Effect of the ortho modification of azobenzene on the photoregulatory efficiency of DNA hybridization and the thermal stability of its eis form

Article abstract of DOI:10.1002/chem.200902789

We synthesized various azobenzenes methylated at their ortho positions with respect to the azo bond for more effective photoregulation of DNA hybridization. Photoregulatory efficiency, evaluated from the change of T _m (ΔT_m) induced by trans-cis isomerization, was significantly improved for all ortho-modified azobenzenes compared with non-modified azobenzene due to the more stabilized trans form and the more destabilized cis form. Among the synthesized azobenzenes, 4-carboxy-2',6'- dimethylazobenzene (2',6'-Me-Azo), in which two ortho positions of the distal benzene ring with respect to carboxyl group were methylated, exhibited the largest ΔTm, whereas the newly synthesized 2,6-Me-Azo (4-carboxy-2,6- dimethylazobenzene), which possesses two methyl groups on the two ortho positions of the other benzene ring, showed moderate improvement of ΔTm. Both NMR spectroscopic analysis and computer modeling revealed that the two methyl groups on 2',6'-Me-Azo were located near the imino protons of adjacent base pairs; these stabilized the DNA duplex by stacking interactions in the trans form and destabilized the DNA duplex by steric hindrance in the cis form. In addition, the thermal stability of cis-2',6'-Me-Azo was also greatly improved, but not that of cis2,6-Me-Azo. Solvent effects on the half-life of the cis form demonstrated that cis-to-trans isomerization of all the modified azobenzenes proceeded through an inversion route. Improved thermal stability of 2',6'-Me-Azo but not 2,6-Me-Azo in the eis form was attributed to the retardation of the inversion process due to steric hindrance between lone pair electrons of the π orbital of the nitrogen atom and the methyl group on the distal benzene ring.

Full text of DOI:10.1002/chem.200902789

DOI: 10.1002/chem.200902789
Effect of the ortho Modification of Azobenzene on the Photoregulatory
Efficiency of DNA Hybridization and the Thermal Stability of its cis Form
Hidenori Nishioka,[a] Xingguo Liang,*[a] and Hiroyuki Asanuma*[a, b]
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Chem. Eur. J. 2010, 16, 2054 – 2062

FULL PAPER
Abstract: We synthesized various azo-
benzenes methylated at their ortho po-
sitions with respect to the azo bond for
more effective photoregulation of
DNA hybridization. Photoregulatory
efficiency, evaluated from the change
of Tm (DTm) induced by trans–cis iso-
merization, was significantly improved
for all ortho-modified azobenzenes
compared with non-modified azoben-
zene due to the more stabilized trans
form and the more destabilized cis
form. Among the synthesized azoben-
zenes, 4-carboxy-2’,6’-dimethylazoben-
zene (2’,6’-Me-Azo), in which two
ortho positions of the distal benzene
ring with respect to carboxyl group
were methylated, exhibited the largest
DTm, whereas the newly synthesized
2,6-Me-Azo (4-carboxy-2,6-dimethyl-
AHCTUNGTRENGNaUN zobenzene), which possesses two
DNA duplex by steric hindrance in the
cis form. In addition, the thermal sta-
bility of cis-2’,6’-Me-Azo was also
greatly improved, but not that of cis-
2,6-Me-Azo. Solvent effects on the
half-life of the cis form demonstrated
that cis-to-trans isomerization of all the
methyl groups on the two ortho posi-
tions of the other benzene ring, showed
moderate improvement of DTm. Both
NMR spectroscopic analysis and com-
puter modeling revealed that the two
methyl groups on 2’,6’-Me-Azo were
located near the imino protons of adja-
cent base pairs; these stabilized the
DNA duplex by stacking interactions
in the trans form and destabilized the
modified
azobenzenes
proceeded
through an inversion route. Improved
thermal stability of 2’,6’-Me-Azo but
not 2,6-Me-Azo in the cis form was at-
tributed to the retardation of the inver-
sion process due to steric hindrance be-
tween lone pair electrons of the p
orbitACTHNUGRTNEUNGal of the nitrogen atom and the
methyl group on the distal benzene
ring.
Keywords: DNA · intercalations ·
isomerization · modified azoben-
zenes · photoswitches
Introduction
cause deterioration or unfavorable chemical reactions; and
3) facile synthetic procedures allow a variety of modifica-
tions of azobenzene that can diversify the photoswitching
properties.[6] In fact, we found that even simple alkylation
greatly improves the photoregulatory efficiency of DNA hy-
bridization. Modification at the ortho position with respect
to the N=N bond stabilizes the duplex in the trans form,
whereas the cis form is destabilized compared with the non-
modified azobenzene.[7] As a result, the DTm (the change in
melting temperature, Tm) induced by trans–cis isomerization
is significantly enhanced. This improvement was successful
only at the ortho position, and modification at the para posi-
tion destabilized the duplex even in the trans form.[8] Signifi-
cantly, methylation at the two ortho positions of the distal
benzene ring of azobenzene (4-carboxy-2’,6’-dimethylazo-
benzene, 2’,6’-Me-Azo, see Figure 1 for the structure) in-
creased DTm by three-fold compared with the nonmodified
Recently, DNA has come to be regarded not only as a carri-
er of genetic information but also as an excellent nanomate-
rial that can perform mechanical motions and construct
static nanostructures in a predetermined fashion due to its
simple hybridization rule.[1] However, because achievable
performance is still limited by only four natural nucleotides,
a variety of functional DNAs involving non-natural mole-
cules have been proposed.[2] We have developed photores-
ponsive DNAs involving azobenzenes on acyclic threoninols
as scaffolds, and the key feature of DNA, the formation and
dissociation of DNA duplexes, can be efficiently photoregu-
lated to create a photon-driven nanomachine as well as pho-
toswitching of enzymatic reactions.[3]
Among reversible photoswitching molecules, we have
been particularly interested in azobenzenes as a photoswitch
of DNA for the following reasons: 1) Photoisomerization in-
duces planar/nonplanar structural changes that crucially
affect duplex stability;[4] 2) unlike other photoresponsive
molecules, such as spiropyran,[5] azobenzenes are sufficiently
stable in water that repeated photoirradiation does not
[a] H. Nishioka, Prof. Dr. X. G. Liang, Prof. Dr. H. Asanuma
Graduate School of Engineering, Nagoya University
Furo-cho, Chikusa-ku
Nagoya 464-8603 (Japan)
Fax : (+81)52-789-2528
E-mail: asanuma@mol.nagoya-u.ac.jp
liang@mol.nagoya-u.ac.jp
[b] Prof. Dr. H. Asanuma
Core Research for Evolution Science and Technology (CREST)
ACHTUNGTRENNUNG(Japan) Science and Technology Agency (JST),
Kawaguchi, Saitama 332-0012 (Japan)
Figure 1. Structures of ortho-methylated azobenzenes and the DNA se-
quences used in this study. N1 and N2 represent natural nucleotides (A,
G, C, T), and N1 and N2 represent their complementary ones. X indicates
azobenzene and its derivatives.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902789.
Chem. Eur. J. 2010, 16, 2054 – 2062
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H. Nishioka, X. G. Liang and H. Asanuma
form. In addition, we unexpectedly discovered that the cis
form of 2’,6’-Me-Azo has excellent thermal stability, with a
half-life about 10-fold longer than that of nonsubstituted
azobenzene (Azo).[7]
Although numerous ortho-modified azobenzenes have
been synthesized to date,[9] there have been few reports on
improved thermal stability of the cis form.[10]
We investigated in detail the effect of modification of azo-
benzene at the ortho position to improve the photoregulato-
ry efficiency (DTm) of DNA hybridization, as well as the
thermal stability of the cis form. Because azobenzenes are
widely used for photoregulation of various biomolecules and
materials,[11] our analysis potentially improves these applica-
tions and clarifies the mechanism of photo- and thermal
Unlike the trans form, all the ortho-methylated azoben-
zenes had somewhat lower Tm values in the cis form and
showed almost the same Tm (around 408C) except for 2’,6’-
Me-Azo. Interestingly, introduction of methyl groups at two
ortho positions of the distal benzene ring greatly lowered
the Tm to as low as 36.38C, which is 78C lower than that of
cis-Azo. However, such a large drop in Tm was not observed
for 2,6-Me-Azo methylated at the two ortho positions of the
proximal benzene ring. Thus, of the ortho substituents we
examined, 2’,6’-Me-Azo showed the largest DTm, which was
three times as large as that of nonmodified Azo due to
more stabilization in the trans form and more destabilization
in the cis form.
isom
N
Dependency of the Tm of the duplex involving 2’,6’-Me-Azo
on DNA sequence: Because the stabilizing effect of the in-
tercalator generally depends on the neighboring base
pairs,[15] we examined the sequence dependence of the Tm of
2’,6’-Me-Azo, which exhibited maximum photoregulatory ef-
ficiency, in CXG/GC. Irrespective of the sequence, all du-
plexes were stabilized in the trans form and destabilized in
the cis form compared with native DNA duplexes without
azobenzene (Table 2). In all combinations of neighboring
base pairs, the Tm values of the trans forms were about 58C
higher than that of native DNA, whereas those of the cis
Results
Effect of the position of methylation on photoregulatory ef-
ficiency: Structures of ortho-substituted azobenzene deriva-
tives are shown in Figure 1, together with the sequences that
were evaluated for Tm. We synthesized all possible azoben-
zenes mono- and dimethylated at the ortho positions with
respect to the N=N bond.[13] As listed in Table 1, all modifi-
cations of azobenzene in the CXG/GC duplex improved the
Table 2. Effect of neighboring bases on the Tm values of N1XN2/N1N2 du-
plexes involving trans and cis forms of 2’,6’-Me-Azo.[a]
Table 1. Effect of different alkyl groups in azobenzene on the Tm of
CXG/GC in the trans and cis forms.
Duplex
Tm [8C]
[c]
Native duplex[b]
trans
cis
DTm
Tm [8C][a]
cis
[b]
44.1 (+7.2)[d]
41.3 (+3.0)
47.1 (+5.9)
42.5 (+5.7)
47.2 (+4.6)
47.8 (+4.2)
50.9 (+3.2)
46.4 (+5.1)
48.0 (+6.7)
48.0 (+1.9)
50.5 (+4.0)
47.7 (+5.7)
42.8 (+5.9)
42.2 (+1.9)
48.0 (+4.9)
43.1 (+4.8)
24.6 (ꢀ12.3)[d]
25.7 (ꢀ12.6)
30.3 (ꢀ10.9)
21.1 (ꢀ15.7)
28.8 (ꢀ13.8)
28.6 (ꢀ15.0)
36.3 (ꢀ11.4)
26.3 (ꢀ15.0)
29.2 (ꢀ12.1)
32.8 (ꢀ13.3)
36.3 (ꢀ10.2)
28.7 (ꢀ13.3)
24.8 (ꢀ12.1)
28.0 (ꢀ12.3)
31.9 (ꢀ11.2)
21.9 (ꢀ16.4)
19.5
15.6
16.8
21.4
18.4
19.2
14.6
20.1
18.8
15.2
14.2
19.0
18.0
14.2
16.1
21.2
trans
DTm
AXA/TT
AXC/TG
AXG/TC
AXT/TA
CXA/GT
CXC/GG
CXG/GC
CXT/GA
GXA/CT
GXC/CG
GXG/CC
GXT/CA
TXA/AT
TXC/AG
TXG/AC
TXT/AA
36.9
38.3
41.2
36.8
42.6
43.6
47.7
41.3
41.3
46.1
46.5
42.0
36.9
40.3
43.1
38.3
Azo
48.9
48.8
50.7
51.4
48.6
50.9
43.2
39.3
40.1
39.9
40.0
36.3
5.7
9.5
10.6
11.5
8.6
2-Me-Azo
2’-Me-Azo
2,2’-Me-Azo
2,6-Me-Azo
2’,6’-Me-Azo
14.6
[a] Solution conditions: [DNA]=5 mm, [NaCl]=100 mm, pH 7.0 (10 mm
phosphate buffer). [b] Change in Tm induced by cis–trans isomerization.
DTm compared with the unmodified Azo, mainly due to the
decrease in Tm in the cis form.[14] However, the methylated
position slightly but distinctly affected the Tm in the trans
form. For example, 2’-Me-Azo, in which the distal benzene
ring was monomethylated, showed a 28C higher Tm than 2-
Me-Azo, which was methylated at the proximal ring, where-
as the difference in Tm in the cis forms was within 18C. As a
result, the DTm of 2’-Me-Azo was slightly higher than that of
2-Me-Azo. Similarly, for dimethylated azobenzene the Tm
values of 2,2’-Me-Azo and 2’,6’-Me-Azo were 2–38C higher
than that of 2,6-Me-Azo, which had no methyl group on the
distal benzene ring. For either mono- or disubstitution,
modification at the proximal benzene ring only exhibited
almost the same Tm as Azo (compare 2-Me-Azo and 2,6-
Me-Azo with Azo in Table 1). Thus, modification at the
ortho position of the distal benzene ring is more effective
for stabilizing the duplex in the trans form.
[a] Solution conditions: [DNA]=5 mm, [NaCl]=100 mm, pH 7.0 (10 mm
phosphate buffer). [b] Melting temperature of the corresponding native
duplex without X residue. [c] Change in Tm induced by trans-to-cis isom-
AHCTUNGTERGeNNUN rization of 2’,6’-Me-Azo. [d] The data in parentheses show the difference
in Tm between native duplexes and modified duplexes containing trans-
or cis-2’,6’-Me-Azo.
forms were 10–158C lower. As a result, DTm increased by as
much as 14–208C, clearly demonstrating that 2’,6’-Me-Azo is
a superior photoregulator of DNA hybridization. Note that
DTm of CXG/GC with 2’,6’-Me-Azo in Table 2 was as large
as 14.68C, but the DTm of nonmodified Azo was only
5.78C.[16]
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Photoregulated DNA Hybridization
FULL PAPER
NMR spectroscopic analysis and computer modeling of
duplex involving 2’,6’-Me-Azo: To better understand the su-
periority of 2’,6’-Me-Azo as a photoregulator, both NMR
spectroscopic analysis and computer modeling were per-
formed on the 5’-GCGAXGTCC-3’/3’-CGCTCAGG-5’
duplex, in which X represents 2’,6’-Me-Azo (see Figure 2a).
Figure 2. a) Sequence of modified DNAs used for NMR measurements
and computer modeling and b) the one-dimensional NMR spectrum at
the imino region in H2O/D2O (9:1) at 278 K (mixing time=150 ms),
pH 7.0 (20 mm phosphate buffer), in the presence of 200 mm NaCl. The
concentration of each DNA was 560 mm. Assignments of the imino pro-
tons and the residue numbers are denoted at the top of the peaks.
Figure 3. 2D NOESY spectra of the duplex containing trans-2’,6’-Me-Azo
between a) aromatic protons and the imino proton and b) methyl groups
and imino proton regions. Solution conditions: [DNA]=560 mm, [NaCl]=
200 mm, pH 7.0 (20 mm phosphate buffer).
In trans-2’,6’-Me-Azo: First, the position of trans-2’,6’-Me-
Azo in the duplex was determined by NMR spectroscopy.
To monitor the imino protons that are exchangeable with
water molecules, NMR spectroscopic measurements were
carried out in water (H2O/D2O 9:1) with a 3-9-19 WATER-
GATE for H2O suppression. A one-dimensional spectrum at
the region of the imino protons (d=11–14 ppm) is depicted
in Figure 2b; seven signals corresponding to eight base pairs
were exhibited. Except for the terminal G1 (G1-C16 pair) and
G9 (C8-G9 pair) that broadened due to rapid exchange with
water, all the internal imino protons could be assigned from
NOESY, DQF-COSY (double quantum-filtered correlation
spectroscopy), and TOCSY (total-correlation spectrosco-
py).[17] The signal assigned to G5 and T13 adjacent to azoben-
zene (X residue) showed a distinct upfield shift compared
with those of G15, G3, or T6 that stayed distant from azoben-
zene. This upfield shift was attributed to the ring current
effect of trans-azobenzene, and demonstrates that these pro-
tons were located at the axial position.[18] Figure 3a depicts
the NOESY between the imino proton (d=11–14 ppm) and
the aromatic proton (d=5.5–8.0 ppm) regions. Weak NOEs
were observed between H9 (H11) of the distal ring of 2’,6’-
Me-Azo, and T13 and G5 imino protons. In addition, the G5
imino proton had relatively strong NOE with the H2 (H6)
aromatic proton on the proximal ring. Methyl protons on
2’,6’-Me-Azo also showed distinct NOE with imino protons
as shown in Figure 3b; strong NOEs were observed for both
T13 and G5 with almost equal intensity. These NOEs clearly
demonstrated that 2’,6’-Me-Azo is intercalated between A4-
T13 and G5-C12. Computer modeling of this duplex involving
trans-2’,6’-Me-Azo also supported an intercalated structure,
as depicted in Figure 4a,b. Two methyl groups of the distal
ring were located between the hydrogen-bonded imino pro-
tons of G5 and T13 as depicted in Figure 4b, which was con-
sistent with the NMR spectroscopic results.
In cis-2’,6’-Me-Azo: We also measured the NMR spectrum
of this duplex in the cis form. However, broadened signals
assignable to the cis-duplex and contaminated sharp peaks
of the trans-duplex has made the assignment of the NMR
signals, even imino proton signals, quite difficult.[19] Accord-
ingly, structural analysis for the cis form was performed only
by computer modeling. Because the preliminary analysis in-
dicated that even cis-azobenzene is partially intercalated,[20]
we started the calculation with intercalated cis-2’,6’-Me-Azo
as an initial structure. Figure 4c,d shows the converged
structure of the duplex with non-planar cis-2’,6’-Me-Azo,
which was located between adjacent base pairs. The proxi-
mal benzene ring covalently bound to the main-chain of
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H. Nishioka, X. G. Liang and H. Asanuma
Table 3. Half lives (t1/2) of thermal cis-to-trans isomerization of azoben-
zenes in various solvents at 608C.[a]
t1/2 [h]
DMF
Toluene
Buffer[b]
Azo
0.28
0.34
0.47
0.29
0.14
3.9
1.3
0.79
1.1
0.95
0.73
9.2
3.4 (3.3)[c]
3.7 (2.6)
3.5 (3.0)
3.3 (2.1)
10 (2.0)
36 (25)
2-Me-Azo
2’-Me-Azo
2,2’-Me-Azo
2,6-Me-Azo
2’,6’-Me-Azo
[a] Azobenzene in the carboxylic acid form before introduction into
DNA was used for the t1/2 measurement. [b] [NaCl]=100 mm, pH 7.0
(10 mm phosphate buffer). [c] The data in parentheses show the t1/2 of
cis-azobenzene tethered to single-stranded DNA (CXG) on d-threoninol
through an amide bond.
which was five times longer than that tethered to DNA
(2.0 h), but still shorter than 2’,6’-Me-Azo (36 h). Other azo-
benzenes had the same or slightly longer t1/2 compared with
those in DNA, indicating that the improved thermal stability
of 2’,6’-Me-Azo was intrinsic, and not due to the effect of
DNA.
We also investigated the effect of the polarity of the sol-
vent on the t1/2 value to clarify the thermal isomerization
pathway. As the polarity of the solvent decreased (water
(buffer)!DMF!toluene), the t1/2 of the cis form decreased.
For example, the t1/2 of 2’-Me-Azo was as long as 3.5 h in
buffer solution, but only 0.79 h in DMF and 0.47 h in tolu-
ene. This solvent effect indicated that thermal isomerization
of all the cis-azobenzenes proceeds through an inversion
mechanism, not a rotation mechanism (vide infra).[22] 2’,6’-
Me-Azo also showed the same solvent effect on the t1/2. Ir-
respective of the solvent, however, 2’,6’-Me-Azo had about
a 10-fold longer t1/2 than other azobenzenes.
Figure 4. Energy-minimized structure of
a modified duplex involving
trans-2’,6’-Me-Azo viewed a) from the side of azobenzene and b) from
the 5’-side of the helix axis. For the top view (b), only the 5’-A4XG5-3’/3’-
T13C12-5’ part is depicted and trans-2’,6’-Me-Azo is drawn in stick form
for clarity. c) Whole energy-minimized structure of a modified duplex in-
volving cis-2’,6’-Me-Azo viewed from the side of azobenzene and d) the
same structure magnified around cis-2’,6’-Me-Azo. The distance between
the imino protons of G5-C12 and A4-T13, depicted with dotted yellow line
in (d), was calculated to be 7.3 ꢁ.
threoninol was largely parallel to the A4-T13 base pair,
whereas the distal ring was twisted in the duplex, and the
two methyl groups protruded to both adjacent base pairs.
As a result, the distance between the imino protons of A4-
T13 and G5-C12 increased to as far as 7.3 ꢁ, resulting in a
kink in the duplex at cis-2’,6’-Me-Azo.
Comparison of the activation enthalpy (DH°) with the ac-
tivation entropy (DS°) of the thermal cis-to-trans isomeriza-
tion rate further highlighted the uniqueness of 2’,6’-Me-Azo
(Figure 5). We evaluated DH° and DS° of thermal isomeri-
zation of all the azobenzenes in single-stranded CXG in
buffer solution based on the dependence of thermal isomeri-
zation rate on temperature.[23] As shown in Figure 5, all the
Thermal isomerization of cis-azobenzene: To evaluate the
thermal stability of the cis-azobenzenes, half-lives (t1/2) of
the thermal cis-to-trans isomerization of the carboxylic acid
derivatives were measured in various solvents at 608C.[21]
Table 3 shows the half-lives of thermal cis-to-trans isomeri-
zation of these derivatives in the same buffer solution as
was used for the Tm measurement and those of the azoben-
zenes tethered to DNA (CXG) on d-threoninol through an
amide bond. As reported previously,[7] the t1/2 of 2’,6’-Me-
Azo tethered to DNA was 25 h, which was about 10 times
longer than those of other azobenzenes. We found that
newly synthesized 2,6-Me-Azo or 2,2’-Me-Azo, which was
methylated on the other two ortho positions, did not show
such enhanced stability of the cis form in DNA. This re-
markable effect was observed only when the two ortho posi-
tions of the distal benzene were methylated. The carboxylic
acid form (without DNA) also showed similar results in
buffer solution except for 2,6-Me-Azo (not 2’,6’-Me-Azo).
The t1/2 of 2,6-Me-Azo in the carboxylic form was 10 h,
Figure 5. Plot of activation entropy (DS°) as a function of activation en-
thalpy (DH°) at T=333 K for the thermal cis-to-trans isomerization of
azobenzenes tethered to single-stranded CXG on d-threoninol in buffer
solution.
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Photoregulated DNA Hybridization
FULL PAPER
azobenzenes except for 2’,6’-Me-Azo were aligned almost
on the same straight line, demonstrating the typical compen-
sation law of enthalpy and entropy. Interestingly, 2’,6’-Me-
Azo deviated greatly from this line, indicating that its ther-
mal isomerization mechanism was different from other azo-
benzenes.
ed conformation similar to cis-2’,6’-Me-Azo (see Figure S5
of the Supporting Information). However, the absence of
two methyl groups on the twisted distal benzene ring al-
lowed for a smaller space between the G5-C12 and A4-T13
pairs (the distance was 5.7 ꢁ). Furthermore, because the
methyl groups on the proximal ring were far from the bases,
steric hindrance might be minimized in the cis form. Ac-
cordingly, cis-2,6-Me-Azo less effectively destabilized the
duplex than cis-2’,6’-Me-Azo.
Discussion
Although 2,2’-Me-Azo involved two methyl groups, free
ꢀ
2’,6’-Me-Azo as an efficient photoregulator: As shown in
Table 1, all the ortho-methylated azobenzenes improved
photoregulatory efficiency. In particular, 2’,6’-Me-Azo but
not 2,6-Me-Azo showed remarkable improvement due to
stabilization of duplex in the trans form and destabilization
in the cis form compared with other azobenzenes, irrespec-
tive of the type of adjacent base pairs (see Table 2). For sta-
bilization of the trans form by ortho modification, methyla-
tion at the distal benzene ring, such as in 2’-Me-Azo, 2,2’-
Me-Azo, and 2’,6’-Me-Azo, was more effective than at the
proximal ring. NMR spectroscopic analysis of the duplex
with trans-2’,6’-Me-Azo demonstrated that the H2(6) proton
at the proximal side had NOE only with imino proton G5,
whereas both G5 and T13 had distinct NOE with CH3 pro-
tons; this indicates that the ortho positions on the distal side
were located on the axial positions of the center of the G5-
C12 and A4-T13 pairs. Consistently, computer modeling also
showed that the distal ortho-methyl groups were located in
close proximity to the adjacent G5-C12 and A4-T13 pairs,
whereas the proximal H2(6) positions were rather distant
from these base pairs and located near the N-glycosidic link-
age between the nucleobase and deoxyribose. We also con-
ducted computer modeling of the duplex containing trans-
2,6-Me-Azo (see Figure S4 of the Supporting Information),
and found that two methyl groups were far from the bases.
Thus, distal modification at the ortho positions made the
trans-azobenzene a more effective intercalator; this facilitat-
ed stacking or hydrophobic interactions between the adja-
cent base pairs and stabilized the duplex.
On the other hand, destabilization by cis isomerization
was remarkable only with 2’,6’-Me-Azo. Computer modeling
of the duplex with cis-2’,6’-Me-Azo (see Figure 4c,d) re-
vealed that the distal ring was twisted by cis isomerization,
and the proximal side was fairly parallel to the adjacent A4-
T13 pairs. Because the proximal ring was directly bound to
the main chain of threoninol through the rigid amide bond,
its orientation might be restricted in the duplex. In contrast,
the distal ring was flexible enough to be twisted with respect
to the N=N bond by cis isomerization. Consequently, the
two ortho positions protruded close to the base pairs (G5-C12
and A4-T13) so that they had to be separated to accept the
twisted benzene ring with two methyl groups; the distance
between the imino protons of G5-C12 and A4-T13 was calcu-
lated to be 7.3 ꢁ as shown with dotted yellow line in Fig-
rotation around the C N bond avoided simultaneous inter-
ruption of both G5-C12 and A4-T13 pairs by forming a proper
orientation of these two methyl groups, thus minimizing de-
stabilization.
Unusual thermal stability of cis-2’,6’-Me-Azo: As summar-
ized in Table 3, 2’,6’-Me-Azo showed unusually slow thermal
cis-to-trans isomerization irrespective of the solvent used.
2,6-Me-Azo, which possessed two methyl groups on the
other benzene ring, did not exhibit improved thermal stabili-
ty of the cis form. Generally, push–pull modifications of azo-
benzene accelerates thermal isomerization. Because the car-
boxyl group pulled the electron and the alkyl group weakly
pushed it, the ortho modification slightly decreased the t1/2
compared with Azo in buffer solution. Furthermore, com-
pensation of DH° and DS° indicated that the decreased t1/2
by ortho methylation was caused by a similar electronic
effect.[24] However, cis-2’,6’-Me-Azo exclusively showed dif-
ferent thermal isomerization behavior, as evidenced from
the definite deviation in results (see Figure 5).
Asano et al. reported two different routes of thermal cis-
to-trans isomerization: inversion and rotation.[25] The inver-
sion route proceeds through a transition state in which one
of the nitrogen atoms is sp hybridized, whereas the rotation
route involves the disruption of a nitrogen–nitrogen p bond
and rotation around the remaining s bond, as shown in
Figure 6. These two routes could be discriminated from the
t1/2 dependence on the polarity of the solvent. Because the
transition state of the inversion route, an sp-hybridized ni-
trogen, is stabilized in the apolar solvent due to its hydro-
phobicity, the t1/2 decreases as the polarity of the solvent de-
creases. In contrast, the transition state of the rotation route
ꢀ
has a single N N (not a double N=N) bond that is stabilized
in a polar environment, resulting in a smaller t1/2 with a
polar solvent.[22] The solvent dependence listed in Table 3
clearly demonstrates that the present ortho-modified azo-
benzenes proceeded via an inversion mechanism as the in-
creased polarity of the solvent raised the thermal stability of
cis-azobenzene.
Nishimura et al. reported on the effect of substituents on
the inversion center. They proposed that an electron-accept-
ing substituent enhances the s character of the C N bond so
that an N atom near the electron-accepting substituent be-
comes the inversion center.[26] In contrast, an electron-donat-
ing substituent allows a distant N atom as the inversion
center. According to this hypothesis, an electron-accepting
carboxylate group on the distal ring enhances the s charac-
ꢀ
ACHTUNGTRENNUNGure 4c; this should significantly hinder their base pairing and
thus destabilize the duplex as shown in Figure 4c,d. In the
case of cis-2,6-Me-Azo, computer modeling showed a twist-
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2059

H. Nishioka, X. G. Liang and H. Asanuma
formation). Consequently, the
cis-to-trans isomerization of 2,6-
Me-Azo occurred smoothly via
an inversion mechanism. In the
case of both monosubstituted
2’-Me-Azo and disubstituted
2’,2’-Me-Azo, this hindrance
could be avoided through the
smaller hydrogen on other
ortho position of the distal ring.
In buffer solution, the t1/2 of
2,6-Me-Azo in the carboxylic
acid form was 5-fold longer
than that tethered to DNA
(2.0 h), whereas other modified
azobenzenes did not elongate
the t1/2 in the carboxylate form.
Figure 6. Two possible pathways of cis-to-trans thermal isomerization. Upper pathway: inversion route through
At pH 7, carboxylic acid is
mostly deprotonated to form a
carboxylate anion thus reducing
the electron-accepting property.
a transition state in which one of the nitrogen atoms is sp hybridized. Lower pathway: Rotation route through
the rupturing of a nitrogen–nitrogen p bond and rotation around the remaining s bond.
[26]
ꢀ
ter of C Na (see Figure 7), which facilitates inversion on
the Na atom rather than on Nb. This hypothesis explains the
transition states of 2’,6’-Me-Azo and 2,6-Me-Azo as depicted
In addition, methyl groups on the proximal ring in 2,6-Me-
Azo act as a weak electron donor, which prefers the Nb
atom to the Na atom as an inversion center. Presumably,
both the weakened electron-accepting property of the car-
boxylate anion and the electron-donating methyl groups on
the same benzene ring allowed Nb as well as Na to be inver-
sion centers, and so the thermal isomerization of cis-2,6-Me-
Azo became slower in pH 7 buffer solution. Consistently,
the t1/2 decreased from 10 to 4.8 h when the pH of the solu-
tion was lowered from 7 to 5 (see Table S2 of the Supporting
Information).[27]
On the other hand, the t1/2 of 2’,6’-Me-Azo in the carbox-
ylate form did not depend on the pH; the t1/2 was as long as
32 h at pH 5.0, which was even longer than that of 2’,6’-Me-
Azo tethered to DNA. Although deprotonation of the car-
boxyl group similarly reduced its electron-donating property,
the electron-donating effect of the methyl groups on the
distal benzene ring fixed the inversion center at the Na
atom even under neutral to basic conditions. In other organ-
ic solvents or by conjugation with DNA, however, only the
Na atom functioned as an inversion center due to the ab-
sence of deprotonation.
Figure 7. Possible transition states of a) 2’,6’-Me-Azo and b) 2,6-Me-Azo,
both of which have an inversion center at the Na atom. (Sphere-images
of transition states of both 2’,6’-Me-Azo and 2,6-Me-Azo constructed
from Insight II/Discover are shown in Figures S6 and S7 of the Support-
ing Information).
Conclusions
in Figure 7 due to the electron-accepting property of the car-
boxylic group, thereby leading to the enhanced thermal sta-
bility of 2’,6’-Me-Azo. At the transition state, lone pair elec-
trons of the p orbital of the Na atom cause steric hindrance
with the methyl groups on the twisted distal benzene ring
(see Figure 7a and Figure S6 of the Supporting Information),
which could markedly retard the inversion to the trans form.
However, the two methyl groups on 2,6-Me-Azo did not
induce such steric hindrance with the inversion center of the
Na atom (see Figure 7b and Figure S7 of the Supporting In-
We have synthesized various ortho-methylated azobenzenes
and found that 2’,6’-Me-Azo, in which two methyl groups
were introduced to the ortho positions of the distal benzene
ring, showed the most efficient photoregulatory ability in all
combinations of neighboring base pairs. NMR spectroscopic
analysis and computer modeling revealed that the two
methyl groups located near the adjacent imino protons sta-
bilized the DNA duplex by stacking interactions in the trans
form and destabilized the DNA duplex by steric hindrance
in the cis form. The solvent effect on the t1/2 indicated that
2060
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Chem. Eur. J. 2010, 16, 2054 – 2062

Photoregulated DNA Hybridization
FULL PAPER
Photoisomerization of azobenzene: The light source for photo-irradiation
was a 150 W Xenon lamp. For the trans!cis isomerization, a UV-D36C
filter (Asahi Tech.) was used, and UV light (l=300–400 nm:
5.3 mWcmꢀ2) was used to irradiate the duplex solution at 608C for 5 min.
Using this procedure, 70–80% of the total azobenzene was isomerized to
the cis form for all azobenzene derivatives except for 2,6-Me-Azo and
2’,6’-Me-Azo, in which about 60% was isomerized to the cis form. The
cis!trans isomerization was carried out by irradiation with visible light
(l>400 nm) through an L-42 filter (Asahi Tech.) at 608C for 5 min. In
both cases, a water filter was used to cut off infrared light.
cis-to-trans isomerization of all the modified azobenzenes
proceeded through an inversion mechanism. The improved
thermal stability of the cis form of 2’,6’-Me-Azo but not 2,6-
Me-Azo was attributed to steric hindrance between the un-
paired electrons of the p orbital of the N atom of the inver-
sion center and the methyl group on the distal benzene ring.
By using the present 2’,6’-Me-Azo as a “photon-engine”, a
photon-driven DNA nanomachine and photoregulation of
bioreaction with high “light-fuel cost” can be expected.[3a,c]
Generally, a push–pull (or push–push) modification of
azobenzene shifts the absorption maximum to the visible
region (l>400 nm) and allows potential trans-to-cis isomeri-
zation with visible light irradiation. However, if thermal cis-
to-trans isomerization is too fast, these azobenzenes cannot
be used practically. Our findings might lead to new modifi-
cations of azobenzene that do not accelerate thermal iso-
merization.
Half-life measurement of thermal isomerization of cis-azobenzene to the
trans form: UV light (l=300–400 nm: 5.3 mWcmꢀ2) was used to irradi-
ate a solution of single-stranded DNA containing azobenzene at 608C for
5 min to isomerize trans-azobenzene to the cis form. Then the solution
was measured by using
a JASCO V-530 UV/Vis spectrophotometer
equipped with a temperature controller, and spectra were monitored at
608C at predetermined intervals. Half-lives were obtained from the
change in absorption maximum of trans-azobenzene (lꢁ340 nm). Condi-
tions: [NaCl]=100 mm, pH 7.0 (10 mm phosphate buffer), [DNAs]=
20 mm. Half-life measurements of modified azobenzenes in the carboxylic
acid form (without tethering to DNA) were also carried out in the same
manner as that with DNA, except for the concentration of azobenzenes
was 60 mm.
Calculation of the activation entropy and enthalpy: DS° and DH° were
calculated based on the following equation:
Experimental Section
Materials: Mesitylene, fuming nitric acid, absolute ethanol, and chromic
acid were purchased from Kishida Chemical Co. (Osaka, Japan). Nitroso-
benzene, 4,4-dimethoxytrityl chloride, and 2-methyl aniline were pur-
chased from Tokyo Chemical Industry (Tokyo, Japan). d-Threoninol was
purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). 2-Cyanoethyl
N,N,N’,N’-tetraisopropylphosphordiamidite was purchased from Chem-
Genes (Wilmington, MA, U.S.A.).
DH° ¼ EaꢀRT, DS° ¼ R½ln Aꢀln ðkT=hÞꢀ1:00ꢂ
in which Ea is the activation energy of thermal isomerization, A is the
frequency factor of thermal isomerization, R is the gas constant, k is the
Boltzmann constant, and h is the Planck constant.[23]
NMR spectroscopic analysis of DNA tethering 2’,6’-Me-Azo: NMR spec-
troscopy samples were prepared by dissolving three-times-lyophilized
DNAs to a solution of duplex (560 mm) in H2O/D2O (9:1) containing
sodium phosphate (20 mm, pH 7.0). NaCl was added to give a final con-
centration of 200 mm. NMR spectra were measured by using a Varian
INOVA (700 MHz) equipped with triple resonance at a probe tempera-
ture of 278 K. Resonance was assigned with the standard method by
using a combination of 1D, TOCSY (60 ms of mixing time), DQF-COSY,
and NOESY (150 ms of mixing time) experiments. All spectra in the
H2O/D2O solution were recorded by using a 3-9-19 WATERGATE pulse
sequence for water suppression.
Synthesis of DNA involving modified azobenzenes: Modified DNAs con-
taining modified azobenzenes were synthesized by using an automated
DNA synthesizer (ABI-3400, Applied Biosystems) from conventional
and azobenzene-carrying phosphoramidite monomers. The modified azo-
benzenes, 2-Me-Azo, 2’-Me-Azo, and 2’,6’-Me-Azo, were synthesized and
converted to the corresponding phosphoramidite monomers according to
a previous report.[7] Other modified azobenzenes, 2,6-Me-Azo and 2,2’-
Me-Azo, were synthesized and converted to phosphoramidite monomers
as described in the Supporting Information (Schemes S1–3). All modified
DNAs were purified by reversed-phase HPLC and characterized by
MALDI-TOFMS (Autoflex Linear, Bruker Daltonics). DNAs containing
only native bases were supplied by Integrated DNA Technologies (Coral-
ville, IA. U.S.A.).
Molecular modeling: The insight II/Discover 98.0 program package was
used for molecular modeling to obtain energy-minimized structures by
conformation-energy minimization. The azobenzene residue was built up
by using a graphical program. The results of the NMR spectroscopic
analyses served as a starting point for the modeling. For the analysis, the
duplex was prepared by positioning the modified azobenzene in the trans
or cis form between the adjacent base pairs. The effect of water and
counterions was simulated by a sigmoidal, distance-dependent, direct
function. The B-type duplex was used, and the AMBER force field was
used for calculation. Computation was carried out by using a Silicon
Graphics O2 workstation with the operating system IRIX64 release 6.5.
MALDI-TOF MS for CXG with Azo=X: m/z calcd for protonated
form: 4020; found: 4021; 2’-Me-Azo=X: m/z calcd: 4034; found: 4035;
2-Me-Azo=X: m/z calcd: 4034; found: 4032; 2,6-Me-Azo=X: m/z calcd:
4048; found: 4047; 2,2’-Me-Azo=X: m/z calcd: 4048; found: 4048; 2’,6’-
Me-Azo=X: m/z calcd: 4048; found: 4048.
MALDI-TOF MS for DNA containing 2’,6’-Me-Azo=X: AXA: m/z
calcd for protonated form: 4056; found: 4057; AXC: m/z calcd: 4032;
found: 4034; AXG: m/z calcd: 4072; found: 4072; AXT: m/z calcd: 4047;
found: 4048; CXA: m/z calcd: 4032; found: 4031; CXC: m/z calcd: 4008;
found: 4009; CXG: m/z calcd: 4048; found: 4049; CXT: m/z calcd: 4023;
found: 4021; GXA: m/z calcd: 4072; found: 4070; GXC: m/z calcd: 4048;
found: 4050; GXG: m/z calcd: 4088; found: 4091; GXT: m/z calcd: 4063;
found: 4062; TXA: m/z calcd: 4047; found: 4051; TXC: m/z calcd: 4023;
found: 4023; TXG: m/z calcd: 4063; found: 4063; TXT: m/z calcd: 4038;
found: 4037; 5’-GCGAXGTCC-3’ (used for NMR analysis): m/z calcd for
protonated form: 2814; found: 2814.
Acknowledgements
This work was supported by JSPS Research Fellow (to H.N.), the Core
Research for Evolution Science and Technology (CREST), the Japan Sci-
ence and Technology Agency (JST) (to H.A.) Partial support was provid-
ed by a Grant-in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan (to H.A. and X.L.).
Tm measurements: Tm values were determined from the maximum in the
first derivative of the melting curve, which was obtained by measuring
the absorbance at l=260 nm as a function of temperature. The tempera-
ture ramp was 1.08Cminꢀ1. Both the heating and cooling curves were
measured, and obtained Tm values agreed within 2.08C. Conditions of the
sample solutions: [NaCl]=100 mm, pH 7.0 (10 mm phosphate buffer),
[DNA]=5 mm.
[1] Y. Ito, E. Fukusaki, J. Mol. Catal. B 2004, 28, 155–166, and referen-
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Chem. Eur. J. 2010, 16, 2054 – 2062
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2061

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ther methylation might not improve the photoregulatory efficiency
based on our present finding; modification of the distal benzene
ring, but not the proximal one was effective for the significant im-
provement of photoregulatory efficiency.
[14] Actual Tm curves were depicted in Figure S1 in the Supporting In-
formation.
[15] R. E. Holmlin, E. D. A. Stemp, J. K. Barton, Inorg. Chem. 1998, 37,
29–34.
[16] Our group previously also investigated the sequence dependency of
non-modified Azo to obtain the nearest-neighbor parameter (H.
Asanuma, D. Matsunaga, M. Komiyama, Nucleic Acids Symp. Ser.
2005, 49, 283–284). It was found that the duplex involving Azo
showed almost the same dependency of Tm on the adjacent base
pair as that of 2’,6’-Me-Azo in both the trans and cis forms. In addi-
tion, although the length of the present sequence involving 2’,6’-Me-
Azo was four bases longer than that of Azo, which our group previ-
ously used, the DTm of 2’,6’-Me-Azo was higher than that of Azo in
all the sequences, demonstrating the superiority of 2’,6’-Me-Azo
(see the Supporting Information, Figure S2 and Table S1).
[17] The weak signal at d=12.7 ppm was assignable to one of the termi-
nal base pairs (G1 or G9). Other terminal imino proton signals might
be overlapped with the signal at d=12.8 ppm.
[18] X. G. Liang, H. Asanuma, H. Kashida, A. Takasu, T. Sakamoto, G.
Kawai, M. Komiyama, J. Am. Chem. Soc. 2003, 125, 16408–16415.
[19] As described above, the DNA duplex that contain cis-2’,6’-Me-Azo
was much more unstable than that of trans form.
[20] M. Liu, H. Asanuma, M. Komiyama, J. Am. Chem. Soc. 2006, 128,
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[8] Modification at the para position led to the reversed photoswitch
with the combination of l-threoninol, see X. G. Liang, N. Takenaka,
H. Nishioka, H. Asanuma, Chem. Asian J. 2008, 3, 553–560.
[9] a) J. P. Ortruba III, R. G. Weiss, J. Org. Chem. 1983, 48, 3448–3453;
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[21] Typical UV/Vis spectra of the thermal cis-to-trans isomerization are
depicted in Figure S3 in the Supporting Information.
[10] M. R. Han, D. Hashizume, M. Hara, New J. Chem. 2007, 31, 1746–
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di, J. Am. Chem. Soc. 2008, 130, 5216–5230.
[13] We did not synthesize tri- or tetramethylated azobenzenes because
of the following reasons: Synthesis of tri- or tetraalkylated azoben-
zenes at ortho positions is rather difficult due to the steric hindrance
between the methyl groups on both sides of the benzene rings (C. J.
Byrne, D. A. R. Happer, M. P. Hartshorn, H. K. J. Powell, J. Chem.
Soc., Perkin Trans. 1 1987, 1649–1653). According to the literature
(ref. [9b]), tri- or tetraalkylation at the ortho positions makes the
trans-to-cis photoisomerization slower, and accordingly the cis con-
tent after UV light irradiation becomes much less, which is unfavor-
able for the efficient photoregulation. In addition, we think that fur-
[22] a) P. Haberfield, P. M. Block, M. S. Lux, J. Am. Chem. Soc. 1975, 97,
5804–5806; b) K. Baba, H. Ono, E. Itoh, S. Itoh, K. Noda, T. Usui,
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Hasegawa, Bull. Chem. Soc. Jpn. 1976, 49, 1381–1387.
[27] Note that the pKa of benzoic acid is 4.20, indicating that 14–15% of
2,6-Me-Azo is protonated at pH 5. Therefore, t1/2 did not increase to
2 h like that of 2,6-Me-Azo tethered to DNA. We also tried to mea-
sure t1/2 at pH values lower than 4, the pKa of benzoic acid. But nei-
ther 2’,6’-Me-Azo nor 2,6-Me-Azo would dissolve at all under stron-
ger acidic conditions.
Received: October 9, 2009
Published online: January 26, 2010
2062
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Chem. Eur. J. 2010, 16, 2054 – 2062