Formation of highly selective and efficient interstrand cross-linking to thymine without photo-irradiation

Article abstract of DOI:10.1039/b915381k

Interstrand cross-linking (ICL) forming oligodeoxynucleotides (ODNs) have been expected to ensure the inhibition of gene expression. In this communication, we report a highly efficient and selective ICL reaction to thymine using a 4-amino-2-vinyl-6-oxopyr

Full text of DOI:10.1039/b915381k

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COMMUNICATION
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Formation of highly selective and efficient interstrand cross-linking to
thymine without photo-irradiationw
Keiichi Hattori, Tomoya Hirohama, Shuhei Imoto, Shuhei Kusano and Fumi Nagatsugi*
Received (in College Park, MD, USA) 5th August 2009, Accepted 26th August 2009
First published as an Advance Article on the web 24th September 2009
DOI: 10.1039/b915381k
Interstrand cross-linking (ICL) forming oligodeoxynucleotides
(ODNs) have been expected to ensure the inhibition of gene
expression. In this communication, we report a highly efficient
and selective ICL reaction to thymine using a 4-amino-2-vinyl-6-
oxopyrimidine derivative.
Interstrand cross-linking (ICL) reactions between comple-
mentary duplexes or triplexes have provided an important
method to improve the stability of the complexes by covalent
bond formation. ICL forming oligodeoxynucleotides (ODNs)
are expected to enhance the antisense activity by irreversibly
binding to the target RNA utilizing steric blocking.1 Recently,
microRNAs (miRNAs) endogenously expressing small regu-
latory non-coding RNAs2 have been recognized as playing a
critical role in regulating gene expression, and significant
concerns have been raised about efficient antisense ODNs
against miRNAs.3,4 There are a number of reports on ICL
forming ODNs by using triggering signals such as photo-
irradiation5–7 or chemical reactions.8–10 We have developed
2-amino-6-vinylpurine nucleoside (1) as a selective cross-
linking agent to cytidine, activated by duplex formation to
the target DNA.11–13 In this communication, we describe a
highly efficient and selective ICL reaction to thymine using
pyrimidine derivative (2), which is designed based on the
structure of 1.
Fig. 1 Design of the novel cross-linking agents based on 2-amino-6-
vinylpurine.
Scheme
1 Reagents and conditions: (a) (1) nBu3SnCHCH2,
The unique structural features of 1 include both the
hydrogen bond donor–acceptor site as recognition sites, and
the vinyl group as a reactive moiety in a single molecule. In
addition, the reactive vinyl moiety of 1 can be regenerated
from stable precursors, sulfide derivatives, by hybridization
with the target gene.12,13 We have shown that this inducible
alkylation system functions in an intracellular environment
to promote efficient and selective antisense activity.11 Based
on these features, we have designed 4-amino-6-oxo-2-
vinylpyrimidine (2) as a reactive base. It is expected that 2a
can form a complex with guanine using the two hydrogen
bonds shown in Fig. 1. In a previous study, the nucleoside
derivatives with a butyl or ethyl spacer between the sugar part
and the 2-amino-6-vinylpurine selectively reacted with cyto-
sine or adenine by forcing them into close proximity with the
target bases in the triple helix formation.14,15 We therefore
(PPh3)2PdCl2, DMF; (2) C8H17SH, 77% for two steps. (b) LDA,
dioxane, reflux, 37%
designed the novel nucleoside derivative (2b) with an ethyl
spacer between the sugar part and the 4-amino-6-oxo-2-
vinylpyrimidine motif as a selective cross-linking agent to
thymine.
The vinyl group of the pyrimidine was anticipated to not
tolerate the ODN synthesis conditions and so was protected
with a sulfide group. The Pd(0)-catalyzed cross-coupling
reaction of 316 with nBu3SnCHCH2 gave the pyrimidine
derivative, which was, without purification, reacted with
octanethiol to afford 4 (77% for 2 steps; Scheme 1).
We attempted to perform the coupling reaction between 4
and various D-ribose derivatives under a variety of conditions
for the synthesis of 2a, but the target compound was not
obtained. Next, we examined the synthesis of nucleoside
derivatives (2b) with an ethyl spacer between the sugar part
and the 4-amino-6-oxo-2-vinylpyrimidine motif. The sugar
part (5) was synthesized from 2-deoxy-D-ribose as described
previously.15 The coupling reaction between the pyrimidine
base (4) and the sugar (5) was carried out under basic
conditions, to give N1- and O6-alkylated products. The
reaction conditions were screened in order to optimize the
Institute of Multidisciplinary Research for Advanced Materials,
Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai-shi, Miyagi,
980-8577, Japan. E-mail: nagatugi@tagen.tohoku.ac.jp;
Fax: 001-81-22-(217)-5633; Tel: 001-81-22-(217)-5633
w Electronic supplementary information (ESI) available: Synthetic
procedures for 4-amino-6-oxo-2-vinylpyrimidine containing ODN,
experimental procedures for cross-linking reactions, and spectroscopic
structural determination of adducts 17 and 18. See DOI: 10.1039/b915381k
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Fig. 3 Site selectivity of ICL reactions between 11 and target RNA.
ICL reactions were performed under the same conditions as in Fig. 2.
comparison of the reactivity of 11 towards the different bases
at the target site of the DNA (12a) or RNA (12b). It appears
that the ICL reactions to RNA are a little faster than to DNA.
The extremely rapid ICL reactions of 11 to the target DNA or
RNA were observed with dT or U at the target site, and 11
produced adducts to these targets in over 50% yields after only
1.5 h. It was confirmed that the ICL reactions of 11 took place
at the opposing thymine in the target strand by hydroxyl
radical cleavage of the purified cross-linked product (see the
ESIw).17 These results have demonstrated that 4-amino-6-oxo-
2-vinylpyrimidine exhibits highly selective and very fast ICL
reactions to thymine for DNA and uracil for RNA. The ICL
reaction using 11 with target RNA (13–16) with U at the non-
target site did not occur, as shown in Fig. 3. Similar results of
ICL reactions were obtained using DNA target and 11.
These results indicate that the highly selective reactivity of
11 is due to the efficient proximity effect between the vinyl
group of 4-amino-6-oxo-2-vinylpyrimidine and the target U or
dT in the duplex.
Scheme 2 Reagents and conditions: (a) (1) PhOCH2COCl, 1-HBT,
pyridine, CH3CN, 78%; (2) BF3ꢁEt2O, Me2S, CH2Cl2, 94%. (b) (1)
DMTrCl, pyridine, 90%; (2) iPr2NP(Cl)OC2H4CN, iPr2NEt, CH2Cl2,
63%. (c) Automated DNA synthesizer. (d) 3.0 eq. of MMPP, pH 10.
(e) 400 mM NaOH.
formation of the N1-alkylated products (6), and the use of
LDA in dioxane at 100 1C resulted in a considerable amount
of N1-alkylation with a ratio of N1 : O6 = 2 : 1. The
regioisomers of the N1-alkylation and the O6-alkylation were
determined by 2D NMR. After protection of the 4-amino
group with phenoxyacetyl, the conventional procedure
produced phosphoramidite precursor (8) in good yield. The
amidite precursor was applied to an automated DNA
synthesizer that produced the sulfide-protected ODN in good
yield. ODN (9) was then smoothly converted to the sulfoxide
by oxidation with magnesium monoperphthalate (MMPP),
followed by elimination under alkaline conditions to give
ODN (11) (Scheme 2).13
The ICL reactions using 11 and target DNA or RNA were
also followed by HPLC analysis. Fig. 4A shows the HPLC
analysis of the ICL reaction between 11 and the non-labeled
target DNA (12c: N = dT). After 2 h, a new peak related to
the ICL product (17) appeared at around 80% along with the
disappearance of target DNA (12c) and reactive ODN (11).
The isolated species (17) was found to have a mass consistent
with a cross-linked duplex for DNA (calcd. 9789.548, found
9788.979). The purified ODN (17) was digested with snake
venom phosphodiesterase I and bacterial alkaline phosphatase
to produce a new product (18) (Fig. 4B), which was purified by
HPLC for determining the structure. The ESI-TOF MS
spectra indicated that this new product consisted of three
bases, two dT derivatives and 4-amino-6-oxo-2-ethylpyrimidine,
which were produced by inhibition of the enzymatic digestion
The structures of the ODNs were confirmed by UV and
MALDI-TOF mass measurements.
The ICL reaction was investigated under neutral conditions
using reactive ODN (11) and target DNA (12a) (N = dG, dA,
dC, dT) or RNA (12b) (N = G, A, C, U) labeled with
fluorescein at the 50 end.
Each reaction mixture was analyzed by gel electrophoresis
with 20% denaturing gel, and the yields of the ICL reaction
were calculated from these analysis data. Fig. 2 shows a
1
1
near the cross-linked sites. The H NMR and H–1H COSY
spectra of this new product suggested that the selective ICL
reaction between 11 and thymine would occur on the
complementary target thymine at the O2 position, as shown
in 18, by comparing the chemical shifts of 18 to those of the
thymine adducts as reported in the literature18,19 (see Table 1
in the ESIw). These results have indicated that the novel
nucleoside derivative (2b) might selectively react with thymine
due to the effective proximity effect, forming hydrogen bonds
between 2b and thymine at the complementary site as shown in
Fig. 1. It is unclear whether or not the structure shown as 18 is
the primary product during the ICL reactions and further
study will be necessary for determining the ICL reaction
Fig. 2 Comparison of the reaction yields calculated from the gel
electrophoresis analysis of the cross-linking using 11 and target DNA
(12a) (N = dT, dG, dC, dA) or RNA (12b) (N = U, G, C, A). The
reaction was performed with 10 mM ODN 11 and 5 mM target ODN 12
in 0.1 M NaCl, 50 mM MES, pH 7.0, 30 1C.
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derivatives of 2b, have the potential to provide effective
antisense activity by inducible alkylation in the cell. Further
studies of these ICL reactions and their applications are
currently ongoing.
This work was supported by a Grant-in-Aid for Scientific
Research (B) from Japan Society for the Promotion of Science
(JSPS), CREST from Japan Science and Technology Agency.
Notes and references
1 J. M. Kean, A. Murakami, K. R. Blake, C. D. Cushman and
P. S. Miller, Biochemistry, 1988, 27, 9113–9121.
2 L. Ma, J. Teruya-Feldstein and R. A. Weinberg, Nature, 2007, 449,
682–689.
3 C. C. Esau, Methods, 2008, 44, 55–60.
4 S. Davis, S. Propp, S. M. Freier, L. E. Jones, M. J. Serra,
G. Kinberger, B. Bhat, E. E. Swayze, C. F. Bennett and C. Esau,
Nucleic Acids Res., 2009, 37, 70–77.
5 M. Higuchi, A. Kobori, A. Yamayoshi and A. Murakami, Bioorg.
Med. Chem., 2009, 17, 475–483.
6 X. H. Peng, I. S. Hong, H. Li, M. M. Seiclman and
M. M. Greenberg, J. Am. Chem. Soc., 2008, 130, 10299–10306.
7 Y. Yoshimura and K. Fujimoto, Org. Lett., 2008, 10, 3227–3230.
8 E. E. Weinert, R. Dondi, S. Colloredo-Melz, K. N. Frankenfield,
C. H. Mitchell, M. Freccero and S. E. Rokita, J. Am. Chem. Soc.,
2006, 128, 11940–11947.
Fig. 4 HPLC analysis of the ICL reactions between 11 and DNA
(12c) (50AGAAAGGAGAATAAAG30) in (A) and the adducts from
the enzymatic hydrolysate (B). The ICL reaction was performed under
the same conditions as in Fig. 2. HPLC conditions for A: ODS
column, 1.0 mL minꢂ1; solvent A 0.1 M TEAA; solvent B CH3CN,
linear gradient from 10–40% over 20 min and monitored at 254 nm.
Peak 17 was isolated and digested with snake venom phospho-
diesterase I and bacterial alkaline phosphatase in buffer (50 mM
Tris-HCl, 10 mM MgCl2). HPLC conditions for part B: ODS column,
1 mL minꢂ1; solvent A, 50 mM ammonium formate, solvent B CH3CN,
linear gradient from 5–30% over 30 min and monitored at 260 nm. Peak
18 was isolated and its structure determined as shown in (C).
9 Z. Qiu, L. Lu, X. Jian and C. He, J. Am. Chem. Soc., 2008, 130,
14398–14399.
10 K. Stevens and A. Madder, Nucleic Acids Res., 2009, 37,
1555–1565.
11 M. M. Ali, M. Oishi, F. Nagatsugi, K. Mori, Y. Nagasaki,
K. Kataoka and S. Sasaki, Angew. Chem., Int. Ed., 2006, 45,
3136–3140.
12 F. Nagatsugi, T. Kawasaki, D. Usui, M. Maeda and S. Sasaki,
J. Am. Chem. Soc., 1999, 121, 6753–6754.
13 T. Kawasaki, F. Nagatsugi, M. M. Ali, M. Maeda, K. Sugiyama,
K. Hori and S. Sasaki, J. Org. Chem., 2005, 70, 14–23.
14 F. Nagatsugi, Y. Matsuyama, M. Maeda and S. Sasaki,
Bioorg. Med. Chem. Lett., 2002, 12, 487–489.
15 F. Nagatsugi, D. Usui, T. Kawasaki, M. Maeda and S. Sasaki,
Bioorg. Med. Chem. Lett., 2001, 11, 343–345.
pathways. The structure determination of the cross-linked
product in ODNs is in progress. The experimental studies on
cross-linking reaction pathways are quite often very limited20
and future combined experimental and computational studies
of the cross-linking reaction pathways must be suggested with
the ultimate goal of identifying all the relevant transition and
product structures. A computational study of the cross-linking
reaction using 4-amino-6-oxo-2-vinylpyrimidine is indispensable
to discuss the reaction pathway.
16 T. Hirayama, M. Kamada, H. Tsurumi and M. Mimura,
Chem. Pharm. Bull., 1976, 24, 26–35.
In conclusion, we have demonstrated a highly selective and
very fast ICL reaction to thymine using the novel nucleoside
derivative 2b without photo-irradiation. New cross-linking
motifs based on 2b will be generally useful for site-directed
chemical modification of thymine within a selected target.
It is expected that the stable precursors, sulfide protected
17 M. M. Paz and P. B. Hopkins, J. Am. Chem. Soc., 1997, 119,
5999–6005.
18 M. Y. Wang, Y. B. Lao, G. Cheng, Y. L. Shi, P. W. Villalta and
S. S. Hecht, Chem. Res. Toxicol., 2007, 20, 625–633.
19 H. Borowy-Borowski and R. W. Chambers, Biochemistry, 1989,
28, 1471–1477.
20 P. M. Mitrasinovic, Bioconjugate Chem., 2005, 16, 588–597.
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Chem. Commun., 2009, 6463–6465 | 6465