Base pair recognition ability of 2-(methylamino)pyrimidin-4-yl nucleobase in parallel triplex dna

Article abstract of DOI:10.3987/COM-14-S(K)41

A phosphoramidite bearing a 2-(methylamino)pyrimidin-4-yl nucleobase was synthesized and the modified oligonucleotide (triplex-forming oligonucleotide, TFO) was successfully obtained using the phosphoramidite on an automated DNA synthesizer. UV-melting ex

Full text of DOI:10.3987/COM-14-S(K)41

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HETEROCYCLES, Vol. 90, No. 1, 2015
HETEROCYCLES, Vol. 90, No. 1, 2015, pp. 432 - 441. © 2015 The Japan Institute of Heterocyclic Chemistry
Received, 23rd June, 2014, Accepted, 16th July, 2014, Published online, 23rd July, 2014
DOI: 10.3987/COM-14-S(K)41
BASE PAIR RECOGNITION ABILITY OF
2-(METHYLAMINO)PYRIMIDIN-4-YL NUCLEOBASE IN PARALLEL
TRIPLEX DNA
Yoshiyuki Hari,* Satoshi Kashima, Yuya Matsuda, Akihiro Sakata, Ryutaro
Takamine, Shin Ijitsu, and Satoshi Obika*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
Suita, Osaka 565-0871, Japan; E-mail: hari@phs.osaka-u.ac.jp and
obika@phs.osaka-u.ac.jp
Dedicated to Professor Dr. Isao Kuwajima on the occasion of his 77th birthday
Abstract – A phosphoramidite bearing a 2-(methylamino)pyrimidin-4-yl
nucleobase was synthesized and the modified oligonucleotide (triplex-forming
oligonucleotide, TFO) was successfully obtained using the phosphoramidite on an
automated DNA synthesizer. UV-melting experiment of triplex DNA between the
synthesized TFO and duplex DNA targets indicated that the 2-aminopyrimidin-4-yl
unit in TFO could be a useful core structure for recognition of a TA base pair in
duplex DNA.
INTRODUCTION
Triplex DNA formation is a basic strategy for targeting duplex DNA by an oligonucleotide (triplex-forming
oligonucleotide, TFO).1 The targeting is achieved by Hoogsteen or reverse-Hoogsteen hydrogen bonding
between duplex DNA and TFO. However, the duplex DNA sequences capable of forming triplex DNA are
limited to homopurine regions without any pyrimidine interruption. For example, in the case of triplex
DNA formation via Hoogsteen hydrogen bonds, T and the protonated C in TFO can specifically recognize
A and G in the homopurine regions (Figure 1). Therefore, many researchers have tried to develop modified
nucleobases that can recognize pyrimidine interruption, namely pyrimidine-purine base pairs such as CG
and TA base pairs, within duplex DNA.2 However, it is still hard to target an arbitrary sequence of duplex
DNA with sequence selectivity and strong affinity.

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Figure 1. Structures of base triplets of triplex DNA via Hoogsteen hydrogen bonds (H-bonds)
We have also engaged in the development of modified nucleobases recognizing CG and TA base pairs,3-5
and very recently, a 4-[(3S)-3-guanidinopyrrolidino]-5-methylpyrimidin-2-one nucleobase (GP) was
developed as a counterpart for the CG base pair.3 On the other hand, concerning the recognition of the TA
base pair, it was found that the 3-hydroxybenzene nucleobase (3H) might recognize T in the TA base pair in
such a way that avoids sterically the steric hindrance of the 5-methyl group of T (Figure 2a).4 However,
base pair recognition by 3H was non-selective, probably because the hydroxyl group of 3H acted not only
as a hydrogen donor but also as a hydrogen acceptor.6 Herein, the 2-(methylamino)pyrimidin-4-yl
nucleobase (MeAPm) was designed as a new modified nucleobase on the basis of the findings on 3H
(Figure 2b). It is considered that the 2-amino group works only as a hydrogen donor, suitable for recogning
the 4-carbonyl group of T, and the electron-deficient pyrimidine ring increases the hydrogen-donating
ability of the 2-amino group. Moreover, replacing the N-methyl group in MeAPm with various substituents
would be an effective approach to search for the structure (R in Figure 2b) capable of recognizing A in the
TA base pair. We considered comparing the ability of MeAPm with that of 3H in recognizing the TA base
pair. In this study, we describe the synthesis of TFO containing MeAPm and the base pair recognition
ability of the modified nucleobase in the triplex DNA formed.
Figure 2. (a) Proposed structure of 3H-TA base triplet and (b) design of nucleobase for the TA base pair

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HETEROCYCLES, Vol. 90, No. 1, 2015
RESULTS AND DISCUSSION
The synthetic route of the desired phosphoramidite 1 bearing 2-(methylamino)pyrimidin-4-yl nucleobase
was shown in Scheme 1. Initially, compound 2 bearing the 2-(methylthio)pyrimidin-4-yl nucleobase was
synthesized from compound 37 in reference to the letter reported by Baldwin’s group.8 Compound 3
underwent a reaction with TMSCN and TMSOTf to give -isomer 4 and -isomer 5 in 25% and 67% yield,
respectively. Weinreb amide 6 was obtained by acid treatment of 4 followed by condensation with
MeNHOMe. Reaction of 6 with TMSC≡CLi and successive annulation with S-methylisothiourea gave
desired 2 in 35% yield for two steps. The stereochemistry of 2 at the 1´-position was confirmed to be
-configuration by NOE correlation between 1´-H and 4´-H. After oxidation of 2 into sulfone 7 in
quantitative yield, debenzylation of 7 by BCl3 resulted in diol 8, the dimethoxytritylation of which led to
compound 9 in 93% yield. Substitution of the methylsulfonyl moiety in 9 with a methylamino group
yielded 10, the phosphitylation of which resulted in the desired 1, a suitable building block for
oligonucleotide synthesis. On an automated DNA synthesizer using a common phosphoramidite chemistry
for oligonucleotide synthesis, the desired TFO 11 containing the 2-(methylamino)pyrimidin-4-yl
nucleobase (MeAPm) was successfully synthesized.
Scheme 1. Reagents and conditions: (i) TMSCN, TMSOTf, CH2Cl2, rt, 25% (4) and 67% (5); (ii) 35% HCl
aq., 1,4-dioxane, reflux, then MeNHOMe•HCl, EDC, DMAP, CH2Cl2, rt, 66% for two steps; (iii)
TMSC≡CLi, THF, -78 ºC, then (S-methylisothiourea)2•H2SO4, K2CO3, MeCN-H2O, reflux, 35% for two
steps; (iv) mCPBA, CH2Cl2, rt, quant.; (v) BCl3, CH2Cl2-hexane, rt, 57%; (vi) DMTrCl, pyridine, rt, 93%;
(vii) MeNH2, THF-H2O, rt, 93%; (viii) i-Pr2NP(Cl)OCH2CH2CN, i-Pr2NEt, CH2Cl2, rt, 76%; (ix)
oligonucleotide synthesis.
Next, the base pair recognition ability of MeAPm in the synthesized TFO was measured by UV-melting
experiment and was compared with that of 3H. The results are summarized in Table 1. The affinity of

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MeAPm to the TA base pair was same as that of 3H, and the Tm value of the triplexes (YZ = TA) was 26 ºC.
These results indicate that like 3H, MeAPm can form a hydrogen bond with T in a TA base pair in duplex
DNA. On the contrary, MeAPm led to a significant decrease in the stability of triplex DNA with other
duplex DNA targets (YZ = CG, AT and GC) compared with that shown by 3H. As a result, it was found that
the MeAPm in TFO was able to selectively recognize a TA base pair within duplex DNA although further
improvement of the affinity to the TA base pair is required.9 The selective TA base pair recognition by
MeAPm occurred as MeAPm behaved only as a hydrogen donor by using the N-H of the methylamino
group, as we expected. In addition, it was shown that the methyl group in MeAPm would not prevent the
hydrogen bond formation between the N-H in MeAPm and the 4-carbonyl group of T in the TA base pair.
This observation strongly suggests that the recognition of A in the TA base pair via a suitable substituent in
place of the methyl group of MeAPm might be feasible in accordance with our plan shown in Figure 2b.
Table 1. Tm values (ºC) of triplexes between TFOs and hairpin-loop duplex DNAa
YZ
X
TA
26
26
CG
27
AT
28
19
GC
25
3Hb
MeAPm
22
21
a
Condition : 10 mM sodium cacodylate buffer (pH 6.8) containing 100 mM KCl, 50 mM MgCl2 and 1.89 μM of each
oligonucleotide. C : 2´-Deoxy-5-methylcytidine. b The data are shown in ref 4.
Figure 3. UV-melting curves of triplex DNA between TFO 11 (X = MeAPm) and duplex DNA targets