2′,6′-Dimethylazobenzene as an efficient and thermo-stable photo-regulator for the photoregulation of DNA hybridization

Article abstract of DOI:10.1039/b708952j

The introduction of methyl groups into two ortho positions (2′ and 6′ positions) of the same benzene ring in an azobenzene remarkably raised both its photoregulation ability and the thermal stability of the cis-form. The Royal Society of Chemistry.

Full text of DOI:10.1039/b708952j

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2
9,69-Dimethylazobenzene as an efficient and thermo-stable photo-
regulator for the photoregulation of DNA hybridization{
a
Hidenori Nishioka, Xingguo Liang, Hiromu Kashida and Hiroyuki Asanuma*
a
a
ab
Received (in Cambridge, UK) 13th June 2007, Accepted 26th July 2007
First published as an Advance Article on the web 13th August 2007
DOI: 10.1039/b708952j
The introduction of methyl groups into two ortho positions (29
and 69 positions) of the same benzene ring in an azobenzene
remarkably raised both its photoregulation ability and the
thermal stability of the cis-form.
cis-forms. Obviously, the photoregulation of DNA hybridization
with fewer azobenzenes is favorable for bio-applications. For this
purpose, a new molecule that further stabilizes the duplex in the
trans-form and destabilizes it in the cis-form is highly desirable. In
the present paper, we found that the introduction of methyl groups
The photoregulation of biologically active compounds can be used
as a robust tool for the investigation of particular biological
phenomena and mechanisms in living cells, which are otherwise
to the ortho positions of azobenzene enhanced the value of DT . In
m
particular, 29,69-dimethylazobenzene gave a three-times larger
change in DTm than the unmodified form used previously.
Unexpectedly, we also found that this dimethylation significantly
suppressed the thermal isomerization of cis-azobenzene to the
trans-form.
1
very difficult to achieve. The most commonly used approach is to
covalently attach a light-responsive molecule to the target
biological compound so that the light signal becomes an efficient
trigger for the function under investigation. Recently, a large
number of efficient light-switchable systems involving photore-
sponsive nucleic acids, proteins, cellular signalling molecules, or
Azobenzene was modified with methyl or ethyl groups with the
aims of further stabilizing the duplex in the trans-form by stacking
interactions and of destabilization of the cis-form due to steric
hindrance. The modified azobenzene on D-threoninol was inserted
2,3
lipids have been constructed.
1
4
We have previously introduced azobenzenes into DNA on
D-threoninol as a linking molecule, and have efficiently photo-
regulated primer extension, transcription, and the RNase H
at the centre of a 12 nt natural oligodeoxyribonucleotide. The
structures of the synthesized azobenzenes and the sequences of the
azobenzene-modified oligodeoxyribonucleotides (Da) used in this
study are shown in Fig. 1. Photo-isomerization of trans-
azobenzene to the cis-form was performed by irradiating with
UV light (300 nm , l , 400 nm) before the Tm measurement.{§
By this procedure, 60–80% of the total azobenzene was isomerized
4–7
reaction. These photoregulatory bioreactions are mostly based
on the reversible formation and dissociation of the DNA duplex
by irradiating with either UV or visible light: planar trans-
azobenzene (visible light irradiation) is intercalated between the
adjacent base-pairs and thus stabilizes the duplex, whereas non-
planar cis-azobenzene (UV light irradiation) destabilizes the duplex
1
5
to the cis-form. When the para-position of the azobenzene was
m
substituted with a methyl group (49-Me-Azo), T of the trans-form
8
–12
by steric hindrance.
Since photoregulatory efficiencies
) induced
became 46.8 uC, which was 2 uC lower than that of the non-
substituted Azo (see Table 1). In contrast, Tm of the cis-form was
depended on the change in melting temperature (DT
m
by trans–cis isomerization, the enhancement of DTm has been
crucially important in achieving still more effective photoregula-
tion. One of the methods for raising DTm is to introduce multiple
increased by para-substitution. As a result, DTm became smaller.
azobenzene moieties. We found that DT
with the number of azobenzenes that were introduced, and even a
0-bp-long DNA duplex could be efficiently photoregulated by
m
increased uniformly
2
6,11,13
tethering 9 azobenzene moieties.
However, this causes great
changes in the structure of the DNA duplex far from the B-form
due to the enhanced asymmetry of the strands, and prevents
its interaction with protein or enzymes both in the trans- and
a
Department of Molecular Design and Engineering, Graduate School of
Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan.
E-mail: asanuma@mol.nagoya-u.ac.jp; Fax: +81-52-789-2528;
Tel: +81-52-789-2488
b
Core Research for Evolution Science and Technology (CREST), Japan
Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012,
Japan
{
Electronic supplementary information (ESI) available: experimental
procedures for the synthesis of alkylated azobenzenes and modified
oligodeoxyribonucleotides involving modified azobenzene, Energy-mini-
mized structures of Da/Dc duplex involving 29,69-Me-Azo, change of UV-
Vis spectra of 29,69-Me-Azo by the thermal cis A trans isomerization. See
DOI: 10.1039/b708952j
Fig. 1 Structures of modified azobenzenes and the DNA sequences used
in this study.
4
354 | Chem. Commun., 2007, 4354–4356
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Table 1 Effect of the insertion position of alkyl group on azobenzene
of Da/Dc duplex in the trans- and cis-forms
incorporated methyl group with a phosphodiester linkage and
on the T
m
deoxyribose at the counter strand Dc. It should be noted that the
1
6
a
˚
size of the Watson–Crick base-pair in this case is around 11 A,
m
T /uC
b
Azobenzene
DT
m
˚
17
whereas for unmodified azobenzene it is 11–12 A. 49-Me-Azo, in
which the para-position of the azobenzene is methylated, was too
long to intercalate effectively between the base-pairs, and thus the
duplex was destabilized in the trans-form. In the case of ortho- (or
meta-) substitution, such destabilization did not occur because the
methyl group was located far from the counter strand in the
duplex. Rather, such alkylation stabilized the duplex by hydro-
phobic interaction. However in the cis-form, methyl groups on the
trans
cis
Azo
48.9
46.8
49.7
50.7
49.6
48.8
49.0
50.9
43.2
45.4
44.8
40.1
39.8
39.3
44.4
36.3
5.7
1.4
4.9
10.6
9.8
9.5
4.6
4
3
2
2
2
3
2
a
9-Me-Azo
9-Me-Azo
9-Me-Azo
9-Et-Azo
-Me-Azo
9,59-Me-Azo
9,69-Me-Azo
14.6
29-position (or 2-position) protruded towards the base-pair and
Solution conditions: [Da] 5 [Dc] 5 5 mM, [NaCl] 5 0.1 M, pH 7.0
b
inhibited base-pairing due to steric hindrance (see Supplemental
Fig. 1{). Such inhibition of base-pairing would be enhanced by the
double-methylation of the 29-(ortho-)positions. As a result, a
m
(10 mM phosphate buffer). Change of T induced by the cis–trans
isomerization.
much larger DT
9,69-Me-Azo.
Not only visible-light irradiation, but also heat isomerizes cis-
m
was induced by cis–trans isomerization of
The meta-substitution of an azobenzene group (39-Me-Azo) did
not significantly affect the stability of the duplex: DTm was 4.9 uC,
which was almost the same as that of Azo. However, in the case of
2
azobenzene to the trans-form. One of the problems caused by the
2
9-methylazobenzene (29-Me-Azo), in which a methyl group is
attached at an ortho position of the benzene ring far from the
DNA backbone, DT increased significantly compared with the
modification of azobenzene is degradation of the thermal stability
18
of cis-azobenzene. In particular, donor–acceptor (‘‘push–pull’’)
m
modification at the para- (or ortho-position) of azobenzene usually
lowers the thermal stability of cis-azobenzene. In our cases,
carboxyl groups in the vicinity of the threoninol pull and alkyl
groups push the electron, so thermal isomerization should be
accelerated. In fact, the mono-methylation of azobenzene (49-, 39-,
and 29-Me-Azo) accelerated the thermal isomerization: in a single-
stranded Da, the half-life of cis-49-Me-Azo was one-third of that of
unmodified cis-Azo at 60 uC (Fig. 3). Similarly, ortho-substitution
value for Azo: Tm of the trans-form (50.7 uC) was 1.8 uC higher
than that of non-substituted trans-azobenzene (trans-Azo), whereas
the value of the cis-form (40.1 uC) was 3.1 uC lower. As a result,
DT
higher than that of Azo. Both methylation and ethylation (29-Et-
Azo) raised the value of DT . Similarly, 2-Me-Azo methylated at
m
for 29-Me-Azo increased to 10.6 uC, which was about 5 uC
m
an ortho-position on the benzoyl side also showed an increase in
DT . Interestingly, the di-substitution of azobenzene at the ortho-
m
m
(cis-29-Me-Azo), which enhanced DT , also accelerated the
positions (29,69-Me-Azo) exhibited even larger DT : the Tm of the
m
thermal isomerization, although the acceleration effect in this case
trans-form was 50.9 uC whereas that of the cis-form was as low as
was smaller than that of para-substitution. Unexpectedly,
3
6.3 uC. Consequently, this DT (14.6 uC) was the largest among
m
29,69-Me-Azo, which contains two methyl groups at both ortho
all of the azobenzenes that were synthesized in this study. The
m
potions and which displays the largest DT , showed very slow
melting curves of unmodified (Azo) and 29,69-Me-Azo are shown in
thermal isomerization. Its half-life was as long as 25 h (rate
21
1
5
Fig. 2. Large improvements were only observed at the ortho-
position: azobenzene di-substituted at the meta-positions
constant was 0.028 h ) at 60 uC, which is as much as 8-times
slower than unmodified cis-Azo. The half-life of cis-29,69-Me-Azo
19
m
(39,59-Me-Azo) did not show a change in DT . Thus, the
at 37 uC was estimated by extrapolation of the Arrhenius plots to
be 200–400 h, indicating that its thermal cis-to-trans isomerization
could be practically suppressed under physiological conditions.
Thus, methylation of the two ortho-positions exhibited both
photoregulation ability was improved by modifying the ortho-
positions of azobenzene.
According to molecular modelling, methylation of the para-
position inhibits hybridization due to the steric hindrance of the
Fig. 3 Half-lives of cis-azobenzene to trans-form in the single-stranded
Da at 60 uC in the presence of 0.1 M NaCl at pH 7.0 (10 mM phosphate
buffer). [Da] 5 20 mM. All the thermal isomerizations are first order (See
Supplemental Fig. 2{ for the change of UV-Vis spectra of cis-29,69-Me-
Azo).
Fig. 2 Melting curves of a Da/Dc duplex involving non-substituted (Azo:
gray line) and 29,69-dimethylazobenzene (29,69-Me-Azo) either in the trans-
(solid line) or the cis-forms (black line).
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with a temperature controller (JASCO model V-530 or V-550), and spectra
were monitored at 60 uC at a predetermined interval. The half-lives were
obtained from the changes in the absorbance at the absorption maximum
of trans-azobenzene (around 340 nm). It should be noted that all of the
thermal cis A trans isomerizations were first-order (See Supplemental
Fig. 2{).
effective photo-regulation of hybridization and thermal durability
of the cis-form.
According to Asano, the thermal isomerization of the cis to the
2
0
trans-form has two routes. The first of these is inversion, which
proceeds through a transition state in which one of the nitrogen
atoms is sp hybridized. The other is a rotation mechanism which
involves the rupturing of a nitrogen–nitrogen p-bond and rotation
around the remaining s-bond. Presumably, the introduction of
methyl groups on the two ortho-positions would restrict either the
rotation around the nitrogen–nitrogen bond or the inversion
process due to the close proximity of the benzene ring to the two
1
(a) G. Mayer and A. Heckel, Angew. Chem., Int. Ed., 2006, 45,
900–4921; (b) I. Willner and S. Rubin, Angew. Chem., Int. Ed. Engl.,
996, 35, 367–385.
4
1
2 (a) M. Simons, E. M. Kramer, C. Thiele, W. Stoffel and J. Trotter,
J. Cell Biol., 2000, 151, 143–153; (b) E. A. Mintzer, B. L. Warrts,
J. Wilschut and R. Bittman, FEBS Lett., 2002, 39, 181–184.
3
D. M. Rothman, M. D. Shults and B. Imperiali, Trends Cell Biol., 2005,
5, 502–510.
4 A. Yamazawa, X. G. Liang, T. Yoshida, H. Asanuma and
21
methyl groups, and thus suppress the cis A trans isomerization.
1
In conclusion, the introduction of two methyl groups into the
ortho positions of the same benzene ring greatly raised its
photoregulation ability and concurrently suppressed the thermal
isomerization of the cis-form. A robust photoregulator, 29,69-Me-
Azo, was developed, and the clear-cut photoregulation of various
DNA functions now becomes promising.
M. Komiyama, Angew. Chem., Int. Ed., 2000, 39, 2356–2357.
5
H. Asanuma, D. Tamaru, A. Yamazawa, M. Liu and M. Komiyama,
ChemBioChem, 2002, 3, 786–789.
D. Matsunaga, H. Asanuma and M. Komiyama, J. Am. Chem. Soc.,
6
2
004, 126, 11452–11453.
7 M. Liu, H. Asanuma and M. Komiyama, J. Am. Chem. Soc., 2006, 128,
009–1015.
1
This work was supported by Core Research for Evolution
Science and Technology (CREST), Japan Science and Technology
Agency (JST). Partial support was provided by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan, and The Mitsubishi
Foundation (for H. A.) is also acknowledged.
8
H. Asanuma, T. Ito, T. Yoshida, X. G. Liang and M. Komiyama,
Angew. Chem., Int. Ed., 1999, 38, 2393–2395.
9
H. Asanuma, T. Yoshida, T. Ito and M. Komiyama, Tetrahedron Lett.,
1999, 40, 7995–7998.
10 H. Asanuma, X. G. Liang, T. Yoshida and M. Komiyama,
ChemBioChem, 2001, 2, 39–44.
1
1 H. Asanuma, T. Takarada, T. Yoshida, X. G. Liang and
M. Komiyama, Angew. Chem., Int. Ed., 2001, 40, 2671–2673.
2 X. G. Liang, H. Asanuma, H. Kashida, A. Takasu, T. Sakamoto,
1
Notes and references
G. Kawai and M. Komiyama, J. Am. Chem. Soc., 2003, 125,
16408–16415.
13 H. Asanuma, X. G. Liang, H. Nishioka, D. Matsunaga, M. Liu and
M. Komiyama, Nat. Protocols, 2007, 2, 203–212.
14 See Supplemental Scheme 1{ for the syntheses of modified azobenzenes
and oligodeoxyribonucleotides.
{
zenes
See Supplemental Information{ for syntheses of alkylated azoben-
22,23
and their phosphoramidite monomers. All of the modified
oligonucleotides were synthesized on an ABI 3400 DNA/RNA Synthesizer
by using the corresponding phosphoramidite monomer and other
conventional precursors. The purification was achieved by using Poly-
Pak cartridges and then by a reversed-phase HPLC (Merck LiChrospher
15 In the case of 29,69-Me-Azo, about 65% of the total azobenzene was
1
00 RP-18(e) column, with a linear gradient of a mixture of acetonitrile and
isomerized to the cis-form under the conditions employed (See
Supplemental Fig. 2{ for the UV-Vis spectra). The melting curve of a
Da/Dc duplex involving 29,69-Me-Azo showed two sigmoids because not
only the cis-form, but also 35% of the trans-form was involved in the
solution. The upper sigmoid (minor one) in Fig. 2 corresponds to trans-
21
2
H O containing 50 mM ammonium formate, 0.5 mL min , detection at
13
260 nm). The purified DNAs were then characterized by MALDI-
TOFMS. MALDI-TOFMS for Da with Azo: obsd. 4021 (calcd. for
protonated form: 4020), 49-Me-Azo: obsd. 4035 (calcd. 4034), 39-Me-Azo:
obsd. 4035 (calcd.: 4034), 29-Me-Azo: obsd. 4035 (calcd. 4034), 29-Et-Azo:
obsd. 4048 (calcd. 4048), 2-Me-Azo: obsd. 4032 (calcd. 4034), 39,59-Me-Azo:
obsd. 4049 (calcd. 4048), 29,69-Me-Azo: obsd. 4049 (calcd. 4048).
2
9,69-Me-Azo, whereas the lower sigmoid (major one) corresponds to the
cis-form. The T of the cis-form was determined from this lower
sigmoid.
6 S. Arnott, S. D. Dover and A. J. Wonacott, Acta Crystallogr., Sect. B:
Struct. Crystallogr. Cryst. Chem., 1969, 25, 2192–2206.
m
§
The T
of the melting curves, which were obtained by measuring the absorbance at
60 nm as a function of temperature. A JASCO model V-530 or V-550
m
values were determined from the maxima in the first derivatives
1
2
1
7 The distance between the 49-positioned hydrogen and the 4-positioned
carbonyl carbon was estimated from the energy-minimized structure
calculated by use of Spartan 902 (Wavefunction, Inc.).
spectrophotometer equipped with a programmable temperature-controller
was used. Both the heating and cooling curves were measured, and the
values of T
m
that were obtained coincided with each other to within 2.0 uC.
The T values presented here are an average of 2–4 independent
1
8 (a) N. Nishimura, S. Kosako and Y. Sueishi, Bull. Chem. Soc. Jpn.,
1984, 57, 1617–1625; (b) D.-M. Shin and D. G. Whitten, J. Am. Chem.
Soc., 1988, 110, 5206–5208.
m
21
experiments. The temperature ramp was 1.0 uC min . The conditions of
the sample solutions were as follows: [NaCl] 5 0.1 M, pH 7.0 (10 mM
phosphate buffer), [Da] 5 [Dc] 5 5 mM.
1
9 As far as we have been able to search, the effect of ortho-substitution on
the thermal cis A trans isomerization has not been systematically
investigated.
Photo-isomerization of azobenzene: The light source for the photo-
irradiation was a 150 W Xenon lamp. For the trans A cis isomerization, a
UV-D36C filter (Asahi Tech. Co.) was used, and UV light (l 5 300 y
2
0 (a) T. Asano and T. Okada, J. Org. Chem., 1986, 51, 4454–4458; (b)
T. Baba, H. Ono, E. Itoh, S. Itoh, K. Noda, T. Usui, K. Ishihara,
M. Inamo, H. D. Takagi and T. Asano, Chem.–Eur. J., 2006, 12,
2
2
4
00 nm: 5.3 mW cm ) was irradiated to Da/Dc solution at 60 uC for 5 min.
The cis A trans isomerization was carried out by irradiating with visible
light (l . 400 nm) through an L-42 filter (Asahi Tech. Co.) at 60 uC for
5328–5333.
5
min. In both cases, a water filter was used to cut off the infrared light.
Half-life of thermal isomerization of cis-azobenzene to the trans-form: UV
21 The mechanism to explain why thermal isomerization is suppressed is
now under investigation.
22 H. Kagechika, T. Himi, K. Namikawa, E. Kawachi, Y. Hashimoto and
K. Shudo, J. Med. Chem., 1989, 32, 1098–1108.
23 W. H. Nuttig, R. A. Jewell and H. Rapoport, J. Org. Chem., 1970, 35,
505–508.
22
light (l 5 300 y 400 nm: 5.3 mW cm ) was irradiated to a solution of Da
involving azobenzene ([NaCl] 5 0.1 M, pH 7.0 (10 mM phosphate buffer),
[
Da] 5 20 mM) at 60 uC for 5 min to isomerize trans-azobenzene to the cis-
form. Then the solution was inserted into a UV-Vis spectrometer equipped
4
356 | Chem. Commun., 2007, 4354–4356
This journal is ß The Royal Society of Chemistry 2007