Synthesis of 6-N-(benzothiazol-2-yl)deoxyadenosine and its exciton-coupled circular dichroism

Article abstract of DOI:10.1016/j.bmc.2009.12.009

6-N-(Benzothiazol-2-yl)deoxyadenosine (A^BT) was synthesized and incorporated into DNAs. Although, the multipoint benzothiazole (BT) modification of oligodeoxynucleotides reduced the stability of duplexes with their complementary strands, it ind

Full text of DOI:10.1016/j.bmc.2009.12.009

Bioorganic & Medicinal Chemistry 18 (2010) 567–572
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 6-N-(benzothiazol-2-yl)deoxyadenosine and its
exciton-coupled circular dichroism
*
Yoshiaki Masaki, Akihiro Ohkubo, Kohji Seio, Mitsuo Sekine
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
6-N-(Benzothiazol-2-yl)deoxyadenosine (ABT) was synthesized and incorporated into DNAs. Although,
the multipoint benzothiazole (BT) modification of oligodeoxynucleotides reduced the stability of
duplexes with their complementary strands, it induced the strong exciton coupling between BT moieties.
The circular dichroism (CD) spectra of this exciton coupling interaction were observed at wavelengths
above 300 nm and overlapping with the UV absorption bands of nucleotides could be avoided. The shapes
of the CD spectra due to this interaction were strongly influenced by the helicity of two BT groups.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 19 October 2009
Revised 2 December 2009
Accepted 3 December 2009
Available online 11 December 2009
Keywords:
Exciton coupling
Circular dichroism
Modified nucleic acid
Benzothiazole
1. Introduction
convenient synthesis of 6-N-(benzothiazol-2-yl)deoxyadenosine
(ABT) and unique properties of its exciton-coupled circular dichro-
Circular dichroism (CD) spectra are widely used to study confor-
mation of DNAs and their changes.1–3 The conformational change
of DNAs was observed below 300 nm in their CD spectra. However,
the presence of other forms of DNAs or the binding with other UV-
active biomolecules makes it difficult to detect the change of local
conformation.4–6 To overcome this problem, the chiroptical probes
that absorb above 300 nm were desired. The porphyrins4–6 and
stilbene7–9 were well known as the exciton-coupled CD (ECCD)
probes that absorb above 300 nm, and the porhyrins were used
as probes for the B–Z transition of DNAs.6 These chromophores
were attached to the terminal position of DNA strands. However,
there is no paper to incorporate such chromophores into the intra-
strand position to investigate the local conformation change of
long DNA by ECCD. In this case, the chromophore should not cause
to distort the duplex and should have an exciton coupling property
at wavelengths above 300 nm to avoid the effect of the UV absor-
bance bands of the nucleobases.
The benzothiazole moiety usually gives UV absorption bands
above 300 nm and has been used to attach optical properties to
biomolecules.10–12 Because benzothiazole derivatives were often
used as the intercalators, it was expected that a benzothiazole
(BT)-modified adenine base could be used like a universal base
with the minimum distortion on DNA duplex structures. In addi-
tion, 6-N-modification of the adenine moiety could be attained
using the Buchwald–Hartwig reaction. Herein, we report the
ism upon incorporation into DNA oligomers.
2. Results and discussion
2.1. Synthesis of 6-N-(benzothiazol-2-yl)deoxyadenosine and
its derivatives
The synthesis of 6-N-(benzothiazol-2-yl)deoxyadenosine (3) is
shown in Scheme 1. The reaction of compound 113 with 2-amino-
benzothiazole in the presence of Pd(dba)2, Xantphos, and Cs2CO3 at
80 °C produced the N-arylated product 2 in 93% yield. Treatment of
compound 2 with 3HFꢀTEA produced the deprotected product 3 in
89% yield. Compound 3 was converted to the phosphoramidite
derivatives 4 in 72% yield (two steps) by the successive 50-O-
dimethoxytritylation and 30-phosphytilation. The synthesis of oli-
gonucleotides was performed according to the phosphoramidite
approach using an ABI 392 DNA/RNA automated synthesizer. The
base sequences of the synthesized oligodeoxynucleotides are
summarized in Figure 1.
2.2. Properties of modified oligonucleotides (ODN1, ODN21,
ODN22, and ODN3)
2.2.1. UV spectra of single stranded oligodeoxynucleotides
modified with ABT
The normalized UV spectra of the modified oligodeoxynucleo-
tides (ODNs) are shown in Figure 2. To compare the unmodified
* Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924 5772.
E-mail address: msekine@bio.titech.ac.jp (M. Sekine).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.009

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Y. Masaki et al. / Bioorg. Med. Chem. 18 (2010) 567–572
Scheme 1. Reagents and conditions: (a) 2-aminobenzothiazole, 5 mol % Pd(dba)2, 10 mol % Xantphos, Cs2CO3, 1,4-dioxane, 80 °C, 3 h, 93%; (b) 3HFꢀTEA, TEA, THF, rt, 16 h,
89%; (c) DMTrCl, pyridine, rt, 12 h, 89%; (d) (CEO)P(NiPr2)2, 1H-tetrazole, iPr2NH, CH2Cl2, rt, 2.5 h, 80%.
in 300–350 nm region were increased proportionally with an in-
crease in the number of BT moieties.
2.2.2. Hybridization properties of multi-modified DNAs
The hybridization properties of BT modified oligodeoxynucleo-
tides toward the target DNA were determined by measuring the
melting temperature (Tm) values from the temperature-dependent
UV profiles of the duplexes with their complementary oligodeoxy-
nucleotides. The Tm value of ODN1 with a target strand (Y = T) was
39.3 °C and that of the control duplex was 43.6 °C (Table 1). This
means that incorporation of the ABT moiety into DNA destabilized
the thermal stability of the resulting duplex. On the other hand, the
Tm values of three mismatch strands with ODN1 were 37.2 °C for
A
BT:G (29.9 °C for A:G), 36.5 °C for ABT:A (26.5 °C for A:A), and
41.7 °C for ABT:C (27.3 °C for A:C). The base-recognition ability
was determined by the difference in Tm value between the match
and mismatch duplexes. The base-recognition ability of ODN1
strand was only +2.4 to ꢁ2.8 °C and control strand was ꢁ13.7 to
ꢁ17.1 °C. It seems that the ABT was not paired with the counterpart
base.
2.2.3. CD spectra of duplexes with ABT
To clarify the influence of the BT moiety on the duplex struc-
ture, the CD spectra of the modified and unmodified duplexes were
measured (Fig. 3). Their spectra were almost similar in the region
of 220–300 nm without relatively stronger positive cotton effects
in 260–300 nm. It should be noted that there were almost no sig-
nals at 300–350 nm where the UV absorption bands arising from
the BT moiety were observed.
Figure 1. 6-N-(Benzothiazol-2-yl)deoxyadenosine and base sequences of oligode-
oxynucleotides incorporating this modified nucleoside.
Next, we measured the CD spectra of the multi-modified du-
plexes of ODN21, ODN22, and ODN3 with their complementary
strands. These results are summarized in Figure 4. Interestingly,
the multi-modification of the BT moiety induced new circular
dichroism signals in the 300–350 nm region, which was not ob-
served in the duplex containing ODN1. In addition, although there
Table 1
The base-recognition ability of ABT
Xa
Yb
Tm (°C)
D
Tm (°C)
c
d
Entry
1
2
3
4
ABT
T
39.3
37.2
36.5
41.7
—
G
A
C
ꢁ2.1
ꢁ2.8
+2.4
Figure 2. Normalized UV spectra of modified oligodeoxynucleotides containing ABT
.
The sequence of ODN1: 50-d(TACCTAABTATCCAT), ODN21: 50-d(TACCTABTABTATC
CAT), ODN22: 50-d(TABTCCTAABTATCCAT), ODN3: 50-d(TACCTABTABTABTTCCAT). The
control means unmodified oligodeoxynucleotide: 50-d(TACCTAAATCCAT).
5
6
7
8
A
T
43.6
29.9
26.5
27.3
—
G
A
C
ꢁ13.7
ꢁ17.1
ꢁ16.3
a
b
c
50-d(TACCTAXATCCAT)30, ODN1 (X = ABT), Control (X = A).
ODN (control) with a one-point BT modified ODN (ODN1), the
additional absorption of the BT moiety was observed at a 300–
350 nm region. The UV intensities of two-point BT modified ODNs
(ODN21 and ODN22) and a three-point BT modified ODN (ODN3)
50-d(ATGGATYTAGGTA)-30.
Tm values were measured in 10 mM sodium phosphate buffer (pH 7.0), con-
M of each oligomer.
taining 0.1 M NaCl, 0.1 mM EDTA, 2.0
l
d
Y = T
D
Tm = Tm ꢁ Tm
.

Y. Masaki et al. / Bioorg. Med. Chem. 18 (2010) 567–572
569
standard B-form base-pair step and that of ODN22 was expected
to be near 180°, that is, fivefold wider than the twist of the base-pair
step. The mirror image CD shape suggested that the angle (h) be-
tween the chromophores was below 180°.
The ODN3 duplex also showed a significant positive Cotton ef-
fect at 317–350 nm, a negative Cotton effect at 300–317 nm, and
the same shape as that of the nucleobase region. The intensity of
the BT region (300–350 nm) was increased, but that of the nucleo-
base region (220–300 nm) was decreased. This result suggests that
the consecutive three-point BT modification would destabilize the
whole duplex structure and the Cotton effect due to the right-
handed helical structure of three benzothiazoles was markedly ob-
served. Thus, the two-point BT modification is more suitable for
the detection of local conformation change.
2.3. Properties of modified oligonucleotides (A1–A6)
Figure 3. Circular dichroism spectra of 50-d(TACCTAABTATCCAT)/50-d(ATGGATX
TAGGTA) duplexes (X = T, G, A, or C) at 10 °C in 10 mM sodium phosphate buffer
2.3.1. Hybridization properties of two-point modified DNAs
Next, we studied the dependency of the distance and helicity
between BT moieties. The base sequences and Tm values of the
modified oligonucleotides synthesized in this study are summa-
rized in Table 2. Consequently, the DNA oligomer (A1) with a con-
secutively arranged ABTABT sequence showed the Tm value of
42.0 °C that was lower by 8.2 °C than the unmodified oligomer,
when hybridized with the complementary DNA oligomer. The du-
plexes involving dispersedly modified oligomers (A2–A5) showed
the Tm values of 45.6, 46.6, 44.4, and 40.2 °C, respectively. The du-
plex of the dangling-end modified oligomer (A6) with the same
complementary DNA oligomer showed the Tm value of 59.5 °C.
The additional stabilizing effect (+9.3 °C) of this duplex could be
explained by the stacking effect of the dangling-end modified
nucleobase.14
(pH 7.0), containing 0.1 M NaCl, 0.1 mM EDTA, 5.0
means unmodified duplex.
lM of each oligomer. The control
are the only two or three BT moieties in the 13 mer duplex (total 26
nucleobases), the intensities of CD spectra in the 300–350 nm re-
gion were comparable with those in the region of 220–300 nm,
which was originated from the nucleobases. These results indi-
cated that the exciton coupling existed between the BT moieties.
The duplex of ODN21 with the complementary oligomer
showed a positive Cotton effect at 320–350 nm and a negative Cot-
ton effect at 300–320 nm. The modification did not affect the CD
signal in the nucleobase region (220–300 nm). The shape of the
CD spectrum at 300–350 nm was quite similar to that observed
in the nucleobase region. This suggested that the BT moieties were
aligned along the helical geometry. Interestingly, the duplex con-
taining ODN22 showed a negative Cotton effect at 320–340 nm
and a positive Cotton effect at 300–320 nm, which is opposite to
the spectrum of the ODN21-containing duplex. This phenomenon
was agreed with the previous studies.7–9 An important factor of
the intensity of the Cotton effect is the angle (h) between the elec-
tronic transition dipole moments of two chromophores. The effect
of the angle was expressed by the following formula:8
Table 2
The melting temperature of two-point ABT modified ODNs
a
b
Entry
Sequence
Tm (°C)
DTm (°C)
1
2
3
4
5
6
A1
A2
A3
A4
A5
A6
50-d(GGCABTABTAAAACGG)
50-d(GGCABTAABTAAACGG)
50-d(GGCABTAAABTAACGG)
50-d(GGCABTAAAABTACGG)
50-d(GGCABTAAAAABTCGG)
50-d(ABTGGCAAAAAACGGABT
42.0
45.6
46.6
44.4
40.2
59.5
ꢁ8.2
ꢁ4.6
ꢁ3.6
ꢁ5.8
ꢁ10.0
+9.3
p
4k
)
D
e
ꢂ ꢃ li2lj2Riꢁj 2 sinð2hÞ;
7
Control
50-d(GGCAAAAAACGG)
50.2
—
where li and lj are the transition dipole moments of the two chro-
mophores and Rij is the distance between them. The angle (h) of
ODN21 was expected to be near the twist value (36°) of the
a
Tm values were measured in 10 mM sodium phosphate buffer (pH 7.0), con-
taining 0.1 M NaCl, and 0.1 mM EDTA, 2.0 M of each oligomer.
l
b
control
D
Tm = Tm ꢁ Tm
.
Figure 4. (a) Circular dichroism spectra of the duplexes containing ODN1, ODN2, and ODN3 strands at 10 °C in 10 mM sodium phosphate buffer (pH 7.0), containing 0.1 M
NaCl, and 0.1 mM EDTA, 5.0
M of each oligomer. The control means unmodified duplex. (b) The direction of transition dipole moment of the ABT analogue was calculated at
the TDDFT/B3LYP/6-31+G(d,p) level by using GAUSSIAN03.15
l

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Y. Masaki et al. / Bioorg. Med. Chem. 18 (2010) 567–572
2.3.2. Dependency of the helicity between BT moieties
ism (ECCD). Although the benzothiazole (BT) moiety destabilizes
the thermal stability of duplexes, the CD spectra indicated that
the BT moiety does not markedly distort the duplex structure. In
addition, multipoint BT modifications induced strong ECCD at the
300–350 nm region, which was not overlapped with the absorp-
tion region of nucleotidic UV bands. The shapes of CD spectra were
strongly influenced by the helicity of benzothiazole chromophores.
Figure 5 shows the CD spectra of the duplexes of A1–A6 with
the complementary DNA 12 mer. The A1 duplex with consecu-
tively two-point modified bases showed a strong Cotton effect at
320–350 nm and a relatively weak negative Cotton effect at 300–
320 nm. The CD intensity at 300–350 nm was comparable with
that of the nucleobase region. The CD spectrum of the A2 duplex
having an ABTAABT sequence was similar to that of the A1 duplex
except for the blue shift of the maximum of the positive Cotton ef-
fect at 320–350 nm (from 337 nm (A1) to 333 nm (A2)).
Surprisingly, the shapes of the CD spectra of the A3 and A4 du-
plexes with two and three deoxyadenosines (As) between two ABT
deoxynucleosides were changed. The A3 and A4 duplexes showed
only the positive Cotton effect at 300–350 nm region and the neg-
ative Cotton effect disappeared. The maxima of the positive Cotton
effect were blue shifted (326 nm for A3 and 321 nm for A4 duplex).
In addition, the shapes of the CD spectra of the A3 and A4 duplexes
also changed in the nucleobase region (220–300 nm), especially at
260–280 nm. The NMR study of this sequence showed that the
middle part of this sequence forms the A-tract structure.16 This
change of the nucleobase region suggests that the BT moieties
incorporated in the mid-part of the A-tract sequence interfered
with the formation of the A-tract structure, possibly causing the
change in the orientation of the BT moieties. These factors might
cause the unusual behavior of CD spectra.
Interestingly, the A5 duplex with four deoxyadenosines in-
serted between two ABT nucleosides showed almost no Cotton ef-
fect at 300–350 nm. The angle (h) between the BT moieties in A5
duplex is expected to be approximately 182°, which was the sum
of the twist angles of base-pair step parameters of the NMR struc-
ture (PDB: 1FZX).16 As discussed above (Section 2.2.3), the inten-
sity of the CD spectrum is proportional to sin(2h). Thus, the A5
duplex would have almost no signal of the Cotton effect.
The A6 duplex still showed the Cotton effect. A negative Cotton
effect was observed at 300–350 nm (negative absorption minimum
at 327 nm). The BT moiety could show the ECCD spectra even when
separated by 12 base pairs. It is said that the intensity of interac-
tion between chromophores decreases sharply with an increase
in the distance. For example, the intensity of fluorescence reso-
nance energy transfer (FRET) and photo-induced electron transfer
(PET) decreased with an increase in distance R (1/R,6 and exp
(1/R), respectively).7,17,18 Our results indicate that the ECCD spectra
were also a powerful tool to observe the long-distance interaction.
4. Experimental procedures
4.1. General remarks
The dry solvents were purchased and stored over molecular
sieves 4A. 1H, 13C, and 31P NMR spectra were obtained at 500, 126,
and 203 MHz, respectively. The chemical shifts were measured from
CDCl3 (7.26 ppm), DMSO-d6 (2.50 ppm) for 1H NMR, CDCl3
(77.0 ppm), DMSO-d6 (39.5 ppm) for 13C NMR and 85% phosphoric
acid (0.0 ppm) for 31P NMR. UV spectra were recorded with a U-
2810 spectrometer. Column chromatography was performed with
silica gel C-200 (purchased from Wako Co. Ltd). High performance
liquid chromatography (HPLC) was performed using the following
systems: Reversed exchange HPLC was done on a Waters Alliance
system with a Waters 3D UV detector and a Waters XTerra MS C18
column (4.6 ꢄ 150 mm); a linear gradient (0–30%) of solvent I
(0.1 M ammonium acetate buffer (pH 7.0)) in solvent II (CH3CN)
was used at 50 °C at a flow rate of 1.0 mL/min for 30 min; anion-ex-
change HPLC was done on a Shimadzu LC-10 AD VP with a Shimadzu
3D UV detector and a Gen-Pak FAX column (Waters, 4.6 ꢄ 100 mm);
a linear gradient (10–67%) of solvent III (1 M NaCl in 25 mM phos-
phate buffer (pH 6.0)) in solvent IV (25 mM phosphate buffer (pH
6.0)) was used at 50 °C at a flow rate of 1.0 mL/min for 40 min. ESI
mass was performed using Mariner™ (PerSeptive Biosystems Inc.).
MALDI-TOF mass was performed using a Bruker Daltonics (Matrix:
3-hydoroxypicolinic acid (100 mg/mL) in H2O-diammoniumhydro-
gen citrate (100 mg/mL) in H2O (10 : 1, v/v)).
4.2. Synthesis of phosphoramidite unit
4.2.1. 30,50-Di-tert-butyldimehylsilyl-6-N-(benzothiazoyl-2-yl)-
deoxyadenosine 2
Pd(dba)2 (57.8 mg, 0.1 mmol) and Xantphos (116 mg, 0.2 mmol)
were dissolved in dry 1,4-dioxane (20 mL). The mixture was stirred
at room temperature for 30 min. To the solution were added Cs2CO3
(982 mg, 3.01 mmol), 2-aminobenzothiazole (603 mg, 4.02 mmol),
and compound 1 (1.00 g, 2.01 mmol) in dry 1,4-dioxane (20 mL).
After being stirred at 80 °C for 3 h, the mixture was filtered using
Celite. The solvent was evaporated in vacuo and the residue was
chromatographed on a silica gel column with CHCl3–MeOH (99:1,
3. Conclusions
We synthesized the 6-N-(benzothiazol-2-yl)deoxyadenosine
(ABT) and clarified its property of exciton-coupled circular dichro-
Figure 5. Circular dichroism spectra at 10 °C in 10 mM sodium phosphate buffer (pH 7.0), containing 0.1 M NaCl, and 0.1 mM EDTA, 5.0 lM of each oligomer.

Y. Masaki et al. / Bioorg. Med. Chem. 18 (2010) 567–572
571
v/v) to give compound 2 as pale yellow oil (1.11 g, 93%): 1H NMR
(CDCl3, 500 MHz): d 8.72 (1H, s), 8.38 (1H, s), 7.83 (1H, d, J = 7.5),
7.80 (1H, d, J = 7.5), 7.43 (1H, t, J = 7.5), 7.29 (1H, t, J = 7.5), 6.52
(1H, t, J = 6.0), 4.65–4.62 (1H, m), 4.04 (1H, d, J = 3.5), 3.88 (1H,
dd, J = 3.5, 11.0), 3.78 (1H, dd, J = 3.0, 11.0), 2.68 (1H, dt,
J = 6.0,12.5), 2.49 (1H, ddd, J = 3.5, 6.0, 12.5) 0.92 (9H, s), 0.91
(9H, s), 0.11 (6H, s), 0.08 (6H, s); 13C NMR (CDCl3, 126 MHz): d
158.3, 151.5, 150.2, 149.0, 148.6, 141.7, 133.2, 125.9, 123.2,
121.0, 120.8, 120.6, 88.1, 84.7, 72.0, 62.8, 41.1, 25.8, 25.7, 18.3,
18.0, ꢁ4.7, ꢁ4.8, ꢁ5.5, ꢁ5.6; MS (ESI) calcd for C29H44N6O3SSi2
[M+H]+ 613.2807, found 613.2837.
126 MHz): d 158.4, 151.3, 150.2, 148.9, 148.7, 144.5, 142.2, 142.1,
135.6, 133.1, 130.0, 129.9, 128.1, 128.0, 127.7, 126.8, 125.9,
123.1, 120.9, 120.5, 117.5, 117.4, 113.0, 86.3, 86.0, 85.8, 85.8,
84.9, 74.2, 74.0, 73.5, 73.3, 63.5, 63.3, 58.4, 58.2, 58.1, 55.1, 45.3,
45.2, 44.4, 44.4, 43.3, 43.2, 39.1, 24.6, 24.5, 24.5, 24.4, 23.6, 22.9,
22.8, 22.6, 20.3, 20.3, 20.2, 20.1, 20.0, 19.9; 31P NMR (CDCl3) d
149.4, 149.3; MS (ESI) calcd for C47H52N8O6PS [M+H]+ 887.3463,
found 887.3445.
4.3. Synthesis of oligonucleotides
The synthesis of ODNs was performed in an ABI 392 DNA syn-
thesizer by the standard 1.0-lmol-scale phosphoramidite ap-
4.2.2. 6-N-(Benzothiazoyl-2-yl)deoxyadenosine 3
Compound 2 (1.11 g, 1.81 mmol) was dissolved in dry THF
(20 mL). To the solution were added Et3Nꢀ3HF (0.59 mL, 3.62 mmol)
and triethylamine (0.51 mL, 3.62 mmol). After being stirred at room
temperature for 16 h, the reaction mixture was filtered. The residue
was dissolved in pyridine and precipitated by CHCl3 to give com-
pound 3 as a pale yellow solid (0.618 g, 89%): 1H NMR (DMSO,
500 MHz): d 12.45 (1H, br s), 8.67 (1H, s), 8.65 (1H, s), 7.95 (1H, d,
J = 7.5), 7.68 (1H, d, J = 7.5), 7.41 (1H, t, J = 7.5), 7.26 (1H, t, J = 7.5),
6.46 (1H, t, J = 6.5), 5.36 (1H, br s), 5.05 (1H, br s), 4.44 (1H, br s),
3.90 (br s, 1H), 3.65–3.62 (1H, m), 3.55–3.52 (1H, m), 2.77 (1H, dt,
J = 6.5, 13.0), 2.37–2.32 (1H, m); 13C NMR (DMSO, 126 MHz): d
150.7, 150.3, 142.0, 126.1, 122.9, 121.4, 88.0, 83.8, 70.7, 61.6; MS
(ESI) calcd for C17H16N6O3S [M+H]+ 385.1077, found 385.1091.
proach, which consists of detritylation, coupling, capping, and
iodine oxidation steps. The universal supports III (Glen Research
Inc.) was used for the synthesis of the A6 sequence. Then, the syn-
thesized oligomers were released from the resin by treatment with
a solution of 28% aq NH3 solution at room temperature for 4 h. The
polymer supports were removed by filtration and washed with
0.1 M ammonium acetate buffer (1 mL ꢄ 3). The filtrates were
purified by anion-exchange HPLC to give ODNs.
Oligonucleotide: ODN1: MALDI-TOF Mass (M+H) calcd for
C133H165N46O75P12S+: 4009.2; found: 4006.0.
Oligonucleotide: ODN21: MALDI-TOF Mass (M+H) calcd for
C140H168N47O75P12S2+: 4142.7; found: 4139.6.
Oligonucleotide: ODN22: MALDI-TOF Mass (M+H) calcd for
C140H168N47O75P12S2+: 4142.7; found: 4137.0.
4.2.3. 50-O-(4,40-Dimethoxytrityl)-6-N-(benzothiazoyl-2-yl)deoxy-
adenosine 3a
Oligonucleotide: ODN3: MALDI-TOF Mass (M+H) calcd for
C147H171N48O75P12S3+: 4275.7; found: 4273.4.
Compound 3 (0.716 g, 1.86 mmol) was co-evaporated three
times with dry pyridine and finally dissolved in dry pyridine
(20 mL). To the solution was added 4,40-dimethoxytrityl chloride
(0.757 g, 2.24 mmol). After being stirred at room temperature for
12 h, the mixture was quenched by adding methanol and evapo-
rated in vacuo. The residue was dissolved with CHCl3. The solution
was washed with water. The organic layer was concentrated in va-
cuo. The residue was chromatographed on a column of silica gel
with CHCl3–MeOH (95:5, v/v) containing 0.5% pyridine to give
compound 3a as white foam (1.144 g, 89%): 1H NMR (CDCl3,
500 MHz): d 11.2 (1H, br s), 8.66 (1H, s), 8.55 (1H, s), 7.84 (1H, d,
J = 8.0), 7.74 (1H, d, J = 8.0), 7.10–7.40 (11H, m), 6.69–6.76 (4H,
m), 6.57 (1H, t, J = 6.0), 4.78 (1H, d, J = 3.5), 4.26–4.32 (1H, m),
3.70 (6H, s), 3.44–3.51 (1H, m), 3.38–3.44 (1H, m), 2.96 (1H, dt,
J = 6.5, 13.0), 2.64–2.72 (1H, m); 13C NMR (CDCl3, 126 MHz): d
158.5, 158.4, 151.3, 150.1, 148.8, 148.6, 144.4, 141.9, 135.6,
135.5, 133.0, 129.9, 129.9, 128.0, 127.7, 126.8, 125.9, 123.1,
120.9, 120.7, 120.5, 113.0, 86.5, 86.1, 84.6, 72.3, 63.6, 55.1, 39.9;
MS (ESI) calcd for C38H35N6O5S [M+H]+ 687.2384, found 687.2347.
Oligonucleotide: A1: MALDI-TOF Mass (M+H) calcd for
C132H152N58O64P11S2+: 3977.7; found: 3972.9.
Oligonucleotide: A2: MALDI-TOF Mass (M+H) calcd for
C132H152N58O64P11S2+: 3977.7; found: 3978.0.
Oligonucleotide: A3: MALDI-TOF Mass (M+H) calcd for
C132H152N58O64P11S2+: 3977.7; found: 3974.5.
Oligonucleotide: A4: MALDI-TOF Mass (M+H) calcd for
C132H152N58O64P11S2+: 3977.7; found: 3972.7.
Oligonucleotide: A5: MALDI-TOF Mass (M+H) calcd for
C132H152N58O64P11S2+: 3977.7; found: 3976.0.
Oligonucleotide: A6: MALDI-TOF Mass (M+H) calcd for
C152H176N68O74P13S2+: 4603.8; found: 4599.7.
4.4. Tm measurement
The unmodified oligodeoxynucleotides were purchased from
SIGMA GENOSYS. An appropriate ODN (2
lM) and its complemen-
tary ssDNA (2 M) were dissolved in a buffer consisting of 100 mM
l
NaCl, 10 mM sodium phosphate, and 0.1 mM EDTA adjusted to pH
7.0. The solution was kept at 85 °C for 10 min for complete disso-
ciation of the duplex to single strands, cooled at the rate of
ꢁ1.0 °C/min, and kept 10 °C for 10 min. Then, the melting temper-
atures (Tm) were determined at 260 nm using a UV spectrometer
(Pharma Spec UV-1700™, Shimadzu) by increasing the tempera-
ture at the rate of 1.0 °C/min.
4.2.4. 30-O-[(N,N-Diisopropylamino)-2-cyanoethoxyphosphinyl]-
50-O-(4,40-dimethoxytrityl)-6-N-(benzothiazoyl-2-yl)deoxyade-
nosine 4
Compound 3a (671 mg, 0.98 mmol) was dissolved in dry CH2Cl2
(10 mL). To the solution were added diisopropylamine (82.4
0.58 mmol) and bis-(N,N-diisopropylamino)(2-cyanoethoxy)phos-
phine (324 L) in dry CH2Cl2 (10 mL). After being stirred at room
lL,
l
4.5. CD spectra
temperature for 2.5 h, the mixture was washed with saturated
NaHCO3 solution. The organic layer was evaporated in vacuo. The
residue was chromatographed on a column of silica gel with
CHCl3–MeOH (97:3, v/v) containing 0.5% pyridine to give com-
CD spectra were measured on J-725 Spectroplarimeter, JASCO.
Each oligodeoxynucleotide solutions (5 lM)) were prepared in
pound
4
as white foam (692 mg, 80%): 1H NMR (CDCl3,
10 mM sodium phosphate buffer pH 7.0, 0.1 mM EDTA, and
100 mM NaCl. The solutions were heated to 85 °C and annealed
by slowly cooling to room temperature over a period of 12 h. Spec-
tra were recorded between 220 and 370 nm. Spectra were aver-
aged over 8 scans, which were recorded at 100 nm minꢁ1 with a
response time of 4 s and a bandwidth of 1.0 nm.
500 MHz): d 8.67 (1H, s), 8.54–8.64 (1H, m), 7.85 (1H, d, J = 7.0),
7.75 (1H, d, J = 7.5), 7.30–7.40 (11H, m), 6.69–6.81 (4H, m), 6.48–
6.56 (1H, br s), 4.78–4.8 (1H, m), 4.32–4.42 (1H, m), 3.50–3.92
(10H, m), 3.34–3.48 (2H, m), 2.98–3.09 (1H, m), 2.58–2.82 (2H,
m), 2.44–2.53 (1H, m), 1.13–1.38 (12H, m); 13C NMR (CDCl3,

572
Y. Masaki et al. / Bioorg. Med. Chem. 18 (2010) 567–572
5. Balaz, M.; Bitsch-Jensen, K.; Mammana, A.; Ellestad, G. A.; Nakanishi, K.;
4.6. Computational method
Berova, N. Pure Appl. Chem. 2007, 79, 801.
6. Balaz, M.; Li, B. C.; Steinkruger, J. D.; Ellestad, G. A.; Nakanishi, K.; Berova, N.
Org. Biomol. Chem. 2006, 4, 1865.
7. Lewis, F. D.; Liu, X.; Wu, Y.; Zuo, X. J. Am. Chem. Soc. 2003, 125, 12729.
8. Lewis, F. D.; Zhang, L.; Liu, X.; Zuo, X.; Tiede, D. M.; Long, H.; Schatz, G. C. J. Am.
Chem. Soc. 2005, 127, 14445.
9. Burin, A. L.; Armbruster, M. E.; Hariharan, M.; Lewis, F. D. Proc. Natl. Acad. Sci.
U.S.A. 2009, 106, 989.
10. Ono, M.; Hayashi, S.; Kimura, H.; Kawashima, H.; Nakayama, M.; Saji, H. Bioorg.
Med. Chem. 2009, 17, 7002.
11. Abbotto, A.; Beverina, L.; Bozio, R.; Facchetti, A.; Ferrante, C.; Pagani, G. A.;
Pedron, D.; Signorini, R. Org. Lett. 2002, 4, 1495.
12. Mashraqui, S. H.; Kumar, S.; Vashi, D. J. Inclusion Phenom. Macrocycl. Chem.
2004, 48, 125.
The structure of the 6-N-benzothiazolyl adenine analogue was
optimized at the B3LYP/6-31+G(d,p) level by using the GAUSSIAN03
software.15 The vibrational frequency was calculated to confirm
the equilibrium geometries that correspond to energy the mini-
mum. After that, the excited state calculation was performed on
the optimized structure at the TDDFT/B3LYP/6-31+G(d,p) level.
The direction of the transition dipole moment was depicted by
using the GaussView.
Acknowledgments
13. Dai, Q.; Ran, C.; Harvey, R. G. Org. Lett. 2005, 7, 999.
14. Isaksson, J.; Chattopadhyaya, J. Biochemistry 2005, 44, 5390.
15. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A.; Stratmann, R. E.; Burant,
J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas,
O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.;
Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. A.; Cui, Q.;
Morokuma, K.; Salvador, P.; Dannenberg, J. J.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.;
Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres,
J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. GAUSSIAN03,
Revision E.01, Gaussian, Inc., Wallingford CT, 2004.
16. MacDonald, D.; Herbert, K.; Zhang, X.; Polgruto, T.; Lu, P. J. Mol. Biol. 2001, 306,
1081.
17. Lewis, F. D.; Wu, T.; Zhang, Y.; Letsinger, R. L.; Greenfield, S. R.; Wasielewski, M.
R. Science 1997, 277, 673.
18. Cardullo, R. A.; Agrawal, S.; Flores, C.; Zamecnik, P. C.; Wolf, D. E. Proc. Natl.
Acad. Sci. U.S.A. 1988, 85, 8790.
This work was supported by a grant from Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan and in part by Health Sciences
Research Grants for Research on Psychiatric and Neurological
Diseases and Mental Health from the Ministry of Health, Labor
and Welfare of Japan, and by the global COE project.
References and notes
1. Muhuri,S.;Mimura,K.;Miyoshi,D.;Sugimoto,N.J.Am.Chem.Soc.2009,131,9268.
2. Schonhoft, J. D.; Bajracharya, R.; Dhakal, S.; Yu, Z.; Mao, H.; Basu, S. Nucleic Acids
Res. 2009, 37, 3310.
3. Westrate, L.; Mackay, H.; Brown, T.; Nguyen, B.; Kluza, J.; David Wilson, W. D.;
Lee, M.; Hartley, J. A. Biochemistry 2009, 48, 5679.
4. Balaz, M.; Holmes, A. E.; Benedetti, M.; Rodriguez, P. C.; Berova, N.; Nakanishi,
K.; Proni, G. J. Am. Chem. Soc. 2005, 127, 4172.