J. Am. Chem. Soc. 1999, 121, 6753-6754
6753
Scheme 1a
Highly Efficient and Selective Cross-Linking to
Cytidine Based on a New Strategy for
Auto-Activation within a Duplex
Fumi Nagatsugi, Takeshi Kawasaki, Daisaku Usui,
Minoru Maeda, and Shigeki Sasaki*
4
Graduate School of Pharmaceutical Sciences
Kyushu UniVersity, 3-1-1 Maidashi
Higashi-ku, Fukuoka 812-8582, Japan
ReceiVed February 5, 1999
Sequence-selective cross-linking between complementary du-
plexes or triplexes provides an important method for improving
the stability of the hybridized complexes.1-11 This strategy has
been expected to effect inhibition of gene expression in the
antisense and antigene methods. The basic concept of cross-linking
includes the incorporation of a reactive functional group into the
oligomers, and there are a number of reports on alkylating groups,
such as haloacetyl amide,4,5,9 aziridine,1,3,8 and psolaren deriva-
tives.7,10 Recently, interest in cross-linking has been expanding
as a tool for site-directed chemical modification which may induce
point mutation of a genetic code.12 However, the existing
aklylating agents do not seem to be satisfactory for general
application, and there is an urgent requirement for new cross-
linking agents that exhibit efficient and selective reactivity toward
a chosen target site. The use of functional groups with intrinsic
high reactivity may cause considerable instability, while those
requiring ultraviolet activation may limit the target site. In our
approach, we have developed a new strategy for cross-linking in
which a reactive species is auto-generated within a duplex. Here
we wish to report a highly efficient and selective cross-linking
reaction toward cytidine with the use of derivatives of a 2-amino-
6-vinylpurine nucleoside (1).
a (a) (1) PhSH, CH2Cl2 rt, 1 h, 74%, (2) PhOCH2COCl, 1-hydroxy-
benzotriazole, 78%, (3) nBu4NF, 76%, (4) DMTrCl, pyridine, 75%, (5)
iPr2EtN, CH2Cl2, iPr2NP(Cl)OCH2CH2CN, 64%. (b) (1) synthesis with
an automated DNA synthesizer, (2) 0.1 N NaOH, (3) neutralized with
CH3COOH, 70-80%, (3) 10% CH3COOH. (c) 2 equiv MMPP (magne-
sium monoperphthalate), pH 10, 30 min, quantitative. (d) 10 equiv MMPP,
pH 10, 1 d, quantitative, (e) 470 mM NaOH, 30 min, quantitative.
We have previously designed a 2-amino-6-vinylpurine nucleo-
side (1) as a selective alkylating agent to cytidine. The complex
resembling a natural G-C pair would favor the attack of the
4-amino group of cytidine, a weak nucleophile, leading to a cross-
linked product (2).13 Model experiments in organic solvents and
investigations with ODNs bearing 1 have demonstrated that 1 is
a cytidine-selective alkylating agent.14 To achieve high reactivity
without incurring chemical stability of 1, we next designed a new
strategy in which less reactive precursors (3, 4, or 5) would be
auto-activated within duplexes to generate 1.
The 2′-deoxy nucleoside derivative of 2-amino-6-vinylpurine
(1) was synthesized from 2′-deoxyguanosine as described previ-
ously,14 protected as phenylsulfide, and then transformed to the
amidite precursor (3). An example of the synthesis of function-
alized ODNs incorporating a 6-substituted 2-aminopurine nucleo-
side is illustrated in Scheme 1. The sulfide functional group of 6
was converted to the sulfoxide form (7) as a sole product by
oxidation with 2 equiv of magnesium monoperphthalate (MMPP).
The transformation of 6 to the sulfone form (8) was also a
quantitative reaction with an excess of MMPP. Both 7 and 8 were
smoothly transformed to 9, incorporating the 2-amino-6-vinylpu-
rine nucleoside under mild alkaline conditions. The structures of
the ODNs (6-9) were confirmed by UV and MALDI-TOF mass
measurements.15
The cross-linking was investigated with the functionalized
ODNs (6-9) and the target ODN (10) in the presence of 32P-
labeled 10 as a tracer and analyzed by gel electrophoresis with
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(13) The actual position of protonation is uncertain and could possibly be
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10.1021/ja990356e CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/03/1999