Synthesis and triplex-forming properties of oligonucleotides containing thio-substituted C-nucleoside 4-thiopseudoisocytidine

Article abstract of DOI:10.1016/j.tetlet.2010.11.065

We synthesized the 3′-phosphoramidite building block of 4-thiopseudoisocytidine (s⁴ΨiC) and incorporated it into triplex-forming oligonucleotides (TFOs). The results of thermal denaturation of triplexes incorporating s⁴ΨiC showed tha

Full text of DOI:10.1016/j.tetlet.2010.11.065

Tetrahedron Letters 52 (2011) 407–410
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and triplex-forming properties of oligonucleotides containing
thio-substituted C-nucleoside 4-thiopseudoisocytidine
⇑
Shi-Qi Cao, Itaru Okamoto, Hirosuke Tsunoda, Akihiro Ohkubo, Kohji Seio, Mitsuo Sekine
Department of Life Science, Tokyo Institute of Technology, 4295 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
We synthesized the 30-phosphoramidite building block of 4-thiopseudoisocytidine (s4WiC) and incorpo-
Article history:
Received 1 October 2010
Revised 10 November 2010
Accepted 12 November 2010
Available online 18 November 2010
rated it into triplex-forming oligonucleotides (TFOs). The results of thermal denaturation of triplexes
incorporating s4
W
iC showed that s4
WiC could be used as a nucleoside component of TFOs to increase
the thermal stability of triplexes at pH 7.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Triplex-forming oligonucleotides
Antigene
C-Nucleoside
Disulfide bond
4-Thiopseudoisocytidine
In recent years, triplex-forming oligonucleotides (TFOs) have
been extensively studied because they can be used in the antigene
strategy targeting cellular double helical DNAs.1–4 Researchers
have shown that, under suitable conditions, TFOs can bind to the
major groove of DNA duplexes and form triplexes in parallel and
antiparallel orientations.1–4 In the parallel orientation, triple he-
lixes can be constructed by the TꢀA–T and C+ꢀG–C triad formations
of thymine (T) and protonated cytosine (C+) in TFOs and A–T and
G–C base pairs in DNA duplexes, respectively, through Hoogsteen
hydrogen bonds. However, there is a major drawback in the
C+ꢀG–C motif.5,6 Protonation of the N3-position of C in TFOs is nec-
essary for the formation of its hydrogen bond with N7 of G in DNA
duplexes, requiring an optimized range of pH 5–6, which does not
meet the physiological conditions required for the antigene ther-
apy. To overcome this restriction, several modified nucleosides
have been developed to mimic the structure of the N3-protonated
C.7–11 For example, 20-O-methylated pseudoisocytidine with 2-
On the other hand, TFOs containing thionucleosides such as 20-
O-methyl-2-thiouridine or 2-thiothymidine formed relatively sta-
ble parallel triplexes when they were bound to DNA duplexes.12
The enhancement of the thermal stability of these triplexes can
be explained by the strong stacking interaction of the thiocarbonyl
group.
Accordingly, we designed 4-thiopseudoisocytidine (s4
which the thiocarbonyl group at position 4 was replaced with a
WiC) in
carbonyl group (see Fig. 1, left). Previously, we synthesized s4
WiC
successfully.13 From 1H NMR spectral analysis, we determined that
the preferred sugar conformation of this nucleoside was a C30-endo
pucker. As it is well known that RNA-type TFOs form more stable
triplexes than their deoxy counterparts,14 we expected that s4
WiC
would be favorable for the stabilization of triplexes when we used
it as the nucleoside component of TFOs. However, to the best
of our knowledge, no group has reported the synthesis of the
amino-4-oxopyrimidin-5-yl moiety (WiC) as an aglycone was used
to form a triad with a G–C base pair under neutral conditions when
incorporated into TFOs.10,11 As indicated in the base pair motif
shown in the right side of Figure 1, there is a proton at the N3 posi-
tion of
W
iC (X = O) which is available for a Hoogsteen hydrogen
bond with the N7 position of G. TFOs containing
W
iC could stabilize
the triplex structure more than those containing unmodified C or
5-methylcytosine at neutral pH.
⇑
Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924 5772.
E-mail address: msekine@bio.titech.ac.jp (M. Sekine).
Figure 1. Structure of 4-thiopseudoisocytidine (left); proposed hydrogen bonding
schemes of
W
iCꢀG–C and s4
iCꢀG–C base triads (right).
W
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.11.065

408
S.-Q. Cao et al. / Tetrahedron Letters 52 (2011) 407–410
as the activator for the coupling step.17 Because s4
WiC is a ribonu-
cleoside, this step was performed for 20 min, according to the
usual RNA synthetic protocol.18 We used the 0.02 M I2 in THF/pyr-
idine/H2O as the oxidation reagent.19 Capping occurred with Ac2O
in 0.1 M DMAP–pyridine (1:9, v/v). From the DMTr cation assay, we
calculated the overall coupling yield as 35%. According to the
method reported by Ogilvie and co-workers,20 we attempted to
cleave the dimer intermediates from the CPG supports via treat-
ment with 3:1 ammonium hydroxide–ethanol (3:1, v/v) for 2 h at
room temperature to minimize the removal of the TBDMS group
at this step. Before removal of the TBDMS group, the crude mixture
was analyzed via reversed-phase HPLC–MS.
However, we observed several unexpected products. As shown
in Figure 2, in addition to the desired intermediate 60 at 21.5 min
and the unreacted deoxythymidine (dT) at 6.7 min, we observed
compounds containing a disulfide bond and one or two Bz groups
at longer retention times. For example, compound 7 at 23.4 min
was assigned as the dimer of 60 with a disulfide bond. Compound
8 at 25.1 min was the mono-benzoylated product of 7. Compound
9 at 26.0 min was the di-benzoylated product of 7. In addition, we
found a small amount of mono-benzoylated compound 10 at
29.3 min.
Therefore, in order to remove the residual Bz group, we treated
the mixture again with 3:1 ammonium hydroxide–ethanol at 55 °C
for 2 h. In addition, NaSH was added to the reaction mixture to
convert the disulfide bond to two thiocarbonyl groups.21 After
Scheme 1. Reagents and conditions: (i) (a) TMSCl (10 equiv), pyridine, rt, 1 h; (b)
PhC(O)Cl (2.0 equiv), pyridine, rt, 2 h; (c) 1.0 M TBAF/THF (10 equiv), rt, overnight,
53% (three steps); (ii) DMTrCl (1.2 equiv), pyridine, rt, 5 h, 61%; (iii) TBDMSCl
(1.2 equiv), imidazole (3 equiv), rt, 4 h, 26%; (iv) NCCH2CH2OPClN(i-Pr)2 (2.5 equiv),
2,4,6-collidine (7.5 equiv), N-methylimidazole (0.5 equiv), THF, rt, 40 min, 80%.
corresponding thio-modified C-ribonucleoside phosphoramidite
and oligonucleotides incorporating this modified nucleoside.
In order to incorporate s4
W
iC into TFOs and study their triplex
formation, we developed a practical approach for the synthesis of
the 30-phosphoramidite derivative (5) of s4
iC as shown in Scheme
W
1. We began the synthesis of 5 from compound 1, which we syn-
thesized via a four-step reaction from pseudouridine.13 According
to the procedure described by Jones and co-workers,15 compound
1 was first treated with chlorotrimethylsilane in pyridine for tran-
sient protection of the hydroxyl groups and then with benzoyl
chloride to protect the exocyclic amino group.
Treatment with 1.0 M tetrabutylammonium fluroride (TBAF) in
THF removed the trimethylsilylethyl (TMSE) group and the TMS
groups to give 2 in 53% yield. Separately, we also tried to use the
TMSE group as the protecting group of the thiocarbonyl group dur-
ing chain elongation required for the synthesis of oligonucleotides
incorporating compound 1. However, the TMSE group was too sta-
ble to easily remove after the incorporation to the oligonucleotides
without damaging the phosphodiester backbones, and the 4-thio-
carbonyl group was rather labile (date not shown).
Subsequently, we selectively protected the 50-OH group of 2
with the DMTr group to give 3 in 61% yield. For the protection of
the 20-OH group, we protected it using TBDMSCl, and the 20-pro-
tected derivative 4 could be isolated in 26% yield from a mixture
with the 30-protected and 20,30-diprotected products. Finally, the
reaction of 4 with NCCH2CH2OPClN(i-Pr)2 in the presence of
2,4,6-collidine and N-methylimidazole16 gave the phosphorami-
dite 5 in 80% yield. We should note that the unprotected thiocar-
bonyl group did not react with NCCH2CH2OPClN(i-Pr)2 under the
conditions used in this procedure.
In order to examine the stability of s4
DNA synthesis, we first synthesized the dimer (s4
W
iC during the solid phase
iC)pT (6) on the
W
solid supports, using the phosphoramidite 5. According to the
usual protocol of DNA and RNA synthesis, we used 1H-tetrazole
Figure 2. RP-HPLC profile and products of the synthesis of (s4
WiC)pT dimers.

S.-Q. Cao et al. / Tetrahedron Letters 52 (2011) 407–410
409
After removal of ammonia and the solvents, according to the
method reported by Sekine and co-workers,22 we added 20% AcOH
in H2O to compound 60 to remove the TBDMS group. The use of
these acidic conditions was essential for the removal of this TBDMS
group because the thiocarbonyl group was converted to the car-
bonyl group if usual desilylating agents such as TBAF were used
(data not shown). The reaction mixture was kept at 30 °C for 2 h,
and then NH4OAc buffer was added to quench the reaction. After
lyophilization, we obtained the target molecule (s4
quantitatively (see Fig. 3, lower panel).
WiC)pT (6)
Based on the results of the dimer synthesis, we successfully
synthesized 14mer TFO-1, 50TTTTTTs4 iCTTTCTTT 30containing
s4
iC using the above mentioned ammonia treatment in the pres-
W
W
ence of NaSH and acid treatment for removal of the TBDMS group.
After purification, TFO-1 was analyzed via reversed-phase HPLC as
shown in Figure 4, upper panel. From the UV absorption spectrum
of TFO-1 at 8.9 min, we observed an absorbance at 350 nm (see
Fig. 4, lower panel). This result suggested that a thiocarbonyl group
existed in this oligonucleotide. The structure was also confirmed
by MALDI-TOF-MS analysis. The observed molecular weight
(4197.96) was consistent with the calculated mass (4197.67).
Finally, to assess the triplex-forming ability of TFO-1, we
conducted melting temperature (Tm) experiments using a hairpin
DNA molecule, 50CAAAAAAGAAAGAAACTTTTGTTTCTTTCTTTTT-
TG30, which contains a stem region shown by the underlined por-
tions and a four base internal loop region. For comparison, control
TFO, 50TTTTTTXTTTCTTT 30 with deoxycytidine (TFO-2: X = dC;
Figure 3. RP-HPLC profiles of compound 60 (top) and compound 6 (bottom).
TFO-3: X = C) in place of s4
WiC was also hybridized with the hairpin
oligonucleotide.
Table 1 lists the results of the Tm experiments using TFO-1 and
TFO-2 under deferent conditions (pH 7.0, 5.7, and 5.0). At pH 7.0,
the TFO-2 with unmodified dC showed a Tm value of 29 °C. On
the other hand, the Tm value (45 °C) of a one-point modified triplex
that resulted from TFO-1 having s4
WiC was much greater (DTm
16 °C) than those (29 °C and 26 °C) of the triplexes derived from
TFO-2 and TFO-3. We measured the pH dependency of Tm in the
triplexes containing TFO-1 and TFO-2. With a decrease of pH, the
Tm values of the former significantly increased. On the other hand,
the Tm values of the latter moderately increased up to 55 °C. At pH
5.0 where the two dCs in TFO-2 could be completely protonated,
the triplex containing TFO-1 showed a somewhat higher Tm value
than that containing TFO-2. At pH 5.7, the triplex containing TFO-1
showed a significantly higher Tm value than that containing TFO-2.
These results indicated that TFO-1 containing one s4
WiC residue
could form a more stable triplex with the hairpin duplex than the
unmodified one under the neutral conditions because the Hoogs-
teen base pair of s4
WiC and guanine does not require protonation
at position 3, as shown in Figure 1.
Figure 4. RP-HPLC profile of TFO-1 (top) and UV spectra of TFO-1 (bottom).
The stabilization effects of the triplex derived from TFO-2 under
acidic conditions are explained in terms of protonation at the N3-
position capable of formation of Hoogsteen hydrogen bonds and
neutralization of the neighboring anionic phosphate residues by
generation of the positive cation on the cytosine moiety. It is likely
that the triplex containing TFO-1 that might be protonated at pH
these reactions, we purified the mixture with a C-18 cartridge col-
umn. As expected, the Bz group was removed, and the disulfide
bonds were also successfully reduced to give the 20-O-TBDMS pro-
tected compound 60 (see Fig. 3, upper panel).
Table 1
Tm analysis of DNA triplexes
TFOs
X
pH 7.0
pH 5.7
pH 5.0
a
b
a
b
a
b
Tm (°C)
D
Tm (°C)
Tm (°C)
D
Tm (°C)
Tm (°C)
D
Tm (°C)
TFO-1
TFO-2
s4
dC
W
iC
45
29
+16
52
44
+8
55
52
+3
a
Tm values are accurate within 0.5 °C. The Tm measurements were conducted with 2
10 mM MgCl2 adjusted at pH 7.0, 5.7, or 5.0.
lM triplex in a buffer containing 10 mM sodium cacodylate buffer, 500 mM NaCl,
b
D
Tm is the difference in the value between the modified triplex containing TFO-1 and the unmodified triplex containing TFO-2.

410
S.-Q. Cao et al. / Tetrahedron Letters 52 (2011) 407–410
5.0 exhibited an additional stacking effect of the thiocarbonyl
group toward the nearby bases and/or a stronger effect of the mod-
ified base on the Hoogsteen hydrogen bonds.
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In conclusion, we synthesized s4 iC 30-phosphoramite 5 and
W
succeeded in incorporating this thio-substituted C-nucleoside into
TFOs. In the synthesis of the modified oligonucleotides, we found
that the deprotection and cleavages by ammonia should be per-
formed in the presence of NaSH and that the removal of the
TBDMS groups should be performed under acidic conditions to
suppress the side reactions at the thiocarbonyl group. Further-
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ited a significant increase in triplex stability as compared to the
unmodified TFOs. More detailed studies of the mechanism associ-
ated with the stabilization of triplexes with s4
progress.
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´
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This study was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. This study was also supported in part by
the global COE project. The ESI-TOF mass spectra of the dimers
and the MALDI-TOF mass spectra of the oligonucleotides were
kindly measured by Mr. Koizumi, M. from the Center for Advanced
Materials Analysis, Technical Department, TIT.