Selective reaction to a flipping cytidine of the duplex DNA mediated by triple helix formation

Article abstract of DOI:10.1016/S0960-894X(00)00666-1

A new nucleoside derivative (2) with a butyl spacer between the sugar part and the 2-amino-6-vinylpurine motif has been synthesized. The triplex-forming oligodeoxynucleotide incorporating 2 has achieved strand- and cytidine-selective cross-linking reaction to the G-C target site mediated by triple helix formation. It has been suggested that 2 reacts with a flipping cytidine at the target site.

Full text of DOI:10.1016/S0960-894X(00)00666-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 343±345
Selective Reaction to a Flipping Cytidine of the Duplex DNA
Mediated by Triple Helix Formation
Fumi Nagatsugi, Daisaku Usui, Takeshi Kawasaki, Minoru Maeda and Shigeki Sasaki*
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Received 11 October 2000; accepted 15 November 2000
AbstractÐA new nucleoside derivative (2) with a butyl spacer between the sugar part and the 2-amino-6-vinylpurine motif has been
synthesized. The triplex-forming oligodeoxynucleotide incorporating 2 has achieved strand- and cytidine-selective cross-linking
reaction to the G-C target site mediated by triple helix formation. It has been suggested that 2 reacts with a ¯ipping cytidine at the
target site. # 2001 Elsevier Science Ltd. All rights reserved.
Gene manipulation at the base level within a speci®c site
of DNA has attracted much attention.1 Covalent bond
formation within duplexes or triplexes has been applied
to improve the e€ectiveness of the antisense or antigene
oligonucleotides (ODN).2 Also, covalent modi®cation
of a base theoretically should become a potential che-
mical method to cause a point mutation that may result
in activation or modi®cation of the functional expres-
sion of the gene. A recent example has been demon-
strated in the study of site-directed mutation with the
psoralen-conjugate of the triplex-forming oligonucleo-
tide (TFO).3 Alkylation of nucleobases with functional
TFOs might have potential for such purpose. Pre-
viously, oligodeoxynucleotides and related oligomers
were conjugated with haloacetyl amide,4 aryl nitrogen
mustard,5 aziridine units6 or a minor groove-reactive
compound7 and were used for interstrand cross-linking
of a duplex or triplex. In order to develop such reactive
oligonucleotides as a useful tool for manipulation of a
gene at the base level, it is apparent that further
improvement of the selectivity and eciency of the
reactive group is needed. In our own approach to selec-
tive functional oligonucleotides, we have developed a 2-
amino-6-vinylpurine nucleoside derivative as a cytidine
selective cross-linking agent within a duplex.8,9 Here we
wish to report the highly selective reaction to a ¯ipping
cytidine of the duplex mediated by triplex formation by
using TFO conjugating the new 2-amino-6-vinylpurine
derivative.
It has been already demonstrated that the oligonucleo-
tide incorporating 2-amino-6-vinylpurine (1) exhibits
selective and ecient alkylation to a cytidine at the tar-
get site. A further remarkable point of 1 is that the
alkylating activity can be auto-generated within the
duplex from its stable precursor, a phenylsul®de or
phenylsulfoxide derivative (Fig. 1).10 Proximity e€ect
between the 2-amino-6-vinylpurine motif and a cytidine
apparently plays a key role in the selectivity and
eciency of the new cross-linking.
Accordingly, we hypothesized that, if the 2-amino-6-
vinylpurine motif of TFO is forced into proximity with
a cytidine of a duplex, selective cross-linking should
take place. Thus, a new nucleoside derivative (2) having
a butyl spacer between the sugar part and the 2-amino-
6-vinylpurine motif was designed (Fig. 2). The 2-amino-
6-vinylpurine motif of 2 would react selectively with the
cytidine in the opposite strand when the cytidine is ¯ip-
ped out from a G-C base pair (Fig. 2). Based on a
molecular model, the length of the butyl spacer seemed
*Corresponding author. Tel.: +81-92-642-6651; fax: +81-92-642-
6654; e-mail: sasaki@phar.kyushu-u.ac.jp
Figure 1. Synchronous activation within duplex.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00666-1

344
F. Nagatsugi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 343±345
Figure 2. Design of 2 to achieve selective reaction to the ¯ipping cytidine within the triplex.
to be suitable to bring the 2-amino-6-vinylpurine motif
into close proximity to the cytidine in the pyrimidine
strand of a parallel-type triplex (Fig. 2).
group with phenoxyacetyl, the conventional procedure13
produced the phosphoramidite precursor (6) in good
yield. The sul®de-protected ODN (7) was obtained by
applying 6 to an automated DNA synthesizer in good
yield after puri®cation with RP-HPLC. The ODN (7)
was then smoothly converted to 8a by oxidation with
magnesium monoperphthalate (MMPP) following elim-
ination under an alkaline condition.10 Addition of thio-
phenol to 8a and following oxidation with MMPP gave
8b in good yield. The synthesis of 9 containing 1 was
done according to the reported procedure.10 The struc-
ture of ODN was con®rmed by UV and MALDI-TOF
mass measurements.
The sugar part 3 was synthesized by b-selective C-gly-
coside formation via Wittig±Honor±Emmons reaction
from 20-deoxy-d-ribose (Scheme 1).11 Several transfor-
mation reactions with 3 a€orded the tosylate (4) having
the butyl spacer. 9-N-Alkylation of 2-amino-6-chloro-
purine12 with 4 yielded the desired product as a major
isomer, which was used for the Pd(II)-catalyzed cross-
coupling reaction with nBu3SnCHˆCH2 to a€ord 2-
amino-6-vinylpurine derivative (5). After protection of
the vinyl group with methylsul®de and the 2-amino
The cross-linking was investigated with the functiona-
.
lized ODNs (8a,b or 9) and the target duplexes (10 11).
In order to clarify the strand of the reaction, one of the
two strands (10 or 11) labeled with 32P at the 50-end was
used as a tracer. The results were analyzed by gel elec-
trophoresis with 15% denaturing gel and the cross-
linked products were identi®ed as slower moving bands
relative to the unreacted radiolabeled duplex strand
(Fig. 3).
Scheme 1. (a) 1) TrCl, DMAP, pyridine, 2) t-BuOK, THF,
(EtO)2P(O)CH2CO2Et, (b) 1) i-Pr2NEt, MOMCl, 2) DIBAL,
CH2Cl2, 3) NaH, (EtO)2P(O)CH2CO2Et, THF, 4) H2, Pd±C, EtOH,
5) LAH, THF, 6) TsCl, pyridine, (c) 1) 2-amino-6-chloropurine, t-
BuOK, DMSO, 2) n-Bu3SnCHˆCH2, (Ph3P)2PdCl2, dioxane, (d) 1)
MeSNa, CH3CN, 2) PhOCH2COCl, 1-HBT, CH3CN, pyridine, 3)
BF3-Et2O, Me2S, 4) DMTrCI, pyridine, 5) i-Pr2NEt, CH2Cl2, i-
Pr2NP(Cl)OC2H4CN, (e) 1) automated DNA synthesizer, 2) 28%
NH3, 3) 10% AcOH, (f) 1) 3.0 equiv MMPP, pH 10, 2) 470 mM
NaOH, (g) 1) PhSH, 2) 3.0 equiv MMPP, pH 10.
Figure 3. Comparison of the cross-linking reactivity by auto-
radiogram of gel electrophoresis (15% denaturing gel). A with 9
(Z=1), B with 8a (Z=2). The reaction was done using 10 mM of
.
ODNs (9: Z=1 or 8a: Z=2), 1 mM of target duplex 10 11 in a bu€er
including 10 mM cacodylate, 0.25 mM spermine, 100 mM NaCl, pH
4.5 Æ 0.3 at 30 ꢀC. The reaction mixture contained either 50-32P-labeled
10 or 11 as a tracer. The reaction was stopped by the addition of for-
mamide after 20 h.

F. Nagatsugi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 343±345
345
It is clearly shown that 1-bearing ODN (9) produces no
Acknowledgements
adducts with any target site (Fig. 3, A). On the other
hand, 2-bearing ODN (8a) reacted only to the cytidine
within the pyrimidine strand and did not produce any
adducts with other target sites (Fig. 3, B, lane 6). All
ODN combinations using 7, 10 and 11 showed triplex
melting temperatures higher than 45 ꢀC, indicating that
the major part of them are in triplex form under the
reaction temperature at 30 ꢀC. Thus, it is clear that the
selectivity is not due to di€erence in triplex stability.
These results indicated that 2 exhibited high selectivity
to cytidine only at the target site of the pyrimidine
strand (11) of the duplex, and that the reaction of the 2-
amino-6-vinylpurine motif needs precise proximity with
the target cytidine. A similar selective reaction to the
cytidine in the pyrimidine strand (11) was also obtained
with the TFO 8b incorporating the phenylsulfoxide
derivative of 2. Considering that the pyrimidine strand
(11) contains ®ve cytidines as possible reaction sites, the
high selectivity achieved by 2-bearing ODN (8a) is
remarkable. The high selectivity is probably ascribed to
the complex formation between 2 and the ¯ipping-cyti-
dine as we anticipated in the molecular design of 2. This
speculation has been supported by the fact that higher
yields of the cross-linking reactions were obtained with
duplexes containing the mismatch pairs (X±Y in 10ꢁ 11:
A±C or T±C), in which a higher ratio of the ¯ipping-
cytidine was expected compared to a matched G±C pair.
That is, the yield of cross-linked products increased
from 25% for G±C (a match pair) to 40% for A±C and
T±C (a mismatch pair). As it is clear from the estima-
tion with the electrophoresis using nature gel that the
duplex containing a mismatch pair of A±C or T±C is
only slightly less stable than that with a G±C pair at
room temperature, increase in the cross-linking yield
may be attributable to a higher ratio of the ¯ipping
cytidine of these mismatch pairs. In contrast, lower
cross-linking yield of 16% was obtained at the C±C
mismatch site probably due to thermal instability of the
duplex containing the C±C mismatch as evidenced by
the electrophoresis.14
The authors are grateful for support by Grant-in-Aid
for Scienti®c Research on Priority Area (A), Area No.
404 to SS and for Young Scientist to FN from the Min-
istry of Education, Science, Sports and Culture, Japan.
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In conclusion, we have successfully demonstrated a
highly selective cross-linking reaction to a ¯ipping cyti-
dine within the triplex using TFO bearing 2-amino-6-
vinylpurine derivatives (2). The selective reactivity of 2
should be useful in application to site-directed mod-
i®cation of a cytidine within a selected target. Further
work is now under way in the search for the appropriate
structure having higher reactivity in the triplex forming
cross-linking reaction.
14. This accords with the reported fact, Peyret, N.; Senevir-
atne, P. A.; Allawi, H. T.; SantaLucia, J., Jr. Biochemistry
1999, 38, 3468.