Synthesis and biochemical properties of oligodeoxynucleotides acylated by the chemically stable 2-(trimethylsilyl)benzoyl (TMSBz) group at the 5′ or 3′ terminus

Article abstract of DOI:10.1016/j.tetlet.2010.07.121

Oligodeoxynucleotides acylated with a 2-(trimethylsilyl)benzoyl (TMSBz) group at the 5′ or 3′ terminus were synthesized according to the general method used for DNA synthesis. The acylated DNA oligomers could be easily purified due to the high lipophilicity of the TMSBz group and showed enhanced hybridization ability and resistance to exonucleases.

Full text of DOI:10.1016/j.tetlet.2010.07.121

Tetrahedron Letters 51 (2010) 5173–5176
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Tetrahedron Letters
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Synthesis and biochemical properties of oligodeoxynucleotides acylated by
the chemically stable 2-(trimethylsilyl)benzoyl (TMSBz) group at the 50 or
30 terminus
*
Ken Yamada, Haruhiko Taguchi, Akihiro Ohkubo, Kohji Seio, Mitsuo Sekine
Department of Life Science, Tokyo Institute of Technology, J2-12, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Oligodeoxynucleotides acylated with a 2-(trimethylsilyl)benzoyl (TMSBz) group at the 50 or 30 terminus
were synthesized according to the general method used for DNA synthesis. The acylated DNA oligomers
could be easily purified due to the high lipophilicity of the TMSBz group and showed enhanced hybrid-
ization ability and resistance to exonucleases.
Article history:
Received 31 May 2010
Revised 20 July 2010
Accepted 22 July 2010
Available online 25 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
In recent years, much attention has been paid to nucleic acid-
based gene-targeting strategies.1 A variety of chemically modified
oligonucleotides were synthesized to increase the hybridization
affinity for the target DNA or RNA genes.2 Many efforts have also
been paid to improve the resistance against nuclease degradation
and the cell membrane permeability which have proved to be of
great importance for expression of their antisense activities.3
Chemical modification of oligonucleotides at their 50 and 30 ter-
minus is one of the strategies widely used to improve their original
properties.4 As a new method, we focused on the 50 or 30 terminal
modification of oligonucleotides with acyl groups that could sup-
press their degradation due to cellular exonucleases. However, it
is necessary to use suitably designed acyl groups that tolerate basic
conditions such as NH3 aq generally used in chemical synthesis of
oligonucleotides.5
In the previous Letter, we reported that the 2-(trimethyl-
silyl)benzoyl (TMSBz) group attached to the 50, 30, or 20-hydroxyl
group of nucleosides that was found to be extremely stable under
basic conditions.6 For example, the TMSBz group substituted at the
50-hydroxyl group of thymidine was stable under the basic condi-
tions of 28% ammonia at 55 °C for 24 h. These results led us to syn-
thesize TMSBz-modified oligonucleotides using the general
method where removal of the protecting groups and cleavage of
oligonucleotides from CPG resins were carried out simultaneously
by treatment with 28% aqueous ammonia.
protection of the 30-hydroxyl group, as reported previously by
us.6 50-O-TMSBz-thymidine-30-O-phosphoramidite unit (2) was
synthesized in 65% yield by the reaction of compound 1 with
1.2 equiv of 2-cyanoethoxy[bis(diisopropylamino)]phosphine in
CH3CN in the presence of 0.6 equiv of diisopropylammonium 1H-
tetrazolide at room temperature for 2 h according to the general
method, as shown in Scheme 1.
To examine the stability of the TMSBz group in oligodeoxynu-
cleotides modified with the TMSBz group, we synthesized 50-
d([TMSBzT]GACTGACTGACT)-30 (ODN1) using the phosphoramidite
unit 2 and the other building units (where the phenoxyacetyl
(Pac) group was used for A and G and the acetyl group was used
for C) in an automated DNA synthesizer. The chain elongation cycle
consisted of (i) detritylation, (ii) coupling, (iii) capping with phe-
noxyacetic anhydride (Pac2O), and (iv) iodine oxidation.5 After
the final chain elongation, ODN1 having the TMSBz group at the
50-terminal site was cleaved from the solid support by treatment
with aqueous ammonia at room temperature for 1 h. The protect-
ing groups of the base and phosphate groups were simultaneously
removed during this treatment. The hydrophobic property of the
TMSBz group enabled us to easily purify the 50-O-TMSBz-capped
oligonucleotide using a Sep-Pak Plus C18 cartridge (Waters Corp.).
In this Letter, we report a new method for the synthesis of oli-
gonucleotides acylated by the TMSBz group and their chemical and
enzymological properties.
50-O-TMSBz-thymidine (1: TMSBzT) was prepared by Mitsunobu
reaction of 2-(trimethylsilyl)benzoic acid with thymidine without
* Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924 5772.
E-mail address: msekine@bio.titech.ac.jp (M. Sekine).
Scheme 1. Reagents: (a) 1H-tetrazole, (i-Pr)2NH, 2-cyanoethoxy[bis(diisopropyl-
amino)]phosphine.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.07.121

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K. Yamada et al. / Tetrahedron Letters 51 (2010) 5173–5176
The reversed-phase HPLC profile of the ODN1 obtained after the
Sep-Pak C18 purification suggested that ODN1 was obtained as
sufficiently pure material in 42% yield so that further purification
using HPLC was no longer needed, as shown in Figure 1. It should
be noted that the TMSBz group remained unchanged in this succes-
sive purification procedure. ODN1 was characterized by MALDI-
TOFF mass analysis (found 4123.5, calcd 4123.8 [H+M]).
We examined if the sterically hindered TMSBz group affected
the hybridization property of the original structure. As a result, it
was found that the Tm value of the duplex formed between
ODN1 and its complementary oligomer was higher by 2.3 °C than
that of the duplex formed between ODN2 and its complementary
strand (Table 1). This result implies that the TMSBz group at the
50-terminus of the oligonucleotide does not disturb the formation
of the duplex.
To evaluate the stabilizing effect of the TMSBz group on enzy-
matic digestion, the stability of the ODN1 toward a 50-exonuclease,
bovine spleen phosphodiesterase (BSP, Sigma), was examined. The
enzyme assay using BSP (0.2 U/mL) was performed in a buffer of
Figure 2. Time course of degradation of ODN1 (closed diamonds) and ODN2 with
BSP (closed box).
30 mM NaOAc at pH 6.0 at 37 °C by use of 50
lM oligonucleotide.
After the enzyme was deactivated by heating 100 °C for 2 min, the
solution was diluted and filtered by a 45 lm filter (Millex-HV, Mil-
lipore).7 The mixture was analyzed by anion-exchange HPLC, as
shown in Figure 2. The unmodified oligonucleotide was completely
degraded in few minutes. On the other hand, the modified oligonu-
cleotide ODN1 remained unchanged under the same conditions
even after 8 h.
To see if the chemically stable TMSBz group is also stable to
esterases, the enzyme assay was carried out using pig liver esterase
(PLE, Sigma). ODN1 (11 lM) was incubated with PLE (8.9 U/mL) in
a 0.2 M potassium phosphate buffer (pH 7.4 at 37 °C).8 After the
enzyme was deactivated by heating 100 °C for 2 min, the solution
was diluted and filtered by a 45 lm filter (Millex-HV, Millipore).
The mixture was analyzed by reversed-phase HPLC, as shown in
Figure 3. It was found that the modified oligomer with the reten-
tion time of 21 min was completely resistant to PLE even after
incubation for 24 h (Fig. 3). The unmodified oligomer 50-
Figure 3. Reversed-phase HPLC profiles of the enzymatic reaction and alkali
hydrolysis of ODN1: (a) ODN1, (b) the mixture obtained after incubation of ODN1
with PLE in 0.2 M potassium phosphate buffer at 37 °C for 24 h, (c) the mixture
obtained after treatment of ODN1 with 2 M NaOH at room temperature for 2 h, (d)
ODN2 as the authentic sample.
d(TGACTGACTGACT)-30 (ODN2) appeared at the retention time of
10.5 min and no new peak was observed at this area.
Previously, we reported that the TMSBz group could be re-
moved from compound 1 by treatment with 2 M NaOH in ethanol
at room temperature for 4 h. Therefore, we tested whether the
TMSBz group can be removed from ODN1 by treatment with 2 M
NaOH in aqueous solution at room temperature for 2 h. As a result,
the hydrolysis of the TMSBz group was completed in 2 h and the
deacylated product was observed as a single peak at 10.5 min, as
shown in Figure 3.
The marked resistance of the 50-O-TMSBz group against BSP
led us to synthesize a 30-O-TMSBz-capped oligonucleotide 50-
d(TTTTTTTTTTTT[TTMSBz])-30 (ODN3) using 30-O-DMTr-thymidine
50-phosphoramidite unit and a 30-O-TMSBz-50-O-phosphoramidite
unit 6 in the reverse 50 ? 30 direction in the solid phase synthesis.
The unit 6 required in the last coupling reaction was synthesized as
shown in Scheme 2.
Figure 1. RP-HPLC of ODN1 after the Sep-Pak C18 purification.
Table 1
Hybridization properties of oligonucleotides having TMSBz group at 50-hydroxyl
terminus
30-O-[2-(Trimethylsilyl)benzoyl]thymidine (5), which was ob-
tained by a two-step reaction from 50-O-(4,40-dimethoxytrityl)thy-
midine (3) according to the method previously reported,6 was
allowed to react with 2-cyanoethoxy[bis(diisopropylamino)]phos-
phine to give the 50-phosphoramidite derivative 6 in 42% yield. In a
manner similar to that described in the synthesis of ODN1, ODN3
a
ODN
Sequence
Tm (°C)
DTm (°C)
1
2
50-d([TMSBzT]GACTGACTGACT)-30
50-d(TGACTGACTGACT)-30
55.9
53.6
+2.3
none
a
Measured in a buffer containing 10 mM sodium phosphate buffer (pH 7.0),
100 mM NaCl, 0.1 mM EDTA, and 2.0 M duplex.
l

K. Yamada et al. / Tetrahedron Letters 51 (2010) 5173–5176
5175
Figure 5. Time course of degradation of ODN3 (closed diamonds) and ODN4 with
SVP (closed box).
Scheme 2. Reagents: (a) 2-(trimethylsilyl)benzoic acid chloride, pyridine-CH2Cl2
(1:1, v/v); (b) 3% TCA/CH2Cl2; (c) 1H-tetrazole, (i-Pr)2NH, 2-cyanoethoxy[bis(diiso-
propylamino)]phosphine.
was synthesized and isolated in 60% yield, as shown in Figure 4,
and characterized by MALDI-TOFF mass analysis (found 4069.7;
calcd: 4066.7[H+M]).
The nuclease stability of ODN3 was evaluated by treatment
with snake venom phosphodiesterase (SVP, Sigma). The enzyme
assay using snake venom phosphodiesterase (5 ꢀ 10ꢁ5 U/mL) was
performed in a buffer of 50 mM Tris–HCl at pH 8.5, 72 mM NaCl,
and 14 mM MgCl2 at 37 °C by use of 50 l
M oligonucleotide.9 After
workup similar to that described in the enzyme assay using BSP,
the mixture was analyzed by anion-exchange HPLC. As shown in
Figure 5 the half-life t1/2 of ODN3 was ca. 23 min whereas the
unmodified oligomer 50-d(TTTTTTTTTTTTT)-30 (ODN4) degraded
more rapidly with t1/2 of 10 min. The difference in stability against
SVP between ODN3 and ODN4 was observed, but the 30-terminal
TMSBz group was not so effective against SVP as the 50-terminal
one against BSP. In the previous studies, it was reported that when
a modified base was incorporated in DNA, the ‘skipping’ of the
modified base by snake venom phosphodiesterase was often ob-
served.10 On the other hand, the HPLC profile, shown in Figure 6,
T12 appeared at 21.8 min, suggesting the predominant enzymatic
cleavage of the 30-terminal phosphodiester bond. A dimer fragment
of 50-(T[TTMSBz])-30 that should be generated by the skipping was
not observed in our case (Fig. 6).
Figure 6. Anion exchange HPLC profiles of ODN3 and ODN4 incubated at 37 °C in
the presence of snake venom phosphodiesterase (5 ꢀ 10ꢁ5 U/mL) for 30 min.
In summary, we successfully introduced the chemically stable
TMSBz group at 50- or 30-hydroxy terminus of oligonucleotides
via the general synthetic protocol using 28% aqueous ammonia.
Very high lipophilicity of TMSBz group enables facile purification
by Sep-Pak Plus C18 cartridge. TMSBz group attached to 50-hydro-
xyl group of the oligonucleotide did not disturb the forming of DNA
duplex and was found to enhance the 50-exonuclease resistance;
however, modification at 30-hydroxyl group with TMSBz group
did not show predominant increase of 30-exonuclease resistance.
Further investigation of TMSBz-modified oligonucleotides is cur-
rently in progress in our laboratory.
Acknowledgments
This work was supported by a grant from a Grant-in-Aid for Sci-
entific Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan, and in part by a grant from by
Health Sciences Research Grants for Research on Psychiatric and
Neurological Diseases and Mental Health from the Ministry of
Health, Labor and Welfare of Japan, and by the global COE project.
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Figure 4. RP-HPLC of ODN3 after the Sep-Pak C18 purification.

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