Nucleosides and nucleotides. Part 214: Thermal stability of triplexes containing 4′α-C-aminoalkyl-2′-deoxynucleosides

Article abstract of DOI:10.1016/S0968-0896(02)00141-4

In order to develop novel antigene molecules forming thermally stable triplexes with target DNAs and having nuclease resistance properties, we synthesized oligodeoxynucleotides (ODNs) with various lengths of aminoalkyl-linkers at the 4′α position of thymi

Full text of DOI:10.1016/S0968-0896(02)00141-4

Bioorganic & Medicinal Chemistry 10 (2002) 2933–2939
Nucleosides and Nucle0otides. Part 214: Thermal Stability of
Triplexes Containing 4 ꢀ-C-Aminoalkyl-20-deoxynucleosidesy
Naoko Atsumi, Yoshihito Ueno,{ Makiko Kanazaki,
Satoshi Shuto and Akira Matsuda*
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, Japan
Received 8 March 2002; accepted 17 April 2002
Abstract—In order to develop novel antigene molecules forming thermally stable triplexes with target DNAs and having nuclease
resistance properties, we synthesized oligodeoxynucleotides (ODNs) with various lengths of aminoalkyl-linkers at the 40a position
of thymidine and the aminoethyl-linker at the 40a position of 20-deoxy-5-methylcytidine. Thermal stability of triplexes between these
ODNs and a DNA duplex was studied by thermal denaturation. The ODNs containing the nucleoside 2 with the aminoethyl-linker
or the nucleoside 3 with the aminopropyl-linker thermally stabilized the triplexes, whereas the ODNs containing the nucleoside 1
with the aminomethyl-linker or the nucleoside 4 with the 2-[N-(2-aminoethyl)carbamoyl]oxy]ethyl-linker thermally destabilized the
triplexes. The ODNs containing 2 were the most efficient at stabilizing the triplexes with the target DNA. The ODNs containing
40a-C-(2-aminoethyl)-20-deoxy-5-methylcytidine (5) also efficiently stabilized the triplexes with the target DNA. Stability of the
ODN containing 5 to nucleolytic hydrolysis by snake venom phosphodiesterase (a 30-exonuclease) was studied. It was found that
the ODN containing 5 was more resistant to nucleolytic digestion by the enzyme than an unmodified ODN. In a previous paper, we
reported that the ODNs containing 2 were more resistant to nucleolytic digestion by DNase I (an endonuclease) than the unmodi-
fied ODNs. Thus, it was found that the ODNs containing 40a-C-(2-aminoethyl)-20-deoxynucleosides were good candidates for
antigene molecules. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
DNA duplex is thermodynamically weaker than duplex
formation itself. Thus, much effort has been made to
increase the affinity of the third strand for its target.
Triplex forming oligodeoxynucleotides (ODNs) have
attracted a great deal of attention because of their
potential use in gene therapy.2 In intermolecular trip-
lexes, a third strand of ODN binds to the major groove
of the DNA. To date, two major classes of triplexes
have been identified based on the orientation of the
third strand. When the third strand consists mainly of
pyrimidines, Hoogsteen-type Pyꢀ PuPy base triplets
(Tꢀ AT and C+ꢀ GC) are formed, in which the third
strand is parallel to the purine strand of the target
duplex.3,4 When the third strand is predominantly pur-
ines, Puꢀ PuPy-type base triplets (Gꢀ GC and Aꢀ AT) are
formed, where the third strand is antiparallel to the
purine strand of the target duplex.5,6 However, in gen-
eral, the binding of a third-strand ODN to a target
On the other hand, naturally occurring polyamines,
such as spermidine and spermine, are known to bind
strongly to DNA7ꢁ9 and stabilize duplex10,11 and triplex
formation,12ꢁ14 although the precise mode of binding is
not clear. Their enhanced thermal stability can be
explained by the reduction of the anionic electrostatic
repulsion between the phosphate moieties by the cati-
onic polyamines. Consequently, ODN analogues carry-
ing various polyamines have been synthesized,15ꢁ21
some of which have been shown to increase the thermal
stability of duplexes and triplexes and to be more resis-
tant to nucleases than unmodified ODNs.
Recently, Cuenoud et al. reported a synthesis of
ODNs containing 20-O-aminoethyl-5-methyluridine and
20-O-aminoethyl-5-methylcytidine.22ꢁ24 They showed
that the ODNs containing the 20-O-aminoethylnucleo-
sides thermally stabilized triplexes with target DNAs.
The thermal stabilization of the triplexes by the
*Corresponding author. Tel.: +81-11-706-3228; fax: +81-11-706-
4980; e-mail: matuda@pharm.hokudai.ac.jp
yFor Part 213 of this series, see ref 1.
{Present address: Faculty of Engineering, Gifu University, Yanagido
1-1, Gifu 501–1193, Japan.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00141-4

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N. Atsumi et al. / Bioorg. Med. Chem. 10 (2002) 2933–2939
Table 1. Sequences of ODNs used in this studya
ODNs
12
50-MTMTMTMTMTMTMTMTMT-30
13-1, -2,-3, or -4
14-1, -2, -3, or -4
15-1, -2, -3, or -4
16-1, -2, -3, or -4
17-1, -2, -3, or -4
18-1, -2, -3, or -4
19-1, -2, -3, or -4
50-MXMTMTMTMTMTMTMTMT-30
50-MTMTMTMTMTMTMTMTMX-30
50-MTMTMTMTMXMTMTMTMT-30
50-MTMTMXMTMTMTMXMTMT-30
50-MXMTMTMTMXMTMTMTMX-30
50-MXMTMXMTMTMXMTMTMX-30
50-MXMTMXMTMXMTMXMTMX-30
20
21
22
23
24
25
26
27
50-5TMTMTMTMTMTMTMTMT-30
50-MTMTMTMTMTMTMTMT5T-30
50-MTMTMTMT5TMTMTMTMT-30
50-MTMT5TMTMTMTMT5TMT-30
50-MT5TMTMT5TMTMT5TMT-30
50-MT5TMT5TMT5TMT5TMT-30
50-MT5T5TMT5TMT5TMT5T-30
50-AG(TC)9C(T)5AG(GA)9CT-30
Figure 1. Structures of the modified nucleoside analogues.
a1 series (X=1); 2 series (X=2); 3 series (X=3); 4 series (X=4); T,
thymidine; M, 20-deoxy-5-methylcytidine.
20-O-aminoethylnucleosides can be explained by an
electrostatic interaction between the positively charged
aminoethyl residue of the nucleosides and a pro-R oxy-
gen of a negatively charged phosphate at the second
strand of the target DNA. Recently, we also synthesized
ODNs containing 40a-C-aminoalkylthymidines 1–4
(Fig. 1).25ꢁ27 We found that the ODNs containing
40a-C-aminoethylthymidine formed thermally stable
duplexes with the complementary DNA and RNA, and
were much more resistant to nucleolytic hydrolysis by
snake venom phosphodiesterase (a 30-exonuclease) and
DNase I (an endonuclease) than the unmodified parent
ODNs. On the basis of this background information
and our own experimental results, we envisaged that if
the distal ammonium cation of the aminoalkyl-linkers
of the 40a-C-aminoalkyl-20-deoxynucleosides interacted
with the phosphate group of the target DNA in the tri-
plex, as in the 20-O-aminoethyl-nucleoside, and ther-
mally stabilized the triplex, the ODNs containing
40a-C-aminoalkylthymidines would be good candidates
for antigene molecules.
Table 2. Hybridization dataa
ODNs
Tm (ꢂC)
Á Tm (ꢂC)
ODNs
Tm (ꢂC)
Á Tm (ꢂC)
12
44.1—
13-1
14-4
15-1
16-1
17-1
18-1
19-1
42.3
42.8
41.3
41.3
43.8
42.9
44.0
ꢁ1.8
ꢁ1.3
ꢁ2.8
ꢁ2.8
ꢁ0.3
ꢁ1.2
ꢁ0.1
13-4
14-4
15-4
16-4
17-4
18-4
19-4
43.6
44.9
44.6
43.6
41.8
42.1
41.4
ꢁ0.5
+0.8
+0.5
ꢁ0.5
ꢁ2.3
ꢁ2.0
ꢁ2.7
13-2
14-2
15-2
16-2
17-2
18-2
19-2
45.2
44.4
44.2
46.5
47.2
47.9
46.7
+1.1
+0.3
+0.1
+2.4
+3.1
+3.8
+2.6
20
21
22
23
24
25
26
44.2
44.3
44.5
44.7
45.4
47.2
46.5
+0.1
+0.2
+0.4
+0.6
+1.3
+3.1
+2.4
13-3
14-3
15-3
16-3
17-3
18-3
19-3
44.2
44.6
45.3
45.4
46.0
46.0
46.3
+0.1
+0.5
+1.2
+1.3
+1.9
+1.9
+2.2
In this paper, we report thermal stabilities of triplexes
between the ODNs containing the 40a-C-aminoalkyl-
thymidines 1–4 and 40a-C-aminoethyl-20-deoxy-5-methyl-
cytidine (5) and the target DNA. The resistance of the
ODNs containing 40a-C-aminoethyl-20-deoxy-5-methyl-
cytidine (5) to nucleolytic hydrolysis by snake venom
phosphodiesterase was also studied.
aExperimental conditions are described in the Experimental.
(4) (Fig. 1) were synthesized according to the methods
reported previously.25,26 The sequences of the syn-
thesized ODNs are shown in Table 1. The thermal sta-
bility of the triplexes between these ODNs and the
target duplex 27 was studied by thermal denaturation in
a buffer containing 0.5 M NaCl at pH 7.0. Two tran-
sitions were observed in the melting profile of each tri-
plex (data not shown): the transition with the higher Tm
was due to melting of the target duplex 27 (88 ꢂC), and
the transition with the lower Tm corresponded to dis-
sociation of the third strand from the triplex. The Tms
are listed in Table 2. The stability of the triplexes was
dependent on the number and length of the aminoalkyl-
linkers of the modified nucleosides. The Tm of the con-
trol triplex was 44.1 ꢂC. The ODNs containing 2 or 3
Results
Thermal stability of triplexes containing 40ꢀ-C-
aminoalkylthymidines
To study the thermal stability of triplexes consisting of
ODNs containing 40a-C-aminoalkylthymidines at var-
ious positions and in different numbers and the target
DNA, 50-[AG(TC)9C(T)5AG(GA)9CT]-30 (27) (Table
1), the ODNs containing 40a-C-aminomethyl (1), 40a-C-
(2-aminoethyl) (2), 40a-C-(3-aminopropyl) (3), and
40a-C-[2-[N-(2-aminoethyl)carbamoyl]oxy]ethyl]thymidine

N. Atsumi et al. / Bioorg. Med. Chem. 10 (2002) 2933–2939
2935
Scheme 1. Conditions: (a) (1) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, 1h; (2) concd NH 4OH, 0 ꢂC–rt, 2 h, 75%;
(b) Bz2O, pyridine, rt, 24 h, 93%; (c) H2, Pd/C, EtOCOCF3, Et3N, EtOAc, rt, 3 h, 56%; (d) TBAF, THF, rt, 4 h, 95%; (e) i-Pr2NP(Cl)O(CH2)2CN,
i-Pr2NEt, CH2Cl2, rt, 10 min, 62%.
thermally stabilized the triplexes, whereas the ODNs
containing 1 or 4 thermally destabilized the triplexes.
The triplexes containing 2 or 3 became more stable as
the number of the modified nucleosides increased except
for the triplex containing five molecules of 2. When the
same numbers of the modified nucleosides were incor-
porated into the ODNs at the same positions, the order
of ÁTm values [Tm (each ODN) ꢁTm (the control ODN
12)] for the ODNs was as follows: the ODNs containing
2 >the ODNs containing 3 >the ODNs containing 1
>the ODNs containing 4 except for the ODNs con-
taining one residue of the modified nucleosides at their
30-end or center. ÁTm values for the ODNs containing
four molecules of 2 (ODN 18–2) or five molecules of 3
(ODN 19–3) were +3.8 and +2.2 ꢂC, respectively.
Therefore, it was found that the ODNs containing 2
with the aminoethyl-linker were the most efficient at
stabilizing the triplexes with the target DNA 27.
follows: 40a-C-azidoethyl-30-O-tert-butyldimethylsilyl-
20-deoxy-50-O-dimethoxytritylthymidine (6), which was
prepared according to the reported method,26 was trea-
ted with 2,4,6-triisopropylbenzenesulfonyl chloride in
the presence of DMAP and Et3N, followed by NH4OH,
to afford the 20-deoxy-5-methylcytidine derivative 7 in
75% yield. After protection of the exo-amino function
of 7 with benzoyl group, the azide group in 8 was
hydrogenated with Pd/C in the presence of ethyl tri-
fluoroacetate to give the N-protected 40a-C-aminoethyl-
20-deoxy-5-methylcytidine derivative 9 in 56% yield.
After removal of the TBS group, 10 was phosphitylated
by a standard procedure30 to give the corresponding
nucleoside phosphoramidite 11 in 62% yield (Scheme 1).
The ODNs containing 5 were prepared on a DNA syn-
thesizer by the phosphoramidite method.31 The sequen-
ces of the synthesized ODNs are shown in Table 1. The
ODNs obtained were analyzed by matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF)
mass spectrometry, and the observed molecular weights
supported their structures (see Experimental).
Synthesis of ODNs containing 40ꢀ-C-(2-aminoethyl)-20-
deoxy-5-methylcytidine
When the various lengths of aminoalkyl-linkers were
incorporated into the ODNs at the 40a-position of the
thymidine residues, the thymidine analogue 2 with the
aminoethyl-linker stabilized the triplexes very efficiently.
The parallel-type triplexes are formed by Hoogsteen-
type Tꢀ AT and C+ꢀ GC base triplets.2 Thus, we next
planned to synthesize a cytidine analogue with the
aminoethyl-linker at its 40a-position. Formation of the
parallel-type triplexes requires conditions of low pH
(pH <6), because unmodified cytosine residues, if pre-
sent in the third strand, must be protonated to bind with
guanine of the G/C duplex.2,28 It is known that the
ODNs containing 20-deoxy-5-methylcytidine form stable
parallel-type triplexes under neutral conditions.29 Thus,
we synthesized 40a-C-(2-aminoethyl)-20-deoxy-5-methyl-
cytidine (5). A phosphoramidite of 5 was synthesized as
Thermal stability of the triplexes containing 40ꢀ-C-(2-
aminoethyl)-20-deoxy-5-methylcytidine
The thermal stability of the triplexes between the ODNs
(20–26)
containing
40a-C-(2-aminoethyl)-20-deoxy-
5-methylcytidine (5) at various positions and in different
numbers and the target duplex 27 was also studied by
thermal denaturation in the buffer containing 0.5 M
NaCl at pH 7.0. Two transitions were observed in the
melting profile of each triplex (data not shown): the
transition with the higher Tm was due to melting of the
target duplex, and the transition with the lower Tm cor-
responded to dissociation of the third strand from the
triplex. The Tms are summarized in Table 2. The stabi-
lity of the triplexes was dependent on the position and

2936
N. Atsumi et al. / Bioorg. Med. Chem. 10 (2002) 2933–2939
Discussion
The ODNs with the various lengths of the aminoalkyl-
linkers at the 40a position of thymidine and the amino-
ethyl-linker at the 40a position of 20-deoxy-5-methyl-
cytidine were synthesized using the phosphoramidite
method. The thermal stability of the triplexes between
these ODNs and the target duplex was studied by ther-
mal denaturation. It was found that the stability of the
triplexes was dependent on the number and length of the
aminoalkyl-linkers of the modified nucleosides. The
ODNs containing 2 with the aminoethyl-linker or 3 with
the aminopropyl-linker thermally stabilized the triplexes,
whereas the ODNs containing 1 with the aminomethyl-
linker or
4
with 2-[N-(2-aminoethyl)carbamoyl]-
oxy]ethyl-linker thermally destabilized the triplexes. The
ODNs containing 2 with the aminoethyl-linker most
efficiently stabilized the triplexes with the target DNA.
Recently, Cuenoud et al. reported that the ODNs con-
taining the 20-O-aminoethylnucleosides also enhanced
thermal stability of triplexes with DNA duplexes.22ꢁ24
Based on the molecular dynamics (MD) simulation and
an NMR study, they concluded that the enhanced ther-
mal stability of the triplexes upon incorporation of
20-O-aminoethylnucleosides was due to an electrostatic
interaction between the positively charged aminoethyl
chain of the nucleoside and a pro-R oxygen of a nega-
tively charged phosphate of the second strand of the
target DNA. To understand the effect of the 40a-C-
(2-aminoethyl)thymidine 2 on the triplex formation, we
also performed an MD simulation of the triplex con-
taining 2. The model of the triplex containing 2 derived
from the MD simulation showed that the ammonium
ion at the end of the aminoethyl chain of 2 could form
an intermolecular ionic bond with the pro-R-oxygen
atom of the phosphate group of the second strand of the
target duplex (data not shown). Thus, we reasoned a
priori that the aminomethyl-linker of 1 was too short to
stabilize the triplexes while the 2-[[N-(2-amino-
ethyl)carbamoyl]oxy]ethyl-linker of 4 was too long to
stabilize the triplexes.
Figure 2. Polyacrylamide gel electrophoresis of 50-32P-labeled ODNs
hydrolyzed by snake venom phosphodiesterase: (a) 12; (b) 21. ODNs were
incubated with snake venom phosphodiesterase for 0 min (lane 1), 10 min
(lane 2), 20 min (lane 3), 30 min (lane 4), 60 min (lane 5), and 120 min (lane
6). Experimental conditions are described in the Experimental.
number of the modified nucleoside 5. The ODNs con-
taining 5 thermally stabilized the triplexes with the target
DNA relative to the control ODN 12. The ODN, which
contained one residue of 5 at the center, stabilized the
triplex more than the ODNs containing one residue of 5
at its 50-end or near its 30-end. The triplexes containing 5
became thermally more stable as the number of the mod-
ified nucleosides increased except for the triplex contain-
ing five molecules of 5. The ÁTm values for the ODNs
containing four molecules of 5 (ODN 25) was +3.1 ꢂC.
Nuclease resistance of the ODN containing 40ꢀ-C-
The 40a-C-(2-aminoethyl)thymidine (2) and 40a-C-
(2-aminoethyl)-20-deoxy-5-methylcytidine (5) thermally
stabilized the triplexes with the target DNAs. However,
the stabilization of the triplexes by the 40a-C-(2-amino-
ethyl)-20-deoxynucleosides seems to be less efficient than
the stabilization by the 20-O-aminoethylnucleoside. An
increase of the Tm value of +0.95 ꢂC per modification
was observed when four molecules of 2 were incorpor-
ated into the ODN, whereas an increase of Tm value of
the +0.78 ꢂC per modification was observed when four
molecules of 5 were incorporated into the ODN. On the
other hand, the ODNs containing 20-O-aminoethylnu-
cleosides stabilize the triplexes by an increase of the Tm
value of +3.5 ꢂC per modification.22 It has been repor-
ted that an ODN consisting of a nucleoside adopting an
N-type sugar conformation thermally stabilizes a triplex
with a DNA duplex.32ꢁ34 The 20-O-aminoethylnucleo-
side is known to adopt predominantly an N-type sugar
conformation,23,24 whereas the 40a-C-(2-aminoethyl)-
20-deoxynucleoside adopts predominantly an S-type
(2-aminoethyl)-20-deoxy-5-methylcytidine
Recently, we found that the ODN containing 2 was
more resistant to nucleolytic digestion both by snake
venom phosphodiesterase (a 30-exonuclease) and DNase
I (an endonuclease) than the unmodified ODN.26 We
next examined the stability of the ODNs containing 21
against snake venom phosphodiesterase. The ODN 21
containing 5 was labeled at the 50-end with 32P and
incubated with the enzyme, and the reactions were ana-
lyzed by polyacrylamide gel electrophoresis under
denaturing conditions. The control ODN 12 was
hydrolyzed randomly by the enzyme after 120 min of
incubation (Fig. 2a). In contrast, the phosphodiester
linkage at the 50-side of 5 was highly resistant to the
enzyme, and the phosphodiester linkage at the 30-side of
5 was also slightly resistant to the enzyme (Fig. 2b).
Thus, it was found that the ODN containing 5 was more
resistant to nucleolytic digestion by snake venom phos-
phodiesterase than the unmodified ODN.

N. Atsumi et al. / Bioorg. Med. Chem. 10 (2002) 2933–2939
2937
sugar conformation.26 Thus, the difference in their sugar
conformation should influence the thermal stability of
the triplexes with the DNA duplex.
40ꢀ-C-Azidoethyl-30-O-tert-butyldimethylsilyl-20-deoxy-
50-O-dimethoxytrityl-5-methylcytidine (7). A solution of
40a-C-azidoethyl-30-O-tert-butyldimethylsilyl-20-deoxy-50-
O-dimethoxytritylthymidine (6)26 (1.6 g, 2.2 mmol),
Et3N (1.2 mL, 8.8 mmol), DMAP (0.81 g, 6.6 mmol) and
The triplexes containing 40a-C-(2-aminoethyl)thymidine
(2) and 40a-C-(2-aminoethyl)-20-deoxy-5-methylcytidine
(5) became thermally more stable as the number of the
modified nucleosides increased until four molecules of 2
or 5 were incorporated into the ODNs. However, the
thermal stabilities of the triplexes containing five mole-
cules of 2 or 5 decreased relative to those of the triplexes
containing four molecules of 2 or 5. This phenomenon
might be due to the sequence of the ODNs used, in
which 20-deoxy-5-methylcytidine derivatives were alter-
natively incorporated into the ODNs. When the paral-
lel-type triplexes are formed, the N3 position of the
cytosine residues, if present in the third strand, must be
protonated to bind with the guanine of the G/C duplex.
Therefore, the triplexes of this sequence might already
be stabilized by positive charges in the M+/G/C base
triplets; thus, additional positive charges can not further
stabilize them.
2,4,6-triisopropylbenzenesulfonyl
chloride
(1.33 g,
4.4 mmol) in CH3CN (100 mL) was stirred at room
temperature for 1h. The mixture was cooled in an ice-
bath. Concentrated NH4OH (25%, 160 mL) was added,
and the mixture was stirred at room temperature for 2 h.
The mixture was evaporated, and the residue was parti-
tioned between EtOAc and H2O. The organic layer was
washed brine, dried (Na2SO4), and evaporated. The
residue was purified by column chromatography (SiO2,
1–4% EtOH in CHCl3) to give 7 (1.2 g, 75% as a yellow
1
foam): H NMR (400 MHz, CDCl3) d 8.27 (br s, 2H),
7.20 (s, 1H), 7.41–6.82 (m, 13H), 6.21 (dd, 1H, J=5.9,
6.3), 4.60 (m, 1H), 3.80 (s, 6H), 3.42–3.35 (m, 2H), 3.26
(m, 1H), 3.07 (d, 1H, J=10.3), 2.45 (m, 1H), 2.19 (m,
1H), 1.98 (m, 1H), 1.68 (m, 1H), 1.44 (s, 3H), 0.83 (s,
9H), 0.02, ꢁ0.05 (each s, each 3H); 13C NMR
(100 MHz, CDCl3) d 165.46, 158.60, 144.04, 137.93,
135.11, 130.04, 130.00, 128.10, 127.87, 127.07, 113.17,
113.13, 101.37, 87.23, 86.79, 84.75, 72.29, 65.12, 55.25,
46.98, 41.37, 31.14, 25.70, 17.93, 12.61, ꢁ4.55, ꢁ5.07.
Resistance of the ODNs to nucleolytic hydrolysis by
nucleases is an important factor in antigene studies.2
Thus, the stability of the ODNs containing 40a-C-
(2-aminoethyl)-20-deoxy-5-methylcytidine (5) to nucleo-
lytic hydrolysis by snake venom phosphodiesterase (a
30-exonuclease) was studied. It was found that the
ODN containing 5 was more resistant to nucleolytic
digestion by the enzyme than the unmodified ODN.
In our previous paper, we reported that ODNs con-
taining 40a-C-(2-aminoethyl)thymidine (2) were more
resistant to nucleolytic digestion by DNase I (an endo-
nuclease) than the unmodified ODN.26 Thus, it was
found that the ODNs containing 40a-C-(2-amino-
ethyl)nucleosides were resistant against both 30-exo and
endo nucleases.
.
Anal. calcd for C39H50N6O6Si 1/2H2O: C, 64.10; H,
7.43; N, 10.69. Found: C, 64.20; H, 7.17; N, 10.58.
N4-Benzoyl-40ꢀ-C-azidoethyl-30-O-tert-butyldimethylsilyl-
20-deoxy-50-O-dimethoxytrityl-5-methylcytidine (8).
A
solution of 7 (86 mg, 0.12 mmol) and Bz2O (54 mg,
0.24 mmol) in pyridine (5 mL) was stirred at room tem-
perature for 24 h. The mixture was partitioned between
EtOAc and aqueous saturated NaHCO3. The organic
layer was washed with H2O and then brine, dried
(Na2SO4), and evaporated. The residue was purified by
column chromatography (SiO2, 4–13% EtOAc in hex-
ane) to give 8 (93 mg, 93% as a white foam): FAB-MS
m/z 831(MH +); 1H NMR (400 MHz, CDCl3) d 13.3 (br
s, 1H), 8.29 (m, 2H), 7.77 (s, 1H), 7.41–6.84 (m, 16H),
6.24 (dd, 1H, J=6.3, 6.6), 4.63 (dd, 1H, J=4.6, 6.8),
3.80 (s, 6H), 3.42–3.36 (m, 2H), 3.26 (m, 1H), 3.12 (d,
1H, J=10.3), 2.40 (ddd, 1H, J=4.6, 6.3, 13.7), 2.27
(ddd, 1H, J=6.6, 6.8, 13.7), 2.00 (m, 1H, J=14.6), 1.70
(m, 1H, J=14.6), 1.65 (s, 3H), 0.86 (s, 9H), 0.05, ꢁ0.01
(each s, each 3H); 13C NMR (100 MHz, CDCl3) d
179.28, 159.59, 158.65, 147.75, 143.91, 137.00, 136.57,
134.97, 134.93, 132.26, 129.98, 129.73, 128.04, 127.97,
127.92, 127.15, 113.22, 113.19, 111.85, 87.84, 87.04,
84.47, 72.74, 65.39, 55.23, 46.91, 41.19, 31.28, 25.70,
17.96, 12.98, ꢁ4.58, ꢁ5.04; HRMS calcd for
C46H55O7N6Si: 831.3901. Found: 831.3918.
In conclusion, we have found that the ODNs containing
40a-C-(2-aminoethyl)thymidine (2) and 40a-C-(2-amino-
ethyl)-20-deoxy-5-methylcytidine (5) thermally stabilized
the triplexes with the DNA duplex, and that the ODNs
containing 5 were more resistant to nucleolytic digestion
by snake venom phosphodiesterase than the unmodified
ODN. Thus, the ODNs containing 40a-C-(2-amino-
ethyl)-20-deoxynucleosides appear to be good candidates
for antigene molecules.
Experimental
General remarks
N4-Benzoyl-30-O-tert-butyldimethylsilyl-20-deoxy-50-O-di-
methoxytrityl-5-methyl-40ꢀ-C-(2-N-trifluoroacetyl)amino-
ethylcytidine (9). A solution of 8 (0.415 g, 0.5 mmol),
Et3N (0.49 mL, 3.5 mmol), ethyl trifluoroacetate
(0.30 mL, 2.5 mmol) and Pd/C (10%, 85 mg) in EtOAc
(45 mL) was stirred under atmospheric pressure of H2 at
room temperature for 3 h. The catalyst was filtered off
with Celite, and the filtrate was evaporated. The residue
was purified by column chromatography (a neutralized
SiO2, 15% EtOAc in hexane) to give 9 (0.25 g, 56% as a
NMR spectra were recorded at 400 MHz (1H), at
100 MHz (13C) and at 202 MHz (31P). Chemical shifts
(d) are reported in ppm downfield from TMS or 85%
H3PO4. J values are given in Hertz. Mass spectra were
obtained by fast atom bombardment (FAB) method.
Thin-layer chromatography was done on Merck coated
plates 60F254. The silica gel or the neutralized silica gel
used for column chromatography were Merck silica gel
5715 or ICN silica 60A, respectively.

2938
N. Atsumi et al. / Bioorg. Med. Chem. 10 (2002) 2933–2939
white foam): FAB-MS m/z 901(MH +); 1H NMR
(400 MHz, CDCl3) d 13.3 (br s, 1H), 8.29 (m, 2H), 7.61
(s, 1H), 7.54–7.24 (m, 12H), 6.93 (m, 1H), 6.86–6.84 (m,
4H), 6.28 (dd, 1H, J=6.3, 6.8), 4.55 (m, 1H), 3.79 (m,
8H), 3.50 (m, 1H), 3.38 (m, 1H), 3.31 (d, 1H, J=10.3),
3.17 (d, 1H, J=10.3), 2.34 (ddd, 1H, J=3.9, 6.3, 13.7),
2.26 (m, 1H), 2.03 (m, 1H), 1.72 (m, 1H), 1.71 (s, 3H),
0.86 (s, 9H), 0.05, 0.01(each s, each 3H); 13C NMR
(100 MHz, CDCl3) d 206.91, 179.12, 162.51, 162.13,
161.78, 161.46, 159.44, 158.61, 143.77, 136.70, 135.06,
134.74, 132.34, 130.34, 130.23, 129.96, 129.62, 129.19,
129.02, 128.91, 128.03, 127.88, 127.13, 116.38, 113.19,
113.16, 111.96, 88.24, 87.04, 84.80, 72.74, 64.83, 55.13,
39.87, 36.01, 31.03, 30.95, 30.88, 30.79, 25.52, 17.79,
12.82, ꢁ4.89, ꢁ5.26; HRMS calcd for C48H56F3N4
O8Si: 901.3820. Found: 901.3817.
NH4OH at 55 ꢂC for 16 h, and the released ODN pro-
tected by a DMTr group at the 50-end was chroma-
tographed on a C-18 silica gel column (1 ꢃ 10 cm,
Waters) with a linear gradient of CH3CN (from 0 to
30%) in 0.1M TEAA buffer (pH 7.0). The fractions
were concentrated, and the residue was treated with
aqueous 80% AcOH at room temperature for 20 min,
then the solution was concentrated, and the residue was
coevaporated with H2O. The residue was dissolved in
H2O and the solution was washed with Et2O, then the
H2O layer was concentrated to give a deprotected
ODN. The ODN was further purified by reversed-phase
HPLC, using a J’sphere ODN M80 column (4.6 ꢃ
150 mm, YMC) with a linear gradient of CH3CN (from
10 to 35% over 20 min) in 0.01 M TEAA buffer (pH 7.0)
to give a highly purified ODNs 20 (6), 21 (7), 22 (7), 23
(4), 24 (4), 25 (4), and 26 (2). The yields are indicated in
parentheses as OD units at 260 nm starting from 1 mmol
scale.
N4-Benzoyl-20-deoxy-50-O-dimethoxytrityl-5-methyl-40ꢀ-
C-(2-N-trifluoroacetyl) aminoethylcytidine (10). A solu-
tion of 9 (0.254 g, 0.28 mmol), TBAF (1.0 M in THF,
0.56 mL, 0.56 mmol) in THF (15 mL) was stirred at
room temperature for 4 h. The mixture was evaporated,
and the residue was purified by column chromato-
graphy (SiO2, 20–50% EtOAc in hexane) to give 10
(0.21g, 95% as a white foam): FAB-MS m/z 786
Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry
Spectra were obtained on a Voyager Elite reflection
time-of-flight mass spectrometry (PerSeptive Bio-
systems, Inc., Framingham, MA, USA) equipped with a
nitrogen laser (337 nm, 3-ns pulse) in the negative ion
mode. 3-Hydroxypicolinic acid (HPA), dissolved in
H2O to give a saturated solution at room temperature,
was used as the matrix. Time-to-mass conversion was
achieved by calibration by using the peak representing
the C+ cation of the charged derivative to be analyzed.
ODN 20: calculated mass, 5444.1; observed mass,
5446.2. ODN 21: calculated mass, 5444.1; observed
mass, 5445.9. ODN 22: calculated mass, 5444.1;
observed mass, 5444.5. ODN 23: calculated mass,
5487.1; observed mass, 5486.5. ODN 24: calculated
mass, 5530.1; observed mass, 5530.3. ODN 25: calcu-
lated mass, 5573.2; observed mass, 5573.7. ODN 26:
calculated mass, 5616.2; observed mass, 5615.9.
1
(MH+); H NMR (400 MHz, CDCl3) d 13.3 (br s, 1H),
8.25 (m, 2H), 7.69 (s, 1H), 7.60 (br s, 1H), 7.54–6.83 (m,
16H), 6.32 (dd, 1H, J=5.9, 7.3), 4.55 (m, 1H), 3.77 (m,
7H), 3.43–3.33 (m, 2H), 3.29 (t, 2H, J=10.5), 2.51 (ddd,
1H, J=2.9, 5.9, 13.7), 2.43 (m, 1H), 2.10–1.95 (m, 2H),
1.71 (s, 3H); 13C NMR (100 MHz, CDCl3) d 207.29,
179.41, 159.56, 158.65, 157.03, 148.19, 143.86, 136.72,
134.88, 134.79, 132.49, 129.96, 129.81, 129.67, 128.06,
127.98, 127.20, 115.75, 113.27, 112.24, 88.60, 87.39,
84.94, 84.87, 73.54, 73.41, 66.47, 55.27, 55.21, 40.98,
35.75, 30.76, 13.06; HRMS calcd for C42H42F3N4 O8:
787.2955. Found: 787.2969.
N4-Benzoyl-20-deoxy-50-O-dimethoxytrityl-5-methyl-40ꢀ-
C-(2-N-trifluoroacetyl)aminoethylcytidine 30-O-(2-cya-
noethyl) N,N-diisopropylphosphoramidite (11). A solu-
tion of 10 (0.844 g, 1.1 mmol), N,N-diisopropylethylamine
(0.77 mL, 4.4 mmol) and chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine (0.49 mL, 2.2 mmol) in
CH2Cl2 (35 mL) was stirred at room temperature for
10 min. The mixture was partitioned between CHCl3
and aqueous saturated NaHCO3. The organic layer was
washed with H2O and then brine, dried (Na2SO4), and
evaporated. The residue was purified by column
chromatography (a neutralized SiO2, 10–30% EtOAc in
hexane) to give 11 (0.67 g, 62% as a white foam): FAB-
MS m/z 987 (MH+); 31P NMR (202 MHz, CDCl3) d
150.44, 149.92; HRMS calcd for C51H59F3N6O9P:
987.4033. Found: 987.4017.
Thermal denaturation
The solution, that contained the oligodeoxynucleotide
(3 mM) and the target duplex (3 mM) in a buffer of
0.01M sodium cacodylate (pH 7.0) and 0.5 M NaCl,
was heated at 60 ꢂC for 5 min and then cooled gradually
to an appropriate temperature, and used for the thermal
denaturation study. Thermal-induced transitions of
each mixture were monitored at 260 nm on a Beckman
DU 650 spectrophotometer. Sample temperature was
increased 0.5 ꢂC/min.
Partial hydrolysis of ODN with snake venom
phosphodiesterase
Synthesis of ODNs
Each ODN labeled with 32P at the 50-end (5 pmol) was
incubated with snake venom phosphodiesterase
(0.32 mg) in the presence of Torula RNA (0.15 OD units
at 260 nm) in a buffer containing 37.5 mM Tris–HCl
(pH 8.0) and 7.5 mM MgCl2 (total 20 mL) at 37 ꢂC. At
appropriate periods, aliquots of the reaction mixture
were separated and added to a solution of EDTA
ODNs were synthesized on a DNA synthesizer (Applied
Biosystem Model 392) by the phosphoramidite
method.31 The fully protected ODNs were then
deblocked and purified by the same procedure as for the
purification of normal ODNs.35 That is, each ODN
linked to the resin was treated with concentrated

N. Atsumi et al. / Bioorg. Med. Chem. 10 (2002) 2933–2939
2939
(5 mM, 10 mL), then mixture were heated for 3 min at
90 ꢂC. The solutions were analyzed by electrophoresis
on 20% polyacrylamide gel containing 7 M urea.36
16. Prakash, T. P.; Barawkar, D. A.; Kumar, V.; Ganesh,
K. N. Bioorg. Med. Chem. Lett. 1994, 4, 1733.
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Biophys. Res. Comm. 1994, 205, 1665.
18. Schmid, N.; Behr, J.-P. Tetrahedron Lett. 1995, 36, 1447.
19. Nara, H.; Ono, A.; Matsuda, A. Bioconjugate Chem. 1995,
6, 54.
Acknowledgements
20. Sund, C.; Puri, N.; Chattopadhyaya, J. Tetrahedron 1996,
52, 12275.
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This investigation was supported in part by a Grant-in-
Aid for Encouragement of Young Scientists from the
Ministry of Education, Science, Sports, and Culture of
Japan and a Grant from ‘Research for the Future’ Pro-
gram of the Japan Society for the Promotion of Science
(JSPS-RFTF97I00301). We thank Y. Misawa (Hok-
kaido University) for technical assistance.
23. Blommers, M. J. J.; Natt, F.; Jahnke, W.; Cuenoud, B.
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