Formation of pyrene dimer radical cation at the minor groove of DNA

Article abstract of DOI:10.1246/bcsj.79.312

To arrange pyrene at the minor groove of DNA, we synthesized a nucleoside derivative possessing a pyrene group on guanine N2. Doubly pyrene-modified duplex DNA was synthesized and the formation rates of the pyrene dimer radical cation were measured upon o

Full text of DOI:10.1246/bcsj.79.312

312 Bull. Chem. Soc. Jpn. Vol. 79, No. 2, 312–316 (2006)
ꢀ 2006 The Chemical Society of Japan
Formation of Pyrene Dimer Radical Cation at the Minor Groove of DNA
ꢀ
Sachiko Tojo, Mamoru Fujitsuka, and Tetsuro Majima
Kiyohiko Kawai, Takumi Kimura, Hiroko Yoshida, Akira Sugimoto,
ꢀ
The Institute of Scientific and Industrial Research (SANKEN), Osaka University,
8-1 Mihogaoka, Ibaraki, Osaka 567-0047
Received August 12, 2005; E-mail: majima@sanken.osaka-u.ac.jp
To arrange pyrene at the minor groove of DNA, we synthesized a nucleoside derivative possessing a pyrene group
on guanine N2. Doubly pyrene-modified duplex DNA was synthesized and the formation rates of the pyrene dimer radi-
cal cation were measured upon one-electron oxidation during pulse radiolysis. For all of the DNA studied here, the for-
mation of a pyrene dimer radical cation was accomplished quickly, within 20 micro seconds after a 8-ns electron pulse
during pulse radiolysis. The results show that the minor groove of DNA offers a good space for the formation of pyrene
dimer radical cations.
with water and then with brine. The organic layer was dried over
anhydrous Na2SO4 and evaporated to dryness. The residue was
subjected to silica gel flash chromatography (CH2Cl2/MeOH =
50/1 (v/v)) to yield 2 (84 mg, 17% yield). 1H NMR (270 MHz)
(CDCl3) ꢁ 0.00–0.02 (m, 12H), 0.78–0.88 (m, 18H), 2.11–2.22
(m, 3H), 2.24–2.50 (m, 1H), 2.82–2.99 (m, 2H), 3.36–3.52 (m,
4H), 3.67–3.74 (m, 2H), 3.86–3.89 (m, 1H), 4.28–4.40 (m, 2H),
4.46–4.50 (m, 1H), 6.18–6.23 (dd, 1H, J ¼ 7:0, 6.2 Hz), 7.14–7.19
(m, 2H), 7.76–8.20 (m, 12H). FAB MS (positive ion): m=z 887.9
(M þ H).
DNA is a dynamic structure with several transient confor-
mations appearing in a short period of time, and the transiently
existing conformations are thought to play a variety of impor-
tant biological roles.1–5 However, the low population and short
lifetime of the transient conformations make their characteri-
zation very difficult. One way to identify less frequent motions
of biomolecules is to chemically trap such transient conforma-
tions. We recently reported the use of a pyrene (Py) dimer rad-
ical cation (Py2ꢁþ), which is stabilized by the charge resonance
(CR) between the two aromatics.6,7 Two Pys were introduced
at the end or at the internal site of a duplex DNA, and then the
Synthesis of 3. To a solution of 2 (80 mg, 0.09 mmol) in THF
(5 mL) was added a THF solution (5 mL) of triethylamine 3HF
ꢃ
ꢁþ
formation rates of Py2 were measured upon the one-electron
ꢁꢂ
(0.15 mL, 0.45 mmol), and the solution was stirred at room tem-
perature for 24 h. After evaporation of the solvent, the residue
was diluted with AcOEt and the organic phase was washed with
saturated NaHCO3 solution and then with brine. The organic layer
was dried over anhydrous Na2SO4 and evaporated to dryness. The
residue was subjected to silica gel flash chromatography (CH2Cl2/
oxidation by sulfate radical anions (SO4 ) generated from
2ꢂ
dissociative electron attachment of S2O8 during the pulse
radiolysis of an aqueous solution of Py-modifieꢁdþDNA in the
presence of K2S2O8. The formation rate of Py2 reflects the
frequency of the occurrence of DNA conformations in which
Pyꢁþ and Py can associate, and was discussed with relation
to the DNA dynamics that allow the interaction between
Pyꢁþ and Py. As for the structure of the transiently trapped
DNA, it was suggested thatꢁþDNA unwinding and bending
allowed the formation of Py2 at the minor groove of DNA.
The minor groove of DNA has been demonstrated to serve as
a good candidate for arranging heterocyclic compounds, and
allows ꢀ–ꢀ dimer formaꢁtiþon.8–11 Herein, to test the plausibility
of the formation of Py2 at the minor groove of DNA, we
synthesized a nucleoside derivative possessing a Py group on
guanine N2 to arrange Py at the minor groove of DNA. Doubly
Py-modified oligodeoxynucleotiꢁdþes (ODNs) were synthesized
and the formation rates of Py2 were measured upon one-
electron oxidation during pulse radiolysis.
1
MeOH = 50/2 (v/v)) to yield 3 (42.8 mg, 72% yield). H NMR
(270 MHz) (CDCl3) ꢁ 2.14–2.23 (m, 3H), 2.66–2.80 (m, 2H),
2.96–3.07 (m, 1H), 3.42–3.54 (m, 4H), 3.75–3.98 (m, 2H), 4.13–
4.21 (m, 3H), 4.66–4.72 (m, 1H), 6.14–6.20 (dd, 1H, J ¼ 9:6, 5.5
Hz), 7.01–7.06 (m, 2H), 7.54 (s, 1H), 7.85–8.27 (m, 11H). FAB
MS (positive ion): m=z 659.6 (M þ H).
Synthesis of 4. 3 (40 mg, 0.061 mmol) was dried by coevapo-
ration with dry pyridine three times. The residue was dissolved
in 4 mL of dry pyridine, and then 4,40-dimethoxytrityl chloride
(DMTrCl) (31 mg, 0.091 mmol) was added. The solution was stir-
red at room temperature for 48 h. The reaction was quenched with
MeOH (1 mL) and evaporated to dryness. The residue was diluted
with CH2Cl2 and the organic phase was washed with saturated
NaHCO3 solution (two times). The organic layer was dried over
anhydrous Na2SO4 and evaporated to dryness. The residue was
subjected to silica gel flash chromatography (CH2Cl2/MeOH =
50/1 (v/v)) to yield 4 (18 mg, 31% yield). 1H NMR (270 MHz)
(CDCl3) ꢁ 2.15–2.21 (m, 2H), 2.28–2.37 (m, 1H), 2.69–2.79 (m,
1H), 2.91–3.01 (m, 2H), 3.31–3.50 (m, 6H), 3.73 (s, 6H), 3.99–
4.05 (m, 1H), 4.34–4.44 (m, 2H), 4.54–4.60 (m, 1H), 6.19–6.23
(dd, 1H, J ¼ 6:8, 6.5 Hz), 6.76–8.24 (m, 27H). FAB MS (positive
ion): m=z 961.8 (M þ H).
Experimental
Synthesis of 2. Pyrenepropylamine (180 mg, 0.72 mmol)12
and 1 (390 mg, 0.60 mmol, synthesized as previously reported13,14
)
were dissolved in pyridine and the solution was stirred at room
temperature for 3 days. After evaporation of the solvent, the res-
idue was diluted with CH2Cl2 and the organic phase was washed
Published on the web February 10, 2006; DOI 10.1246/bcsj.79.312

K. Kawai et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
313
NO
NO
2
2
O
N
O
N
N
N
N
N
Et3N-3HF
DMTr-Cl
DBU
N
N
NH2
R O
1
R O
1
THF, rt
24 h, (3:72%)
pyridine, rt
48 h, (4:31%) 18 h, (61%)
pyridine, rt
pyridine, rt
3 days, (17%)
F
NH
(CH )
O
O
2 3
OR
OR
2
1
2
2
1, 2: R1, R2 = TBDMS
3: R1, R2 = H
4: R1 = DMTr, R2 = H
O
O
N
Cl
O P N(i-Pr)2
NC
N
N
NH
NH
(CH )
NH
DMTrO
DMTrO
DIEA, CH2Cl2
rt, 2 h
N
N
N
NH
O
O
(CH )
2 3
2 3
O
OH
5
NC
O P N(i-Pr)
6
2
Scheme 1. Synthetic route of the PyG-phosphoramidite 6. TBDMS denotes tert-butyldimethylsilyl.
Synthesis of 5. 4 (204 mg, 0.21 mmol) was dissolved in 15 mL
of dry pyridine, and then DBU (263 mg, 1.73 mmol) was added.
The solution was stirred at room temperature for 18 h. After evap-
oration of the solvent, the residue was diluted with CH2Cl2
(50 mL), and the organic phase was washed with a 0.5 M citric
acid solution (30 mL, three times), and then with a 5% NaHCO3
solution (30 mL, two times). The organic layer was dried over an-
hydrous Na2SO4 and evaporated to dryness. The residues were
subjected to flash chromatography (CH2Cl2/MeOH/Et3N = 95/
5/1 (v/v)) to yield 5 (105 mg, 61% yield). 1H NMR (270 MHz)
(CDCl3) ꢁ 1.94–2.22 (m, 3H), 2.32–2.48 (m, 1H), 3.14–3.52 (m,
6H), 3.70 (s, 6H), 3.82–3.93 (m, 1H), 4.27–4.39 (m, 1H), 5.91–
6.00 (m, 1H), 6.74–8.20 (m, 23H). ESI MS (positive ion): m=z
812.2 (M þ H).
mass spectra, and by complete digestion with s. v. PDE, nuclease
P1, and AP to 20-deoxyribonucleosides.
Melting Temperatures. Thermal denaturation profiles were
recorded on a JASCO V-530 spectrometer equipped with a Peltier
temperature controller (ETC-505T). The absorbance of the sample
was monitored at 260 nm from 10 to 70 ꢄC with a heating rate of
1 ꢄC minꢂ1. The Tm value was determined as the maximum in a
plot of ꢀA260=ꢀT versus temperature.
Fluorescence Spectra. The fluorescence spectra of Py-mod-
ified ODNs were measured in an Ar-saturated aqueous solution
in the presence of a 20 mM Na phosphate buffer (pH 7.0) at a total
strand concentration of 8 mM at 20 ꢄC on a Hitachi 850 spectro-
fluorometer.
Pulse Radiolysis. Pyꢁþ was formed from the reaction with
SO4ꢁꢂ, which was generated during the pulse radiolysis (28 MeV,
8 ns) of Ar-saturated D2O solution containing 10 mM K2S2O8,
100 mM t-BuOH, 20 mM Na phosphate buffer (pH 7.0), and
0.1 mM (strand conc.) ODN.6,7,15,16 A xenon flash lamp (Osram,
XBO-450), which was synchronized with the electron pulse, was
focused through the sample as a probe light for the transient
absorption measurement. The monitor light for the measurement
of time profiles of the transient absorption in the NIR region was
passed through an interference filter (CVI, transmittance of 40%,
band width of 10 nm) and its intensity was monitored with a fast
InGaAs PIN photodiode equipped with an amplifier (Thorlabs,
PDA255) and a digital oscilloscope (Tektronix, TDS 580D).
Synthesis of 6. 5 (106 mg, 0.13 mmol) was dried by coevap-
oration with pyridine (three times) and dissolved in 5.1 mL of
CH2Cl2. To this solution 38 mL (52 mg, 0.4 mmol) of diisopro-
pylamine and 84 mL (99 mg, 0.42 mmol) of 2-cyanoethyl N,N-di-
isopropylchlorophosphoramidite were added. The mixture was
stirred for 2 h at room temperature under Ar. The reaction was
quenched with MeOH (1 mL) and filtered. The reaction mixture
was diluted with CH2Cl2 (30 mL), the organic phase was washed
with 5% NaHCO3 solution (20 mL, three times), and then was
dried over anhydrous Na2SO4 and evaporated to dryness to yield
crude 6 (90 mg). Crude 6 was dried by coevaporation with CH3CN
(three times), dissolved in CH3CN/CH2Cl2 = 1/1 (2 mL) and
used in an automated DNA synthesizer without further purifica-
tion. ESI MS (positive ion): m=z 1034 (M þ Na).
Results and Discussion
DNA Synthesis. All of the reagents for DNA synthesis were
purchased from Glen Research. The DNA used in this study was
synthesized with an Expedite 8909 DNA synthesizer (Applied
Biosystems) according to the standard synthetic protocol. After
the automated synthesis, the DNA was detached and deprotected
by treating it with 28% NH3aq for 24 h at rt. Crude DNA was
purified by reverse phase HPLC and lyophilized. All of the Py-
modified DNA studied here was characterized by MALDI-TOF
In order to arrange Py at the minor groove of DNA, a nucleo-
side derivative that possesses a Py group on guanine N2 (PyG)
was synthesized as shown in Scheme 1. PyG was converted to
the phosphoramidite derivative 6 and PyG-modified ODNs
were synthesized by a DNA synthesizer according to the stan-
dard procedure. To check the duplex stability of the PyG-modi-
fied ODNs, melting temperatures were measured (Table 1).

314 Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
Pyrene Dimer Cation at the Minor Groove
Table 1. Calculated and Measured Mass Values, Melting Temperatures, and Formation Rate
Constants (kdimer) of Pyrene Dimer Radical Cation of ODNs
aÞ
cÞ
kdimer
ODNs
Mass
Tm
nbÞ
Calcd
Found
/ꢄC
42
/105 sꢂ1
0
50 GCAPyGACACG
2975.1
2948.1
2975.4
2948.5
1
4.4
py1
py2
py3
py4
pyr2
m2
3 CGTCTPyGTGC
0
50 GCAPyGAACACG
3288.3
3252.3
3289.3
3251.8
45
46
48
56
41
41
2
4.5
4.6
4.7
3.6
—
3 CGTCTTPyGTGC
0
50 GCAPyGAAACACG
3601.6
3556.5
3602.3
3556.9
3
3 CGTCTTTPyGTGC
0
50 GCAPyGAAAACACG
3914.8
3860.6
3914.9
3860.7
4
3 CGTCTTTTPyGTGC
0
50 GCAPyGCGCACG
3280.3
3262.3
3279.1
3261.5
2
3 CGTCGCPyGTGC
0
50 GCAGAACACG
nd
3252.3
nd
3251.8
—
—
3 CGTCTTPyGTGC
0
50 GCAGAACACG
nd
nd
nd
nd
—
n2
3 CGTCTTGTGC
a) UV melting measurements were carried out in a pH 7.0 Na phosphate buffer 20 mM at a
total strand concentration of 8 mM. b) Number of intervening A–T base pairs between two
PyGs. c) Determined from the time profiles of transient absorption in Fig. 2.
e−
1.0
0.8
0.6
0.4
0.2
0.0
H2O
H•
,
e−
,
•OH
< 10 ns
< 20 ns
< 2 µs
aq
py1
py2
py3
py4
pyr2
S2O8
+
e−
SO42− + SO4•−
2−
aq
Py-Py-ODN + SO4•−
Py-Py-ODN•+ + SO4
Py-Py•+-ODN
Py-Py•+-ODN
Py2•+-ODN
2−
Py-Py-ODN•+
Py-Py•+-ODN
< 10 µs
kdimer
Scheme 2. Mechanistic scheme for pulse radiolysis of dou-
bly PyG-modified ODN.
the population of the sandwich conformation between two
Pys is low in the non-oxidized state.
400
450
500
550
Wavelength (nm)
To measure the intramolecular rates of association between
Pyꢁþ and Py in DNA, Py-modified ODNs were oxidized by
SO4ꢁꢂ, which was generated during the pulse radiolysis of a
D2O solution in the presence of K2S2O8. As previously report-
ed, Pyꢁþ was formed by two processes as shown in Scheme 2:
Fig. 1. Fluorescence spectra (ꢂex ¼ 345 nm) for doubly
Py-modified ODNs in aqueous solution in the presence
of 20 mM Na phosphate buffer (pH 7.0) at a total strand
concentration of 8 mM.
Singly PyG-modified ODN (m2) showed a similar Tm value
with that of the corresponding unmodified ODN (n2). Incorpo-
ration of two PyG (py2) showed a slightly higher thermostabil-
ity compared to that of n2. Thus, PyG was incorporated into
DNA without a large disturbance of the duplex stability.
To identify the population of the DNA conformation in
which two Pys associate close to each other at the non-oxi-
dized state, the fluorescence spectra of the doubly Py-modified
ODNs were measured (Fig. 1). Since formation of the Py
excimer requires close cofacial contact between the excited
The direct oxidation of Py by SO4 and the hole transfer from
ꢁꢂ
Gꢁþ generated in DNA to Py.15,16,21 Here, if thereꢁiþs a DNA
motion that brings Pyꢁþ and Py into contact, Py2 will be
formed (Scheme 3). The pulse radiolysis of the doubly Py-
modified DNA led to the formaꢁtiþon of an absorption around
1600 nm that is assigned to Py2 (CR band22–24), as shown
in the inset of Fig. 2. In order to investigate the plausibility of
ꢁþ
the formation of Py2 at the minor groove of DNA, we stud-
ꢁþ
ied the Py2 formation process for the five ODNs shown in
Table 1. Interestingly, for all of the DNA studied here, the for-
ꢀ
ꢁþ
fluorescent probe (1Py ) and its neutral counterpart (Py) within
mation of Py2 was accomplished quickly, within 20 micro
the short lifetime of Py in the DNA conformations,17–20 Py
excimer emission can provide structural information about
the two Pys. For all of the doubly Py-modified ODNs studied
here, excimer emission was not apparent, demonstrating that
seconds after a 8-ns electron pulse during pulse radiolysis
(Fig. 2), showing that the minor groove of DNA offers a good
space for the formation of Py2ꢁþ. This was in strong contrast to
the results of the previous report, in which Pys were modified
1
ꢀ

K. Kawai et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
315
accomplished quickly, within 20 micro seconds after an elec-
tron pulse during pulse radiolysis for all the doubly Py-modi-
fied ODNs studied here. The results clearly demonstrate that
the minor groove of DNA offers a good space for the forma-
ꢁþ
tion of Py2
.
·+
·+
·+
Py2
·+
DNA dynamics
DNA dynamics
We thank the members of the Radiation Laboratory of ISIR
(SANKEN), Osaka Univ. for running the linear accelerator,
and Prof. K. Nakatani and Mr. G. Hayashi for the measure-
ment of MALDI-TOF mass spectra. This work has been partly
supported by a Grant-in-Aid for Scientific Research (Project
No. 17105005, Priority Area (417), 21st Century COE Re-
search and others) from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of the Japanese Gov-
ernment.
ꢁþ
Scheme 3. DNA dynamics and the formation of Py2
.
py1
0.04
0.03
0.02
0.01
0.00
py2
py3
py4
pyr2
m2
References
1
A. P. McCaffrey, L. Meuse, T.-T. T. Pham, D. S. Conklin,
G. J. Hannon, M. A. Kay, Nature 2002, 418, 38.
V. A. Soldatenkov, S. Chasovskikh, V. N. Potaman, I.
2
Time
Trofimova, M. E. Smulson, A. Dritschilo, J. Biol. Chem. 2002,
277, 665.
3 E. Zazopoulos, E. Lalli, D. M. Stocco, P. Sassone-Corsi,
Nature 1997, 390, 311.
4 P. Catasti, X. Chen, R. K. Moyzis, E. M. Bradbury, G.
Gupta, J. Mol. Biol. 1996, 264, 534.
0.015
0.010
0.005
1300 1400 1500 1600 1700 1800
Wavelength (nm)
5
D. B. Roth, P. B. Nakajima, J. P. Menetski, M. J. Bosma,
M. Gellert, Cell 1992, 69, 41.
K. Kawai, K. Miyamoto, S. Tojo, T. Majima, J. Am. Chem.
Soc. 2003, 125, 912.
0
2
4
6
8
10
Time (µs)
6
ꢁþ
Fig. 2. Time profiles of the transient absorption of Py2
monitored at 1550 nm during pulse radiolysis of doubly
Py-modified ODNs in Ar-saturated D2O solution in the
presence of 10 mM K2S2O8, 100 mM t-BuOH, 20 mM
Na phosphate buffer (pH 7.0), at a total strand concentra-
tion of 200 mM (100 mM in the case of m2). The inset
shows the transient absorption spectra for py2 (total strand
conc. of 100 mM) observed at 2, 5, 10, 20, and 50 ms after
the electron pulse during the pulse radiolysis.
7 K. Kawai, H. Yoshida, T. Takada, S. Tojo, T. Majima,
J. Phys. Chem. B 2004, 108, 13547.
8
P. B. Dervan, E. J. Fechter, B. S. Edelson, J. M.
Gottesfeldr, in Pseudo-Peptides in Drug Discovery, Wiley-VCH,
Weinheim, 2004, pp. 121–152.
9 M. Wang, G. L. Silva, B. A. Armitage, J. Am. Chem. Soc.
2000, 122, 9977.
10 M. Eriksson, S. K. Kim, S. Sen, A. Graslund, B. Jernstrom,
B. Norden, J. Am. Chem. Soc. 1993, 115, 1639.
11 H. Sugiyama, C. Y. Lian, M. Isomura, I. Saito, A. H. J.
Wang, Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14405.
12 J. Oxelbark, S. Claeson, Tetrahedron: Asymmetry 2002,
13, 2235.
at the 20-sugar posiꢁtiþon of the nucleoside (PyU), where the for-
mation rate of Py2 strongly depended on the number of in-
tervening base-pairs between the two PyUs and no formation
ꢁþ
of Py2 was observed for ODNs with more than three inter-
13 K. Nakatani, C. Dohno, I. Saito, J. Am. Chem. Soc. 2000,
122, 5893.
14 A. Adib, P. F. Potier, S. Doronina, I. Huc, J.-P. Behr,
Tetrahedron Lett. 1997, 38, 2989.
15 K. Kawai, T. Takada, S. Tojo, N. Ichinose, T. Majima,
J. Am. Chem. Soc. 2001, 123, 12688.
16 T. Takada, K. Kawai, S. Tojo, T. Majima, J. Phys. Chem. B
2003, 107, 14052.
17 T. L. Netzel, M. Zhao, K. Nafisi, J. Headrick, M. S.
Sigman, B. E. Eaton, J. Am. Chem. Soc. 1995, 117, 9119.
18 M. Manoharan, K. L. Tivel, M. Zhao, K. Nafisi, T. L.
Netzel, J. Phys. Chem. 1995, 99, 17461.
19 N. Amann, E. Pandurski, T. Fiebig, H.-A. Wagenknecht,
Angew. Chem., Int. Ed. 2002, 41, 2978.
vening A–T base pairs between the two PyUs in the time scale
of ꢅ1 ms. This is probably because larger conformational
changes are needed for PyU-modified ODNs to bring two Pys
in contact in the minor groove of DNA. The change of inter-
vening A–T base pairs to G–C base pairs between two Pys
(py2/pyr2) leads to a slight decrease in the formation rate
of Py2ꢁþ. Since G–C base pairs are known to have a more rigid
structure, as can be seen from the higher Tm value for pyꢁrþ2
compared to that for py2, the slower formation rate of Py2
can be interpreted in terms of the reduced flexibility of DNA.
Since the formation of Py2ꢁþ was not obvious in the case of the
singly Py-modified m2, the observed results are accounted for
by intramolecular processes as shown in Scheme 3.
In conclusion, to test the plausibility of the formation of
ꢁþ
20 R. Huber, T. Fiebig, H.-A. Wagenknecht, Chem. Commun.
2003, 1878.
21 K. Kobayashi, S. Tagawa, J. Am. Chem. Soc. 2003, 125,
10213.
Py2 at the minor groove of DNA, we synthesized a nucleo-
side derivative possessing a Py group on guanine N2 to arrange
ꢁþ
Py at the minor groove of DNA. The formation of Py2 was

316 Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
Pyrene Dimer Cation at the Minor Groove
22 A. Tsuchida, T. Ikawa, M. Yamamoto, A. Ishida, S.
Takamuku, J. Phys. Chem. 1995, 99, 14793.
23 A. Tsuchida, Y. Tsujii, M. Ohoka, M. Yamamoto, J. Phys.
Chem. 1991, 95, 5797.
24 A. Kira, S. Arai, M. Imamura, J. Chem. Phys. 1971, 54,
4890.