A new reactive nucleoside analogue for highly reactive and selective cross-linking reaction to cytidine under neutral conditions

Article abstract of DOI:10.1016/S0960-894X(01)00505-4

We have already demonstrated that the oligonucleotides DNA (ODNs) bearing a 2-amino-6-vinylpurine derivative (1) exhibited efficient interstrand cross-linking to cytidine selectively. In this study, a new reactive nucleoside analogue, 2-amino-6-(1-ethylsulfoxy)vinylpurine derivative (7), was designed based on a computational method to achieve high and selective alkylation with cytidine under neutral conditions. It has been demonstrated that the ODN (13) bearing 2-amino-6-(1-ethylsulfoxy)vinylpurine achieved highly selective and efficient cross-linking to cytidine under neutral conditions.

Full text of DOI:10.1016/S0960-894X(01)00505-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2577–2579
A New Reactive Nucleoside Analogue for Highly Reactive and
Selective Cross-Linking Reaction to Cytidine under Neutral
Conditions
Fumi Nagatsugi, Natsuko Tokuda, Minoru Maeda and Shigeki Sasaki*
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Received 30 May 2001; accepted 14July 2001
Abstract—We have already demonstrated that the oligonucleotides DNA (ODNs) bearing a 2-amino-6-vinylpurine derivative (1)
exhibited efficient interstrand cross-linking to cytidine selectively. In this study, a new reactive nucleoside analogue, 2-amino-6-(1-
ethylsulfoxy)vinylpurine derivative (7), was designed based on a computational method to achieve high and selective alkylation with
cytidine under neutral conditions. It has been demonstrated that the ODN (13) bearing 2-amino-6-(1-ethylsulfoxy)vinylpurine
achieved highly selective and efficient cross-linking to cytidine under neutral conditions. # 2001 Elsevier Science Ltd. All rights
reserved.
The cross-linking reaction within complementary
duplexes and triplexes has been expected as a reliable
strategy to ensure inhibition of gene expression in the
antisense1ꢀ7 and antigene method.8ꢀ11 The strategy of
cross-linking is based on the use of oligodeoxynucleo-
tides (ODNs) incorporating a reactive functional, and
there are a number of reports on alkylating agents such
as psolaren,7,10 haloacetyl amide4,5,9 or aziridine1,3,8
units, etc. Recently, the cross-linking reaction has
attracted new attention as a tool for site-directed chem-
ical reaction to nucleobases of DNA or RNA.12 How-
ever, the existing alkylating agents do not seem to be
satisfactory for general application, and there is an
urgent need of new alkylating agents for application
in either in vitro or in vivo study. Thus, we have
established a new principle in the designing of cross-
linking agents, and developed a new nucloside ana-
logue 2-amino-6-vinylpurine derivative (1) that
exhibited efficient and selective reactivity toward the cyti-
dine at the target site.13,14 The principle demonstrated
with 2-amino-6-vinylpurine derivatives is characteristic in
that high reactivity is exhibited by the proximity effect
within the DNA hybrids without incurring chemical
instability of the functional nucleoside analogue. The
reactivity of 1 is also unique in that a weak nucleophilic
amino group of cytidine can be selectively alkylated.
High reactivity of 2-amino-6-vinylpurine skeleton to a
cytosine has been successfully applied to triplex-orien-
ted cross-linking.15 However, application of 1 to a living
system was arrested because of its need of acidic condi-
tions. Here, we wish to report that a new derivative of 2-
amino-6-vinylpurine with a double-activation structure
exhibited dramatic improvement in the yield compared
to that with 1, under neutral conditions.
Previously, theoretical assumption of the reactivity
between 9-methyl-6-(1-substituted vinyl)-2-aminopurine
and 1-methyl-cytosine showed good agreement with the
experimental data. That is, protonation to N1 of the
purine base is essential for alkylation of the 4-amino
group of cytosine, and methyl or TMS substitution at
the 2-position of the vinyl group decreased the reactiv-
ity.13 We expected that introduction of an electron
withdrawing group to the vinyl group would increase
the reactivity. Estimation by a semi-empirical MO cal-
culation [MOPAC (PM3)] suggested that the introduc-
tion of a methylsulfoxy or methylsulfonyl group to the
vinyl group decreased the activation energy compared
to the non-substituted one (Table 1, R=SOMe, SO2Me
vs R=H). Thus, the sulfoxy- or sulfonylvinyl deriva-
tives (2 or 4) were expected to achieve efficient reaction
even when the concentration of the N1-protonated form
of the purine analogue is low under neutral conditions.
The active species (7) would be obtained by either
chemical reaction or in situ activation within the duplex
from the stable precursor (5 or 6) (Fig. 1).
*Corresponding author. Tel.:+81-92-642-6651; fax:+81-92-642-6654;
e-mail: sasaki@phar.kyushu-u.ac.jp
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00505-4

2578
F. Nagatsugi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2577–2579
The synthesis of ODNs incorporating a reactive
nucleoside analogue is summarized in Scheme 1. 2-
Amino-6-vinylpurine (8) was synthesized from 20-deoxy-
guanosine as described previously,13 The vinyl group of
8 was tranformed into the dibromide, followed by dis-
placement with ethanethiol to produce a bisethylsulfide
derivative (9). The 2-amino group of 9 was protected
with the phenoxyacetyl group,16 followed by depro-
tection of the TBDMS group. The phosphoramidite
precursor (10) was obtained in good yield by the con-
ventional method,17 and applied to an automated DNA
synthesizer. The ODN (50-CTTT-X-TTCTCCTTTCT)
was cleaved from the support with 0.1 M NaOH,18 and
the solution was neutralized with CH3COOH instantly,
followed by purification by reverse-phase HPLC to give
DMTr-protected ODN in good yields. The purified oli-
gomers were treated with 10% CH3COOH to give the
deprotected ODN (11). Oxidation of 11 with MMPP
gave a mixture of several compounds. Interestingly,
mild alkaline treatment of 11 produced the ethylthio-
vinyl-bearing ODN (12) in good yield. The ODN (12)
was treated with two equivalents of MMPP to give the
corresponding ethanesulfoxyvinyl derivative (13). By
the treatment of a large excess MMPP for 24h, the
ethanesulfonylvinyl-bearing ODN (14) was obtained.
The structures of all ODNs were determined by
MALDI-TOF measurements. The synthesis of 15 was
carried out according to the reported procedure.14
Figure 1. In situ activation to sulfonylvinyl derivative (7).
Table 1. Estimated heat of formation (kcal/mola)
Compd
R
GS
TS
ÁG
Pr
1
2
3
4
H
185.7
159.2
186.3
123.0
208.7
177.7
.2110.424 189.3
141.70 18.7
23.0
18.5
205.4
174.5
The cross-linking was investigated under neutral condi-
tions with the functionalized ODNs (11–14 and 15) and
the target ODN (16) in the presence of 32P-labeled 16 as
a tracer, and analyzed by gel electrophoresis with 19%
denaturing gel. The formation of a higher molecular
weight band is indicative of the cross-linked adduct, and
the yield of the adduct can be obtained by quantifica-
tion of both the lower and higher bands. The reactivities
of the four functionalized ODNs (12–14 and 15) toward
the ODN bearing cytidine at the target site (16, N=C)
are compared in Fig. 2. The ODN (11) did not give the
adduct at all, and the reactivity of ODN (12) incorpor-
SOMe
SMe
SO2Me
140.1
aMOPAC96(PM3). GS, ground state; TS, transition state; Pr, product.
Structures of ground state and product of the complex are schemati-
cally shown.
Figure 2. Comparison of the cross-linking reactivity. Cross-linking
was done using using 10 mM reactive ODN (12–15), 1 mM target oli-
gomer (16) in the buffer containing 100mM NaCl, 50 mM MES, pH 7.0,
37 ꢁC, and analyzed by electrophoresis with 19% denaturing poly-
acrylamide gel at the indicated time. Yields were determined by quan-
tification of the bands on the gels by BAS 2500 using the imaging plate.
Scheme 1. (a) (1) Br2 aq, CHCl3, 0 ꢁC, 2 h, 65%; (2) DBU, CHCl3,
0 ꢁC, 1 h, then EtSH, 83%, (b) (1) PhOCH2COCl 1-hydroxy-benzo-
triazole, pyridine–CH3CN, 95%; (2) nBu4NF, 73%; (3) DMTrCl, pyr-
idine, 86%; (4) iPr2EtN, CH2Cl2, iPr2NP(Cl)OCH2CH2CN, 44%; (c)
(1) synthesis with an automated DNA synthesizer; (2) 0.1 N NaOH;
(3) neutralized with CH3COOH, 20–30%; (3) 10% CH3COOH; (d) 0.5
M NaOH, 24h; (e) 2 equiv MMPP (magesium monoperphthalate),
pH 10, 30 min, quant; (f) 6 equiv MMPP, pH 10, 1 day, quant.

F. Nagatsugi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2577–2579
2579
ating the ethylthiovinyl nucleoside is negligible. In con-
References and Notes
trast, the ODN (13) incorporating the ethylsulfoxyvinyl
nucleoside exhibited the most efficient reaction, and the
yield was as high as 50% at 1 h and reached 80% after
12 h. Although the phenylsulfoxide analogue (15) pro-
duced the adduct in over 80% yield under acidic condi-
tions, only slow reaction was observed under the neutral
condition. Thus, it has been proved that the new ethyl-
sulfoxyvinyl nucleoside may be applicable under
physiological conditions. The ODN (14) with an ethyl-
sulfonylvinyl analogue gave the adduct in a lower
yield than the ODN (13), which differs from the
theoretical assumption that has suggested similar ÁG
values for both the methylsulfoxy- and methylsulfonyl-
vinyl (Table 1). As the primary product after the addi-
tion reaction is only slightly more stable than the
transition state in the reaction with the sulfonylvinyl
compound, a reverse reaction to the starting structure
might impede the promotion to adduct formation.
1. (a) Webb, T. R.; Matteucci, M. D. J. Am. Chem. Soc. 1986,
108, 2764. (b) Webb, T. R.; Matteucci, M. D. Nucleic Acids
Res. 1986, 14, 7661.
2. (a) Zeng, Q.; Rokita, S. E. J. Org. Chem. 1996, 61, 9080. (b)
Chatterjee, M.; Rokita, S. E. J. Am. Chem. Soc. 1994, 116,
1690. (c) Li, T.; Rokita, S. E. J. Am. Chem. Soc. 1991, 113,
7771. (d) Chatterjee, M.; Rokita, S. E. J. Am. Chem. Soc.
1991, 113, 5116.
3. Cowart, M.; Benkovic, S. J. Biochemistry 1991, 30, 788.
4. (a) Coleman, R. S.; Pires, R. M. Nucleic Acids Res. 1997,
25, 4771. (b) Coleman, R. S.; Kesicki, E. A. J. Org. Chem.
1995, 60, 6252.
5. (a) Tabone, J. C.; Stamm, M. R.; Gamper, H. B.; Meyer,
R. B., Jr. Biochemistry 1994, 33, 375. (b) Meyer, R. B., Jr.;
Tabone, J. C.; Hurst, G. D.; Smith, T. M.; Gamper, H. B. J.
Am. Chem. Soc. 1989, 111, 8517.
6. Lukhtanov, E. A.; Podyminogin, M. A.; Kutyavin, I. V.;
Meyer, R. B., Jr.; Gamper, H. B. Nucleic Acids Res. 1996, 24,
683.
7. (a) Chang, E. H.; Miller, P. S.; Cushman, C.; Devadas, K.;
Pirollo, K. F.; Ts’o, P. O. P.; Yu, Z. P. Biochemistry 1991, 30,
8283. (b) Kean, J. M.; Murakami, A.; Blake, K. R.; Cushman,
C. D.; Miller, P. S. Biochemistry 1988, 27, 9113. (c) Lee, B. L.;
Murakami, A.; Blake, K. R.; Lin, S.-B.; Miller, P. S. Bio-
chemistry 1988, 27, 3197.
8. Shaw, J.-P.; Milligan, J. F.; Krawczyk, S. H.; Matteucci,
M. D. J. Am. Chem. Soc. 1991, 113, 7765.
9. (a) Grant, K. B.; Dervan, P. B. Biochemistry 1996, 65,
12313. (b) Povsic, T. J.; Strobel, S. A.; Dervan, P. B. J. Am.
Chem. Soc. 1992, 114, 5934. (c) Povsic, T. J.; Dervan, P. B. J.
Am. Chem. Soc. 1990, 112, 9428.
10. Takasugi, M.; Guenouz, A.; Chassignol, M.; Deout, J. L.;
Lohmme, J.; Thuong, N. T.; Helene, C. Proc. Natl. Acad. Sci.
U.S.A. 1991, 88, 5602.
Figure 3. Base selectivity of the cross-linking. Reaction conditions are
recorded in the caption of Figure 2.
11. Lukhtanov, E. A.; Mills, A. G.; Kutyavin, I. V.; Vladimir,
V. V.; Reed, M. W.; Meyer, R. B., Jr. Nucleic Acids Res. 1997,
25, 5077.
12. Woolf, T. M. Nature Biotechnol. 1998, 16, 341; and refer-
ences cited therein.
Figure 3 illustrates high cytidine selectivity of the ODN
(13) bearing the ethylsulfonylvinyl nucleoside in the
reaction with the target ODN (16, N=C, G, A, or T).
In all cases, adduct formation was observed only to the
ODN (16) with C at the target site.
13. (a) Nagatsugi, F.; Uemura, K.; Nakashima, S.; Maeda,
M.; Sasaki, S. Tetrahedron Lett. 1995, 36, 421. (b) Nagatsugi,
F.; Uemura, K.; Nakashima, S.; Maeda, M.; Sasaki, S. Tetra-
hedron 1997, 53, 3035.
14. (a) Nagatsugi, F.; Kawasaki, T.; Usui, D.; Maeda, M.;
Sasaki, S. J. Am. Chem. Soc. 1999, 121, 6753. (b) Kawasaki,
T.; Nagatsugi, F.; Maeda, M.; Sasaki, S. Nucleic Acids Sym-
posium Series 1999, 42, 43. (c) Nagatsugi, F.; Kawasaki, T.;
Tanaka, Y.; Maeda, M.; Sasaki, S. Nucleic Acids Symposium
Series 1998, 39, 103. (d) Nagatsugi, F.; Kawasaki, T.; Maeda,
M.; Sasaki, S. Nucleic Acids Symposium Series 1997, 37, 67. (e)
Nagatsugi, F.; Kawasaki, T.; Maeda, M.; Sasaki, S. Nucleic
Acids Symposium Series 1996, 35, 79.
Finally, we have successfully demonstrated that
2-amino-6-(1-ethylsulfoxyvinyl)purine exhibited selec-
tive and efficient cross-linking to a cytidine at the target
site under neutral conditions. As this new reactive motif
is stable under the reaction conditions, 2-amino-6-(1-
ethylsulfoxyvinyl)purine will be useful in the antisense
strategy as well as for site-directed chemical modifica-
tion of a cytidine within a selected target. Application of
the 2-amino-6-(1-ethylsulfoxyvinyl)purine motif to
TFOs for triplex cross-linking is now under study.
15. Nagatsugi, F.; Usui, D.; Kawasaki, T.; Maeda, M.;
Sasaki, S. Bioorg Med. Chem. Lett. 2001, 11, 343.
16. Schulhof, J. C.; Dolko, D.; Teoule, R. Nucleic Acids Res.
1987, 15, 397.
Acknowledgements
This study was partially supported by a Grant-in-Aid
for Scientific Research [Priority Area (A)(2), No.
13022250] from the Ministry of Education, Science,
Sports, Science and Technology, Japan.
17. Nagai, H.; Fujiwara, T.; Fujii, M.; Sekine, M.; Hata, T.
Nucleic Acids Res. 1987, 17, 8581.
18. Koh, J. S.; Dervan, P. B. J. Am. Chem. Soc. 1992, 114,
1470.