Recognition of T·A interruption by 2′,4′-BNAs bearing heteroaromatic nucleobases through parallel motif triplex formation

Article abstract of DOI:10.1016/j.bmc.2007.12.043

2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA) monomers bearing novel unnatural nucleobases, 4-(3-benzamidophenyl)-2-pyridone and 2-(N-methylbenzamido)thiazole, were synthesized and successfully incorporated into oligonucleotides. UV melting experim

Full text of DOI:10.1016/j.bmc.2007.12.043

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2945–2954
Recognition of TÆA interruption by 20,40-BNAs
bearing heteroaromatic nucleobases through parallel
motif triplex formation
Satoshi Obika,a,b Hiroyasu Inohara,a Yoshiyuki Haria,ꢀ and Takeshi Imanishia,*
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
bPRESTO, JST, Sanbancho Building, 5-Sanbancho, Chiyodaku, Tokyo 102-0075, Japan
Received 17 November 2007; revised 19 December 2007; accepted 20 December 2007
Available online 25 December 2007
Abstract—20-O,40-C-methylene bridged nucleic acid (20,40-BNA) monomers bearing novel unnatural nucleobases, 4-(3-benzamid-
ophenyl)-2-pyridone and 2-(N-methylbenzamido)thiazole, were synthesized and successfully incorporated into oligonucleotides.
UV melting experiments showed that the corresponding oligonucleotide derivatives formed stable triplexes with dsDNA targets even
in the presence of a TÆA interruption.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
2',4'-BNA/LNA
Oligonucleotides with a homopyrimidine sequence are
able to form triplexes with double-stranded DNA
O
Base
(dsDNA) in a sequence-specific manner via Hoogs-
teen-type hydrogen bonding.1–3 Sequence-specific triplex
formation would be useful in genetic therapy (e.g., the
antigene approach) and/or diagnosis. In addition, it
could be a fascinating and powerful tool in molecular
biology. However, two major drawbacks in triplex for-
mation limit the practical use of triplex-forming oligo-
nucleotides (TFOs). One problem is the relatively low
stability of the triplex under neutral pH conditions. To
enhance the stability of the triplex, chemical modifica-
tions of TFOs have been carried out.4 These studies
have shown that the 20,40-BNA5–10/LNA,11–14 which
has a locked N-type sugar conformation, shows high
binding affinity toward dsDNA target in a sequence-spe-
cific manner (Fig. 1).10,15,16 The second problem is limi-
tations of the target sequence. Pyrimidine nucleobases in
TFOs interact with purineÆpyrimidine base pairs in tar-
O
N
O
O
O
O
O
O
O
(Base: natural or
unnatural nucleobase)
PB
N-type sugar conformation
Figure 1. Structures of 20,40-BNA/LNA and PB.
get dsDNA to form T AÆT and C+ GÆC base triads.17
*
*
It is well known that the presence of pyrimidineÆpurine
interruptions (CÆG or TÆA interruptions) in the dsDNA
target drastically decrease the stability of the triplex.1–3
To overcome this problem, considerable effort has been
devoted to the synthesis of nucleobase analogues that
recognize CÆG interruptions.4,18 We have developed no-
vel 20,40-BNAs bearing unnatural nucleobases,19–23 and
found that the 20,40-BNA incorporating 2-pyridone
(PB: Fig. 1) effectively recognized CÆG interruptions in
the dsDNA target.19,20 On the other hand, various base
analogues have also been developed for recognizing TÆA
interruptions.4,18,24–31 Among these nucleobase ana-
logues, the phenylimidazole derivative, D3,24–26 and
aminothiazole derivatives, S27,29,30,32 and Bt,28,29
Keywords: Bridged nucleic acid (BNA); Locked nucleic acid (LNA);
Triplex; Oligonucleotide.
*
Corresponding author. Tel.: +81 6 6879 8200; fax: +81 6 6879
8204; e-mail: imanishi@phs.osaka-u.ac.jp
ꢀ
Present address: Graduate School of Pharmaceutical Sciences,
Nagoya City University, 3-1 Tanabe-dori Mizuho-ku, Nagoya 467-
8603, Japan.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.043

2946
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
D3
S
S
O
O
N
O
N
H
N
H
Me
N
DNA
N
N
DNA
H
Bt
N
Me
N
S
N
N
H
DNA
H
O
O
bPB
TzB
N
N
H
N
Me
N
S
2',4'-BNA
2',4'-BNA
O
Figure 2. Structures of D3, S, Bt, bPB, and TzB.
showed effective interaction with a TÆA interruption in a
parallel motif triplex. The non-fused heteroaromatic
structure of these nucleobase analogues is likely to con-
tribute to base stacking, hydrogen bonding and/or inter-
OH
BnO
BnO
O
a
b
N
O
O
OAc
O
TsO
BnO
BnO
OAc
TsO
BnO
BnO
BnO
OAc
1
2
OH
OTs
c
N
O
N
d
O
O
BnO
O
O
3
4
NHBz
R1
DMTrO
R2O
N
O
N
O
i
O
O
O
P
O
R3O
O
i-Pr2N
OCH2CH2CN
5: R1 = NO2, R2 = R3 = Bn
6: R1 = NH2, R2 = R3 = Bn
7: R1 = NHBz, R2 = R3 = Bn
8: R1 = NHBz, R2 = R3 = H
e
f
10
g
h
9: R1 = NHBz, R2 = DMTr, R3 = H
Scheme 1. Reagents and conditions: (a) 4-hydroxy-2-pyridone, N,O-bis(trimethylsilyl)acetamide, TMSOTf, 1,2-dichloroethane, reflux, 2.5 h, 96%;
(b) K2CO3, MeOH, rt, 9 h, 98%; (c) TsCl, Et3N, CH2Cl2, ꢀ50 ꢁC, 3 h, 98%; (d) m-nitrophenylboronic acid, Pd(PPh3)4, K3PO4, KBr, dioxane, 90 ꢁC,
24 h, 64%; (e) H2, PtO2, EtOH–AcOEt, rt, 18 h, quant.; (f) BzCl, pyridine, rt, 2 h, 99%; (g) 20% Pd(OH)2–C, cyclohexene, EtOH, reflux, 8 h, 86%; (h)
DMTrCl, pyridine, rt, 2.5 h, 95%; (i) (i-Pr2N)2POCH2CH2CN, diisopropylammonium tetrazolide, MeCN–THF, rt, 21 h, 94%.

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
2947
TBDPSO
a, b
BnO
c
O
O
OMe
OMe
TBDPSO
O
BnO
O
12
11
Bz
N
Me
N
S
TBDPSO
TBDPSO
d
e
OH
OH
CHO
OH
TBDPSO
O
TBDPSO
O
13
14
Bz
N
Bz
N
Me
Me
N
N
S
S
R1O
DMTrO
h
O
O
R2O
O
O
P
O
i-Pr2N
OCH2CH2CN
15: R1 = R2 = TBDPS
16: R1 = R2 = H
f
18
g
17: R1 = DMTr, R2 = H
Scheme 2. Reagents and conditions: (a) 20% Pd(OH)2–C, H2, rt, 1.5 h; (b) TBDPSCl, imidazole, DMF, rt, 12 h, 79% over 2 steps; (c) 10% HCl aq,
THF, rt, 4 h, 96%; (d) 2-(N-methylbenzamido)thiazole, LDA, THF, ꢀ78 ꢁC, 1 h, 53%; (e) 1,10-azobis(N,N-dimethylformamide), n-Bu3P, THF, rt,
2 h, 64%; (f) TBAF, THF, rt, 2 h, 72%; (g) DMTrCl, pyridine, rt, 12 h, 96%; (h) i-Pr2NP(Cl)OCH2CH2CN, i-Pr2NEt, CH2Cl2, rt, 3 h, 89%.
calation in the process of recognizing of the TÆA base
pair.
The other 20,40-BNA analogue, TzB, was synthesized as
shown in Scheme 2. The Bn groups of compound 1137
were converted to the TBDPS groups,38 and treated with
10% HCl aq in THF to give aldehyde 13. 2-(N-Methyl-
benzamido)thiazole was treated with LDA in THF and
the resulting litho-derivative was allowed to react with
aldehyde 13 to give the coupling product 14 stereoselec-
tively. Mitsunobu reaction of 14 gave bicyclic nucleoside
15, and TBDPS groups were removed by treatment with
TBAF to give 16. Dimethoxytritylation of 16 followed
by phosphitylation afforded the phosphoramidite 18.
We have designed heteroaromatic compounds, 4-(3-
benzamidophenyl)-2-pyridone and 2-(N-methylbenzam-
ido)thiazole, as novel artificial nucleobases. Here, we
describe the synthesis of 20,40-BNAs bearing the unnat-
ural nucleobases (bPB and TzB: Fig. 2) and demonstrate
successful recognition of the TÆA interruption by bPB
and TzB through parallel motif triplex formation.
2. Results and discussion
The obtained phosphoramidites 10 and 18 were incorpo-
rated into 15-mer oligonucleotides by a standard phos-
phoramidite protocol on an automated DNA
synthesizer. The obtained oligonucleotides were purified
by reversed-phase HPLC, and the composition of the
oligonucleotides was confirmed by MALDI-TOF-MS
analysis (Table 1).
Synthesis of the phosphoramidite derivative of bPB was
accomplished as shown in Scheme 1. As per Vorbruggen’s
¨
method,33 diacetate 134 was coupled with 4-hydroxy-2-
pyridone in the presence of N,O-bis(trimethylsilyl)acet-
amide and TMSOTf to give the desired b-anomer 2.
Treatment of 2 with K2CO3 resulted in deacetylation
and subsequent ring-closure reaction to afford 3 in good
yield.35 The obtained 20,40-BNA derivative 3 bearing 4-
hydroxy-2-pyridone nucleobase was tosylated to give 4,
which was subjected to a palladium-catalyzed cross-cou-
pling reaction with m-nitrophenylboronic acid in the pres-
ence of K3PO4 and KBr to provide 5.36 Catalytic
hydrogenation of 5 over PtO2 furnished the correspond-
ing amino compound 6, which was then treated with BzCl
in pyridine to give 7. Hydrogenolysis of 7 followed by
dimethoxytritylation provided 9. Compound 9 was phos-
phitylated to give the phosphoramidite 10.
Triplex-forming ability of the TFOs containing bPB and
TzB was evaluated by UV melting experiments under
physiological pH conditions (Tables 2 and 3). As shown
in Table 2, the triplexes containing bPB TÆA and
*
TzB TÆA triads showed T values of 37 ꢁC and 30 ꢁC,
respectively, which are higher than those of the triplexes
*
m
containing G TÆA and GB TÆA triads. Thus, bPB and
*
*
TzB effectively stabilized the triplex containing a TÆA
interruption. However, bPB showed only moderate abil-
ity to discriminate between the TÆA base pair and other

2948
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
Table 1. Sequences and MALDI-TOF-mass data of TFOs
noteworthy that the stability of the triplex containing
*
including the G TÆA triad. Thus, the locked N-type su-
*
gar conformation and G nucleobase were found to be an
the GB TÆA triad was similar to that of the triplex
TFO
MALDI-TOF-mass
[MꢀH]ꢀ
Calcd
Found
inadequate combination for TÆA recognition. On the
50-TTTTTCTbPB TCTCTCT-30
50-TTTTTCTTzB TCTCTCT-30
50-TTTTTCTbPB CTCTCT-30
50-TTTTTCbPB TCTCTCT-30
50-TTTTTCTTzB CTCTCT-30
50-TTTTTCTzB TCTCTCT-30
4688.3
4616.5
4687.3
4687.3
4612.8
4612.8
4688.6
4617.2
4686.4
4686.8
4612.8
4612.2
other hand, the triplex containing the bPB CÆG triad
*
had a Tm value of 33 ꢁC, which is the same as that of
the triplex involving the PB CÆG triad. This means that
*
the additional benzoylaminophenyl group in bPB had no
effect on the interaction with a CÆG base pair. It was pre-
viously reported that D3 intercalates between the TÆA
base pair and the 30 neighboring triad when D3 recog-
nizes the TÆA interruption in a pyrimidine motif tri-
plex.26 In addition, it was also demonstrated that the
30 neighboring triad affected triplex stability.25 Such
effects of a neighbor base have been discussed with re-
spect to G TÆA25 and S TÆA29 triads. To investigate
C means 20-deoxy-5-methylcytidine.
base pairs (DTm values for the CÆG, AÆT, and GÆC base
pairs were ꢀ4 ꢁC, ꢀ6 ꢁC, and ꢀ9 ꢁC, respectively). In
contrast, the Tm value of the triplex containing TzB
TÆA is much higher than those of the triplexes contain-
*
*
*
ing TzB CÆG, TzB AÆT, and TzB GÆC triads
the stabilization effect of bPB in more detail, we exam-
ined the neighbor effect with the triplexes containing
bPB TÆA and TzB TÆA triads. As shown in Table 3,
*
*
*
(DTm = ꢀ11 ꢁC, ꢀ14 ꢁC, and ꢀ7 ꢁC, respectively). Elec-
trostatic surface potential analysis of the TzB TÆA triad
*
*
*
suggested that the selective affinity of TzB to the TÆA
base pair was likely to involve favorable van der Waals
interactions between TzB and the TÆA base pair,
although there is no appropriate hydrogen bonding be-
tween TzB and TÆA base pair (Fig. 3, below). However,
the shape of the polyheteroaromatic ring of bPB is a
poor fit to the TÆA base pair. A part of the polyhetero-
aromatic ring would be overlapped with the TÆA base
pair (Fig. 3, above), which suggests that the heteroaro-
matic part of bPB acts as an intercalator to stabilize
replacement of the T AÆT triad with C GÆC at the 50
* *
or 30 neighboring site (X1 Y1Z1 or X2 Y2Z2) decreases
*
*
the triplex0s stability, even if the triplex contains no
TÆA interruption (X = T and YZ = AÆT: DTm = ꢀ14 to
ꢀ12 ꢁC). A similar decrease in Tm value was observed
for the triplexes containing TzB TÆA (DT = ꢀ14 to
*
m
ꢀ13 ꢁC) and G TÆA triads (DT = ꢀ13 to ꢀ12 ꢁC). In
*
m
these cases, no obvious 30- or 50-C GÆC side effect was
*
observed. However, the adjacent 30-C GÆC resulted in
*
greater destabilization of the triplex in the case of bPB
the triplex involving the bPB TÆA triad. Here, it is also
(DT s for 30- and 50-C GÆC = ꢀ15 and ꢀ10 ꢁC, respec-
*
*
m
Table 2. Tm values (ꢁC) of triplexa,b
TFO
dsDNA targets (YZ)
(X)
TÆA
CÆG
AÆT
GÆC
bPB
TzB
G
GB
PB
37
30
27
26
14d
33 (ꢀ4)
19 (ꢀ11)
20 (ꢀ7)
19, 28c
33d
31 (ꢀ6)
16 (ꢀ14)
16 (ꢀ9)
16, 22c
23d
28 (ꢀ9)
23 (ꢀ7)
23 (ꢀ4)
24 (ꢀ2)
19d
50-TTTTTCTXTCTCTCT-30 TFO.
50-GCTAAAAAGAYAGAGAGATCG-30 dsDNA.
30-CGATTTTTCTZTCTCTCTAGC-50.
a UV melting profiles were measured in 7 mm sodium phosphate buffer (pH 7.0) containing 140 mm KCl and 10 mm MgCl2 at a scan rate of 0.5 ꢁC/
min with detection at 260 nm. Tm values were obtained as the maxima of the first derivative of the melting curves. Concentration of each
oligonucleotide was 1.5 lm. C and GB mean 20-deoxy-5-methylcytidine and 20,40-BNA/LNA-G, respectively.
b DTm values (Tm value of the triplex involving the X YÆZ triad—that which contains X TÆA) are shown in parentheses.
*
*
c Two transitions were observed for triplex dissociation.
d Our previous work. See Refs. 19 and 20.
Table 3. Neighbor effect of the triplexes containing bPB TÆA and TzB TÆA triada,b
*
*
X1 Y1ÆZ1
*
X2 Y2ÆZ2
*
Tm (ꢁC)
X = bPB (Y = T, Z = A)
X = TzB (Y = T, Z = A)
X = G (Y = T, Z = A)
X = T (Y = A, Z = T)
T AÆT
T AÆT
37
22 (ꢀ15)
27 (ꢀ10)
30
16 (ꢀ14)
17 (ꢀ13)
27
14 (ꢀ13)
15 (ꢀ12)
44
30 (ꢀ14)
32 (ꢀ12)
*
*
T AÆT
C GÆC
*
*
C GÆC
*
T AÆT
*
50-TTTTTCX1XX2CTCTCT-30 TFO.
50-GCTAAAAAGY1YY2GAGAGATCG-30 dsDNA.
30-CGATTTTTCZ1ZZ2CTCTCTAGC-50.
a See footnote in Table 2.
b DTm values [Tm value of the triplex (X1XX2 = TXC or CXT)—that of the triplex (X1XX2 = TXT)] are shown in parentheses.

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
2949
O
bPB
N
bPB
HO
HO
N
H
N
H
N
O
dR
H
N
O
N
N
N
O
H
O
Me
T
A
O
N
A
T
dR
TzB
O
TzB
HO
H
N
N
N
S
N
N
Me
O
dR
H
O
N
N
HO
H
O
Me
O
N
A
A
N
T
T
dR
Figure 3. Proposed structures and electrostatic surface potential of bPB TÆA and TzB TÆA. Calculated with Spartan’04 (Wavefunction, Inc.).
*
*
tively). This result indicates a similar nearest neighbor
effect with regard to stabilization of bPB TÆA and
67.8 MHz) and 31P NMR spectra were recorded on a
Varian VXR-200 (31P, 81.0 MHz). Mass spectra of
nucleoside analogues were recorded on a JEOL JMS-
D300 or JMS-600 mass spectrometer. Fuji Silysia BW-
300 (200–400 mesh) was used for flash chromatography.
MALDI-TOF-Mass spectra were recorded on an Ap-
plied Biosystems Voyagerꢂ-DE.
*
D
TÆA25 triads, and it is therefore probable that bPB,
*
3
like D3, intercalates between the TÆA base pair and the
30 flanking base triad.
3. Conclusion
4.1.1. 1-(2-O-Acetyl-3,5-di-O-benzyl-4-p-toluenesulfonyl-
oxymethyl-b-D-ribofuranosyl)-4-hydroxy-2-pyridone (2).
In conclusion, 20,40-BNA analogues bearing novel
unnatural nucleobases, 4-(3-benzamidophenyl)-2-pyri-
done and 2-(N-methylbenzamido)thiazole, were synthe-
sized and incorporated into oligonucleotides. The
TFOs containing the 20,40-BNA analogues, bPB and
TzB, formed a stable triplex with the dsDNA targets
incorporating a TÆA interruption under physiological
pH conditions. The stability of the triplexes containing
Under
a
N2 atmosphere, 4-hydroxy-2-pyridone
(300 mg, 2.70 mmol) and N,O-bis(trimethylsilyl)acetam-
ide (1.47 ml, 5.95 mmol) were added to a solution of
compound 1 (1.08 g, 1.80 mmol) in anhydrous 1,2-
dichloroethane (20 ml) at room temperature and the
mixture was refluxed for 1 h. TMSOTf (0.26 ml,
1.44 mmol) was added to the reaction mixture at room
temperature and the mixture was refluxed for 2.5 h.
The reaction was quenched by addition of saturated
aqueous NaHCO3 solution. The mixture was extracted
with AcOEt. The organic phase was washed with water
and brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash silica
gel column chromatography [n-hexane/AcOEt (1:4, v/v)]
a bPB TÆA triad was much higher than that of the cor-
*
responding triplexes containing a G TÆA triad. TzB
*
effectively discriminated the TÆA interruption from other
base pairs. Electrostatic surface potential analysis and
observation of the 30-C GÆC side effect suggest that
*
bPB, like D3, works as an intercalator when it recognizes
a TÆA interruption, whereas specific binding of TzB with
a TÆA base pair involves shape recognition, i.e., van der
Waals interaction.
to give compound 2 (1.13 g, 96%). White powder. Mp
22
D
72–74 ꢁC. ½aꢁ +27.9 (c 0.81, CHCl3). IR mmax (KBr):
1749, 1650, 1552, 1494, 1364, 1227, 1180, 1106 cmꢀ1
.
1H NMR (CDCl3) d 2.01 (3H, s), 2.40 (3H, s), 3.46,
3.82 (2H, AB, J = 10 Hz), 4.14, 4.26 (2H, AB,
J = 11 Hz), 4.31, 4.41 (2H, AB, J = 12 Hz), 4.31, 4.51
(2H, AB, J = 12 Hz), 4.38 (1H, d, J = 6 Hz), 5.24 (1H,
dd, J = 3, 6 Hz), 5.63 (1H, dd, J = 2, 8 Hz), 5.88 (1H,
d, J = 2 Hz), 5.91 (1H, d, J = 3 Hz), 7.18–7.34 (12H,
m), 7.62 (1H, d, J = 8 Hz), 7.76 (2H, d, J = 8 Hz),
11.20 (1H, br s). 13C NMR (CDCl3) d 20.7, 21.7, 69.3,
69.4, 73.5, 74.1, 75.1, 76.1, 85.4, 88.1, 98.7, 103.0,
127.8, 127.9, 128.0, 128.3, 128.4, 129.7, 132.0, 133.9,
136.9, 137.0, 145.1, 164.0, 168.9, 169.1. Mass (FAB):
4. Experimental
4.1. General
All melting points were measured on a Yanagimoto mi-
cro melting points apparatus and are uncorrected. Opti-
cal rotations were recorded on a JASCO DIP-370
instrument. IR spectra were recorded on a JASCO FT/
IR-200 spectrometer. 1H and 13C NMR spectra were re-
corded on a JEOL EX-270 (1H, 270 MHz; 13C,

2950
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
m/z 650 (MH+). Anal. Calcd for C34H35NO10S: C,
62.85; H, 5.43; N, 2.16; S, 4.94. Found: C, 62.73; H,
5.49; N, 2.10; S, 4.85.
perature and the mixture was stirred at 90 ꢁC for 24 h.
The mixture was filtered through a pad of Celiteꢂ and
the filtrate was concentrated under reduced pressure.
The residue was purified by flash silica gel column chro-
matography [n-hexane/AcOEt (1:1, v/v)] to give com-
4.1.2. 1-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-D-ribo-
furanosyl)-4-hydroxy-2-pyridone (3). To a solution of
compound 2 (700 mg, 1.08 mmol) in MeOH (15 ml)
was added K2CO3 (434 mg, 0.52 mmol) at room temper-
ature and the mixture was stirred for 9 h. After neutral-
ization by addition of 10% HCl aqueous solution, the
mixture was extracted with AcOEt. The organic phase
was washed with water and brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue
was purified by flash silica gel column chromatography
pound 5 (253 mg, 64%). Pale brown powder. Mp 49–
22
50 ꢁC. ½aꢁ +145.1 (c 0.74, CHCl3). IR mmax (KBr):
D
1
1661, 1592, 1531, 1350, 1098 cmꢀ1. H NMR (CDCl3)
d 3.86, 3.89 (2H, AB, J = 11 Hz), 3.91, 4.08 (2H, AB,
J = 8 Hz), 4.01 (1H, s), 4.48, 4.62 (2H, AB, J = 12 Hz),
4.66, 4.68 (2H, AB, J = 12 Hz), 4.71 (1H, s), 5.93 (1H,
s), 6.34 (1H, dd, J = 2, 7 Hz), 6.75 (1H, d, J = 2 Hz),
7.21–7.39 (10H, m), 7.66 (1H, dd, J = 8, 8 Hz), 7.86–
7.95 (2H, m), 8.28–8.32 (1H, m), 8.40–8.42 (1H, m).
13C NMR (CDCl3) d 64.5, 72.0, 72.1, 73.6, 75.6, 76.2,
87.3, 88.0, 104.5, 117.2, 121.5, 123.9, 127.3, 127.4,
127.7, 128.2, 128.3, 130.0, 132.3, 132.8, 136.8, 137.4,
138.9, 148.4, 149.1, 161.4. Mass (EI): m/z 540 (M+,
7.5), 91 (100). Anal. Calcd for C31H28N2O7: C, 68.88;
H, 5.22; N, 5.18. Found: C, 68.52; H, 5.27; N, 5.17.
[n-hexane/AcOEt (1:4, v/v)] to give compound
3
(462 mg, 98%). Colorless crystals. Mp 117–118 ꢁC
24
(AcOEt). ½aꢁ +171.5 (c 1.07, CHCl3). IR mmax (KBr):
D
1648, 1546, 1489, 1244, 1094, 1052 cmꢀ1
.
1H NMR
(CDCl3) d 3.81, 3.83 (2H, AB, J = 11 Hz), 3.88, 4.04
(2H, AB, J = 8 Hz), 3.97 (1H, s), 4.43, 4.58 (2H, AB,
J = 12 Hz), 4.60 (1H, s), 4.60 (2H, s), 5.86 (1H, s), 5.94
(1H, s), 5.96 (1H, d, J = 7 Hz), 7.20–7.36 (10H, m),
7.70 (1H, d, J = 7 Hz), 11.86 (1H, br s). 13C NMR
(CDCl3) d 64.7, 72.1, 72.2, 73.7, 75.7, 76.7, 87.2, 87.9,
98.9, 103.2, 127.4, 127.6, 127.8, 127.9, 128.3, 128.4,
132.9, 136.9, 137.5, 164.0, 169.2. Mass (FAB): m/z 436
(MH+). Anal. Calcd for C25H25NO6: C, 68.95; H,
5.79; N, 3.22. Found: C, 68.85; H, 5.85; N, 3.23.
4.1.5. 4-(3-Aminophenyl)-1-(3,5-di-O-benzyl-2-O,4-C-
methylene-b-D-ribofuranosyl)-2-pyridone (6). Under a
H2 atmosphere PtO2 (8.0 mg, 35 lmol) was added to a
solution of compound 5 (100 mg, 0.18 mmol) in anhy-
drous EtOH/AcOEt (1:1, 2 ml) at room temperature
and the mixture was stirred at room temperature for
18 h. The mixture was filtered and the filtrate was con-
centrated under reduced pressure. The residue was puri-
fied by flash silica gel column chromatography [n-
4.1.3. 1-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-D-ribo-
furanosyl)-4-p-toluenesulfonyloxy-2-pyridone (4). Under
a N2 atmosphere, TsCl (175 mg, 0.92 mmol) and Et3N
(148 ll, 1.06 mmol) were added to a solution of com-
pound 3 (308 mg, 0.71 mmol) in anhydrous CH2Cl2
(8 ml) at ꢀ50 ꢁC and the mixture was stirred at
ꢀ50 ꢁC for 3 h. After addition of water, the mixture
was extracted with AcOEt. The organic phase was
washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was
purified by flash silica gel column chromatography [n-
hexane/AcOEt (1:2, v/v)] to give compound 6 (94 mg,
quant.). Pale yellow powder. Mp 52–54 ꢁC. ½aꢁ
22
D
+149.2 (c 1.48, CHCl3). IR mmax (KBr): 3446, 3353,
2944, 2877, 1656, 1585, 1523, 1455, 1268, 1205, 1097,
1
1052 cmꢀ1. H NMR (CDCl3) d 3.08 (2H, br s), 3.85,
3.87 (2H, AB, J = 11 Hz), 3.91, 4.06 (2H, AB,
J = 8 Hz), 4.00 (1H, s), 4.46, 4.60 (2H, AB, J = 12 Hz),
4.65, 4.66 (2H, AB, J = 12 Hz), 4.70 (1H, s), 5.93 (1H,
s), 6.35 (1H, dd, J = 2, 7 Hz), 6.69 (1H, d, J = 1 Hz),
6.80 (1H, dd, J = 1, 8 Hz), 6.92 (1H, s), 6.96 (1H, d,
J = 7 Hz), 7.21–7.36 (11H, m), 7.81 (1H, d, J = 7 Hz).
13C NMR (CDCl3) d 64.7, 72.0, 73.6, 75.7, 76.4, 87.2,
88.0, 105.6, 113.1, 116.1, 116.4, 116.9, 127.4, 127.6,
127.8, 127.9, 128.3, 128.5, 129.9, 131.8, 136.9, 137.6,
138.4, 146.9, 152.1, 162.1. Mass (EI): m/z 510 (M+,
5.2), 91 (100). Anal. Calcd for C31H30N2O5Æ1/4H2O: C,
72.29; H, 5.97; N, 5.44. Found: C, 72.34; H, 6.00; N,
5.42.
hexane/AcOEt (3:2, v/v)] to give compound 4 (413 mg,
22
D
98%). Colorless oil. ½aꢁ +156.4 (c 0.64, CHCl3). IR mmax
(KBr): 1663, 1598, 1543, 1379, 1087 cmꢀ1
.
1H NMR
(CDCl3) d 2.46 (3H, s), 1.32 (3H, s), 3.80, 3.83 (2H,
AB, J = 11 Hz), 3.85, 4.03 (2H, AB, J = 8 Hz), 3.91
(1H, s), 4.45, 4.58 (2H, AB, J = 12 Hz), 4.56 (1H, s),
4.61, 4.62 (2H, AB, J = 12 Hz), 5.78 (1H, s), 6.03–6.07
(2H, m), 7.19–7.40 (12H, m), 7.77–7.81 (3H, m). 13C
NMR (CDCl3) d 21.8, 64.4, 72.0, 72.1, 73.6, 75.4, 76.2,
87.3, 88.0, 101.8, 109.7, 127.3, 127.6, 127.9, 127.9,
128.2, 128.3, 128.4, 130.0, 131.9, 133.8, 136.7, 137.4,
146.0, 158.9, 161.9. Mass (EI): m/z 589 (M+, 6.3), 91
(100). Anal. Calcd for C32H31NO8S: C, 65.18; H, 5.30;
N, 2.38; S, 5.44. Found: C, 64.84; H, 5.41; N, 2.31; S,
5.33.
4.1.6. 4-(3-Benzamidophenyl)-1-(3,5-di-O-benzyl-2-O,4-
C-methylene-b-D-ribofuranosyl)-2-pyridone (7). Under a
N2 atmosphere, BzCl (0.17 ml, 1.46 mmol) was added
to a solution of compound 6 (508 mg, 0.99 mmol) in
anhydrous pyridine (5 ml) at room temperature and
the mixture was stirred at room temperature for 2 h.
After addition of water, the mixture was extracted with
AcOEt. The organic phase was washed with water and
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The residue was purified by flash silica
gel column chromatography [n-hexane/AcOEt (2:3,
4.1.4. 1-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-D-ribo-
furanosyl)-4-(3-nitrophenyl)-2-pyridone (5). Under a N2
atmosphere, m-nitrophenylboronic acid (146 mg,
0.87 mmol), K3PO4 (232 mg, 1.09 mmol), KBr
(104 mg, 0.87 mmol), and Pd(PPh3)4 (84 mg, 73 lmol)
were added to a solution of compound 4 (508 mg,
0.99 mmol) in anhydrous dioxane (10 ml) at room tem-
v/v)] to give compound 7 (606 mg, 99%). White powder.
22
D
(KBr): 3296, 3062, 3030, 2946, 2876, 1656, 1588, 1544,
Mp 80–83 ꢁC. ½aꢁ +113.4 (c 1.68, CHCl3). IR mmax

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
2951
1
1257, 1098, 1052 cmꢀ1. H NMR (CDCl3) d 3.83, 3.86
136.3, 136.5, 138.4, 140.8, 145.7, 152.0, 159.5, 162.1,
166.3. Mass (FAB): m/z 737 (MH+). Mass (FAB): m/z
759 (MNa+). Anal. Calcd for C45H40N2O8H2O: C,
71.60; H, 5.61; N, 3.71. Found: C, 71.41; H, 5.62; N,
3.61.
(2H, AB, J = 11 Hz), 3.88, 4.05 (2H, AB, J = 8 Hz),
4.00 (1H, s), 4.44, 4.57 (2H, AB, J = 11 Hz), 4.64, 4.67
(2H, AB, J = 12 Hz), 4.66 (1H, s), 5.90 (1H, s), 6.44
(1H, d, J = 7 Hz), 6.70 (1H, s), 7.20–7.53 (15H, m),
7.75–8.00 (5H, m), 8.60 (1H, s). 13C NMR (CDCl3) d
64.5, 72.1, 72.2, 73.6, 75.7, 76.4, 87.2, 88.0, 105.8,
116.6, 118.7, 121.4, 122.6, 127.2, 127.5, 127.6, 127.9,
127.9, 128.4, 128.5, 128.7, 129.6, 131.9, 132.1, 134.7,
136.9, 137.6, 138.1, 139.0, 151.8, 162.0, 166.1. Mass
(FAB): m/z 615 (MH+). Anal. Calcd for
C38H34N2O6Æ1/4H2O: C, 73.71; H, 5.62; N, 4.52. Found:
C, 73.64; H, 5.76; N, 4.46.
4.1.9. 4-(3-Benzamidophenyl)-1-[3-O-[2-cyanoethoxy-(diiso-
propylamino)phosphino]-5-O-(4,40-dimethoxy-trityl)-2-O,
4-C-methylene-b-D-ribofuranosyl]-2-pyridone (10). Under
a
N2 atmosphere, (i-Pr2N)2POCH2CH2CN (38 ll,
0.12 mmol) was added to a solution of compound 9
(30 mg, 41 lmol), diisopropylammonium tetrazolide
(12 mg, 70 lmol) in anhydrous MeCN/THF (1:1, 2 ml)
at room temperature and the mixture was stirred at
room temperature for 21 h. The solvent was concen-
trated under reduced pressure. The residue was purified
by flash silica gel column chromatography [n-hexane/
AcOEt (1:1, v/v)] to give compound 10 (36 mg, 94%).
Colorless oil. 31P NMR (acetone-d6) d 148.8, 149.6.
4.1.7. 4-(3-Benzamidophenyl)-1-(2-O,4-C-methylene-b-D-
ribofuranosyl)-2-pyridone (8). A solution of compound 7
(520 mg, 0.85 mmol), 20% Pd(OH)2–C (300 mg), and
cyclohexene (4.30 ml, 42.5 mmol) in EtOH (25 ml) was
refluxed for 8 h. After filtration of the mixture, silica
gel (2 g) was added to the filtrate and the mixture was
concentrated under reduced pressure. The residue was
purified by flash silica gel column chromatography
4.1.10. Methyl 3,5-di-O-tert-butyldiphenylsilyl-2-O,4-C-
methylene-D-ribofuranoside (12). Under a H2 atmosphere
at room temperature, a solution of compound 11
(847 mg, 2.38 mmol) and 20% Pd(OH)2–C (400 mg) in
AcOEt (15 ml) was stirred for 1.5 h. The mixture was fil-
tered and concentrated under reduced pressure. Under a
N2 atmosphere, the residue was dissolved in anhydrous
DMF (5 ml), and TBDPSCl (1.3 ml, 5.00 mmol) and
imidazole (1.20 g, 18.2 mmol) were added at room tem-
perature. After stirring for 12 h, saturated aqueous
NaHCO3 was added to the mixture, and the organic
phase was extracted with ethyl acetate, washed with
water and brine, dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by sil-
ica gel column chromatography [n-hexane/CHCl3 (4:1,
[CHCl3/MeOH (12:1, v/v)] to give compound
(315 mg, 86%). White powder. Mp 277–278 ꢁC. ½aꢁ
8
28
D
+94.7 (c 1.10, DMSO). IR mmax (KBr): 3303, 2970,
1675, 1570 cmꢀ1 1H NMR (DMSO-d6) d 3.72, 3.88
.
(2H, AB, J = 8 Hz), 3.82 (2H, d, J = 6 Hz), 3.95 (1H,
d, J = 4 Hz), 4.22 (1H, s), 5.22 (1H, dd, J = 6, 6 Hz),
5.64 (1H, d, J = 4 Hz), 5.68 (1H, s), 6.64 (1H, d,
J = 2 Hz), 6.67 (1H, dd, J = 2, 7 Hz), 7.47–7.64 (5H,
m), 7.89–8.00 (4H, m), 8.16 (1H, s), 10.37 (1H, s). 13C
NMR (DMSO-d6) d 56.1, 68.3, 71.2, 78.8, 87.1, 89.0,
104.4, 115.2, 118.5, 121.5, 122.1, 127.7, 128.5, 129.6,
131.8, 133.0, 134.8, 137.0, 139.9, 150.9, 161.1, 165.8.
Mass (FAB): m/z 435 (MH+). Anal. Calcd for
C24H22N2O6: C, 66.35; H, 5.10; N, 6.45. Found: C,
65.98; H, 5.21; N, 6.31.
v/v)] to give compound 12 (1.23 g, 79% 2 steps from
26
D
11). Colorless oil. ½aꢁ ꢀ45.2 (c 2.67, CHCl3). IR mmax
(KBr): 3063, 2939, 1468, 1368, 1106 cmꢀ1
.
1H NMR
4.1.8. 4-(3-Benzamidophenyl)-1-[5-O-(4,40-dimethoxy-tri-
tyl)-2-O,4-C-methylene-b-D-ribofuranosyl]-2-pyridone
(CDCl3) d 1.03 (9H, s), 1.04 (9H, s), 3.23 (3H, s), 3.60
(1H, s), 3.77, 4.10 (2H, AB, J = 7 Hz), 3.94 (2H, s),
4.32 (1H, s), 4.67 (1H, s), 7.23–7.42 (12H, m), 7.57–
7.72 (8H, m). 13C NMR (CDCl3) d 19.2, 19.4, 26.8,
26.9, 54.9, 60.8, 72.1, 73.4, 79.1, 86.8, 104.5, 127.6,
127.6, 127.6, 129.5, 129.6, 129.7, 129.8, 132.5, 133.1,
133.3, 133.3, 135.5, 135.5, 135.6. Mass (FAB): m/z 675
(MNa+). HRMS Calcd for C39H48O5Si2Na: 675.2938.
Found: 675.2938.
(9). Under
a N2 atmosphere, DMTrCl (62 mg,
0.18 mmol) was added to a solution of compound 8
(43 mg, 99 lmol) in anhydrous pyridine (3 ml) at room
temperature and the mixture was stirred at room tem-
perature for 2.5 h. After addition of saturated aqueous
NaHCO3 solution, the mixture was extracted with
AcOEt. The organic phase was washed with water and
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The residue was purified by flash silica
gel column chromatography [CHCl3/AcOEt (4:5, v/v)]
4.1.11. 3,5-Di-O-tert-butyldiphenylsilyl-2-O,4-C-methy-
lene-D-ribofuranose (13). At room temperature, 10%
HCl aqueous solution (0.2 ml) was added to a solution
of compound 12 (1.00 g, 1.53 mmol) in THF (15 ml)
and the mixture was stirred for 4 h. After the addition
of saturated aqueous NaHCO3 solution, the organic
phase was extracted with AcOEt, washed with water
and brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by silica gel
to give compound 9 (69 mg, 95%). White powder. Mp
21
D
(KBr): 3301, 1655, 1577, 1512, 1301, 1253, 1179,
153–155 ꢁC. ½aꢁ +32.2 (c 0.80, CHCl3). IR mmax
1046 cmꢀ1 1H NMR (acetone-d6) d 3.55, 3.63 (2H,
.
AB, J = 11 Hz), 3.78 (6H, s), 3.82, 3.96 (2H, AB,
J = 8 Hz), 4.43 (1H, s), 4.47 (1H, d, J = 5 Hz), 4.90
(1H, d, J = 5 Hz), 5.82 (1H, s), 6.55 (1H, dd, J = 2,
7 Hz), 6.65 (1H, d, J = 2 Hz), 6.91–6.94 (4H, m), 7.27–
7.58 (14H, m), 7.96–8.05 (3H, m), 8.18 (1H, d,
J = 7 Hz), 8.28 (1H, s), 9.74 (1H, s). 13C NMR (ace-
tone-d6) d 55.4, 59.5, 70.3, 72.4, 79.9, 87.1, 88.6, 88.6,
105.1, 113.8, 116.6, 119.1, 121.7, 122.6, 127.6, 128.2,
128.5, 128.8, 129.1, 130.1, 130.8, 132.3, 133.5, 135.8,
column chromatography [n-hexane/CHCl3 (2:1, v/v)] to
give compound 13 (940 mg, 96%). Colorless oil. ½aꢁ
26
D
ꢀ3.4 (c 1.83, CHCl3). IR mmax (KBr): 3434, 3063, 2939,
1
2862, 1728, 1467, 1108 cmꢀ1. H NMR (CDCl3) d 0.97
(9H, s), 1.07 (9H, s), 2.64 (1H, br s), 3.79, 3.90 (2H,
AB, J = 10 Hz), 3.99, 4.07 (2H, AB, J = 9 Hz), 4.06

2952
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
(1H, s), 4.29 (1H, s), 7.25–7.47 (12H, m), 7.54–7.61 (8H,
m), 9.35 (1H, s). 13C NMR (CDCl3) d 19.2, 19.4, 26.9,
27.0, 63.3, 76.0, 80.9, 82.3, 89.6, 127.7, 127.8, 127.8,
129.9, 130.0, 130.0, 131.9, 132.3, 132.4, 132.9, 135.4,
135.6, 135.6. Mass (FAB): m/z 661 (MNa+). HRMS
Calcd for C38H46O5Si2Na: 661.2781. Found: 661.2780.
ride in THF (2.4 ml, 2.40 mmol) was added to a solution
of compound 15 (680 mg, 3.22 mmol) in anhydrous
THF (30 ml) and the mixture was stirred for 2 h. The
mixture was concentrated under reduced pressure and
the residue was purified by silica gel column chromatog-
raphy [AcOEt/EtOH (30:1, v/v)] to give compound 16
(840 mg, 72%). Colorless crystals. Mp 180–182 ꢁC. ½aꢁ
26
D
4.1.12. 2-(N-Methylbenzamido)-5-(3,5-di-O-tert-butyldi-
phenylsilyl-2-O,4-C-methylene-D-ribitol-1-yl)thiazole
(14). Under a N2 atmosphere at ꢀ78 ꢁC, a solution of 2-
(N-methylbenzamido)thiazole (1.71 g, 7.85 mmol) in
anhydrous THF (10 ml) was added to lithium diisopro-
pylammonium (67.0 mM in THF, 130 ml, 8.64 mmol).
After stirring for 30 min, the mixture was added to a
solution of compound 13 (1.0 g, 1.57 mmol) in anhy-
drous THF (10 ml) at ꢀ78 ꢁC and stirred for 1 h. After
the addition of a saturated aqueous NH4Cl solution, the
organic phase was extracted with AcOEt, washed with
water and brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by sil-
ica gel column chromatography [n-hexane/CHCl3 (2:1,
v/v)] to give compound 14 (710 mg, 53%). White pow-
ꢀ23.7 (c 0.55, MeOH). IR mmax (KBr): 3041, 2949,
1496, 1378, 1027 cmꢀ1 1H NMR (MeOH-d4) d 3.82
.
(3H, s), 3.89 (2H, s), 3.93, 4.03 (2H, AB, J = 8 Hz),
4.16 (1H, s), 4.26 (1H, s), 5.06 (1H, s), 7.32 (1H, d,
J = 1 Hz), 7.40–7.50 (3H, m), 8.22–8.26 (2H, m). 13C
NMR (MeOH-d4) d 36.6, 59.9, 73.1, 73.8, 80.1, 84.2,
89.1, 102.3, 126.1, 126.4, 129.4, 130.4, 133.1, 138.2,
169.0, 175.6. Mass (EI): m/z 362 (M+, 87.2), 247
(26.4), 105 (100). HRMS Calcd for C17H18N2O5S:
362.0936. Found: 362.0933.
4.1.15. 2-(N-Methylbenzamido)-5-[5-O-(4,40-dimethoxy-
trityl)-2-O,4-C-methylene-b-D-ribofuranosyl]thiazole
(17). Under a N2 atmosphere, DMTrCl (282 mg,
2.48 mmol) was added to a solution of compound 16
(201 mg, 1.65 mmol) in anhydrous pyridine (5 ml) at
room temperature and the mixture was stirred at room
temperature for 12 h. After addition of saturated aque-
ous NaHCO3 solution, the mixture was extracted with
AcOEt. The organic phase was washed with water and
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The residue was purified by silica gel
column chromatography [n-hexane/AcOEt (1:1, v/v)]
der. Mp 100–102 ꢁC. ½aꢁ26 +72.2 (c 1.04, CHCl3). IR mmax
D
(KBr): 3330, 3064, 2938, 2861, 1495, 1372, 1107 cmꢀ1
.
1H NMR (CDCl3) d 0.83 (9H, s), 1.02 (9H, s), 3.28
(1H, broad s), 3.59 (3H, s), 3.63 (1H, s), 3.78 (1H, s),
3.80, 3.89 (2H, AB, J = 12 Hz), 3.88 (2H, s), 4.16 (1H,
s), 6.50 (1H, s), 7.16–7.21 (3H, m), 7.29–7.46 (16H,
m), 7.54–7.57 (4H, m), 9.35 (1H, dd, J = 2, 6 Hz). 13C
NMR (CDCl3) d 19.1, 19.3, 26.8, 26.9, 35.5, 64.7, 67.4,
76.1, 79.5, 82.5, 90.6, 123.4, 126.8, 127.7, 127.7, 127.8,
127.9, 129.0, 129.8, 129.9, 130.0, 130.1, 131.1, 132.0,
132.3, 132.8, 135.3, 135.3, 135.5, 135.6, 136.7, 167.2,
173.2. Mass (FAB): m/z 857 (MH+). HRMS Calcd for
C49H57N2O6SSi2: 857.3476. Found: 857.3491.
to give compound 17 (352 mg, 96%). White powder.
22
D
Mp 104–107 ꢁC. ½aꢁ +9.3 (c 1.70, CHCl3). IR mmax
(KBr): 3332, 3063, 2943, 1492, 1372, 1105 cmꢀ1
.
1H
NMR (CDCl3) d 2.88 (1H, d, J = 5 Hz), 3.48 (2H, s),
3.72 (3H, s), 3.77 (6H, s), 4.00 (2H, s), 4.17 (1H, s),
4.45 (1H, d, J = 5 Hz), 5.08 (1H, s), 6.84 (4H, d,
J = 7 Hz), 6.88 (1H, d, J = 1 Hz), 7.18–7.50 (11H, m),
8.31 (2H, dd, J = 1, 8 Hz). 13C NMR (CDCl3) dC:
35.7, 55.1, 59.6, 72.3, 72.6, 78.4, 82.0, 85.9, 86.2, 113.0,
123.7, 126.7, 127.7, 127.8, 127.9, 129.0, 129.8, 131.4,
135.4, 136.3, 144.3, 158.3, 167.0, 173.6. Mass (FAB):
m/z 665 (MH+). HRMS Calcd for C38H37N2O7S:
665.2243. Found: 665.2321.
4.1.13. 2-(N-Methylbenzamido)-5-(3,5-di-O-tert-butyldi-
phenylsilyl-2-O,4-C-methylene-b-D-ribofuranosyl)thiazole
(15). Under a N2 atmosphere at room temperature, 1,10-
azobis(N,N-dimethylformamide) (370 mg, 2.13 mmol)
and tri-n-butylphosphine (0.53 ml, 2.13 mmol) were
added to a solution of compound 14 (1.22 g, 1.42 mmol)
in anhydrous THF (15 ml) and the mixture was stirred
for 2 h. The mixture was concentrated under reduced
pressure and the residue was purified by silica gel col-
umn chromatography [n-hexane/AcOEt (6:1, v/v)] to
4.1.16. 2-(N-Methylbenzamido)-5-[3-O-[2-cyanoethoxy
(diisopropylamino)phosphino]-5-O-(4,40-dimethoxytrityl)-
2-O,4-C-methylene-b-D-ribofuranosyl] thiazole (18). Un-
der a N2 atmosphere, i-Pr2NEt (0.17 ml, 0.96 mmol)
and i-Pr2NP(Cl)OCH2CH2CN (40 ll, 0.19 mmol) were
added to a solution of compound 9 (64 mg, 96 lmol)
in anhydrous CH2Cl2 (4 ml) at 0 ꢁC and the mixture
was stirred at room temperature for 3 h. After addition
of saturated aqueous NaHCO3 solution, the mixture
was extracted with AcOEt. The organic phase was
washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was
purified by silica gel column chromatography [n-hex-
ane/AcOEt (3:1, v/v)] to give compound 18 (84 mg,
89%). Colorless oil. 31P NMR (CDCl3) d 148.2, 149.0.
give compound 15 (763 mg, 64%). White powder. Mp
26
D
79–82 ꢁC. ½aꢁ +23.9 (c 0.56, CHCl3). IR mmax (KBr):
3063, 2940, 2862, 1600, 1565, 1506, 1364, 1108 cmꢀ1
.
1H NMR (CDCl3) d 1.04 (9H, s), 1.06 (9H, s), 3.50
(3H, s), 3.76 (1H, s), 4.01 (2H, s), 4.01, 4.19 (2H, AB,
J = 8 Hz), 4.14 (1H, s), 4.91 (1H, s), 6.42 (1H, d,
J = 1 Hz), 7.17–7.75 (23H, m), 8.34 (1H, dd, J = 2,
8 Hz). 13C NMR (CDCl3) d 19.1, 19.4, 26.8, 27.0, 35.4,
60.2, 72.8, 73.3, 78.4, 81.2, 87.3, 122.8, 124.0, 127.6,
127.7, 127.7, 127.9, 129.1, 129.7, 129.8, 130.0, 131.4,
132.3, 132.7, 133.0, 135.4, 135.5, 135.5, 135.6, 136.7,
166.9, 173.4. Mass (FAB): m/z 839 (MH+). HRMS
Calcd for C49H55N2O5SSi2: 839.3370. Found: 839.3387.
4.1.14. 2-(N-Methylbenzamido)-5-(2-O,4-C-methylene-b-
D- ribofuranosyl)thiazole (16). Under a N2 atmosphere at
room temperature, 1 M tetra-n-butyl ammonium fluo-
4.1.17. Oligonucleotide synthesis. Synthesis of oligonucle-
otides containing bPB andTzB was performedona0.2 lmol
scale on an automated DNA synthesizer (Applied Biosys-

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
2953
tems, Expediteꢂ 8909) using the standard phosphorami-
dite protocol. The oligonucleotide synthesis was per-
formed on DMTr-ON mode. Cleavage from the CPG
support and removal of protecting groups were accom-
plished using 28% aqueous ammonia (55 ꢁC for 12 h).
The crude oligonucleotides bearing a DMTr group were
detritylated and purified with NENSORBTM PREP
according to the manufacturer’s protocol. The obtained
oligonucleotides were again purified by reversed-phase
HPLC (ChemcoPakꢂ CHEMCOSORB 300-5C18,
4.6 mm · 250 mm or Waters Xterraꢂ MS C18 2.5 lm,
10 mm · 50 mm). The composition of the oligonucleo-
tides was confirmed by MALDI-TOF-Mass analysis: 50-
TTTTTCTbPB TCTCTCT-30: [MꢀH]ꢀ calcd 4688.3;
found: 4688.6; 50-TTTTTCTTzB TCTCTCT-30: [MꢀH]ꢀ
calcd 4616.5; found: 4617.2; 50-TTTTTCTbPB CTCTCT-
30: [MꢀH]ꢀ calcd 4687.3; founꢀd: 4686.4; 50-
TTTTTCTTzB CTCTCT-30: [MꢀH] calcd 4612.8;
found: 4612.8; 50-TTTTTCbPB TCTCTCT-30: [MꢀH]ꢀ
9. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.;
Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401–
5404.
10. Obika, S.; Uneda, T.; Sugimoto, T.; Nanbu, D.; Minami,
T.; Doi, T.; Imanishi, T. Bioorg. Med. Chem. 2001, 9,
1001–1011.
11. The 20,40-BNA was independently synthesized by the
group of Wengel et al. immediately after our first report
(reference 8), and it is called a locked nucleic acid (LNA).
See references.12–14
12. Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J.
Chem. Commun. 1998, 455–456.
13. 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. Jepsen, J. S.; Wengel, J. Biochemistry 2004, 43, 13233–
13244.
15. Obika, S.; Hari, Y.; Sugimoto, T.; Sekiguchi, M.; Iman-
ishi, T. Tetrahedron Lett. 2000, 41, 8923–8927.
16. Torigoe, H.; Hari, Y.; Sekiguchi, M.; Obika, S.; Imanishi,
T. J. Biol. Chem. 2001, 276, 2354–2360.
17. The symbols and indicate Watson–Crick and Hoogsteen
*
hydrogen bonds, respectively.
18. Luyten, I.; Herdewijn, P. Eur. J. Med. Chem. 1998, 33,
515–576.
19. Obika, S.; Hari, Y.; Sekiguchi, M.; Imanishi, T. Angew.
Chem. Int. Ed. Engl. 2001, 40, 2079–2081; Angew. Chem.
2001, 113, 2149–2151.
20. Obika, S.; Hari, Y.; Sekiguchi, M.; Imanishi, T. Chem.
Eur. J. 2002, 8, 4796–4802.
21. Obika, S.; Hari, Y.; Morio, K.; Imanishi, T. Tetrahedron
Lett. 2000, 41, 221–224.
22. Hari, Y.; Obika, S.; Sakaki, M.; Morio, K.; Yamagata, Y.;
Imanishi, T. Tetrahedron 2002, 58, 3051–3063.
23. Hari, Y.; Obika, S.; Sekiguchi, M.; Imanishi, T. Tetrahe-
dron 2003, 59, 5123–5128.
24. Griffin, L. C.; Kiessling, L. L.; Beal, P. A.; Gillespie,
P.; Dervan, P. B. J. Am. Chem. Soc. 1992, 114, 7976–
7982.
calcd
4687.3;
fouꢀnd:
4686.8;
50-TTTTTCTzB
TCTCTCT-30: [MꢀH] calcd 4612.8; found: 4612.2.
4.2. Tm measurements
The UV melting experiments were carried out on a
Beckman DU-650 spectrophotometer equipped with a
Tm analysis accessory using quartz cuvettes with 1 cm
optical path length. The UV melting profiles were re-
corded in 7 mm sodium phosphate buffer (pH 7.0) con-
taining 140 mm KCl and 10 mm MgCl2 at a scan rate of
0.5 ꢁC/min with detection at 260 nm. The final concen-
tration of each oligonucleotide was 1.5 lm. The melting
temperatures were obtained as the maxima of the first
derivative of the melting curves.
25. Kiessling, L. L.; Griffin, L. C.; Dervan, P. B. Biochemistry
1992, 31, 2829–2834.
Acknowledgments
26. Koshlap, K. M.; Gillespie, P.; Dervan, P. B.; Feigon, J.
J. Am. Chem. Soc. 1993, 115, 7908–7909.
27. Guianvarc’h, D.; Benhida, R.; Fourrey, J.-L.; Maurisse,
R.; Sun, J.-S. Chem. Commun. 2001, 1814–1815.
28. Guianvarc’h, D.; Fourrey, J.-L.; Maurisse, R.; Sun, J.-S.;
Benhida, R. Org. Lett. 2002, 4, 4209–4212.
29. Guianvarc’h, D.; Fourrey, J.-L.; Maurisse, R.; Sun, J.-S.;
Benhida, R. Bioorg. Med. Chem. 2003, 11, 2751–2759.
30. Wang, Y.; Rusling, D. A.; Powers, V. E. C.; Lack, O.;
Osborne, S. D.; Fox, K. R.; Brown, T. Biochemistry 2005,
44, 5884–5892.
31. Efficient recognition of the TÆA interruption in an
antiparallel motif triplex was also reported: Parel, S. P.;
Leumann, C. J. Nucleic Acids Res. 2001, 29, 2260–2267;
Sasaki, S.; Taniguchi, Y.; Takahashi, R.; Senko, Y.;
Kodama, K.; Nagatsugi, F.; Maeda, M. J. Am. Chem.
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This work was supported in part by PRESTO from Ja-
pan Science and Technology Agency (J.S.T.), a SUN-
BOR Grant from 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, Culture, Sport, Science, and
Technology of Japan.
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retrieving.html (or from the Cambridge Crystallographic

2954
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 2945–2954
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
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