2′,4′-BNA bearing a 2-pyridine nucleobase for CG base pair recognition in the parallel motif triplex DNA

Article abstract of DOI:10.1039/c004895j

We succeeded in the synthesis of triplex-forming oligonucleotides (TFOs) that contain a deoxyribonucleotide (Py) bearing a 2-pyridine nucleobase or the 2′,4′-BNA congener (Py^B). By UV melting experiments, it was found that 2-pyridine was a very

Full text of DOI:10.1039/c004895j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
2¢,4¢-BNA bearing a 2-pyridine nucleobase for CG base pair recognition in the
parallel motif triplex DNA†
Yoshiyuki Hari,*a Sachiko Matsugu,a Hiroyasu Inohara,a Yuri Hatanaka,a Masaaki Akabane,a
Takeshi Imanishia and Satoshi Obika*a,b
Received 6th April 2010, Accepted 16th June 2010
First published as an Advance Article on the web 21st July 2010
DOI: 10.1039/c004895j
We succeeded in the synthesis of triplex-forming oligonucleotides (TFOs) that contain a
deoxyribonucleotide (Py) bearing a 2-pyridine nucleobase or the 2¢,4¢-BNA congener (PyB). By UV
melting experiments, it was found that 2-pyridine was a very promising nucleobase for the
sequence-selective recognition of a CG base pair within double-stranded DNA (dsDNA) in a parallel
motif triplex. Moreover, PyB in TFOs showed stronger affinity to a CG base pair than Py with further
increase in the selectivity. Using TFO including multiple PyB units, triplex formation with dsDNA
containing three CG base pairs was observed.
Introduction
Triplex-forming oligonucleotides (TFOs) that sequence-
specifically bind to double-stranded DNA (dsDNA) have
received considerable attention because of the possibility of their
application to various technologies such as gene therapy and
gene diagnosis.1 There are two motifs for recognition of dsDNA
by TFOs, namely parallel and antiparallel ones. In the former,
Fig. 1 T–AT and C+H–GC base triplets.
homopyrimidine TFOs bind to the homopurine tract of dsDNA
through Hoogsteen hydrogen bonds in the major groove to form
T-AT and protonated C (C+H)-GC base triplets (Fig. 1). In the
latter, purine-rich TFOs form A–AT (or T–AT) and G–GC base
triplets with the homopurine tract of dsDNA by making reverse-
Hoogsteen hydrogen bonds. In other words, in either motif, the
recognition is restricted to the homopurine tract within dsDNA.
Thus, over the past few decades, the development of nucleic acids
bearing artificial nucleobases that sequence-selectively and stably
form base triplets with pyrimidine-purine (CG or TA) base pairs
in dsDNA has been carried out.2 However, to date, successful
examples number only a few and are limited to 2-pyridone (P),3,4
5-methylpyrimidin-2-one (4HT)5 and a pyrrolo[2,3-d]pyrimidin-
2-one derivative (APP),6 developed by us and other groups on
the basis of the fact that T has a degree of affinity to CG base
pairs (Fig. 2).7 These nucleobases can sequence-selectively bind
to a CG base pair, though the affinity is not high. For practical
applications, the development of nucleobases having stronger
binding affinity and higher selectivity to a CG base pair, e.g., T
for an AT base pair, is still required.
Fig. 2 T–CG base triplet and nucleobases for CG base pair recognition.
Fig. 3 Oligonucleotides designed and used in the present study.
In our exploration for a new nucleobase toward the recognition
of a CG base pair, we designed 2-pyridine (Fig. 3). Since the
nitrogen of pyridine is basic, pyridine as a nucleobase is expected
to act as a strong hydrogen acceptor compared to pyridone- or
pyrimidinone-based nucleobases shown in Fig. 2. Meanwhile, we
previously discovered that a 2¢,4¢-BNA (or LNA) modification in
TFO (Fig. 3) dramatically improved triplex-forming ability with
dsDNA in terms of both sequence-selectivity and stability.8,9 Thus,
we synthesized TFOs containing a deoxyribonucleotide (Py) with
2-pyridine or the 2¢,4¢-BNA congener (PyB) and evaluated the
sequence-selectivity and affinity of the TFOs to dsDNA targets in
the parallel motif triplex formation. The details of our results are
described in this paper.10
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6
Yamadaoka, Suita, Osaka, 565-0871, Japan. E-mail: hari@phs.osaka-u.
ac.jp, obika@phs.osaka-u.ac.jp; Fax: +81 6 6879 8201, +81 6 6879 8204;
Tel: +81 6 6879 8201, +81 6 6879 8200
bPRESTO, JST, Sanbancho Building, 5-Sanbancho, Chiyodaku, Tokyo,
102-0075, Japan
† Electronic supplementary information (ESI) available: 1H and 13C (or
31P) spectra for new compounds. See DOI: 10.1039/c004895j
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Fig. 4 TFOs and dsDNA used in the present study. mC indicates 2¢-deoxy-5-methylcytidine.13
Results and discussion
Synthesis
The phosphoramidite 1 combined with a deoxyribose and a
2-pyridine nucleobase was synthesized as shown in Scheme
1. Coupling of the dibenzylated 2¢-deoxy-D-ribose 211 with 2-
pyridyllithium, prepared from 2-bromopyridine and n-BuLi,
followed by the Mitsunobu reaction using 1,1¢-azobis(N,N-
dimethylformamide) and n-Bu3P gave a 2 : 1 separable mixture
of desired b-isomer 3 and the a-isomer 4 in 60% yield from
2. The structure of each isomer was confirmed by the NOESY
correlations shown in Scheme 1. Debenzylation of 3 gave the
desired monomer 5 in 60% yield. Compound 5 was reacted with
DMTrCl in pyridine to afford 6 in 49% yield, which was phosphity-
lated to give the phosphoramidite 1, a suitable building block for
the oligonucleotide synthesis, in 78% yield. The synthesis of the
2¢,4¢-BNA phosphoramidite 7 bearing pyridine as an unnatural
nucleobase was achieved from the 2¢,4¢-BNA monomer 8 that we
previously reported (Scheme 2).12 Compound 9 was obtained
Scheme 2 Reagents and conditions: (i) DMTrCl, pyridine, rt, 3 h
(90%); (ii) (i-Pr2N)2POCH2CH2CN, diisopropylammonium tetrazolide,
CH3CN–THF, rt, 5 h (89%).
Table 1 Tm values (◦C) of triplexes between TFOs 10 and four dsDNA
targets 15.a,b
XY
TFO
CG
TA
GC
AT
10-T
25 (44)
24 (41)
36 (56)
17 (33)
17 (34)
22 (33)
20 (35)
15 (35)
18 (38)
44 (59)
19 (35)
21 (44)
10-Py
10-PyB
a Conditions: 7 mM sodium phosphate buffer (pH 7.0), 140 mM KCl and
10 mM MgCl2. The final concentration of each oligonucleotide used was
1.5 mM. b Tm values, shown in the parenthesis, were obtained when 5 mM
spermine instead of 10 mM MgCl2 was used.
reversed-phase HPLC and their compositions were determined by
MALDI-TOF-MS analysis.
Evaluation
Triplex-forming ability of the TFOs 10–14 containing Py or PyB
was evaluated on the basis of a Tm value calculated by UV melting
experiments. Initially, the UV melting experiment using TFOs 10-
T, 10-Py and 10-PyB, and four dsDNA targets 15 (XY = CG, TA,
GC and AT), was performed in 7 mM sodium phosphate buffer
(pH 7.0), 140 mM KCl and 10 mM MgCl2, and the results are
summarized in Table 1. As described in the introduction, T has an
affinity for CG base pairs. The TFO 10-T had a Tm value of 25
◦C against dsDNA 15 (XY = CG). However, the triplex between
10-T and 15 (XY = AT) containing a stable T–AT base triplet
naturally showed a higher Tm value (44 ◦C). By contrast, TFO
10-Py, which bears a 2-pyridine nucleobase, showed the highest
Tm value against 15 (XY = CG) among four targets 15 and the
stability was as much as that of 10-T and 15 (XY = CG). This result
suggests that a 2-pyridine nucleobase effectively recognizes a CG
base pair in dsDNA (Fig. 5). Next, the properties of PyB, combined
with 2¢,4¢-BNA modification and a 2-pyridine nucleobase, instead
of Py, were examined (Table 1 and Fig. 6). The Tm value of 10-PyB
Scheme 1 Reagents and conditions: (i) 2-pyridyllithium, Et2O–THF, -78
◦C, 1.5 h; (ii) 1,1¢-azobis(N,N-dimethylformamide), n-Bu3P, CH2Cl2, rt,
1.5 h (60% for 2 steps, 3 : 4 = 2 : 1); (iii) 20% Pd(OH)2-C, cyclohexene,
EtOH, reflux, 3 h (60%); (iv) DMTrCl, pyridine, rt, 2.5 h (49%); (v)
i-Pr2NP(Cl)OCH2CH2CN, i-Pr2NEt, CH2Cl2, rt, 1.5 h (78%). Two-headed
arrows indicate observed NOESY correlations.
in 90% yield by treatment of 8 with DMTrCl in pyridine and
phosphitylation of 9 gave the desired phosphoramidite 7 in 89%
yield. The obtained phosphoramidites 1 and 7 were introduced
into TFOs using a standard phosphoramidite protocol on an
automated DNA synthesizer. The purity of the prepared TFOs
10–14 including Py or PyB, shown in Fig. 4, was verified using
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Table 2 UV melting experiments of TFOs 10–14 and dsDNA targets
15–19.a
TFO
dsDNA target
Tm value/◦C
10-T
15 (XY = CG)
44
56
39
49
34
46
15
34
10-PyB
11-T
15 (XY = CG)
16
16
17
17
18
18
19
19
11-PyB
12-T
Fig. 5 Plausible recognition pattern of a CG base pair by 2-pyridine
nucleobase (Py).
12-PyB
13-T
13-PyB
14-T
b
—
22
14-PyB
a Conditions: 7 mM sodium phosphate buffer (pH 7.0), 140 mM KCl and
5 mM spermine. The final concentration of each oligonucleotide used was
1.5 mM. b No triplex formation was observed.
value was 22 ◦C, equal to that of TFO containing PB in the same
context.3b
Conclusions
We have synthesized TFOs that contain a deoxyribonucleotide
(Py) bearing a 2-pyridine nucleobase or the 2¢,4¢-BNA congener
(PyB) and their triplex-forming ability was evaluated by UV melt-
ing experiments. From the results, PyB is a promising candidate for
recognition of a CG base pair in triplex formation though more
detailed experiments are naturally needed. The ability of PyB is
the same or more than that of 2¢,4¢-BNA bearing a 2-pyridone
nucleobase, PB, which we previously developed.3 It is interesting
to note that 2-pyridine has a new type of nucleobase structure,
differing from the pyridone- or pyrimidinone-based nucleobases,
like P, 4HT and APP. This indicates that 2-pyridine is attractive
as a nucleobase core to improve recognition ability by additional
introduction of functional groups, i.e., to sequence-selectively and
stably recognize a CG base pair through two or more hydrogen
bonds.
Fig. 6 UV melting profiles (260 nm) for triplexes 10-PyB·15 (XY = CG,
TA, GC and AT). The presented melting curves were normalized.
with 15 (XY = CG) was 36 ◦C, an increase of 12 ◦C compared to
that of 10-Py. On the sequence-selectivity of 10-PyB, the Tm value
with 15 (XY = CG) was 14–18 ◦C higher than those with the other
targets 15 (XY = TA, GC or AT), while, on the sequence-selectivity
of 10-Py, the Tm difference, DTm, was only 5–9 ◦C. The dramatic
improvement of selectivity and affinity to a CG base pair by PyB
is attributed to the synergy between recognition of a CG base
pair by 2-pyridine and stabilization of the triplex by 2¢,4¢-BNA
modification. Interestingly, under the same conditions, the ability
of PyB slightly exceeded that of 2¢,4¢-BNA bearing a 2-pyridone
nucleobase, PB, which we previously developed.14,15 On the other
hand, under conditions using 5 mM spermine in place of 10 mM
MgCl2, the Tm and DTm values of the triplex between 10-PyB and
15 (XY = CG), 56 ◦C and over 12 ◦C respectively, were found to
be almost comparable to those of natural triplex between 10-T to
15 (XY = AT).
In view of the potential general applications, we examined the
influence of neighboring sites of a PyB-CG base triplet under
neutral conditions containing 5 mM spermine (Table 2). Triplexes
between 11 and 16, 12 and 17, and 13 and 18 have C–GC base
triplets at the 3¢-site, 5¢-site, and both 3¢- and 5¢-sites, respectively.
Although these triplexes were less stable than that of TFO 10 with
15 (XY = CG) because of an increase in the number of labile mC–
GC base triplets at neutral pH, it was clarified, by comparison
with the corresponding T–CG base triplet, that PyB can effectively
recognize a CG base pair.
Experimental
General
All chemicals were purchased from chemical suppliers. For column
chromatography, Fuji Silysia silica gel PSQ-100B and FL-100D
were used. All melting points were measured on a Yanagimoto
micro melting point apparatus and are uncorrected. 1H NMR, 13
C
NMR and 31P NMR spectra were recorded on a JEOL ECS400
spectrometer. IR spectra were recorded on JASCO FT/IR-
200 and JASCO FT/IR-4200 spectrometers. Optical rotations
were recorded on a JASCO DIP-370 instrument. Mass spectra
were measured on a JEOL JMS-600 or JEOL JMS-700 mass
spectrometer. MALDI-TOF mass spectra were recorded on a
Bruker Daltonics Autoflex II TOF/TOF mass spectrometer.
2-(2-Deoxy-3,5-di-O-benzyl-b-D-ribofuranosyl)pyridine (3) and
2-(2-deoxy-3,5-di-O-benzyl-a-D-ribofuranosyl)pyridine (4)
Finally, to verify the practicality of PyB further, we investigated
the stability of the triplexes of TFO 14-PyB with dsDNA 19,
including three PyB-CG base triplets (Table 2). In the case of the
triplex between 14-T and 19 including three T-CG base triplets,
triplex formation was not observed at all. However, TFO 14-PyB
formed the relatively stable triplex with dsDNA 19 and the Tm
Under a nitrogen atmosphere, a solution of compound 212 (321 mg,
1.02 mmol) in anhydrous THF (5 mL) was added to a solution
of 2-pyridyllithium, prepared from 2-bromopyridine (0.60 mL,
6.12 mmol) and n-BuLi (1.53 M in hexane, 4 mL, 6.12 mmol) in
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◦
anhydrous Et2O (19 mL), at -78 C and the mixture was stirred
anhydrous pyridine (1 mL) at room temperature and the mixture
was stirred for 2.5 h. After addition of saturated aqueous NaHCO3
solution, the mixture was extracted with AcOEt. The organic
extracts were washed with water and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by flash silica
gel column chromatography (n-hexane–AcOEt–Et3N = 50 : 50 : 1)
to give compound 6 (70 mg, 49%) as a white powder. M.p. 45–49
◦C; [a]2D3 +32.59 (c 1.00, CHCl3); IR (KBr) n: 3379, 2931, 1606,
1509, 1250, 1177 cm-1; 1H NMR (400 MHz, CDCl3) d 2.01 (1H, br
s), 2.22 (1H, ddd, J = 6.2, 9.0 and 13.2 Hz), 2.41 (1H, ddd, J = 2.9,
6.4 and 13.2 Hz), 3.29 (1H, dd, J = 5.5 and 9.7 Hz), 3.36 (1H, dd, J =
4.4 and 9.7 Hz), 3.78 (6H, s), 4.10–4.14 (1H, m), 4.39–4.42 (1H, m),
5.29 (1H, dd, J = 6.6 and 9.0 Hz), 6.82 (4H, d, J = 8.6 Hz), 7.15–7.46
(10H, m), 7.53 (1H, d, J = 7.9 Hz), 7.64 (1H, m), 8.53 (1H, d, J =
4.6 Hz); 13C NMR (101 MHz, CDCl3) d 42.11, 55.18, 64.34, 74.12,
80.56, 86.16, 86.22, 113.07, 120.17, 122.27, 126.74, 127.78, 128.14,
130.05, 135.96, 135.98, 136.69, 144.80, 148.75, 158.41, 161.78; MS
(FAB): m/z 520 (MH+); HRMS (FAB): 520.2100 (MNa+, calcd
for C31H31NNaO5: 520.2100).
at -78 ◦C for 1.5 h. After addition of water, the mixture was
extracted with AcOEt. The organic extracts were washed with
brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by flash silica gel column chromatography (n-hexane–
AcOEt = 1 : 2) to give appropriate compounds (294 mg, Rf = ca.
0.2), which were dissolved in CH2Cl2 (8 mL) and 1,1¢-azobis(N,N-
dimethylformamide) (193 mg, 1.12 mmol) and n-Bu3P (0.28 mL,
1.12 mmol) were added at room temperature. After being stirred
at room temperature for 1.5 h, the mixture was diluted with Et2O
R
and filtered through a pad of Celiteꢀ. The filtrate was concentrated
in vacuo and the residue was purified by flash silica gel column
chromatography (n-hexane–AcOEt = 5 : 1) to give a mixture of b-
isomer 3 and a-isomer 4 (230 mg, 60%, 3 : 4 = 2 : 1). Compound
3, pale yellow oil; [a]2D4 +50.84 (c 1.00, CHCl3); IR (KBr) n: 2861,
1592, 1454, 1436, 1100, 1028 cm-1; 1H NMR (400 MHz, CDCl3) d
2.06 (1H, ddd, J = 6.0, 10.1 and 13.3 Hz), 2.56 (1H, ddd, J = 1.8,
6.0 and 13.3 Hz), 3.60 (1H, dd, J = 5.0 and 10.1 Hz), 3.67 (1H, dd,
J = 4.6 and 10.1 Hz), 4.15–4.17 (1H, m), 4.34 (1H, ddd, J = 2.3,
4.6 and 5.0 Hz), 4.53, 4.59 (2H, AB, J = 12.0 Hz), 4.58 (2H, s),
5.26 (1H, dd, J = 6.0 and 10.1 Hz), 7.14–7.18 (1H, m), 7.26–7.36
(10H, m), 7.51 (1H, d, J = 7.8 Hz), 7.64 (1H, dt, J = 1.8 and 7.8
Hz), 8.53 (1H, m); 13C NMR (101 MHz, CDCl3) d 39.05, 70.96,
71.05, 73.39, 81.00, 81.12, 84.11, 120.33, 122.31, 127.60, 127.62,
127.65, 128.34, 128.39, 136.63, 138.06, 138.15, 148.85, 161.55; MS
(FAB): m/z 376 (MH+); HRMS (FAB): 376.1912 (MH+, calcd for
C24H26NO3: 376.1913). Compound 4, pale yellow oil; [a]2D4 +5.89
(c 1.00, CHCl3); IR (KBr) n: 2861, 1591, 1454, 1361, 1102 cm-1;
1H NMR (400 MHz, CDCl3) d 2.34 (1H, ddd, J = 4.6, 6.4 and
12.8 Hz), 2.70 (1H, ddd, J = 6.4, 7.3 and 12.8 Hz), 3.62 (2H, d, J =
4.6 Hz), 4.19–4.22 (1H, m), 4.37–4.45 (3H, m), 4.59, 4.61 (2H, AB,
J = 11.9 Hz), 5.25 (1H, dd, J = 6.4 and 7.3 Hz), 7.10–7.16 (3H,
m), 7.23–7.35 (8H, m), 7.59 (1H, d, J = 7.8 Hz), 7.67 (1H, dt, J =
1.8 and 7.8 Hz), 8.52 (1H, d, J = 4.6 Hz); 13C NMR (101 MHz,
CDCl3) d 38.69, 70.76, 71.16, 73.45, 80.52, 80.95, 83.52, 119.83,
121.86, 127.50, 127.59, 128.23, 128.24, 128.34, 136.54, 137.90,
138.09, 148.67, 162.80; MS (FAB): m/z 376 (MH+); HRMS (FAB):
376.1912 (MH+, calcd for C24H26NO3: 376.1913).
2-[3-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-2-deoxy-5-
O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]pyridine (1)14
Under a nitrogen atmosphere, 2-cyanoethyl N,N-diisopropy-
lchlorophosphoramidite (94 mL, 0.54 mmol) was added to a
solution of compound 9 (211 mg, 0.40 mmol) and i-Pr2NEt
(94 mL, 0.54 mmol) in anhydrous CH2Cl2 (1 mL) at 0 ◦C and the
mixture was stirred for 1.5 h. After addition of saturated aqueous
NaHCO3 solution, the mixture was extracted with AcOEt. The
organic extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash silica gel
column chromatography (n-hexane–AcOEt = 3 : 1 to 2 : 1) to give
compound 1 (46 mg, 78%) as a colorless oil.31P NMR (162 MHz,
CDCl3) d 147.93, 148.47.
2-[5-O-(4,4¢-Dimethoxytrityl)-2-O,4-C-methylene-b-D-
ribofuranosyl]pyridine (9)
Under a nitrogen atmosphere, DMTrCl (197 mg, 0.58 mmol)
was added to a solution of compound 8 (100 mg, 0.45 mmol) in
anhydrous pyridine (2 mL) at room temperature and the mixture
was stirred for 3 h. After addition of saturated aqueous NaHCO3
solution, the mixture was extracted with AcOEt. The organic
extracts were washed with water and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by flash
silica gel column chromatography (n-hexane–AcOEt = 1 : 1) to
give compound 9 (212 mg, 90%) as a white powder. M.p. 68–73
◦C; [a]2D8 -5.60 (c 0.77, CHCl3); IR (KBr) n: 3334, 3007, 2944,
1605, 1508, 1251, 1035 cm-1; 1H NMR (400 MHz, CDCl3) d 2.00
(1H, br s), 3.50, 3.53 (2H, AB, J = 11.5 Hz), 3.80 (6H, s), 4.01,
4.10 (2H, AB, J = 8.2 Hz), 4.22 (1H, d, J = 6.0 Hz), 4.47 (1H, s),
5.18 (1H, s), 6.85 (4H, d, J = 8.7 Hz), 7.18–7.52 (10H, m), 7.65
(1H, d, J = 8.2 Hz), 7.69–7.73 (1H, m), 8.56 (1H, d, J = 4.1 Hz);
13C NMR (101 MHz, CDCl3) d 55.21, 60.06, 72.20, 72.80, 82.58,
83.93, 86.29, 86.37, 113.18, 120.77, 122.40, 126.88, 127.89, 128.12,
130.08, 130.11, 135.69, 135.75, 136.74, 144.71, 149.07, 158.53,
158.83; MS (FAB): m/z 548 (MNa+); HRMS (FAB): 548.2050
(MNa+, calcd for C32H31NNaO6: 548.2049)
2-(2-Deoxy-b-D-ribofuranosyl)pyridine (5)16
A solution of compound 3 (82 mg, 0.22 mmol), 20% Pd(OH)2-C
(85 mg) and cyclohexene (1.6 mL, 16 mmol) in EtOH (2 mL) was
refluxed for 3 h. The mixture was filtered through a paper filter and
the filtrate was concentrated in vacuo. The residue was purified by
flash silica gel column chromatography (AcOEt–MeOH = 20 : 1
to 10 : 1) to give compound 5 (26 mg, 60%) as a colorless oil.1H
NMR (400 MHz, CD3OD) d 2.04 (1H, ddd, J = 5.9, 9.6 and 13.3
Hz), 2.31 (1H, ddd, J = 1.8, 6.4 and 13.3 Hz), 3.67 (1H, dd, J = 4.3
and 11.9 Hz), 3.73 (1H, dd, J = 3.7 and 11.9 Hz), 4.01–4.02 (1H,
m), 4.35–4.36 (1H, m), 5.20 (1H, dd, J = 6.4 and 9.6 Hz), 7.32 (1H,
dd, J = 3.7 and 7.3 Hz), 7.58 (1H, d, J = 7.8 Hz), 7.83 (1H, dd, J =
7.3 and 7.8 Hz), 8.47 (1H, d, J = 3.7 Hz).
2-[2-Deoxy-5-O-(4,4¢-dimethoxytrityl)-b-D-ribofuranosyl]-
pyridine (6)
Under a nitrogen atmosphere, DMTrCl (200 mg, 0.59 mmol)
was added to a solution of compound 5 (58 mg, 0.29 mmol) in
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2-[3-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4¢-
dimethoxytrityl)-2-O,4-C-methylene-b-D-ribofuranosyl]pyridine
Acknowledgements
This work was supported in part by Special Coordination Funds
for Promoting Science and Technology of the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan (MEXT)
and by a Grant-in-Aid for Science Research (KAKENHI) from
the Japan Society for the Promotion of Science (JSPS).
(7)
Under
a
nitrogen atmosphere, 2-cyanoethyl-N,N,N¢,N¢-
tetraisopropylphosphordiamidite (0.19 mL, 0.60 mmol) was
added to a solution of compound 9 (211 mg, 0.40 mmol)
and diisopropylammonium tetrazolide (97 mg, 0.48 mmol) in
anhydrous MeCN–THF (3 : 1, 10 mL) at room temperature and
the mixture was stirred for 5 h. The solvent was removed under
reduced pressure and the residue was purified by flash silica
gel column chromatography (n-hexane–AcOEt = 1 : 1) to give
compound 7 (256 mg, 89%) as a white powder. M.p. 48–51 ◦C;
1H NMR (400 MHz, CDCl3) d 0.83, 0.93 (6H, 2d, J = 6.4 Hz
and J = 6.9 Hz), 1.07, 1.08 (6H, 2d, J = 6.9 Hz and J = 6.9 Hz),
2.31, 2.45 (2H, 2ddd, J = 1.4, 6.0 and 6.4 Hz and J = 2.7, 6.4
and 6.9 Hz), 3.37–3.72 (6H, m), 3.79, 3.80 (6H, 2 s), 3.93, 3.96
(1H, 2d, J = 7.3 Hz and J = 7.8 Hz), 4.10–4.33 (2H, m), 4.56,
4.67 (1H, 2 s), 5.23, 5.24 (1H, 2 s), 6.82–6.86 (4H, m), 7.17–7.33
(4H, m), 7.39–7.43 (4H, m), 7.51–7.54 (2H, m), 7.67–7.76 (2H,
m), 8.54–8.56 (1H, m); 31P NMR (162 MHz, CDCl3) d 147.71,
148.39; MS (FAB): m/z 726 (MH+); HRMS (FAB): 726.3307
(MH+, calcd for C41H49N3O7P: 726.3308).
Notes and references
1 For reviews: (a) S. Buchini and C. J. Leumann, Curr. Opin. Chem. Biol.,
2003, 7, 717; (b) M. Duca, P. Vekhoff, K. Oussedik, L. Halby and P. B.
Arimondo, Nucleic Acids Res., 2008, 36, 5123.
2 For reviews: (a) S. O. Doronina and J.-P. Behr, Chem. Soc. Rev., 1997,
26, 63; (b) D. M. Gowers and K. R. Fox, Nucleic Acids Res., 1999, 27,
1569.
3 (a) S. Obika, Y. Hari, M. Sekiguchi and T. Imanishi, Angew. Chem., Int.
Ed., 2001, 40, 2079; (b) S. Obika, Y. Hari, M. Sekiguchi and T. Imanishi,
Chem.–Eur. J., 2002, 8, 4796; (c) H. Torigoe, Y. Hari, S. Obika and T.
Imanishi, Nucleosides, Nucleotides Nucleic Acids, 2003, 22, 1097.
4 We also reported a 1-isoquinolone nucleobase, a 2-pyridone analog,
for CG base pair recognition: (a) Y. Hari, S. Obika, M. Sekiguchi
and T. Imanishi, Tetrahedron, 2003, 59, 5123; (b) H. Torigoe, Y. Hari, S.
Obika and T. Imanishi, Nucleosides, Nucleotides Nucleic Acids, 2003, 22,
1571.
5 (a) I. Pre´vot-Halter and C. J. Leumann, Bioorg. Med. Chem. Lett., 1999,
9, 2657; (b) S. Buchini and C. J. Leumann, Tetrahedron Lett., 2003, 44,
5065; (c) S. Buchini and C. J. Leumann, Angew. Chem., Int. Ed., 2004,
43, 3925; (d) S. Buchini and C. J. Leumann, Eur. J. Org. Chem., 2006,
3152.
6 (a) R. T. Ranasinghe, D. A. Rusling, V. E. C. Powers, K. R. Fox and T.
Brown, Chem. Commun., 2005, 2555; (b) D. A. Rusling, V. E. C. Powers,
R. T. Ranasinghe, Y. Wang, S. D. Osborne, T. Brown and K. R. Fox,
Nucleic Acids Res., 2005, 33, 3025.
7 (a) K. Yoon, C. A. Hobbs, J. Koch, M. Sardaro, R. Kuntny and
A. L. Weis, Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 3840; (b) I.
Radhakrishnan and D. J. Patel, J. Mol. Biol., 1994, 241, 600.
8 (a) S. Obika, Y. Hari, T. Sugimoto, M. Sekiguchi and T. Iminishi,
Tetrahedron Lett., 2000, 41, 8923; (b) H. Torigoe, Y. Hari, S. Obika and
T. Imanishi, J. Biol. Chem., 2001, 276, 2354; (c) S. Obika, T. Uneda, T.
Sugimoto, D. Nanbu, T. Minami, T. Doi and T. Imanishi, Bioorg. Med.
Chem., 2001, 9, 1001.
9 (a) B.-W. Sun, B. R. Babu, M. D. Sørensen, K. Zakrzewska, J. Wengel
and J.-S. Sun, Biochemistry, 2004, 43, 4160; (b) E. Brunet, P. Alberti, L.
Perrouault, R. Babu, J. Wengel and C. Giovannangeli, J. Biol. Chem.,
2005, 280, 20076.
10 A part of the results was reported at the 4th International Symposium
on Nucleic Acids Chemistry. S. Matsugu, H. Inohara, S. Obika and T.
Imanishi, Nucleic Acids Symp. Ser., 2005, 49, 159.
Synthesis of TFOs 10–14
The synthesis of TFOs 10–14 was performed on a 0.2 mmol
scale on an automated DNA synthesizer (Applied Biosystems
ExpediteTM 8909) using the common phosphoramidite protocol
except for a prolonged coupling time of 5–30 min for unnatural
phosphoramidites. TFOs synthesized on DMTr-ON mode were
cleaved from the CPG resin by treatment with 28% aqueous NH3
solution at room temperature for 1.5 h. Additional treatment with
28% aqueous NH3 solution at 55 ◦C for 15 h underwent the
removal of all protecting groups on TFOs. The obtained crude
R
TFOs were purified with Sep-Pakꢀ Plus C18 cartridges (Waters)
followed by reversed-phase HPLC (Waters XTerraꢀ MS C18 2.5
R
mm, 10 ¥ 50 mm). The composition of the TFOs was confirmed
by MALDI-TOF-MS analysis. MALDI-TOF-MS data ([M -
H]-) for TFOs 10–14: 10-Py, found 4449.57 (calcd 4448.97); 11-
PyB, found 4475.83 (calcd 4476.04); 12-PyB, found 4475.07 (calcd
4475.07); 13-PyB, found 4475.04 (calcd 4475.07); 14-PyB, found
4475.03 (calcd 4474.08); 15-PyB, found 4441.88 (calcd 4442.05).
11 W. Wierenga and H. I. Skulnick, Carbohydr. Res., 1981, 90, 41.
12 Y. Hari, S. Obika, M. Sakaki, K. Morio, Y. Yamagata and T. Imanishi,
Tetrahedron, 2002, 58, 3051.
13 It is known that mC has higher binding affinity to a GC base pair than
C at physiological pH. T. J. Povsic and P. B. Dervan, J. Am. Chem. Soc.,
1989, 111, 3059.
UV melting experiments (Tm measurements)
UV melting experiments were carried out on a Shimadzu UV-
1650PC spectrophotometer equipped with Tm analysis accessory.
The UV melting profiles were recorded in 7 mM sodium phosphate
buffer (pH 7.0), 140 mM KCl and 10 mM MgCl2 or 10 mM
14 Under the same conditions, selectivity (DTm) and affinity (Tm) of PB to
a CG base pair were 10–19 ◦C and 33 ◦C, respectively3a.
15 Although TB showed the Tm value of 35 ◦C for a CG base pair under
the same conditions, TB has no selectivity to a CG base pair because
the Tmvalue of TBfor an AT base pair was 57 ◦C. S. Obika, Y. Hari, T.
Sugimoto, M. Sekiguchi and T. Imanishi, Tetrahedron Lett., 2000, 41,
8923.
spermine from 5 ◦C to 85 ◦C at a scan rate of 0.5 C min-1 at
◦
260 nm. The final concentration of each oligonucleotide used was
1.5 mM. A Tm value was designated the maximum of the first
derivative calculated from the profile.
16 Y. Kim, A. M. Leconte, Y. Hari and F. E. Romesberg, Angew. Chem.,
Int. Ed., 2006, 45, 7809.
4180 | Org. Biomol. Chem., 2010, 8, 4176–4180
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