the resulting mixture was stirred at room
temperature for 1 h. After addition of aqueous saturated NaHCO3 to
the reaction mixture, the contents were extracted with CH2Cl2. The
organic layer was washed with H2O and brine, dried over Na2SO4,
and evaporated to give a crude mixture as a yellow oil. To a solution
of the crude mixture in THF (29 mL) was added TBAF (1.0 M solu-
tion in THF, 5.77 mL, 5.77 mmol), and the solution was stirred at
room temperature for 1 h. After concentration, the resulting crude
mixture was purified by silica gel column chromatography (CHCl3/
4. Experimental
4.1. Material and methods
Unless otherwise mentioned, all chemicals from commercial
sources were used without further purifications. Acetonitrile
(MeCN), dichloromethane (CH2Cl2), pyridine, and triethylamine
(Et3N) used in reactions were distilled from calcium hydride.
o-Xylene was dried over calcium hydride, and was distilled. Tetra-
hydrofuran (THF) was distilled from lithium aluminum hydride just
EtOH¼15:1) to afford 2 (1.02 g, 2.30 mmol, 85% over two steps) as
21
a white powder. Mp 122–124 ꢁC. [
a
]
D
þ33.5 (c 0.98, MeOH). IR nmax
before use. All reactions were performed under
a
nitrogen
(KBr): 1090, 1181, 1336, 1703, 2939, 3312 cmꢀ1 1H NMR (CD3OD)
.

2120
T. Kodama et al. / Tetrahedron 65 (2009) 2116–2123
d
3.24 (3H, s), 3.75 (1H, dd, J¼2, 13 Hz), 3.94 (1H, d, J¼13 Hz), 4.03
was stirred at room temperature for 2 h. The reaction mixture was
diluted with EtOAc, washed with H2O and brine, dried over Na2SO4,
and evaporated to give a crude product, which was purified by silica
gel column chromatography (CHCl3 then CHCl3/MeOH¼50:1) to
(1H, m), 4.33 (1H, dd, J¼5, 6 Hz), 4.65 (2H, s), 5.09 (1H, dd, J¼3,
5 Hz), 5.46 (2H, s), 5.73 (1H, d, J¼8 Hz), 6.03 (1H, d, J¼3 Hz), 7.24–
7.32 (5H, m), 8.05 (1H, d, J¼8 Hz). 13C NMR (CDCl3) dC 38.8, 61.1,
69.2, 71.5, 73.1, 82.7, 85.8, 89.6, 102.2, 128.5, 128.6, 129.2, 139.3,
140.8, 152.3, 164.5. MS (FAB): m/z 443 (MHþ). High-resolution MS
(FAB): calcd for C18H23O9N2S (MHþ) 443.1124, found: 443.1127.
afford 3b (509 mg, 0.737 mmol, 65%) as anwhite solid. Mp 54–56 ꢁC.
21
[a
]
þ18.5 (c 0.99, CHCl3). IR nmax (KBr): 1096, 1360, 1667, 1717,
D
3445 cmꢀ1. 1H NMR (CDCl3)
d 1.46 (9H, s), 3.23 (3H, s), 3.50 (1H, d,
J¼15 Hz), 4.10 (1H, d, J¼15 Hz), 4.21 (2H, m), 4.68 (2H, s), 5.05 (1H, d,
J¼3 Hz), 5.18 (1H, d, J¼12 Hz), 5.24 (1H, d, J¼12 Hz), 5.45 (2H, s), 5.69
(1H, d, J¼8 Hz), 5.79 (1H, d, J¼1 Hz), 7.24–7.41 (11H, m). 13C NMR
(CDCl3) dC 27.5, 38.8, 51.5, 68.9, 69.8, 70.2, 72.2, 80.0, 81.4, 86.2, 89.9,
102.3,127.6,127.6,127.9,128.2,128.4,134.8,137.6,138.2,150.6,151.7,
156.0, 162.2. MS (FAB): m/z 692 (MHþ). High-resolution MS (FAB):
calcd for C31H38O13N3S (MHþ) 692.2125, found: 692.2106.
4.3. 3-N-Benzyloxymethyl-50-N,O-bis(tert-butoxy-
carbonyl)hydroxyamino-50-deoxy-20-O-
methanesulfonyluridine (3a)
To a solution of
2 (1.78 g, 4.02 mmol) and Ph3P (1.59 g,
6.03 mmol) in THF (25 mL) was added a solution of Boc-NH-O-Boc
(2.14 g, 9.17 mmol) in THF (5 mL) and di-tert-butyl azodicarboxylate
(1.56 g, 6.78 mmol) in THF (4 mL) at 0 ꢁC, and the resulting mixture
was stirred at room temperature for 1 h. The reaction mixture was
diluted with EtOAc, washed with H2O and brine, dried over Na2SO4,
and evaporated to give a crude product, which was purified by silica
gel column chromatography (CHCl3 then CHCl3/MeOH¼50:1) to
4.6. 50-(N-Benzyloxycarbonyl)hydroxyamino-3-N-benzyloxy-
methyl-30-O-tert-butyldiphenylsilyl-50-deoxy-20-O-
methanesulfonyluridine (4b)
TBDPSCl (0.425 mL, 1.64 mmol) was added to a solution of 3b
(377 mg, 0.545 mmol) and imidazole (186 mg, 2.73 mmol) in DMF
(3 mL) at 0 ꢁC. After stirring at room temperature for 10 h, the re-
action mixture was diluted with Et2O, washed with H2O and brine,
dried over Na2SO4, and was concentrated to give a crude product.
To a solution of the obtained product in CH2Cl2 (10 mL) was added
TFA (1.1 mL) and the mixture was stirred at room temperature for
2 h. The reaction mixture was basified with aqueous saturated
NaHCO3 at 0 ꢁC, and then extracted with CH2Cl2. The organic layer
was washed with H2O and brine, dried over Na2SO4, and evapo-
rated. The resulting crude mixture was purified by silica gel column
chromatography (hexane/EtOAc¼2:3) to afford 4b (291 mg,
afford 3a (1.88 g, 2.86 mmol, 71%) as a white solid. Mp 60–61 ꢁC.
24
[
a]
þ38.3 (c 1.00, CHCl3). IR nmax (KBr): 1096,1171,1277,1362,1455,
D
1668, 1716, 1783, 2980, 3444 cmꢀ1. 1H NMR (CDCl3)
d 1.48 (9H, s),
1.52 (9H, s), 2.61 (1H, br s), 3.23 (3H, s), 3.99 (2H, m), 4.17–4.24 (2H,
m), 4.68 (2H, s), 5.05 (1H, dd, J¼2, 5 Hz), 5.46 (2H, s), 5.77 (1H, d,
J¼8 Hz), 5.84 (1H, d, J¼2 Hz), 7.22–7.37 (5H, m), 7.51 (1H, d, J¼8 Hz).
13C NMR (CDCl3) dC 27.6, 28.0, 38.9, 51.2, 69.7, 70.2, 72.3, 80.3, 81.6,
83.7, 85.8, 89.7, 102.2, 127.5, 127.6, 128.2, 137.6, 138.3, 150.6, 152.0,
155.4, 162.2. MS (FAB): m/z 658 (MHþ). High-resolution MS (FAB):
calcd for C28H40O13N3S (MHþ) 658.2282, found: 658.2261.
4.4. 3-N-Benzyloxymethyl-30-O-tert-butyldiphenylsilyl-50-
deoxy-50-hydroxyamino-20-O-methanesulfonyluridine (4a)
0.352 mmol, 64% over two steps) as a white solid. Mp 47–50 ꢁC.
24
[a
]
þ31.0 (c 1.00, CHCl3). IR nmax (KBr): 1105, 1355, 1457, 1671,
D
1715, 2938, 3292 cmꢀ1. 1H NMR (CDCl3)
d 1.03 (9H, s), 2.65 (3H, s),
TBDPSCl (2.00 mL, 7.69 mmol) was added to a solution of 3a
(3.54 g, 5.38 mmol) and imidazole (1.07 g, 15.7 mmol) in DMF
(13.5 mL) at 0 ꢁC, and the mixture was stirred at room temperature
for 11 h. The reaction mixture was then diluted with Et2O, washed
with H2O and brine, dried over MgSO4, and was concentrated to
give a crude product. This was dissolved in CH2Cl2 (50 mL), TFA
(6.20 mL, 83.3 mmol) was added, and the mixture was stirred at
room temperature for 4 h. The reaction mixture was basified with
aqueous saturated NaHCO3 and was extracted with EtOAc. The or-
ganic layer was washed with aqueous saturated NaHCO3, H2O and
brine, dried over Na2SO4, and evaporated. The resulting crude
mixture was purified by silica gel column chromatography (hexane/
3.27 (1H, dd, J¼15, 5 Hz), 3.41 (1H, dd, J¼15, 4 Hz), 4.14 (1H, d,
J¼8 Hz), 4.47 (1H, dd, J¼4, 5 Hz), 4.54 (2H, s), 4.94 (1H, m), 4.95 (1H,
d, J¼12 Hz), 5.02 (1H, d, J¼12 Hz), 5.33 (2H, s), 5.54 (1H, d, J¼8 Hz),
5.87 (1H, d, J¼6 Hz), 7.08–7.37 (17H, m), 7.54–7.61 (4H, m). 13C NMR
(CDCl3) dC 19.4, 26.8, 37.9, 51.4, 68.2, 70.2, 71.9, 76.9, 82.7, 90.0,
102.6, 127.6, 127.7,127.8,127.9,127.9,128.2,128.3, 128.4, 130.1, 130.3,
132.1, 132.4, 135.3, 135.7, 135.7, 137.5, 139.8, 151.1, 157.3, 162.1. MS
(FAB): m/z 830 (MHþ). High-resolution MS (FAB): calcd for
C42H48O11N3SSi (MHþ) 830.2779, found: 830.2754.
4.7. (2S,8R,10R,11S,12S)-5-(Benzyloxymethyl)-11-tert-butyldi-
phenylsilyloxy-9,13-dioxa-1,5,7-triazatetracyclo-
[8.3.1.02,7.08,12]tetradecane-4,6-dione (6)
EtOAc¼1:1 and 1:3) to afford 4a (2.93 g, 4.21 mmol, 78% over two
24
steps) as a white solid. Mp 54–56 ꢁC. [
a]
þ23.2 (c 0.97, CHCl3). IR
D
nmax (KBr): 1104, 1176, 1355, 1672, 1717, 2938 cmꢀ1. 1H NMR (CDCl3)
1.12 (9H, s), 2.65 (1H, dd, J¼5, 14 Hz), 2.81 (3H, s), 2.93 (1H, d,
d
To a solution of 4a (1.42 g, 2.04 mmol) in THF (20 mL) at 0 ꢁC was
added KHMDS (0.5 M in toluene, 6.45 mL, 3.23 mmol), and the
mixture was stirred at the same temperature for 30 min. Aqueous
saturated NH4Cl was added to the reaction mixture, and the
resulting mixture was diluted with EtOAc and separated. The or-
ganic layer was washed with H2O and brine, dried over Na2SO4, and
evaporated. The obtained crude product was purified by silica gel
J¼14 Hz), 4.20 (1H, d, J¼4 Hz), 4.60 (1H, m), 4.64 (3H, s), 5.14 (1H,
dd, J¼5, 5 Hz), 5.45 (2H, s), 5.73 (1H, d, J¼8 Hz), 5.95 (1H, d, J¼5 Hz),
7.25–7.48 (12H, m), 7.65–7.72 (4H, m). 13C NMR (CDCl3) dC 19.4,
26.8, 38.1, 53.9, 70.2, 72.0, 72.0, 77.7, 82.6, 90.4, 102.5, 127.6, 127.7,
127.8, 127.9, 128.2, 130.1, 130.2, 132.2, 132.7, 135.7, 135.8, 137.6, 140.1,
150.9, 162.2. MS (FAB): m/z 696 (MHþ). High-resolution MS (FAB):
calcd for C34H42O9N3SSi (MHþ) 696.2411, found: 696.2430.
column chromatography (CHCl3) to afford 6 (655 mg, 1.09 mmol,
24
53%) as a white solid. Mp 184–186 ꢁC. [
a
]
ꢀ44.5 (c 0.98, CHCl3). IR
D
4.5. 50-(N-Benzyloxycarbonyl-O-tert-butoxycarbonyl)-
hydroxyamino-3-N-benzyloxymethyl-50-deoxy-20-O-
methanesulfonyluridine (3b)
nmax (KBr): 1111, 1274, 1357, 1444, 1681, 1728, 2861, 2939 cmꢀ1. 1H
NMR (CDCl3)
d
1.08 (9H, s), 2.68 (1H, dd, J¼4, 16 Hz), 2.91 (1H, d,
J¼14 Hz), 2.93 (1H, d, J¼16 Hz), 3.32 (1H, d, J¼14 Hz), 4.11–4.17 (3H,
m), 4.22 (1H, d, J¼4 Hz), 4.67 (2H, AB, J¼13 Hz), 5.31 (1H, d,
J¼10 Hz), 5.41 (1H, d, J¼10 Hz), 6.48 (1H, d, J¼5 Hz), 7.24–7.48 (11H,
m), 7.64–7.68 (4H, m). 13C NMR (CDCl3) dC 19.0, 26.7, 38.0, 60.2, 69.1,
70.2, 72.0, 75.2, 77.6, 80.4, 81.7,127.4,127.5,127.9,128.0,128.4,130.1,
130.2, 132.6, 132.9, 135.5, 138.5, 151.5, 168.2. MS (FAB): m/z 600
To a solution of 2 (500 mg, 1.13 mmol) and Ph3P (445 mg,
1.70 mmol) in THF (5 mL) was added a solution of Cbz-NH-O-Boc
(616 mg, 2.30 mmol) in THF (2.5 ml) and di-tert-butyl azodi-
carboxylate (391 mg, 1.70 mmol) at 0 ꢁC, and the resulting mixture

T. Kodama et al. / Tetrahedron 65 (2009) 2116–2123
2121
(MHþ). High-resolution MS (FAB): calcd for C33H38O6N3Si (MHþ)
600.2530, found: 600.2521.
s), 2.25 (1H, d, J¼12 Hz), 3.00 (1H, dd, J¼4, 12 Hz), 3.40 (1H, d,
J¼13 Hz), 3.72 (3H, s), 3.67–3.77 (2H, m), 4.06–4.13 (1H, m), 4.20
(1H, d, J¼4 Hz), 4.66 (2H, s), 4.81 (1H, s), 5.33 (1H, d, J¼10 Hz), 5.44
(1H, d, J¼10 Hz), 5.64 (1H, d, J¼8 Hz), 5.98 (1H, d, J¼3 Hz), 6.69–
6.99 (4H, m), 7.25–7.70 (15H, m), 7.79 (1H, d, J¼8 Hz). 13C NMR
(CDCl3) dC 19.1, 26.8, 55.1, 58.4, 61.5, 69.9, 71.9, 79.2, 79.9, 89.4, 98.9,
113.3, 126.9, 127.5, 127.7, 127.8, 127.9, 128.1, 130.0, 130.1, 130.7, 132.2,
133.1, 135.4, 135.5, 137.9, 141.4, 150.6, 158.9, 163.1. MS (FAB): m/z 720
(MHþ). High-resolution MS (FAB): calcd for C41H46O7N3Si (MHþ)
720.3105, found: 720.3124.
4.8. (2S,8R,10R,11S,12S)-5-(benzyloxymethyl)-11-hydroxy-
9,13-dioxa-1,5,7-triazatetracyclo[8.3.1.02,7.08,12]tetradecane-
4,6-dione (7)
To a solution of 6 (36.0 mg, 0.060 mmol) in THF (2 mL) was
added TBAF (1.0 M solution in THF, 0.09 mL, 0.09 mmol), and the
solution was stirred at room temperature for 30 min. After con-
centration of the reaction mixture, the resulting crude mixture was
purified by silica gel column chromatography (CHCl3 and CHCl3/
4.11. (2S,8R,10R,11S,12S)-11-tert-butyldiphenylsilyloxy-9,13-
dioxa-1,5,7-triazatetracyclo[8.3.1.02,7.08,12]tetradecane-4,6-
dione (10)
EtOH¼15:1) to afford 7 (21.2 mg, 0.059 mmol, 98%) as a white solid.
21
Mp 179–182 ꢁC. [
a]
ꢀ17.7 (c 0.99, CHCl3). IR nmax (KBr): 1274,1446,
D
1680, 1728, 2961, 3303 cmꢀ1. 1H NMR (CDCl3)
d
2.45 (1H, d, J¼7 Hz),
2.68 (1H, dd, J¼4, 16 Hz), 2.98 (1H, d, J¼16 Hz), 3.08 (1H, dd, J¼4,
14 Hz), 3.54 (1H, d, J¼14 Hz), 4.09 (2H, m), 4.25 (1H, d, J¼4 Hz), 4.33
(1H, d, J¼5 Hz), 4.67 (2H, s), 5.31 (1H, d, J¼10 Hz), 5.40 (1H, d,
J¼10 Hz), 6.35 (1H, d, J¼5 Hz), 7.16–7.29 (5H, m). 13C NMR (CDCl3) dC
38.1, 60.2, 69.1, 70.3, 72.1, 73.7, 77.4, 79.8, 81.3, 127.4, 127.5, 128.3,
138.3, 151.5, 168.1. Some of the white solid 7 were recrystallized
from acetone for X-ray crystallographic analysis.
Crystallographic data (excluding structure factors) of 7 has been
deposited at the Cambridge Crystallographic Data Center as sup-
plementary publication no. CCDC 704648. Copy of the data can be
obtained, free of charge via www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi
(or from the Cambridge Crystallographic Data Center, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: þ44 1223 336033; or
deposit@ccdc.cam.ac.uk).
A mixture of 6 (78.2 mg, 0.130 mmol), 20% Pd(OH)2 on carbon
(64.5 mg), cyclohexene (0.54 mL, 5.34 mmol), and ethanol (4 mL)
was refluxed for 3.5 h. After cooling to room temperature, the re-
action mixture was filtered, and the filtrate was concentrated to give
a crude product. To a solution of the crude product was added an
aqueous solution of NaOH (16 mg in 0.25 mL of H2O) over an ice
bath, and the resulting mixture was stirred for 2 h at the same
temperature before being diluted AcOEt, washed with H2O and
brine, dried over Na2SO4, and evaporated. The resulting crude
product was purified by silica gel column chromatography (EtOAc/
n-hexane¼1:2 and 1:1) to afford 10 (47.8 mg, 99.7 mmol, 75% over
25
two steps) as a white solid. Mp 107–110 ꢁC. [
a
]
ꢀ45.8 (c 1.00,
D
CHCl3). IR nmax (KBr): 1115, 1286, 1446, 1713, 2937, 3046, 3221 cmꢀ1
.
1H NMR (CDCl3)
d
1.06 (9H, s), 2.75 (1H, dd, J¼4,17 Hz), 2.98 (1H, m),
3.04 (1H, dd, J¼12, 17 Hz), 3.36 (1H, d, J¼15 Hz), 4.11 (1H, m), 4.16
(1H, s), 4.27 (1H, d, J¼5 Hz), 4.66 (1H, dd, J¼4, 12 Hz), 6.46 (1H, d,
J¼5 Hz), 7.39–7.73 (10H, m). 13C NMR (CDCl3) dC 19.1, 26.8, 37.6, 60.3,
70.6, 75.2, 77.7, 79.6, 81.6, 127.8, 127.9, 130.0, 130.1, 132.4, 132.8,
135.4, 150.8, 168.4. MS (FAB): m/z 480 (MHþ). High-resolution MS
(FAB): calcd for C25H30O5N3Si (MHþ): 480.1955, found: 480.1931.
4.9. 50-Benzylamino-3-N-benzyloxymethyl-30-O-tert-
butyldiphenylsilyl-20-O,50-N-cyclo-50-
deoxyarabinouridine (8a)
A
mixture of 6 (32.5 mg, 50.1 mmol), BnBr (0.034 mL,
0.250 mmol) and o-xylene (0.4 mL) was refluxed for 48 h. The
resulting mixture was mixed with aqueous saturated NaHCO3 over
an ice bath and was diluted with EtOAc. The organic layer was
washed with H2O and brine, dried over Na2SO4, and evaporated.
The resulting crude product was purified by silica gel column
4.12. 30-O-tert-Butyldiphenylsilyl-20-O,50-N-cyclo-50-deoxy-50-
(4-methoxyphenyl)methylaminoarabinouridine (11)
A mixture of 10 (20.0 mg, 41.7 mmol), MPMCl (0.045 mL,
0.334 mmol), and o-xylene (0.3 mL) was refluxed for 32 h. After
cooling to room temperature, the reaction mixture was diluted
with EtOAc, and washed with aqueous saturated NaHCO3, H2O and
brine, dried over Na2SO4, and evaporated. The resulting crude
product was purified by silica gel column chromatography (EtOAc/
chromatography (CHCl3) to afford 8a (16.0 mg, 0.0232 mmol, 46%)
21
as colorless oil. [
a
]
þ58.6 (c 0.71, CHCl3). IR nmax (KBr): 1077, 1110,
D
1453,1665,1711, 2939 cmꢀ1. 1H NMR (CDCl3)
d 1.08 (9H, s), 2.28 (1H,
d, J¼12 Hz), 3.04 (1H, dd, J¼6, 12 Hz), 3.48 (1H, d, J¼12 Hz), 3.75
(1H, d, J¼12 Hz), 4.07 (1H, d, J¼2 Hz), 4.21 (1H, d, J¼5 Hz), 4.65 (2H,
s), 4.81 (1H, s), 5.34 (1H, d, J¼10 Hz), 5.44 (1H, d, J¼10 Hz), 5.67 (1H,
d, J¼8 Hz), 5.99 (1H, d, J¼2 Hz), 7.04–7.69 (20H, m), 7.83 (1H, d,
J¼8 Hz). 13C NMR (CDCl3) dC 18.9, 26.8, 58.5, 62.4, 69.9, 71.9, 72.8,
79.3, 79.8, 89.5, 99.0, 122.4, 127.5, 127.6, 127.7, 127.8, 128.0, 128.1,
129.4, 129.9, 130.1, 130.1, 132.2, 133.0, 135.1, 135.4, 135.5, 137.7, 141.6,
150.7, 163.2. MS (EI): m/z 689 (Mþ, 7), 400 (100). High-resolution
MS (EI): calcd for C40H43O6N3Si 689.2921, found: 689.2917.
n-hexane¼1:2) to afford 11 (11.0 mg, 18.3 mmol, 45%) as pale yel-
24
low oil. [
a]
þ126.5 (c 1.01, CHCl3). IR nmax (KBr): 1112, 1271, 1462,
D
1686, 2945, 3045, 3165 cmꢀ1. 1H NMR (CDCl3)
d 1.08 (9H, s), 2.25
(1H, d, J¼12 Hz), 3.01 (1H, dd, J¼5,12 Hz), 3.41 (1H, d, J¼12 Hz), 3.74
(1H, d, J¼12 Hz), 3.76 (3H, s), 4.07 (1H, s), 4.21 (1H, d, J¼5 Hz), 4.76
(1H, s), 5.62 (1H, dd, J¼2, 8 Hz), 6.00 (1H, d, J¼3 Hz), 6.76 (2H, d,
J¼9 Hz), 7.00 (2H, d, J¼9 Hz), 7.37–7.86 (10H, m), 7.85 (1H, d,
J¼8 Hz), 8.89 (1H, s). 13C NMR (CDCl3) dC 19.1, 26.8, 55.2, 58.1, 61.6,
79.2, 79.8, 89.1, 99.0, 113.5, 113.6, 126.9, 127.8, 127.9, 129.6, 130.0,
130.1, 130.7, 132.2, 132.9, 135.4, 135.5, 142.8, 149.9, 159.0, 163.7. MS
(FAB): m/z 600 (MHþ). High-resolution MS (FAB): calcd for
C33H38O6N3Si (MHþ): 600.2530, found: 600.2512.
4.10. 3-N-Benzyloxymethyl-30-O-tert-butyldiphenylsilyl-20-
O,50-N-cyclo-50-deoxy-50-(4-methoxyphenyl)methyl-
aminoarabinouridine (8b)
A mixture of 6 (21.0 mg, 0.035 mmol), MPMCl (0.047 mL,
0.334 mmol), and o-xylene (0.3 mL) was refluxed for 22 h. After
cooling to room temperature, the reaction mixture was diluted
with EtOAc, and washed with aqueous saturated NaHCO3, H2O and
brine, dried over Na2SO4, and evaporated. The resulting crude
product was purified by silica gel column chromatography (EtOAc/
4.13. 3-N-Benzyloxymethyl-30-O-tert-butyldiphenylsilyl-20-
O,50-N-cyclo-50-deoxy-(4-nitrophenoxy)carbonyl-
aminoarabinouridine (8c)
A mixture of 7 (256 mg, 0.427 mmol), 2,6-di-tert-butyl-4-
methylpyridine (146 mg, 0.711 mmol), tetra-n-butylammonium
iodide (27 mg, 0.072 mmol), 4-nitrophenyl chloroformate (347 mg,
1.72 mmol), and o-xylene (0.2 mL) was refluxed for 66 h. After
n-hexane¼1:5 and 1:3) to afford 8b (12.0 mg, 16.7 mmol, 50%) as
25
a pale yellow oil. [
a]
þ115.3 (c 0.80, CHCl3). IR nmax (KBr): 1078,
D
1110, 1249, 1452, 1665, 1710, 2941 cmꢀ1. 1H NMR (CDCl3)
d 1.08 (9H,

2122
T. Kodama et al. / Tetrahedron 65 (2009) 2116–2123
cooling to room temperature, the reaction mixture was diluted
with EtOAc, and washed with aqueous saturated NaHCO3, H2O and
brine, dried over Na2SO4, and evaporated. The resulting crude
product was purified by silica gel column chromatography (CHCl3)
164.2. MS (FAB): m/z 778 (MHþ). High-resolution MS (FAB): calcd
for C37H44O12N5Si (MHþ) 778.2756, found: 778.2726.
4.16. 50-O-Dimethoxytritylthymidylyl-(30-50-carbamoyl)-50-
amino-30-O-tert-butyldiphenylsilyl-20-O,50-N-cyclo-50-
deoxyarabinouridine (15)
to afford 8c (182 mg, 0.238 mmol, 55%) as a white solid. Mp 80–
25
82 ꢁC. [
a]
þ67.0 (c 1.10, CHCl3). IR nmax (KBr): 1082, 1120, 1351,
D
1149, 1671, 1722, 2973 cmꢀ1. 1H NMR (CDCl3)
d 1.12 (9H, s), 3.17 (1H,
d, J¼13 Hz), 4.18 (1H, dd, J¼5, 13 Hz), 4.27 (1H, d, J¼1 Hz), 4.41 (1H,
d, J¼5 Hz), 4.59 (2H, s), 5.13 (1H, s), 5.47 (2H, s), 5.77 (1H, d, J¼8 Hz),
6.09 (1H, d, J¼3 Hz), 7.05 (2H, d, J¼9 Hz), 7.26–7.73 (16H, m), 8.11
(2H, d, J¼9 Hz). 13C NMR (CDCl3) dC 19.1, 26.7, 49.8, 69.9, 71.9, 75.7,
78.7, 80.2, 88.7, 100.6, 121.7, 125.1, 127.5, 127.5, 128.0, 128.1, 128.1,
130.4, 130.5, 131.5, 135.4, 137.4, 138.3, 145.0, 150.1, 154.6, 162.4. MS
(FAB): m/z 765 (MHþ). High-resolution MS (FAB): calcd for
C40H41O10N4Si (MHþ) 765.2592, found: 765.2579.
To a solution of 14 (161 mg, 0.207 mmol) in pyridine (1.5 mL)
was added 4,40-dimethoxytrityl chloride (103 mg, 0.305 mmol),
and the mixture was stirred at room temperature for 16 h. The
reaction solution was mixed with aqueous NaHCO3 over an ice bath
and then extracted with AcOEt. The organic layer was washed with
H2O and brine, dried over Na2SO4, and evaporated. The resulting
crude product was purified by silica gel column chromatography
(EtOAc/n-hexane¼1:1 and 2:1) to give 15 (137 mg, 0.130 mmol,
24
63%) as a white solid. Mp 160–163 ꢁC. [
a
]
þ65.1 (c 0.98, CHCl3). IR
D
4.14. 3-N-Benzyloxymethyl-50-O-dimethoxytritylthymidylyl-
(30-50-carbamoyl)-50-amino-3-N-benzyloxymethyl-30-O-tert-
butyldiphenylsilyl-20-O,50-N-cyclo-50-deoxyarabinouridine (13)
nmax (KBr): 1108, 1260, 1456, 1696, 2942, 3063, 3198 cmꢀ1. 1H NMR
(CDCl3) 0.99 (9H, s), 1.30 (3H, s), 2.28–2.39 (2H, m), 2.81 (1H, d,
d
J¼13 Hz), 3.33 (1H, d, J¼8 Hz), 3.42 (1H, d, J¼8 Hz), 3.69 (6H, s), 3.89
(1H, m), 3.96 (1H, s), 4.09 (1H, s), 4.27 (1H, d, J¼4 Hz), 4.96 (1H, s),
5.00 (1H, s), 5.49 (1H, d, J¼8 Hz), 5.97 (1H, d, J¼3 Hz), 6.21 (1H, dd,
J¼5, 9 Hz), 6.72–6.75 (4H, m), 7.12–7.60 (21H, m), 10.26 (1H, br s),
10.59 (1H, br s). 13C NMR (CDCl3) dC 11.7, 19.0, 26.7, 37.5, 49.5, 55.2,
63.7, 75.8, 78.6, 78.8, 79.6, 83.9, 84.1, 87.1, 88.4, 100.8, 111.7, 113.2,
127.0, 127.8, 127.9, 128.0, 129.9, 130.2, 130.4, 131.5, 132.3, 135.0,
135.0, 135.1, 135.3, 135.3, 139.1, 144.0, 150.0, 151.2, 158.4, 158.4,
158.5, 163.1, 163.8. MS (FAB): m/z 1072 (MNaþ). High-resolution MS
(FAB): calcd for C57H59O13N5SiNa (MNaþ) 1072.3776, found:
1072.3798.
To a mixture of NaH (20.4 mg, 0.51 mmol) in THF (1 mL) was
slowly added a solution of 3-N-benzyloxymethyl-50-O-(4,40-dime-
thoxytrityl)thymidine16 (71.6 mg, 0.108 mmol) in THF (1.2 mL) over
an ice bath, and the mixture was stirred at the same temperature for
30 min. A solution of 8c (69.8 mg, 0.0913 mmol) in THF (1.5 mL) was
added to the reaction mixture, and the mixture was stirred for
further 3 h. The resulting mixture was diluted with Et2O, washed
with H2O and brine, dried over Na2SO4, and evaporated. The
resulting crude mixture was purified by silica gel column chroma-
tography (EtOAc/n-hexane¼1:2) to give 13 (104 mg, 0.0807 mmol,
21
87%) as a white solid. Mp 97–100 ꢁC. [
a]
þ25.6 (c 1.07, CHCl3). IR
4.17. 50-O-Dimethoxytritylthymidylyl-(30-50-carbamoyl)-50-
amino-20-O,50-N-cyclo-50-deoxyarabinouridine (16)
D
nmax (KBr): 1105, 1454, 1667, 1710, 2940 cmꢀ1. 1H NMR (CDCl3)
d 1.10
(9H, s), 1.36 (3H, s), 2.33 (2H, m), 3.02 (1H, d, J¼12 Hz), 3.32 (1H, d,
J¼10 Hz), 3.41 (1H, d, J¼10 Hz), 3.77 (7H, s), 3.92 (1H, s), 4.02 (1H,
dd, J¼4, 12 Hz), 4.17 (1H, s), 4.31 (1H, d, J¼4 Hz), 4.67 (2H, s), 4.69
(2H, s), 5.00 (1H, s), 5.20 (1H, d, J¼4 Hz), 5.46 (4H, s), 6.02 (1H, d,
J¼3 Hz), 6.24 (1H, t, J¼8 Hz), 6.80–6.83 (4H, m), 7.20–7.69 (31H, m).
13C NMR (CDCl3) dC 12.3, 19.1, 26.8, 38.3, 49.9, 55.2, 63.7, 70.0, 70.7,
72.1, 75.8, 78.2, 78.7, 79.8, 83.7, 84.9, 87.4, 88.8, 110.9, 113.2, 127.1,
127.4,127.5,127.6,127.9,128.0,128.1,128.1,128.2,130.0,130.3,130.5,
131.5, 132.4, 134.0, 134.9, 135.1, 135.4, 135.4, 135.8, 137.7, 137.8, 138.3,
143.9, 150.4, 150.5, 150.8, 158.5, 158.5, 163.2, 163.2. MS (FAB): m/z
1312 (MNaþ). High-resolution MS (FAB): calcd for C73H75O15N5SiNa
(MNaþ) 1312.4952, found: 1312.4955.
To a solution of 16 (187 mg, 0.178 mmol) in THF (2 mL) was
added TBAF (1.0 M solution in THF, 0.21 mL, 0.21 mmol), and the
solution was stirred at room temperature for 15 min. After con-
centration of the reaction mixture under reduced pressure, the
resulting crude mixture was purified by silica gel column chro-
matography (EtOAc/EtOH¼15:1) to afford 16 (140 mg, 0.172 mmol,
25
97%) as a white powder. Mp 100–104 ꢁC. [
a]
þ74.7 (c 0.87, CHCl3).
D
IR nmax (KBr): 1110, 1259, 1457, 1694, 2932, 3063, 3200 cmꢀ1
.
1H
NMR (acetone-d6)
d
1.25 (3H, s), 2.36 (1H, d, J¼7 Hz), 2.37 (1H, d,
J¼7 Hz), 3.22 (1H, dd, J¼3, 10 Hz), 3.33 (1H, dd, J¼3, 10 Hz), 3.35
(1H, d, J¼13 Hz), 3.66 (6H, s), 3.96 (1H, s), 4.17 (1H, dd, J¼5, 13 Hz),
4.23 (1H, s), 4.58 (1H, d, J¼5 Hz), 4.78 (1H, s), 5.04 (1H, br s), 5.19
(1H, s), 5.41 (1H, d, J¼8 Hz), 5.69 (1H, d, J¼3 Hz), 6.15 (1H, t, J¼7 Hz),
6.76–6.81 (4H, m), 7.11–7.35 (11H, m), 7.52 (1H, d, J¼1 Hz), 7.67 (1H,
d, J¼8 Hz), 10.12 (2H, br s). 13C NMR (acetone-d6) dC 11.4, 38.2, 50.0,
54.9, 64.0, 74.2, 77.7, 79.2, 80.1, 83.9, 84.6, 87.1, 88.3, 100.0, 110.6,
113.3, 127.0, 128.0, 128.2, 130.2, 130.2, 135.2, 135.5, 135.6, 140.2,
144.9, 150.1, 150.7, 158.9, 158.9, 163.3. MS (FAB): m/z 834 (MNaþ).
High-resolution MS (FAB): calcd for C41H41O13N5Na (MNaþ)
834.2599, found: 834.2589.
4.15. Thymidylyl-(30-50-carbamoyl)-50-amino-30-O-tert-
butyldiphenylsilyl-20-O,50-N-cyclo-50-deoxy-3-N-
hydroxymethylarabinouridine (14)
To a solution of 13 (96.5 mg, 0.0748 mmol) in CH2Cl2 (8 mL) at
ꢀ78 ꢁC was added a solution of BCl3 (1 M in CH2Cl2, 0.748 mL,
0.748 mmol). After stirring for 30 min at ꢀ78 ꢁC, MeOH was added
to the reaction mixture at the same temperature. The resulting
mixture was concentrated to give crude mixture, which was puri-
fied by silica gel column chromatography (CHCl3/MeOH¼15:1) to
4.18. 50-O-Dimethoxytritylthymidylyl-(30-50-carbamoyl)-50-
amino-20-O,50-N-cyclo-50-deoxy-30-O-(N,N-diisopropylamino-
give 14 (48.7 mg, 0.0651 mmol, 84%) as a white solid. Mp 105–
24
110 ꢁC. [
a]
þ79.6 (c 1.01, CHCl3). IR nmax (KBr): 1108, 1462, 1696,
b-cyanoethoxyphosphino)arabinouridine (17)
D
2940, 3439 cmꢀ1. 1H NMR (CDCl3)
d 1.10 (9H, s), 1.90 (3H, s), 2.28
(2H, m), 3.03 (1H, d, J¼13 Hz), 3.76 (1H, s), 3.79 (1H, d, J¼3 Hz), 3.95
(1H, dd, J¼2, 13 Hz), 4.07 (1H, m), 4.19 (1H, s), 4.36 (1H, m), 5.00
(1H, s), 5.06 (1H, s), 5.48 (2H, s), 5.67 (1H, d, J¼8 Hz), 5.74 (1H, d,
J¼8 Hz), 6.05 (1H, dd, J¼3, 13 Hz), 6.12 (1H, t, J¼6 Hz), 7.42–7.53
(7H, m), 7.61–7.70 (5H, m), 9.35 (1H, br s). 13C NMR (CDCl3) dC 13.0,
19.1, 26.7, 37.6, 49.4, 62.2, 64.9, 65.6, 75.8, 78.8, 79.8, 84.6, 85.6, 88.3,
88.7, 100.4, 110.6, 128.0, 128.1, 130.3, 130.4, 131.5, 132.3, 135.0, 135.3,
135.3, 139.0, 140.2, 149.7, 150.3, 150.6, 150.6, 162.9, 163.3, 163.9,
2-Cyanoethyl N,N,N0,N0-tetraisopropylphosphorodiamidite (0.07
mL, 0.219 mmol) was added dropwise to a mixture of 16 (105 mg,
0.130 mmol) and N,N-diisopropylammonium tetrazolide (30.4 mg,
0.151 mmol) in a mixed solution of MeCN and THF (3:1, v/v, 1.6 mL)
at room temperature. After stirring for 18 h, the solvent was
evaporated and the product was purified by silica gel column
chromatography (EtOAc/n-hexane¼2:1) followed by trituration
from n-hexane to afford 17 (102 mg, 0.101 mmol, 78%) as a white

T. Kodama et al. / Tetrahedron 65 (2009) 2116–2123
2123
powder. Mp 129–133 ꢁC. 31P NMR (CDCl3) dP 149.24, 150.88. MS
(FAB): m/z 1034 (MNaþ). High-resolution MS (FAB): calcd for
C50H58O14N7PNa (MNaþ) 1034.3677, found: 1034.3734.
2. (a) Waldner, A.; De Mesmaeker, A.; Lebreton, J. Bioorg. Med. Chem. Lett. 1994, 4,
405–408; (b) Stirchak, E. P.; Summerton, J. E.; Weller, D. D. J. Org. Chem. 1987, 52,
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4.19. Synthesis of oligodeoxynucleotides
4. Imanishi, T.; Obika, S. Chem. Commun. 2002, 1653–1659.
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Miyashita, K.; Imanishi, T. Nucleosides Nucleotides Nucleic Acids 2007, 26, 1625–
1628; (c) Rahman, S. M. A.; Seki, S.; Obika, S.; Haitani, S.; Miyashita, K.; Imanishi,
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4886–4896.
Synthesis of oligodeoxynucleotides 19 was carried out on
0.2 mmol scale using commercially available 2-cyanoethyl
a
phosphoramidites, compound 17 (0.1 M in MeCN), and dicya-
noimidazole as an activator. The synthesis followed the regular
protocol for DNA synthesis: however, for compound 17 a pro-
longed coupling time of 30 min was used. The 50-O-DMTr-Off
oligonucleotides were cleaved from the solid support by treat-
ment with concd ammonia at room temperature for 1.5 h, and
the protecting groups were removed by heating to 70 ꢁC for 3 h.
Purification was carried out using disposable gel filtration car-
tridges (NAPÔ-10 column from GE Healthcare) followed by HPLC
(Waters XTerraÒ MS C18 2.5 mm, 50 ꢁC) to afford the pure oli-
8. (a) Obika, S.; Onoda, M.; Morita, K.; Andoh, J.; Koizumi, M.; Imanishi, T. Chem.
Commun. 2001, 1992–1993; (b) Obika, S.; Nakagawa, O.; Hiroto, A.; Hari, Y.;
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Shibata, N.; Masaki, M.; Hari, Y.; Imanishi, T. Tetrahedron Lett. 2002, 43, 4365–
4368; (c) Obika, S.; Osaki, T.; Sekiguch, M.; Somjing, R.; Harada, Y.; Imanishi, T.
Tetrahedron Lett. 2004, 45, 4801–4804; (d) Osaki, T.; Obika, S.; Harada, Y.;
Mitsuoka, Y.; Sugaya, K.; Sekigchi, M.; Roongjang, S.; Imanishi, T. Tetrahedron
2007, 63, 8977–8986; (e) Osaki, T.; Obika, S.; Harada, Y.; Mitsuoka, Y.; Sugaya,
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2007, 26, 1079–1082; (f) Kodama, T.; Sugaya, K.; Harada, Y.; Mitsuoka Y.; Im-
anishi. T.; Obika, S. Heterocycles, 77, in press; (g) Kodama, T.; Sugaya, K.; Baba, T.;
Imanishi, T.; Obika, S. Heterocycles, 79, in press.
10. (a) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh, S. K.;
Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252–13253; (b) Koshkin, A. A.; Singh, S.
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K.; Kumar, R.; Nielsen, P.; Wengel, J. Angew. Chem., Int. Ed. 2000, 39, 1656–1659.
11. (a) Steffens, R.; Leumann, C. Helv. Chim. Acta 1997, 80, 2426–2439; (b) Steffens,
R.; Leumann, C. J. Am. Chem. Soc. 1999, 121, 3249–3255; (c) Renneberg, D.;
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12. Saenger, W. Principles of Nucleic Acid Structure; Springer: New York, NY, 1984.
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Chem. Commun. 2003, 1234–1235.
gonucleotide 19. MALDI-TOF-MS using
a 1:1 mixture of 3-
hydroxypicolinic acid and diammonium hydrogen citrate as the
matrix gave the following results: calcd for 19 3594.62 (MHꢀ),
found 3594.65.
4.20. UV melting experiments
UV melting experiments were carried out in a medium salt
buffer containing 10 mM sodium phosphate (pH 7.2) and 100 mM
NaCl using 4 mM concentrations of the two complementary se-
quences. The increase in absorbance at 260 nm as a function of time
was recorded while the temperature was raised linearly from 5 to
80 ꢁC at a rate of 0.5 ꢁC minꢀ1. The melting temperature was de-
termined as the local maximum of the first derivatives of the ab-
sorbance versus temperature curve.
Acknowledgements
Part of this work was supported by a Grant-in-Aid for Science
Research from the Japan Society for the Promotion of Science and
the Ministry of Education, Culture, Sports, Science, and Technology
of Japan.
15. Li, H.; Miller, M. J. J. Org. Chem. 1999, 64, 9289–9293.
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References and notes
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18. A small part of this paper was described in the following: Kodama, T.; Matsuo,
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159–160.
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