Nucleosides and nucleotides. 182. Synthesis of branched oligodeoxynucleotides with pentaerythritol at the branch point and their thermal stabilization of triplex formation

Article abstract of DOI:10.1021/jo981831e

To find an oligodeoxynucleotide (ODN) with triplex stabilization capability, we designed and synthesized novel branched ODNs 1 and 2 with pentaerythritols at their branch points. Branched ODNs 1 and 2 were synthesized on a solid support using bis(phosphoramidite) 11. The stability of the triplexes formed by branched ODNs 1 and 2 with (dA)₂₁ or (dT)₂₁, was studied by thermal denaturation. Branched ODN 1 formed a stable parallel T-AT-type triplex with (dA)₂₁ (T(m) = 38.9 °C) in a buffer of 0.01 M sodium phosphate (pH 7.0) containing 0.5 M NaCl and 0.02 M MgCl₂, while branched ODN 2 formed a stable antiparallel A · AT-type triplex with (dT)₂₁ (T(m) = 44.2 °C) in the same buffer. The formation of these triplexes was also confirmed by circular dichroism (CD) measurements and a gel retardation assay.

Full text of DOI:10.1021/jo981831e

J . Org. Chem. 1999, 64, 1211-1217
1211
Nu cleosid es a n d Nu cleotid es. 182. Syn th esis of Br a n ch ed
Oligod eoxyn u cleotid es w ith P en ta er yth r itol a t th e Br a n ch P oin t
a n d Th eir Th er m a l Sta biliza tion of Tr ip lex F or m a tion 1
Yoshihito Ueno, Masako Takeba, Mai Mikawa, and Akira Matsuda*
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, J apan
Received September 8, 1998
To find an oligodeoxynucleotide (ODN) with triplex stabilization capability, we designed and
synthesized novel branched ODNs 1 and 2 with pentaerythritols at their branch points. Branched
ODNs 1 and 2 were synthesized on a solid support using bis(phosphoramidite) 11. The stability of
the triplexes formed by branched ODNs 1 and 2 with (dA)21 or (dT)21 was studied by thermal
denaturation. Branched ODN 1 formed a stable parallel T‚AT-type triplex with (dA)21 (Tm ) 38.9
°C) in a buffer of 0.01 M sodium phosphate (pH 7.0) containing 0.5 M NaCl and 0.02 M MgCl2,
while branched ODN 2 formed a stable antiparallel A‚AT-type triplex with (dT)21 (Tm ) 44.2 °C) in
the same buffer. The formation of these triplexes was also confirmed by circular dichroism (CD)
measurements and a gel retardation assay.
In tr od u ction
binding of a third-strand ODN to a target DNA duplex
is thermodynamically weaker than duplex formation
itself. Thus, many efforts have been made to increase the
affinity of the third strand for its target.2 These include
the synthesis of backbone-, sugar-, and/or base-modified
ODNs as well as ODN-intercalator conjugates.3
On the other hand, since branched RNAs containing
vicinal 2′-5′ and 3′-5′ internucleotidic phosphodiester
linkages have been discovered in the splicing products
of eukaryotic mRNAs, considerable attention has been
paid to the chemical synthesis of this unique structure.4-6
The chemical synthesis of branched RNAs in a solution
phase has been reported in several laboratories.4 On the
other hand, the first automated solid-phase synthesis of
Recently, chemically synthesized oligodeoxynucleotides
(ODNs) and their analogues have been used in studies
of triplex formation and in biological and biochemical
studies, such as in the site-specific cleavage of DNA or
the inhibition of DNA-protein binding.2 In intermolecu-
lar triplexes, an oligopyrimidine‚oligopurine sequence of
a DNA duplex is bound by a third-strand ODN in the
major groove. To date, two major classes of triplexes have
been identified based on the orientation of the third
strand.2 When the third strand consists mainly of pyri-
midines, Hoogsteen-type Py‚PuPy base triplets (T‚AT and
C+‚GC) are formed, in which the third strand is parallel
to the purine strand of the target duplex. On the other
hand, when the third strand is predominantly purines,
Pu‚PuPy-type base triplets (G‚GC and A‚AT) are formed,
in which the third strand is antiparallel to the purine
strand of the target duplex. However, in general, the
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5815. (b) Sekine, M.; Heikkila¨, J .; Hata, T. Tetrahedron Lett. 1987,
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hedron 1988, 44, 6929-6939. (m) Sund, C.; Fo¨ldesi, A.; Yamakage, S.;
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* To whom reprint requests should be addressed. Phone: +81-11-
706-3228. Fax: +81-11-706-4980. E-mail: matuda@pharm.hokudai.ac.jp.
(1) This paper constitutes Part 182 of Nucleosides and Nucleotides.
Part 181: Nishizono, N.; Sumita, U.; Ueno, Y.; Matsuda, A. Nucleic
Acids Res. 1998, 26, 5067-5072.
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P. S. Bioconjugate Chem. 1996, 7, 600-605. (k) Lehmann, T. E.;
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10.1021/jo981831e CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/28/1999

1212 J . Org. Chem., Vol. 64, No. 4, 1999
Ueno et al.
F igu r e 1. Structures of the branched ODNs.
branched RNA was reported by Damha et al.5a,b They
constructed branched RNAs and ODNs with adenosine
at the branch point by reacting adenosine 2′,3′-O-bis-
(phosphoramidite) with the 5′-hydroxyl groups of two
adjacent RNAs or ODNs on a solid support. Furthermore,
they also showed that branched poly(thymidine) ODNs
with adenosine at the branch point can form stable
antiparallel T‚AT triplexes.5d More recently, several kinds
of branched ODNs with 3′-deoxy-â-D-psicothymidine,
1-(2-methyl-â-D-arabinofuranosyl)uracil, or 4′-C-(hy-
droxymethyl)thymidine at their branch points have been
synthesized, and some of these have been shown to form
stable triplexes with single-stranded ODNs.7,8 Branched
(dendrimeric) ODNs have also been used to amplify
radioactive or fluorescent signals in hybridization stud-
ies.9
On the basis of these findings, we designed new
branched ODNs 1 and 2 with pentaerythritol 5 at the
branch point (Figure 1), which form a stable parallel
T‚AT-type triplex with (dA)21 and an antiparallel A‚AT-
type triplex with (dT)21, respectively (Figure 2). In this
paper, we report the synthesis of branched ODNs 1 and
2 and their thermal stabilization of triplex formation.
F igu r e 2. Structures of the triplexes between the branched
ODNs and (dT)21 or (dA)21.
pentaerythritol 5 was modified with a propylcarbamoyl
tether for efficient coupling on a solid support and for
stable triplex formation.
First, pentaerythritol 5 was treated with 2 equiv of tert-
butylchlorodiphenylsilane (TBDPSCl) in the presence of
imidazole in DMF to give mono-, bis-, and tris(TBDPS)
derivatives 6a ,10 6b, and 6c in 13, 55, and 31% yields,
respectively (Scheme 2). Compound 6b was converted
into a carbonylimidazolide, which was reacted with
3-amino-1-propanol to afford 7 in 88% yield. The primary
hydroxy groups of 7 were protected with a DMTr group.
After removal of the TBDPS groups, 9 was converted into
carbonylimidazolide, which was reacted with 3-amino-
1-propanol again to give 10 in 91% yield. Compound 10
was phosphitylated by a standard procedure11 to afford
the bis(phosphoramidite) 11 in 65% yield.
Branched ODNs 1 and 2 were synthesized on a DNA
synthesizer by the phosphoramidite method.12 A low
concentration (0.025 M) of bis(phosphoramidite) 11 was
used in the branching step. The coupling yield of 11 was
112% using a 0.025 M solution of 11 in CH3CN with a
coupling time of 600 s. The fully protected ODNs (1 µmol)
were concomitantly cleaved from the solid support and
deprotected by treatment with concentrated NH4OH at
55 °C for 16 h. Removal of the ammonia solution
furnished the crude ODNs. The crude ODNs were puri-
fied by 20% polyacrylamide gel electrophoresis (20%
PAGE), followed by reversed-phase HPLC. In 20% PAGE,
Resu lts a n d Discu ssion
Syn th esis. Damha et al. constructed branched ODNs
with adenosine at the branch point by reacting a low
concentration of adenosine 2′,3′-O-bis(phosphoramidite)
with the 5′-hydroxyl groups of two adjacent ODNs on a
solid support.5a-d We also adopted this so-called “conver-
gent” method (Scheme 1). For the synthesis of branched
ODNs 1 and 2, we synthesized a bis(phosphoramidite)
11. In the convergent method, the reaction of bis-
(phosphoramidite) 11 with the 5′-hydroxyl groups of two
adjacent ODNs on a solid support is a crucial step. Thus,
(7) (a) Azhayeva, A.; Guzaev, A.; Hovinen, J .; Azhayeva, E.; Lo¨nn-
berg, H. Tetrahedron Lett. 1993, 34, 6435-6438. (b) Azhayeva, E.;
Azhayeva, A.; Guzaev, A.; Hovinen, J .; Lo¨nnberg, H. Nucleic Acids Res.
1995, 23, 1170-1176. (c) Azhayeva, E.; Azhayeva, A.; Guzaev, A.;
Lo¨nnberg, H. Nucleic Acids Res. 1995, 23, 4255-4261.
(8) (a) Brandenburg, G.; Petersen, G. V.; Rasmussen, K.; Wengel,
J . Bioorg. Med. Chem. Lett. 1995, 5, 791-794. (b) von Buren, M.;
Petersen, G. V.; Rasmussen, K.; Brandenburg, G.; Wengel, J . Tetra-
hedron 1995, 51, 8491-8506. (c) Thrane, H.; Fensholdt, J .; Regner,
M.; Wengel, J . Tetrahedron 1995, 51, 10389-10402. (d) Meldgaard,
M.; Nielsen, N. K.; Bremner, M.; Pedersen, O. S.; Olsen, C. E.; Wengel,
J . J . Chem. Soc., Perkin Trans. 1 1997, 1951-1955.
(9) (a) Shchepinov, M. S.; Udalova, I. A.; Bridgman, A. J .; Southern,
E. M. Nucleic Acids Res. 1997, 25, 4447-4454. (b) Horn, T.; Chang,
C.-A.; Urdea, M. S. Nucleic Acids Res. 1997, 25, 4835-4841. (c) Horn,
T.; Chang, C.-A.; Urdea, M. S. Nucleic Acids Res. 1997, 25, 4842-
4849.
(10) The same compound was recently synthesized by Hanessian
et al.: Hanessian, S.; Prabhanjan, H.; Qiu, D.; Nambiar, S. Can. J .
Chem. 1996, 74, 1731-1737.
(11) Gait, M. J ., Ed. In Oligonucleotides Synthesis: a Practical
Approach; IRL Press: Oxford, UK, 1984.
(12) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22,
1859-1862.

Branched Oligodeoxynucleotides
J . Org. Chem., Vol. 64, No. 4, 1999 1213
Sch em e 1
Sch em e 2a
a
(a) TBDPSCl, Imidazole, DMF; (b) (1) N,N-carbonyldiimidazole, DMAP, DMF, (2) 3-amino-1-propanol, DMF; (c) DMTrCl, pyridine;
(d) 0.5 M TBAF, THF; (e) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine, CH2Cl2.
two major products were observed. In the synthesis of
branched ODN 1, ODN 1 and ODN 3 (Figure 1), which
would be derived from the reaction of 11 with one of the
5′-hydroxyl groups of the ODNs on a solid support, were
obtained in 19 and 21 OD260 units, respectively. Similarly,
in the synthesis of branched ODN 2, ODN 2 and ODN 4
were obtained in 17 and 16 OD260 units, respectively.
The structures of ODNs 1-4 were confirmed by elec-
trospray ionization (ESI) mass spectrometry. The ob-
served molecular weights supported their structures (see
Experimental Section).
Stu d ies of Tr ip lex F or m a tion by Th er m a l Den a -
tu r a tion . Branched ODN 1 was designed to form a
parallel T‚AT-type triplex with (dA)21, whereas branched
ODN 2 was designed to form an antiparallel A‚AT-type
triplex with (dT)21 (Figure 2). To study the effect of the
linker groups on triplex formation, we examined the
stability of the triplexes formed by these branched ODNs
with (dA)21 or (dT)21 by thermal denaturation. Their
melting transitions were monitored at both 260 and 284
nm. UV absorbances of A-T Watson-Crick duplexes do
not change when their melting transitions are monitored
at 284 nm, since this wavelength (284 nm) is an isosbestic
point of an A-T Watson-Crick duplex.13 On the other
hand, the UV absorbances of triplexes change at both 260
and 284 nm. Thus, a melting transition of a triplex can
be detected independently of a transition of an A-T
Watson-Crick duplex by measuring the melting profiles
at 284 nm.
transition at around 30 °C was also observed in a melting
curve at 284 nm. Thus, we presumed that the transition
with the higher Tm (47.7 °C) at 260 nm was due to
melting of the A-T Watson-Crick duplex, while that
with the lower Tm (30.1 °C) corresponded to dissociation
of the third strand from the triplex. On the other hand,
in the melting profile of the triplex between ODN 1 and
(dA)21, only one transition was observed at each wave-
length (Tm ) 38.9 °C). Thus, we supposed that the
transition was due to both dissociation of the third strand
from the triplex and melting of the A-T Watson-Crick
duplex. Based on these results, it was found that the
thermal stability of the triplex between branched ODN
1 and (dA)21 is greater than that of the natural triplex
[∆Tm
[Tm of the triplex between branched ODN 1
triplex
and (dA)21 minus Tm of the triplex of the mixture of (dA)21:
2(dT)14] was +8.8 °C], although the thermal stability of
the A-T Watson-Crick duplex between the branched
ODN and A21 was less than that of the natural duplex
[∆Tm
[Tm of the Watson-Crick duplex between
duplex
branched ODN 1 and (dA)21 minus Tm of the duplex
between (dT)14 and (dA)21] was -8.8 °C].
Melting profiles of the triplex between ODN 2 and
(dT)21, and a solution of (dT)21:2(dA)14 in a buffer of 0.01
M sodium phosphate (pH 7.0) containing 0.5 M NaCl and
0.02 M MgCl2, are shown in Figure 3, parts d and c,
respectively. The (dT)21:2(dA)14 solution had a monopha-
sic transition (Tm ) 48.1 °C) at 260 nm, but no observable
transition at 284 nm (Figure 3c). This result indicates
that the mixture of (dT)21:2(dA)14 forms a A-T Watson-
Crick duplex, but not an antiparallel A‚AT-type triplex
under these conditions.13a,14 On the other hand, the
mixture of branched ODN 2 and (dT)21 had a monophasic
transition (Tm ) 44.2 °C) at each wavelength. Thus,
branched ODN 2 formed a thermally stable triplex with
(dT)21 under these conditions. These results show that
the thermal stabilities of triplexes increase due to linking
A melting profile of the triplex between ODN 1 and
(dA)21 in a buffer of 0.01 M sodium phosphate (pH 7.0)
containing 0.5 M NaCl and 0.02 M MgCl2 is shown in
Figure 3b, whereas a melting profile of a natural triplex
composed of (dA)21:2(dT)14 in the same buffer is shown
in Figure 3a. In the melting curve of the natural triplex
at 260 nm, two transitions are observed (Figure 3a). The
(13) (a) Pilch, D. S.; Levenson, C.; Shafer, R. H. Proc. Natl. Acad.
Sci. U.S.A. 1990, 87, 1942-1946. (b) Pilch, D. S.; Brousseau, R.; Shafer,
R. H. Nucleic Acids Res. 1990, 18, 5743-5750. (c) Durand, M.; Peloille,
S.; Thuong, N. T.; Maurizot, J . C. Biochemistry 1992, 31, 9197-9204.
(d) Fossella, J . A.; Kim, Y. J .; Shih, H.; Richards, E. G.; Fresco, J . R.
Nucleic Acids Res. 1993, 21, 4511-4515.
(14) Dagneaus, C.; Gousset, H.; Shchyolkina, A. K.; Ouali, M.;
Letellier, R.; Liquier, J .; Florentiev, V. L.; Taillandier, E. Nucleic Acids
Res. 1996, 24, 4506-4512.

1214 J . Org. Chem., Vol. 64, No. 4, 1999
Ueno et al.
F igu r e 3. UV melting profiles monitored at 260 nm (s) and 284 nm (- - -). (a) (dA)21:2(dT)14. (b) ODN 1:(dA)21. (c) (dT)21:2(dA)14.
(d) ODN 2:(dT)21.
F igu r e 4. CD spectra of the triplexes at 5 °C in a buffer of 0.01 M sodium phosphate (pH 7.0) containing 0.5 M NaCl and 0.02
M MgCl2. (a) An ODN 1:(dA)21 solution (s); the normalized sum of a ODN 1 plus (dA)21 solutions (-‚-); the normalized sum of
a (dT)14 plus (dA)21:(dT)14 solutions (- -); a (dT)14:(dA)21:(dT)14 solution (- - -). (b) An ODN 2:(dT)21 solution (s); the normalized
sum of a ODN 2 plus (dT)21 solutions (-‚-); the normalized sum of a (dA)14 plus (dA)21:(dT)14 solutions (- - -).
between the third strand and one strand of the Watson-
Crick duplex by linker groups.
CD band at 217 nm and negative CD bands at 206, 248,
and 267 nm. The shapes of these CD spectra were similar
to each other. On the other hand, the normalized summed
spectra of branched ODN 1 and (dA)21, and (dT)14 and
(dA)21:(dT)14, exhibited large positive CD bands at around
216 and 280 nm and negative CD bands at around 250
nm. The shapes of these spectra were obviously different
from that of the branched ODN 1:(dA)21 solution. The
positive CD bands at around 215 and 280 nm for the
branched ODN 1:(dA)21 solution were clearly less intense
than those in the normalized summed spectra from the
branched ODN 1 and (dA)21, and (dT)14 and (dA)21:(dT)14
solutions. Recently, Pilch et al. reported that the positive
CD band at around 280 nm of a (dT)10:(dA)10:(dT)10-type
triplex was less intense than that of the normalized
Cir cu la r Dich r oism . The formation of triplexes was
confirmed by circular dichroism (CD) measurements. The
CD spectra of the mixture of a 1:1 molar ratio of branched
ODN 1:(dA)21 in a buffer of 0.01 M sodium phosphate (pH
7.0) containing 0.5 M NaCl and 0.02 M MgCl2 at 5 °C
were compared with the appropriately normalized summed
spectra of branched ODN 1 and (dA)21, and (dT)14 and
(dA)21:(dT)14, and the spectrum of a mixture of a 1:1:1
molar ratio of (dT)14:(dA)21:(dT)14 (Figure 4a).
The CD spectrum (solid line) of a branched ODN
1:(dA)21 solution had positive CD bands at 217 and 283
nm and negative CD bands at 206 and 248 nm. The CD
spectrum of a (dT)14:(dA)21:(dT)14 solution had a positive

Branched Oligodeoxynucleotides
J . Org. Chem., Vol. 64, No. 4, 1999 1215
F igu r e 5. Gel retardation assays were carried out in 90 mM tris-borate buffer (pH ) 7.8) containing 10 mM MgCl2 at room
temperature. The stoichiometry of ODNs is indicated by ratio. 32P-Labeled ODNs are indicated by an asterisk.
summed spectra from (dT)10 and (dA)10:(dT)10 solutions.13a
Therefore, these CD spectral data support the existence
of a triplex between branched ODN 1 and (dA)21.
similar to that of the (dT)14:(dA)21*:(dT)14 triplex were
observed (Figure 5, lanes 9-13). We presumed that the
bands were due to the formation of a (dA)21:ODN 1*
triplex. Similarly, when ODN 1 was added to a solution
containing (dA)21*, bands with mobilities similar to that
of the (dT)14:(dA)21*:(dT)14 triplex were also observed
(Figure 5, lanes 15-19).
Con clu sion . To find ODNs with triplex stabilization
capability, we designed and synthesized novel branched
ODNs 1 and 2 with pentaerythritols at their branch
points. Branched ODNs 1 and 2 were synthesized on a
solid support using bis(phosphoramidite) 11. The stability
of the triplexes formed by branched ODNs 1 and 2 with
(dA)21 or (dT)21 was studied by thermal denaturation.
Branched ODN 1 formed a stable parallel T‚AT-type
triplex with (dA)21 (Tm ) 38.9 °C) in a buffer of 0.01 M
sodium phosphate (pH 7.0) containing 0.5 M NaCl and
0.02 M MgCl2, and branched ODN 2 formed a stable
antiparallel A‚AT-type triplex with (dT)21 (Tm ) 44.2 °C)
in the same buffer. The formation of these triplexes was
also confirmed by circular dichroism (CD) measurements
and a gel retardation assay.
The CD spectra of the mixture of a 1:1 molar ratio of
branched ODN 2:(dT)21 in a buffer of 0.01 M sodium
phosphate (pH 7.0) containing 0.5 M NaCl and 0.02 M
MgCl2 at 5 °C was next compared with the appropriately
normalized summed spectra of branched ODN 2 and
(dT)21, and (dA)14 and (dA)14:(dT)21 (Figure 4b). The CD
spectrum (solid line) of the branched ODN 2:(dT)21
solution had positive CD bands at 218 and 281 nm,
shoulders near 235 and 260 nm, and negative CD bands
at 206 and 248 nm. The shape of the spectrum was
obviously different from that of the normalized summed
spectrum of branched ODN 2 and (dT)21. Furthermore,
the positive CD bands at around 218 and 280 nm and
the negative CD bands at around 206 and 248 nm for
the branched ODN 2:(dT)21 solution were clearly less
intense than those in the normalized summed spectra
from (dA)14 and (dA)14:(dT)21, although the shapes of these
spectra were similar to each other. Recently, Howard et
al. reported that the CD spectrum of a (dT)n:(dA)n:(dA)n-
type triplex had maxima at 218 and 282 nm, a shoulder
near 260 nm, and a minimum at 248 nm.15 Therefore,
these CD spectral data also support the existence of a
triplex between branched ODN 2 and (dT)21.
Gel Reta r d a tion Assa y. The formation of triplexes
was further confirmed by a gel retardation assay. Gel
retardation assays were carried out in a 90 mM Tris-
borate buffer (pH ) 7.8) containing 10 mM MgCl2 at room
temperature. The experiments presented in Figure 5
show the formation of the (dT)14:(dA)21*:(dT)14 triplex
(lanes 1-7), the (dA)21:ODN 1* triplex (lanes 8-13), and
the (dA)21*:ODN 1 triplex (lanes 14-19). The asterisks
indicate 32P-labeled ODNs.
As can be seen in Figure 5, lanes 1-7, as the molar
ratio of (dT)14 increased, the bands corresponding to the
double strand [(dA)21*:(dT)14] (Figure 5, lanes 2-4), which
has a slower mobility than (dA)21*, and those correspond-
ing to the triple strand [(dT)14:(dA)21*:(dT)14], which have
slower mobilities than (dA)21* and (dA)21*:(dT)14, became
more intense. On the other hand, when (dA)21 was added
to a solution containing ODN 1*, bands with mobilities
Recently, several kinds of branched ODNs with 3′-
deoxy-â-D-psicothymidine, 1-(2-methyl-â-D-arabinofura-
nosyl)uracil, or 4′-C-(hydroxymethyl)thymidine at their
branch points have been synthesized, and some of these
have been shown to form stable triplexes with single-
stranded ODNs.7,8 Branched ODNs can target single-
stranded ODNs or RNAs. Therefore, we believe that
branched ODNs will be a new class of antisense ODNs.
Exp er im en ta l Section
NMR spectra were recorded at 270 MHz (1H), at 125 MHz
(13C), and at 202 MHz (31P) and 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.
2-[[(ter t-Bu t yld ip h en ylsilyl)oxy]m et h yl]-2-(h yd r oxy-
m eth yl)-1,3-p r op a n ed iol (6a ), 2,2-Bis[[(ter t-bu tyld ip h en -
ylsilyl)oxy]m eth yl]-1,3-p r op a n ed iol (6b), a n d 3-O-(ter t-
Bu tyld ip h en ylsilyl)-2,2-bis[[(ter t-bu tyld ip h en ysilyl)oxy]-
m eth yl]-1,3-p r op a n ed iol (6c). A mixture of pentaerythritol
5 (4.08 g, 30.0 mmol), TBDPSCl (17.2 mL, 66.1 mmol), and
imidazole (8.99 g, 132 mmol) in DMF (500 mL) was stirred at
(15) Howard, F. B.; Miles, H. T.; Ross, P. D. Biochemistry 1995, 34,
7135-7144.

1216 J . Org. Chem., Vol. 64, No. 4, 1999
Ueno et al.
room temperature. After 30 min, EtOH (10 mL) was added to
the mixture, and the whole was stirred for 10 min. The mixture
was concentrated in vacuo and was taken in AcOEt, which
was washed with saturated aqueous NaHCO3 and brine. The
separated organic phase was dried (Na2SO4) and concentrated
to dryness. The residue was purified by column chromatog-
raphy (SiO2, 0-5% MeOH in CHCl3) to give 6a 10 (1.48 g, 13%),
6b (10.1 g, 55%), and 6c (7.95 g, 31%). The physical data of
6a : mp 69-72 °C; 1H NMR (CDCl3) δ 7.67-7.37 (m, 10H),
3.72 (d, 6H, J ) 4.8), 3.64 (s, 2H), 2.60 (t, 3H, J ) 4.8, D2O
exchangeable), 1.07 (s, 18H); 13C NMR (CDCl3, 13C signals were
assigned on the basis of DEPT experiment) δ 135.59 (CH),
132.64 (C), 130.03 (CH), 127.91 (CH), 65.32 (CH2), 64.83 (CH2),
45.62 (C), 26.91 (CH3), 19.21 (C); HRMS (FAB) calcd for
C21H31O4Si 375.1990, found 375.1974. Anal. Calcd for C21H30O4-
Si: C, 67.34; H, 8.07. Found: C, 67.44; H, 7.90. The physical
(m, 4H), 3.06 (m, 4H), 2.98 (t, 4H), 1.67 (br s, 4H); 13C NMR
(CDCl3, 13C signals were assigned on the basis of DEPT
experiment) δ 158.4 (C), 157.1 (C), 144.8 (C), 136.1 (C), 129.9
(CH), 128.0 (CH), 127.8 (CH), 126.8 (CH), 113.1 (CH), 86.3 (C),
62.4 (CH2), 61.7 (CH2), 61.5 (CH2), 55.2 (CH3), 46.1 (C), 39.4
(CH3), 29.6 (CH2). Anal. Calcd for C55H62N2O12‚H2O: C, 68.73;
H, 6.71; N, 2.91. Found: C, 68.74; H, 6.51; N, 2.93.
1,3-O-Bis[[[N-[3-O-(4,4′-d im eth oxytr ityloxy)p r op yl]ca r -
b a m oyl]-2,2-b is[[N-(3-h yd r oxyp r op yl)ca r b a m oyl]oxy]-
m eth yl]-1,3-pr opan ediol (10). N,N-Carbonyldiimidazole (6.49
g, 40.0 mmol) and DMAP (120 mg, 1.00 mmol) were added to
a solution of 9 (9.43 g, 10.0 mmol) in DMF (40 mL), and the
mixture was stirred at room temperature. After 40 min,
3-amino-1-propanol (11.5 mL, 150 mmol) was added to the
mixture, and it was stirred at room temperature for 1 h. The
mixture was concentrated in vacuo and was taken in AcOEt,
which was washed with H2O and brine. The separated organic
phase was dried (Na2SO4) and concentrated to dryness. The
residue was purified by column chromatography (SiO2, 1-3%
MeOH in CHCl3) to give 10 (10.4 g, 91%): 1H NMR (DMSO-
d6) δ 7.37-7.20 (m, 18H), 7.09 (br s, 4H, D2O exchangeable),
6.89-6.86 (m, 8H), 4.39 (t, 2H, J ) 5.0, D2O exchangeable),
3.94 (br s, 8H), 3.72 (s, 12H), 3.39 (m, 4H), 3.05-2.97 (m, 12H),
1.66 (m, 4H), 1.54 (m, 4H); 13C NMR (CDCl3, 13C signals were
assigned on the basis of DEPT experiment) δ 158.4 (C), 145.0
(C), 136.2 (C), 129.9 (CH), 128.1 (CH), 127.8 (CH), 126.8 (CH),
113.1 (CH), 86.2 (C), 61.6 (CH2), 59.6 (CH2), 55.2 (CH3), 39.2
(CH2), 38.0 (CH2), 32.3 (CH2), 29.7 (CH2); HRMS (FAB) calcd
for C63H77N4O16 1145.5330, found 1145.5410. Anal. Calcd for
1
data of 6b: mp 85-86 °C; H NMR (CDCl3) δ 7.62-7.32 (m,
20H), 3.75 (d, 4H, J ) 6.0), 3.67 (s, 4H), 2.26 (t, 2H, J ) 6.0,
D2O exchangeable), 1.02 (s, 18H); 13C NMR (CDCl3, 13C signals
were assigned on the basis of DEPT experiment) δ 135.6 (CH),
132.8 (C), 129.9 (CH), 127.8 (CH), 64.8 (CH2), 64.6 (CH2), 46.4
(C), 26.9 (CH3), 19.2 (C); HRMS (FAB) calcd for C37H49O4Si2
613.3167, found 613.3141. Anal. Calcd for C37H48O4Si2: C,
72.50; H, 7.89. Found: C, 72.49; H, 7.84. The physical data of
6c: mp 92-94 °C; 1H NMR (CDCl3) δ 7.58-7.25 (m, 30H), 3.81
(d, 2H, J ) 6.0), 3.74 (s, 6H), 2.78 (t, 1H, J ) 6.0, D2O
exchangeable), 0.96 (s, 27H); 13C NMR (CDCl3, 13C signals were
assigned on the basis of DEPT experiment) δ 135.6 (CH), 132.6
(C), 130.0 (CH), 127.9 (CH), 65.3 (CH2), 64.7 (CH2), 64.6 (CH2),
45.6 (C), 25.9 (CH3), 19.2 (C); HRMS (FAB) calcd for C53H67O4-
Si3 851.4343, found 851.4391. Anal. Calcd for C53H66O4Si3: C,
74.77; H, 7.81. Found: C, 74.76; H, 7.97.
C
63H76N4O16‚3/4H2O: C, 65.30; H, 6.74; N, 4.83. Found: C,
65.23; H, 6.60; N, 4.90.
2,2-Bis[[[N-[3-O-[[â-cya n oeth oxy)(N,N-d iisop r op yla m i-
n o)p h osp h in o]oxy]p r op yl]ca r b a m oyl]oxy]m et h yl]-1,3-
bis[N-[3-O-(4,4′-d im eth oxytr ityloxy)p r op yl]ca r ba m oyl]-
1,3-p r op a n ed iol (11). After successive coevaporation with
pyridine, 10 (458 mg, 0.40 mmol) was dissolved in CH2Cl2 (12
mL) containing N,N-diisopropylethylamine (0.28 mL, 1.60
mmol). 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite
(0.27 mL, 1.20 mmol) was added to the solution, and the
reaction mixture was stirred for 10 min at room temperature.
Aqueous saturated NaHCO3 and CHCl3 were added to the
mixture, and the separated organic layer was washed with
aqueous saturated NaHCO3, brine, dried (Na2SO4), and con-
centrated. The residue was purified by column chromatogra-
phy (neutralized SiO2, AcOEt) to give 11 (400 mg, 65%): 1H
NMR (CDCl3) δ 7.42-6.81 (m, 26H), 5.14 (br s, 2H), 4.98 (br
s, 2H), 4.03 (s, 8H), 3.78 (s, 12H), 3.89-3.42 (m, 12H), 3.26
(m, 8H), 3.16 (t, 4H), 2.63 (t, 4H), 1.78 (m, 8H), 1.19 (d, 12H),
1.17 (d, 12H); 31P NMR (CDCl3) δ 148.51.
Syn th esis of ODNs. ODNs were synthesized on a DNA
synthesizer (Applied Biosystem Model 391A) by using a
slightly modified 1-µmol-scale cycle in the “trityl off” mode.
The 1-µmol-scale cycle supplied by ABI was modified in the
following manner: phosphoramidite coupling, a 300-s “wait”
for 5′-O-(dimethoxytrityl)thymidine 3′-O-(2-cyanoethyl N,N-
diisopropylphosphoramidite) and N6-benzoyl-5′-O-(dimethoxy-
trityl)adenosine 3′-O-(2-cyanoethyl N,N-diisopropylphosphora-
midite) and a 600-s “wait” for 11. A 0.025 M solution of 11 in
CH3CN was used in the branching step. Controlled-pore glass
(CPG, 500-Å pore size, Funakoshi Co.) was used as a solid
support. The ODN (1 µmol) linked to the solid support was
treated with concentrated NH4OH (2 mL) at 55 °C for 16 h.
The ammoniacal solutions of ODNs were concentrated under
reduced pressure. The crude ODNs were purified by 20%
polyacrylamide gel electrophoresis under denaturing condi-
tions (7 M urea), followed by reversed-phase HPLC. The
deprotected ODNs 1 and 3 were obtained in 19 and 21 OD260
units, respectively. Similarly, ODNs 2 and 4 were obtained in
17 and 16 OD260 units, respectively.
1,3-O-Bis(ter t-bu tyldiph en ysilyl)-2,2-bis[[[N-(3-h ydr oxy-
p r op yl)ca r ba m oyl]oxy]m eth yl]-1,3-p r op a n ed iol (7). N,N-
Carbonyldiimidazole (9.73 g, 60.0 mmol) and DMAP (370 mg,
3.00 mmol) were added to a solution of 6b (9.19 g, 15.0 mmol)
in DMF (50 mL), and the mixture was stirred at room
temperature. After 40 min, 3-amino-1-propanol (17.2 mL, 225
mmol) was added to the mixture, and the whole was stirred
at room temperature for 1 h. The mixture was concentrated
in vacuo and was taken in AcOEt, which was washed with
H2O and brine. The separated organic phase was dried (Na2-
SO4) and concentrated to dryness. The residue was purified
by column chromatography (SiO2, 1% MeOH in CHCl3) to give
7 (10.9 g, 88%): mp 148-149 °C; 1H NMR (CDCl3) δ 7.64-
7.31 (m, 20H), 4.69 (br s, 2H, J ) 5.8, D2O exchangeable), 4.09
(s, 4H), 3.67 (s, 4H), 3.62 (m, 4H), 3.28 (m, 4H), 2.80 (br s, 2H,
D2O exchangeable), 1.65 (m, 4H), 1.02 (s, 18H); 13C NMR
(CDCl3, 13C signals were assigned on the basis of DEPT
experiment) δ 157.2 (C), 135.7 (CH), 133.2 (C), 129.7 (CH),
127.7 (CH), 65.9 (CH2), 61.4 (CH2), 59.4 (CH2), 50.5 (CH3), 45.8
(C), 37.4 (CH2), 32.5 (CH2), 26.9 (CH3), 19.3 (C); HRMS (FAB)
calcd for C45H63Ν2O8Si2 815.4119, found 815.4087. Anal. Calcd
for C45H62N2O8Si2: C, 66.30; H, 7.67; N, 3.44. Found: C, 66.32;
H, 7.68; N, 3.66.
2,2-Bis[[[N-[3-O-(4,4′-d im et h oxyt r it yloxy)p r op yl]ca r -
ba m oyl]oxy]m eth yl]-1,3-p r op a n ed iol (9). A mixture of 7
(10.0 g, 12.3 mmol) and DMTrCl (9.17 g, 27.1 mmol) in
pyridine (50 mL) was stirred at room temperature for 1 h.
EtOH (15 mL) was added to the mixture, and the whole was
stirred for 10 min. The mixture was evaporated under reduced
pressure. The residue was taken in AcOEt, which was washed
with saturated aqueous NaHCO3 and brine. The separated
organic phase was dried (Na2SO4) and concentrated to dryness.
The residue was dissolved in THF (50 mL). TBAF (1 M in THF,
50 mL, 50 mmol) was added to the solution, and the resulting
mixture was stirred overnight at room temperature. The
mixture was evaporated under reduced pressure and was
taken in AcOEt, which was washed with H2O and brine. The
separated organic phase was dried (Na2SO4) and concentrated
to dryness. The residue was purified by column chromatog-
raphy (SiO2, 0-2% MeOH in CHCl3) to give 9 (9.88 g, 85%):
Electr osp r a y Ion iza tion Ma ss Sp ectr om etr y. Spectra
were obtained on a Quattro II (Micromass, Manchester, UK)
triple quadrupole mass spectrometer equipped with an ESI
source in the negative ion mode. The HPLC-purified ODN
samples were dissolved in aqueous 50% 2-propanol containing
1% triethylamine (10 pmol ODN/µL) and introduced into the
1
mp 148-149 °C; H NMR (DMSO-d6) δ 7.37-7.21 (m, 18H),
6.97 (br s, 2H, D2O exchangeable), 6.89-6.86 (m, 8H), 4.44
(br s, 2H, D2O exchangeable), 3.88 (s, 4H), 3.72 (s, 12H), 3.36

Branched Oligodeoxynucleotides
J . Org. Chem., Vol. 64, No. 4, 1999 1217
ion source through a loop injector with a carrier solvent, 33%
aqueous methanol, flowing at 10 mL/min flow rate. About 15
scans were acquired in approximately 1 min period and
combined to obtain smoothed spectra. All molecular masses
of the ODNs were calculated from the multiple-charge nega-
tive-ion spectra. The observed average molecular masses of 1,
2, 3, and 4 were 9058.6, 9309.0, 7008.9, and 7197.1, respec-
tively, and fit the calculated molecular weights (theoretical
average molecular masses) for these compounds, i.e., 9058.0
(for 1, C301H404N60O208P28), 9310.4 (for 2, C301H376N144O152P28),
procedure used in the thermal denaturation study, and spectra
were measured at 5 °C. The ellipticities of triplexes were
recorded from 200 to 350 nm in a cuvette with a path length
1
mm. CD data were converted into mdeg‚mol of
residues-1‚cm-1
.
Gel Electr op h or esis. For gel retardation assays of the
triplexes, a nondenaturating 15% polyacrylamide gel (99:1
acrylamide/bisacrylamide) containing 90 mM Tris-borate (pH
) 7.8) and 10 mM MgCl2 was prepared and run at room
temperature using same buffer. ODNs were labeled with 32P
at the 5′-end using [γ-32P]ATP and T4 polynucleotide kinase.17
A solution containing each ODN in a 90 mM Tris-borate buffer
(pH ) 7.8) containing 10 mM MgCl2 was heated at 100 °C for
3 min and then gradually cooled to room temperature. The
samples were loaded in 15% Ficoll (Pharmacia type 400).
Electrophoresis was performed for 20 h with 60 V at room
temperature. After electrophoresis, gels were autoradio-
graphed using Kodak X-Omat J B film at -70 °C.
7008.7 (for 3, C231H314N46O162P22), and 7197.9 (for 4, C231H293N109
-
O120P22) within a commonly accepted error range of ESI MS,
0.01%.
Th er m a l Den a tu r a tion a n d CD Sp ectr oscop y. Each
solution containing each branched ODN (3 µM) and each single
stranded DNA (3 µM) in an appropriate buffer was heated at
100 °C for 3 min and then gradually cooled to 4 °C. Thermal-
induced transitions of each mixture were monitored at 260 and
284 nm on a Perkin-Elmer Lambda2S. Sample temperature
was increased 0.5 °C/min. Extinction coefficients of the branched
ODNs were calculated from those of mononucleotides and
dinucleotides according to the nearest-neighbor approxima-
tion.16 Samples for CD spectroscopy were prepared by the same
Ack n ow led gm en t . This investigation was sup-
ported in part by a Grant-in-Aid for the Encouragement
of Young Scientists from the Ministry of Education,
Science, Sports, and Culture of J apan and a Grant from
the “Research for the Future” Program of the J apan
Society for the Promotion of Science (J SPS-RFTF97-
I00301).
(16) Puglisi, J . D.; Tinoco, I., J r. In Methods in Enzymology;
Dahlberg, J . E., Abelson, J . N., Eds.; Academic Press: San Diego,
1989: Vol. 180, pp 304-325.
(17) Maniatis, T.; Fritsch, E. F.; Sambrook, J . In Molecular Clon-
ing: a Laboratory Manual; Cold Spring Harbor University Press: Cold
Spring Harbor, NY, 1982.
J O981831E