Synthesis and properties of thymidines with six-membered amide bridge

Article abstract of DOI:10.1016/j.bmc.2013.04.049

Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DN

Full text of DOI:10.1016/j.bmc.2013.04.049

Bioorganic & Medicinal Chemistry 21 (2013) 4405–4412
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and properties of thymidines with six-membered amide
bridge
⇑
Yoshiyuki Hari, Takashi Osawa, Yutaro Kotobuki, Aiko Yahara, Ajaya R. Shrestha, Satoshi Obika
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized,
and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides pre-
ferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide-
and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to
ssRNA by up to +4.7 and +3.7 °C of the Tm value per modification, respectively, compared with natural
oligonucleotide. In addition, their oligonucleotides showed high stability against 30-exonuclease.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 13 March 2013
Revised 17 April 2013
Accepted 18 April 2013
Available online 27 April 2013
Keywords:
Bridged nucleic acids
Nucleosides
Nucleotides
Oligonucleotides
Sugar modifications
1. Introduction
stituents could be introduced into the nitrogen in the amide bridge
to improve the functions of the oligonucleotides. In this study, two
Artificial nucleic acids that possess strong and sequence-selec-
tive binding affinities with single-stranded RNA (ssRNA) and/or
high nuclease-resistant properties are promising tools for nucleic
acid-based technologies such as the antisense method.1,2 In fact,
a large number of nucleic acid derivatives have been developed
to date.1 The breakthrough was the discovery by us3 and Wengel’s
group4 of 20,40-BNA/LNA with a 20-O,40-C-methylene-bridged struc-
ture in the sugar moiety, (Fig. 1). The 20,40-BNA/LNA modification of
oligonucleotides leads to a great improvement in hybridizing abil-
ity with ssRNA and an increase in nuclease resistance compared
with natural oligonucleotides. Since 20,40-BNA/LNA was reported,
the development of artificial nucleic acids with additional 20,40-
bridged structures has attracted much attention. In particular,
ring-enlargement from a five-membered bridge to a six-membered
one in 20,40-bridged structures would be a useful modification. For
example, ENA, the six-membered analog of 20,40-BNA/LNA, shows
the same level of duplex-hybridizing ability with ssRNA as does
20,40-BNA/LNA, and improved enzymatic stability as compared to
20,40-BNA/LNA (Fig. 1).5,6 Recently, we developed AmNA with
five-membered amide bridge, the duplex-forming ability of which
was comparable to that of 20,40-BNA/LNA, and the nuclease resis-
tance of which was superior to that of 20,40-BNA/LNA.7 Against this
background, we were interested in the properties of the ring-en-
larged analog of AmNA shown in Figure 2. In addition, various sub-
thymidines with a six-membered amide bridge, that is, NH and
NMe analogs, were synthesized, and the duplex-forming abilities
and the nuclease resistances of their oligonucleotides were
evaluated.
O
N
O
N
Me
Me
NH
NH
HO
HO
O
O
O
O
O
O
HO
2',4'-BNA/LNA
HO
ENA
Figure 1. Structures of 20,40-BNA/LNA and ENA monomers.
O
O
Me
Me
NH
NH
HO
O
N
O
HO
O
N
R
O
O
N
HO
N
HO
R
O
AmNA[NR]
⇑
Corresponding author. Tel.: +81 6 6879 8200; fax: +81 6 6879 8204.
E-mail address: obika@phs.osaka-ua.c.jp (S. Obika).
Figure 2. Design of nucleic acid monomers (R = H or Me) used in this study.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.049

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Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 4405–4412
BnO
using Me3P, ring-closed 13 was produced together with 12. Com-
pound 12 was converted to the desired 13 using EDC or MsCl;
the yields were 72% and 86%, respectively.
BnO
O
O
(ii)
(iii)
OAc
O
RO
TBDPSO
BnO
O
Me
The synthesis of phosphoramidite 14 was carried out in three
steps from 13 (Scheme 3). Diol 15 was prepared by hydrogenolysis
of 13. Then, dimethoxytritylation of 15, followed by phosphityla-
tion, yielded the desired phosphoramidite 14. Concerning the
N-methyl congener 17, protection of the imide nitrogen of thymine
in 13, and successive methylation of 18 gave 19, which was hydro-
genolyzed to produce diol 20. Using a similar synthetic route to 14,
20 was converted into the desired phosphoramidite 17 with an
N-methylamide bridge via dimethoxytritylated 21. The phospho-
ramidites 14 and 17 obtained were used to synthesize modified
oligonucleotides 22–29 on an automated DNA synthesizer (see
the Section 4 for details). These amide bridges were stable under
conventional conditions, that is, aqueous ammonia or methanolic
K2CO3, for cleavage from the resin and removal of b-cyanoethyl
groups on the phosphates.
BnO OAc
Me
3
1
)
R = H (
(i)
2
R = TBDPS (
)
O
O
Me
Me
NH
NH
BnO
BnO
N
O
(vi)
N
O
(viii)
O
O
OR
TBDPSO
TBDPSO
(vii)
BnO OR
R = Ac (4)
BnO
R = H (7)
(iv)
(v)
5
8
)
R = H (
)
R = Tf (
6
)
R = Ms (
O
O
The duplex-forming abilities of oligonucleotides 22–27 with
ssDNA or ssRNA were evaluated and the results are summarized
in Table 1. Regardless of whether it was the NH or NMe analog,
the modified oligonucleotides generally showed a significantly de-
creased affinity for ssDNA compared with natural oligonucleotide
30, although in the case of 24, possessing three NH analogs, the
affinity was increased by +1.7 °C per modification. Concerning
ssRNA, the single-modified oligonucleotides 22 and 25 had almost
the same affinities as natural oligonucleotide 30. However, by mul-
tiple modifications, the binding affinity was greatly increased and
Me
Me
NH
NH
BnO
RO
BnO
N
O
(x)
N
O
O
O
O
BnO
N3
BnO
OH
N3
R = TBDPS (9)
R = H (10)
11
(ix)
Scheme 1. Reagents and conditions: (i) TBDPSCl, DMAP, CH2Cl2, rt, 16 h, quant.; (ii)
Ac2O, AcOH, concd H2SO4, rt, 2 h, 91%; (iii) thymine, BSA, TMSOTf, MeCN, reflux,
3.5 h, 84%; (iv) K2CO3, MeOH, rt, 1 h, 95%; (v) MsCl, Et3N, CH2Cl2, 0 °C, 1.5 h, 91%;
(vi) NaOH, EtOH, rt, 12 h, 89%; (vii) Tf2O, pyridine, CH2Cl2, 0 °C, 0.5 h, 83%; (viii)
NaN3, DMF, rt, 13 h, quant.; (ix) TBAF, THF, rt, 17 h, quant.; (x) PDC, MS4Å, DMF, rt,
12 h, 94%.
the changes in Tm value per modification (DTm/mod.) of the NH
(24) or NMe (27) analogs were +4.7 and +3.7 °C, respectively. These
results demonstrate that oligonucleotides containing these thymi-
dines with six-membered amide bridges formed stable duplexes
with ssRNA and recognized ssRNA more selectively than ssDNA
in the duplex formation.
The binding affinities with ssDNA and ssRNA of the oligonucle-
otides modified by AmNA[NH] or AmNA[NMe] with a five-mem-
bered amide bridge, shown in Figure 2, and those of the
oligonucleotides modified by HxNA[NMe] with a six-membered
hydroxamate bridge, shown in Figure 3, were evaluated in our pre-
vious reports.7,8 Although these analogs showed lower duplex-
forming abilities with ssRNA than AmNA[NH] or AmNA[NMe],
one of these analogs, the NH analog, was more stably bound to
ssRNA compared with HxNA[NMe] with a six-membered bridge
similar to these amide bridges.
The stabilities of oligonucleotides 28 and 29 including these
analogs against 30-exonuclease were determined and compared
with those of AmNA[NH]-, AmNA[NMe]-, and HxNA[NMe]-modi-
fied oligonucleotides 31-33 and natural 30 (Fig. 4 and the se-
quences of oligonucleotides used are shown in the legend of
Fig. 4). Under conditions where natural 30 decomposed completely
within 5 min, over 50% and 60% of 28 and 29 remained after
40 min. The NMe analog (29) showed higher stability against
nuclease than the NH analog (28) did. This is probably because ac-
2. Results and discussion
2.1. Synthesis
The silylation of known compound 15 using TBDPSCl gave 2 in
quantitative yield (Scheme 1); 2 was converted to diacetate 3. The
reaction of 3 with silylated thymine, prepared in situ from thymine
and N,O-bis(trimethylsilyl)acetamide (BSA), in the presence of
TMSOTf produced the desired b-isomer 4. Next, introduction of a
nitrogen atom at the 20-position was performed using a double-ste-
reoinversion approach. After deacetylation of 4 on exposure to
K2CO3 in MeOH, stereoinversion of the 20-hydroxyl group was
achieved by mesylation of the resulting 5, followed by treatment
with NaOH. Then, 7 underwent reaction with Tf2O to give 8 in 83%
yield, and compound 9, with a nitrogen atom at the 20-position,
was obtained by azidation. Desilylation of 9, followed by oxidation
of 10 with PDC in DMF, efficiently produced carboxylic acid 11.
Next, reduction of the azide group in 11 was examined
(Scheme 2). Reduction by NaBH4 in i-PrOH gave the corresponding
amine 12 in 55% yield. Fortunately, under Staudinger conditions
O
N
O
O
Me
Me
Me
NH
NH
NH
BnO
BnO
BnO
O
N
O
O
N
O
(i)
O
O
O
+
O
BnO
OH
BnO
OH
NH2
NH
N3
BnO
O
(ii)
12
13
11
Scheme 2. Reagents and conditions: (i) NaBH4, i-PrOH, reflux, 1 h, 55% (12) or Me3P, THF/H2O (5:1), rt, 14 h, ca 35% (12) and 43% (13); (ii) EDC, DMAP, CH2Cl2, rt, 24 h, 72% or
MsCl, Et3N, CH2Cl2, 0 °C to rt, 17 h, 86%.

Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 4405–4412
4407
O
N
O
O
N
Me
Me
Me
NH
NH
NH
BnO
HO
DMTrO
O
N
O
O
O
O
O
(i)
(ii)
NH
13
(iv)
N
N
BnO
HO
HO
R
R
O
O
O
15
: R = H
20: R = Me
16
: R = H
21: R = Me
(i)
(iii)
O
O
O
N
Me
Me
N
OBn
N
OBn
Me
NH
BnO
BnO
N
O
N
O
(v)
DMTrO
O
O
O
O
NH
18
N
i-Pr2N
BnO
BnO
Me
N
O
O
P
O
R
19
O
NCCH2CH2O
14
: R = H
17: R = Me
Scheme 3. Reagents and conditions: (i) H2, 20% Pd(OH)2-C, MeOH, rt, 24 h, 61% (15) or 20% Pd(OH)2-C, cyclohexene, EtOH, reflux, 2 h, quant. (20); (ii) DMTrCl, pyridine, rt, 3–
17 h, 81% (16) and 69% (21); (iii) (i-Pr2N)2POCH2CH2CN, 1H-tetrazole, MeCN/THF (3:1), rt, 12 h, 42% (14) or i-Pr2NP(Cl)OCH2CH2CN, i-Pr2NEt, CH2Cl2, rt, 2 h, 73% (17); (iv)
BnOCH2Cl, DBU, DMF, 0 °C, 0.5 h, 86%; (v) NaH, MeI, DMF, 0 °C, 2 h, 60%.
Table 1
100
Tm values (°C) of duplexes formed between oligonucleotides and ssDNA or ssRNAa,b
Oligonucleotide
ssDNA
ssRNA
80
60
40
20
0
50-TTTTTTTTTT-30 (30)
50-TTTTTTTTTT-30 (22)
50-TTTTTTTTTT-30 (23)
50-TTTTTTTTTT-30 (24)
50-TTTTTTTTTT-30 (25)
50-TTTTTTTTTT-30 (26)
50-TTTTTTTTTT-30 (27)
21
19
16 (ꢀ5.0)
19 (ꢀ1.0)
26 (+1.7)
15 (ꢀ6.0)
13 (ꢀ4.0)
18 (ꢀ1.0)
20 (+1.0)
27 (+4.0)
33 (+4.7)
20 (+1.0)
26 (+3.5)
30 (+3.7)
a
Conditions: 10 mM sodium phosphate buffer (pH 7.2) and 100 mM NaCl. The
final concentration of each oligonucleotide used was 4 lM. The sequences of ssDNA
0
10
20
30
40
and ssRNA are 50-d(AAAAAAAAAA)-30 and 50-r(AAAAAAAAAA)-30, respectively. T
Time (min)
and T indicate NH analog and NMe analog, respectively.
b
The change in Tm value per modification is shown in parentheses.
Figure 4. Enzymatic stability of oligonucleotides 28 and 29. Conditions: 1.75
Crotalus admanteus venom phosphodiesterase (CAVP), 10 mM MgCl2, 50 mM Tris–
HCl (pH 8.0), and 7.5
cleotides used was 50-TTTTTTTTTT-30. T = NH analog (open circle, oligonucleotide
28), NMe analog (closed circle, oligonucleotide 29), AmNA[NH] (open diamond,
oligonucleotide 31), AmNA[NMe] (cross, oligonucleotide 32), HxNA[NMe] (open
triangle, oligonucleotide 33) and natural (open square, oligonucleotide 30).
lg/mL
l
M each oligonucleotide at 37 °C. The sequence of oligonu-
O
Me
NH
HO
N
O
O
cases of these NMe and NH analogs bearing a six-membered amide
bridge, as well as AmNA[NH] and AmNA[NMe] bearing a five-
membered amide bridge, the 50-phosphate moieties were easily
degradable and such a phenomenon was not observed. These re-
sults suggest that resistance against nuclease was greatly affected
by not only the bridge size but also by the composition of the
bridge, for example, whether there is any substituent and/or het-
eroatom, or the location of the substituent and/or heteroatom.
O
N
O
HO
Me
HxNA[NMe]
Figure 3. Structure of HxNA[NMe] monomer.
cess of the nuclease to the phosphodiester linkage was prevented
by the more bulky N-methylamide bridge. In a comparison of 28
and 29 with 31 and 32, respectively, no significant improvement
in nuclease resistance as a result of enlargement of the amide
bridge was observed. However, oligonucleotides 28 and 29 were
quite stable compared with 33, including an HxNA[NMe] with
the same six-membered ring size. In contrast, the six-membered
hydroxamate bridge of HxNA was opened under the nuclease
treatment conditions, and, consequently, the 50-phosphate moiety
of HxNA showed high resistance against nuclease.8 However, in the
3. Conclusion
Two thymidines bearing six-membered bridges, that is, amide
and N-methylamide bridges, were successfully synthesized and
introduced into oligonucleotides, using an automated DNA synthe-
sizer. These oligonucleotides formed stable duplexes with ssRNA,
but the duplexes with ssDNA were destabilized. They also had high
nuclease resistance, which were slightly better than those of

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Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 4405–4412
AmNAs bearing five-membered amide bridges. Moreover, their
nuclease resistances were much higher than that of HxNA[NMe]
with the same bridge size. These findings demonstrated that the
composition of the bridge moiety is a key factor in the develop-
ment of bridged nucleic acids. We believe that the accumulation
of properties such as duplex-forming ability and nuclease resis-
tance through the development of various bridged nucleic acids
will contribute to developing ideal useful tools for nucleic acid-
based technologies.
concentrated. The obtained crude residue (6.18 g) was purified by
column chromatography (silica gel 200 g, n-hexane/EtOAc = 5:1) to
give diacetate 3 as a colorless oil (5.67 g, 91%).
½
a 2D4
ꢂ
ꢀ16.3 (c 1.00, CHCl3). IR (KBr): 3069, 3031, 2931, 2857,
1748, 1496, 1472, 1455, 1428, 1369, 1309, 1219 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 1.02 (s, 9H), 1.83 (s, 3H), 1.93 (s, 3H), 1.96–
2.13 (m, 2H), 3.39 (d, J = 10.0 Hz, 1H), 3.50 (d, J = 10.0 Hz, 1H),
3.81–3.93 (m, 2H), 4.36–4.44 (m, 3H), 4.46 (d, J = 12.0 Hz, 1H),
4.57 (d, J = 12.0 Hz, 1H), 5.28 (d, J = 5.5 Hz, 1H), 6.03 (s, 1H),
7.21–7.67 (m, 20H). 13C NMR (100 MHz, CDCl3): d 19.09, 20.63,
20.93, 26.81, 35.75, 59.70, 73.16, 73.18, 73.44, 74.68, 78.07,
86.66, 97.61, 127.44, 127.49, 127.55, 127.57, 127.70, 127.75,
128.23, 128.32, 129.47, 133.87, 133.91, 135.53, 137.80, 138.15,
169.37, 169.70. MS (FAB): m/z 719 (MNa+). HRMS (FAB): calcd for
4. Experimental
4.1. General
C
41H48O8SiNa (MNa+) 719.3016, found 719.2999.
All chemicals were purchased from chemical suppliers. For col-
umn chromatography, Fuji Silysia silica gel PSQ-100B and FL-100D
were used. All melting points were measured on a Yanagimoto mi-
cro melting point apparatus and are uncorrected. 1H NMR, 13C NMR
and 31P NMR spectra were recorded on a JEOL ECS400 spectrome-
ter. IR spectra were recorded on JASCO FT/IR-200 and JASCO FT/IR-
4200 spectrometers. Optical rotations were recorded on a JASCO
DIP-370 instrument. Mass spectra were measured on a JEOL JMS-
600 or JEOL JMS-700 mass spectrometer. MALDI-TOF mass spectra
were recorded on a Bruker Daltonics Autoflex II TOF/TOF mass
spectrometer.
4.4. 20-O-Acetyl-30,50-di-O-benzyl-40-(2-tert-
butyldiphenylsiloxyethyl)-5-methyluridine (4)
Under nitrogen atmosphere, thymine (1.41 g, 11.2 mmol) and
BSA (6.8 mL, 28 mmol) were added to a solution of diacetate 3
(4.36 g, 11.0 mmol) in MeCN (20 mL) at room temperature. The
reaction mixture was refluxed for 1.5 h. TMSOTf (1.9 mL, 10 mmol)
was added to the resulting mixture at 0 °C. The reaction mixture
was refluxed for 3.5 h. The reaction was then quenched with satd
NaHCO3 aq and extracted with EtOAc. The combined organic layer
was washed with water and brine, dried over Na2SO4, and concen-
trated. The obtained crude residue (5.71 g) was purified by column
chromatography (silica gel 150 g, n-hexane/EtOAc = 3:2) to give
acetate 4 as a white foam (5.14 g, 84%).
4.2. 3,5-Di-O-benzyl-4-(2-tert-butyldiphenylsiloxyethyl)-1,2-O-
isopropylidene-a-D-ribofuranose (2)
Under nitrogen atmosphere, DMAP (350 mg, 2.87 mmol), Et3N
(4.0 mL, 29 mmol), and TBDPSCl (4.0 mL, 15 mmol) were added
to a solution of alcohol 15 (3.96 g, 9.56 mmol) in CH2Cl2 (100 mL)
at 0 °C. The reaction mixture was stirred at room temperature for
16 h. The reaction was then quenched with satd NaHCO3 aq and
extracted with CH2Cl2 (3 ꢁ 100 mL). The combined organic layer
was washed with water and brine, dried over Na2SO4, and concen-
trated. The obtained crude residue (8.82 g) was purified by column
chromatography (silica gel 200 g, n-hexane/EtOAc = 10:1) to give
silyl ether 2 (6.28 g, quant.) as a colorless oil.
Mp: 45–48 °C. ½a 2D5
ꢂ
+10.2 (c 1.00, CHCl3). IR (KBr): 3172, 3068,
2930, 2857, 1747, 1693, 1470, 1428, 1372, 1234 cmꢀ1
.
1H NMR
(400 MHz, CDCl3): d 1.01 (s, 9H), 1.48 (s, 3H), 1.76–1.83 (m, 1H),
2.04 (s, 3H), 2.03–2.11 (m, 1H), 3.41 (d, J = 10.5 Hz, 1H), 3.71–
3.83 (m, 2H), 3.89 (d, J = 10.5 Hz, 1H), 4.35 (d, J = 6.5 Hz, 1H),
4.38 (d, J = 11.5 Hz, 1H), 4.39 (d, J = 9.5 Hz, 1H), 4.44 (d, J = 9.5 Hz,
1H), 4.57 (d, J = 11.5 Hz, 1H), 5.35 (dd, J = 5.0, 6.5 Hz, 1H), 6.08 (d,
J = 5.0 Hz, 1H), 7.20–7.41 (m, 16H), 7.49 (s, 1H), 7.60–7.65 (m,
4H), 7.83 (br s, 1H). 13C NMR (100 MHz, CDCl3): d 11.96, 19.06,
20.82, 26.82, 35.09, 59.27, 73.10, 73.38, 74.22, 75.24, 77.43,
86.03, 87.35, 111.21, 127.58, 127.66, 127.85, 128.01, 128.42,
128.59, 129.67, 133.47, 133.51, 135.51, 135.69, 137.32, 137.49,
150.28, 163.58, 170.05. MS (FAB): m/z 763 (MH+). HRMS (FAB):
calcd for C44H51N2O8Si (MH+) 763.3415, found 763.3402.
½
a 2D5
ꢂ
+16.3 (c 1.00, CHCl3). IR (KBr): 3068, 3031, 2932, 2857,
1496, 1472, 1454, 1428, 1383, 1312, 1256, 1209 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 1.00 (s, 9H), 1.30 (s, 3H), 1.50 (s, 3H), 1.87
(ddd, J = 7.0, 8.0, 14.5 Hz, 1H), 2.43 (ddd, J = 5.0, 6.5, 14.5 Hz, 1H),
3.29 (d, J = 10.5 Hz), 1H, 3.70 (d, J = 10.5 Hz, 1H), 3.82 (ddd,
J = 5.0, 7.0, 12.0 Hz, 1H), 3.94 (ddd, J = 6.5, 8.0, 12.0 Hz, 1H), 4.21
(d, J = 5.5 Hz, 1H), 4.35 (d, J = 12.5 Hz, 1H), 4.47 (d, J = 12.5 Hz,
1H), 4.53 (d, J = 12.0 Hz, 1H), 4.60 (dd, J = 4.0, 5.5 Hz, 1H), 4.74 (d,
J = 12.0 Hz, 2H), 5.74 (d, J = 4.0 Hz, 1H), 7.19–7.67 (m, 20H). 13C
NMR (100 MHz, CDCl3): d 19.08, 26.15, 26.59, 26.80, 34.93, 59.78,
72.41, 73.37, 77.88, 79.26, 86.65, 104.16, 113.01, 127.50, 127.52,
127.57, 127.72, 127.80, 128.29, 128.32, 129.45, 133.87, 135.57,
138.08, 138.21. MS (FAB): m/z 675 (MNa+). HRMS (FAB): calcd for
4.5. 30,50-Di-O-benzyl-40-(2-tert-butyldiphenylsiloxyethyl)-5-
methyluridine (5)
K2CO3 (450 mg, 330 mmol) was added to a solution of com-
pound 4 (5.04 g, 6.61 mmol) in MeOH (10 mL) at room tempera-
ture. The reaction mixture was stirred at room temperature for
1 h. The reaction was then quenched with satd NH4Cl aq and ex-
tracted with EtOAc. The combined organic layer was washed with
water and brine, dried over Na2SO4, and concentrated. The ob-
tained crude residue (4.76 g) was purified by column chromatogra-
phy (silica gel 100 g, n-hexane/EtOAc = 4:3) to give alcohol 5 as a
white foam 4.54 g, 95%).
C
40H48O6SiNa (MNa+) 675.3118, found 675.3115.
4.3. 1,2-Di-O-acetyl-3,5-di-O-benzyl-4-(2-tert-
butyldiphenylsiloxyethyl)- -ribofuranose (3)
D
Mp: 62–64 °C. ½a 2D6
ꢂ
ꢀ10.3 (c 1.00, CHCl3). IR (KBr): 3423, 3179,
Under nitrogen atmosphere, Ac2O (10 mL, 110 mmol) and
H2SO4 (0.1% in AcOH, 4.0 mL) were added to a solution of silyl ether
2 (5.86 g, 8.98 mmol) in AcOH (30 mL) at room temperature. The
reaction mixture was stirred at room temperature for 2 h. The reac-
tion was then quenched with satd NaHCO3 aq and extracted with
EtOAc. The combined organic layer was washed with satd NaHCO3
aq, water and brine, dried over Na2SO4 and concentrated. The com-
bined organic layer was washed with brine, dried over Na2SO4, and
3066, 2928, 2856, 1695, 1471, 1428, 1391, 1362, 1270 cmꢀ1
.
1H
NMR (400 MHz, CDCl3): d 1.06 (s, 9H), 1.56 (s, 3H), 1.77 (ddd,
J = 6.5, 8.0, 14.5 Hz, 1H), 2.14 (dt, J = 5.0, 14.5 Hz, 1H), 2.83 (d,
J = 8.0 Hz, 1H), 3.45 (d, J = 10.5 Hz, 1H), 3.73–3.88 (m, 2H), 3.85
(d, J = 10.5 Hz, 1H), 4.14 (d, J = 6.0 Hz, 1H), 4.30 (m, 1H), 4.46 (d,
J = 12.0 Hz, 1H), 4.49 (d, J = 12.0 Hz, 1H), 4.56 (d, J = 11.0 Hz, 1H),
4.63 (d, J = 11.0 Hz, 1H), 5.82 (d, J = 6.0 Hz, 1H), 7.22–7.65 (m,

Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 4405–4412
4409
21H), 8.12 (br s, 1H). 13C NMR (100 MHz, CDCl3): d 12.04, 19.05,
26.83, 35.36, 59.36, 73.49, 74.05, 74.57, 75.02, 79.41, 87.03,
88.43, 111.93, 127.50, 127.64, 127.66, 128.01, 128.06, 128.25,
128.57, 128.60, 129.65, 129.68, 133.51, 135.52, 135.89, 137.02,
137.33, 150.80, 163.69. MS (FAB): m/z 721 (MH+). HRMS (FAB):
calcd for C42H49N2O7Si (MH+) 721.3309, found 721.3320.
(2.07 g, 2.87 mmol) in CH2Cl2 (30 mL) at 0 °C. The reaction mixture
was stirred at 0 °C for 0.5 h. The reaction was then quenched with
satd NaHCO3 aq at 0 °C and extracted with CH2Cl2. The combined
organic layer was washed with water and brine, dried over Na2SO4,
and concentrated. The obtained crude residue (2.56 g) was purified
by column chromatography (silica gel 100 g, n-hexane/
EtOAc = 2:1) to give triflate 8 as a white foam (2.03 g, 83%).
4.6. 30,50-Di-O-benzyl-40-(2-tert-butyldiphenylsiloxyethyl)-20-O-
methanesulfonyl-5-methyluridine (6)
Mp: 37–40 °C. ½a 2D5
ꢂ
+37.1 (c 1.00, CHCl3). IR (KBr): 3190, 3069,
2930, 2858, 1695, 1455, 1426, 1245, 1215, 1143 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 1.03 (s, 9H), 1.67 (s, 3H), 1.81–1.95 (m, 2H),
3.36 (d, J = 10.5 Hz, 1H), 3.69 (d, J = 10.5 Hz, 1H), 3.72–3.86 (m,
2H), 4.38 (d, J = 12.0 Hz, 1H), 4.43 (d, J = 12.0 Hz, 1H), 4.48 (d,
J = 11.5 Hz, 1H), 4.57 (d, J = 4.5 Hz, 1H), 4.72 (d, J = 11.5 Hz, 1H),
5.35 (dd, J = 4.5, 5.5 Hz, 1H), 6.19 (d, J = 5.5 Hz, 1H), 7.19–7.63
(m, 21H), 8.10 (br s, 1H). 13C NMR (100 MHz, CDCl3): d 12.21,
19.00, 26.77, 34.04, 59.04, 71.23, 73.32, 73.47, 80.48, 81.51,
84.37, 87.53, 110.98, 118.24 (q, J = 318 Hz), 127.64, 127.67,
127.69, 127.98, 128.01, 128.33, 128.45, 128.53, 129.72, 129.79,
133.26, 133.28, 135.47, 135.49, 136.41, 137.12, 150.13, 163.48.
MS (FAB): m/z 853 (MH+). HRMS (FAB): calcd for C43H48N2O9F3SiS
(MH+) 853.2802, found 853.2813.
Under nitrogen atmosphere, Et3N (4.0 mL, 29 mmol) and MsCl
(0.70 mL, 9.0 mmol) were added to
a solution of alcohol 5
(3.26 g, 4.52 mmol) in CH2Cl2 (40 mL) at 0 °C. The reaction mixture
was stirred at 0 °C for 1.5 h. The reaction was then quenched with
satd NaHCO3 aq at 0 °C and extracted with CH2Cl2. The combined
organic layer was washed with water and brine, dried over Na2SO4,
and concentrated. The obtained crude residue (3.80 g) was purified
by column chromatography (silica gel 100 g, n-hexane/
EtOAc = 4:3) to give mesylate 6 as a white foam (3.29 g, 91%).
Mp: 57–62 °C. ½a 2D2
ꢂ
+214.8 (c 1.00, CHCl3). IR (KBr): 3168, 3068,
3031, 2932, 2857, 1694, 1538, 1471, 1428, 1361, 1272 cmꢀ1
.
1H
NMR (400 MHz, CDCl3): d 1.02 (s, 9H), 1.44 (s, 3H), 1.85 (ddd,
J = 6.5, 8.0, 14.5 Hz, 1H), 2.10 (dt, J = 5.0, 14.5 Hz, 1H), 3.07 (s,
3H), 3.44 (d, J = 6.5 Hz, 1H), 3.70–3.86 (m, 2H), 3.95 (d, J = 6.5 Hz,
1H), 4.33 (d, J = 5.0 Hz, 1H), 4.37 (d, J = 12.0 Hz, 1H), 4.42 (d,
J = 12.0 Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H), 4.83 (d, J = 12.0 Hz, 1H),
5.26 (dd, J = 3.0, 5.0 Hz, 1H), 6.01 (d, J = 3.0 Hz, 1H), 7.18–7.63 (m,
21H), 8.29 (br s, 1H). 13C NMR (75.5 MHz, CDCl3): d 11.90, 19.08,
26.85, 35.11, 38.83, 59.13, 72.65, 73.40, 73.78, 76.39, 80.30,
86.74, 87.83, 111.27, 127.60, 127.68, 127.70, 128.11, 128.12,
128.47, 128.64, 129.70, 129.73, 133.42, 133.46, 135.28, 135.53,
137.11, 137.22, 150.46, 163.44. MS (FAB): m/z 799 (MH+). HRMS
(FAB): calcd for C43H48N2O9F3SiS (MH+) 799.3085, found 799.3085.
4.9. (20R)-20-Azido-30,50-di-O-benzyl-40-(2-tert-
butyldiphenylsiloxyethyl)thymidine (9)
Under nitrogen atmosphere, NaN3 (464 mg, 7.14 mmol) was
added to a solution of triflate 8 (2.03 g, 2.38 mmol) in DMF
(20 mL) at room temperature. The reaction mixture was stirred
at room temperature for 13 h. The resulting mixture was added
to water and extracted with Et2O. The combined organic layer
was washed with brine, dried over Na2SO4, and concentrated. The
obtained crude residue (2.20 g) was purified by column chroma-
tography (silica gel 150 g, n-hexane/EtOAc = 2:1) to give azide 9
as a white foam (1.80 g, quant.).
4.7. 30,50-Di-O-benzyl-40-(2-tert-butyldiphenylsiloxyethyl)-5-
methylarabinouridine (7)
Mp: 46–49 °C. ½a 2D5
ꢂ
ꢀ5.1 (c 1.00, CHCl3). IR (KBr): 3179, 3068,
2929, 2857, 2109, 1694, 1470, 1428, 1362, 1268 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 1.03 (s, 9H), 1.58 (d, J = 1.0 Hz, 3H), 1.78
(ddd, J = 6.0, 8.0, 14.5 Hz, 1H), 2.15 (ddd, J = 5.0, 5.5, 14.5 Hz, 1H),
3.44 (d, J = 11.0 Hz, 1H), 3.70–3.85 (m, 2H), 3.91 (d, J = 11.0 Hz,
1H), 3.97 (t, J = 6.0 Hz, 1H), 4.28 (d, J = 6.0 Hz, 1H), 4.43 (d,
J = 11.5 Hz, 1H), 4.49 (d, J = 11.5 Hz, 1H), 4.49 (d, J = 11.5 Hz, 1H),
4.79 (d, J = 11.5 Hz, 1H), 5.99 (d, J = 6.0 Hz, 1H), 7.20–7.64 (m,
20H), 7.49 (d, J = 1.0 Hz, 1H), 7.93 (br s, 1H). 13C NMR (100 MHz,
CDCl3): d 12.10, 19.04, 26.82, 35.22, 59.26, 65.17, 73.41, 73.47,
74.28, 79.06, 86.00, 87.57, 111.09, 127.55, 127.64, 127.66, 128.05,
128.12, 128.21, 128.51, 128.64, 129.66, 129.71, 133.43, 133.44,
135.24, 135.50, 136.90, 137.13, 150.25, 163.66. MS (FAB): m/z
746 (MH+). HRMS (FAB): calcd for C42H48N5O6Si (MH+) 746.3374,
found 746.3404.
1 M NaOH aq (12 mL, 12 mmol) was added to a solution of mes-
ylate 6 (3.16 g, 3.95 mmol) in EtOH (40 mL) at room temperature.
The reaction mixture was stirred at room temperature for 12 h.
The reaction was then quenched with satd NH4Cl aq at 0 °C and ex-
tracted with EtOAc. The combined organic layer was washed with
water and brine, dried over Na2SO4, and concentrated. The ob-
tained crude residue (2.72 g) was purified by column chromatogra-
phy (silica gel 100 g, n-hexane/EtOAc = 1:1) to give alcohol 7 as a
white foam (2.55 g, 89%).
Mp: 56–58 °C. ½a 2D6
ꢂ
+43.5 (c 1.00, CHCl3). IR (KBr): 3357, 3181,
3068, 3031, 2929, 2856, 1695, 1472, 1455, 1428, 1390, 1362, 1281
1206 cmꢀ1 1H NMR (300 MHz, CDCl3): d 1.02 (s, 9H), 1.71 (d,
.
J = 1.0 Hz, 3H), 1.88–1.93 (m, 2H), 3.52 (d, J = 10.5 Hz, 1H), 3.72–
3.86 (m, 2H), 3.91 (d, J = 10.5 Hz, 1H), 4.14 (d, J = 4.0 Hz, 1H), 4.26
(d, J = 8.0 Hz, 1H), 4.39 (ddd, J = 4.0, 5.0, 8.0 Hz, 1H), 4.45 (d,
J = 12.0 Hz, 1H), 4.49 (d, J = 11.5 Hz, 1H), 4.51 (d, J = 11.5 Hz, 1H),
4.74 (d, J = 12.0 Hz, 1H), 5.96 (d, J = 5.0 Hz, 1H), 7.21–7.65 (m,
20H), 7.48 (d, J = 1.0 Hz, 1H), 9.01 (br s, 1H). 13C NMR (100 MHz,
CDCl3): d 12.27, 19.09, 26.86, 35.16, 59.59, 72.65, 73.18, 73.70,
74.66, 83.80, 84.65, 85.07, 109.27, 127.66, 127.83, 127.93, 128.33,
128.39, 128.70, 129.69, 129.72, 133.44, 135.50, 136.62, 137.40,
137.55, 150.67, 163.99. MS (FAB): m/z 721 (MH+). HRMS (FAB): calcd
for C42H49N2O7Si (MH+) 721.3309, found 721.3300.
4.10. (20R)-20-Azido-30,50-di-O-benzyl-40-(2-hydroxyethyl)
thymidine (10)
Under nitrogen atmosphere, TBAF (1.0 M in THF, 2.6 mL,
2.6 mmol) was added to a solution of azide 9 (1.78 g, 2.38 mmol)
in THF (20 mL) at room temperature. The reaction mixture was
stirred at room temperature for 17 h. The resulting mixture was
added to water and extracted with EtOAc. The combined organic
layer was washed with brine, dried over Na2SO4, and concentrated.
The obtained crude residue (2.20 g) was purified by column chro-
matography (silica gel 75 g, n-hexane/EtOAc = 1:1) to give com-
pound 10 as a white foam (1.22 g, quant.).
4.8. 30,50-Di-O-benzyl-40-(2-tert-butyldiphenylsiloxyethyl)-5-
methyl-20-O-(trifluoromethanesulfonyl)arabinouridine (8)
Mp: 45–50 °C. ½a 2D5
ꢂ
+10.2 (c 1.00, CHCl3). IR (KBr): 3441, 3182,
3063, 2927, 2109, 1693, 1496, 1469, 1455, 1364, 1267 cmꢀ1
.
1H
Under nitrogen atmosphere, pyridine (1.9 mL, 17 mmol) and
Tf2O (1.4 mL, 8.6 mmol) were added to a solution of alcohol 7
NMR (400 MHz, CDCl3): d 1.56 (s, 3H), 1.66 (br s, 1H), 1.76 (dt,
J = 6.0, 15.0 Hz, 1H), 2.21 (dt, J = 6.0, 15.0 Hz, 1H), 3.44 (d,

4410
Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 4405–4412
J = 10.5 Hz, 1H), 3.75 (t, J = 6.0 Hz, 2H), 3.82 (d, J = 10.5 Hz, 1H),
4.04 (t, J = 6.0 Hz, 1H), 4.30 (d, J = 6.0 Hz, 1H), 4.47 (d, J = 11.5 Hz,
1H), 4.53 (d, J = 11.5 Hz, 1H), 4.55 (d, J = 12.0 Hz, 1H), 4.83 (d,
J = 12.0 Hz, 1H), 6.10 (d, J = 6.0 Hz, 1H), 7.22–7.38 (m, 10H), 7.44
(s, 1H), 8.34 (br s, 1H). 13C NMR (100 MHz, CDCl3): d 12.12,
34.83, 58.28, 64.83, 73.32, 73.66, 74.50, 79.56, 86.30, 87.81,
111.36, 127.67, 128.11, 128.27, 128.37, 128.61, 128.72, 135.19,
136.72, 136.96, 150.22, 163.39. MS (FAB): m/z 508 (MH+). HRMS
(FAB): calcd for C26H30N5O6 (MH+) 508.2196, found 508.2204.
Condensation using MsCl: Under nitrogen atmosphere, Et3N
(0.32 mL, 2.25 mmol) and MsCl (70 L, 0.0820 mmol) were added
l
to a solution of amino acid 12 (223 mg, 0.450 mmol) in CH2Cl2
(5.0 mL) at 0 °C. The reaction mixture was stirred at room temper-
ature for 17 h. The reaction was then quenched with satd NaHCO3
aq at 0 °C and extracted with CH2Cl2. The combined organic layer
was washed with water and brine, dried over Na2SO4, and concen-
trated. The obtained crude residue (300 mg) was purified by col-
umn chromatography (silica gel 10 g, CHCl3/MeOH = 20:1) to give
lactam 13 as a white foam (185 mg, 86%).
4.11. (20R)-20-Azido-30,50-di-O-benzyl-40-(2-carboxymethyl)
thymidine (11)
Compound 12: Mp: 45–48 °C. ½a D26
ꢂ ꢀ12.2 (c 1.00, CHCl3). IR (KBr):
3426, 3197, 3033, 2925, 2878, 1697, 1497, 1473, 1455, 1390, 1274,
1213 cmꢀ1. 1H NMR (400 MHz, CD3OD): d 1.65 (s, 3H), 2.84 (s, 2H),
3.84 (d, J = 9.5 Hz, 1H), 3.92 (d, J = 9.5 Hz, 1H), 4.19–4.20 (m, 1H),
4.61–4.85 (m, 5H), 6.22 (d, J = 8.0 Hz, 1H), 7.25–7.42 (m, 10H), 7.55
(s, 1H), 7.90 (br s, 1H). 13C NMR (100 MHz, CD3OD): d 12.48, 38.19,
57.92, 74.79, 75.07, 76.89, 81.36, 86.89, 89.23, 112.37, 128.66,
128.79, 128.97, 129.10, 129.19, 129.72, 136.82, 138.01, 138.86,
152.64, 165.92, 173.68. MS (FAB): m/z 496 (MH+). HRMS (FAB): calcd
for C26H30N3O7 (MH+) 496.2084, found 496.2081.
MS4Å (3.0 g) and PDC (10.9 g, 28.8 mmol) were added to a solution
of compound 10 (1.22 g, 2.38 mmol) in DMF (20 mL) at room tempera-
ture. The reaction mixture was stirred at room temperature for 12 h. The
resulting mixture was added to water and AcOH and extracted with
EtOAc. The combined organic layer was washed with 0.4 M (COOH)2
aq, 0.2 M (COONH4)2 aq, and brine, dried over Na2SO4, and concentrated.
The obtained crude residue (3.20 g) was purified by column chromatog-
raphy (silica gel 75 g, n-hexane/EtOAc/AcOH = 50:50:1) to give car-
boxylic acid 11 as a white foam (1.18 g, 94%).
Compound 13: Mp: 112–114 °C. ½a D21
ꢂ
+78.9 (c 1.00, CHCl3). IR
(KBr): 3500, 3169, 3063, 2926, 1685, 1454, 1367, 1273,
Mp: 78–81 °C. ½a 2D5
ꢂ
ꢀ29.4 (c 1.00, CHCl3). IR (KBr): 3510, 3179,
1211 cmꢀ1 1H NMR (300 MHz, CDCl3): d 1.37 (d, J = 1.0 Hz, 3H),
.
3033, 2929, 2108, 1705, 1470, 1455, 1269, 1213 cmꢀ1
.
1H NMR
2.40 (d, J = 18.0 Hz, 1H), 2.58 (d, J = 18.0 Hz, 1H), 3.60 (d,
J = 11.0 Hz, 1H), 3.72 (d, J = 11.0 Hz, 1H), 4.23 (dd, J = 4.0, 6.0 Hz,
2H), 4.32 (d, J = 4.0 Hz, 1H), 4.54 (d, J = 11.5 Hz, 1H), 4.56 (d,
J = 11.5 Hz, 1H), 4.60 (d, J = 11.5 Hz, 1H), 4.71 (d, J = 11.5 Hz, 1H),
5.83 (s, 1H), 7.25–7.35 (m, 11H), 7.94 (d, J = 1.0 Hz, 1H), 9.40 (br
s, 1H). 13C NMR (100 MHz, CDCl3): d 11.96, 38.60, 55.75, 68.71,
70.57, 71.96, 73.57, 83.78, 90.10, 109.61, 127.56, 127.89, 127.97,
128.23, 128.43, 128.66, 135.54, 136.96, 137.07, 150.72, 164.25,
169.56. MS (FAB): m/z 478 (MH+). HRMS (FAB): calcd for
(400 MHz, CDCl3): d 1.56 (s, 3H), 2.71 (d, J = 16.5 Hz, 1H), 2.88 (d,
J = 16.5 Hz, 1H), 3.77 (d, J = 10.0 Hz, 1H), 3.94 (d, J = 10.0 Hz, 1H),
3.99 (dd, J = 5.5, 7.0 Hz, 2H), 4.37 (d, J = 5.5 Hz, 1H), 4.49 (d,
J = 11.0 Hz, 1H), 4.53 (d, J = 10.5 Hz, 1H), 4.61 (d, J = 10.5 Hz, 1H),
4.88 (d, J = 11.0 Hz, 1H), 6.22 (d, J = 7.0 Hz, 1H), 7.20–7.48 (m,
11H), 9.65 (br s, 1H). 13C NMR (100 MHz, CDCl3): d 12.14, 38.05,
64.99, 73.80, 73.99, 75.30, 80.26, 85.30, 86.36, 111.74, 127.81,
128.06, 128.19, 128.46, 128.52, 128.89, 135.59, 136.81, 137.02,
150.67, 164.38, 175.09. MS (FAB): m/z 522 (MH+). HRMS (FAB):
calcd for C26H28N5O7 (MH+) 522.1989, found 522.1979.
C
26H28N3O6 (MH+) 478.1978, found 478.1983.
4.13. (20R)-20,40-(2-Oxo-iminoethano)thymidine (15)
4.12. (20R)-20-Amino-30,50-di-O-benzyl-40-(2-carboxymethyl)
thymidine (12) and (20R)-30,50-Di-O-benzyl-20,40-(2-oxo-
iminoethano)thymidine (13)
Lactam 13 (50.0 mg, 0.105 mmol) in MeOH (2.0 mL) was added
to a suspension of 20% Pd(OH)2 on carbon (50.0 mg, 0.462 mmol)
in MeOH (1.0 mL) at room temperature under nitrogen atmo-
sphere. The reaction mixture was stirred at room temperature for
24 h under hydrogen atmosphere. The resulting mixture was fil-
tered and concentrated. The obtained crude residue (25.4 mg)
was purified by recrystallization from MeOH to give nucleoside
15 as a white solid (19.1 mg, 61%).
Reduction by NaBH4: Under nitrogen atmosphere, NaBH4
(56.2 mg, 1.49 mmol) was added to a solution of carboxylic acid
11 (155 mg, 0.297 mmol) in i-PrOH (3.0 mL) at 0 °C. The reaction
mixture was refluxed for 1 h. The reaction was then quenched with
acetone and the resulting mixture was concentrated. The obtained
crude residue (220 mg) was purified by column chromatography
(silica gel 5.0 g, CHCl3/MeOH = 20:1) to give compound 12 as a
white foam (81.3 mg, 55%).
Mp: 187–188 °C. ½a D23
ꢂ
+36.5 (c 0.200, CH3OH); IR (KBr): 3449,
3318, 3175, 3060, 2926, 2819, 1707, 1651, 1469, 1386, 1272,
1215 cmꢀ1 1H NMR (300 MHz, CD3OD): d 1.85 (s, 3H), 2.29 (d,
.
Staudinger reaction: Under nitrogen atmosphere, Me3P (1.0 M
in toluene, 0.55 mL, 0.55 mmol) was added to a solution of carbox-
ylic acid 11 (241 mg, 0.462 mmol) in THF/H2O (5:1, 6.0 mL) at
room temperature. The reaction mixture was stirred at room
temperature for 24 h. The resulting mixture was concentrated.
The obtained crude residue (293 mg) was purified by column chro-
matography (silica gel 10 g, CHCl3/MeOH = 30:1) to give lactam 13
as a white foam (106 mg, 43%) together with compound 12 (81 mg,
ca. 35%) including a small amount of impurity.
J = 18.0 Hz, 1H), 2.46 (d, J = 18.0 Hz, 1H), 3.68 (d, J = 12.5 Hz, 1H),
3.75 (d, J = 12.5 Hz, 1H), 3.82 (d, J = 4.0 Hz, 1H), 4.33 (d, J = 4.0 Hz,
1H), 5.68 (s, 1H), 8.35 (s, 1H). 13C NMR (100 MHz, CD3OD): d
12.55, 38.80, 60.02, 61.82, 64.42, 85.99, 91.01, 110.08, 137.78,
152.07, 166.63, 173.32. MS (FAB): m/z 298 (MH+). HRMS (FAB):
calcd for C26H28N3O6 (MH+) 298.1039, found 298.1044.
4.14. (20R)-50-O-(4,40-Dimethoxytrityl)-20,40-(2-oxo-
iminoethano)thymidine (16)
Condensation using EDC: Under nitrogen atmosphere, EDC
(157 mg, 0.820 mmol) and DMAP (10.0 mg, 0.0820 mmol) were
added to a solution of amino acid 12 (81.3 mg, 0.164 mmol) in
CH2Cl2 (3.0 mL) at 0 °C. The reaction mixture was stirred at room
temperature for 24 h. The reaction was then quenched with satd
NaHCO3 aq at 0 °C and extracted with CH2Cl2. The combined organ-
ic layer was washed with water and brine, dried over Na2SO4, and
concentrated. The obtained crude residue (65.4 mg) was purified
by column chromatography (silica gel 3.0 g, CHCl3/MeOH = 20:1)
to give lactam 13 as a white foam (56.6 mg, 72%).
Under nitrogen atmosphere, DMTrCl (95.7 mg, 0.283 mmol) was
added to a solution of nucleoside 15 (28.0 mg, 0.0941 mmol) in pyr-
idine (2.0 mL) at 0 °C. The reaction mixture was stirred at room tem-
perature for 3 h. The reaction was then quenched with satd NaHCO3
aq at 0 °C and extracted with EtOAc. The combined organic layer was
washed with water and brine, dried over Na2SO4, and concentrated.
The obtained crude residue (159 mg) was purified by column chro-
matography (silica gel 5.0 g, CHCl3/MeOH = 20:1) to give compound
16 as a white foam (45.9 mg, 81%).

Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 4405–4412
4411
Mp: 204–206 °C. ½a D23
ꢂ
+80.7 (c 1.00, CHCl3). IR (KBr): 3563,
3341, 3062, 2933, 2838, 1705, 1657, 1608, 1509, 1466, 1384,
4.17. (20R)-3-Benzyloxymethyl-30,50-di-O-benzyl-20,40-(N-
methyl-2-oxo-iminoethano)thymidine (19)
1254 cmꢀ1 1H NMR (300 MHz, CD3OD): d 1.20 (d, J = 1.0 Hz, 3H),
.
2.37 (d, J = 18.0 Hz, 1H), 2.44 (d, J = 18.0 Hz, 1H), 3.33 (d,
J = 11.0 Hz, 1H), 3.37 (d, J = 11.0 Hz, 1H), 3.74 (s, 6H), 3.94 (d,
J = 4.0 Hz, 1H), 4.56 (d, J = 4.0 Hz, 1H), 5.66 (s, 1H), 6.83–7.47 (m,
13H), 7.86 (d, J = 1.0 Hz, 1H). 13C NMR (75 MHz, CD3OD): d 12.28,
39.32, 55.72, 59.82, 64.37, 65.71, 85.55, 88.06, 91.70, 110.67,
114.28, 128.19, 129.03, 129.44, 131.45, 136.38, 136.59, 136.87,
145.75, 152.01, 160.32, 160.35, 166.52, 173.00. MS (FAB): m/z
600 (MH+). HRMS (FAB): calcd for C26H28N3O6 (MH+) 600.2346,
found 600.2347.
Under nitrogen atmosphere, 60% NaH (in mineral oil, 10.4 mg,
0.260 mmol) was added to a solution of compound 18 (130 mg,
0.218 mmol) in DMF (2.0 mL) at 0 °C. The reaction mixture was
stirred at 0 °C for 0.5 h. MeI (68 lL, 1.1 mmol) was added to the
resulting mixture at 0 °C. The reaction mixture was stirred at 0 °C
for 2 h. The reaction was then quenched with satd NaHCO3 aq at
0 °C and extracted with Et2O. The combined organic layer was
washed with brine, dried over Na2SO4, and concentrated. The ob-
tained crude residue (139 mg) was purified by column chromatog-
raphy (silica gel 5.0 g, n-hexane/EtOAc = 3:2) to give N-Me lactam
19 as a white foam (79.6 mg, 60%).
4.15. (20R)-30-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-
50-O-(4,40-dimethoxytrityl)-20,40-(2-oxo-iminoethano)thymidine
(14)
Mp: 48–50 °C. ½a 2D8
ꢂ
+67.8 (c 0.760, CHCl3). IR (KBr): 3065, 3031,
1H
1.45 (d, J = 1.0 Hz, 3H), 2.42 (d,
2925, 2819, 1707, 1651, 1496, 1453, 1365, 1279, 1212 cmꢀ1
.
NMR (400 MHz, CDCl3):
d
Under
nitrogen
atmosphere,
1H-tetrazole
(10.1 mg,
J = 17.0 Hz, 1H), 2.60 (d, J = 17.0 Hz, 1H), 3.07 (s, 3H), 3.68 (d,
J = 12.0 Hz, 1H), 3.74 (d, J = 12.0 Hz, 1H), 3.82 (d, J = 4.0 Hz, 1H),
4.16 (d, J = 4.0 Hz, 1H), 4.54–4.61 (m, 4H), 4.70 (s, 2H), 5.43 (d,
J = 9.5 Hz, 1H), 5.47 (d, J = 9.5 Hz, 1H), 5.66 (s, 1H), 7.23–7.39 (m,
15H), 7.91 (d, J = 1.0 Hz, 1H). 13C NMR (100 MHz, CDCl3): d 12.55,
34.17, 38.77, 63.11, 68.51, 70.20, 71.19, 72.27, 72.72, 73.64,
84.24, 88.87, 109.38, 127.52, 127.66, 127.70, 127.89, 128.27,
128.30, 128.32, 128.62, 128.69, 133.93, 136.69, 136.92,
137.81,150.68, 163.35, 167.29. MS (FAB): m/z 612 (MH+). HRMS
(FAB): calcd for C35H38N3O7 (MH+) 612.2710, found 612.2712.
0.144 mmol) and (i-Pr2N)2POCH2CH2CN (46
lL, 0.14 mmol) were
added to a solution of compound 16 (72.0 mg, 0.120 mmol) in
THF/MeCN (3:1, 2.0 mL) at 0 °C. The reaction mixture was stirred
at room temperature for 12 h. The reaction was then quenched
with satd NaHCO3 aq at 0 °C and extracted with CH2Cl2. The com-
bined organic layer was washed with satd NaHCO3 aq, water and
brine, dried over Na2SO4, and concentrated. The obtained crude
residue (99.0 mg) was purified by column chromatography (silica
gel 3.0 g, CHCl3/MeOH = 20:1) to give 14 as
a white foam
(70.2 mg, 73%).
Mp: 127–129 °C. 1H NMR (400 MHz, CDCl3): d 0.99 (d, J = 7.0 Hz,
1.8H), 1.09 (d, J = 7.0 Hz, 4.2 H), 1.10 (d, J = 7.0 Hz, 4.2H), 1.16 (d,
J = 7.0 Hz, 1.8H), 1.23 (s, 0.9H), 1.26 (s, 2.1H), 2.36–2.64 (m, 4H),
3.24–3.81 (m, 12H), 4.18 (dd, J = 4.0, 6.0 Hz, 0.3H), 4.32 (dd,
J = 4.0, 5.5 Hz, 0.7H), 4.62 (dd, J = 4.0, 7.0 Hz, 0.3H), 4.76 (dd,
J = 4.0, 7.0 Hz, 0.7H), 5.74 (s, 0.7H), 5.76 (s, 0.3H), 6.28 (d,
J = 5.5 Hz, 0.7H), 6.33 (d, J = 6.0 Hz, 0.3H), 6.82–7.47 (m, 13H),
7.83 (s, 1H), 8.27 (br s, 1H). 31P NMR (160 MHz, CDCl3): d 148.70,
150.53. MS (FAB): m/z 800 (MH+), HRMS (FAB): calcd for
4.18. (20R)-20,40-(N-Methyl-2-oxo-iminoethano)thymidine (20)
Under nitrogen atmosphere, N-Me lactam 19 (79.6 mg,
0.130 mmol) in EtOH (8.0 mL) and cyclohexene (1.3 mL, 13 mmol)
were added to a suspension of 20% Pd(OH)2 on carbon (91.0 mg) in
EtOH (2.0 mL) at room temperature. The reaction mixture was re-
fluxed for 2 h. The resulting mixture was filtered and concentrated.
The obtained crude residue was purified by column chromatogra-
phy (silica gel 2.0 g, CHCl3/MeOH = 10:1) to give nucleoside 20 as a
white powder (40.8 mg, quant.).
C
42H51N5O9P (MH+) 800.3424, found 800.3406.
Mp: >300 °C. ½a 2D8
ꢂ
+85.0 (c 1.40, CH3OH). IR (KBr): 3430, 3301,
4.16. (20R)-3-Benzyloxymethyl-30,50-di-O-benzyl-20,40-(2-oxo-
3175, 2925, 1705, 1473, 1386, 1273, 1215 cmꢀ1 1H NMR
.
iminoethano)thymidine (18)
(400 MHz, CD3OD): d 1.77 (d, J = 1.0 Hz, 3H), 2.21 (d, J = 18.0 Hz,
1H), 2.39 (d, J = 18.0 Hz, 1H), 3.02 (s, 3H), 3.57 (d, J = 12.5 Hz,
1H), 3.64 (d, J = 12.5 Hz, 1H), 3.80 (d, J = 4.0 Hz, 1H), 4.23 (d,
J = 4.0 Hz, 1H), 5.60 (s, 1H), 8.18 (d, J = 1.0 Hz, 1H). 13C NMR
(100 MHz, CD3OD): d 12.55, 34.92, 39.01, 61.60, 64.86, 67.68,
86.54, 89.33, 110.19, 137.47, 152.07, 166.65, 170.81. MS (FAB):
m/z 312 (MH+). HRMS (FAB): calcd for C13H18N3O6 (MH+)
312.1196, found 312.1207.
Under nitrogen atmosphere, DBU (0.12 mL, 0.79 mmol) and
BOMCl (55 lL, 0.39 mmol) were added to a solution of lactam 13
(94.0 mg, 0.197 mmol) in DMF (2.0 mL) at 0 °C. The reaction mix-
ture was stirred at 0 °C for 0.5 h. The reaction was then quenched
with satd NaHCO3 aq at 0 °C and extracted with EtOAc. The com-
bined organic layer was washed with water and brine, dried over
Na2SO4, and concentrated. The obtained crude residue (112 mg)
was purified by column chromatography (silica gel 5.0 g, n-hex-
ane/EtOAc = 1:1) to give compound 18 as a white foam (101 mg,
86%).
4.19. (20R)-50-O-(4,40-Dimethoxytrityl)-20,40-(N-methyl-2-oxo-
iminoethano)thymidine (21)
Mp: 66–68 °C. ½a 2D8
ꢂ
+79.7 (c 1.00, CHCl3). IR (KBr): 3070, 3032,
1H
1.43 (d, J = 1.0 Hz, 3H), 2.45 (d,
Under nitrogen atmosphere, DMTrCl (175 mg, 0.567 mmol) was
added to a solution of nucleoside 20 (26.8 mg, 0.0861 mmol) in
pyridine (2.0 mL) at 0 °C. The reaction mixture was stirred at room
temperature for 17 h. The reaction was then quenched with satd
NaHCO3 aq at 0 °C and extracted with CH2Cl2. The combined organ-
ic layer was washed with water and brine, dried over Na2SO4, and
concentrated. The obtained crude residue (320 mg) was purified by
column chromatography (silica gel 5.0 g, CHCl3/MeOH = 25:1) to
give compound 21 as a white foam (36.6 mg, 69%).
2924, 2819, 1703, 1661, 1496, 1454, 1364, 1274, 1212 cmꢀ1
.
NMR (400 MHz, CDCl3):
d
J = 17.5 Hz, 1H), 2.60 (d, J = 17.5 Hz, 1H), 3.59 (d, J = 10.5 Hz, 1H),
3.76 (d, J = 10.5 Hz, 1H), 3.96 (dd, J = 4.0, 5.5 Hz, 1H), 4.19 (d,
J = 4.0 Hz, 1H), 4.51 (d, J = 11.0 Hz, 1H), 4.55 (d, J = 11.0 Hz, 1H),
4.60 (d, J = 11.0 Hz, 1H), 4.63 (d, J = 11.0 Hz, 1H), 4.69 (s, 2H),
5.43 (d, J = 9.5 Hz, 1H), 5.46 (d, J = 9.5 Hz, 1H), 5.74 (s, 1H), 6.15
(d, J = 5.5 Hz, 1H), 7.25–7.38 (m, 15H), 7.92 (d, J = 1.0 Hz, 1H). 13C
NMR (100 MHz, CDCl3): d 12.53, 38.68, 56.09, 68.73, 70.27, 70.57,
72.27, 72.37, 73.69, 83.76, 90.53, 109.48, 127.55, 127.64, 127.71,
127.91, 128.30, 128.34, 128.58, 128.73, 133.97, 136.59, 136.88,
137.82, 150.79, 163.29, 169.26. MS (FAB): m/z 598 (MH+). HRMS
(FAB): calcd for C34H36N3O7 (MH+) 598.2553, found 598.2529.
Mp: 155–157 °C. ½a D26
ꢂ
+6.1 (c 2.10, CHCl3). IR (KBr): 3350, 3198,
3087, 3006, 2933, 2836, 1694, 1651, 1508, 1464, 1392, 1350,
1254 cmꢀ1 1H NMR (300 MHz, CDCl3): d 1.30 (s, 3H), 2.44 (d,
.
J = 17.5 Hz, 1H), 2.54 (d, J = 17.5 Hz, 1H), 3.09 (s, 3H), 3.33 (d,
J = 11.0 Hz, 1H), 3.38 (d, J = 11.0 Hz, 1H), 3.75 (s, 3H), 3.76 (s, 3H),

4412
Y. Hari et al. / Bioorg. Med. Chem. 21 (2013) 4405–4412
3.94 (d, J = 4.0 Hz, 1H), 4.28 (br s, 1H), 4.49 (br s, 1H), 5.61 (s, 1H),
6.80–7.42 (m, 13H), 7.75 (s, 1H), 7.90 (br s, 1H). 13C NMR (75 MHz,
CDCl3): d 11.91, 34.51, 38.54, 55.20, 62.87, 65.43, 65.98, 84.96,
86.88, 88.60, 110.39, 111.34, 127.20, 128.07, 130.10, 134.92,
135.08, 144.05, 152.35, 158.69, 164.37, 168.32. MS (FAB): m/z
614 (MH+). HRMS (FAB): calcd for C34H36N3O8 (MH+) 614.2502,
found 614.2496.
with a Tm analysis accessory. Oligonucleotides and ssDNA or ssRNA
were dissolved in 10 mM sodium phosphate buffer (pH 7.2) con-
taining 100 mM NaCl to give a final concentration of each strand
of 4 lM. The samples were annealed by heating at 100 °C followed
by slow cooling to 15 °C. The melting profiles were recorded at
260 nm from 15 to 85 °C at a scan rate of 0.5 °C/min. The two-point
average method was employed to obtain the Tm values and the fi-
nal values were determined by averaging three independent mea-
surements which were accurate to within 1 °C.
4.20. (20R)-30-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-
50-O-(4,40-dimethoxytrityl)-20,40-(N-methyl-2-oxo-
iminoethano)thymidine (17)
4.23. Enzymatic degradation experiments
Under nitrogen atmosphere, DIPEA (10.1 mg, 0.144 mmol) and
Enzymatic degradation experiments were carried out under
i-Pr2NP(Cl)OCH2CH2CN (46
l
L, 0.14 mmol) were added to a solu-
conditions of 1.75
lg/mL Crotalus admanteus venom phosphodies-
tion of compound 21 (72.0 mg, 0.120 mmol) in CH2Cl2 (2.0 mL) at
0 °C. The reaction mixture was stirred at room temperature for
2 h. The reaction was then quenched with satd NaHCO3 aq at
0 °C and extracted with CH2Cl2. The combined organic layer was
washed with satd NaHCO3 aq, water, and brine, dried over Na2SO4,
and concentrated. The obtained crude residue (99.0 mg) was
purified by column chromatography (silica gel 3.0 g, CHCl3/
MeOH = 20:1) to give 17 as a white foam (70.2 mg, 73%).
terase (CAVP), 10 mM MgCl2, 50 mM Tris–HCl (pH 8.0) and 7.5 lM
each oligonucleotide at 37 °C. The amount of intact oligonucleo-
tides was determined by reversed-phase HPLC (Waters XBridgeÒ
MS C18 2.5
lm, 10 ꢁ 50 mm).
Acknowledgments
This work was supported in part by the Program for Promotion
of Fundamental Studies in Health Sciences of the National Institute
of Biomedical Innovation (NIBIO), the Program to Disseminate Ten-
ure Tracking System of the Ministry of Education, Culture, Sports,
Science and Technology, Japan (MEXT), and a Grant-in-Aid for Sci-
entific Research on Innovative Areas (No. 22136006) from MEXT.
Mp: 109–111 °C. 1H NMR (400 MHz, CDCl3): d 0.99 (d, J = 7.0 Hz,
3.0H), 1.07 (d, J = 7.0 Hz, 3.0H), 1.11 (d, J = 7.0 Hz, 3.0H), 1.16 (d,
J = 7.0 Hz, 3.0H), 1.26 (s, 1.5H), 1.29 (s, 1.5H), 2.36–2.64 (m, 4H),
3.17 (s, 3H), 3.22–3.88 (m, 12H), 4.10 (d, J = 4.5 Hz, 0.5H), 4.27 (d,
J = 4.5 Hz, 0.5H), 4.60 (dd, J = 4.5, 7.0 Hz, 0.5H), 4.75 (dd, J = 4.0,
6.5 Hz, 0.5H), 5.67 (s, 0.5H), 5.69 (s, 0.5H), 6.82–7.47 (m, 13H),
7.83 (s, 1H), 8.27 (br s, 1H). 31P NMR (400 MHz, CDCl3): d 148.86,
149.97. MS (FAB): m/z 814 (MH+). HRMS (FAB): calcd for
Supplementary data
C
43H53N5O9P (MH+) 814.3581, found 814.3599.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.049.
4.21. Oligonucleotide synthesis
References and notes
Phosphoramidites 14 and 17 were used and the 0.2 lmol scale
1. Reviews: Shukla, S.; Sumaria, C. S.; Pradeepkumar, P. I. ChemMedChem 2010, 5,
328; Obika, S.; Rahman, S. M. A.; Fujisaka, A.; Kawada, Y.; Baba, T.; Imanishi, T.
Heterocycles 2010, 81, 1347; Yamamoto, T.; Narukawa, K.; Nakatani, M.; Obika,
S. Future Med. Chem. 2011, 3, 339; (d) Prakash, T. P. Chem. Biodiv. 2011, 8, 1616;
(e) Deleavey, G. F.; Damha, M. J. Chem. Biol. 2012, 19, 937.
2. Reviews: Crooke, S. T. Annu. Rev. Med. 2004, 55, 61; Gleave, M. E.; Monia, B. P.
Nat. Rev. Cancer 2005, 5, 468; Chan, J. H.-P.; Lim, S.; Wong, W.-S. F. Clin. Exp.
Pharm. Phys. 2006, 33, 533.
3. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T.
Tetrahedron Lett. 1997, 38, 8735.
4. Nielsen, P.; Wengel, J. Chem. Commun. 1998, 455.
5. Morita, K.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone, J.; Ishikawa, T.;
Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. Lett. 2002, 12, 73; Morita, K.;
Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone, J.; Ishikawa, T.;
Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003, 11, 2211.
synthesis of oligonucleotides was performed on an automated DNA
synthesizer (Gene Design nS-8) using a standard phosphoramidite
protocol (DMTr-ON mode). Oligonucleotides 22–29 were prepared
by cleavage from the CPG supports and deprotection of the phos-
phate moieties (28% NH4 aq, rt, 1.5 h or 50 mM K2CO3 in MeOH,
rt, 2 h). Removal of ammonia was carried out in vacuo. In the case
of K2CO3 treatment, after neutralization with 1% HCl aq, the solvent
was concentrated in vacuo. The crude 22–29 were purified with
Sep-PakÒ Plus C18 cartridges (Waters), followed by reversed-phase
HPLC (Waters XBridgeÒ MS C18 2.5
l
m, 10 ꢁ 50 mm). The compo-
sitions of 22–29 were confirmed by MALDI-TOF mass analysis.
MALDI-TOF mass analysis. MALDI-TOF MS data ([MꢀH]ꢀ) for 22–
29: 22, found 3033.70 (calcd 3034.00); 23, found 3090.36 (calcd
3089.03); 24, found 3145.24 (calcd 3144.07); 25, found 3048.53
(calcd 3048.02); 26, found 3117.32 (calcd 3117.08); 27, found
3187.85 (calcd 3186.25); 28, found 3033.23 (calcd 3034.00); 29,
found 3048.78 (calcd 3048.02).
6. Properties of 20-amino-LNA and its six-membered analog, aza-ENA, were also
reported by Wengel’s group9 and Chattopadhyaya’s group,10 respectively.
However, there is no example in which they were compared.
7. Yahara, A.; Shrestha, A. R.; Yamamoto, T.; Hari, Y.; Osawa, T.; Yamaguchi, M.;
Nishida, M.; Kodama, T.; Obika, S. ChemBioChem 2012, 13, 2513.
8. Shrestha, A. R.; Hari, Y.; Yahara, A.; Osawa, T.; Obika, S. J. Org. Chem. 2011, 76,
9891.
9. Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 10035.
10. Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; Honcharenko, D.;
Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128, 15173.
4.22. UV-melting experiments
UV-melting experiments were carried out using SHIMADZU
UV-1650 and SHIMADZU UV-1800 spectrophotometers equipped