Alteration of cross-linking selectivity with the 2′-OMe analogue of 2-amino-6-vinylpurine and evaluation of antisense effects

Article abstract of DOI:10.1016/j.bmcl.2010.08.027

We previously reported that oligodeoxynucleotides containing 2-amino-6-vinylpurine (2-AVP: 1) exhibit efficient selective cross-linking to cytosine. In this study, the 2′-OMe nucleoside analogue (2) of 2-AVP was designed in order to increase its affinity

Full text of DOI:10.1016/j.bmcl.2010.08.027

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6121–6124
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Alteration of cross-linking selectivity with the 20-OMe analogue
of 2-amino-6-vinylpurine and evaluation of antisense effects
Shuhei Imoto a, Tsuneaki Hori a, Shinya Hagihara a, Yosuke Taniguchi b, Shigeki Sasaki b,c, Fumi Nagatsugi a,c,
*
a Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai-shi, Miyagi 980-8577, Japan
b Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
c CREST, Japan Science and Technology Agency, 4-1-8 Motomachi, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported that oligodeoxynucleotides containing 2-amino-6-vinylpurine (2-AVP: 1) exhibit
efficient selective cross-linking to cytosine. In this study, the 20-OMe nucleoside analogue (2) of 2-AVP
was designed in order to increase its affinity to RNA and enhance metabolic stability. It has been demon-
strated that 20-OMe oligonucleotides bearing 2 achieve highly selective cross-linking to the thymine base
in DNA and show higher antisense effect on luciferase production in cell lysate.
Received 7 June 2010
Revised 3 August 2010
Accepted 5 August 2010
Available online 10 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Oligonucleotide
Cross-linking
Antisense
DNA
Nucleic acids chemistry
An oligonucleotide (ON) with a sequence complementary to a
specific mRNA can inhibit its expression, thereby inhibiting the
transfer of genetic information from DNA to protein as an antisense
strategy.1 This method is therefore very effective not only for ther-
apeutic purposes but also to investigate gene functions.2 The use of
unmodified ON for applications in cells, however, is very limited
because of the following reasons: ON’s poor cellular uptake effi-
ciency and targeted delivery, its low specificity and affinity for
the target sequence, and susceptibility to degradation by nucleas-
es. Many modified ONs containing unnatural bases or modified
sugars have been prepared for improving the efficacy of the anti-
sense method.3 One such example is ONs incorporating a 20-mod-
ified nucleotide. These analogues are known to exhibit high
binding affinity to target RNA because of their enhanced metabolic
stability.3b Recently, 20-modified antisense ONs have been shown
to effectively inhibit microRNAs activity in cells.4 Furthermore,
cross-linking reactions are expected to enhance the antisense
activity by irreversibly binding to the target RNA, based on the
steric blocking mechanism. Several cross-linking ONs have been
reported to react with the target DNA when triggered by photoir-
radiation5 or chemical reactions.6 We previously demonstrated
that the 2-amino-6-vinylpurine (2-AVP) deoxynucleoside analogue
was activated by the duplex formation with the target DNA and
reacted to cytosine selectively.7 In this study, we designed the
20-O-methyl (20-OMe) analogue (2) of 2-AVP (Fig. 1) to check its
reactivity and affinity to RNA. The use of the 20-OMe backbone
was also expected to enhance metabolic stability. Herein, we de-
scribe the synthesis and evaluation of the cross-linking properties
of 2’-OMe ON incorporating the 2-AVP (2), and the higher anti-
sense effect with the use of 2 in non-cell translation assay.
Scheme 1 summarizes the synthesis of 20-OMe ONs incorporat-
ing a reactive nucleoside analogue. 2-AVP derivative (3) was syn-
thesized from 20-OMe guanosine, as described previously.7a After
protection of the vinyl group and the 2-amino group with methyl
sulfide and with phenoxyacetyl, respectively, the conventional
procedure produced the phosphoramidite precursor (5) in good
yield. The sulfide protected 20-OMe ON (6) was then synthesized
from the phosphoramidite precursor through the use of an
automated DNA synthesizer. The sequences of synthesized ONs
were found to be complementary to firefly-luciferase mRNA
(Scheme 1). After deprotection of the DMTr group, the mixture
was purified by gel electrophoresis using denaturing polyacryl-
H
N
N
O
O
N
H
N
N
O
N
H
O
N
N
H
O
N
N
N
H
H
2
O
N
N
DNA
1
O
H
OMe
* Corresponding author.
E-mail address: nagatugi@tagen.tohoku.ac.jp (F. Nagatsugi).
Figure 1. Structure of 20-OMe analog of 2-AVP.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.027

6122
S. Imoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6121–6124
the bands of the target sequence to the cross-linked adducts. Com-
O
N
parison of the reactivity of the 2-AVP-containing 20-OMe ON (7) to-
wards the different bases at the target site of the DNA (8a) or RNA
(8b) is shown in Figure 2. Surprisingly, 7 gave the highest yields in
the reaction with the thymine base in DNA or with uracil base in
RNA under acidic conditions, albeit it gave lower yields in the reac-
tion with dC target (Fig. 2A). When the reactions of 7 with the DNA
target were performed under neutral conditions, the cross-linked
adduct was observed only with the thymine base, and no cross-link
was formed with the cytosine base (Fig. 2B (a: vs DNA)). Cross-
linking reactions of 7 with the RNA target did not occur under neu-
tral conditions (Fig. 2B (b: vs RNA)). The reaction product between
7 and 8a (N = dT) was purified by HPLC and proven to be the cross-
linked one by MALDI-TOF MS measurement (calcd 10071.8 found
10072.3).
N
N
N
N
(b)
NH2
NH
NH2
N
N
(a)
(c)
HO
HO
TBSO
N
O
O
3
OH OMe
N
TBSO
OMe
N
SMe
SMe
(d)
NHPac
N
N
DMTrO
N
N
O
N
NHPac
O
O
5
4
O
P
OMe
OH OMe
CN
(iPr)2N
SMe
N
N
N
(e)
N
N
N
N
NH2
3'
N
NH2
To check whether duplexes were formed under the reaction
conditions, thermal stability was estimated by measuring the
melting temperature (Tm) of the duplex formed between the 20-
OMe ON (6) containing stable precursor of 2-AVP and target DNA
(8a) or RNA (8b) under acidic and neutral conditions (Supplemen-
tary data). The melting profile of 20-OMe ON (6) /DNA or RNA du-
plex suggested that the duplexes of 7/target DNA or 7/RNA would
be formed under the reaction conditions, and that the cross-linking
reactions might occur in each duplex. In the case of the reactions
shown in Figure 2B (b: vs RNA), where no cross-linking was ob-
served, 20-OMe ON/RNA duplexes were clearly formed, as evi-
denced by the melting profiles. However, differences in thermal
stability could not explain the base selectivity of the cross-linking
reactions when 20-OMe ON (7) was used. The circular dichroism
(CD) spectra were then measured to check the conformational
change in the duplex between 6 and the DNA or RNA. These spectra
were similar to that of the duplex between 20-OMe ON containing
adenosine instead of the 2-AVP derivative and target DNA (8a,
N = dT) or RNA (8b, N = U). These CD spectra have indicated that
the 20-OMe ON/DNA duplexes lie between the A- and B-conforma-
tion, whereas the 20-OMe ON/RNA duplexes lie in the A-conforma-
tion.8 As the oligo DNA containing 2’-deoxy 2-AVP cross-linked to
the cytosine base in the DNA/DNA duplex in the B-conformation,7
the alteration of the base selectivity to the thymine base using 2’-
OMe ON might be attributed to the difference of the duplex
conformation.
5'
2'-OMe-r(UCUUUGAUU)
ACGCC
2'-OMe-r(UCUUUGAUU)
ACGCC
6
7
Scheme 1. Reagents and conditions: (a) (1) TBSCl, imidazole, DMF; (2) TsCl, TEA,
DMAP, CH2Cl2; (3) (C2H3BO)3, Pd(0), LiBr, dioxane–H2O; (b) (1) CH3SNa, CH3CN,
CH2Cl2; (2) PhOCH2COCl, HBT, CH3CN, pyridine; (3) n-Bu4NF, THF; (c) (1) DMTrCl,
Py; (2) (i-Pr)2NP(Cl)OC2H4CN; (d) (1) synthesis with an automated DNA synthesizer,
(2) 28% aqueous NH3; (3) PAGE purification; (e) (1) 2 equiv MMPP, (2) aqueous
NaOH.
amide gel to produce the sulfide protected 20-OMe ONs in good
yield. The ON (6) was then smoothly converted to 7 by oxidation
with magnesium monoperphthalate, following elimination of the
sulfoxide group under an alkaline condition. The structures of
20-OMe ONs were confirmed by MALDI-TOF MS measurements.
The cross-linking reaction was then investigated under acidic
conditions using the reactive 20-OMe ON (7) and the target DNA
(8a) (N = dG, dA, dC, dT) or RNA (8b) (N = rG, rA, rC, U) labeled with
fluorescein at the 50 end. The progress of the cross-linking was
monitored by gel electrophoresis with 20% denaturing gel. The
yields of the cross-linking reactions were calculated by comparing
7
8a
8b
(X: 2-AVP)
FAM
FAM5’
(A) pH 5.0
To get more insight into the cross-linking reaction, the cross-
linked nucleoside was isolated by enzymatic hydrolysis, and the
structure of the cross-linked product was determined. After 6 h
incubation of the purified cross-linked adduct with nuclease P1,
snake venom phosphodiesterase and alkaline phosphatase were
added to the mixture. HPLC analysis showed the formation of 8
(a) DNA target
(b) RNA target
7+8b (N=U, G, C, A)
7 +8a (N=dT, dG, dC, dA)
N=
dT
100
50
100
50
N=
U
dC
G
C
A
dG
dA
0
0
(A)
(B)
10
20
10
20
Reaction time (h)
Reaction time (h)
dA
(B) pH 7.0
(a) DNA target
O
4
(b) RNA target
N
HO
100
50
dG
100
N=
N
*each 2’-OMe nucleoside.
7 +8b (N=U, G, C, A)
N
N
N
7 +8a (N=dT, dG, dC, dA)
O
*
rU
N
N
dT
H
O
9
HO
*
rC
dC
O
*
rA
H
*
OMe
rG
HO
dT
50
0
FAM
9
OH
N=
U
dG
G
C A
dC
dA
0
10
20
10
Reaction time (h)
20
Figure 3. (A) HPLC analysis of the enzymatic hydrolysate. The purified adduct was
digested with nuclease P1 in buffer (30 mM NaOAc, 5 mM ZnCl2), snake venom
phosphodiesterase and alkaline phosphatase in buffer (50 mM Tris–HCl, 10 mM
MgCl2). HPLC conditions: ODS column 1 mL/min; solvent; 0.1 M TEAA buffer, B:
CH3CN, B: 5–15%/10 min, 15–40%/20 min. (B) Speculative structure of the adduct
(9).
Reaction time (h)
Figure 2. Comparison of the reaction yields calculated from the gel electrophoresis
analysis of the cross-linking using 7 and target DNA (8a) (N = dT, dG, dC, dA) or RNA
(8b) (N = U, G, C, A). The reaction was performed with 10
lM ODN (7) and 5 lM
target ODN (8) in 0.1 M NaCl, 50 mM MES, pH 5.0 (A) or pH 7.0 (B), at 30 °C.

S. Imoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6121–6124
6123
4.25 ppm
7:5’ 2’-OMe-r(UCUUUGAUU X ACGCC)3’ (X:2-AVP)
8a:
3’d(AGAAACTAA N TGCGG)-FAM 5’
4.19-4.27 ppm
H
3.80 ppm
H
R
Ha
O
O
N
O
N
100
S
4.29 ppm
O
R
N
H
N
NH
N
N
N
N
R
N
N
O
N
O
O
N
O
O
HO
N :
HO
HO
HO
4-thio-dT
O
N
NH2
HO
O
O
O
O
O
60
20
OH
dT
N :
9
10
11
12
OH
OH OMe
OH
OH
NH
R=(CH2)2OH
N
O
O
Figure 4. Structure of the thymine adducts.
NH
N :
2-thio-dT
N
S
4
6
2
Reaction time (h)
(A)
(B)
(C)
4
4
S
O
O
4
Figure 6. Cross-linking ability of 7 for the target DNA with thymine or thiothymine
derivatives at the complementary site. The reaction was performed with 10
ODN (7) and 5 lM target ODN (8a) in 0.1 M NaCl, 50 mM MES, pH 7.0 at 30 °C.
N
N
N
N
H
N
N
H
H
N
N
N
N
lM
N
H
H
2
O
N
2
N
DNA
DNA
2
DNA
N
S
O
N
N
H
N
H
N
N
N
H
N
H
2'-OMe RNA
2'-OMe RNA
2'-OMe RNA
2-AVP:4-thiothymine
2-AVP:2-thiothymine
2-AVP : thymine
~3’
UU UCU CCG)5’ (a:X=guanine, b:X=2-AVP)
UUAGUU UCU)5’
X (a:X=adenine, b:X=2-AVP)
Targ et RNA 5’~
AAAACG CUG GGC GU
13:
14:
UAAU CAA AGA GGC GAACUG UGU
Figure 5. Speculative complex between 2-AVP and thymine or 2-or 4-thiothymine
in duplex.
3’ 2’-OMe –r(CAA UUA
X
3’ 2’-OMe –r (CCG CA
120
100
80
60
40
20
0
nucleosides together with an additional peak labeled with an
arrow in the HPLC chart (Fig. 3).
The HR ESI MS spectrum of the isolated new product indicated
the cross-linking between the thymine base and the 2-AVP deriva-
tive (calcd for C23H31N7O9Na (M+Na): 572.2075 found 572.2073).
In the 1H NMR and 1H–1H cosy spectra of 9 in DMSO-d6, multiplet
peaks were observed around 4.19–4.27 ppm, which are assigned
to Ha (Fig. 4. and Supplementary data).
The methylene proton adjacent to nitrogen in N3-alkylated thy-
mine (10) appeared at 3.80 ppm, and the methylene proton adja-
cent to oxygen in O2 (11) and O4-alkylated thymine (12)
appeared at around 4.3 ppm as has been reported in literature.9
These results showed that the cross-link formed with either the
2- or 4-oxygen, and not with the 3-nitrogen of the thymine base.
The alkylation site at 2-O or 4-O of the thymine base, however,
could not be unambiguously determined. It was anticipated that
2-AVP in the 20-OMe ON might react with the thymine base be-
cause of the effective proximity effect, by forming hydrogen bonds,
as shown in Figure 5A. Based on such a hypothesized complex, the
assumption was made that the cross-linking reactions in the 2-
AVP:4-thiothymine complex would produce higher reactivity be-
cause of the high nucleophilicity of the sulfur atom (Fig. 5B). In
contrast, the 2-thiothymine base would inhibit the cross-link for-
mation because of the steric repulsion between the 2-thio group10
and the 2-amino group of AVP (Fig. 5C).
The reactivities of (7) towards DNA bearing dT, 4-thio, or 2-thio-
thymine are compared in Figure 6. The rate enhancement was ob-
served with the 4-thiothymine base, and reaction with the 2-
thiothymine base was inhibited. These results accord with our
assumption (shown in Fig. 5) that the cross-linking reaction of
the 2-AVP derivative in 20-OMe ON might occur with the O4 of
the thymine base at the target site. More investigation is needed
to clarify the origin of the selectivity change from the cytosine base
in the DNA/DNA duplexes to the thymine base in the 20-OMe ON/
DNA or RNA duplexes observed in this study.
conc.(µM)
1
5
13a
G
10
1
5
10
1
5
14a
A
10
1
5
14b
10
ON
X
13b
2-AVP
2-AVP
Figure 7. Antisense effects on luciferase production in an assay without cells. The
extent of luciferase production relative to that in the control, which is performed
translation in the absence of ON, is shown in the ordinate.
was designed to react with U. Production of luciferase was moni-
tored by measuring luminescence. Antisense inhibitory effects
are summarized in Figure 7. The 20-OMe ONs, (13a) and (14a) as
the non-reactive control sequences and 2-AVP-containing 20-OMe
ON (13b) did not show antisense inhibition. In contrast, 2-AVP-
containing 20-OMe ON (14b) with the uracil-targeting sequence
showed higher antisense inhibition. The effect of antisense inhibi-
tion by 14b accords with the chemical reactivity of 7 to rU in the
RNA target under acidic conditions (Fig. 2A (b, vs RNA)). These re-
sults suggest that the cross-linking reactions between the 20-OMe
analogue of 2-AVP and U might be responsible for enhancement
of antisense inhibition.
In this study, we have synthesized the 20-OMe analogue of 2-
AVP and the ONs containing this derivative. These ONs have exhib-
ited highly selective cross-linking reactivity to dT in the DNA sub-
strate under neutral conditions. It should be noted that change of
the sugar moiety from deoxyribose to 20-OMe caused a drastic
change in base selectivity. We speculate that the conformational
differences of the hetero duplex may contribute to the alteration
of base selectivity.
It is not currently clear, however, why the base selectivity of the
cross-linking reaction is changed. The reactive 20-OMe ONs with U-
targeting sequence showed higher antisense effects on luciferase
production in cell lysates. These results suggest that cross-linking
reactions of 20-OMe ON might increase the antisense inhibition
in cells; this is now being studied in our laboratory.
Antisense effects of the reactive ONs were evaluated by a trans-
lation assay in the cell lysates. The ONs were incubated for 5 h with
target mRNA of firefly-luciferase under acidic conditions (pH 5.0),
and then subjected to the translation reactions with a wheat-germ
extract for 2 h at 30 °C. Two sequences of ONs (13b) and (14b)
complementary to luciferase mRNA, were used in these experi-
ments; the former was designed to react with rC, and the latter

6124
S. Imoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6121–6124
J. K.; Lee, E. J.; Esau, C.; Schmittgen, T. D. Pancreas 2009, 38, E190; (c) Esau, C. C.
Acknowledgments
Methods 2008, 44, 55; (d) Fabani, M. M.; Gait, M. J. Rna-a Publication of the Rna
Soc. 2008, 14, 336; (e) Stenvang, J.; Kauppinen, S. Expert Opin. Biol. Ther. 2008, 8,
59.
The authors are grateful for the support offered by Grant-in-Aid
for Scientific Research (B) from Japan Society for the promotion of
Science and CREST from Japan Science and Technology Agency.
5. (a) Xu, Y.; Ito, K.; Suzuki, Y.; Komiyama, M. J. Am. Chem. Soc. 2010, 132, 631; (b)
Higuchi, M.; Kobori, A.; Yamayoshi, A.; Murakami, A. Bioorg. Med. Chem. 2009,
17, 475; (c) Qiu, Z.; Lu, L.; Jian, X.; He, C. J. Am. Chem. Soc. 2008, 130, 14398; (d)
Peng, X. H.; Hong, I. S.; Li, H.; Seidman, M. M.; Greenberg, M. M. J. Am. Chem. Soc.
2008, 130, 10299; (e) Yoshimura, Y.; Fujimoto, K. Org. Lett. 2008, 10, 3237.
6. (a) Stevens, K.; Madder, A. Nucleic Acids Res. 2009, 37, 1555; (b) Weinert, E. E.;
Dondi, R.; Colloredo-Melz, S.; Frankenfield, K. N.; Mitchell, C. H.; Freccero, M.;
Rokita, S. E. J. Am. Chem. Soc. 2006, 128, 11940.
7. (a) Nagatsugi, F.; Kawasaki, T.; Usui, D.; Maeda, M.; Sasaki, S. J. Am. Chem. Soc.
1999, 121, 6753; (b) Kawasaki, T.; Nagatsugi, F.; Ali, M. M.; Maeda, M.;
Sugiyama, K.; Hori, K.; Sasaki, S. J. Org. Chem. 2005, 70, 14; (c) Ali, M. M.; Oishi,
M.; Nagatsugi, F.; Mori, K.; Nagasaki, Y.; Kataoka, K.; Sasaki, S. Angew. Chem., Int.
Ed. 2006, 45, 3136; (d) Taniguchi, Y.; Kurose, Y.; Nishioka, T.; Nagatsugi, F.;
Sasaki, S. Bioorg. Med. Chem. 2010, 18, 2894.
8. Belova, N.; Nakanishi, K.; Woody, R. W. Circular Dichroism Principles and
Applications; Wiley-VCH, 2000.
9. Wang, M. Y.; Lao, Y. B.; Cheng, G.; Shi, Y. L.; Villalta, P. W.; Hecht, S. S. Chem. Res.
Toxicol. 2007, 20, 625.
10. (a) Kutyavin, I. V.; Rhinehart, R. L.; Lukhtanov, E. A.; Gorn, V. V.; Meyer, R. B., Jr.;
Gamper, H. B., Jr. Biochemistry 1996, 35, 11170; (b) Lohse, J.; Dahl, O.; Nielsen,
P. E. Proc. Natl. Acad. Sci. USA 1999, 21, 11804.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.027.
References and notes
1. Bennett, C. F.; Swayze, E. E. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259.
2. (a) Pan, W. H.; Clawson, G. A. J. Cell. Biochem. 2006, 98, 14; (b) Verma, S.;
Eckstein, F. Annu. Rev. Biochem. 1998, 67, 99.
3. (a) Egli, M.; Gryaznov, S. M. Cell. Mol. Life Sci. 2000, 57, 1440; (b) Prakash, T. P.;
Bhat, B. Curr. Top. Med. Chem. 2007, 7, 641; (c) Faria, M.; Ulrich, H. Curr. Opin.
Mol. Ther. 2008, 10, 168; (d) Gryaznov, S. M. Chem. Biodivers. 2010, 7, 477; (e)
Obika, S. Chem. Pharm. Bull. 2004, 52, 1399.
4. (a) Davis, S.; Propp, S.; Freier, S. M.; Jones, L. E.; Serra, M. J.; Kinberger, G.; Bhat,
B.; Swayze, E. E.; Bennett, C. F.; Esau, C. Nucleic Acids Res. 2009, 37, 70; (b) Park,