Synthesis and triplex formation of oligonucleotides containing 8-thioxodeoxyadenosine

Article abstract of DOI:10.1021/ol802622s

(Chemical Equation Presented) For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides containing 8-thioxode-oxyadenosine (s⁸dA) residues in place of the protonated deoxycytidines requir

Full text of DOI:10.1021/ol802622s

ORGANIC
LETTERS
2009
Vol. 11, No. 3
605-608
Synthesis and Triplex Formation of
Oligonucleotides Containing
8-Thioxodeoxyadenosine
Ken-ichi Miyata, Ryuji Tamamushi, Hirosuke Tsunoda, Akihiro Ohkubo,
Kohji Seio, and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku,
Yokohama 226-8501, Japan
msekine@bio.titech.ac.jp
Received November 13, 2008
ABSTRACT
For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides containing 8-thioxode-
oxyadenosine (s8dA) residues in place of the protonated deoxycytidines required for the third base pairing with DNA duplexes. Consequently,
it was found that s8dA exhibited much stronger hybridization ability than dC under neutral conditions when four s8dA bases were arranged
in a consecutive sequence.
Triplex formation between DNA duplexes and external DNA
single strands has been extensively studied during the past
two decades.1 In naturally occurring triplexes, it is well-
known that two sets of planar triads composed of T-A-T
and C+:G-C, in which Hoogsteen types of hydrogen bonds
are involved, contribute to stabilization of the triplexes in a
parallel manner as shown in Figure 1.2
cytosine base, have been reported.3,4 Among them, 5-me-
thylcytosine (m5C) can bind more strongly to a G-C pair
compared with cytosine in triplexes having discontinuous
C+:G-C sequenes under neutral conditions because the pKa
value of m5C is higher than that of C.5-7 However, a series
of C+:G-C sequences arranged in a straight manner resulted
in a significant decrease in the thermal stability of the
resulting triplexes because of the internal cation repulsion
arising from the protonated cytosine bases.8,9 Although
6-oxocytosine,10-13 pseudoisocytosine,14,15 and 1-deaza-6-
To date, a large number of studies on increasing the
hybridization affinity of triplex-forming oligonucleotides
(TFOs) with the complementary duplex under neutral condi-
tions, using artificially designed nucleobases in place of the
(3) Cuenoud, B.; Casset, F.; Husken, D.; Natt, F.; Wolf, R. M.; Altmann,
K. H.; Martin, P.; Moser, H. E. Angew. Chem., Int. Ed. 1998, 37, 1288–
1291
.
(1) (a) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I., Jr. Nucleic Acids,
University Science Books: Sausalito, CA, 2000. (b) Neidle, S., Ed.; Oxford
Handbook of Nucleic Acid Structure; Oxford University Press: New York,
1999. (c) Purwanto, M. G. M.; Weisz, K. Curr. Org. Chem. 2003, 7, 427–
446. (d) Frank-Kamenetskii, M. D.; Mirkin, S. M. Annu. ReV. Biochem.
1995, 64, 65–95.
(4) Puri, N.; Majumdar, A.; Cuenoud, B.; Miller, P. S.; Seidman, M. M.
Biochemistry 2004, 43, 1343–1351
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(5) De los Santos, C.; Rosen, M; Patel, D. Biochemistry 1989, 28, 7282–
7289
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(6) Asensio, J. L.; Lane, A. N.; Dhesi, J.; Bergvist, S.; Brown, T. J.
Mol. Biol. 1998, 275, 811–822
(7) Povsic, T. J.; Dervan, P. B. J. Am. Chem. Soc. 1989, 111, 3059–
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(2) Soyfer, V. N.; Potaman, V. N. Triple-Helical Nucleic Acids; Springer:
New York, Berlin, Heidelberg, 1996.
.
10.1021/ol802622s CCC: $40.75
Published on Web 01/14/2009
2009 American Chemical Society

Figure 1. Parallel stranded triplex formation by naturally occurring nucleobases.
azacytosine16 derivatives have been proposed as neutral
modified bases capable of triplex formation independent of
pH, a synthesis of these modified bases requires multistep
reactions, and some C-nucleoside derivatives tend to epimer-
ize at the anomeric center.
syn orientation compared with o8A. In addition to this
advantage, the use of the thio-carbonyl group could enhance
the stacking interaction with the downstream bases in TFO
because the thiouracil base showed such an effect upon
incorporation into oligonucleotides.21,22 On the basis of these
results, we thought that s8A would be the choice of modified
adenine for increasing the hybridization ability of TFO
having a sequence of consecutive cytosine bases as shown
in Figure 2.
On the other hand, Miller et al. reported that 8-oxoadenine
(o8A) can be used as a neutral species that can bind to G of
the G-C pair using two N-H···N hydrogen bonds.17 As a
result, the binding ability of oligodeoxyribonucleotides
having o8A toward G of the G-C pair of DNA duplexes at
physiological pH was similar to that of oligodeoxyribonucle-
otides having cytosines. Davison et al. also synthesized
oligodeoxyribonucleotides incorporating a cytosine, 8-oxoad-
enine, or adenine base and showed their effects on the
thermal stability of the triplexes formed with a DNA
duplex.18 These results also showed that 8-oxoadenine base
could bind to the G-C base pair, whereas the unmodified A
could not. The orientation of the glycosyl bond of 8-oxoad-
enosine is known to be syn.19 It is also suggested that this
modified base has a syn conformation in its deoxyribo-
nucleoside counterpart.20 Only the syn conformation, which
is in equilibrium with the anti conformation, allows the
formation of a third base pair between o8A and G-C.
Therefore, if it is possible to make this equilibrium syn
conformation, formation of the triad would be entropically
favorable.
Figure 2.
Structure of 8-thioxodeoxyadenosine (s8A) and triplex
formation with a G-C base pair.
To incorporate this modified deoxyribonucleoside into
DNA oligomers, the phosphoramidite building block 6 was
synthesized, as shown in Scheme 1. The trimethylsilylethyl
Because the sulfur atom is larger than the oxygen atom,
it was expected that 8-thioxoadenine (s8A) should favor the
(8) Roberts, R. W.; Crothers, D. M. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 4320–4325.
Scheme 1
.
Synthesis of 6-N-Carbamoyl-8-thioxodeoxyadenosine
3′-Phosphoramidite 6
(9) James, P. L.; Brown, T.; Fox, K. R. Nucleic Acids Res. 2003, 31,
5598–5606.
(10) Xiang, G.; Soussou, W.; MaLaughlin, L. W. J. Am. Chem. Soc.
1994, 116, 11155–11156.
(11) Berressem, R.; Engel, J. W. Nucleic Acids Res. 1995, 23, 3465–
3472.
(12) Xiang, G.; Robert, B.; McLaughlin, L. W. Nucleic Acids Res. 1996,
24, 1963–1970.
(13) Uwe, P.; Engels, J. W. Chem. Eur. J. 2000, 6, 2409–2424.
(14) Ono, A.; Ts’o, P. O. P.; Kan, L. S. J. Am. Chem. Soc. 1991, 113,
4032–4032.
(15) Ono, A.; Ts’o, P. O. P.; Kan, L. S. J. Org. Chem. 1992, 57, 3225–
3230.
(16) Benner, S. A.; Ulrike, V. K. J. Am. Chem. Soc. 1995, 117, 5361–
5362.
(17) Miller, P. S.; Bhan, P.; Cushman, C. D.; Trapane, T. L. Biochemistry
1992, 31, 6788–6793.
(18) Davison, E. C.; Johnsson, K. Nucleosides Nucleotides 1993, 12,
237–243.
(19) Mizuno, H.; Kitamura, K.; Miyao, A.; Yamagata, Y.; Wakahara,
A.; Tomita, K.; Ikehara, M. Acta Crystallogr. 1980, B36, 902–905.
(20) Wood, M. L.; Esteve, A.; Morningstar, M. L.; Kuziemko, G. M.;
Essigmann, J. M. Nucleic Acids Res. 1992, 20, 6023–6032.
606
Org. Lett., Vol. 11, No. 3, 2009

group was chosen as the protecting group for a thiol group,
which is tautomerized with the 8-thioxo group of s8A,
because 8-[2-(trimethylsilyl)ethylthio]deoxyadenosine 3 has
been synthesized from deoxyadenosine 1 via 8-bromode-
oxyadenosine 2, reported by Chambert et al.,23 as shown in
Scheme 1. Additionally, we also chose the unsubstitued
carbamoyl group as a protecting group of an amino group
at the 6-position of adenine, which could be removed by
treatment with a 1 M tetrabutylammonium fluoride (TBAF)
solution in THF to give the desired oligomers (see Figure
S1 in Supporting Information).
The carbamoyl group could be easily introduced into the
6-amino group by treatment of 3 with phenyl chloroformate
in the presence of trimethylsilyl chloride and triethylamine
followed by ammonolysis to produce the 2-N-carbamoyl
derivative 4 in 85% yield. The usual dimethoxytritylation
of 4 followed by 3′-phosphitylation afforded the phosphora-
midite building block 6 via the 5′-tritylated species 5.
Table 1. Tm Values for DNA Triplexes Containing C, s8A, and
o8A
entry
triplex
X
X′
Y
Z
Tm (°C) ∆Tm (°C)
1
2
3
4
5
6
7
8
9
TFO 1 - HP 1
C
C
C
G
G
G
G
G
G
C
G
C
G
C
C
C
C
C
C
C
G
C
G
C
G
30.5a
TFO 2 - HP 1 s8A
29.0a
27.4a
28.2a
22.4a
41.9b
16.3b
39.7b
8.9b
-1.5c
-3.1c
-2.3c
-8.4c
TFO 3 - HP 1 s8A s8A
TFO 4 - HP 1 o8A
TFO 5 - HP 1 o8A o8A
TFO 1 - HP 1
TFO 1 - HP 2
TFO 2 - HP 1 s8A
TFO 2 - HP 2 s8A
TFO 3 - HP 1 o8A
TFO 3 - HP 2 o8A
C
C
C
C
C
C
C
C
C
-25.6d
-30.8d
-18.5d
10
11
33.8b
15.3b
a Tm values are accurate within (0.5 °C. The Tm measurements were
carried out in a buffer containing 10 mM sodium cacodylate buffer (pH
7.0), 500 mM NaCl, 10 mM MgCl2, and 2 µM triplex. b Tm measurements
were carried out in a buffer containing 10 mM sodium cacodylate buffer
(pH 6.0), 500 mM NaCl, 10 mM MgCl2, and 2 µM triplex. c ∆Tm is the
difference in the Tm value between the unmodified triplex (entry 1) and the
modified triplexes having s8A or o8A (entries 2-5). d ∆Tm is the difference
in the Tm value between the matched triplex (entries 6, 8, 10) and
mismatched triplexes (entries 7, 9, 11).
an oligonucleotide containing an s8Acm residue. Upon
prolonged treatment, this peak was completely converted to
a new peak at 9.96 min. This final product was identified
with the target DNA 14mer containing a s8A residue by
MALDI-TOF mass spectrometry. TFOs 3 and 7 containing
s8A residues were also synthesized by the same treatment
as described above.
To examine the triplex-forming ability of TFOs 2 and 3,
Tm experiments of the triplexes formed between these
oligomers and the DNA duplex HP 1 having a hairpin
structure described in Table 1 were carried out. In addition,
we synthesized an unmodified DNA oligomer (TFO 1) and
two oligomers (TFOs 4 and 5) containing one or two o8As
to compare the effect of the thioxo group on the thermal
Figure 3. Time course of deprotection of the TMSE and carbamoyl
groups by treatment with a 1 M TBAF silution in THF. Each peak
was identified by MALDI-TOF mass spectroscopy.
The synthesis of oligodeoxyribonucleotides (TFOs 2, 3,
and 7, as shown in Tables 1 and 2) containing s8A was
carried out using an automated synthesizer. After the
synthetic cycles were finished, they were isolated in the usual
way. The oligodeoxyribonucleotides having a 5′-terminal
DMTr group were absorbed on a C18 cartridge. The eluate
was treated with a 1 M TABF solution in THF. Figure 3
shows a reverse-phase HPLC analysis of the synthesis of
TFO 2. A somewhat stable but degradable intermediate with
a peak at 10.4 min was observed after 30 min. A mass
spectrographic analysis suggested that this early product was
Table 2. Tm Values for DNA Triplexes Containing Consecutive
Cs, s8As, and o8As.
entry
triplex
X
Tm (°C)a
∆Tm (°C)b
1
2
3
TFO 6 - HP 3
TFO 7 - HP 3
TFO 8 - HP 3
C
s8A
o8A
9.7
36.8
17.4
+27.1
+ 7.7
a Tm values are accurate within (0.5 °C. The Tm measurements were
carried out in a buffer containing 10 mM sodium cacodylate buffer (pH
7.0), 500 mM NaCl, 10 mM MgCl2, and 2 µM triplex. b ∆Tm is the
difference in the Tm values between the unmodified triplex (entry 1) and
the modified triplexes having s8A or o8A (entries 2-3).
(21) Mazumdar, S. K.; Saenger, W.; Scheit, K. H. J. Mol. Biol. 1974,
85, 213–229.
(22) Okamoto, I.; Seio, K.; Sekine, M. Bioorg. Med. Chem. Lett. 2006,
16, 3334–336.
(23) Chambert, S.; G.-Luneau, I.; Fontecave, M.; Decout, J.-L. J. Org.
Chem. 2000, 65, 249–253.
Org. Lett., Vol. 11, No. 3, 2009
607

stability of triplexes. The results of the Tm experiments are
also shown in Table 1.
complementary HP 4, 5′-CAAAGAAGAAGAAGACTTT-
TGTCTTCTTCTTCTTTG. The significant decrease in the
Tm value could be explained in terms of the increasing
electronic repulsion between the protonated cytosine bases.
Similar phenomena have been observed in earlier studies by
Roberts8 and recent studies by James.9 In contrast, the Tm
values of TFOs 7 and 8 having s8A and o8A residues,
respectively, as the protonated cytosine base analogs in-
creased compared with the unmodified TFOs (entries 2 and
3). In particular, the Tm value of TFO 7 having four
consecutive s8A bases was significantly higher (by 27.1 °C)
than that of TFO 6. These results strongly support our
proposal that the hybridization ability of TFO by incorporat-
ing a sequence of consecutive s8A bases would be increased.
In summary, we synthesized the s8A phosphoramidite unit
6 and TFOs having s8A residues for the first time. The
selectivity of a TFO having an s8A residue was higher than
that of the unmodified TFO, whereas the hybridization ability
of a TFO having an s8A was lower than that of the
unmodified TFO. Moreover, it was also found that the
hybridization ability of TFO 7 having four consecutive s8A
bases was significantly higher than those of the corresponding
unmodified TFO 6 or TFO 8 containing four consecutive
o8A as a well-known modified base of a protonated cytosine.
These results indicate that it might be necessary to use the
cytosine analogs properly in sequences of discontinuous and
consecutive cytosine bases. In the case of TFOs containing
discontinuous cytosine bases, the use of 5-methylcytosine
accessible to the protonation5,6 of the cytosine ring in a TFO
under neutral conditions might be better because of its
stronger hydrogen bonding ability. On the other hand, s8A
should be used in place of cytosine to avoid electronic
repulsion and enhance the stacking interaction when a series
of cytosine bases are arranged in a consecutive sequence.
Further studies are now under way in this direction.
The Tm value of TFO 2 containing one s8A in entry 2 was
lower than that of unmodified TFO 1 in entry 1 (29.0 vs
30.5 °C), although the Tm value of TFO 2 containing one
s8A was higher than that of TFO 3 containing one o8A in
entry 3 (29.0 vs 28.2 °C). It was also found that the Tm value
of the TFO decreased further by the addition of a discontinu-
ous s8A as shown in entry 3 (27.4 vs 30.5 °C) though the
Tm values of TFO 3 obtained at various pHs showed the pH-
independence of TFO 3 in Figure S2 in Supporting Informa-
tion. In entry 5, the Tm value of TFO 5 containing two
discontinuous o8As was significantly lower (by 8.4 °C) than
that of unmodified TFO 1. Because the backbone structure
was somewhat disturbed by the presence of a bigger s8A
base in place of the protonated C, the distance between the
C1′ atoms in the neighboring mononucleotide units became
longer only at the modified bases so that the constant
structure could not be preserved around the modified sites.
Subsequently, we examined the selectivity of modified
DNA oligomers to study whether the s8A base can actually
form a third Hoogsteen-type base pair with the guanine base.
In the hairpin duplex (HP 2), we replaced the central C-G
base pair with a base pair of G-C that cannot form hydrogen
bonds with s8A or o8A, as shown in entries 6-11 of Table
1. We carried out the Tm experiments at pH 6.0 since the
triplexes formed between TFO 1 or 2 and HP 2 were very
unstable at pH 7.0. It should be noted that the selectivity of
s8A with ∆Tm of 30.8 °C (difference in the Tm values between
entry 8 and entry 9) was superior to that of the unmodified
cytosine with ∆Tm of 25.6 °C (difference in the Tm values
between entry 6 and entry 7). On the other hand, the o8A
base has a poorer selectivity with ∆Tm of 18.5 °C (difference
in the Tm values between entry 10 and entry 11). Because
the s8A base has a higher selectivity than the protonated C
base, it is likely that the former can form two hydrogen bonds
with G of G-C. The predominance of s8A over o8A could
be explained in terms of the higher contribution of the syn
form around the glycosyl bond of the former.
Acknowledgment. This study was supported by a grant
from CREST of Japan Science and Technology (JST) and a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan
and Research for Promoting Technological Seeds program
of JST. This study was also supported in part by a grant of
the Genome Network Project from the Ministry of Education,
Culture, Sports, Science and Technology, Japan and by the
global COE project.
Next, we synthesized oligodeoxyribonucleotides having
four s8A or o8A bases in a consecutive sequence and studied
their hybridization property to examine if our proposal is
correct. It was expected that when a consecutive sequence
of four s8A bases is incorporated into the DNA oligomer
TFO 7, the stacking effect of two s8A bases was enhanced,
as reported in the case of poly-2-thiouridylates.21 The Tm
values of the oligomers having four consecutive unmodified
and modified (o8A and s8A) bases are summarized in Table
2. The Tm value of TFO 6 with a consecutive sequence of
four cytosines to HP 3 was very low (Tm ) 9.7 °C) compared
with the Tm value (24.4 °C) of TFO 9 having four
discontinuous cytosine bases, 5′-TTTCTTCTTCTTCT, to the
Supporting Information Available: Experimental pro-
cedures and full spectroscopic data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Org. Lett., Vol. 11, No. 3, 2009