Synthesis of branched oligonucleotides with three different sequences using an oxidatively removable tritylthio group

Article abstract of DOI:10.1021/jo071173a

(Chemical Equation Presented) We synthesized a three-way branched oligodeoxynucleotide (ODN) 30-mer using a new branch unit with acid-labile DMTr and oxidatively cleavable TrS groups as orthogonal protecting groups. The branched ODN was successfully synthesized using 5-[3,5-bis(trifluoromethyl) phenyl]-l H-tetrazole and (2R,8aS)-(+)-(camphorylsulfonyl)oxaziridine as the activator of phosphoramidite units and the oxidizing reagent, respectively. We also found that the TrS group was orthogonal to the Lev, TBDMS, and Fmoc groups. These results indicate the possibility of the synthesis of more complex four- and five-way branched ODNs by the combined use of DMTr, TrS, Lev, TBDMS, and Fmoc groups.

Full text of DOI:10.1021/jo071173a

Synthesis of Branched Oligonucleotides with Three Different
Sequences Using an Oxidatively Removable Tritylthio Group
Eri Utagawa,‡,§ Akihiro Ohkubo,‡.§ Mitsuo Sekine,*,‡.§ and Kohji Seio‡.§
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan,
and CREST, Japan Science and Technology Agency (JST), 4259 Nagatsuta, Yokohama 226-8501, Japan
msekine@bio.titech.ac.jp
ReceiVed June 3, 2007
We synthesized a three-way branched oligodeoxynucleotide (ODN) 30-mer using a new branch unit
with acid-labile DMTr and oxidatively cleavable TrS groups as orthogonal protecting groups. The branched
ODN was successfully synthesized using 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole and (2R,8aS)-
(+)-(camphorylsulfonyl)oxaziridine as the activator of phosphoramidite units and the oxidizing reagent,
respectively. We also found that the TrS group was orthogonal to the Lev, TBDMS, and Fmoc groups.
These results indicate the possibility of the synthesis of more complex four- and five-way branched
ODNs by the combined use of DMTr, TrS, Lev, TBDMS, and Fmoc groups.
Introduction
Branched ODNs have also been used in the construction of
programmed nanostructures. In particular, branched ODNs
containing different sequences have often been used as the
vertices of such nanostructures including tetrahedral and cubic
forms.8-14
In general, central phosphoramidite building units having two
hydroxyl groups protected with different protecting groups,
PRO1 and PRO2, have been used to synthesize three-way
branched ODNs, as depicted in Figure 1. For example, first,
the DNA (a) is synthesized in the usual manner on solid
supports. Next, the DMTr group is removed, and a central
phosphoramidite building unit is linked to the resulting 5′-
terminal hydroxyl group of DNA (a). After removing one of
the two hydroxyl protecting groups (PRO1), similar chain
elongation is continued at this branching site until a new DNA
Recently, much interest has been paid to synthetic branched
oligodeoxynucleotides (ODNs).1 For example, branched ODNs
have shown high affinity for single-stranded ODNs to form
alternate-strand triplexes.2,3 Another compound with three
different ODNs was used as an efficient template for the
straightforward synthesis of trimeric structures.4 In addition,
central building units with branching points were used for the
synthesis of dendrimeric ODNs as multilabeled DNA probes
to increase the sensitivity of hybridization experiments.5-7
‡ Tokyo Institute of Technology.
§ CREST, JST.
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10.1021/jo071173a CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/27/2007
J. Org. Chem. 2007, 72, 8259-8266
8259

Utagawa et al.
FIGURE 2. Reversed-phase HPLC profile of the mixture obtained
after treatment of 13 with 3% TCA.
the usefulness of this branch unit in the synthesis of a branched
ODN 30-mer. Although we previously reported the use of the
methoxytritylthio (MMTrS) group, which can be removed by
mild oxidation as a protecting group for the 5′-hydroxyl group
during ODN synthesis,25-28 we chose the TrS group in this study
instead of the MMTrS group because it was stable under the
acidic conditions for the removal of the DMTr group. Since
the DMTr and TrS groups can be removed under mild acidic
and oxidative conditions, respectively, base-labile protecting
groups can remain intact. Moreover, because the TrS group was
proved to be stable toward various basic reagents such as
piperidine, hydrazine, and TBAF, the possibility of using the
TrS group in combination with other protecting groups, such
as Fmoc, Lev, and TBDMS, was indicated as discussed later.
FIGURE 1. General structure of branched building unit 1 and a
designed branched building unit 2.
SCHEME 1. Synthetic Route to 2
Results and Discussion
Branch Unit Synthesis. As a branch unit, we designed 2
which can be synthesized from trimesic acid (3) and two
O-protected 3-amino-1-propanol derivatives 4 and 5 (Scheme
1).
First, the synthesis of O-protected 3-amino-1-propanol deriva-
tives 4 and 5 was examined. Compound 4 was synthesized from
1,3-propanediol in 57% yield in three steps (see Supporting
Information). Compound 5 has already been reported in the
literature.29 The synthesis of the branch unit 2 is shown in
Scheme 2. Condensation of the amine 5 with trimesic acid
dimethyl ester30,31 (6) in the presence of 1,1′-carbonyldiimida-
zole (CDI) gave compound 7 in 90% yield, as shown in Scheme
2. Treatment of 7 with 1 M NaOH gave the monocarboxylic
acid 8 as the triethylammonium salt after purification by column
chromatography using the solvent system containing triethyl-
amine. Similarly, the amine 4 was condensed with compound
8 to give the diamide 9 in a quantitative yield, which, in turn,
was converted to the monocarboxylate 10 in 87% yield by a
similar alkaline treatment as mentioned above. Finally, com-
pound 10 was coupled with 3-amino-1-propanol to give com-
pound 11 in 94% yield. The alcohol 11 was converted in the
usual manner to the phosphoramidite derivative 2 in 63% yield.
Stability of the TrS Group Toward Detritylation Condi-
tions. To confirm the stability of the TrS group toward
chain (b) is elongated. Finally, the remaining protecting group
PRO2 at the branching center is removed, and the third DNA
chain (c) is constructed in a similar manner. In this synthesis
of branched ODN, the PRO1 and PRO2 groups must be cleaved
independently under mild conditions, and the selection is quite
important. Previously, DMTr/Fmoc,15,16 DMTr/levulinyl,17-21
and DMTr/TBDMS22-24 have been reported as PRO1/PRO2 in
the literature.
In this paper, we report a new set of protecting groups, PRO1/
PRO2, for the synthesis of three-way branched molecules with
three different ODNs. As PRO1 and PRO2 on the branched
building unit, we chose the tritylthio (TrS) and DMTr groups,
respectively, as shown in compound 2 of Figure 1 and proved
(15) Avino, A.; Grimau, M. G.; Frieden, M.; Eritja, R. HelV. Chim. Acta
2004, 87, 303-316.
(16) Oliviero, G.; Amato, J.; Borbone, N.; Galeone, A.; Petraccone, L.;
Varra, M.; Piccialli, G.; Mayol, L. Bioconjugate Chem. 2006, 17, 889-
898.
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Bioorg. Med. Chem. Lett. 2000, 10, 1853-1856.
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2333.
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Acids 2005, 24, 927-929.
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Neubauer, G.; Eritja, R. Bioorg. Med. Chem. 1996, 4, 1649-1658.
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1993, 49, 8761-8770.
(19) Ueno, Y.; Shibata, A.; Matsuda, A.; Kitade, Y. Bioconjugate Chem.
2003, 14, 684-689.
(20) Ivanov, S. A.; Volkov, E. M.; Oretskaya, T. S.; Muller, S.
Tetrahedron 2004, 60, 9273-9281.
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(22) Damha, M. J.; Ganeshan, K.; Hudson, R. H. E.; Zabarylo, S. V.
Nucleic Acids Res. 1992, 20, 6565-6573.
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377.
8260 J. Org. Chem., Vol. 72, No. 22, 2007

Synthesis of Branched Oligonucleotides
SCHEME 2. Synthesis of the Branch Unit 2
SCHEME 3. Stability of the TrS Group under Detritylation
Conditions
14 was analyzed by reversed-phase HPLC. As shown in Figure
2, a new compound with the TrS group intact was obtained in
high purity at the retention time of 42 min. This result suggested
that the TrS group was sufficiently stable under the conditions
for the removal of the DMTr group. In addition to the HPLC
analysis, we confirmed the structure of 14 by MALDI-TOF mass
analysis.
Synthesis of Branched ODN 9-mer. Next, we determined
the best conditions for the synthesis of branched ODNs by
synthesizing a simple model compound 15 with three trithymidy-
lates at the three positions, as shown in Scheme 4. In this
structure, two of the three TpTpT strands of 15 are attached to
the trimesic acid branch point at their 3′-ends, whereas the other
TpTpT is attached at its 5′-end. To avoid the cleavage of the
sulfenyl ester by iodine oxidation, the oxidizer should be
changed to other reagents during chain elongation of pTpTpT
in the presence of the TrS group. In this study, we tested two
oxidizers, tert-butyl peroxide (t-BuOOH)32 and (2R,8aS)-(+)-
(camphorylsulfonyl)oxaziridine (CSO).33
In addition, the activating reagents were also optimized by
testing three kinds of activating agents, 1H-tetrazole (Tet),
5-benzylthio-1H-tetrazole (BTT),34 and 5-[3,5-bis(trifluorom-
ethyl)phenyl]-1H-tetrazole (Activator 42: Act42),35 because the
coupling yield was expected to be lowered by the synthesis of
oligonucleotides with unusual structures, such as branched
ODNs.
deprotection conditions of the DMTr group on solid supports,
several experiments were conducted using the solid support 12,
as shown in Scheme 3. This support was prepared by condensa-
tion of the branch unit 2 with the 5′-hydroxyl group of
thymidine-loaded CPG followed by successive capping and
oxidation reactions. First, the DMTr group was removed from
12 by treatment with 3% trichloroacetic acid (3% TCA, 30 s ×
3). Subsequently, the support was exposed again to 3% TCA
at room temperature for 5 min. We chose such a two-step
treatment with 3% TCA in order to simplify the reaction
conditions by removing the still reactive DMTr cation. The
product 13 was treated with aq NH3, and the desired compound
The branch unit 2 (40 equiv) was condensed with the
phosphate-protected trithymidylate 16, which was synthesized
in advance on the CPG support using one of the above-
mentioned activating reagents, by use of 80 equiv of Tet, BTT,
or Act42 for 2 min. After the capping of the remaining hydroxyl
(32) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett. 1986,
27, 4191-4194.
(33) Manoharan, M.; Lu, Y.; Casper, M. D.; Just, G. Org. Lett. 2000, 2,
243-246.
(34) Welz, R.; Mu¨ller, S. Tetrahedron Lett. 2002, 43, 795-797.
(35) Michael, L.; Andreas, W. PCT Int. WO 2006116476, 2006.
J. Org. Chem, Vol. 72, No. 22, 2007 8261

Utagawa et al.
SCHEME 4. Synthesis of the Branched ODN 15 with Three Trithymidylates
function by treatment with Ac2O/pyridine (9:1, v/v) in the
presence of 0.1 M DMAP for 2 min, the oxidation reactions
were carried out using either t-BuOOH (0.5-0.6 M in CH3-
CN, 10 min) or CSO (0.5 M in CH3CN, 3 min) to give 17.
After the detritylation of the branch unit 17 by treatment
with 3% TCA/CH2Cl2 (30 s × 3), similar stepwise condensa-
tions of a thymidine phosphoramidite unit to the resulting
hydroxyl component 18 (three times) gave compound 19.
In this synthesis, Tet, BTT, or Act42 and either t-BuOOH or
CSO were also tested as condensing and oxidizing agents,
respectively. The 5′-hydroxyl group of the terminal thymidine
residue of 19 was capped by acetylation, and then the TrS
group on the branch unit was removed by treatment with
a 1 M iodine in pyridine/H2O (9:1, v/v) for 2 min to give
compound 20. The third trithymidylate chain was synthesized
using an activating reagent, Tet, BTT, or Act42, and iodine
oxidation. Thus, compound 15 was obtained after NH3 treatment.
The resulting product was analyzed by reversed-phase HPLC,
as shown in Figure 3. When Tet was used as the activator in
combination with t-BuOOH as the oxidizer, shorter sequences
resulting from failure coupling were observed, as shown in
Figure 3a. This result indicated that the activation of phos-
phoramidite by Tet was not enough for the synthesis of the
ODNs with an unusual branch structure. Although changing the
activating reagent from Tet to BTT slightly decreased the ratio
of failure sequences to 15 (data not shown), a stronger activating
reagent Act42 gave a better result, as shown in Figure 3b. These
results indicated that the increase of coupling efficiency could
be an essential factor that improves the synthesis of the branched
ODN. Further improvement was achieved by changing the
oxidizer from t-BuOOH to CSO. By this combination, the target
material was synthesized in much better purity. Since CSO is a
stronger oxidizer than t-BuOOH, it could oxidize the phosphite
intermediate more completely and in a shorter reaction time (3
min for CSO, 10 min for t-BuOOH). In these experiments,
structure of 15 was characterized by MALDI-TOF mass
analyses.
We also checked the stability of the TrS group toward CSO
(3 min × 10) in a protocol similar to that described in Scheme
3. Briefly, 12 was prepared on solid phase by use of 0.5 M
CSO/CH3CN (3 min × 10), which corresponded to the oxidation
time required for the synthesis of oligodeoxynucleotide 10-mers,
in place of the t-BuOOH treatment. After the detritylation by
3% TCA (30 s × 3) and the aqueous ammonia treatment, the
resulting mixture containing 14 was analyzed by reversed-phase
HPLC. Although small amounts of byproducts were observed
at around 10 min, the HPLC profile showed the presence of 14
having the intact TrS group with good purity (Figure S20). The
FIGURE 3. Reversed-phase HPLC profiles of the branched ODN oligomer 15. Panel (a): Tet and t-BuOOH were used as the activator and the
oxidizing agent, respectively. Panel (b): Act42 and t-BuOOH were used. Panel (c): Act42 and CSO were used. See Experimental Section.
8262 J. Org. Chem., Vol. 72, No. 22, 2007

Synthesis of Branched Oligonucleotides
SCHEME 5. Synthesis of 23
partial elimination of the TrS group during the CSO treatment
indicated the possible formation of the side products during the
synthesis of the second DNA fragments such as the DNA (b)
described below. However, the byproducts generated by such
side reactions could be easily separated from the target molecule
by a simple DMTr-On purification on C18 reversed-phase
column after the elongation of the third DNA fragments such
as the DNA (c). Considering these results of CSO totally, we
decided to use the protocol using CSO to synthesize a longer
branched DNA 30-mer.
Synthesis of Branched ODN 30-mer. Finally, the synthesis
of a branched ODN 30-mer 23 was achieved from the CPG
support by the combined use of the conventional protocol of
DNA synthesis on an automated DNA synthesizer and the
manual synthesis using Act42 and CSO instead of Tet and
aqueous iodine, respectively, as shown in Scheme 5. In this
structure, DNA (a), DNA (b), and DNA (c) should have the
sequences that did not interact with each other. For this purpose,
we chose a set of three sequences, 5′-GGTGTAGCGC-3′, 5′-
GGGCTACCTC-3′, and 5′-CCGCTAGCAC-3′, which were
reported by Endo et al. in the development of programmable
DNA nanostructures.13 Similarly to 15, DNA (a) was attached
to the branch point at its 5′-end, and DNAs (b) and (c) were
attached at their 3′-ends.
The DNA (a):5′-GGTGTAGCGC-3′ was elongated on a DNA
synthesizer, and the branch unit 2 (40 equiv) was condensed
with the 5′-terminal hydroxyl group of DNA (a) in the presence
of Act42 (80 equiv) to give 21. After the elongation of DNA
(b):5′-GGGCTACCTC-3′ using Act42 and CSO under the
conditions identical to those used for the synthesis of 15, the
5′-end of the terminal G residue was capped using acetic
anhydride to give 22. Finally, the TrS group was removed by
treatment with 1 M iodine solution, and the last strand DNA
(c):5′-CCGCTAGCAC-3′ was elongated on an automated DNA
synthesizer. To simplify the purification procedure, the DMTr
group on the last DNA (c) sequence was not removed before
exposing the aqueous ammonia solution. After the cleavage and
deprotection of the phosphate and base protecting groups by
treatment with aqueous ammonia, the compound with the DMTr
group was isolated from the crude material by reversed-phase
HPLC. After its isolation, the DMTr group was removed by
treatment with aqueous 2% TFA to give the target material 23.
Shown in Figure 4 is the anion-exchange HPLC profile of the
branched 30-mer 23, which showed high purity even after
isolation. The structure of 23 was characterized by the MALDI-
TOF mass analysis.
Hybridization Properties of the Branched ODN 30-mer
23. Hybridization properties of 23 with the complementary DNA
strands were studied. Here, the complementary strand to the
DNA (a) strand of 23 is named DNA (a′):5′-GCGCTACACC-
3′; the one complementary to DNA (b) is DNA (b′):5′-
GAGGTAGCCC-3′; and the one complementary to DNA (c)
is DNA (c′):5′-GTGCTAGCGG-3′. The complementary strands
of DNA (a′), (b′), and (c′) were separately hybridized to 23.
Each oligonucleotide was dissolved in 10 mM sodium cacody-
late (pH 7.0) containing 0.5 M NaCl and 10 mM MgCl2 so that
the final concentration of each ODN became 1.5 µM. The
melting curves of the complexes formed between 23 and its
complementary ODNs showed two inflection points (Figure
5A-C and Figure S21A-C). The Tm values are listed in Table
1. In all cases, melting curves at higher temperatures (around
75 °C) were commonly observed. In contrast, those at lower
temperatures were specific in each case. The DNA (a)/(a′)
duplex showed the lowest melting point (25 °C) compared to
the other duplexes (50 and 51 °C). Interestingly, the branched
ODN 30-mer 23 in the absence of any complementary strands
showed an inflection point at around 75 °C (Figures 4D and
S21D). From those experiments, we presumed that 23 formed
a higher-order structure and the higher inflection point corre-
sponds to the melting point of the higher-order structure. The
lower melting points would be those of the duplexes formed
between 23 and the complementary strands. It should be noted
that the DNA duplexes having the sequences identical to those
of DNA (a)/DNA (a′), DNA (b)/DNA (b′), and DNA (c)/ DNA
(c′) had similar Tm values of 53, 48, and 53 °C, respectively
FIGURE 4. Anion exchange HPLC profile of the branched ODN 30-
mer 23.
J. Org. Chem, Vol. 72, No. 22, 2007 8263

Utagawa et al.
FIGURE 6. Reversed-phase HPLC profiles of 14 obtained by the
successive treatment of 12 with various reagents. Panel (a): (i) 0.5 M
hydrazine monohydrate, Py/Ac2O (4:1, v/v), 20 min; (ii) 3% TCA, CH2-
Cl2, 30 s, 3 times; (iii) aq NH3. Panel (b): (i) 1 M TBAF, THF, 10
min; (ii) 3% TCA, CH2Cl2, 30 s, 3 times; (iii) aq NH3.
protecting groups, such as DMTr/Lev, DMTr/TBDMS, and
DMTr/Fmoc, have been used for the previous branch units
reported in the literature. If the TrS group is stable under the
conditions required for the selective removal of the Lev,
TBDMS, or Fmoc group, it could be used to synthesize branched
ODNs in combination with these protecting groups. Therefore,
we checked the stability of the TrS group under the deprotection
conditions of Lev, TBDMS, and Fmoc groups separately.
Two conditions were tested using the branch unit 2 on solid
supports: The reagents, NH2NH2 and TBAF, used for the
deprotection of the Lev36,37 and TBDMS groups,38 respectively,
were examined. First, compound 12 was exposed to NH2NH2
and TBAF for 20 and 10 min, respectively. The DMTr group
was removed by treatment with 3% TCA. The resulting
nucleotidic materials were cleaved from the solid support by
treatment with aqueous ammonia and then analyzed by reversed-
phase HPLC. As shown in Figure 6a and b, the peak corre-
sponding to compound 14 was clearly observed. These results
suggested that the TrS group was stable under the above
conditions.
FIGURE 5. Melting profile of the complexes formed. Panel (A):
between 23 and DNA (a′). Panel (B): between 23 and DNA (b′). Panel
(C): between 23 and DNA (c′). Panel (D): melting profile of 24. Panel
(E): 23 and DNA (a′) + DNA (b′) + DNA (c′).
The stability of the TrS group to piperidine used for the
deprotection of the Fmoc group16 was also tested in the liquid
phase by treatment of 11 with piperidine/DMF (1:5, v/v). As a
result, the TrS group proved to be stable for over 48 h by TLC
analysis.
TABLE 1. Melting Temperatures at 260 nm and Sequences of
Complementary Strands of DNA (a′), (b′), and (c′)a
lower Tm
(°C)
higher Tm
(°C)
DNA (a′) and 23
DNA (b′) and 23
DNA (c′) and 23
23
25
50
51
n.d.
26, 53
75
75
75
75
73
Conclusion
We synthesized the branch unit 2 with the DMTr and TrS
groups as orthogonal protecting groups. Because the TrS group
was unstable to iodine used for the oxidation of phosphite
intermediates during the usual DNA synthesis, a peroxide-type
oxidizer was tested for the synthesis of the branched ODN. As
a result, CSO proved to be efficient. In addition, Act42 was
more effective than the conventional Tet for the synthesis of
the branched ODN, probably because of its higher acidity.
From the DNA 10-mer on the CPG support, we synthesized
the three-way branched ODN 23 using the branch unit 2. The
three-way branched ODN with three different 10-mers at each
branch point could form duplexes with the complementary
strands. These results clearly demonstrated the usefulness of
the TrS group for the synthesis of the branched ODN in
combination with the DMTr group. Moreover, we also revealed
DNA (a′ + b′ + c′) and 23
a DNA (a′) 5′-GCGCTACACC-3′; DNA (c′) 5′-GTGCTAGCGG-3′;
DNA (b′) 5′-GAGGTAGCCC-3′.
(data not shown). The reasons of the large differences in the
Tm value between DNA (a)/DNA (a′) and the other duplexes in
the branched complexes are not clear. Because the DNA (a)
was attached to the branch point at its 5′-end and the other
strands were attached at their 3′-ends, such structural variations
could be one of the reasons.
We also measured the Tm values of 23 in the presence of all
three complementary strands DNA (a′) + DNA (b′) + DNA
(c′), as shown in Figure 5E and the bottom row of Table 1. The
differentiation of the UV melting curve suggested the presence
of three inflection points at 26, 53, and 73 °C (Figure S21E),
which indicated that the melting of the three duplexes and the
high-order structure occurred independently.
(36) Ueno, Y.; Shibata, A.; Matsuda, A.; Kitade, Y. Bioconjugate Chem.
2003, 14, 684-689.
(37) Van Boom, J. H.; Burgers, P. M. J. Tetrahedron Lett. 1976, 52,
4875-4878.
Stability of the TrS Group Under Various Basic Condi-
tions. As described in the Introduction, combinations of
(38) Damha, M. J.; Braich, R. S. Tetrahedron Lett. 1998, 39, 3907-
3910.
8264 J. Org. Chem., Vol. 72, No. 22, 2007

Synthesis of Branched Oligonucleotides
(500 MHz, CDCl3) δ 1.60 (t, J ) 6.3 Hz, 2H), 1.90 (m, 2H), 3.22-
3.29 (m, 4H), 3.36 (t, J ) 5.6 Hz, 2H), 3.58 (dd, J ) 6.1, 12 Hz,
2H), 3.74 (s, 6H), 3.87 (s, 3H), 6.18 (t, J ) 5.6 Hz, 1H), 6.72-
6.78 (m, 5H), 7.15-7.41 (m, 24H), 8.23 (s, 1H), 8.38 (s, 1H), 8.44
(s, 1H); 13C NMR (67.8 MHz, CDCl3) δ 29.1, 30.1, 37.8, 38.9,
52.5, 55.1, 62.1, 72.0, 76.6, 86.4, 113.1, 126.8, 127.3, 127.9, 128.0,
129.8, 129.8, 129.9, 130.3, 130.4, 130.9, 135.5, 135.8, 136.0, 142.7,
144.7, 158.4, 165.5, 165.6, 165.8. ESI-MS: calcd for C56H54N2O8-
SNa (M + Na)+, 937.3493; found, 937.3939.
Triethylammonium 3-[(3-(4,4′-Dimethoxytrityloxy)propyl)-
carbamoyl}-5-{(3-tritylsulfenyloxypropyl)carbamoyl]-
benzoate (10). Compound 9 (1.6 g, 1.7 mmol) was dissolved in
pyridine (10 mL). To the solution was added 1 M aq NaOH (2.1
mL). The resulting mixture was stirred at room temperature for 5
h, and then the reaction mixture was concentrated under reduced
pressure. The residue was diluted with ethyl acetate (150 mL) and
washed three times with brine (100 mL). The organic layer was
collected, dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was chromatographed on a silica gel
column (70 g) with chloroform/methanol/triethylamine (100:2:1,
v/v/v) to give 10 (1.5 g, 87%): 1H NMR (500 MHz, CDCl3) δ
1.23 (t, J ) 7.3 Hz, 9H), 1.58 (m, 2H), 1.88 (m, 2H), 2.94 (d, J )
6.6 Hz, 6H), 3.21 (m, 4H), 3.31 (t, J ) 5.9 Hz, 2H) 3.56 (dd, J )
6.6, 12 Hz, 2H), 3.74 (s, 6H), 6.17 (t, J ) 5.9 Hz, 1H), 6.45 (t, J
) 5.6 Hz, 1H), 6.79 (d, J ) 9.0 Hz, 4H), 7.14-7.42 (m, 24H),
8.19 (s, 1H), 8.48 (s, 1H), 8.51 (s, 1H); 13C NMR (67.8 MHz,
CDCl3) δ 9.4, 29.7, 30.3, 37.2, 38.2, 45.4, 55.1, 61.3, 71.9, 76.1,
86.1, 113.0, 126.7, 127.3, 127.8, 128.0, 128.1, 129.9, 130.0, 130.1,
134.6, 134.8, 136.2, 138.2, 142.8, 144.9, 158.3, 166.7, 166.8, 171.1.
ESI-MS: calcd for C61H68N3O8S (M + H)+, 1002.4712; found,
1002.4668.
N-[3-(4,4′-Dimethoxytrityloxy)propyl]-N′-(3-hydroxypropyl)-
N′′-(3-tritylsulfenyloxypropyl)benzene-1,3,5-tricarboxamide (11).
To a solution of 10 (1.9 g, 2.8 mmol) in THF (6 mL) was added
CDI (690 mg, 4.3 mmol). The resulting mixture was stirred at room
temperature for 1 h, and then a solution of 3-aminopropan-1-ol (1.5
g, 4.3 mmol) in THF (3 mL) was added. After being stirred at
room temperature for 1 h, the reaction mixture was concentrated
under reduced pressure but was not dried completely. The solution
was diluted with ethyl acetate (70 mL) and washed three times
with water (70 mL). The organic layer was collected, dried over
MgSO4, filtered, and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column (70 g) with
hexane/ethyl acetate (2:3, v/v) to give 11 (1.2 g, 94%): 1H NMR
(500 MHz, CDCl3) δ 1.53 (t, J ) 6.1 Hz, 2H), 1.69 (t, J ) 5.9 Hz,
2H), 1.83 (t, J ) 5.9 Hz, 2H), 3.14-3.19 (m, 4H), 3.27-3.30 (m,
2H), 3.43-3.49 (m, 4H), 3.58-3.61 (m, 2H), 3.69 (s, 6H), 3.79-
3.82 (m, 1H), 6.74-6.77 (m, 4H), 6.91-6.93 (m, 1H), 7.15-7.41
(m, 28H), 7.62-7.65 (m, 1H), 7.92 (s, 1H), 8.00 (s, 1H), 8.02 (s,
1H); 13C NMR (67.8 MHz, CDCl3) δ 29.4, 30.1, 31.8, 37.5, 38.5,
55.1, 59.9, 61.6, 72.0, 76.3, 76.7, 86.2, 113.1, 126.7, 127.3, 127.8,
127.9, 128.0, 129.9, 135.1, 135.3, 135.5, 136.1, 142.7, 144.8, 158.4,
166.1, 166.3, 166.4. ESI-MS: calcd for C58H59N3O8SNa (M +
Na)+, 980.3921; found, 980.4393.
3-[3-(4,4′-Dimethoxytrityloxy)propylcarbamoyl]-5-(3-trityl-
sulfenyloxypropylcarbamoyl)benzamido]propyl 2-Cyanoethyl
N,N-Diisopropylphosphoramidite (2). Compound 11 (1.56 g, 1.63
mmol) was rendered anhydrous by repeated coevaporation with dry
pyridine, toluene, CH2Cl2, and finally dissolved in dry CH2Cl2 (8
mL). To this solution were added 2-cyanoethyl N,N,N′N′-tetraiso-
propylphosphorodiamidite (621 µL, 1.96 mmol), diisopropylamine
(91 µL, 0.65 mmol), and 1H-tetrazole (46 mg, 0.65 mmol). The
resulting solution was stirred for 1.5 h. The reaction was quenched
by addition of H2O/CH3CN (1:1, v/v, 2 mL). The mixture was
diluted with Et2O/AcOEt (3:1, v/v, 40 mL) and washed with 0.2
M aq NaOH (3 × 30 mL). The organic layer was collected, dried
over MgSO4, filtered, and concentrated under reduced pressure. The
residue was chromatographed on an NH silica gel column (40 g)
with hexane/ethyl acetate (1:1, v/v) to give 2 (1.2 g, 63%): 1H
that the TrS group is orthogonal to the Lev, TBDMS, or Fmoc
group. These results indicated that, by the combined use of
DMTr, TrS, and/or Lev, TBDMS, and Fmoc, we could
synthesize more complex four- and five-way branched ODNs,
which can be used as the building units of DNA nanostructures.
Experimental Section
Dimethyl 5-[3-(4,4′-Dimethoxytrityloxy)propylcarbamoyl]-
benzene-1,3-dicarboxylate (7). To a solution of 3,5-bis(methoxy-
carbonyl)benzoic acid (6) (2.3 g, 9.5 mmol) in THF (20 mL) was
added CDI (2.3 g, 14 mmol). The resulting mixture was stirred at
room temperature for 1 h, and then the solution of (4,4′-
dimethoxytrityl)oxypropan-1-amine (5) (5.4 g, 14 mmol) in THF
(30 mL) was added. After being stirred at room temperature for 1
h, the reaction mixture was concentrated under reduced pressure.
During this concentration, care should be taken not to render the
mixture to complete dryness. The residue was diluted with ethyl
acetate (80 mL) and washed three times with water (80 mL). The
organic layer was collected, dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was crystallized
from hexane/ethyl acetate (4:1, v/v), and the crystals were collected
by filtration to give 7 (3.6 g, 64%). The filtrate was concentrated
under reduced pressure, and the residue was chromatographed on
an NH silica gel column (15 g) with hexane/ethyl acetate (3:1, v/v).
The eluted solution was concentrated under reduced pressure. The
residue was crystallized from hexane/ethyl acetate (4:1, v/v), and
the crystals were collected by filtration to give 7 (1.5 g, 26%; total
5.1 g, 90%): 1H NMR (500 MHz, CDCl3) δ 1.91 (m, 2H), 3.31 (t,
J ) 5.3 Hz, 2H), 3.60 (dd, J ) 5.7, 12 Hz, 2H), 3.74 (s, 6H), 3.91
(s, 6H), 6.76 (d, J ) 8.5 Hz, 4H), 6.83 (br, 1H), 7.16-7.30 (m,
9H), 7.39 (d, J ) 8.1 Hz, 2H), 8.46 (s, 2H), 8.76 (s, 1H); 13C NMR
(67.8 MHz, CDCl3) δ 29.0, 39.2, 52.5, 55.1, 62.4, 86.5, 113.1,
126.8, 127.9, 128.0, 129.8, 131.0, 132.1, 132.9, 136.0, 144.7, 158.4,
165.4, 165.8. Anal. Calcd for C35H35NO8‚0.1H2O: C, 70.13; H,
5.92; N, 2.34. Found: C, 69.90; H, 5.93; N, 2.70.
Triethylammonium 3-{3-(4,4′-Dimethoxytrityloxy)propylcar-
bamoyl}-5-(methoxycarbonyl)benzoate (8). Compound 7 (3.6 g,
6.1 mmol) was dissolved in pyridine (30 mL). To the solution was
added 1 M aq NaOH (7.3 mL). The resulting mixture was stirred
at room temperature for 3 h, and then the reaction mixture was
concentrated under reduced pressure. Pyridine was removed by
coevaporation three times with toluene. The residue was chromato-
graphed on a silica gel column (80 g) with chloroform/methanol/
triethylamine (100:1:1, v/v/v) to give 8 (2.6 g, 62%): 1H NMR
(500 MHz, CDCl3) δ 1.33 (t, J ) 7.3 Hz, 9H), 1.89 (m, 2H), 3.14
(dd, J ) 7.3, 15 Hz, 6H), 3.22 (t, J ) 5.6 Hz, 2H), 3.57 (dd, J )
6.1, 6.3 Hz, 2H), 3.74 (s, 6H), 3.88 (s, 3H), 6.57 (t, J ) 5.4 Hz,
1H), 6.78 (d, J ) 8.8 Hz, 4H), 7.16 (t, J ) 7.2 Hz, 1H), 7.22-7.32
(m, 6H), 7.41 (d, J ) 7.3 Hz, 2H), 8.43 (s, 1H), 8.53 (s, 1H), 8.83
(s, 1H); 13C NMR (67.8 MHz, CDCl3) δ 8.6, 29.5, 38.4, 45.0, 52.1,
55.1, 61.6, 86.2, 113.1, 126.7, 127.8, 128.1, 129.9, 130.2, 130.3,
131.7, 133.1, 134.9, 136.2, 137.8, 144.9, 158.3, 166.4, 166.7, 170.9.
ESI-MS: calcd for C40H49N2O8 (M + H)+, 685.3483; found,
685.3473.
Methyl 3-[3-(4,4′-Dimethoxytrityloxy)propylcarbamoyl]-5-(3-
tritylsulfenyloxypropylcarbamoyl)benzoate (9). To a solution of
8 (1.9 g, 2.8 mmol) in THF (6 mL) was added CDI (690 mg, 4.3
mmol). The resulting mixture was stirred at room temperature for
1 h, and then the solution of 4 (1.5 g, 4.3 mmol) and THF (3 mL)
was added. After being stirred at room temperature for 1 h, the
reaction mixture was concentrated under reduced pressure. Care
should be taken not to render the residue to complete dryness. The
solution was diluted with ethyl acetate (70 mL) and washed three
times with water (70 mL). The organic layer was collected, dried
over MgSO4, filtered, and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column (70 g) with
hexane/ethyl acetate (2:3, v/v) to give 9 (2.6 g, quant): 1H NMR
J. Org. Chem, Vol. 72, No. 22, 2007 8265

Utagawa et al.
NMR (500 MHz, CDCl3) δ 1.14-1.16 (m, 12H), 1.59 (m, 2H),
2.61-2.64 (m, 4H), 3.23 (m, 2H), 3.33 (t, J ) 5.9 Hz, 2H), 3.53-
3.12 (m, 6H), 3.74-3.89 (m, 10H), 6.31 (t, J ) 5.6 Hz, 1H), 6.64
(t, J ) 5.6 Hz, 1H), 6.79 (m, 4H), 6.87 (t, J ) 5.6 Hz, 1H), 7.15-
7.41 (m, 24H), 8.16 (s, 1H), 8.24, 8.25 (s, 2H); 13C NMR (67.8
MHz, CDCl3) δ 20.4, 24.5, 29.4, 31.2, 31.5, 37.5, 37.9, 38.5, 43.0,
43.1, 55.1, 55.2, 58.0, 58.2, 61.5, 72.0, 76.3, 86.2, 113.1, 113.1,
118.2, 126.8, 127.3, 127.8, 128.0, 129.9, 130.0, 135.3, 135.4, 136.1,
142.8, 144.9, 158.4, 165.7, 165.7, 165.9; 31P NMR (202.33 MHz,
CDCl3) δ 148.9. ESI-MS: calcd for C67H76N5O9PSNa (M + Na)+,
1180.4994; found, 1180.4628.
the detritylation step of the 10th dG. Chain elongation cycle was
repeated 11 times. The last nucleotide, dG, was detritylated and
then capped twice by use of acetic anhydride. After that, the TrS
group was removed by treatment with a 1 M I2 solution (Py/H2O,
9:1, v/v, 500 µL) for 2 min. Then the solid support was packed
back into the column of the automatic synthesizer to extend the
third sequence. The third sequence was extended under the usual
conditions of the DMTr-On method. After the synthesis, the solid
phase support was exposed to an ammonia solution for 20 h. The
resulting solution was collected and evaporated. The material having
a DMTr group was isolated using reversed-phase HPLC. The
fractions were combined and evaporated under reduced pressure.
The DMTr group was deprotected on a C-18 cartridge column, and
the resulting target material was eluted with CH3CN/H2O, (2:8, v/v).
The fractions were collected, combined, evaporated under reduced
pressure to give 23 in 5.4% yield, and analyzed by anion exchange
HPLC. The yield was determined by the absorbance at 260 nm.
The ꢀ260 was estimated as the sum of the ꢀ260 values of DNA (a),
DNA (b), and DNA (c). MALDI-MS: calcd for C307H393N116O187P30
(M + H)+, 9624.7; found, 9628.2. The sequences of DNA (a), (b),
and (c) were designed according to the literature.13
Melting Curve Analysis. Each oligonucleotide was dissolved
in 10 mM sodium cacodylate (pH 7.0) containing 0.5 M NaCl and
10 mM MgCl2 so that the final concentration of each oligonucleotide
became 1.5 µM. The solutions were separated into quartz cells (10
mm) and incubated at 85 °C. After 10 min, the solutions were
cooled to 5 °C at the rate of 0.5 °C/min and then heated until the
temperature reached 85 °C at the same rate. During this annealing
and melting, the absorptions at 260 nm were recorded and used to
draw UV melting curves. The oligonucleotide concentrations of
DNA were determined as described in the literature.39
The Stability of the TrS Group to Hydrazine. Compound 12
was synthesized by the above procedure. A 0.5 M hydrazine
monohydrate solution (1 mL, in pyridine/acetic acid, 4:1, v/v) was
added, and the reaction mixture was kept at the ambient temperature
for 20 min. After washing, the DMTr group was removed. The
oligomer was released from the polymer support by treatment with
concd aq NH3 (1 mL) for 30 min. The product was analyzed by
reversed-phase HPLC.
The Stability of the TrS Group to TBAF. Compound 12 was
synthesized by the above procedure. A 1 M TBAF solution (1 mL,
in THF) was added, and the reaction mixture was kept at the
ambient temperature for 10 min. After washing, the DMTr group
was removed. The oligomer was released from the polymer support
by treatment with concd aq NH3 (1 mL) for 30 min. The product
was analyzed by reversed-phase HPLC.
Synthesis of 12. Thymidine-loaded CPG (0.5 µmol, 46 µmol/g,
succinyl linker) was used in the manual operation: (1) detritylation
(3% TCA in CH2Cl2, 0.5 mL, 30 s × 3); (2) washing [CH2Cl2 (1
mL × 3), CH3CN (1 mL × 3)]; (3) coupling [phosphoramidite
unit (20 µmol), Activator 42 (0.25 M, 160 µL, 40 µmol), and CH3-
CN (80 µL), 2 min]; (4) washing [CH3CN (1 mL × 3)]; (5) capping
[Ac2O/Py (1:9, v/v, 500 µL) in the presence of 0.1 M DMAP for
2 min]; (6) oxidation [0.5 M CSO, CH3CN (200 µL), 3 min]; and
(7) washing [CH3CN (1 mL × 3)].
The Stability of the TrS Group to 3% TCA. A 3% solution
of TCA in CH2Cl2 (0.5 mL) was added to 12, and the reaction
mixture of compound 13 was kept at ambient temperature for 5
min. After washing, the oligomer was released from the polymer
support by treatment with concd aq NH3 (1 mL) for 30 min. The
product was analyzed by reversed-phase HPLC and ESI-MS: calcd
for compound 14, C47H55N5O13PS (M + H)+, 960.3249; found,
960.3148.
Synthesis of 15 by Use of Tet as an Activator and t-BuOOH
as an Oxidizing Agent. A thymidine-loaded CPG (0.5 µmol, 46
µmol/g, succinyl linker) was used. After the usual elongation of
trithymidylate by use of Tet as an activator and a 0.02 M I2 solution
as an oxidizing reagent, and the branch unit 2 was coupled with
the resin. Furthermore, an additional trithymidylate synthesis was
performed on the resin. Each cycle of the additional chain elongation
consisted of the following steps: (1) detritylation (3% TCA in CH2-
Cl2, 0.5 mL, 30 s × 3); (2) washing [CH2Cl2 (1 mL × 3), CH3CN
(1 mL × 3)]; (3) coupling [phosphoramidite unit (20 µmol), Tet
(40 µmol), and CH3CN (250 µL), 2 min]; (4) washing [CH3CN (1
mL × 3)]; (5) capping [Ac2O/Py (1:9, v/v, 500 µL) in the presence
of 0.1 M DMAP for 2 min]; (6) oxidation [0.5-0.6 M t-BuOOH,
CH3CN (250 µL), 10 min]; (7) washing [CH3CN (1 mL × 3)].
After the chain elongation was finished, the terminal 5′-hydroxyl
group was acetylated by use of acetic anhydride. Then the TrS group
was removed by a 1 M I2 solution (Py/H2O, 9:1, v/v, 250 µL) for
2 min. From the generated hydroxyl group, another trithymidylate
was synthesized by use of Tet and a 0.02 M I2 solution. After the
terminal DMTr group was removed, the oligomer was released from
the polymer support by treatment with concd aq NH3 (1 mL) for
30 min. The polymer support was removed by filtration and washed
with water (1 mL × 3). The filtrate was evaporated and analyzed
by reversed-phase HPLC. The retention time of the target compound
15 was ca. 25 min. Compound 15 was isolated by use of reversed-
phase HPLC, and the structure was characterized by MALDI-MS.
MALDI-MS: calcd for compound 15, C108H145N21O69P9 (M + H)+,
3118.6; found, 3116.8.
The Stability of the TrS Group to Piperidine. Compound 11
(4.1 mg, 4.3 µmol) was dissolved in DMF (84 µL). To the solution
was added piperidine (21 µL, 0.21 mmol). The reaction was
monitored for 48 h by TLC analysis.
Acknowledgment. This work was supported by a Grant-
in-Aid to CREST of JST (Japan Science and Technology) and
the Ministry of Education, Science, Sports and Culture, Grant-
in-aid for Scientific Research (A). This study was also supported
by Industrial Technology Research Grant Program in ’05 from
New Energy and Industrial Technology Development Organiza-
tion (NEDO) of Japan.
Synthesis of 15 by Use of Act42 as the Activator and
t-BuOOH as the Oxidizing Agent. The activator was changed from
Tet to Act42 (0.25 M, 160 µL, 80 µmol) in the method described
above.
Synthesis of 15 by Use of Act42 as the Activator and 0.5 M
CSO as the Oxidizing Agent. The oxidizing reagent was changed
from t-BuOOH to CSO (0.5 M, 200 µL, 3 min) during the synthesis
of the second trithymidylate from the method described above.
Synthesis of Branched ODN 23. First, DNA (a) sequence was
synthesized in an automatic synthesizer by using the standard
procedure of the DMTr-On method. After the 10th dG was oxidized,
the CPG resin was removed from the column and placed in a glass
syringe for the manual operation. Manual synthesis was started from
Supporting Information Available: List of experimental
procedures for the synthesis of the products other than those
described in the Experimental Section. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO071173A
(39) Borer, P. N. Handbook of Biochemistry and Molecular Biology:
Nucleic Acids, 3rd ed.; CRC Press: Cleveland, OH, 1975.
8266 J. Org. Chem., Vol. 72, No. 22, 2007