Synthesis and properties of nucleic acid analogues consisting of a benzene-phosphate backbone

Article abstract of DOI:10.1021/jo050635m

The synthesis and properties of a nucleic acid analogue consisting of a benzene-phosphate backbone are described. The building blocks of the nucleic acid analogue are composed of bis(hydroxymethyl)-benzene residues connected to nucleobases via the biaryl-like axis. Stabilities of the duplexes were studied by thermal denaturation. It was found that the thermal stabilities of the duplexes composed of the benzene-phosphate backbone are highly dependent on their sequences. The duplexes with the benzene-phosphate backbone comprised of the mixed sequences were thermally less stable than the natural DNA duplexes, whereas that composed of the homopyrimidine and homopurine sequences was thermally and thermodynamically more stable than the corresponding natural DNA duplex. It was suggested that the analogues more efficiently stabilize the duplexes in a B-form duplex rather than in an A-form duplex. Thus, the duplexes consisting of the benzene-phosphate backbone, especially composed of the homopyrimidine and homopurine sequences, may offer a novel structural motif useful for developing novel materials applicable in the fields of bio- and nanotechnologies.

Full text of DOI:10.1021/jo050635m

Synthesis and Properties of Nucleic Acid Analogues Consisting of
a Benzene-Phosphate Backbone
Yoshihito Ueno, Takumi Kato, Kumiko Sato, Yasutomo Ito, Mahito Yoshida, Takumi Inoue,
Aya Shibata, Masahiro Ebihara, and Yukio Kitade*
Department of Biomolecular Science, Faculty of Engineering, Gifu University,
Yanagido, Gifu 501-1193, Japan
kitade@biomol.gifu-u.ac.jp
Received April 1, 2005
The synthesis and properties of a nucleic acid analogue consisting of a benzene-phosphate backbone
are described. The building blocks of the nucleic acid analogue are composed of bis(hydroxymethyl)-
benzene residues connected to nucleobases via the biaryl-like axis. Stabilities of the duplexes were
studied by thermal denaturation. It was found that the thermal stabilities of the duplexes composed
of the benzene-phosphate backbone are highly dependent on their sequences. The duplexes with
the benzene-phosphate backbone comprised of the mixed sequences were thermally less stable
than the natural DNA duplexes, whereas that composed of the homopyrimidine and homopurine
sequences was thermally and thermodynamically more stable than the corresponding natural DNA
duplex. It was suggested that the analogues more efficiently stabilize the duplexes in a B-form
duplex rather than in an A-form duplex. Thus, the duplexes consisting of the benzene-phosphate
backbone, especially composed of the homopyrimidine and homopurine sequences, may offer a novel
structural motif useful for developing novel materials applicable in the fields of bio- and
nanotechnologies.
Introduction
On the other hand, it is known that biaryl compounds
have various interplanar angles depending on the sub-
stituents on their aromatic rings.5 Recently, Sauter and
Leumann reported the synthesis of an ONA consisting
of a building block composed of a uracil base attached to
a thiophene residue via the biaryl-like axis.6,7 The ONA
is expected to possess a novel structure and properties
One major goal of biotechnology and nanotechnology
is the assembly of novel biomaterials that can be used
for analytical, industrial, and therapeutic purposes. So
far, a wide variety of oligonucleotide analogues (ONAs)
have been synthesized, and their properties have been
extensively investigated in relation to such purposes. For
instance, ONAs consisting of six-membered sugar units
instead of a natural nucleoside have been synthesized,1
and their properties have been intensively studied by
Eschenmoser’s and Herdewijn’s groups.2 Peptide nucleic
acids (PNAs) composed of a (2-aminoethyl)glycine back-
bone developed by Nielsen’s group were shown to form
thermally stable duplexes with a complementary DNA
or RNA,3,4 and have been applied to antisense studies.
(2) For examples: (a) Mu¨ller, D.; Pitsch, S.; Kittaka, A.; Wagner,
E.; Winter, C. E.; Eschenmoser, A. Helv. Chim. Acta 1990, 73, 1410-
1468. (b) Krishnamurthy, R.; Pitsch, S.; Minton, M.; Miculka, C.;
Windhab, N.; Eschenmoser, A. Angew. Chem., Int. Ed. Engl. 1996, 35,
1537-1541. (c) Scho¨ning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.;
Krishnamurthy, R.; Eschenmoser, A. Science 2000, 290, 1347-1351.
(d) Herdewijn, P. Angew. Chem. Int. Ed. 2001, 40, 2249-2251. (e)
Lescrinier, E.; Froeyen, M.; Herdewijn, P. Nucleic Acids Res. 2003,
31, 2975-2989.
(3) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497-1500.
* To whom correspondence should be addressed. Phone & Fax: +81-
58-293-2640.
(1) For examples: (a) Zhang, Y.; Seeman, N. C. J. Am. Chem. Soc.
1994, 116, 1661-1669. (b) Trotta, P. P.; Beutel, B. A.; Sherman, M. I.
Med. Res. Rev. 1995, 15, 277-298. (c) Praseuth, D.; Guieysse, A. L.;
He´le`ne, C. Biochim. Biophys. Acta 1999, 1498, 181-206. (d) Leumann,
C. J. Bioorg. Med. Chem. 2002, 10, 841-854.
(4) Egholm, M.; Buchardt, O.; Christensen, L.; Bejrens, C.; Freier,
S.; Driver, D.; Berg, R. H.; Kim, S. K.; Norde´n, B.; Nielsen, P. E. Nature
1993, 365, 566-568.
(5) Adams, R.; Yuan, H. C. Chem. Rev. 1993, 12, 261-338.
(6) Sauter, G.; Stulz, E.; Leumann, C. Helv. Chim. Acta 1998, 81,
14-34.
(7) Sauter, G.; Leumann, C. Helv. Chim. Acta 1998, 81, 916-931.
10.1021/jo050635m CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/27/2005
J. Org. Chem. 2005, 70, 7925-7935
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Ueno et al.
Treatment of 11 with 1 M Cl3CCO2H/CH2Cl2 afforded 2
in 92% yield. To synthesize a phosphoramidite, the
hydroxy group of the mono-DMTr derivative 8 was
protected with a TBDMS group to give 9 in 95% yield.
Compound 9 was converted to a cytosine derivative 12
by the same method described above. After protection of
the exo-amino function of 12 with a benzoyl (Bz) group,
the silyl group was removed by treating with TBAF to
afford a mono-DMTr derivative 14 in 97% yield.
The adenosine analogue AB was synthesized according
to Scheme 2. Treatment of 5 with 5-amino-4,6-dichloro-
pyrimidine in a solution of H2O/EtOH/concentrated HCl
produced 16 in 86% yield. Compound 16 was treated with
a mixture of ethyl orthoformate and acetic anhydride,
followed by methanolic ammonia in a steel sealed tube
at 90°C, to give the adenosine analogue 3 in 36% yield.
To synthesize a phosphoramidite, the hydroxyl functions
of 16 were protected with a TBDMS group to produce 17
in 70% yield. Compound 17 was treated with a mixture
of ethyl orthoformate and acetic anhydride to give 18 in
77% yield. After 18 was treated with methanolic am-
monia in a steel sealed tube at 90°C, the exo-amino group
of 19 was protected with a Bz group to afford 20 in 49%
yield. Deprotecting of the silyl groups with TBAF in a
solution of THF/CH2Cl2 gave a mono-hydroxyl derivative
21 in 53% yield with a recovery of 20 in 44% yield. After
the hydroxyl group of 21 was protected with a DMTr
group, the TBDMS group was deprotected to afford a
mono-DMTr derivative 22 in 84% yield.
FIGURE 1. Structures of nucleoside analogues.
different from those of a natural ON, since it has a
π-electron-rich backbone instead of a natural ribose-
phosphate backbone. They attempted to synthesize a
homo-hexamer consisting of the uridine analogue but
have not yet succeeded. It was revealed that an un-
welcome side reaction occurred and produced a mixture
of N-terminal-modified derivatives with various chain
lengths.7 N-Phenyl-substituted nucleobases, such as 9-phe-
nyladenine, 9-phenylguanine, 1-phenylcytosine, and 1-phe-
nyluracil, are also thought to possess interplanar angles
between the phenyl groups and the nucleobases. Thus,
we planned to synthesize ONAs consisting of a benzene-
phosphate backbone, of which the building blocks are
built from nucleobases attached to the benzene residues
via the biaryl-like axis.
The guanosine analogue GB was synthesized according
to Scheme 3. Treatment of 5 with 2-amino-4-chloro-6-
hydroxy-5-phenylazopyrimidine (23)10 in EtOH produced
24 in 70% yield. After cleavage of the diazo linkage of 24
with Zn in an acidic medium, the resulting triamino-
pyrimidine derivative was treated with N,N-dimethyl-
formamide dimethyl acetal and (EtO)3CH to give the
guanosine analogue 4 in 25% yield. To synthesize a
phosphoramidite, the hydroxyl functions of 24 were
protected with a TBDMS group to produce 25 in 99%
yield. Treatment of 25 with Zn in the acidic medium,
followed by N,N-dimethylformamide dimethyl acetal and
(EtO)3CH, afforded a guanine derivative 26 protected
with a dimethylaminomethylene (dmf) group in 76%
yield. Deprotecting of the silyl group of 26 with TBAF
gave a mono-hydroxyl derivative 27 in 59% yield. After
the hydroxyl function of 27 was protected with a DMTr
group, the TBDMS group was deprotected to afford a
mono-DMTr derivative 28 in 86% yield.
The mono-DMTr derivatives 8, 12, 22, and 28 were
phosphitylated by the standard procedure11 to give the
corresponding phosphoramidites 29, 30, 31, and 32 in 81,
96, 88, and 60% yields, respectively (Scheme 4). To
incorporate the analogues to ends of oligomers, the mono-
DMTr derivatives 8, 12, 22, and 28 were modified to the
corresponding succinates, which were reacted with con-
trolled pore glasses (CPGs) to produce solid supports 33-
36 containing 8 (67 µmol/g), 12 (82 µmol/g), 22 (54 µmol/
g), and 28 (70 µmol/g), respectively.
In this paper, we report the synthesis and properties
of novel ONAs consisting of the benzene-phosphate
backbone, of which the building blocks AB, GB, CB, and
UB are built from benzene-core units connected via the
biaryl-like axis to the nucleobases (Figure 1).
Results and Discussion
Synthesis of Monomers. To synthesize the ONAs by
the phosphoramidite method, appropriately protected
phosphoramidites of the analogues AB, GB, CB, and UB
were synthesized. First, the uridine and cytidine ana-
logues UB and CB were synthesized according to Scheme
1. 3,5-Bis(hydroxymethyl)aniline (5)8 was treated with
TBDMSCl to give the corresponding bis-TBDMS deriva-
tive 6 in 74% yield. Compound 6 was reacted with
3-methoxy-2-propenoylisocyanate9 in DMF and then
treated with a solution of 2 M NaOH/EtOH to afford 1
in 45% yield. To synthesize a phosphoramidite, one of
the two hydroxyl groups of 1 was protected with a DMTr
group to produce a mono-DMTr derivative 8 in 59% yield.
The cytidine analogue 2 was derived from the uridine
analogue 1. After protection of two hydroxyl functions of
1 with a DMTr group, the di-DMTr derivative 10 was
treated with 2,4,6-triisopropylbenzenesulfonyl chloride
(TPSCl) in the presence of DMAP and Et3N, followed by
NH4OH, to produce a cytosine derivative 11 in 97% yield.
Structures of Monomers. To gain insight into the
conformational preference of the monomer UB, X-ray
(8) Behrens, C.; Egholm, M.; Buchardt, O. Synthesis 1992, 1235-
1236.
(10) Boon, W. R.; Leigh, T. J. Chem. Soc. 1951, 1497-1501.
(11) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22,
1859-1862.
(9) Santana, L.; Teijeira, M.; Uriarte, E. J. Heterocycl. Chem. 1999,
36, 293-295.
7926 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis and Properties of Nucleic Acid Analogues
SCHEME 1a
a Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt, 74%; (b) OdCdN-C(dO)-CHdCH-O-CH3, benzene/DMF, -20 °C to
room temperature, 86%; (c) 2 M NaOH/EtOH, 60 °C, 45%; (d) DMTrCl, pyridine, rt, 59%; (e) DMTrCl, DMAP, pyridine, rt, 66%; (f)
2,4,6-triisopropylbenzenesulfonyl chloride, Et3N, DMAP, CH3CN, rt, then concd NH4OH, 0 °C to room temperature, 97%; (g) 1 M CCl3CO2H,
CH2Cl2/THF, rt, 92%; (h) TBDMSCl, imidazole, DMF, rt, 95%; (i) 2,4,6-triisopropylbenzenesulfonyl chloride, Et3N, DMAP, CH3CN, rt,
then concd NH4OH, 0 °C to room temperature, 72%; (j) BzCl, pyridine, rt, 92%; (k) TBAF, THF, rt, 97%.
SCHEME 2a
a Reagents and conditions: (a) concd HCl, EtOH, reflux, 86%; (b) TBDMSCl, imidazole, DMF, rt, 70%; (c) (1) (EtO)3CH, Ac2O, reflux,
(2) NH3/MeOH, 90 °C, 36%; (d) (EtO)3CH, Ac2O, reflux, 77%; (e) NH3/MeOH, 90 °C, 63%; (f) BzCl, pyridine, rt, 49%; (g) TBAF, THF/
CH2Cl2, rt, 53%; (h) (1) DMTrCl, DMAP, pyridine, rt, (2) TBAF, THF, rt, 84%.
SCHEME 3a
a Reagents and conditions: (a) EtOH, reflux, 70%; (b) TBDMSCl, imidazole, DMF, rt, 99%; (c) (1) Zn, AcOH/H2O/EtOH/THF, reflux, (2)
(EtO)3CH, 1 M HCl, DMF, rt, 25%; (d) (1) Zn, AcOH/H2O/EtOH/THF, reflux, (2) N,N-dimethylformamide dimethyl acetal, DMF, rt, (3)
(EtO)3CH, Ac2O, rt, 76%; (e) TBAF, THF, rt, 59%; (f) (1) DMTrCl, DMAP, pyridine, rt, (2) TBAF, THF, rt, 86%.
analysis was performed. As shown in Figure 2, the uracil
ring forms the plane that is propeller-twisted relative to
the benzene plane. It was found that the interplanar
angle between the uracil and the benzene moiety is 53.5-
(4)°. Structures of the low-energy conformers of the
analogues UB, CB, AB, and GB were also calculated by
MOE using ESFF force field.12 The interplanar angle
between the uracil and the benzene moiety was 56.5°,
which was similar to that obtained from X-ray analysis.
The interplanar angle between the cytosine and benzene
moiety was 54.8°. On the other hand, the interplanar
angles between the adenine and the guanine and the
benzene moieties were 32.8° and 34.1°, respectively.
These values are slightly smaller than those of the
pyrimidine analogues.
Structures of base pairs between T and dA, and UB and
AB based on the X-ray analysis and molecular modeling
are shown in Figure 3. Although the conformation of the
analogues is thought not to be rigid in oligomers, the
interplanar angles between the bases and benzene moi-
eties are supposed to influence distances between neigh-
boring base pairs in duplexes.
(12) Halgren, T. A. J. Comput. Chem. 1999, 20, 720-729.
J. Org. Chem, Vol. 70, No. 20, 2005 7927

Ueno et al.
SCHEME 4a
a Reagents and conditions: (a) Chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, N,N-diisopropylethylamine, THF, rt, 81% (29),
96% (30), 88% (31), 60% (32); (b) (1) succinic anhydride, DMAP, pyridine, rt, (2) CPG, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, DMF, rt, 67 µmol/g (33), 82 µmol/g (34), 54 µmol/g (35), 70 µmol/g (36).
TABLE 1. pKa Values of Uridine and its Analoguea
compound
pKa
rU
UB
9.3 (9.2)b
9.3
a A 0.2 M NaCl solution was used for all pH ranges instead of
buffer. b Data taken from Dawson et al.14
sequences. The ONAs 9-11 contain one base mismatch
against the complementary ONA 7. The ONAs 14 and
15 are composed of homopurine and homopyrimidine
sequences, respectively. The ONAs 18-21 contain one
or three analogues AB or UB in the middle of oligodeoxy-
nucleotides (ONs). The ONAs 23 and 24 contain one or
three analogues AB in the middle of oligonucleotides
(2′-O-Me-ONs) composed of 2′-O-methylribonucleosides.
The fully protected ONAs were treated with concentrated
NH4OH at 55°C, and the released ONAs were purified
by denaturing 20% polyacrylamide gel electrophoresis to
afford deprotected ONAs. These ONAs were analyzed by
MALDI-TOF/MS and the observed molecular weights
supported their structures.
FIGURE 2. ORTEP drawing of UB.
UV Melting Studies of Duplexes. The thermal
stabilities of the duplexes composed of the benzene-
phosphate backbone were compared with those of the
natural ONs. Thermal denaturation was performed in a
buffer of 10 mM sodium phosphate (pH 7.0) containing
1.0 M NaCl.
The thermal stabilities of the duplexes were highly
dependent on their sequences. The UV melting profiles
of the duplexes are shown in Figure 4. The melting
temperatures (Tms) of the duplexes 4 and 5 comprised of
the benzene-phosphate backbone were 38.4 and 48.8 °C
(Figure 4a and b), whereas those of the duplexes 1 and 2
consisting of the natural nucleosides were 49.1 and 51.7
°C, respectively. Thus, it was found that the duplexes
with the benzene-phosphate backbone composed of the
mixed sequences are thermally less stable than the
natural DNA duplexes. However, as shown in Figure 4d,
the Tm value of the duplex 4 was apparently higher than
those of the duplexes between the ONA 7 and ONA 9,
10, or 11 containing one mismatch base pair. The result
implies that the ONAs composed of the benzene-
phosphate backbone retain enough base selectivity.
FIGURE 3. Structure of base pairs between T and dA (a) and
UB and AB (b).
pKa Measurement of UB. Next, we determined the
pKa of the base moiety of UB since the pKa is thought to
be an important parameter for understanding the base-
pairing properties of the analogues.13 The pKa values are
listed in Table 1. The pKa of UB was 9.3, which was the
same as that of rU.
Design of Oligomers. ONAs were synthesized using
the phosphoramidite method with a DNA/RNA synthe-
sizer. Sequences of ONAs used in this study are listed
in Table 2. The ONAs 7-13 are comprised of mixed
On the other hand, the Tm value of the duplex 6
between (UB)12 and (AB)12 was 48.3 °C, whereas that of a
natural T12:dA12 duplex was 41.8 °C (Figure 4c). Thus, it
was revealed that the duplex between (UB)12 and (AB)12
composed of the homopyrimidine and homopurine se-
(13) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I., Jr. Nucleic Acidss
Structure, Properties, and Functions; University Science Books: Sau-
salito, CA, 2000.
7928 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis and Properties of Nucleic Acid Analogues
TABLE 2. Sequences of Oligomersa
a b indicates the oligomers composed of the benzene-phosphate backbone; underlined letters show the mismatch bases against ONA7.
quences was thermally more stable than the correspond-
ing natural DNA duplex. Thermodynamic parameters of
the duplexes 3 and 6 on duplex formations were deter-
mined by calculations based on the slope of a 1/Tm versus
ln(CT/4) plot, where CT is the total concentration of the
single strands.15 The parameters are summarized in
Table 3. The ∆G°37 values of the duplexes 3 and 6 were
-9.1 and -10.8 kcal/mol, respectively. Both the ∆H° and
∆S° values of the duplex 6 were smaller than those of
the duplex 3. This implies that the duplex formation
between the ONs 14 and 15 is less favorable in entropy
but more favorable in enthalpy than that between the
ONs 5 and 6 consisting of the natural nucleosides. The
disadvantage in entropy in the duplex formation between
the ONAs 14 and 15 is compensated by the enthalpy
term. The ∆∆H° value between the duplexes 6 and 3 was
15.6 kcal/mol. These results suggest that the duplex 6
between the ONAs 14 and 15 with the benzene-
phosphate backbone is thermally and thermodynamically
stabilized by the stacking interaction of the benzene
moiety of each unit with the neighboring benzene or base
moieties.
Global Conformations of Duplexes. To investigate
the influence of the AB:UB base pair on the duplex
formation in detail, we next prepared the oligodeoxy-
nucleotides, ONAs 18-21, and the oligonucleotides com-
prised of 2′-O-methylribonucleotides, 2′-O-Me-ONAs 23
and 24, which contain the analogue AB or UB in the
middle of their strands (Table 2). To study the global
conformations of the duplexes 8, 9, 11, and 12 between
ONA and ONA, and 2′-O-Me-ONA and ONA, CD spectra
of the duplexes were measured. Generally, a B-form
duplex shows a positive CD band around 280 nm and a
negative band around 240 nm, whereas an A-form duplex
reveals a positive peak around 270 nm and a negative
peak at 210 nm.
The spectrum of the duplex 7 between the ONs 16 and
17 showed the positive CD band at 278 nm and the
negative band at 248 nm, which were attributable to the
B-form duplex (Figure 5). Although the intensity of the
negative CD band of the duplex 8 containing one AB:UB
(14) Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M. Data
for Biochemical Research, 3rd ed.; Clarendon Press: Oxford, UK, 1986.
(15) Marky, L. A.; Breslauer, K. L. Biopolymer 1987, 26, 1601-1620.
J. Org. Chem, Vol. 70, No. 20, 2005 7929

Ueno et al.
FIGURE 4. UV melting profiles of duplexes and their Tms. Thermal denaturation was performed in a buffer comprised of 10 mM
sodium phosphate (pH 7.0) and 1.0 M NaCl.
TABLE 3. Thermodynamic Parametersa
scribed above. The parameters are summarized in Table
3. In the case of the ONA/ONA duplex, the Tm and
-∆G°37 values of the duplexes 8 and 9 were smaller than
those of the duplex 7. Thus, it was found that the ONA/
ONA duplexes are thermally and thermodynamically
destabilized by introducing the AB:UB base pair. How-
ever, the -∆G°37 value of the duplex 9 was greater than
that of the duplex 8. This implies that the duplex 9
containing three AB:UB base pairs is thermodynamically
more stable than the duplex 8 containing one AB:UB base
pair. The -∆H° values became greater as the numbers
of the AB:UB base pairs increased, although the -∆S°
values also became larger as the numbers of the AB:UB
base pairs increased. The ∆∆H° value between the
duplexes 9 and 7 was 48.7 kcal/mol. The result indicates
that the benzene moieties of the analogues interact with
the neighboring benzene or base moieties by the stacking
interaction in the ONA/ONA duplex.
In the case of the 2′-O-Me-ONA/ONA duplex, the Tm
and -∆G°37 values became smaller as the numbers of the
AB:UB base pairs increased. Thus, it was found that the
2′-O-Me-ONA/ONA duplexes are also thermally and
thermodynamically destabilized by introducing the AB:
UB base pair. It was revealed that the ∆∆H° value (8.3
kcal/mol) between the duplexes 10 and 12 is not so large
as compared to that between the duplexes 7 and 9,
although the ∆∆S° value (33.3 cal/K‚mol) between the
duplexes 10 and 12 is smaller than that (163.0 cal/K‚
mol) between the duplexes 7 and 9. The result suggests
that the stacking interaction of the benzene moiety of
the analogues in the 2′-O-Me-ONA/ONA duplex is weaker
than that in the ONA/ONA duplex.
b
Tm
∆Tm
(°C)
-∆H°
(kcal/mol)
-∆S°
-∆G°37
(°C)
(cal/K‚mol) (kcal/mol)
duplex 3
duplex 6
duplex 7
duplex 8
duplex 9
duplex 10 59.3
duplex 11 52.8
duplex 12 49.2 -10.1
41.5
48.3
63.6
56.4 ( 1.1 152.5 ( 2.9
72.0 ( 2.0 197.3 ( 5.4
114.2 ( 5.1 312.6 ( 13.9
9.1
6.8
10.8
17.3
14.6
15.5
13.5
12.5
11.5
53.2 -10.4 126.6 ( 5.3 361.2 ( 15.0
51.3 -12.3 162.9 ( 9.0 475.6 ( 26.2
78.8 ( 1.3 210.5 ( 3.6
86.4 ( 1.6 238.5 ( 4.3
87.1 ( 1.3 243.8 ( 3.5
-6.5
a Thermal denaturation was performed in a buffer (10 mM
sodium phosphate, pH 7.0) containing 1.0 M NaCl for duplexes 3
and 6, or containing 0.1 M NaCl for duplexes 7-12. The standard
deviations for ∆H° and ∆S° were estimated from the linearity of
-1
the Tm
versus ln(CT/4) plots. b Tm values at 3 µM duplex
concentrations.
base pair was slightly less than that of the duplex 7, the
duplex was found to retain the B-like conformation. When
three AB:UB base pairs were introduced into the duplex,
intensities of both the positive and negative CD bands
decreased as compared to those of the duplex 7. In
addition, the negative CD band shifted to around 254 nm,
and a new CD band appeared around 240 nm. This
indicates that the global conformation of the DNA B-form
duplex is disordered by introducing three AB:UB base
pairs.
On the other hand, the spectrum of the duplex 10
revealed the positive CD band at 267 nm and the
negative band at 209 nm, which were attributable to the
A-form duplex. Although the intensities of the positive
CD bands of the duplexes 11 and 12 containing the
AB:UB base pairs were slightly less than that of the
duplex 10, those duplexes were found to retain the A-like
conformation.
In conclusion, we have demonstrated the synthesis of
nucleic acid analogues consisting of a benzene-phosphate
backbone. It was found that the thermal stabilities of the
duplexes composed of the benzene-phosphate backbone
are highly dependent on their sequences. The duplexes
Thermodynamic Parameters of Duplexes. Ther-
modynamic parameters of the duplexes 7-12 on duplex
formations were determined by the same method de-
7930 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis and Properties of Nucleic Acid Analogues
FIGURE 5. CD spectra of duplexes. (a) ONA/ONA duplex; (b) 2′-O-Me-ONA/ONA duplex.
(38 mL) at -20 °C with 4 Å molecular sieves. After the
with the benzene-phosphate backbone comprised of the
mixed sequences were thermally less stable than the
natural DNA duplexes, whereas the duplex between
(UB)12 and (AB)12 composed of the homopyrimidine and
homopurine sequences was thermally and thermo-
dynamically more stable than the corresponding natural
DNA duplex. Additionally, it was suggested that the
benzene moieties of the analogues more efficiently inter-
act with the neighboring benzene or base moieties in the
ONA/ONA duplex than in the 2′-O-Me-ONA/ONA duplex.
Thus, the duplexes consisting of the benzene-phosphate
backbone, especially those composed of the homopyrimi-
dine and homopurine sequences, may offer a novel
structural motif useful for developing novel materials and
probes, such as DNA-based nanowires16 and molecular
beacons,17 applicable to the fields of bio- and nanotech-
nologies.
addition, the mixture was allowed to warm to room temper-
ature while stirring overnight. The molecular sieves were
filtered off, the solvent was evaporated in vacuo, and the
resulting residue was purified by column chromatography
(SiO2, 10-25% EtOAc in hexane) to give 7 (4.22 g, 86% as a
white solid): mp 150 °C; 1H NMR (CDCl3) δ 10.78 (s, 1H), 9.07
(s, 1H), 7.76 (d, 1H, J ) 12.4), 7.36-7.11(s, 3H), 5.36 (d, 1H,
J ) 12.4), 4.71 (s, 4H), 3.75 (s, 3H), 0.94 (s, 18H), 0.10 (s, 12H);
13C NMR (DMSO-d6) δ 168.2, 163.6, 151.2, 142.1, 137.5, 118.5,
115.4, 97.7, 64.1, 58.2, 25.8, 18.0, -5.3; LRMS (FAB) m/z 509
(MH+); HRMS (FAB) calcd for C25H45N2O5Si2 509.2789, found
509.2880. Anal. Calcd for C25H44N2O5Si2: C, 59.02; H, 8.72;
N, 5.51. Found: C, 58.81; H, 8.87; N, 5.51.
1-[3,5-Bis(hydroxymethyl)phenyl]uracil (1). A mixture
of 7 (0.322 g, 0.63 mmol), 2 M NaOH (2.6 mL), and EtOH (2.6
mL) was stirred at 60 °C for 1 h. After the solution was cooled,
it was neutralized with AcOH. The solvent was evaporated in
vacuo, and the resulting residue was purified by column
chromatography (SiO2, 6-9% MeOH in CHCl3) to give 1 (71
mg, 45% as a white solid): mp 201-203 °C; UV λmax (H2O)
220 nm, 273 nm; 1H NMR (DMSO-d6) δ 11.42 (s, 1H), 7.65 (d,
1H, J ) 8.0), 7.32-7.17 (s, 3H), 5.65 (d, 1H, J ) 8.0), 5.31 (t,
2H, J ) 5.6), 4.52 (d, 4H, J ) 5.6); 13C NMR (DMSO-d6) δ
163.7, 150.4, 145.5, 143.7, 138.7, 124.0, 122.8, 101.6, 62.7;
LRMS (EI) m/z 248 (M+); HRMS (EI) calcd for C12H12N2O4
248.0797, found 248.0790. Anal. Calcd for C12H12N2O4: C,
58.06; H, 4.87; N, 11.29. Found: C, 58.00; H, 4.87; N, 11.24.
1-[3-(4,4′-Dimethoxytrityloxymethyl)-5-(hydroxymeth-
yl)phenyl]uracil (8). A mixture of 1 (0.23 g, 0.93 mmol) and
DMTrCl (0.38 g, 1.12 mmol) in pyridine (5 mL) was stirred at
room temperature for 4 h. The mixture was partitioned
between CHCl3 and H2O. The organic layer was washed with
aqueous NaHCO3 (saturated) and brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatog-
raphy (SiO2, 0-9% MeOH in CHCl3) to give 8 (0.302 g, 59%):
mp 85-87 °C; 1H NMR (DMSO-d6) δ 11.41 (s, 1H), 7.67 (d,
1H, J ) 8.0), 7.44-6.89 (m, 16H), 5.64 (d, 1H, J ) 8.0), 5.32
(t, 1H, J ) 5.4), 4.53 (d, 2H, J ) 5.4), 4.09 (s, 2H), 3.72 (s,
6H); 13C NMR (DMSO-d6) δ 163.7, 158.1, 150.4, 145.5, 144.8,
144.0, 139.6, 138.8, 135.5, 129.7, 128.0, 127.6, 126.8, 124.3,
123.4, 123.3, 113.4, 101.6, 86.0, 79.2, 64.6, 62.3, 55.0; LRMS
(FAB) m/z 551 (MH+); HRMS(FAB) calcd for C33H31N2O6
551.2104, found 551.2176. Anal. Calcd for C33H30N2O6‚
1/2H2O: C, 70.83; H, 5.58; N, 5.01. Found: C, 70.62; H, 5.78;
N, 4.74.
Materials and Methods
General Remarks. NMR spectra were recorded at 400
MHz (1H), at 100 MHz (13C), and at 162 MHz (31P) and are
reported in ppm downfield from TMS or 85% H3PO4. J values
are given in hertz. Mass spectra were obtained by electron
ionization (EI) or fast atom bombardment (FAB) method.
3,5-Bis(tert-butyldimethylsilanyloxymethyl)aniline (6).
A mixture of 3,5-bis(hydroxymethyl)aniline (5)8 (1.35 g, 8.82
mmol), TBDMSCl (2.92 g, 19.4 mmol), and imidazole (2.64 g,
38.8 mmol) in DMF (18 mL) was stirred at room temperature.
After 22 h, EtOH (2 mL) was added to the mixture, and the
whole was stirred for 10 min. The mixture was partitioned
between EtOAc and H2O. The organic layer was washed with
brine, dried (Na2SO4), and concentrated. The residue was
purified by column chromatography (SiO2, 10-50% EtOAc in
hexane) to give 6 (2.50 g, 74% as a yellow oil): 1H NMR (CDCl3)
δ 6.66 (s, 1H), 6.55 (s, 2H), 4.65 (s, 4H), 3.64 (s, 2H), 0.95 (s,
18H), 0.09 (s, 12H); 13C NMR (DMSO-d6) δ 148.3, 141.5, 111.7,
110.4, 64.7, 25.8, 18.0, -5.3; LRMS (EI) m/z 381 (M+); HRMS
(EI) calcd for C20H39NO2Si2 381.2519, found 381.2516.
N-[3,5-Bis(tert-butyldimethylsilanyloxymethyl)phen-
ylcarbamoyl]-3-methoxy-2-propenamide (7). A solution of
3-methoxy-2-propenoylisocyanate9 in benzene (78 mL, 31.3
mmol) was added to a solution of 6 (3.70 g, 9.71 mmol) in DMF
1-[3-(tert-Butyldimethylsilanyloxymethyl)-5-(4,4′-dimeth-
oxytrityloxymethyl)phenyl]uracil (9). A mixture of 8 (0.44
g, 0.79 mmol), TBDMSCl (0.13 g, 0.87 mmol), and imidazole
(16) Tanaka, K.; Tengeiji, A.; Kato, T.; Toyama, N.; Shionoya, M.
Science 2003, 299, 1212-1213.
(17) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308.
J. Org. Chem, Vol. 70, No. 20, 2005 7931

Ueno et al.
(0.12 g, 1.74 mmol) in DMF (1.6 mL) was stirred at room
temperature for 1 h. The mixture was partitioned between
CHCl3 and H2O. The organic layer was washed with aqueous
NaHCO3 (saturated) and brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography
(SiO2, 10% EtOAc in hexane) to give 9 (0.501 g, 95%): mp 62-
was partitioned between CHCl3 and aqueous NaHCO3 (satu-
rated). The organic layer was washed with brine, dried (Na2-
SO4), and concentrated. The residue was purified by column
chromatography (SiO2, 25-100% EtOAc in hexane) to give 10
(1.4 g, 66%):1H NMR (DMSO-d6) δ 11.42 (s, 1H), 7.66 (d, 1H,
J ) 7.6), 7.51-6.86 (m, 29H), 5.64 (d, 1H, J ) 7.6), 4.14 (s,
4H), 3.71 (s, 12H); 13C NMR (DMSO-d6) δ 163.1, 158.5, 150.0,
144.8, 144.7, 141.4, 138.1, 136.0, 130.0, 128.1, 127.9, 126.9,
125.7, 123.1, 113.2, 102.4, 86.7, 64.9, 55.2; LRMS (FAB) m/z
853 (MH+); HRMS (FAB) calcd for C54H49N2O8 853.3489, found
853.3505.
1-[3,5-Bis(4,4′-dimethoxytrityloxymethyl)phenyl]-
cytosine (11). Compound 10 (0.20 g, 0.23 mmol) was treated
as described in the preparation of 12 to give 11 (0.19 g, 97%):
1H NMR (DMSO-d6) δ 7.77 (d, 1H, J ) 7.4), 7.60-6.86 (m,
31H), 5.75 (d, 1H, J ) 7.4), 4.13 (s, 4H), 3.71 (s, 12H); 13C NMR
(DMSO-d6) δ 166.0, 158.5, 156.3, 145.8, 144.9, 140.7, 140.6,
136.1, 130.0, 128.1, 127.9, 126.8, 124.9, 123.4, 113.2, 94.4, 86.6,
65.1, 55.2; LRMS (FAB) m/z 852 (MH+); HRMS (FAB) calcd
for C54H50N3O7 852.3649, found 852.3642.
1-[3,5-Bis(hydroxymethyl)phenyl]cytosine (2). A 1 M
solution ofCCl3CO2H in CH2Cl2 (10 mL) was added to a
solution of 11 (0.15 g, 0.176 mmol) in THF (1.8 mL). The
mixture was stirred at room temperature for 18 h. The mixture
was partitioned between CHCl3 and H2O. The water layer was
concentrated, and the product was washed with H2O to give 2
(40 mg, 92%): mp 132-135 °C; UV λmax (H2O) 202 nm, 246
nm, 286 nm; 1H NMR (DMSO-d6) δ 7.58 (d, 1H, J ) 7.2), 7.25
(s, 1H), 7.09 (s, 1H), 5.79 (s, 1H), 5.27 (t, 2H, J ) 5.8), 4.50 (d,
4H, J ) 5.8); 13C NMR (DMSO-d6) δ 166.1, 154.9, 145.8, 143.3,
141.1, 123.1, 122.6, 93.9, 62.5; LRMS (EI) m/z 247 (M+); HRMS
(EI) calcd for C12H13N3O3 247.0957, found 247.0967. Anal.
Calcd for C12H13N3O3: C, 58.29; H, 5.30; N, 17.00. Found: C,
58.11; H; 5.49, N, 16.89.
5-Amino-4-[3,5-bis(hydroxymethyl)anilino]-6-chloro-
pyrimidine (16). A mixture of 5 (0.728 g, 4.76 mmol),
5-amino-4,6-dichloropyrimidine (15) (0.702 g, 4.28 mmol),
concentrated HCl (0.18 mL), and EtOH (1.8 mL) was refluxed
for 7 h. H2O (10 mL) was added to the solution, and the
mixture was allowed to stand overnight in the refrigerator.
The product was filtered and washed with H2O to give 16 (1.03
g, 86%): mp 243-245 °C; 1H NMR (DMSO-d6) δ 8.57 (s, 1H),
7.86 (s, 1H), 7.56 (s, 2H), 6.93 (s, 1H), 5.44 (s, 2H), 5.18 (s,
2H), 4.47 (s, 4H); 13C NMR (DMSO-d6) δ 148.8, 144.7, 143.7,
142.7, 139.5, 138.3, 124.7, 118.9, 116.8, 63.0; LRMS (EI) m/z
280 (M+); HRMS (EI) calcd for C12H13N4ClO2 280.0727, found
280.0723. Anal. Calcd for C12H13N4ClO2: C, 51.34; H, 4.67; N,
19.96. Found: C, 51.09; H, 4.67; N, 19.87.
1
64 °C; H NMR (DMSO-d6) δ 11.42 (s, 1H), 7.67 (d, 1H, J )
8.0), 7.49-6.89 (m, 16H), 5.65 (q, 1H, J ) 2.0 and 8.0), 4.76
(s, 2H), 4.10 (s, 2H), 3.73 (s, 6H), 0.91 (s, 9H), 0.11 (s, 6H);
LRMS (FAB) m/z 665 (MH+); HRMS (FAB) calcd for C39H45-
N2O6Si, 665.3047 found 665.3043. Anal. Calcd for C39H44N2O6-
Si‚3/4H2O: C, 69.05; H, 6.76; N, 4.13. Found: C, 69.14; H, 6.83;
N, 3.76.
1-[3-(tert-Butyldimethylsilanyloxymethyl)-5-(4,4′-di-
methoxytrityloxymethyl)phenyl]cytosine (12). A mixture
of 9 (0.45 g, 0.68 mmol), Et3N (0.19 mL, 1.35 mmol), DMAP
(0.17 g, 1.35 mmol), and 2,4,6-triisopropylbenzenesulfonyl
chloride (0.41 g, 1.35 mmol) in CH3CN (3.4 mL) was stirred
at room temperature for 1 h. The mixture was cooled in an
ice-bath. Concentrated NH4OH (25%, 1.6 mL) was added, and
the mixture was stirred at room temperature for 19 h. The
mixture was evaporated, and the residue was partitioned
between CHCl3 and H2O. The organic layer was washed with
aqueous NaHCO3 (saturated) and brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatog-
raphy (SiO2, 2-4% MeOH in CHCl3) to give 12 (0.32 g, 72%):
1H NMR (DMSO-d6) δ 7.58 (d, 1H, J ) 7.0), 7.44-6.88 (m,
16H), 5.74 (d, 1H, J ) 7.0), 4.75 (s, 2H), 4.08 (s, 2H), 3.72 (s,
6H), 0.90 (s, 9H), 0.10 (s, 6H); 13C NMR (DMSO-d6) δ 166.1,
158.1, 154.8, 145.7, 144.9, 142.2, 141.1, 139.5, 135.5, 129.6,
127.9, 127.6, 126.8, 123.2, 122.9, 122.8, 113.3, 94.1, 86.0, 64.5,
63.8, 55.0, 25.8, 18.0, -5.3; LRMS (FAB) m/z 664 (MH+);
HRMS (FAB) calcd for C39H46N3O5Si 664.3207, found 664.3211.
Anal. Calcd for C39H45N3O5Si: C, 70.56; H, 6.83; N, 6.33.
Found: C, 70.38; H, 6.81; N, 6.13.
4-N-Benzoyl-1-[3-(tert-Butyldimethylsilanyloxymethyl)-
5-(4,4′-dimethoxytrityloxymethyl)phenyl]cytosine (13).
A mixture of 12 (0.25 g, 0.38 mmol) and BzCl (0.13 mL, 0.77
mmol) in pyridine (2 mL) was stirred at room temperature
for 1 h. The mixture was partitioned between CHCl3 and
aqueous NaHCO3 (saturated). The organic layer was washed
with brine, dried (Na2SO4), and concentrated. The residue was
purified by column chromatography (SiO2, 25-100% EtOAc
in hexane) to give 13 (0.27 g, 92%): 1H NMR (DMSO-d6) δ
11.34 (s, 1H), 8.15 (d, 1H, J ) 6.4), 8.02 (d, 2H, J ) 7.2), 7.65-
6.89 (m, 20H), 4.79 (s, 2H), 4.13 (s, 2H), 3.73 (s, 6H), 0.92 (s,
9H), 0.12 (s, 6H); 13C NMR (DMSO-d6) δ 158.1, 144.9, 142.6,
140.0, 139.8, 135.5, 132.8, 129.7, 128.5, 128.4, 127.9, 127.6,
126.8, 123.7, 123.2, 122.7, 113.3, 86.0, 64.5, 63.7, 59.7, 55.0,
25.8, 20.7, 18.0, 14.1, -5.3; LRMS (FAB) m/z 768 (MH+);
HRMS (FAB) calcd for C46H50N3O6Si 768.3469, found 768.3476.
Anal. Calcd for C46H49N3O6Si‚2/3H2O: C, 70.83; H, 6.50; N,
5.39. Found: C, 70.68; H, 6.51; N, 5.22.
5-Amino-4-[3,5-bis(tert-butyldimethylsilanyloxymethyl)-
anilino]-6-chloropyrimidine (17). A mixture of 16 (1.08 g,
3.83 mmol), TBDMSCl (1.27 g, 8.45 mmol), and imidazole (1.14
g, 16.7 mmol) in DMF (8 mL) was stirred at room temperature
for 24 h. The mixture was partitioned between EtOAc and
H2O. The organic layer was washed with aqueous NaHCO3
(saturated) and brine, dried (Na2SO4), and concentrated. The
residue was purified by column chromatography (SiO2, 20-
50% EtOAc in hexane) to give 17 (1.33 g, 70%): mp 167-169
4-N-Benzoyl-1-[3-(4,4′-dimethoxytrityloxymethyl)-5-
(hydroxymethyl)phenyl]cytosine (14). A mixture of 13
(0.31 g, 0.41 mmol) and TBAF (1 M in THF, 0.82 mL, 0.82
mmol) in THF (2.4 mL) was stirred at room temperature for
19 h. The solvent was evaporated, and the resulting residue
was purified by column chromatography (SiO2, 2% MeOH in
CHCl3) to give 14 (0.26 g, 97%): 1H NMR (DMSO-d6) δ 11.35
(s, 1H), 8.17 (d, 1H, J ) 7.2), 8.02 (d, 2H, J ) 7.6), 7.65-6.90
(m, 20H), 5.37 (t, 1H, J ) 6.0), 4.56 (d, 2H, J ) 6.0), 4.12 (s,
2H), 3.73 (s, 6H); 13C NMR (DMSO-d6) δ 158.1, 144.8, 143.9,
139.6, 135.5, 132.8, 129.7, 128.5, 128.0, 127.6, 126.8, 124.4,
123.1, 122.9, 113.3, 86.1, 79.2, 64.6, 62.3, 55.0; LRMS (FAB)
m/z 654 (MH+); HRMS (FAB) calcd for C40H36N3O6 654.2604,
found 654.2610. Anal. Calcd for C40H35N3O6‚5/4H2O: C, 70.81;
H, 5.61; N, 6.19. Found: C, 71.19; H, 5.67; N, 5.77.
1
°C; H NMR (DMSO-d6) δ 8.57 (s, 1H), 7.70 (s, 2H), 6.94 (s,
1H), 5.45 (s, 2H), 4.68 (s, 4H), 0.90 (s, 18H), 0.08 (s, 12H); 13
C
NMR (DMSO-d6) δ 149.2, 145.0, 142.0, 140.1, 139.0, 125.3,
118.5, 117.2, 64.9, 26.3, 18.5, 4.8; LRMS (EI) m/z 508 (M+);
HRMS (EI) calcd for C24H41N4ClO2Si2 508.2457, found 508.2463.
Anal. Calcd for C24H41N4ClO2Si2: C, 56.61; H, 8.12; N, 11.00.
Found: C, 56.48; H, 8.07; N, 10.95.
9-[3,5-Bis(tert-butyldimethylsilanyloxymethyl)phenyl]-
6-chloropurine (18). A mixture of 17 (0.303 g, 0.61 mmol),
(EtO)3CH (1.4 mL), and Ac2O (1.4 mL) was refluxed for 6 h.
The solvent was evaporated in vacuo, and the resulting residue
was purified by column chromatography (SiO2, 17% EtOAc in
hexane) to give 18 (0.242 g, 77%): mp 91-93 °C; 1H NMR
(CDCl3) δ 8.80 (s, 1H), 8.41 (s, 1H), 7.58 (s, 2H), 7.34 (s, 1H),
4.84 (s, 4H), 0.96 (s, 18H), 0.13 (s, 12H); 13C NMR (CDCl3) δ
1-[3,5-Bis(4,4′-dimethoxytrityloxymethyl)phenyl]-
uracil (10). A mixture of 1 (0.62 g, 2.5 mmol), DMTrCl (1.69
g, 5.0 mmol), and DMAP (15 mg, 0.125 mmol) in pyridine (13
mL) was stirred at room temperature for 18 h. The mixture
7932 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis and Properties of Nucleic Acid Analogues
152.6, 151.6, 151.5, 144.2, 144.0, 134.0, 132.2, 123.4, 119.2,
64.2, 25.9, 18.4, -5.3; LRMS (FAB) m/z 519 (MH+); HRMS
(FAB) calcd for C25H40N4ClO2Si2 519.2378, found 519.2388.
Anal. Calcd for C25H39N4ClO2Si2: C, 57.83; H, 7.57; N, 10.79.
Found: C, 57.85; H, 7.50; N, 10.68.
8.31 (s, 1H), 8.06 (d, 2H, J ) 7.6), 7.69-6.82 (m, 19H), 4.84
13C NMR (CDCl3) δ
164.7, 158.6, 153.2, 151.8, 149.9, 149.5, 144.7, 143.3, 142.1,
142.0, 135.9, 134.4, 133.6, 132.8, 130.0, 128.9, 128.1, 128.0,
127.9, 126.9, 125.0, 123.3, 120.9, 120.3, 64.9, 64.4, 55.2, 53.6,
39.0, 20.7, 14.0; LRMS (FAB) m/z 678 (MH+); HRMS (FAB)
calcd for C41H36N5O5 678.2716, found 678.2711.
9-[3,5-Bis(hydroxymethyl)phenyl]adenine (3). A mix-
ture of 16 (0.764 g, 2.72 mmol), (EtO)3CH (3.4 mL), and Ac2O
(3.4 mL) was refluxed for 16 h. The solvent was evaporated in
vacuo. The resulting residue was treated with methanolic
ammonia (100 mL, saturated at 0 °C) in a steel sealed tube at
90 °C for 16 h. After the tube was cooled to room temperature
then degassed, the solvent was evaporated in vacuo. The
resulting residue was purified by column chromatograph (SiO2,
1% MeOH in CHCl3) to give 3 (0.265 g, 36% as a white solid):
mp 244-246 °C; UV λmax (H2O) 213 nm, 243 nm, 270 nm; 1H
NMR (DMSO-d6) δ 8.53 (s, 1H), 8.20 (s, 1H), 7.66 (s, 2H), 7.37
(m, 3H), 5.35 (t, 2H, J ) 5.2), 4.58 (d, 4H, J ) 5.2); 13C NMR
(DMSO-d6) δ 156.3, 153.1, 149.2, 144.0, 139.6, 134.8, 123.2,
119.2, 119.1, 62.5; LRMS (EI) m/z 271 (M+); HRMS (EI) calcd
for C13H13N5O2 271.1069, found: 271.1075. Anal. Calcd for
C13H13N5O2‚1/10H2O: C, 57.18; H, 4.87; N, 25.65. Found: C,
57.16; H, 4.73; N, 25.52.
(d, 2H, J ) 5.6), 4.33 (s, 2H), 3.79 (s, 6H);
9-[3,5-Bis(tert-butyldimethylsilanyloxymethyl)phenyl]-
adenine (19). A solution of 18 (101 mg, 0.19 mmol) in
methanolic ammonia (10 mL, saturated at 0 °C) in a steel
sealed tube was heated to 90 °C for 6 h. After the tube was
cooled to room temperature then degassed, the solvent was
evaporated in vacuo. The resulting residue was purified by
column chromatography (SiO2, 20-100% EtOAc in hexane) to
give 19 (59 mg, 63%): mp 122-124 °C; 1H NMR (CDCl3) δ
8.40 (s, 1H), 8.08 (s, 1H), 7.54 (s, 2H), 7.36 (s, 1H), 5.85 (s,
2H), 4.83 (s, 4H), 0.96 (s, 18H), 0.13 (s, 12H); 13C NMR (CDCl3)
δ 155.6, 153.6, 150.0, 143.6, 139.7, 134.7, 122.9, 120.1, 119.3,
64.4, 25.9, 18.4, -5.3; LRMS (EI) m/z 499 (M+); HRMS (EI)
calcd for C25H41N5O2Si2 499.2799, found: 499.2803. Anal. Calcd
for C25H41N5O2Si2‚1/2H2O: C, 59.01; H, 8.32; N, 13.76.
Found: C, 59.19; H, 8.17; N,13.76.
6-N-Benzoyl-9-[3,5-bis(tert-butyldimethylsilanyloxy-
methyl)phenyl]adenine (20). A mixture of 19 (0.87 g, 1.73
mmol) and BzCl (0.24 mL, 2.08 mmol) in pyridine (9 mL) was
stirred at room temperature. After 2 h, BzCl (40 µL, 0.35
mmol) was further added to the mixture. After 1 h, the mixture
was partitioned between EtOAc and aqueous NaHCO3 (satu-
rated). The organic layer was washed with brine, dried (Na2-
SO4), and concentrated. The residue was purified by column
chromatography (SiO2, 20-100% EtOAc in hexane) to give 20
(0.506 g, 49% as a white solid): mp 210-212 °C; 1H NMR
(CDCl3) δ 9.08 (s, 1H), 8.87 (s, 1H), 8.31 (s, 1H), 8.07-7.40
(m, 8H), 4.86 (s, 4H), 0.97 (s, 18H), 0.15 (s, 12H); 13C NMR
(CDCl3) δ 165.6, 154.3, 150.8, 144.9, 143.0, 135.3, 134.6, 133.8,
129.9, 128.8, 124.5, 124.1, 120.3, 65.3, 26.9, 19.4, -4.3; LRMS
(EI) m/z 603 (M+); HRMS (EI) calcd for C32H45N5O3Si2 603.3061,
found 603.3068. Anal. Calcd for C32H45N5O3Si2‚1/2H2O: C,
62.71; H, 7.56; N, 11.43. Found: C, 62.80; H, 7.55; N,
11.39.
6-N-Benzoyl-9-[3-(tert-butyldimethylsilanyloxymethyl)-
5-(hydroxymethyl)phenyl]adenine (21). A mixture of 20
(0.215 g, 0.30 mmol), TBAF (1 M in THF, 0.15 mL, 0.15 mmol),
THF (2.8 mL), and CH2Cl2 (2.8 mL) was stirred at room
temperature. After 4 h, TBAF (1 M in THF, 0.3 mL, 0.3 mmol)
was further added to the solution. After 4 h, the mixture was
partitioned between CHCl3 and aqueous NaHCO3 (saturated).
The organic layer was washed with brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatog-
raphy (SiO2, 2.5% MeOH in CHCl3) to give 20 (96 mg, 44%)
and 21 (92 mg, 53% as a white solid): mp 184-186 °C; 1H
NMR (CDCl3) δ 9.10 (s, 1H), 8.84 (s, 1H), 8.29 (s, 1H), 8.05-
7.39 (m, 8H), 5.46 (s, 1H), 4.84 (m, 4H), 0.95 (s, 9H), 0.13 (s,
6H); 13C NMR (CDCl3) δ 164.6, 153.3, 149.8, 144.3, 143.2,
141.9, 134.5, 132.9, 128.9, 127.9, 124.0, 123.4, 120.1, 120.0,
64.5, 64.2, 25.9, 18.4, -5.3; LRMS (EI) m/z 489 (M+); HRMS
(EI) calcd for C26H31N5O3Si 489.2196, found 489.2207. Anal.
Calcd for C26H31N5O3Si‚1/2H2O: C, 62.62; H, 6.47; N, 14.04.
Found: C, 62.70; H, 6.45; N, 13.57.
6-N-Benzoyl-9-[3-(4,4′-dimethoxytrityloxymethyl)-5-
(hydroxymethyl)phenyl]adenine (22). A mixture of 21
(0.38 g, 0.78 mmol), DMTrCl (0.53 g, 1.56 mmol), and DMAP
(5 mg, 40 µmol) in pyridine (4 mL) was stirred at room
temperature for 16 h. The mixture was partitioned between
CHCl3 and aqueous NaHCO3 (saturated). The organic layer
was washed with brine, dried (Na2SO4), and concentrated. The
residue was dissolved in THF (4 mL). TBAF (1 M in THF, 1.56
mL, 1.56 mmol) was added to the solution, and the whole was
stirred at room temperature for 17 h. The mixture was
partitioned between CHCl3 and aqueous NaHCO3 (saturated).
The organic layer was washed with brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatog-
raphy (SiO2, 1-2% MeOH in CHCl3) to give 22 (0.45 g, 84%
as a white solid): 1H NMR (CDCl3) δ 9.03 (s, 1H), 8.89 (s, 1H),
2-Amino-4-[3,5-bis(hydroxymethy)anilino]-6-hydroxy-
5-phenylazopyrimidine (24). A mixture of 5 (0.50 g, 3.30
mmol) and 2-amino-4-chloro-6-hydroxy-5-phenylazopyrimidine
(23)10 (1.24 g, 4.95 mmol) in EtOH (9 mL) was refluxed. After
7 h, the mixture was allowed to stand overnight in the
refrigerator. The product was filtered and washed with Et2O
1
to give 24 (0.84 g, 70%): mp 210-212 °C; H NMR (DMSO-
d6) δ 7.86 (s, 1H), 7.51-7.16 (m, 8H), 5.26 (s, 2H), 4.53 (s, 4H),
3.33 (s, 2H); LRMS (EI) m/z 366 (M+); HRMS (EI) calcd for
C18H18O3N6 366.1440, found 366.1449.
2-Amino-4-[3,5-bis(tert-butyldimethylsilanyloxymethyl)-
anilino]-6-hydroxy-5-phenylazopyrimidine (25). A mix-
ture of 24 (0.50 g, 1.37 mmol), TBDMSCl (0.62 g, 4.10 mmol),
and imidazole (0.56 g, 8.20 mmol) in DMF (3 mL) was stirred
at room temperature for 18 h. The mixture was partitioned
between EtOAc and H2O. The organic layer was washed with
aqueous NaHCO3 (saturated) and brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatog-
raphy (SiO2, 4% MeOH in CHCl3) to give 25 (0.804 g, 99%):
1
mp 216-218 °C; H NMR (CDCl3) δ 7.79-7.06 (m, 8H), 4.78
(d, 4H, J ) 7.6), 0.98 (s, 18H), 0.15 (s, 12H); LRMS (EI) m/z
594 (M+); HRMS (EI) calcd for C30H46N6O3Si2 594.3170, found
594.3185.
9-[3,5-Bis(tert-butyldimethylsilanyloxymethyl)phenyl]-
2-N-[(dimethylamino)methylene]guanine (26). A mixture
of 25 (5.05 g, 8.50 mmol), Zn powder (5.53 g, 84.6 mmol), AcOH
(6.4 mL), H2O (63 mL), EtOH (72 mL), and THF (72 mL) was
refluxed for 1 h. After the excess Zn was filtered off, the solvent
was partitioned between EtOAc and H2O. The organic layer
was washed with aqueous NaHCO3 (saturated) and brine,
dried (Na2SO4), and evaporated in vacuo. The resulting residue
was mixed with N,N-dimethylformamide dimethyl acetal (14.5
mL, 0.11 mmol) and DMF (125 mL), and the whole was stirred
at room temperature for 15 h. The solvent was evaporated in
vacuo. The resulting residue was mixed with (EtO)3CH (19
mL, 113 mmol) and Ac2O (19 mL, 198 mmol), and the whole
was stirred at room temperature for 7 h. The mixture was
partitioned between EtOAc and H2O. The organic layer was
washed with aqueous NaHCO3 (saturated) and brine, dried
(Na2SO4), and concentrated. The residue was purified by
column chromatography (SiO2, 2.5% MeOH in CHCl3) to give
1
26 (3.70 g, 76%): mp 89-90 °C; H NMR (CDCl3) δ 8.64 (s,
1H), 8.51 (s, 1H), 7.80 (s, 1H), 7.42 (s, 2H), 7.34 (s, 1H), 4.81
(s, 4H), 3.12 (s, 3H), 3.09 (s, 3H), 0.95 (s, 18H), 0.12 (s, 12H);
13C NMR (CDCl3) δ 158.2, 158.0, 156.8, 150.2, 143.3, 137.8,
135.0, 122.8, 121.0, 120.1, 64.4, 41.3, 35.2, 25.9, 18.4, -5.3;
LRMS (EI) m/z 570 (M+); HRMS (EI) calcd for C28H46N6O3Si2
J. Org. Chem, Vol. 70, No. 20, 2005 7933

Ueno et al.
570.3170, found 570.3164. Anal. Calcd for C28H46N6O3Si2‚1/
2H2O: C, 57.99; H, 8.17; N, 14.49. Found: C, 58.34; H, 8.33;
N, 14.15.
(Na2SO4), and concentrated. The residue was purified by
column chromatography (a neutralized SiO2, EtOAc) to give
29 (0.88 g, 81%): 31P NMR (CDCl3) δ 148.9.
4-N-Benzoyl-1-[3-[[(2-cyanoethoxy)(N,N-diisoprop-
ylamino)phosphinyl]oxymethyl]-5-(4,4′-dimethoxy-
trityloxymethyl)phenyl]cytosine (30). Compound 12 (0.66
g, 1.01 mmol) was phosphitylated as described in the prepara-
tion of 29 to give 30 (0.83 g, 96%): 31P NMR (CDCl3) δ 149.4.
6-N-Benzoyl-9-[3-[[(2-cyanoethoxy)(N,N-diisoprop-
ylamino)phosphinyl]oxymethyl]-5-(4,4′-dimethoxy-
trityloxymethyl)phenyl]adenine (31). Compound 22 (0.45
g, 0.66 mmol) was phosphitylated as described in the prepara-
tion of 29 to give 31 (0.51 g, 88%): 31P NMR (CDCl3) δ 149.4.
9-[3-[[(2-Cyanoethoxy)(N,N-diisopropylamino)phos-
phinyl]oxymethyl]-5-(4,4′-dimethoxytrityloxymethyl)-
phenyl]-2-N-[(dimethylamino)methylene]guanine (32).
Compound 28 (0.78 g, 1.21 mmol) was phosphitylated as
described in the preparation of 29 to give 32 (0.62 g, 60%):
31P NMR (CDCl3) δ 149.3.
Solid Support Synthesis. A mixture of 8 (0.15 g, 0.27
mmol), succinic anhydride (90 mg, 0.90 mmol), and DMAP (33
mg, 0.27 mmol) in pyridine (3 mL) was stirred for 72 h at room
temperature. The solution was partitioned between CHCl3 and
H2O, and the organic layer was washed with H2O and brine.
The separated organic phase was dried (Na2SO4) and concen-
trated to give the corresponding succinate. Aminopropyl
controlled pore glass (0.50 g, 45 µmol) was added to a solution
of the succinate and 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (63 mg, 0.33 mmol) in DMF (8 mL), and
the mixture was kept for 72 h at room temperature. After the
resin was washed with pyridine, a capping solution (8 mL, 0.1
M DMAP in pyridine:Ac2O ) 9:1, v/v) was added and the whole
mixture was kept for 16 h at room temperature. The resin was
washed with MeOH and acetone, and dried in vacuo. The
amount of loaded compound 8 to the solid support was 67
µmol/g from calculation of released dimethoxytrityl cation by
a solution of 70% HClO4:EtOH (3:2, v/v). In a similar manner,
the solid supports with 12, 22, and 28 were obtained in 82,
54, and 70 µmol/g loading amounts, respectively.
X-ray Crystallography. Crystal data for 1: Colorless plate
(0.51 × 0.40 × 0.03 mm); C12H12N2O4, monoclinic space group
Cc, Z ) 4. a ) 4.354(4) Å, b ) 22.74(2) Å, c ) 12.12(1) Å, â )
105.022(8)°. X-ray diffraction measurements were carried out
on a diffractometer with a Mercury CCD area detector (Mo-
KR λ ) 0.71069 Å). Of the 4743 measured reflections (θ <
27.48°), 1330 were unique (Rint ) 0.036). All calculations were
performed using the teXsan crystallographic software package.
The structure was solved by the direct methods SIR92. The
final least-squares refinement included non-hydrogen and
hydrogen atoms with anisotropic and isotropic thermal pa-
rameters, respectively. The refinement converged at R1 ) 0.035
for 1093 reflections with I > 2σ(I) and Rw ) 0.068 for all
reflections.
Oligomer Synthesis. The synthesis was carried out with
a DNA/RNA synthesizer by the phosphoramidite method. For
the incorporation of the analogues into the oligomers, a 0.12
M solution of each analogue phosphoramidite in THF with a
coupling time of 15 min was used. Deprotection of the bases
and phosphates was performed in concentrated NH4OH at 55
°C for 16 h. The deprotected ONAs and 2′-O-Me-ONAs were
purified by 20% PAGE containing 7 M urea to give the highly
purified ONAs 7 (5), 8 (5), 9 (3), 10 (3), 11 (6), 12 (3), 13 (3),
14 (12), 15 (6), 18 (4), 19 (7), 20 (2), 21 (5), 2′-O-Me-ONAs 23
(20), and 24 (7). The yields are indicated in parentheses as
OD units at 260 nm starting from 1 µmol scale.
9-[3-(tert-Butyldimethylsilanyloxymethyl)-5-(hydroxy-
methyl)phenyl]-2-N-[(dimethylamino)methylene]-
guanine (27). A mixture of 26 (0.204 g, 0.36 mmol) and TBAF
(1 M in THF, 0.18 mL, 0.18 mmol) in THF (5 mL) was stirred
at room temperature for 30 min. The mixture was partitioned
between CHCl3 and aqueous NaHCO3 (saturated). The organic
layer was washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography
(SiO2, 3-5% MeOH in CHCl3) to give 27 (95 mg, 59% as a
white solid): mp 216-217 °C; 1H NMR (CDCl3) δ 8.61 (s, 1H),
8.53 (s, 1H), 7.83 (s, 1H), 7.61 (s, 1H), 7.52 (s, 1H), 7.34 (s,
1H), 4.81 (s, 4H), 3.13 (s, 3H), 3.07 (s, 3H), 0.95 (s, 9H), 0.12
(s, 6H); 13C NMR (CDCl3) δ 158.2, 156.9, 150.2, 143.4, 137.4,
135.2, 123.3, 120.8, 120.7, 119.4, 64.4, 64.3, 41.5, 35.0, 25.9,
18.4, -5.3; LRMS (FAB) m/z 457 (MH+); HRMS (FAB) calcd
for C22H33N6O3Si1 457.2305, found 457.2393.
9-[3-(4,4′-Dimethoxytrityloxymethyl)-5-(hydroxymeth-
yl)phenyl]-2-N-[(dimethylamino)methylene]guanine (28).
A mixture of 27 (0.95 g, 2.09 mmol), DMTrCl (1.42 g, 4.18
mmol), and DMAP (13 mg, 0.11 mmol) in pyridine (10 mL)
was stirred at room temperature for 18 h. The mixture was
partitioned between CHCl3 and aqueous NaHCO3 (saturated).
The organic layer was washed with brine, dried (Na2SO4), and
concentrated. The residue was dissolved in THF (11 mL).
TBAF (1 M in THF, 4.18 mL, 4.18 mmol) was added to the
solution, and the mixture was stirred at room temperature for
5 h. The mixture was partitioned between CHCl3 and aqueous
NaHCO3 (saturated). The organic layer was washed with
brine, dried (Na2SO4), and concentrated. The residue was
purified by column chromatography (SiO2, 3-5% MeOH in
CHCl3) to give 28 (1.16 g, 86% as a white solid): 1H NMR
(CDCl3) δ 8.78 (s, 1H), 8.45 (s, 1H), 7.89-6.82 (m, 17H), 4.78
(s, 2H), 4.25 (s, 2H), 3.78 (s, 6H), 2.92 (s, 3H), 2.53 (s, 3H); 13
C
NMR (CDCl3) δ 158.6, 158.1, 158.0, 156.8, 150.2, 144.9, 143.1,
141.2, 137.3, 136.0, 135.4, 129.9, 128.0, 126.9, 124.0, 121.0,
120.5, 120.1, 113.2, 86.5, 64.9, 64.4, 55.2, 40.7, 35.0; LRMS
(FAB) m/z 645 (MH+); HRMS (FAB) calcd for C37H37N6O5
645.2747, found 645.2819. Anal. Calcd for C37H36N6O5 ‚5/
4H2O: C, 66.99; H, 5.99; N, 12.34. Found: C, 66.83; H, 5.73;
N, 12.09.
9-[3,5-Bis(hydroxymethyl)phenyl]guanine (4). A mix-
ture of 24 (0.70 g, 1.91 mmol), Zn powder (1.25 g, 19.1 mmol),
AcOH (1.4 mL), H2O (14 mL), EtOH (16 mL), and THF (16
mL) was refluxed for 30 min. After the excess Zn was filtered
off, the solvent was evaporated in vacuo. The resulting residue
was mixed with (EtO)3CH (26 mL, 157 mmol), DMF (13 mL),
and 1 M HCl (1.2 mL), and the whole was stirred at room
temperature for 18 h. The solvent was evaporated in vacuo.
The resulting residue was dissolved in 0.5 M HCl (36 mL),
and the whole was stirred at room temperature for 1 h. The
product was filtered and recrystallized from MeOH to give 4
(0.27 g, 25%): UV λmax (H2O) 210 nm, 242 nm, 262 nm; 1H
NMR (DMSO-d6) δ 10.67 (s, 1H), 7.93 (s, 1H), 7.42 (s, 2H),
7.35 (s, 1H), 6.48 (s, 2H), 5.31 (t, 2H, J ) 5.4), 4.55 (d, 4H, J
) 5.4); 13C NMR (DMSO-d6) δ 157.5, 154.1, 151.5, 144.2, 137.4,
135.0, 124.2, 120.8, 117.3, 62.8; LRMS (FAB) m/z 288 (MH+);
HRMS (FAB) calcd for C13H14N5O3 288.1097, found 288.1101.
Anal. Calcd for C13H13N5O3‚1/7H2O: C, 53.87; H, 4.62; N,
24.16. Found: C, 54.11; H, 4.64; N, 24.07.
1-[3-[[(2-Cyanoethoxy)(N,N-diisopropylamino)phos-
phinyl]oxymethyl]-5-(4,4′-dimethoxytrityloxymethyl)-
phenyl]uracil (29). Compound 8 (0.80 g, 1.45 mmol) was
dissolved in THF (9.7 mL) containing N,N-diisopropylethy-
lamine (1.46 mL, 8.72 mmol). Chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine (0.65 mL, 2.9 mmol) was added
to the solution, and the mixture was stirred at room temper-
ature for 1 h. Aqueous NaHCO3 (saturated) and CHCl3 were
added to the mixture, and the separated organic layer was
washed with aqueous NaHCO3 (saturated) and brine, dried
MALDI-TOF/MS Analyses of Oligomers. Spectra were
obtained with a time-of-flight mass spectrometer. ONA 7:
calculated mass, 3828.7; observed mass, 3828.2. ONA 8:
calculated mass, 3828.7; observed mass, 3831.4. ONA 9:
calculated mass, 3804.7; observed mass, 3807.5. ONA 10:
calculated mass, 3844.7; observed mass, 3845.7. ONA 11:
calculated mass, 3827.7; observed mass, 3830.2. ONA 12:
7934 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis and Properties of Nucleic Acid Analogues
calculated mass, 3874.8; observed mass, 3875.2. ONA 13:
calculated mass, 3784.6; observed mass, 3781.9. ONA 14:
calculated mass, 3936.9; observed mass, 3938.2. ONA 15:
calculated mass, 3660.4; observed mass, 3662.5. ONA 18:
calculated mass, 5186.4; observed mass, 5184.8. ONA 19:
calculated mass, 5220.4; observed mass, 5221.3. ONA 20:
calculated mass, 5226.5; observed mass, 5227.7. ONA 21:
calculated mass, 5232.4; observed mass, 5234.0. 2′-O-Me-ONA
23: calculated mass, 5666.9; observed mass, 5659.6. 2′-O-Me-
ONA 24: calculated mass, 5646.9; observed mass, 5641.8.
Hyperchromicities and Extinction Coefficients of the
Oligomers. Each oligomer (0.2 OD unit at 260 nm) was
incubated with snake venom phosphodiesterase (1.0 unit),
nuclease P1 (1.0 unit), and alkaline phosphatase (1.0 unit) in
a buffer containing 100 mM Tris-HCl (pH 7.7) and 2 mM
MgCl2 (total 600 µL) at 37 °C for 48 h. Hyperchromicity of each
oligomer was determined by comparing UV absorbances at 260
nm of the solutions before and after hydrolyses. The extinction
coefficients (at 260 nm) of each oligomer was determined using
the following equation: ꢀoligomer ) the sum of ꢀnucleoside/hyper-
chromicity. The extinction coefficient (at 260 nm) of the natural
nucleosides used for calculations were as follows: dA, 15400;
dC, 7300; dG, 11700; T, 8800. The extinction coefficients of
the analogues at 260 nm were determined to be the following:
AB, 18500; CB, 9050; GB, 15300; UB, 13100. The extinction
coefficients of the natural ONs were calculated from those of
mononucleotides and dinucleotides according to the nearest-
neighbor approximation method.18
sodium phosphate (pH 7.0) and 1.0 or 0.1 M NaCl was heated
at 95 °C for 3 min, cooled gradually to an appropriate
temperature, and then used for the thermal denaturation
study. The thermal-induced transition of each mixture was
monitored at 260 nm with a spectrophotometer. The sample
temperature was increased by 0.5 °C/min. CD spectra were
measured by a spectropolarimeter. Samples for CD spectros-
copy were prepared by the same procedure used in the thermal
denaturation study, and spectra were measured at 10 °C. The
molar ellipticitiy was calculated from the equation [θ] ) /cL,
where θ is the relative intensity, c the sample concentration,
and L the cell path length in centimeters.
Acknowledgment. This research was supported in
part by a Grant-in-Aid for Scientific Research (C)
(KAKENHI 16590082) from the Japan Society for the
Promotion of Science (JSPS). We thank Dr. R. Ishiguro
(Gifu University) for helpful discussion.
Supporting Information Available: Crystallographic
information file for the compound 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO050635M
(18) Puglisi, J. D.; Tinoco, I., Jr. In Methods in Enzymology;
Dahlberg, J. E., Abelson, J. N., Eds.; Academic Press: San Diego,
1989: Vol. 180, pp 304-325.
Thermal Denaturation Study and CD Spectroscopy.
A solution containing the ONs in a buffer comprised of 10 mM
J. Org. Chem, Vol. 70, No. 20, 2005 7935