Synthesis and properties of 3′-amino-2′,4′-BNA, a bridged nucleic acid with a N3′→P5′ phosphoramidate linkage

Article abstract of DOI:10.1016/j.bmc.2008.09.013

The synthesis and properties of a bridged nucleic acid analogue containing a N3′→P5′ phosphoramidate linkage, 3′-amino-2′,4′-BNA, is described. A heterodimer containing a 3′-amino-2′,4′-BNA thymine monomer, and thymine and methylcytosine monomers of 3′-am

Full text of DOI:10.1016/j.bmc.2008.09.013

Bioorganic & Medicinal Chemistry 16 (2008) 9230–9237
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and properties of 30-amino-20,40-BNA, a bridged nucleic acid
with a N30?P50 phosphoramidate linkage
a,
Satoshi Obika a, , S. M. Abdur Rahman , Bingbing Song a, Mayumi Onoda a, Makoto Koizumi b,
*
Koji Morita c, Takeshi Imanishi a
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
b Exploratory Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa, Tokyo 140-8710, Japan
c Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
The synthesis and properties of a bridged nucleic acid analogue containing a N30?P50 phosphoramidate
linkage, 30-amino-20,40-BNA, is described. A heterodimer containing a 30-amino-20,40-BNA thymine mono-
mer, and thymine and methylcytosine monomers of 30-amino-20,40-BNA and their 50-phosphoramidites,
were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by con-
ventional 30?50 assembly, and 50?30 reverse assembly phosphoramidite protocols, respectively. Com-
pared to a natural DNA oligonucleotide, modified oligonucleotides containing the 30-amino-20,40-BNA
residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded
RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting tempera-
ture (Tm) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 °C, +5.0 to +7.0 °C, and +5.0 to +11.0 °C,
respectively. These increases are comparable to those observed for 20,40-BNA-modified oligonucleotides.
In addition, an oligonucleotide modified with a single 30-amino-20,40-BNA thymine residue showed
extraordinarily high resistance to nuclease degradation, much higher than that of 20,40-BNA and substan-
tially higher even than that of 30-amino-DNA and phosphorothioate oligonucleotides. The above proper-
ties indicate that 30-amino-20,40-BNA has significant potential for antisense and antigene applications.
Ó 2008 Elsevier Ltd. All rights reserved.
Article history:
Received 16 July 2008
Revised 1 September 2008
Accepted 5 September 2008
Available online 9 September 2008
Keywords:
Bridged nucleic acids
Locked nucleic acids
Oligonucleotide N30?P50
phosphoramidates
1. Introduction
O
O
O
Recently much attention has been focused on chemically
modified oligonucleotides due to their potential application in
antisense and antigene technologies,1–3 diagnostics,4 and nucleic
acid nanotechnology.5,6 In order to be effective and practically
useful in the above technologies, modified oligonucleotides must
have specific characteristics such as high target-binding affinity,
sequence specificity and nuclease resistance. Although numerous
modified oligonucleotides have been developed in the past few
decades, most do not exhibit properties satisfying the above crite-
ria, thus severely restricting their utility in oligonucleotide-based
therapy and nucleic acid nanotechnology.
B
B
B
O
O
O
O
O
HN
O P O
O
HN
O P O
O P O
3'-amino-DNA
2',4'-BNA
3'-amino-2',4'-BNA
Figure 1. Structures of 30-amino-DNA, 20,40-BNA and 30-amino-20,40-BNA.
dsDNA.7–11 The increased hybridization characteristics of these
analogues result from their RNA-like structures. The resemblance
to RNA is due to the dominant N-type sugar pucker generated by
the electronegativity of the nitrogen atom,12,13 which is further
enforced by 20-substitutions (20-fluoro or 20-OH, a ribo sugar). As
a result of these promising properties, N30?P50 oligonucleotides
act as potent antisense molecules in cells and in vivo.14
In 1994, Gryaznov et al. reported N30?P50 phosphoramidate
linked oligonucleotides (30-amino-DNA, Fig. 1)7 in which the 30-O
was replaced by a 30-N atom. A number of structural analogues
of N30?P50 phosphoramidate were synthesized, and all were
shown to possess excellent nuclease resistance along with superior
hybridizing properties with complementary ssDNA, ssRNA and
Several years ago, our group and Wengel et al. independently
discovered a novel modified nucleic acid, 20-O,40-C-bridged nucleic
acid (20,40-BNA, also named locked nucleic acid, or LNA)15–17
* Corresponding author. Tel.: +81 6 6879 8200; fax: +81 6 6879 8204.
E-mail address: obika@phs.osaka-u.ac.jp (S. Obika).
Present address: Faculty of Pharmacy, University of Dhaka, Bangladesh.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.013

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 9230–9237
9231
which has a fixed N-type sugar-conformation. This nucleic acid
2. Results and discussion
derivative shows unprecedented hybridizing ability with comple-
mentary strands of ssRNA, ssDNA and dsDNA,15–26 and somewhat
increased resistance to nuclease degradation compared to natural
DNA.21,27,28 Due to these interesting properties, 20,40-BNA/LNA is
one of the most promising and popular nucleic acid analogues
currently being utilized in various genomic technologies,29–31
and is now commercially available. However, this nucleic acid is
not sufficiently resistant to nucleases, and is significantly less
resistant than phosphorothioate oligonucleotide.32,33 Hence, the
nuclease resistance ability of phosphorothioate oligonucleotide
is even considered as suboptimal,34 further enhancement of
nuclease resistance of 20,40-BNA/LNA is required for practical
in vivo use.
2.1. Synthesis of 30-amino-20,40-BNA heterodimer unit 10
Since 30-amino-20,40-BNA contains a N30?P50 phosphoramidate
linkage instead of an O30?P50 linkage, conventional synthesis via
the 30-phosphoramidite approach is not possible because the P–N
bond in the 30-phosphorodiamidite group would be simulta-
neously cleaved by the common activators used for DNA synthesis.
To overcome this problem, we initially synthesized heterodimer 10
for use in typical phosphoramidite protocols. The synthesis of 10
was accomplished from the known compound, 3-azido-3-deoxy-
furanose 1,36 as shown in Scheme 1. Removal of the benzoyl groups
of 1 by base-mediated hydrolysis afforded diol 2, which was trans-
formed to compound 3 via selective silylation with tert-butyldi-
phenylsilyl chloride (TBDPSCl), followed by tosylation with p-
toluenesulphonyl chloride (TsCl) in the presence of 4-dimethyl-
aminopyridine (DMAP) and triethylamine (Et3N). Acetolysis of 3
with acetic acid, acetic anhydride and concd sulfuric acid provided
an anomeric mixture of diacetate intermediate37 which was cou-
pled with thymine in the presence of N,O-bis(trimethylsilyl)acet-
amide (BSA) and trimethylsilyl triflate (TMSOTf) to give the
nucleoside analogue 4 in very good yield. Treatment of 4 with
potassium carbonate yielded the desired bicyclic nucleoside 5
quantitatively. Desilylation by tetrabutyl ammonium fluoride
(TBAF) furnished a known nucleoside derivative38 (Supplementary
Data), the hydroxyl group of which was tritylated by 4,40-dime-
thoxytrityl chloride (DMTrCl) to afford the trityl derivative 6. Then,
the 30-azido group was reduced to an amino group by the action of
triphenylphosphine (PPh3), pyridine and NH4OH to give compound
7 in excellent yield. Following reported procedures,8,39 compound
7 was coupled with the methyl phosphonate derivative 839 to give
the heterodimer 9. Desilylation followed by phosphitylation in the
presence of 2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodi-
amidite and disopropylammonium tetrazolide provided the de-
sired heterodimer phosphoramidite 10.
To optimize the nuclease resistance and hybridizing properties
of 20,40-BNA, we aimed to develop a bridged nucleic acid (BNA)
with
a
N30?P50 phosphoramidite linkage, 30-amino-20,40-BNA
(Fig. 1). In order to incorporate 30-amino-20,40-BNA into oligonu-
cleotides easily by the conventional phosphoramidite protocols,
we initially synthesized a heterodimer unit of 30-amino-20,40-
BNA.35 However, the synthesis of 30-amino-20,40-BNA oligonucleo-
tides via the heterodimer approach (Fig. 2)35 has some serious
drawbacks: (i) only limited oligonucleotide sequences can be syn-
thesized by heterodimer unit 10, and oligonucleotides represent-
ing all sequence combinations require the separate synthesis of
16 different heterodimers; (ii) oligonucleotides containing consec-
utive modifications cannot be synthesized via the dimer approach.
Due to these limitations, we could not investigate the hybridizing
characteristics of 30-amino-20,40-BNA in detail. To overcome these
problems, we planned a synthetic approach based on the 50?30
reverse assembly protocol shown in Fig. 2, and developed a facile
synthetic route for 50-phosphoramidites of 30-amino-20,40-BNA
thymine and methyl cytosine monomer analogues. These com-
pounds were incorporated into a variety of oligonucleotides. In
our preliminary report, we communicated the synthesis of 30-ami-
no-20,40-BNA oligonucleotides via a dimer unit, showed one exam-
ple of hybridization with DNA and RNA, and presented
preliminary data showing the nuclease resistance of the BNA oli-
gonucleotides.35 Herein, we report full details of the synthesis of
30-amino-20,40-BNA phosphoramidites via both approaches, the
utilization of these compounds in the synthesis of various oligo-
nucleotides, and give a detailed account of their hybridization
and nuclease resistance profiles.
2.2. Synthesis of 30-amino-20,40-BNA monomers 50-
phosphoramidites
As the synthesis of a variety of 30-amino-20,40-BNA oligonucleo-
tides via the heterodimer approach (Fig. 2) is problematic, synthe-
sis via 50?30 reverse assembly was undertaken. Synthesis of
DMTrO
T
O
O
B
O
O
NH
MeO P O
O
O
5' 3'
B
3' 5'
NH2
O
T
O
elongation
elongation
O
O
iPr2N
heterodimer
unit
O
P O
O
P
10
HN
O P O
B
O
CN
O
iPr2N
NC
HO
O
B
MMTrHN
O
3'-amino-2',4'-BNA
oligonucleotides
3'-amino-2',4'-BNA
5'-phosphoramidite
R
O
Figure 2. Synthetic strategies for 30-amino-20,40-BNA oligonucleotides.

9232
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 9230–9237
BzO
BzO
HO
HO
TBDPSO
TsO
TBDPSO
iv, v
T
ii, iii
i
O
O
O
O
(86%)
(63%)
(86%)
O
O
O
T
TsO
O
O
O
T
N3
N3
N3
N3 OAc
4
3
2
1
(quant.)
vi
DMTrO
DMTrO
DMTrO
TBDPSO
vii, viii
(65%)
T
O
ix
(97%)
O
O
O
7
O
O
O
MeO P O
H
NH2
x
N3
N3
5
6
T
O
(39%)
DMTrO
T
OTBS
T
O
O
8
O
O
NH
MeO P O
NH
MeO P O
O
vii, xi
(46%)
O
T
T
O
O
O
O
P
10
OTBS
9
CN
iPr2N
Scheme 1. Reagents and conditions: (i) K2CO3, MeOH, 0 °C; (ii) TBDPSCl, Et3N, CH2Cl2, rt; (iii) TsCl, DMAP, Et3N, CH2Cl2, rt; (iv) concd H2SO4, Ac2O, AcOH, rt; (v) thymine, BSA,
TMSOTf, DCE, 80 °C; (vi) K2CO3, MeOH, rt; (vii) TBAF, THF, rt; (viii) DMTrCl, DMAP, pyridine, rt; (ix) PPh3, pyridine, rt, then NH4OH aq rt; (x) CCl4, Et3N, MeCN, rt; (xi)
(iPr2N)2POCH2CH2CN, diisopropylammonium tetrazolide, MeCN-THF, rt. TBDPSCl, tert-butyldiphenylsilyl chloride; DMAP, 4-dimethylaminopyridine, BSA, N,O-bis(trimeth-
ylsilyl)acetamide; DCE, dichloroethane; DMTrCl, 4,40-dimethoxytrityl chloride.
oligonucleotides following this approach requires 50-phosphorami-
dites instead of the conventional 30-phosphoramidites; this process
is called phosphoramidite transfer reaction.9,10,40 In order to apply
this reverse assembly strategy to the synthesis of 30-amino-20,40-
BNA oligonucleotides, we synthesized 30-amino-20,40-BNA -50-
phosphoramidites derivatives 14 and 20 (Schemes 2 and 3). The
synthesis of 30-amino-20,40-BNA-thymine 50-phosphoramidite 14
is shown in Scheme 2. The azido group of intermediate 5 was
reduced to an amino group quantitatively to give the 30-amino
derivative 11. Protection of the amino functionality with 40-mono-
methoxytrityl chloride (MMTrCl) in the presence of pyridine
afforded compound 12 in excellent yield. Desilylation with TBAF
produced alcohol 13 smoothly, which was phosphitylated to give
the desired 50-phosphoramidite 14 in 91% yield.
to aqueous ammonia delivered amine 17, whose amino group was
protected with a benzoyl group via conventional benzoylation to
give compound 18 in good yield. Deacetylation by lithium hydrox-
ide followed by phosphitylation furnished the desired 50-phospho-
ramidite 20 in good yield.
2.3. Synthesis of oligonucleotides
Using the phosphoramidites 10, 14, and 20, and natural DNA
building blocks, a number of different 30-amino-20,40-BNA-modi-
fied oligonucleotides (oligonucleotides 21–27, Table 1) were syn-
thesized using an automated DNA synthesizer (see
Experimental). A set of 12-mer oligonucleotides 21–24, 15-mer
polypyrimidine oligonucleotides 25 and 26, and 10-mer oligothy-
midylate 27, were synthesized in order to study duplex and triplex
forming properties and nuclease resistance properties, respec-
tively. Heterodimer phosphoramidite 10 was used for the synthesis
of oligonucleotides21, 25, and 27, and monomer phosphoramidites
14 and 20 were used for the synthesis of the remaining oligonucle-
otides. The oligonucleotides were purified by RP-HPLC and
The synthesis of 30-amino-20,40-BNA-5-methylcytosine 50-phos-
phoramidite 20 is outlined in Scheme 3. The primary hydroxyl
group of 13 was protected with an acetyl group to afford com-
pound 15 in 93% yield. Compound 15 was transformed to the tria-
zole derivative 16 by the treatment of 1H-triazole, phosphoryl
chloride and triethylamine41 in nearly quantitative yield. Exposure
NC
O
TBDPSO
i
TBDPSO
ii
P O
HO
T
T
T
T
iPr2N
O
O
O
O
iii
(88%)
MMTr NH
13
iv
5
(91%)
(97%)
(quant.)
O
O
O
O
NH2
MMTr NH
MMTr NH
14
12
11
Scheme 2. Reagents and conditions: (i) PPh3, pyridine, rt, then NH4OH, rt; (ii) MMTrCl, pyridine, rt; (iii) TBAF, THF, rt; (iv) (iPr2N)2POCH2CH2CN, diisopropylammonium
tetrazolide, MeCN-THF, rt.

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 9230–9237
9233
N
N
O
NH2
N
N
Me
Me
Me
NH
N
AcO
AcO
AcO
N
O
N
O
N
O
i
iii
ii
O
O
O
13
(93%)
(99%)
(89%)
O
O
O
MMTr NH
MMTr NH
MMTr NH
17
15
16
NHBz
N
NHBz
N
NHBz
N
Me
Me
Me
NC
O
P O
AcO
HO
N
O
N
O
N
O
O
O
O
iv
iPr2N
v
vi
(87%)
(89%)
(97%)
MMTr NH
19
O
O
O
MMTr NH
MMTr NH
20
18
Scheme 3. Reagents and conditions: (i) Ac2O, pyridine, rt; (ii) 1H-triazole, POCl3, Et3N, MeCN, 0 °C; (iii) NH4OH aq, 1,4-dioxane, rt; (iv) BzCl, Et3N, CH2Cl2, rt; (v) LiOH–H2O
(2:1), rt; (vi) (iPr2N)2POCH2CH2CN, diisopropylammonium tetrazolide, MeCN-THF, rt.
Table 2
Table 1
30-Amino-20,40-BNA oligonucleotides and MALDI-TOF mass dataa
Tm values of duplexes formed by 30-amino-20,40-BNA oligonucleotides with ssDNA and
ssRNAa,b
Oligonucleotides (50-d. . .. . .-30)
Mass [M-H]ꢀ Found/Calcd.
Oligonucleotides (50-d. . .. . .-30)
Tm
(D
Tm/mod.) (°C)
ssRNA
GCGTTTTTTGCT (21)
GCGTTTTTTGCT (22)
GCGTTTTTTGCT (23)
GCGTTTTTTGCT (24)
TTTTTmCTTTmCTmCTmCT (25)
TTTTTmCTTTmCTmCTmCT (26)
TTTTTTTTTT (27)
3024.6/3024.1
3717.2/3714.4
3716.6/3714.4
3799.8/3795.5
4523.3/4523.1
4634.4/4631.2
3010.7/3009.5
ssDNA
47
GCGTTTTTTGCT (28)
GCGTTTTTTGCT (21)
GCGTTTTTTGCT (22)
GCGTTTTTTGCT (23)
GCGTTTTTTGCT (24)
45
51 (+4.0)
53 (+2.0)
53 (+2.0)
63 (+2.7)
52 (+7.0)
63 (+6.0)
61 (+5.3)
75 (+5.0)
a
m
T = 30-amino-20,40-BNA-T, C = 30-amino-20,40-BNA-mC.
GCGTTtTTTGCT (29)
GCGtTtTtTGCT (30)
GCGTTtttTGCT (31)
GCGttttttGCT (32)
53 (+6.0)
56 (+3.0)
54 (+2.3)
67 (+3.3)
52 (+7.0)c
62 (+5.7)c
60 (+5.0)c
80 (+5.8)d
characterized and verified by their MALDI-TOF mass spectra. MAL-
DI-TOF mass data are summarized in Table 1.
a
Targets: ssDNA, 50-d(AGCAAAAAACGC)-30; ssRNA, 50-r(AGCAAAAAACGC)-30.
Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl;
b
strand concentration = 4
l
M. T = 30-amino-20,40-BNA-T, t = 20,40-BNA-T.
c
2.4. Duplex formation and duplex thermal stability
Data from Ref. 42.
Data from Ref. 21.
d
The ability of 30-amino-20,40-BNA-modified oligonucleotides to
form duplexes in physiological salt and buffer conditions, and the
thermal stability of duplexes formed with ssDNA and ssRNA com-
plements, was studied. Thermal stability was evaluated by melting
temperature (Tm). The results obtained with natural DNA and the
20,40-BNA-modified analogues are compared in Table 2. Modifica-
tion of natural DNA oligonucleotide 28 by a single 30-amino-20,40-
BNA residue (oligonucleotide 21) led to an increase in Tm of 4
and 7 °C against ssDNA and ssRNA, respectively. Duplex thermal
stability further improved upon increasing the number of modifi-
cations. Incorporating three 30-amino-20,40-BNA residues either
consecutively or separated by natural DNA units (oligonucleotides
22 and 23, respectively) resulted in duplexes with very high ther-
mal stability. The most prominent enhancement in thermal stabil-
ity was observed in duplexes formed with complementary ssRNA.
stability (Tm values of 63 and 75 °C for ssDNA and ssRNA, respec-
tively). Compared with 20,40-BNA-modified oligonucleotides 29 to
32,21,42 the 30-amino-20,40-BNA-modified oligonucleotides showed
slightly decreased affinity towards ssDNA, but affinity to ssRNA
was similar to that of 20,40-BNA except for oligonucleotide 24 (com-
pare the Tm value of 24 with that of 32). These results indicate that
30-amino-20,40-BNA is more RNA-selective than 20,40-BNA. The
equal or comparable RNA-binding affinity of 30-amino-20,40-BNA
to 20,40-BNA indicates that N-type conformational characteristics
are already optimized by the 20-O,40-C bridge, and that 30-N does
not contribute to the restriction of sugar puckering, in contrast to
findings using non-bridged natural nucleic acids.7–11 The relatively
lower affinity of oligonucleotide 24 compared to 20,40-BNA oligonu-
cleotide 32 indicates that consecutive backbone modifications re-
duce duplex stability of bridged nucleic acids.
The increase in Tm per modification (
+5.3 °C for 22 and 23, respectively. Against ssDNA, the
D
Tm/mod.) was +6.0 and
Tm/mod.
D
was found to be +2.0 °C for both 22 and 23, implying that 30-ami-
no-20,40-BNA oligonucleotides have preferential RNA-binding affin-
ity. A modified oligonucleotide containing six consecutive 30-
amino-20,40-BNA residues (oligonucleotide 24) also formed du-
plexes with ssRNA and ssDNA with remarkably improved thermal
2.5. Triplex-forming properties
Next, the triplex forming ability of 30-amino-20,40-BNA-modified
triplex-forming oligonucleotides (TFOs) was investigated under

9234
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 9230–9237
neutral conditions with or without 10 mM MgCl2 (Table 3). Two
dsDNA targets (targets A and B) were used for singly and multiply
modified TFOs (TFOs 25 and 26, respectively). The reason for using
two different targets is that the target A duplex has a Tm value (ca.
57 °C) substantially lower than the Tm of the triplex formed with
26. Therefore, a hairpin DNA (target B) was used for TFO 26, and
for direct comparison with the 30-amino-20,40-BNA-TFOs, we mea-
sured the Tms of triplexes formed by the natural TFO 33 against
both targets. As shown in Table 3, replacement of only a single nat-
ural DNA-nucleotide of 33 by 30-amino-20,40-BNA-nucleotide (TFO
25) resulted in significant stabilization of the triplex (Tm = 44 and
55 °C in the absence and presence of MgCl2, respectively;
20,40-BNA nucleotides of thymine and methyl cytosine bases (TFO
26) resulted in the formation of a triplex with thermal stability
27 °C (ꢀMgCl2) and 32 °C (+MgCl2) higher (
DTm/mod. = +5.4 and
+6.4 °C, respectively) than that obtained for natural TFO 33 with
the same target (target B). These increased Tms are comparable to
those found for the corresponding 20,40-BNA-modified TFOs 34
and 35 (compare Tms of 25 and 26 with 34 and 35, respectively).
Mismatch discrimination by 30-amino-20,40-BNA oligonucleo-
tide 25 was examined against dsDNA targets containing a mis-
matched base in the homopurine strand. The results are
summarized in Table 4. It was found that the Tm values of triplexes
formed by 30-amino-20,40-BNA-TFO 25 with mismatched dsDNAs
having G:C, C:G, and T:A arrangements decreased significantly
compared to matched DNA (A:T arrangement). Against dsDNA tar-
gets containing central G:C, C:G, and T:A arrangements, the Tm val-
ues decreased by 24, 23, and 39 °C, respectively. Except in the case
of the G:C arrangement, these decreases are larger than those
exhibited by natural DNA-TFO 33. These values are also compara-
ble to those found for 20,40-BNA-TFO 34. These results show that,
like 20,40-BNA, 30-amino-20,40-BNA also has excellent mismatch dis-
crimination ability.
D
Tm = +11 °C in both cases). The incorporation of five 30-amino-
Table 3
Tm Values of triplexes formed by 30-amino-20,40-BNA oligonucleotides with dsDNAa,b
Oligonucleotides (50-d. . .. . .-30)
Targets
Tm
(
D
Tm/mod.) (°C)
+MgCl2
b
ꢀMgCl2
TTTTTmCTTTmCTmCTmCT (33)
TTTTTmCTTTmCTmCTmCT (33)
TTTTTmCTTTmCTmCTmCT (25)
TTTTTmCTTTmCTmCTmCT (26)
A
B
A
B
33
32
44 (+11)
59 (+5.4)
44
39
55 (+11)
71 (+6.4)
TTTTTmCTtTmCTmCTmCT (34)
TTTtTmcTtTmcTmcTmCT (35)
A
B
44 (+11)
60 (+5.6)
57 (+13)
72 (+6.6)
2.6. Nuclease resistance properties
Resistance to degradation by nucleases is extremely important
for the in vivo application of oligonucleotides. Our preliminary re-
port showed the superior nuclease resistance of the 30-amino-20,40-
BNA oligonucleotide over natural DNA, 20,40-BNA and phosphorth-
iate oligonucleotides at low nuclease (snake venom phosphodies-
terase or SPVDE, Boehringer Mannheim) concentration.35 In the
present study, in order to understand the relative nuclease resis-
tance of 30-amino-20,40-BNA with N30?P50-natural DNA (or 30-ami-
no-DNA) and other structurally related analogues, we conducted
nuclease resistance studies at high nuclease concentration using
Crotalus admanteus venom phosphodiesterase (CAVP) (Pharmacia).
Figure 3 shows the collective nuclease resistance profiles of 30-ami-
no-20,40-BNA oligonucleotide 27, 30-amino-DNA oligonucleotide
39, and other nucleic acid analogues (oligonucleotides 36–38). Un-
der the experimental conditions (see Experimental), the natural
oligothymidylates 36 and 20,40-BNA oligonucleotide 37 were com-
pletely digested within 3 and 10 min, respectively, upon exposure
a
Target dsDNAs: Target A = 50-d(GCTAAAAAGAAAGAGAGATCG)-30/30-d(CGATTT
TTCTTTCTCTCTAGC)-50, Target B = 50-d(CGATCTCTCTTTCTTTTTAGCCCCCGCTAAA
AAGAAAGAGAGATCG)-30; underlined portion indicates the target site for triplex
formation.
b
Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl in
the absence or the presence of MgCl2 (10 mM); strand concentration = 1.5 lM.
T = 30-amino-20,40-BNA-T, C = 30-amino-20,40-BNA-mC, t = 20,40-BNA-T, c = 20,40-
m
m
BNA-mC.
Table 4
Sequence-specific triplex formation by 30-amino-20,40-BNA-modified TFOsa
T (TFO)
Tm
(D
Tm = Tm (mismatch) ꢀ Tm (match)) (°C)
X:Y = A:T (match)
G:C
C:G
T:A
Natural (33)
30-Amino-20,40-BNA (25)
20,40-BNA (34)
44
55
57
20 (ꢀ24)
31 (ꢀ24)
31 (ꢀ26)
25 (ꢀ19)
32 (ꢀ23)
35 (ꢀ22)
17 (ꢀ25)
16 (ꢀ39)
16 (ꢀ41)
Modified TFO: 50-d(TTTTTmCTTTmCTmCTmCT)-30.
Target dsDNA: 50-d(GCTAAAAAGAXAGAGAGATCG)-30.
to 0.8 lg of CAVP (Fig. 3A). Phosphorothioate oligonucleotide 38
degraded gradually (about 50% decomposed after 60 min) and 30-
amino-DNA-oligonucleotide 39 degraded only slightly. On the
other hand, 30-amino-20,40-BNA oligonucleotide 27 remained intact
30-d(CGATTTTTCTYTCTCTCTAGC)-50.
a
Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl
M.
and 10 mM MgCl2; strand concentration = 1.5
l
Figure 3. Nuclease resistance of T8XT oligonucleotides against CAVP; X = natural-T (36) (magenta), 20,40-BNA-T (37) (black), phosphorthioate-T (38) (cyan), 30-amino-DNA-T
(39) (red) and 30-amino- 20,40-BNA-T (27) (green). Hydrolysis of the oligonucleotides (3 nmol) was carried out at 37 °C in buffer (400
l) containing 50 mM Tris–HCl (pH 8.0),
10 mM MgCl2 and CAVP (0.8 g for experiment A and 1.6 g for experiment B).
l
l
l

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 9230–9237
9235
even after 60 min exposure. Next, the CAVP concentration was in-
creased two fold (1.6 g for 3 nmol oligonucleotide, Fig. 3B) and
ization-time of flight (MALDI-TOF) mass spectra were recorded on
an Applied Biosystems Voyager DE mass spectrometer.
Full experimental details and characterization data for com-
pounds 2–10 are described in Supplementary Data. The experi-
mental procedures and data for compounds 11–20 are given
below:
l
resistance to nuclease degradation was examined. The 20,40-BNA-
modified oligonucleotide 37 decomposed quantitatively within
2 min, and 30-amino-DNA oligonucleotide 39 also decomposed at
a constant rate depending on the time of exposure. In marked con-
trast, 30-amino-20,40-BNA oligonucleotide 27 was essentially intact
even at that very high nuclease concentration. This result clearly
indicates that 30-amino-20,40-BNA has extremely high nuclease
resistance, much higher than that of natural and 20,40-BNA-modi-
fied oligonucleotides, and substantially higher even than that of
phosphorothioate oligonucleotides and 30-amino-DNA oligonu-
cleotide bearing the same N30?P50-linkage. The extraordinarily
high nuclease resistance of 30-amino-20,40-BNA results from the
combined effects of the N30?P50 linkage and the 20-O,40-C-bridged
moiety.
4.1.1. 30-Amino-50-O-tert-butyldiphenylsilyl-30-deoxy-5-
methyl-20-O,40-C-methyleneuridine (11)
Triphenylphosphine (5.41 g, 29.6 mmol) was added to a solu-
tion of compound 5 (5.49 g, 10.3 mmol) (for details of the synthesis
of 5, see Supplementary Data) in pyridine (90.7 ml) and the mix-
ture was stirred at room temperature for 6 h. After compound 5
disappeared, 28% ammonium solution (227 ml) was added and
the resulting mixture was stirred at room temperature for 19 h.
The solvent was evaporated under reduced pressure, and the resi-
due was purified by column chromatography using ethyl acetate
and n-hexane (2:1, v/v) as eluents to give compound 11 (5.26 g,
3. Conclusions
quant.) as a white foam: mp 89–92 °C. ½a D21
ꢁ
+49.51 (c = 1.00,
CDCl3). IR mmax (KBr): 3175, 3046, 2955, 2858, 1700, 1467,
In conclusion, we have synthesized 30-amino-20,40-BNA dimer
and thymine and methyl cytosine monomer residues and incorpo-
rated them into oligonucleotides via conventional 30?50 assembly
(for dimer residue) and 50?30 reverse assembly (for monomer res-
idues) phosphoramidate protocols. The synthesis via the dimer res-
idue has some limitations, such as oligonucleotides with
consecutive modifications or containing residues with a variety
of nucleobase combinations, cannot be synthesized. On the other
hand, the synthetic protocol via the monomer residues eliminates
these shortcomings and a variety of oligonucleotides, including a
consecutively modified oligonucleotide, were synthesized effi-
ciently. 30-Amino-20,40-BNA-modified oligonucleotides exhibit very
strong binding affinity for ssDNA, ssRNA, and dsDNA targets, com-
parable to that of 20,40-BNA. Moreover, 30-amino-20,40-BNA shows
better RNA selective binding affinity than 20,40-BNA. The mismatch
discrimination of 30-amino-20,40-BNA is similar to that of 20,40-BNA.
The nuclease resistance of 30-amino-20,40-BNA is excellent, much
higher than that of natural and 20,40-BNA oligonucleotides, and
substantially higher even than that of 30-amino-DNA and phosp-
horothioate oligonucleotides. Given these superior properties, 30-
amino-20,40-BNA oligonucleotide has significant potential for prac-
tical antisense and antigene applications. Biological application of
30-amino-20,40-BNA-modified oligonucleotides and synthesis of
the 30-amino-20,40-BNA-adenine and -guanine monomers are now
in progress.
1430 cmꢀ1 1H NMR (CDCl3): d 1.05 (9H, s), 1.62 (3H, s), 3.27 (1H,
.
s), 3.75, 3.68 (2H, AB, J = 9 Hz), 3.88, 4.00 (2H, AB, J = 12 Hz), 4.25
(1H, s), 5.54 (1H,s), 7.31–7.46 (6H, m), 7.61–7.66 (4H, m), 8.19
(1H, br). 13C NMR (CDCl3): d 12.4, 19.5, 27.0, 53.6, 59.1, 70.9,
77.2, 80.5, 86.7, 90.2, 110.1, 127.9, 130.0, 130.1, 132.3, 132.6. MS
(FAB): m/z 508 (M+H)+. Anal. Calcd for C27H33N3O5Siꢂ1/10 H2O: C,
63.65; H, 6.56; N, 8.25. Found: C, 63.36; H, 6.58; N, 8.03.
4.1.2. 50-O-tert-Butyldiphenylsilyl-30-deoxy-30-(4-methoxytri-
phenylmethylamino)-5-methyl-20-O,40-C-methyleneuridine (12)
4-Methoxytriphenylmethyl chloride (MMTrCl) (15.0 g, 48.5 mmol)
was added to a solution of compound 11 (16.4 g, 32.3 mmol) in
pyridine (191 ml). The reaction mixture was stirred at room tem-
perature for 11 h. After the mixture was quenched with saturated
NaHCO3 (aq), the combined organic layers were extracted with
dichloromethane, washed with water and brine, dried over sodium
sulfate, and concentrated under reduced pressure. The residue was
purified by column chromatography by eluting with ethyl acetate
and n-hexane (1:2, v/v) to yield compound 12 (24.3 g, 97%) as a
white foam: mp 102–105 °C. ½a D22
ꢁ
+20.03 (c = 1.00, CH3COCH3). IR
mmax (KBr): 3168, 3064, 2934, 2250, 1687, 1507, 1464 cmꢀ1
.
1H
NMR (CDCl3): d 1.13 (9H, s), 1.62 (3H, s), 1.94 (1H, d, J = 9.7 Hz),
2.47 (1H, s), 2.74 (1H, d, J = 10 Hz), 3.73 (3H, s), 3.82, 3.90 (2H,
AB, J = 8.6 Hz), 4.25, 4.35 (2H, AB, J = 12 Hz), 5.36 (1H, s), 6.68–
7.74 (10H, m), 8.63 (1H, br). 13C NMR (CDCl3): d 12.2, 19.6, 27.1,
55.2, 56.9, 60.7, 70.5, 72.2, 77.2, 77.7, 86.4, 89.3, 109.5, 113.3,
126.7, 127.8, 128.0, 129.4, 130.3, 132.6, 134.0, 135.5, 137.2,
145.5, 145.6, 148.9, 158.1, 163.5. MS (FAB): m/z 780 (M+H)+. Anal.
Calcd for C47H49N3O6Siꢂ3/4H2O: C, 71.14; H, 6.41; N, 5.30. Found: C,
71.21; H, 6.53; N, 5.05.
4. Experimental
4.1. Synthesis and characterization of compounds: general
aspects and instrumentation
4.1.3. 30-Deoxy-30-(4-methoxytriphenylmethylamino)-5-
methyl-20-O,4-C-methyleneuridine (13)
Melting points are uncorrected. All moisture-sensitive reactions
were carried out in well-dried glassware under a N2 atmosphere.
Dichloromethane, dichloroethane, acetonitrile, triethylamine, and
pyridine were distilled from CaH2. Tetrahydrofuran (THF) was
dried by CaH2 and LiAlH4. 1H NMR (270 or 300 MHz), 13C NMR
(67 or 75 MHz), and 31P NMR spectra (202 MHz) were recorded
on a JEOL EX-270, JEOL-AL-300, and JEOL GX-500 spectrometer,
respectively. Chemical shifts are reported in parts per million
downfield from internal tetramethylsilane for 1H spectra, CHCl3
(d = 77.0 ppm) or methanol (49.0) for 13C NMR, and 85% H3PO4
(d = 0 ppm) for 31P spectra. IR spectra were recorded on a JASCO
FT/IR-200 spectrometer. Optical rotations were recorded on a JAS-
CO DIP-370 instrument. Mass spectra were measured on JEOL JMS-
600 or JMS-700 mass spectrometers. For column chromatography,
silica gel FL 100 D was used. Matrix-assisted laser desorption ion-
To a solution of compound 12 (24.3 g, 31.2 mmol) in THF
(282 ml) was added TBAF (1M in THF) (34.3 ml, 34.3 mmol) and
the mixture was stirred at room temperature for 4 h. The reaction
mixture was concentrated under reduced pressure. The residue
was purified by column chromatography, eluting with ethyl ace-
tate and n-hexane (3:1, v/v) to give compound 13 (14.7 g, 88%)
as white foam: mp 136–141 °C. ½a D22
ꢁ
+40.69 (c = 1.00, MeOH). IR
mmax (KBr): 3479, 3188, 3025, 1687, 1508, 1465, 1254 cmꢀ1
.
1H
NMR (CDCl3): d 1.68 (3H, s), 2.01 (1H, s), 2.22 (1H, s), 2.95 (1H,
s), 3.69, 3.92 (2H, AB, J = 8.1 Hz), 3.77 (3H, s), 4.13, 4.27 (2H, AB,
J = 12 Hz), 5.28 (1H, s), 6.77 (2H, d, J = 8.6 Hz), 7.12–7.31 (8H, m),
7.47–7.49 (4H, m), 7.94 (1H, s). 13C NMR (CDCl3): d 12.4, 55.7,
57.3, 58.2, 71.8, 72.9, 78.4, 87.6, 90.9, 110.1, 114.1, 127.6, 128.8,

9236
S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 9230–9237
129.3, 129.5, 130.9, 136.6, 138.8, 147.0, 147.6, 151.1, 159.8. MS
(FAB): m/z 542 (M+H)+. Anal. Calcd for C31H31N3O6ꢂ1H2O: C,
66.53; H, 5.94; N, 7.51. Found: C, 66.20; H, 5.96; N, 7.42.
under reduced pressure and the residue was purified by column
chromatography using ethyl acetate and methanol (10:1, v/v) to
yield compound 17 (781 mg, 89%) as a white solid: mp 154–
158 °C. ½a 2D2
ꢁ
+120.62 (c = 0.52, CHCl3). IR mmax (KBr): 3087, 2250,
4.1.4. 50-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-30-
dexoy-30-(4-methoxytriphenylmethylamino)-5-methyl-20-O,40-
C-methyleneuridine (14)
1745, 1666, 1610, 1488, 1250 cmꢀ1 1H NMR (CDCl3): d 1.80 (3H,
.
s), 2.01 (1H, d, J = 10 Hz), 2.10 (3H, s), 2.44 (1H, s), 2.85 (1H, d,
J = 8.0 Hz), 3.78 (3H, s), 4.03, 4.10 (2H, AB, J = 8.0 Hz), 4.86, 4.90
(2H, AB, J = 13 Hz), 5.34 (1H, s), 6.73 (2H, d, J = 9.0 Hz), 7.18–7.30
(8H, m), 7.42–7.44 (4H, m), 8.35 (1H, d, J = 7.0 Hz). 13C NMR
(CDCl3): d 13.5, 21.1, 55.2, 56.5, 60.0, 70.5, 71.7, 77.2, 77.3, 86.5,
86.8, 101.0, 113.3, 126.8, 127.9, 128.0, 129.3, 136.7, 137.1, 145.2,
145.4, 155.1, 158.1, 165.4, 170.0.
2-Cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodiamidite
(70.7
(500 mg, 928
l
l, 2.23 mmol) was added to a suspension of compound 13
mol) and diisopropylammonium tetrazolide
l
(261 mg, 1.3 mmol) in acetonitrile (6 ml) and THF (2 ml). The mix-
ture was stirred at room temperature for 2 h. The solvent was re-
moved under reduced pressure and the residue was purified by
column chromatography using ethyl acetate and n-hexane
(2:3?1:1, v/v) to give compound 14 (624 mg, 91%) as a white so-
lid: mp 101–103 °C. 31P NMR (CDCl3): d 148.8, 148.9.
4.1.8. 50-O-Acetyl-N4-benzoyl-30-deoxy-30-(4-methoxytriphenyl-
methylamino)-5-methyl-20-O,40-C-methylenecytidine (18)
Trimethylamine (324
(136 l, 1.17 mmol) were added to a solution of compound 17
(575 mg, 987 mol) in dichloromethane (10 ml). The mixture
ll, 2.34 mmol) and benzoyl chloride
l
4.1.5. 50-O-Acetyl-30-deoxy-30-(4-methoxytriphenylemethyl-
amino)-5-methyl-20-O,40-C-methyleneuridine (15)
l
was stirred at room temperature for 12 h. The reaction was
quenched by the addition of saturated NaHCO3 (aq), and the mix-
ture was extracted with ethyl acetate. The combined organic layer
was washed with water, then brine, and dried over sodium sulfate.
Evaporation to dryness under reduced pressure, followed by col-
umn chromatography using ethyl acetate and n-hexane (1:2, v/v),
gave compound 18 (581 mg, 89%) as a white solid: mp 107–
Acetic anhydride (1.56 ml, 16.6 mmol) was added to a solution
of compound 13 (8.11 g, 15.0 mmol) in pyridine (150 ml) and the
mixture was stirred for 28 h at room temperature. The reaction
mixture was quenched with 28% NH4OH, then concentrated under
reduced pressure and extracted with ethyl acetate. The combined
organic layers were washed with water, brine, and then dried over
sodium sulfate. After the solvent was removed, the residue was
purified by column chromatography by eluting with ethyl acetate
and n-hexane (1:1, v/v) to give compound 15 (15.1 g, 93%) as a
110 °C. ½a 2D1
ꢁ
+156.11 (c = 0.57, CHCl3). IR mmax (KBr): 3074, 2054,
1706, 1565, 1247 cmꢀ1
.
1H NMR (CDCl3): d 1.99 (3H, s), 2.03 (1H,
d, J = 10 Hz), 2.15 (3H, s), 2.44 (1H, s), 2.88 (1H, d, J = 11 Hz), 3.78
(3H, s), 3.82, 3.94 (2H, AB, J = 8.0 Hz), 4.59, 4.82 (2H, AB,
J = 13 Hz), 5.40 (1H, s), 6.74 (2H, d, J = 9.0 Hz), 7.09–7.25 (8H, m),
7.34–7.37 (4H, m), 8.35 (1H, d, J = 7.0 Hz). 13C NMR (CDCl3): d
13.6, 21.2, 55.3, 56.6, 70.5, 71.7, 77.1, 77.2, 86.5, 86.9,
110.5,113.3, 126.9, 127.9, 128.0, 128.1, 129.3, 129.8,132.5, 135.3,
136.9, 145.1, 146.6, 158.2, 159.5, 169.9,179.5. Anal. Calcd for
C40H38N4O7ꢂ1/3H2O: C, 69.35; H, 5.63; N, 8.09. Found: C, 69.28;
H, 5.64; N, 8.00.
white solid: mp 136–139 °C. ½a D22
ꢁ
+46.66 (c = 0.50, CHCl3). IR mmax
(KBr): 3193, 3060, 2251, 1746, 1687, 1606, 1507, 1254 cmꢀ1
.
1H
NMR (CDCl3): d 1.52 (3H, s), 1.91 (1H, d, J = 10 Hz), 2.05 (3H, s),
2.33 (1H, s), 2.80 (1H, d, J = 11 Hz), 3.71 (3H, s), 3.73, 3.86 (2H,
AB, J = 9 Hz), 4.50, 4.72 (2H, AB, J = 12 Hz), 5.28 (1H, s), 6.68 (2H,
d, J = 9 Hz), 7.09–7.25 (8H, m), 7.34–7.37 (4H, m), 8.01 (1H, d,
J = 5 Hz). 13C NMR (CDCl3): d 2.6, 21.0, 55.3, 56.7, 60.0, 70.5, 71.7,
77.2, 77.3, 86.2, 86.8, 109.3, 113.3, 126.8, 127.9, 128.0, 129.4,
134.1, 137.0, 145.2, 145.4,148.8, 158.1, 163.3, 169.9. MS (EI): m/z
583 (M+, 0.7), 372 (100). Anal. Calcd for C33H33N3O7ꢂ1/2H2O: C,
66.88; H, 5.78; N, 7.09. Found: C, 66.73; H, 5.91; N, 6.80.
4.1.9. N4-Benzoyl-30-deoxy-30-(4-methoxytriphenylmethyl-
amino)-5-methyl-20-O,40-C-methylenecytidine (19)
Lithium hydroxide (210 mg, 4.99 mmol) was added to a solu-
tion of compound 18 (685 mg, 999 lmol) in a mixture of THF
4.1.6. 50-O-Acetyl-30-deoxy-30-(4-methoxytriphenylmethyl-
amino)-4-(1,2,4-triazol-1-yl)-5-methyl-20-O,40-C-methyl-
eneuridine (16)
and water (9.53 ml, 2:1, v/v), and the mixture was stirred for 4 h
at room temperature. After dilution with water, the mixture was
extracted with dichloromethane, the organic phase was washed
with water, then brine, and dried over sodium sulfate. The solvents
were removed under reduced pressure and the residue was puri-
fied by column chromatography by eluting with ethyl acetate
and n-hexane (1:1, v/v) to provide compound 19 (623 mg, 97%)
Triethylamine (25.5 ml, 184 mmol) was added dropwise to a
stirred mixture of 1H-1,2,4-triazole (11.7 g, 169 mmol) and phos-
phoryl chloride (3.48 ml, 37.3 mmol) in acetonitrile (50.0 ml) at
0 °C. A solution of 15 (1.67 g, 2.38 mmol) in acetonitrile (19 ml)
was then added to the mixture at 0 °C. The resulting mixture was
stirred at room temperature for 6 h. The reaction mixture was
quenched by the addition of NaHCO3 (aq), and was concentrated
under reduced pressure. The residue was diluted with dichloro-
methane, washed with saturated NaHCO3 (aq), water and brine,
and dried over sodium sulfate. The solvent was evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy using ethyl acetate and n-hexane (1:1?2:1, v/v) to give com-
pound 16 (1.79 g, 99%) as a yellow foam: 1H NMR (CDCl3): d 2.03
(1H, d, J = 11 Hz), 2.14 (3H, s), 2.34 (3H, s), 2.65 (1H, s), 2.83 (1H,
d, J = 10 Hz), 3.77 (3H, s), 3.87, 3.99 (2H, AB, J = 9 Hz), 4.58, 4.84
(2H, AB, J = 12 Hz), 5.52 (1H, s), 6.70 (2H, d, J = 9 Hz), 7.18–7.29
(8H, m), 7.37–7.40 (4H, m), 7.78 (1H, s), 8.16 (1H, s) 9.33 (1H, s).
as a white solid: mp 215–218 °C. ½a D21
ꢁ
+110.68 (c = 0.55, CHCl3).
IR mmax (KBr): 3468, 3065, 2954, 2250, 1702, 1644, 1565 cmꢀ1
.
1H
NMR (CDCl3): d 1.90 (3H, s), 2.33 (1H, s), 2.95 (1H, s), 2.85 (1H,
d, J = 8.0 Hz), 3.75 (3H, s), 3.71, 3.94 (2H, AB, J = 9.0 Hz), 4.15,
4.30 (2H, AB, J = 12 Hz), 5.35 (1H, s), 6.74 (2H, d, J = 9.0 Hz), 7.12–
7.29 (8H, m), 7.46–7.48 (4H, m), 8.25 (2H, br). 13C NMR (CDCl3):
d 13.6, 55.3, 56.4, 58.5, 70.6, 71.7, 77.2, 77.3, 86.5, 89.1, 110.7,
113.4, 127.0, 127.9, 128.1, 129.3, 129.8, 132.5, 135.6, 136.9,137.0,
145.3,
145.4,
146.7,
158.3,
159.6.
Anal.
Calcd
for
C38H36N4O6ꢂ1AcOEt: C, 68.84; H, 6.05; N, 7.64. Found: C, 68.57;
H, 5.87; N, 7.97.
4.1.10. N4-Benzoyl-50-O-[2-cyanoethoxy(diisopropylamino)-
phosphino]-30-deoxy-30-(4-methoxytriphenylmethylamino)-5-
methyl-20-O,40-C-methylenecytidine (20)
4.1.7. 50-O-Acetyl-30-deoxy-30-(4-methoxytriphenylmethyl-
amino)-5-methyl-20-O,40-C-methylenecytidine (17)
Compound 16 (954 mg, 1.51 mmol) was dissolved in a mixture
of 28% ammonium solution and dioxane (35.0 ml, 1:6, v/v) and
stirred at room temperature for 1.5 h. The solvent was removed
2-Cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodiamidite
(59.5
(502 mg, 780
l
l, 1.87 mmol) was added to a suspension of compound 19
mol) and diisopropylammonium tetrazolide
l

S. Obika et al. / Bioorg. Med. Chem. 16 (2008) 9230–9237
9237
(220 mg, 1.09 mmol) in acetonitrile (6 ml) and THF (2 ml). The
Acknowledgements
mixture was stirred at room temperature for 2 h. The solvent
was removed under reduced pressure and the residue was purified
by column chromatography using ethyl acetate and n-hexane (1:2,
v/v) to give compound 20 (575 mg, 87%) as a white solid: mp 96–
98 °C. 31P NMR (CDCl3): d 148.7, 148.8.
A part of this work was supported by PRESTO from Japan Sci-
ence and Technology Agency (JST); Grant-in-Aid from Japan Soci-
ety for the Promotion of Science; Grant-in-Aid from the Ministry
of Education, Culture, Sports, Science, and Technology of Japan.
4.2. Oligonucleotide synthesis and purification
Supplementary data
Synthesis of 30-amino-20,40-BNA-modified oligonucleotides 21–
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.09.013.
27 was performed on a 0.2 l
mol scale following the 50?30 reverse
assembly protocol using phosphoramidites 14 and 20 (and/or the
30–50 conventional phosphoramidite assembly protocol using het-
erodimer 10 (for oligonucleotides 21 and 25 only)) on an Expedite
(TM) 8909 Nucleic Acid Synthesis System. All reagents were
assembled and the oligonucleotides were synthesized according
to the standard synthesis cycle (trityl off mode) with the exception
of a prolonged coupling time for 30-amino-20,40-BNA monomers
(320 s instead of 80 s used for natural monomers). Dicyanoimidaz-
ole was found to be a more efficient activator than 1-H tetrazole.
Standard CPG-solid supports from Glen Research were used for
the synthesis. Using a trityl monitor, the coupling efficiency of 30-
amino-20,40-BNA monomers was estimated to be 91–94%. After
synthesis, the solid-supported oligonucleotides were treated with
28% ammonium hydroxide solution (1 ml) at room temperature
for 1.5 h, then at 55 °C for 17–20 h. The ammonia solutions were
then concentrated, and the crude oligonucleotides were initially
purified by NAPTM 10 columns (Amersham Biosciences) followed
by further purification by reverse phase high performance liquid
chromatography (RP-HPLC) on a Waters X-Terra (10 ꢃ 50 mm) col-
umn using 6–14% MeCN in 0.1 M triethylammonium acetate buffer
(pH 7.0) at 50 °C. The oligonucleotides were analyzed for purity by
HPLC and characterized by MALDI-TOF mass spectroscopy.
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Crotalus admanteus venom phosphodiesterase (CAVP) (0.8
lg
for experiment A and 1.6 g for experiment B, Fig. 3) was added
l
to a solution of oligonucleotides 27 and 36–39, (3 nmol each) in
50 mM Tris–HCl buffer (pH 8.0) containing 10 mM MgCl2. The
cleavage reaction was carried out at 37 °C. At several time points,
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