Synthesis and ESR studies of nitronyl nitroxide-tethered oligodeoxynucleotides

Article abstract of DOI:10.1016/j.tetlet.2004.12.012

We report on the development of a nucleoside labeled with a nitronyl nitroxide group, ^NNU, and the synthesis of oligodeoxynucleotides containing ^NNU. The spin signals of ^NNU-containing oligodeoxynucleotides varied with the

Full text of DOI:10.1016/j.tetlet.2004.12.012

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 791–795
Synthesis and ESR studies of nitronyl nitroxide-tethered
oligodeoxynucleotides
Akimitsu Okamoto,a,* Takeshi Inasakia and Isao Saitob,*
aDepartment of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Kyoto 615-8510, Japan
bNEWCAT Institute, School of Engineering, Nihon University, and SORST, Japan Science and Technology Corporation,
Tamura, Koriyama 963-8642, Japan
Received 22 October 2004; revised 1 December 2004; accepted 3December 2004
Available online 15 December 2004
Abstract—We report on the development of a nucleoside labeled with a nitronyl nitroxide group, NNU, and the synthesis of oligo-
deoxynucleotides containing NNU. The spin signals of NNU-containing oligodeoxynucleotides varied with the degree of hybridiza-
tion of the complementary strand and the distance between nitronyl nitroxide spins.
Ó 2004 Elsevier Ltd. All rights reserved.
Recent progress in spin labeling techniques makes ESR
a promising technique for investigating many biological
systems. In particular, site-directed spin labeling
(SDSL) is emerging as a powerful technique for explor-
ing the structure and dynamics of biomolecules.1 The
SDSL technique of the labeled biomolecules provides
much information on the dynamics of the nitroxide
group, the collision rate between a nitroxide group
and a freely diffusing paramagnetic agent, and the dis-
tance between a nitroxide group and another paramag-
netic species fixed in the biomolecular structure. Many
nucleosides labeled with nitroxide radicals, such as
2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)2 and
2,2,5,5-tetramethylpyrrolidine 1-oxyl,3 have been devel-
oped to analyze DNA structures and DNA–protein
interactions. However, there is still no precedent for
nucleosides labeled with a nitronyl nitroxide group,
which are known to be a key structure of the nitric
oxide (NO) scavenger, 2-phenyl-4,4,5,5-tetramethylimi-
dazoline-3-oxide 1-oxyl (PTIO).4 When nitronyl nitrox-
ides are incorporated into selected DNA sites, then
they are expected to be a new powerful SDSL method,
as well as being an NO trap. Here, we report on the
development of a novel nucleoside labeled with a nitro-
nyl nitroxide group, NNU, and the synthesis of oligode-
oxynucleotides (ODNs) containing NNU. The change in
O
N
OHC
N
O
1)
HOHN NHOH
O
O
N
O
N
•H2SO4
I
NH
NH
NH
MeOH
reflux, 3 h
(HO)2B
CHO
N
O
O
O
HO
HO
HO
Pd(PPh3)4, NaOH
MeOH-H2O (5:1)
reflux, 24 h
2) NaIO4
MeOH-H2O (5:1)
r.t., 1 h
O
O
O
OH
OH
OH
NNU
28%
21%
d
1
2
Scheme 1.
Keywords: Pyrene; Oligodeoxynucleotide; Fluorescence; Quadruplex.
*
Corresponding authors. Tel.: +81 75 383 2757; fax: +81 75 383 2759; e-mail: okamoto@sbchem.kyoto-u.ac.jp
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.12.012

792
A. Okamoto et al. / Tetrahedron Letters 46 (2005) 791–795
the spin signals of NNU-containing ODNs caused
by the hybridization with the complementary strand
and the distance between adjacent nitronyl nitroxide
spins have been investigated.
We synthesized NNU from 5-iodo-20-deoxyuridine 1
(Scheme 1). The Suzuki–Miyaura coupling of 1 with 4-
formylphenylboronic acid yielded the aldehyde-possess-
ing nucleoside 2. The aldehyde 2 was then treated with
2,3-bis(hydroxyamino)-2,3-dimethylbutane, and sub-
sequently oxidized with sodium periodate to give
the NNU deoxynucleoside (dNNU).5
The ESR spectrum of dNNU was measured, and is
shown in Figure 1. The ESR spectrum was recorded at
27 °C on a Bruker EMX spectrometer operating in the
X-band using a glass capillary tube. The ESR data
shows that the ESR spectrum of dNNU exhibits a char-
acteristic five-line signal, with a typical intensity ratio of
1:2:3:2:1 (aN = 8.11 G), and is close to that of PTIO
(aN = 8.2 G).4a
Figure 1. ESR spectrum of dNNU. A solution of dNNU (100 lM) in
50 mM sodium phosphate and 0.1 M sodium chloride (pH 7.0) was
examined at 27 °C. The ESR spectrum was obtained using a Bruker
EMX spectrometer, in the X-band, with a modulation amplitude of
2.0 G, a time constant of 20 ms, and a microwave power of 1 mW. The
sample holder was a glass capillary tube. g = 2.0065, and aN = 8.11 G.
OTBS
N
OHC
O
N
O
N
TBSO
•H2SO4
HOHN NHOH
1)
NH
NH
O
N
O
Ac2O
MeOH
pyridine
reflux, 2 h
AcO
AcO
2
O
O
70 ˚C, 2 h
82%
2) TBSCl
imidazole, DMF
80 ˚C, 4 h
22%
OAc
OAc
3
4
OTBS
OTBS
N
N
N
N
O
O
TBSO
TBSO
NH
NH
1) DMTrCl
pyridine
r.t., 24 h
71%
N
O
N
O
NH4OH
MeOH
1) DNA synthesizer
HO
DMTrO
CN
O
O
P
2)
2) NH4OH
55 ˚C, 3 h
r.t., 3 h
quant.
O
(iPr2N)2P
OH
O
O
1H-tetrazole
MeCN-toluene (1:4)
r.t., 2 h
CN
NiPr2
5
6
quant.
OTBS
O
N
N
N
N
O
O
N
O
TBSO
NH
O
NH
1) TBAF
THF-H2O (1:9)
r.t., 24 h
O
P
O
P
N
O
O
O
O
O
O
O
O
O
2) 16.7 mM NaIO4
r.t., 10 min
O
O
7
NNU
Scheme 2.

A. Okamoto et al. / Tetrahedron Letters 46 (2005) 791–795
Table 1. NNU-containing ODN duplexes used in this study
793
Sequences
ODN1/ODN10
50-d(CGCAATNNUTAACGC)-30
30-d(GCGTTA A ATTGCG)-50
ODN0/ODN10
50-d(CGCAATATAACGC)-30
30-d(GCGTTAAATTGCG)-50
ODN2(0)/ODN2(0)0
ODN2(1)/ODN2(1)0
ODN2(2)/ODN2(2)0
ODN2(3)/ODN2(3)0
ODN2(4)/ODN2(4)0
ODN2(8)/ODN2(8)0
50-d(CGCAATNNUNNUAACGC)-30
30-d(GCGTTA A A TTGCG)-50
50-d(CGCAANNUANNUAACGC)-30
30-d(GCGTT A T A TTGCG)-50
50-d(CGCAANNUACNNUAACGC)-30
30-d(GCGTT A TG A TTGCG)-50
50-d(CGCAANNUACGNNUAACGC)-30
30-d(GCGTT A TGC A TTGCG)-50
50-d(CGCAANNUACGTNNUAACGC)-30
30-d(GCGTT A TGCA A TTGCG)-50
50-d(CGCAANNUACGTACGTNNUAACGC)-30
30-d(GCGTT A TGCATGCA A TTGCG)-50
Direct incorporation of dNNU into the oligodeoxynucleo-
tides (ODN) using the conventional phosphoramidite
method was found to be very difficult, because the prepa-
ration of the corresponding phosphoramidite was pro-
hibited by the high oxidizing ability of the nitronyl
nitroxide group. Therefore, we chose a synthetic route
in which the oxidation to nitronyl nitroxide was carried
out after the DNA synthesis step. The synthetic route to
obtain NNU-containing ODN is shown in Scheme 2. The
two hydroxy groups of the aldehyde 2 were protected
using acetyl groups. The aldehyde 3 was treated with
2,3-bis(hydroxyamino)-2,3-dimethylbutane, and the
two hydroxylamine groups of the resulting compound
were immediately protected using tert-butyldimethylsilyl
groups, to give compound 4. The hydrolysis of 4 affor-
ded the NNU precursor 5,6 which was protected by a
4,40-dimethoxytrityl group, and quantitatively converted
to cyanoethyl phosphoramidite 6. The amidite 6 was
employed in a conventional solid phase synthesis of
the ODNs. After ODN synthesis, deprotection was con-
ducted using aqueous ammonia at 55 °C for 3h to give
the ODN 7. The silyl group of 7 was removed using tetra-
butylammonium fluoride, and then oxidized with
sodium periodate to quantitatively yield NNU-contain-
ing ODNs. The crude NNU-containing ODNs were
then purified using reversed phase HPLC. The composi-
tion of the ODNs was determined using MALDI-TOF
mass spectrometry.7 A summary of the ODNs synthe-
sized is shown in Table 1.
Before the ESR studies on the NNU-containing ODNs
were carried out, we examined the structure and struc-
tural stability of the 13-mer ODN duplex ODN1/
ODN10 containing an NNU base using CD spectroscopy
and melting temperature (Tm) measurements. In the CD
spectrum of ODN1/ODN10, a negative peak at 253nm
and a positive peak at 277 nm were observed. This
spectrum was close to that observed for the natural
duplex ODN0/ODN10, indicating that the NNU-contain-
ing ODN maintains a B-DNA duplex. In the Tm
measurements of ODN1/ODN10, a sigmoidal curve at
the A254 change was observed, and the value of Tm
was determined to be 46.0 °C from the first deriva-
tive of the sigmoidal curve. This value of Tm is
4.8 °C lower than that observed for ODN0/ODN10
(50.8 °C).
Figure 2. ESR spectra of the NNU-containing ODNs. A solution of the duplexes (100 lM) in 50 mM sodium phosphate and 0.1 M sodium chloride
(pH 7.0) was examined at 27 °C. The ESR spectrum was measured using a Bruker EMX spectrometer, in the X-band, with a modulation amplitude
of 2.0 G, a time constant of 20 ms, and a microwave power of 1 mW. The sample holder was a glass capillary tube. (a) Single-stranded ODN1; (b)
ODN1/ODN10; and (c) ODN2(0)/ODN2(0)0.

794
A. Okamoto et al. / Tetrahedron Letters 46 (2005) 791–795
Next, an ESR spectral analysis of the single-stranded
ODN1 and the duplex ODN1/ODN10 was carried out,
and the results are shown in Figure 2a and b. The
ESR spectrum of the single-stranded ODN1 exhibited
the characteristic five-line pattern, with a typical hyper-
fine coupling constant of aN = 8.21 G, whereas the value
of aN for the spin probe in ODN1/ODN10 (8.25 G) was
slightly higher than that of the single-stranded state.
This result reflects the increased micropolarity at the
binding site of the nitronyl nitroxide group in the major
groove of the duplex. In addition, the duplex formation
caused the line broadening. This line broadening is
attributed to the motional restriction of the NNU spin
label within duplex DNA.2f,8
the spins was separated over two base pairs, then the sig-
nal intensity increased markedly. When the distance be-
tween the spins was separated over four base pairs, then
the interaction between two NNU spin labels was elimi-
nated. The change in ESR signal intensities of doubly
NNU-labeled ODNs will be a useful tool for monitoring
spatial proximity at room temperature.
In conclusion, we have designed a nucleoside labeled
with a nitronyl nitroxide group, dNNU, and have synthe-
sized ODNs containing one or two NNUs groups. The
spin signals from the NNU-containing ODNs varied be-
cause of the degree of hybridization of the complemen-
tary strand and the distance between the nitronyl
nitroxide spins. Therefore, NNU spin labels will be useful
as a powerful SDSL tool for exploring the structure and
dynamics of nucleic acids.
Using our synthetic protocol for an NNU-containing
ODN, we prepared a doubly NNU-labeled ODN
ODN2(0), which contained an NNUNNU sequence.
In the ESR spectrum of ODN2(0) hybridized with a
complementary strand ODN2(0)0, the signal intensity
was observed to be much lower than that of ODN1/
ODN10 in spite of this duplex having two spin labels
(Fig. 2c). The remarkable line broadening of ODN2(0)/
ODN2(0)0 is assumed to originate from the spin–spin
interaction and motional restriction between the labeled
side chains due to the close proximity of the spin labels
within the duplex.9,10
Acknowledgements
We are grateful to Dr. T. Nishinaga, Dr. A. Wakamiya,
and Professor K. Komatsu (Kyoto University) for tech-
nical assistance and for useful discussions on the ESR
data.
References and notes
We also prepared other doubly NNU-labeled ODNs with
different distances between two nitronyl nitroxide
groups (Table 1), and measured the resulting ESR sig-
nals. The ESR signal intensities observed for each
duplex are shown in Figure 3. As the distance between
the two NNU sites increased, the signal intensities grad-
ually increased. In particular, when the distance between
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Figure 3. Comparison of the signal intensities obtained from the ESR
spectra of NNU-containing duplexes. A solution of the duplexes
(100 lM) in 50 mM sodium phosphate and 0.1 M sodium chloride
(pH 7.0) was examined at 27 °C. The ESR spectrum was performed
using a Bruker EMX spectrometer, in the X-band, with a modulation
amplitude of 2.0 G, a time constant of 20 ms, and a microwave power
of 1 mW. The sample holder was a glass capillary tube. The ESR peak
intensity of ODN1/ODN10 was normalized to 1.0.

A. Okamoto et al. / Tetrahedron Letters 46 (2005) 791–795
795
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ꢀ4.0, ꢀ5.2; FABMS, m/e 993[(M+H) +]; HRMS calcd for
C55H77N4O9Si2 [(M+H)+] 993.5229, found 993.5220.
7. MALDI-TOF mass, ODN1, [MꢀOH]ꢀ calcd 4118.82, found
4118.59; ODN2(0), [MꢀO2H]ꢀ, calcd 4320.06, found 4320.19;
ODN2(1), [MꢀO2H]ꢀ, calcd 4329.08, found 4329.34;
ODN2(2), [MꢀO2H]ꢀ, calcd 4618.26, found 4617.80; ODN2(3),
[MꢀO2H]ꢀ, calcd 4947.46, found 4946.91; ODN2(4), [Mꢀ
O2H]ꢀ, calcd 5251.66, found 5251.83; ODN2(8), [M–
O2H]ꢀ, calcd 6487.45, found 6487.82.
5. Spectroscopic data for dNNU: blue foam; Rf 0.27 (CHCl3–
MeOH = 20:1); kmax [50 mM sodium phosphate, pH = 7.0,
0.1 M NaCl/nm (e)] 293.4 (18180), 570.0 (750); FABMS,
m/e 461 [(M+2H)+]; HRMS calcd for C22H29N4O7
[(M+2H)+] 461.2036, found 461.2036; ESR (50 mM
sodium phosphate,
aN(1) = aN(3) = 8.11 G.
pH = 7.0,
0.1 M
NaCl)
8. Keyes, R. S.; Bobst, A. M. Biochemistry 1995, 34, 9265–
9276.
1
6. Spectroscopic data for 5: white foam; H NMR (CDCl3,
400 MHz) d 11.56 (s, 1H), 7.64 (s, 1H), 7.43(d, J = 7.7 Hz,
2H), 7.37 (d, J = 7.7 Hz, 2H), 7.28–7.16 (m, 9H), 6.83(d,
J = 8.7 Hz, 4H), 6.18 (t, J = 6.6 Hz, 1H), 5.34 (d,
J = 4.2 Hz, 1H), 4.48 (s, 1H), 4.22–4.12 (m, 1H), 3.92–
3.87 (m, 1H), 3.74–3.64 (m, 8H), 2.31 (ddd, J = 6.6, 6.9,
13.3 Hz, 1H), 2.17 (ddd, J = 3.9, 6.6, 13.3 Hz, 1H), 1.11 (s,
12H), 0.74 (s, 9H), 0.73(s, 9H), ꢀ0.09 (s, 3H), ꢀ0.12 (s,
3H); 13C NMR (DMSO-d6, 125 MHz) d 170.3, 161.9,
149.7, 144.7, 139.4, 136.8, 135.5, 135.4, 132.4, 129.9, 129.6,
129.6, 127.7, 127.6, 126.5, 113.1, 113.0, 93.4, 85.6, 85.5,
84.9, 70.5, 67.5, 67.5, 63.7, 54.9, 39.5, 26.0, 24.1, 17.5, 17.0,
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10. Both the exchange interaction and the dipolar interaction
can contribute to the line broadening. Therefore, quanti-
tative analysis is difficult in this case. Matsuda, K.; Stone,
M. T.; Moore, J. S. J. Am. Chem. Soc. 2002, 124, 11836–
11837.