Effect of the terminal amino group of a linker arm and its length at the C5 position of a pyrimidine nucleoside on the thermal stability of DNA duplexes

Article abstract of DOI:10.1006/bioo.2001.1212

2′-Deoxyuridine derivatives bearing a substituent at the C5-position, which has a different chain length and a different functional group (methyl or amino), were synthesized and incorporated into oligodeoxyribonucleotides. The effect of the substituent groups in the major groove on the stability of the duplexes was investigated by UV melting experiments. It was found that the stabilization of these duplexes by a terminal amino group depended on the length of a linker arm.

Full text of DOI:10.1006/bioo.2001.1212

Bioorganic Chemistry 29, 187–197 (2001)
doi:10.1006/bioo.2001.1212, available online at http://www.idealibrary.com on
Effect of the Terminal Amino Group of a Linker Arm and Its Length
at the C5 Position of a Pyrimidine Nucleoside on the Thermal
Stability of DNA Duplexes
Hiroaki Ozaki, Masayuki Mine, Yoshinori Ogawa, and Hiroaki Sawai1
Department of Chemistry, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu,
Gunma 376-8515, Japan
Received November 2, 2000; published online August 9, 2001
2Ј-Deoxyuridine derivatives bearing a substituent at the C5-position, which has a different
chain length and a different functional group (methyl or amino), were synthesized and incorpo-
rated into oligodeoxyribonucleotides. The effect of the substituent groups in the major groove
on the stability of the duplexes was investigated by UV melting experiments. It was found that
the stabilization of these duplexes by a terminal amino group depended on the length of a
linker arm.
᭧ 2001 Academic Press
Key Words: oligodeoxyribonucleotide; C5-substituted 2Ј-deoxyuridine; thermal stability.
INTRODUCTION
There is great interest in modified oligodeoxyribonucleotides (ODN) as research
tools for molecular biology and antisense molecules. An appropriate chemical modifi-
cation of ODN leads to improved hybridization properties, nuclease resistance, and
cell permeability. To date, various chemically modified ODNs have been synthesized
and their ability for the antisense effect has been investigated. The modification at
the 5Ј-end and 3Ј-end, internucleotide phosphodiester of ODNs, the 1Ј-position and
2Ј-position of the sugar, C5 of a pyrimidine, or N4 of cytosine has been reported (1).
The C5-position of a pyrimidine is a suitable site for the modification of oligonucleo-
tides. The substituent at C5 of a pyrimidine does not inhibit the base pairing with
adenine on a complementary strand, but the thermal stability of the duplex depends
on the kind of tethers and functional groups. For example, C5-propagyl substitution
of 2Ј-deoxyuridine conferred significant stability to duplexes and triplexes (2). This
indicates that their modification should bring about perturbation to the stability of a
duplex DNA. In a previous paper (3), we reported the effect of acridine-modification
at the C5-position on duplex stability. The acridine modification causes stabilization
1 To whom correspondence should be addressed. Fax: 0277-30-1224. E-mail: sawai@chem.
gunma-u.ac.jp
187
0045-2068/01 $35.00
Copyright ᭧ 2001 by Academic Press
All rights of reproduction in any form reserved.

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OZAKI ET AL.
of a duplex-form by interaction between the acridine moiety and DNA, and the length
of the spacer arm has a slight effect on the interaction. However, the relationship
between the duplex stability and the length of spacer remains to be clarified. The
structure of a tether to introduce a functional group on DNA is important for the
preparation of hybridization probes or antisense molecules. Here we investigated the
effect of the length of a tether between an oligodeoxyribonucleotide and a functional
group on the duplex stability. An amino group was attached on the tether as the
functional group because the amino group can interact with the DNA backbone and
is useful for further functionalization.
RESULTS AND DISCUSSION
Synthesis of Modified Nucleotides
The C5-Substituted nucleosides bearing an amine linker were prepared from 5-
methoxycarbonylmethyl-2Ј-deoxyuridine (1) as described previously (4). Most of the
amines reacted with 1 in satisfactory yields by a direct aminolysis of a methyl ester
group as shown in Scheme 1. In the reactions with diamines, the terminal amino group
at the C5-substituent was protected with a trifluoroacetyl group without purification.
Heptylamine could not react with 1 by aminolysis because of its low nucleophilicity.
Therefore, the corresponding nucleoside was synthesized by another route as shown
in Scheme 2. The methoxycarbonyl group in 4 was hydrolyzed after protection of
the 5Ј-hydroxyl group, followed by condensation of the resulting carboxyl group with
heptylamine using diphenylphosphoryl azide (DPPA) as a condensing reagent. The
5Ј-hydroxyl group of the modified nucleosides was protected with a dimethoxytrityl
SCHEME 1.

C5-SUBSTITUTED PYRIMIDINE NUCLEOSIDE
189
SCHEME 2.
group and all 5Ј-protected modified nucleosides were converted to nucleoside 3Ј-
phosphoramidites as shown in Scheme 3.
Synthesis of the Modified Oligodeoxyribonucleotides
The modified oligodeoxyribonucleotides (ODNs) were synthesized on a DNA
synthesizer. The coupling yields of the modified nucleoside 3Ј-phosophoramidites on
a CPG support are shown in Table 1, with abbreviations and retention times of ODNs
on a reversed-phase HPLC. The coupling yields decreased with an increase in the
chain length of the C5-substituent, particularly for the nucleosides bearing a terminal
methyl group (7d–f ). The synthesized ODNs were deprotected and purified in the
usual manner. The longer retention time on a reversed-phase HPLC column correlates
SCHEME 3.

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OZAKI ET AL.
TABLE 1
Abbreviations, Coupling Yields, and Retention Times of Modified ODNs
Abbreviations
Xa
Coupling yield (%)b
TR/minc
N-ODN
T
99
100
97
96
100
97
16.5d
15.8
15.9
16.7
18.0
21.1
26.0
ODN-2N
ODN-4N
ODN-6N
ODN-2C
ODN-4C
ODN-6C
2aЈ
2cЈ
2eЈ
2f
2g
2h
87
Note. ODN sequence: 5Јd(CATAGGAGAXGCCTA)3Ј.
a 2aЈ, 2cЈ, and 2eЈ were the deprotected nucleosides of 2a, 2c, and 2d by an alkaline treatment,
respectively.
b Coupling yields are for incorporation of the modified nucleosides.
c HPLC condition is in the experimental section except the gradient. Gradient: 10% B to 35% B in 35 min.
d HPLC condition is in the experimental section except the gradient. Gradient: 3% B to 53% B in 35 min.
with the stronger hydrophobicity of the substituent. The structure of the ODNs was
confirmed by a nuclease digestion.
Thermal Stability of the Duplexes Consisted of the Modified ODNs and Its
Complementary DNA
Thermal stabilities of the ODN/DNA duplexes were investigated by UV melting
experiments. The melting temperatures (Tm) of the duplexes are summarized in Table
2. For the ODN/DNA duplexes, most of the ODNs, except ODN-2N, showed lower
Tm values than a normal ODN (N-ODN). The Tm values decreased with increasing
chain length of the side arm, especially for ODN-2N, -4N, and -6N, which has a
terminal amino group on the C5-substituent. In comparison of the different terminal
groups with the same linker length, the duplexes formed from the ODN bearing a
terminal amino group always had higher Tm values than those from the ODN bearing
a terminal methyl group. This result suggests that the alkyl linker arm interfered with
TABLE 2
Melting Points and Thermodynamic Parameters of ODN/DNA Duplexes
ODN
Tm(ЊC)
⌬H0(kJ molϪ1
)
⌬S0(J molϪ1 KϪ1
)
⌬G37(kJ molϪ1
)
N-ODN
50.6
53.7
49.8
47.9
47.6
46.1
47.1
Ϫ496
Ϫ516
Ϫ521
Ϫ559
Ϫ526
Ϫ481
Ϫ414
Ϫ1420
Ϫ1460
Ϫ1500
Ϫ1630
Ϫ1530
Ϫ1390
Ϫ1180
Ϫ55.5
Ϫ63.1
Ϫ55.7
Ϫ53.4
Ϫ51.4
Ϫ49.8
Ϫ48.0
ODN-2N
ODN-4N
ODN-6N
ODN-2C
ODN-4C
ODN-6C
Note. Measurement condition under Experimental.

C5-SUBSTITUTED PYRIMIDINE NUCLEOSIDE
191
the duplex formation and the terminal amino group stabilized the duplexes. This
effect of the amino linker arm on the duplex stability is consistent with our previous
results (5), which indicated that the stability of the duplexes depends on the number
of amino groups in the linker arm and the length of the arm.
To gain detailed information on the stability of duplexes formed from the ODNs,
thermodynamic parameters for the duplex formation were estimated from the curve-
fitting for each melting curve. The calculation was performed by a nonlinear, least
squares calculation program, “TMSPEC,” which was developed by Kodama et al.
(6). The results are shown in Table 2. In the ODNs bearing a terminal amino group
on the linker arm, ODNs bearing the longer linker arm showed a more negative ⌬H,
which suggests the stabilization of the duplex, and a more negative ⌬S, which suggests
the destabilization of the duplexes. Hence, this destabilization is governed by a
decrease in ⌬S for the ODN bearing the longer linker arm. The ODNs bearing a
terminal methyl group and the longer linker arm showed a less negative ⌬H and ⌬S.
Since their duplexes had almost the same Tm values, an unfavorable ⌬H of the duplex
formation bearing a longer alkyl group was compensated by a favorable ⌬S. This
suggests that the long alkyl linker destabilizes the duplex in terms of enthalpy, but
the long linker with a terminal amino group stabilizes the duplex in terms of enthalpy.
In comparison of the different terminal groups bearing the same linker length, the
differences between the enthalpy change of a terminal amino group and a methyl
group (⌬⌬H0), are ϩ10 kJ molϪ1, for ODN-2N and -2C, Ϫ40 kJ molϪ1 for ODN-
4N and -4C, and Ϫ145 kJ molϪ1 for ODN-6N and -6C from Table 2. It is suggested
that the duplexes consisting of ODN-4N or -6N, which have a long linker, were
stabilized by the contribution of enthalpy change compared with those consisting of
ODN-4C or -6C, although the alkyl group is disadvantageous for the duplex formation
in terms of entropy change. On the other hand, the ODN-2N/DNA duplex was
stabilized by an increase in ⌬S value compared with the ODN-2C/DNA. That is to
say, the large negative ⌬⌬H0 in the long linker arms suggests that the terminal amino
groups at the side chain interact with moieties of the complementary strand on
the DNA duplex. Dande et al. reported that the side chain of 5-(aminohexyl)-2Ј-
deoxyuridine was placed at the 3Ј-side in the major groove could have an electrostatic
interaction with an electronegative atom in the major groove (7). In their model, the
terminal amino group at the linker arm interacted with O6 of 5Ј-side G on the
complementary strand. Therefore, in our duplexes it is also estimated that the terminal
amino group at the side chain of ODN could interact with electronegative atoms in
the major groove such as O6 and N7 of G-12 and G-13 and N7 of A14 in the
complementary stand. One of the possible structures of each duplex, which were
constructed by modeling of the modified ODN/DNA duplex based on Dande’s report,
is shown in Fig. 1.
In conclusion, the side chain at the C5-position of a pyrimidine nucleoside could
interact with the DNA backbone and affect the duplex stability according to the length
of the linker arm. The terminal amino group on the linker arm has a stabilization
effect by the entropy for the ODN bearing a short linker and by the enthalpy for the
ODN bearing a long linker. This knowledge is useful in the development of DNA
probes or antisense molecules.

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OZAKI ET AL.
FIG. 1. Parts of possible structures of ODN-2N/DNA (a), ODN-4N/DNA (b), and ODN-6N/DNA
(c) duplexes. Eight base-pairs from 3Ј-side of the modified ODN strand were illustrated.
EXPERIMENTAL
General. Thin-layer chromatography (TLC) was performed on Kieselgel 60F254
(Art. 5554, E. Merck). Silica-gel column chromatography was performed on Wako
gel C-200 (Wako Pure Chemical Industries Ltd., Wako), silica gel 60 (63–200 or
40–63 ␮m, Merck). High-performance liquid chromatography (HPLC) was carried
out on Wakosil 5C18 columns (4 mm i.d. ϫ 250 mm length or 10 mm i.d. ϫ 250
mm length, Wako) by use of a system consisting of JASCO 880-PU pump, 875-UV
UV/vis detector, 801-SC system controller, and Shimadzu C-R5A chromatopac. The
eluent was acetonitrile gradient in 50 mM triethylammonium acetate (TEAA, pH
7.0). 1H NMR spectra and 31P NMR spectra were obtained with a Varian Gemini 200
1
or a JEOL ␣-500 spectrometer. H NMR spectra were recorded relative to internal
tetramethylsilane and 31P NMR spectra were recorded relative to external 85% H3PO4.
Mass spectra were measured by a Perkin Elmer Sciex API-100 instrument in ESI
mode. Molecular modeling studies were carried out using MacroModel Ver. 6.0
(Schro¨dinger, Inc.). The AMBER* algorithm (8) was used during minimization of
oligonucleotide conformations. Oligodeoxyribonucleotides were synthesized by a
phosphoramidite chemistry on an Applied Biosystems 381A DNA synthesizer. Normal
nucleoside phosphoramidites were purchased from Glen Research. Dichloro(2-cya-
noethoxy)phosphine and chloro(diisopropylamino)-2-cyanoethoxyphosphine were
prepared by the described method (9). 3Ј,5Ј-Di-O-acetyl-5-(methoxycarbonylmethyl)-
2Ј-deoxyuridine (1) was prepared by the method described previously (4). The follow-
ing compounds were also prepared by the previously reported methods (3,5): 5-[N-
(2-trifluoroacetylaminoethyl)]carbamoylmethyl-2Ј-deoxyuridine (2a), 5-[N-(4-triflu-
oroacetylaminobutyl)]carbamoylmethyl-2Ј-deoxyuridine (2b), 5-[N-(6-trifluoroacety-
laminohexyl)]carbamoylmethyl-2Ј-deoxyuridine (2c), 5Ј-O-(4,4Ј-dimethoxytrityl)-
5[N-(2-trifluoroacetylaminoethyl)]carbamoylmethyl-2Ј-deoxyuridine (3a), 5Ј-O-
(4,4Ј-dimethoxytrityl)-5-[N-(6-trifluoroacetylaminohexyl)]carbamoylmethyl-2Ј-de-
oxyuridine (3c), 3Ј-O-[(2-cyanoethyl)(diisopropylamino)]phosphino-5Ј-O-(4,4Ј-di-
methoxytrityl)-5-[N-(2-trifluoroacetylaminoethyl)]carbamoylmethyl-2Ј-deoxyuridine

C5-SUBSTITUTED PYRIMIDINE NUCLEOSIDE
193
(7a), 3Ј-O-[(2-cyanoethyl)(diisopropylamino)]phosphino-5Ј-O-(4,4Ј-dimethoxytri-
tyl)-5-[N-(6-trifluoroacetylaminohexyl)]carbamoylmethyl-2Ј-deoxyuridine (7c).
Snake venom phosphodiesterase (SVPD) was purchased from Worthinton. Nuclease
P1 was purchased from Yamasa Co. Alkaline phosphatase (AP) was purchased from
Boehringer Mannheim BmbH. All other reagents were purchased from Wako or Kanto
Chemical Co., Inc. All organic solvents for reactions were dried and distilled in the
usual manner.
5-(N-Propyl)carbamoylmethyl-2Ј-deoxyuridine (2d). A mixture containing 1 (0.500
g, 1.30 mmol), propylamine (1.29 mL, 15.6 mmol), and 4-dimethylaminopyridine
(0.020 g, 0.16 mmol) in methanol (5 mL) was stirred at 50ЊC for 23 h. After the
reaction solution was evaporated and coevaporated with methanol, the residue was
dissolved in a small amount of methanol and added dropwise to benzene to precipitate
5-(N-propyl)carbamoylmethyl-2Ј-deoxyuridine (2d) as an oily residue. Further, the
purified product from the supernatant was obtained by silica-gel column chromatogra-
phy using 15% methanol in dichloromethane as an eluent. Yield of 2d was 86.2%
(0.367 g). 1H NMR (CD3OD) ␦ 7.89 (s, 1H, H6), 6.27 (t, 1H, H1Ј, J ϭ 6.6 Hz), 4.38
(m, 1H, H3Ј), 3.90 (m, 1H, H4Ј), 3.75 (m, 2H, H5Ј), 3.19 (m, 2H, C5–CH2–), 3.12
(t, 2H, –NHCH2–, J ϭ 7.2 Hz), 2.26 (m, 2H, H2Ј), 1.51 (q, 2H, –CH2CH2CH3, J ϭ
7.2 Hz), 0.91 (t, 3H, –CH3, J ϭ 7.5 Hz); Mass m/z 328.3 ([M ϩ H]+, 328.2 Calcd
for C14H22N3O6).
5-(N-Pentyl)carbamoylmethyl-2Ј-deoxyuridine (2e). 2e was similarly prepared
1
from 1 and 1-aminopentane. Yield 83.5%. H NMR (D2O) ␦ 7.75 (s, 1H, H6), 6.26
(t, 1H, H1Ј, J ϭ 6.6 Hz), 4.42 (q, 1H, H3Ј, J ϭ 5.2 Hz), 3.99 (m, 1H, H4Ј), 3.73
(m, 2H, H5Ј), 3.66 (m, 2H, C5–CH2–), 3.13 (t, 2H, –NHCH2–, J ϭ 6.7 Hz), 2.35
(m, 2H, H2Ј), 1.48–1.18 (m, 6H, –(CH2)3–), 0.82(t, 3H, –CH3, J ϭ 6.7 Hz); Mass
m/z 356.2 ([M ϩ H]+, 356.2 Calcd for C16H26N3O6).
5Ј-O-(4,4Ј-Dimethoxytrityl)-5-[N-(4-trifluoroacetylaminobutyl)]carbamoylmethyl-
2Ј-deoxyuridine (3b). 2b (0.354 g, 0.780 mmol) was reacted with 4,4Ј-dimethoxytrityl
chloride (DMTr-Cl, 0.317 g, 0.94 mmol) in pyridine (4.0 mL) containing 4-dimethy-
laminopyridine (10 mg, 0.082 mmol) at room temperature for 10 h. The reaction
mixture was poured into cold water and extracted 5 times with dichloromethane (5
ϫ 50 mL). After the organic layer was dried with anhydrous sodium sulfate, the
solvent was removed by evaporation and coevaporation with toluene. The product
was purified by silica-gel column chromatography using 5–8% methanol in dichloro-
methane containing 0.2% triethylamine as an eluent. The appropriate fractions were
collected, evaporated, and precipitated with a small amount of dichloromethane into
1
hexane to give white precipitates, 3b. Yield of 3b was 45.0% (0.266 g). H NMR
(CDCl3) ␦ 9.09 (br.s, 1H, imido-NH), 7.75 (s, 1H, H6), 7.38–6.81 (m, 13H, Ar), 6.25
(t, 1H, H1Ј, J ϭ 5.1 Hz), 4.56 (m, 1H, H3Ј), 4.00 (m, 1H, H4Ј), 3.78 (s, 6H, CH3O–),
3.38–3.09 (m, 8H, H5Ј, –NHCH2– ϫ 2, and C5–CH2–), 2.36 (m, 2H, H2Ј), 1.57–1.26
(m, 8H, –(CH2)2–); 13C NMR (CDCl3) ␦ 169.93 (C ϭ O), 164.03 (C4), 158.67
(DMTr), 157.44 (J ϭ 37 Hz, COCF3), 150.27 (C2), 144.29 (DMTr), 138.86 (C6),
135.54 (DMTr), 135.35 (DMTr), 130.08 (DMTr), 128.11 (DMTr), 127.14 (DMTr),
115.99 (J ϭ 288 Hz, COCF3), 113.37 (DMTr), 109.02 (C5), 86.93 (DMTr), 86.15
(C4Ј), 84.95 (C1Ј), 72.24 (C3Ј), 63.48 (C5Ј), 55.27 (OCH3 in DMTr), 41.12 (C2Ј),

194
OZAKI ET AL.
39.47 (CH2), 38.85 (CH2), 34.53 (C5–CH2), 26.57 (CH2), 25.53 (CH2); Mass m/z
753.5 ([M Ϫ H]Ϫ, 753.3 Calcd for C35H40F3N4O9).
5Ј-O-(4,4Ј-Dimethoxytrityl)-5-(N-propyl)carbamoylmethyl-2Ј-deoxyuridine (3d).
Protection of the 5Ј-OH of 2d with 4,4Ј-dimethoxytrityl group was carried out in a
1
similar manner. Yield of 3d was 53.9%; H NMR (CDCl3) ␦ 8.14 (br.s, 1H, imido-
NH), 7.65 (s, 1H, H6), 7.43–6.85 (m, 13H, Ar), 6.31 (t, 1H, H1Ј, J ϭ 6.7 Hz), 4.54
(m, 1H, H3Ј), 3.99 (m, 1H, H4Ј), 3.79 (s, 6H, CH3O–), 3.40 (d, 2H, H5Ј, J ϭ 3.7
Hz), 3.11 (dd, 2H, C5–CH2–, J ϭ 6.8 and 13.1 Hz), 2.72 (m, 2H, –NHCH2–), 2.37
(m. 2H, H2Ј), 1.98 (d, 1H, –CONHCH2–, J ϭ 3.7 Hz), 1.45 (m, 2H, –NHCH2CH2–),
0.87 (t, 3H, CH3–, J ϭ 7.4 Hz); 13C NMR (CDCl3) ␦ 169.59 (C ϭ O), 163.95 (C4),
158.56 (DMTr), 150.29 (C2), 144.45 (DMTr), 138.59 (C6), 135.42 (DMTr), 130.06
(DMTr), 128.00 (DMTr), 126.97 (DMTr), 113.27 (DMTr), 109.17 (C5), 86.75 (DMTr),
85.99 (C4Ј), 84.92 (C1Ј), 71.99 (C3Ј), 63.59 (C5Ј), 55.19 (OCH3 in DMTr), 41.42
(CH2), 40.75 (C2Ј), 34.51 (C5–CH2), 22.53 (CH2), 11.32 (CH3); Mass m/z 630.4
([M ϩ H]+, 630.28 Calcd for C35H40N3O8).
5Ј-O-(4,4Ј-Dimethoxytrityl)-5-(N-pentyl)carbamoylmethyl-2Ј-deoxyuridine (3e).
Protection of the 5Ј-OH of 2e with 4,4Ј-dimethoxytrityl group was carried out in a
1
similar manner. Yield of 3e was 16.4%; H NMR (CDCl3) ␦ 8.05 (br.s, 1H, imido-
NH), 7.65 (s, 1H, H6), 7.43–6.75 (m, 13H, Ar), 6.32 (t, 1H, H1Ј, J ϭ 6.9 Hz), 4.53
(m, 1H, H3Ј), 3.99 (m, 1H, H4Ј), 3.79 (s, 6H, CH3O–), 3.39 (d, 2H, H5Ј, J ϭ 3.7
Hz), 3.12 (dd, 2H, C5–CH2–, J ϭ 6.9 and 13.0 Hz), 2.71 (m, 2H, –NHCH2–), 2.35
(m. 2H, H2Ј), 1.92 (d, 1H, –CONHCH2–, J ϭ 3.7 Hz), 1.42 (m, 2H, –NHCH2CH2–),
1.26 (m, 4H, –(CH2)2–), 0.87 (t, 3H, CH3–, J ϭ 6.7 Hz); 13C NMR (CDCl3) ␦ 169.06
(C ϭ O), 165.81 (C4), 158.70 (DMTr), 149.54 (C2), 144.43 (DMTr), 138.24 (C6),
135.47 (DMTr), 130.07 (DMTr), 128.06 (DMTr), 127.09 (DMTr), 113.36 (DMTr),
109.28 (C5), 86.55 (DMTr), 85.67 (C4Ј), 84.86 (C1Ј), 72.23 (C3Ј), 63.49 (C5Ј), 55.25
(OCH3 in DMTr), 40.71 (C2Ј), 39.70 (CH2), 34.75 (C5–CH2), 29.07 (CH2), 29.00
(CH2), 22.29 (CH2), 13.97 (CH3); Mass m/z 656.65 ([M Ϫ H]Ϫ, 656.30 Calcd for
C37H42N3O8).
5-Methoxycarbonylmethyl-2Ј-deoxyuridine (4). A mixture of 3Ј,5Ј-di-O-acetyl-5-
(methoxycarbonylmethyl)-2Ј-deoxyuridine (1, 512 mg, 1.30 mmol) and sodium meth-
oxide (84 mg, 1.56 mmol) was stirred at room temperature for 2 h. The reaction
mixture was evaporated, redissolved in water, and passed through a cation-exchange
resin (Diaion SKB1B, Mitsubishi Chemical Corp.). The effluent was evaporated to
dryness and the product was recrystallized from chloroform–ethanol. Yield of 4 was
1
79.9% (0.312 g); H NMR (D2O) ␦ 7.74 (s, 1H, H6), 6.19 (t, 1H, H1Ј, J ϭ 6.8 Hz),
4.36 (q, 1H, H3Ј, J ϭ 5.1 Hz), 3.94 (q, 1H, H4Ј, J ϭ 4.2 Hz), 3.69 (m, 2H, H5Ј),
3.62 (s, 3H, –OCH3), 3.35 (s, 2H, C5–CH2–), 2.30 (m, 2H, H2Ј); Mass m/z 301.3
([M ϩ H]+, 301.1 Calcd for C12H17N2O7).
5Ј-O-(4,4Ј-Dimethoxytrityl)-5-methoxycarbonylmethyl-2Ј-deoxyuridine (5). 4 (0.5
g, 1.67 mmol) was reacted with 4,4Ј-dimethoxytrityl chloride (DMTr-Cl, 0.691 g,
2.404 mmol) in pyridine (3.0 mL) containing 4-dimethylaminopyridine (10 mg, 0.082
mmol) at room temperature for 13 h. The reaction mixture was poured into cold water
and extracted five times with dichloromethane (5 ϫ 50 mL). After the organic layer
was dried with anhydrous sodium sulfate, the solvent was removed by evaporation

C5-SUBSTITUTED PYRIMIDINE NUCLEOSIDE
195
and coevaporation with toluene. The product was purified by silica-gel column chro-
matography using 8% methanol in dichloromethane containing 0.2% triethylamine
as an eluent. The appropriate fractions were collected, evaporated, and precipitated
with a small amount of dichloromethane into hexane to give white precipitates, 5.
1
Yield of 5 was 80.8% (0.813 g). H NMR (CDCl3) ␦ 7.76 (s, 1H, H6), 7.35–6.81
(m, 13H, Ar), 6.44 (t, 1H, H1Ј, J ϭ 6.7 Hz), 4.60 (m, 1H, H3Ј), 4.05 (m, 1H, H4Ј),
3.79 (s, 6H, CH3O–), 3.58 (s, 3H, CH3O–), 3.43 (m, 2H, H5Ј), 2.71–2.39 (m, 4H,
H2Ј and C5–CH2–); Mass m/z 603.2 ([M ϩ H]+, 603.2 Calcd for C33H35N2O9).
5Ј-O-(4,4Ј-Dimethoxytrityl)-5-carboxymethyl-2Ј-deoxyuridine triethylammonium
salt (6). 1 M aq. Lithium hydroxide (9.0 mL) was added to a solution of 5 (0.540
g, 0.90 mmol) in 0.5 mL of methanol. After the solution was stirred at room temperature
for 1 h, the reaction mixture was applied on a cation-exchange resin (Dowex 50W-
X8, 100–200 mesh, triethylammonium form) and washed by water. The product was
eluted by 1% aq. thriethylamine solution and the effluent was evaporated to dryness.
1
Yield of 6 was 83% (0.510 g); H NMR (CDCl3) ␦ 7.44 (s, 1H, H6), 7.41–6.79 (m,
13H, Ar), 6.30 (t, 1H, H1Ј, J ϭ 6.6 Hz), 4.42 (m, 1H, H3Ј), 3.96 (m, 1H, H4Ј), 3.75
(s, 6H, CH3O–), 3.33 (m, 2H, H5Ј), 2.99–2.75 (m. 8H, (CH2 CH3)3 and C5–CH2–),
2.30 (m, 1H, H2Ј) 1.16 (t, 9H, –(CH2CH3)3, J ϭ 7.2 Hz); Mass m/z 690.3
([M Ϫ H]Ϫ, 690.3 Calcd for C38H48N3O9).
5Ј-O-(4,4Ј-Dimethoxytrityl)-5-(N-heptyl)carbamoylmethyl-2Ј-deoxyuridine (3f). A
solution of 6 (0.20 g, 0.29 mmol), heptylamine (0.126 mL, 0.86 mmol), diphenylpho-
sphoryl azide (0.182 mL, 0.86 mmol), and triethylamine (0.118 ml) in DMF (3.0 mL)
was stirred at 0ЊC for 3 h and then stirred at room temperature for an additional 3
days. The reaction mixture was evaporated and coevaporated with methanol. The
residue was redissolved in ethyl acetate, washed with 5% aq. sodium bicarbonate and
sat. aq. sodium chloride, and the organic layer was dried over anhydrous sodium
sulfate. The product was purified by silica-gel column chromatography using 8%
methanol in dichloromethane containing 0.2% triethylamine as an eluent. The appro-
priate fractions were collected, evaporated, and precipitated with a small amount of
dichloromethane into hexane to give white precipitates, 3f. Yield of 3f was 60.3%
(0.120 g). 1H NMR (CDCl3) ␦ 8.20 (br.s., 1H, imido-NH), 7.66 (s, 1H, H6), 7.43–6.85
(m, 13H, Ar), 6.32 (t, 1H, H1Ј, J ϭ 6.6 Hz), 4.54 (m, 1H, H3Ј), 3.99 (m, 1H, H4Ј),
3.79 (s, 6H, CH3O–), 3.39 (d, 2H, H5Ј, J ϭ 3.6 Hz), 3.12 (dd, 2H, C5–CH2–, J ϭ
7.1 and 12.7 Hz), 2.72 (m, 2H, –NHCH2–), 2.35 (m, 2H, H2Ј), 2.04 (d, 1H, –
CONHCH2–, J ϭ 3.7 Hz), 1.42 (m, 2H, –NHCH2CH2–), 1.24 (m 8H, –(CH2)4–),
0.86 (t, 3H, CH3–, J ϭ 6.5 Hz); 13C NMR (CDCl3) ␦ 169.29 (C ϭ 0), 163.61 (C4),
158.65 (DMTr), 149.96 (C2), 144.49 (DMTr), 138.39 (C6), 135.53 (DMTr), 130.08
(DMTr), 128.02 (DMTr), 127.04 (DMTr), 113.33 (DMTr), 109.26 (C5), 86.86 (DMTr),
85.84 (C4Ј), 84.88 (C1Ј), 72.13 (C3Ј), 63.56 (C5Ј), 55.24 (OCH3 in DMTr), 40.73
(C2Ј), 39.77 (CH2), 34.66 (C5–CH2), 31.71 (CH2), 29.37 (CH2), 28.90 (CH2), 26.82
(CH2), 22.55 (CH2), 14.05 (CH3); Mass m/z 684.65 ([M Ϫ H]Ϫ, 684.3 Calcd for
C39H46N3O8).
3Ј-O-[(2-Cyanoethyl)(diisopropylamino)]phosphino-5Ј-O-(4,4Ј-dimethoxytrityl)-5-
[N-(3-tri-fluoroacetylaminobutyl)]carbamoylmethyl-2Ј-deoxyuridine (7b). Chloro-
(2-cyanoethoxy)diisopropylaminophosphine (0.13 mL, 0.57 mmol) was added drop-
wise to a solution of 3b (0.200 g, 0.260 mmol) in dry dichloromethane (5 mL)

196
OZAKI ET AL.
containing N-ethyl-N,N-diisopropylamine (0.11 mL, 0.65 mmol) under Ar atmosphere
at room temperature. After this mixture was stirred for 0.5 h, ethyl acetate (prewashed
with 5% aq. sodium hydrogencarbonate solution) was added to the reaction mixture,
the solution was washed with 5% aq. sodium hydrogencarbonate solution and sat.
aq. sodium chloride, dried with anhydrous sodium sulfate, and evaporated to dryness.
The crude product was purified by silica-gel column chromatography using ethyl
acetate/methanol/triethylamine (85/5/10, v/v/v) as an eluent. The appropriate fractions
were collected, evaporated, and precipitated with a small amount of dichloromethane
into hexane to give precipitates. Yield of 7b was 73.1% (0.185 g); 31P NMR (CDCl3)
␦ 149.44 and 149.55 ppm.
3Ј-O-[(2-Cyanoethyl)(diisopropylamino)]phosphino-5Ј-O-(4,4Ј-dimethoxytrityl)-5-
(N-propyl)carbamoylmethyl-2Ј-deoxyuridine (7d). The phosphitylation of 3d was car-
ried out in the same method as described above. Yield 30%; 31P NMR (CDCl3) ␦
149.31 and 149.63 ppm.
3Ј-O-[(2-Cyanoethyl)(diisopropylamino)]phosphino-5Ј-O-(4,4Ј-dimethoxytrityl)-5-
(N-pentyl)carbamoylmethyl-2Ј-deoxyuridine (7e). The phosphitylation of 3e was car-
ried out in the same method as described above. Yield 36%; 31P NMR (CDCl3) ␦
149.32 and 149.66 ppm.
3Ј-O-[(2-Cyanoethyl)(diisopropylamino)]phosphino-5Ј-O-(4,4Ј-dimethoxytrityl)-5-
(N-heptyl)carbamoylmethyl-2Ј-deoxyuridine (7f). The phosphitylation of 3f was car-
ried out in the same method as described above. Yield 64.9% 31P NMR (CDCl3) ␦
149.31 and 149.66 ppm.
Oligodeoxyribonucleotide synthesis. Oligodeoxyribonucleotides and their analogs
were prepared using normal phosphoramidite coupling procedure on a DNA synthe-
sizer. The oligodeoxyribonucleotides bearing the C5-substituted 2Ј-deoxyuridine in
place of thymidine were synthesized along with the normal and complementary
oligodeoxyribonucleotides. The modified nucleoside phosphoramidite was incorpo-
rated into the oligodeoxyribonucleotides at the appropriate position by using the
normal synthetic cycle except that the reaction time for the coupling step was 6 min.
For the synthesis of an oligodeoxyribonucleotide containing the modified base (ODN-
2N, ODN-4N, ODN-6N, ODN-2C, ODN-4C, or ODN-6C), normal deoxyribonucleo-
side phosphoramidites and modified deoxyribonucleoside phosphoramidite (7a, 7c,
7e, 7f, 7g, or 7h) were used. The deprotection and cleavage from CPG support was
carried out by treatment with conc. aq. ammonia solution at 50ЊC for 18 h. All
modified oligodeoxyribonucleotides with 5Ј-(4,4Ј-dimethoxytrityl) group were iso-
lated by reversed-phase HPLC on a Wakosil 5C18 column (10 mm i.d. ϫ 250 mm
length) using 50 mM TEAA (pH 7.0) with a gradient of 15.0–40.0% acetonitrile in
25 min. The isolated compound was treated with 10% acetic acid by the usual
procedure to remove a 4,4Ј-dimethoxytrityl group followed by desalting on a Sephadex
G-25 column. The modified oligodeoxyribonucleotides were further purified by re-
versed-phase HPLC after deprotection of 5Ј-(4,4Ј-dimethoxytrityl) group. Isolated
yields: ODN-2N, 3.6% ODN-4N, 2.6%; ODN-6N, 4.8% ODN-2C, 2.4%; ODN-4C,
11.5%; ODN-6C, 4.8%.
Nuclease digestion of oligodeoxyribonucleotides containing the modified base. The
modified oligomers (ca. 0.5 OD260 nm) were treated overnight with snake venom
phosphodiesterase (0.5 units) and alkaline phosphatase (1 units) in 20 mM Tris–HCl

C5-SUBSTITUTED PYRIMIDINE NUCLEOSIDE
197
(pH 8.0) containing 10 mM MgCl2 at 37ЊC. To the reaction mixtures, nuclease P1
(ca. 4 units) and 0.1 M sodium acetate (pH 4.75) were added, then the reaction
mixtures were incubated at 37ЊC. The reaction mixtures were analyzed by reversed-
phase HPLC. The nucleoside composition ratios were calculated from areas of the
peaks in the HPLC chart.
Tm measurements. UV absorbance was measured with a Hitachi UV-3000 spectro-
photometer equipped with a Hitachi SPR-10 temperature controller. The solution
temperature in a cuvette was measured directly with a temperature data collector AM-
7002 (Anritsu Meter Co., Ltd.). Absorbance and temperature data were recorded on
an NEC PC-9821 personal computer. The rate of heating or cooling was 0.5ЊC/min.
Tm values were obtained in 10 mM sodium phosphate buffer (pH 7.0) containing 150
mM sodium chloride at a duplex concentration of 2 ϫ 10Ϫ6 M. Tm values were
determined by the described method (10) from the absorbance vs temperature plot
using an Igor graphing and data analysis program (WaveMatrics, Inc.) and a Power-
Macintosh 8500 computer (Apple Computer, Inc.).
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