Visible-light photoresponsivity of a 4-(dimethylamino)azobenzene unit incorporated into single-stranded DNA: Demonstration of a large spectral change accompanying isomerization in DMSO and detection of rapid (Z)-to-(E) isomerization in aqueous solution

Article abstract of DOI:10.1002/ejoc.200600935

We demonstrate significant visible-light photoresponsivity in a synthesized oligonucleotide containing a built-in pseudonucleotide possessing a 4-(dimethylarnino)azobenzene (4-DMAzo) side chain. In dry DMSO as solvent, two clearly distinguishable spectra corresponding to the (E) and (Z) forms of the 4-DMAzo moiety tethered to the oligonucleotide were recorded with a conventional spectrophotometer before and after irradiation with 420 nm wavelength light, which induced (E)-to-(Z) isomerization. In addition, (Z)-to-(E) isomerization was accelerated by irradiation with either visible (λ = 550 nm) or UV (λ = 350 nm) light, demonstrating reversible photoresponsivity of the pseudo-ohgonucleotide. In aqueous solutions the (Z)-to-(E) thermal isomerization of the photoresponsive pseudo-ohgonucleotide was very rapid and was only detectable by laser flash photolysis. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejoc.200600935

FULL PAPER
DOI: 10.1002/ejoc.200600935
Visible-Light Photoresponsivity of a 4-(Dimethylamino)azobenzene Unit
Incorporated into Single-Stranded DNA: Demonstration of a Large Spectral
Change Accompanying Isomerization in DMSO and Detection of Rapid
(Z)-to-(E) Isomerization in Aqueous Solution
Takashi Kamei,[a] Masabumi Kudo,[b] Haruhisa Akiyama,[b] Momoyo Wada,[b]
Jun’ichi Nagasawa,[b] Masahiro Funahashi,[b] Nobuyuki Tamaoki,[b] and
Taro Q. P. Uyeda*[a]
Keywords: Photoresponsivity / Oligonucleotides / 4-(Dimethylamino)azobenzene / (E)/(Z) isomerization / DNA nano-
devices
We demonstrate significant visible-light photoresponsivity in
a synthesized oligonucleotide containing a built-in pseudo-
nucleotide possessing a 4-(dimethylamino)azobenzene (4-
DMAzo) side chain. In dry DMSO as solvent, two clearly dis-
tinguishable spectra corresponding to the (E) and (Z) forms
of the 4-DMAzo moiety tethered to the oligonucleotide were
recorded with a conventional spectrophotometer before and
after irradiation with 420 nm wavelength light, which in-
duced (E)-to-(Z) isomerization. In addition, (Z)-to-(E) isomer-
ization was accelerated by irradiation with either visible (λ =
550 nm) or UV (λ = 350 nm) light, demonstrating reversible
photoresponsivity of the pseudo-oligonucleotide. In aqueous
solutions the (Z)-to-(E) thermal isomerization of the photore-
sponsive pseudo-oligonucleotide was very rapid and was
only detectable by laser flash photolysis.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
of the parent azobenzene should permit more sophisticated
photoregulation triggered by light of different wavelengths.
As one example, tethering of oligonucleotides to two dif-
ferent azo-based photoresponsive moieties that could be in-
duced to undergo (E)-to-(Z) and (Z)-to-(E) photoconver-
sions independently of one another should allow four meta-
stable conformations of the device, enabling site-selective or
multi-step regulation of hybridization in DNA devices.
In this regard, it would be useful to develop oligonucleo-
tides incorporating tethered azo-based photoresponsive
components that would undergo reversible conformational
changes in response to visible-light irradiation. Synthetic
oligonucleotides conjugated with methyl red [4Ј-carboxy-4-
(dimethylamino)azobenzene][9–12] have been shown to ab-
sorb visible light, and it has been suggested that methyl red
conjugated oligonucleotides could be useful as DNA bea-
cons thanks to their unique H-aggregation and stacking
properties. When these oligonucleotides were irradiated
with visible light in aqueous solution, however, no photore-
sponsivity of the oligonucleotides could be detected with a
conventional spectrophotometer,[11,13] so little is currently
Introduction
Azobenzene, a photochromic system, is widely used for
syntheses of organic molecular devices that will undergo
conformational changes by (E)/(Z) isomerization.[1–4] Syn-
thetic oligonucleotides conjugated with azobenzene moie-
ties[5,6] are promising examples of photoregulatable molecu-
lar devices. Asanuma et al. demonstrated reversible photo-
induced isomerization of an azobenzene moiety incorpo-
rated in the form of an oligonucleotide side chain on irradi-
ation with UV light (300 Ͻ λ Ͻ 400 nm) followed by visible
light (λ Ͼ 400 nm), to induce (E)-to-(Z) and (Z)-to-(E)
isomerization, respectively.[6] These authors further demon-
strated that hybridization of the pseudo-oligonucleotide
with a complementary oligonucleotide is destabilized by
UV-induced structural changes in the azobenzene moiety,[7]
and introduced a prototype DNA device in which an intra-
molecular hairpin structure could be opened by irradiation
with UV light.[8] Oligonucleotides bearing tethered azoben-
zene derivatives with optical properties different from those
[a] Research Institute for Cell Engineering, National Institute of known about the visible-light photoresponsivity of oligonu-
Advanced Industrial Science and Technology (AIST),
cleotides incorporating tethered azobenzene derivatives
with a dimethylamino group at the 4Ј-position, either in
aqueous or in organic solutions.
In the work reported here we studied the physicochemi-
cal properties of a photoresponsive oligonucleotide incor-
Higashi 1-1-1, Tsukuba, Ibaraki 305-8562, Japan
Fax: +81-29-861-3049
E-mail: t-uyeda@aist.go.jp
[b] Nanotechnology Research Institute, National Institute of Ad-
vanced Industrial Science and Technololgy (AIST),
Higashi 1-1-1, Tsukuba, Ibaraki 305-8565, Japan
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Photoresponsivity of 4-(Dimethylamino)azobenzene in Single-Stranded DNA
FULL PAPER
porating a 4-DMAzo unit upon visible-light irradiation (λ
= 420 nm). In well dried DMSO as solvent, two different
spectra representing the (Z) and (E) forms of 4-DMAzo
tethered to the synthesized oligonucleotide were recorded
with a conventional spectrophotometer. Furthermore, pho-
toinduced reversible isomerization was demonstrated upon
irradiation with light of different wavelengths (550 or
350 nm). In aqueous solution, though, the (Z)-to-(E) ther-
mal isomerization was so rapid that it was detectable only
by a flash photolysis technique.
Results and Discussion
Synthesis of Phosphoramidite Monomer 5
Prior to preparation of the photoresponsive oligonucleo-
tides, a phosphoramidite monomer bearing a 4-DMAzo
moiety was synthesized as shown in Scheme 1. Firstly, N,N-
dimethyl-p-nitrosoaniline (1) was treated with p-phenylene-
diamine to form 4Ј-amino-4-DMAzo (2).[14] Then, in a
manner similar to that described previously,[6,15] 2 and 2,2-
bis(hydroxymethyl)propionic acid were condensed with eli-
mination of water by use of dicyclohexylcarbodiimide and
1-hydroxybenzotriazole to form 3. One of the two hydroxy
groups in 3 was then protected by treatment with 4,4Ј-di-
methoxytrityl chloride in the presence of 4-(dimethyl-
amino)pyridine to form 4, and finally, phosphitylation of 4
was performed with 2-cyanoethyl N,N,NЈ,NЈ-tetraisopro-
pylphosphorodiamidite in the presence of 1H-tetrazole to
form 5, which was used for the synthesis of a photorespon-
sive oligonucleotide.
Scheme 1. Synthetic pathway for the phosphoramidite monomer
5. (a) p-Phenylenediamine, glacial acetic acid, benzene, 42% yield.
(b) 2,2-Bis(hydroxymethyl)propionic acid, 1-hydroxybenzotriazole,
dicyclohexylcarbodiimide, DMF, 27% yield. (c) 4,4Ј-Dimethoxytri-
tyl chloride (DMTr), 4-(dimethylamino)pyridine, pyridine, CH2Cl2,
53% yield. (d) 2-Cyanoethyl N,N,NЈ,NЈ-tetraisopropylphosphorod-
iamidite, 1H-tetrazole, acetonitrile, 36% yield.
mated DNA synthesizer by use of 5. The resultant pseudo-
oligonucleotide 6 was purified by reversed-phase HPLC,
and two major peaks were observed, indicating the exis-
tence of two stereoisomers of 6,[6,16] both of which con-
tained the thermally stable (E) form of 4-DMAzo. These
Synthesis and HPLC Purification of Photoresponsive
Pseudo-Oligonucleotides with 4-DMAzo Side Chains (6)
An oligonucleotide containing a built-in pseudo-nucleo- two peaks were isolated and analyzed separately by
tide possessing a 4-DMAzo side chain (5Ј-AAAXAAAA- MALDI-TOF mass spectrometry. The observed molecular
3Ј, where A is an adenosine residue and X is the residue masses of both compounds were 2549Ϯ1, consistent with
bearing 4-DMAzo; Scheme 2) was prepared with an auto- the calculated mass of [5Ј-AAAXAAAA-3Ј + H+] (2549).
Scheme 2. Photoresponsive oligonucleotide 6 containing a built-in pseudonucleotide with a 4-DMAzo side chain. Reversible (E) (left)/
(Z) (right) isomerization of the 4-DMAzo moiety (residue X) in 6.
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The two fractions from the reversed-phase HPLC were then Table 1. λmax (nm) and half-bandwidth (cm–1) values for the 4-
DMAzo derivatives (2, 7, 8) and the photoresponsive oligonucleo-
freeze-dried under vacuum and stored. We used the fraction
with the longer retention time in the experiments described
below, although the other fraction behaved similarly with
regard to photoresponsivity in a preliminary experiment
(see Exp. Sect.).
tide 6 in cyclohexane, DMSO, and water containing ammonium
formate (50 m). The spectra of compounds 2, 6, 7, and 8 in
DMSO and of 6 in the aqueous solution are shown in Figure 1.
The spectra in cyclohexane are not shown.
Compound
Solvent
Cyclohexane
DMSO
Water
7
2
8
6
399
(4137)
403
(4870)
409
(4826)
insoluble
424
(5129)
449
(5432)
428
(5381)
426
insoluble
Spectral Profiles of 6 and 4-DMAzo Derivatives
insoluble
insoluble
Figure 1(a) shows spectra of the synthesized pseudo-oli-
gonucleotide tethered to the (E) form of 4-DMAzo (6) in
DMSO (solid line) and in water containing ammonium for-
mate (50 mM, dotted line) before irradiation with visible
light. Two major peaks were observed in each spectrum:
one at ca. 260 nm and the other either at 426 nm (in
DMSO) or at 462 nm (in aqueous solution). These peaks
in the visible region were absent from the spectra of pure
oligonucleotide solutions, indicating that they reflect ab-
462
(6093)
(5270)
sorption by the tethered 4-DMAzo in 6. Hereafter, when of 7; Table 1) caused by the amino group at the 4Ј-position
we refer to “λmax” for the pseudo-oligonucleotide, we are acting as an auxochrome. Unlike the amino group in 2, the
referring to the λmax value for the 4-DMAzo moiety in the amide bonds in 6 and 8 linking the DMAzo moiety either
visible region. Table 1 summarizes the values of λmax and to DNA (6) or to a methyl group (8) did not have large
half-bandwidth at the π–π* band for 4-DMAzo, the two chromic effects on the π–π* band in the absorption spectra
derivatives of 4-DMAzo (4Ј-amino-4-DMAzo and 4Ј-ace- of the (E) form of 4-DMAzo.
toamide-4-DMAzo) (Scheme 3; 7, 2, and 8, respectively),
and 6 in organic and aqueous solutions before visible-light DMAzo derivatives (2, 7, and 8) are as follows. In DMSO,
irradiation. which is more polar than cyclohexane, the λmax values for
The solvent effects on the spectra of 6 and on the 4-
All compounds (2, 6, 7, and 8) were soluble in DMSO, 4-DMAzo and its derivatives were shifted to longer wave-
allowing comparison of the spectral properties of 6 [Fig- lengths than were seen in cyclohexane, accompanied by
ure 1 (b)] with those of the 4-DMAzo derivatives 7, 2, and widening of the half-bandwidths by 550–1000 cm–1
8 [Figure 1(c–e), respectively]. The spectral profile and (Table 1). The redshift of λmax with increasing polarity of
characteristic values (λmax and half-bandwidth) for 6 the solvent is consistent with an earlier result obtained with
(428 nm and 5381 cm–1) in DMSO were very similar to 4-(diethylamino)azobenzene.[17] In addition, although 4-
those of 8 (426 nm and 5270 cm–1) [gray solid lines in Fig- DMAzo is insoluble in water, a redshift in the λmax value
ure 1(b) and (e), respectively; Table 1], except for the ab- for 4-DMAzo was observed in an organic/water co-solvent
sorption due to the oligonucleotide bases of 6 in the UV mixture.[18] The even larger redshift for the λmax value of 6
region. This suggests that the optical properties of 6 in the in aqueous solution, accompanied by widening of the half-
visible-light region are similar to those of 8. The profile of bandwidth (from 5270 cm–1 in DMSO to 6093 cm–1 in the
2 at the π–π* band is clearly different from those of 6, 7, aqueous solution, Table 1), is also consistent with the idea
and 8 [gray solid lines in Figure 1(b–e)], with the large that larger redshifts are associated with higher solvent po-
bathchromic shift (λmax = 449 nm, 25 nm larger than that larity.[17]
Scheme 3. 4-DMAzo derivatives: 4-DMAzo 7, 4Ј-amino-4-DMAzo 2, and 4Ј-acetoamide-4-DMAzo 8. (a) Acetyl chloride, triethylamine,
CH2Cl2, 85% yield.
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Photoresponsivity of 4-(Dimethylamino)azobenzene in Single-Stranded DNA
FULL PAPER
Figure 1. Photoresponsive spectral profiles of the oligonucleotide 6 and 4-DMAzo derivatives (2, 7, and 8). (a) Spectral profiles of 6 in
DMSO (7.1 µ; solid line) and in water containing ammonium formate (50 m) (9.2 µ; dotted line) before irradiation with visible
(420 nm) light. (b–d) Time course of the spectral changes of the 4-DMAzo moiety in 6 and 4-DMAzo derivatives in DMSO at controlled
temperature (20.0Ϯ0.1 °C) after irradiation with 420 nm light, corresponding to thermal (Z)-to-(E) transition. (b) Spectral profiles of 6
(7.1 µ) in the visible region are shown (with rising absorbance at ca. 400 nm in the visible region) 0, 10, 30, 60, and 90 min after and
before irradiation, respectively. (c) 4-DMAzo 7 (8.7 µ, with rising absorbance at ca. 400 nm) at 0, 90, 270, 540, and 810 min after and
before irradiation, respectively. (d) 4Ј-Amino-4-DMAzo 2 (7.1 µ, with rising absorbance at ca. 400 nm) at 0, 1, 3, 10, and 20 min after
and before irradiation, respectively. (e) 4Ј-Acetamido-4-DMAzo 8 (8.0 µ, with rising absorbance at ca. 400 nm) at 0, 10, 30, 90, and
180 min after and before irradiation, respectively.
Photoresponsivity of 6 to Visible Light in DMSO
line in Figure 1(b)] and the (Z) form, which has clearly dis-
tinguishable n–π* and π–π* bands [black solid line in Fig-
ure 1(b)]. Clear isosbestic points at λ ≈ 317, 374, and
505 nm were obtained by tracing the spectra during thermal
recovery isomerization [Figure 1(b)]. Figure 1(c–e) shows
the significant spectral changes seen in the 4-DMAzo deriv-
atives 7, 2, and 8, respectively, upon visible-light irradiation
[(E) and (Z) forms shown in solid gray and black lines,
respectively]. The spectral profile and isosbestic points of 6
incorporating the (Z) form of 4-DMAzo are similar to
those of the (Z) form of 8 [isosbestic points at λ ≈ 320, 374,
and 505 nm in 8, as determined from Figure 1 (e)]. This re-
sult is consistent with the similarity of the (E) forms of 6
and 8, as described above.
As mentioned in the Introduction, no (E)/(Z) transitions
in aqueous solutions had been detected in oligonucleotides
bearing azobenzene derivatives with dimethylamino groups
at their 4-positions.[11,13] We speculated that, in aqueous
solutions, the dimethylamino group of the 4-DMAzo moi-
ety in 6 undergoes protonation, similarly to the parent 4-
DMAzo or 4Ј-carboxy-4-DMAzo,[18] so that no significant
spectral change in 6 is detectable with a conventional spec-
trophotometer due to the rapid thermal (Z)-to-(E) transi-
tion. For this reason we attempted to observe changes in
the absorption spectra with a conventional spectrophoto-
meter by conducting measurements in DMSO, an aprotic
solvent that should suppress protonation of the dimeth-
ylamino group. Figure 1(b) shows spectral changes of 6 be-
fore and after visible-light (λ = 420 nm) irradiation in
DMSO that had been thoroughly dried to avoid proton-
The rate constants (k(Z)–(E)) of thermal (Z)-to-(E) isom-
erization of 6 and of the 4-DMAzo derivatives (2, 7, and 8)
in DMSO were estimated by first-order kinetic analysis of
ation from water molecules. The two spectra were signifi- the time courses of spectral changes in the recovery transi-
cantly different, corresponding to the (E) form [gray solid tions of these compounds as shown in Figure 1(b–e)
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T. Q. P. Uyeda et al.
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(Table 2). The k(Z)–(E) value of 6 was larger than that of 7, cycles in Figure 2, bottom) on irradiation with 420 nm light
smaller than that of 2, and similar to that of 8. It is known and either 350 nm or 550 nm light (each absorbance value
that the rates of thermal (Z)-to-(E) isomerization of para- is the average of 10 cycles).
donor/paraЈ-acceptor-substituted (“push-pull-substituted”)
azobenzenes such as 4Ј-nitro-4-DMAzo and “push-push”-
substituted azobenzenes such as 4Ј-(dialkylamino)-4-
DMAzo compounds are faster than that of 4-DMAzo.[19,20]
The electron transition state of 2 should be most similar
to those of “push-push” azobenzenes, consistent with the
finding that the rate for 2 was the largest of all four com-
pounds examined in this experiment. The k(Z)–(E) values of
6 and 8 were significantly smaller than that of 2, suggesting
that the amide bonds and attached DNA or the methyl
group at the 4Ј-positions are poor donors or acceptors rela-
tive to the 4Ј-amino group in 2, not to mention the 4Ј-nitro
or 4Ј-(dialkylamino) groups. The k(Z)–(E) value for 6, as well
as its spectral properties, were similar to those of 8, indicat-
ing that the optical properties of 6 are similar to those of
8. Nonetheless, the k(Z)–(E) value for 6 is slightly larger than
that for 8, implying that protons of the DNA backbone
affect the rate of thermal (Z)-to-(E) isomerization to some
extent.
Table 2. Rate constants (102k(Z)–(E) min–1; averageϮs.d.) for ther-
mal (Z)-to-(E) isomerization of the 4-DMAzo derivatives (2, 7, and
8) and the photoresponsive oligonucleotide
20.0Ϯ0.1 °C.
6
in DMSO at
Compound
7
2
8
6
Figure 2. Photoinduced reversible isomerization of 6 (3.9 µ) in
DMSO. Top: Spectral changes of the 4-DMAzo moiety in 6 before
(gray solid line) and after (gray dotted line) irradiation with 420 nm
light, followed by irradiation either with visible (λ = 550 nm) light
(black solid line) or with UV (λ = 350 nm) light (black dotted line).
Thermal recovery that had occurred after 7 min in the dark is
shown by the gray dashed-dotted line. Bottom: The cycles of alter-
nating reversible changes in absorbance at λ = 425 nm for (E)-to-
(Z) isomerization upon 420 nm irradiation (squares), followed by
(Z)-to-(E) photoinduced isomerization upon irradiation either with
550 nm (circles) or with 350 nm (triangles) light. The sample was
irradiated for 7 min in a fluorescence spectrophotometer immedi-
ately before each measurement at 20.5Ϯ1 °C.
102 k(Z)–(E) 0.224Ϯ0.033 12.4Ϯ0.20 1.13Ϯ0.01 1.54Ϯ0.19
Photoinduced Reversible Isomerization of 6 in DMSO
Figure 2 shows the photoinduced reversible spectral
changes of 6 in DMSO. A proportion of 4-DMAzo tethered
in 6 underwent (E)-to-(Z) isomerization upon irradiation
with 420 nm light (from the gray solid line to the gray dot-
ted line in Figure 2, top), followed by photoinduced (Z)-to-
(E) recovery upon 7 min of irradiation with 550 nm light in
the visible region (from the gray dotted line to the black
solid line in Figure 2, top) or with 350 nm light in the UV
region (from the gray dotted line to the black dotted line in
Figure 2, top). The extent of thermal (Z)-to-(E) recovery in
Detection of the Photoresponsivity of 6 to Visible Light in
Aqueous Solution
With a conventional spectrophotometer we were unable
7 min (from the gray dotted line to the gray dashed-dotted to detect changes in the spectral profile of 6 in an aqueous
line in Figure 2, top) corresponded to ca. 20% of the total solution after irradiation with visible light (420 nm) [Fig-
conversion induced by 7 min of irradiation with visible or ure 3(a), solid and dotted lines], consistent with previous
UV light. The photoresponsive properties of 6 were distinct reports,[11,13] so we attempted to detect (E)/(Z) isomeriza-
from those of the parent azobenzene-conjugated oligonucleo- tion by laser flash photolysis.[17] Figure 3(b) shows the
tide, in which the (E)-to-(Z) isomerization is triggered by changes in absorbance at 460 nm after a laser (λ = 532 nm)
UV light and the (Z)-to-(E) isomerization is triggered only photoflash. Upon triggering of the flash, which lasted for
by visible light.[6] The extent of reversible isomerization had 3–5 ns (arrow), the absorbance decreased instantly, indicat-
not decayed after 10 cycles of alternate irradiation with ing photoinduced (E)-to-(Z) isomerization. This ab-
420 nm and either 550 nm or 350 nm light (Figure 2, bot- sorbance decrease was followed by recovery within ca. 1 ms,
tom). The absorbance at 425 nm decreased to ca. 26% at representing rapid (Z)-to-(E) thermal isomerization [Fig-
the photostationary state (rectangles in Figure 2, bottom) ure 3(b), dark gray line]. Fitting to a first-order exponential
and recovered to ca. 80% (circles and triangles in Figure 2, equation yielded
a time constant of 0.14Ϯ0.01 ms
bottom) of that of the pure (E) state (the initial point at 0 (averageϮs.d., n = 3) [Figure 3 (b), solid black curve], or a
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Photoresponsivity of 4-(Dimethylamino)azobenzene in Single-Stranded DNA
FULL PAPER
Figure 3. Photoresponsivity of 6 in an aqueous solution (50 m ammonium formate) at room temperature. The concentration of 6 was
9.2 µ, as estimated from the absorbance at 260 nm. (a) Absorbance spectra of 6 observed with a conventional spectrophotometer before
(solid line) and after (dotted line) irradiation with visible light (420 nm) for 30 min. (b) Profiles of absorbance changes at 460 nm in 6
after a laser photoflash (arrow, λ = 532 nm). The traces shown in dark and pale gray are the profiles obtained for 6 and for a control 8-
mer adenosine oligonucleotide (normalized), respectively. The solid black line is the fit with a first-order exponential equation [y = a·{1 –
exp(–t/b)} + c; t = time (ms); fitting parameters: a = 0.015, b = 0.15, c = 0.014; R = 0.95].
rate constant of k(Z)–(E) ≈ 4.3ϫ105 min–1. The contribution Okahata et al.[21] Efforts in that direction are currently un-
made to the exponential recovery by diffusion was negligi- derway.
ble, because the detection area (φ ≈ 1 mm) was much larger
than the diffusion field covered by 6 in 1 ms.
Availability of pseudo-nucleotides bearing different azo-
benzene derivatives or other photochromic molecules, as
As described above, the electron transition state of 6 is well as different methods of incorporating the photochro-
distinct from those of the “push-push” or “push-pull” azo- mic systems into the nucleotides, should expand the reper-
benzene derivatives because the rate of thermal (Z)-to-(E) toire of optical properties of photoresponsive oligonucleo-
isomerization of 6 in DMSO is significantly smaller than tides.[5,22,23] This should in turn facilitate the development
those of “push-push” or “push-pull” compounds (for in- of a variety of photoregulated and photoprogrammable
stance, k(Z)–(E) = 7.2ϫ103 min–1 for 4Ј-nitro-4-DMAzo in DNA nanodevices.
DMSO,[19] in comparison to ca. 1.5ϫ10–2 min–1 for 6). We
therefore speculate that the rapid thermal (Z)-to-(E) isom-
Experimental Section
erization of 6 in aqueous solutions is mostly due to proton-
ation by water molecules rather than the high polarity of
the solvent.
General: All solvents and reagents except for DMSO were obtained
from commercial sources and were used for syntheses and spectro-
scopic measurements without further purification. DMSO (Tokyo
Kasei) was used after drying with ca. 1/3 of its volume of molecular
sieves (Wako). Thin-layer chromatography (TLC) was performed
on silica gel pre-coated onto an aluminium sheet (silica gel 60 F254,
Merck). The silica gel used for flash column chromatography was
purchased from Kanto Chemical (silica gel 60N, spherical, neutral,
40–50 µm). The paper filters used for filtration were purchased
from Kiriyama (Nos. 5B or 5C). For identification of the synthe-
sized products, 1H NMR spectra were measured with a Gemini-
2000/300BB (300 MHz) (Varian).
Conclusions
We have demonstrated substantial optical responsivity of
an oligonucleotide incorporating a 4-DMAzo unit – an azo-
benzene derivatized with a dimethylamino group – to irradi-
ation with visible light. We are now able to regulate the
structural changes of a 4-DMAzo moiety in a photorespon-
sive oligonucleotide both in aqueous solutions and in apro-
tic solvents such as DMSO. Although thermal (Z)-to-(E)
recovery in aqueous solution was too rapid to allow light-
induced (Z)-to-(E) recovery in this study, it might in theory
be possible to restrain a controlled proportion of 4-DMAzo
moieties in the (Z) form in aqueous solutions by continuous
irradiation with visible light. With regard to the use of 4-
DMAzo moieties tethered to oligonucleotides in aprotic
solvents, it is known that the secondary structure of double-
stranded DNA is unstable in DMSO, so the assembly of
DNA-based devices driven by photoregulated hybridization
involving 4-DMAzo in organic solvents would require de-
velopments of aprotic solvents that would allow the forma-
4Ј-Amino-4-DMAzo (2): A mixture of N,N-dimethyl-p-nitrosoani-
line (Wako, 0.920 g, 6.1 mmol) and p-phenylenediamine (Wako,
0.661 g, 6.1 mmol) in benzene (60 mL) containing glacial acetic
acid (0.5 mL) was heated at reflux under a Dean–Stark trap at
85 °C for more than 24 h. The reaction mixture was allowed to cool
to room temperature, filtered, and concentrated to provide the
crude product, which was purified by flash column chromatog-
raphy on silica gel (0.61 g, 41.5% yield). 1H NMR [CDCl3 (TMS)]:
δ = 7.82–6.71 (m, 8 H, aromatic protons of azobenzene), 3.91 (s, 2
H, NH2), 3.06 [s, 6 H, N(CH3)2] ppm. Rf = 0.17 (CH2Cl2).
N-(4-{[4-(Dimethylamino)phenyl]azo}phenyl)-3-hydroxy-2-(hydroxy-
methyl)-2-methylpropionamide (3): In dry DMF (12.5 mL), 2,2-
bis(hydroxymethyl)propionic
acid
(Sigma–Aldrich,
2.09 g,
tion of stable double helixes, such as that demonstrated by 15.6 mmol), 4Ј-amino-4-DMAzo (3.12 g, 13.0 mmol), anhydrous 1-
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hydroxybenzotriazole (HOBt, Dojindo, 2.11 g, 15.6 mmol), and di-
cyclohexylcarbodiimide (Sigma–Aldrich, 3.21 g, 15.6 mmol) were
stirred under nitrogen at room temperature overnight (ca. 16 h) and
then at 100 °C for 4 h. Then, ethyl acetate (225 mL) was added,
and the ethyl acetate solution was filtered and washed once each
with 225 mL of saturated aqueous solutions of NaHCO3 and
NaCl. The organic layer was dried with anhydrous Na2SO4, fil-
tered, and concentrated, and the crude product was dried in vacuo
and purified by silica gel flash column chromatography (CH2Cl2/
MeOH, 95:5) to provide 1.23 g (26.6% yield). 1H NMR [300 MHz,
CDCl3(TMS)]: δ = 9.22 (s, 1 H, NHCO), 7.88–6.74 (m, 8 H, aro-
matic protons of azobenzene), 3.91–3.87 (m, 4 H, CH2), 3.09 [s, 6
H, N(CH3)2], 2.81 (m, 2 H, OH), 1.23 (s, 3 H, CH3) ppm. Rf =
0.26 (CH2Cl2/MeOH, 95:5). ESI MS: m/z = 357.2 [M + H]+ (calcd.
357.2).
was obtained after incubation at 55 °C for 12 h and freeze-drying.
All reagents for the synthesis of the oligonucleotide were purchased
from ABI, except for ammonia solution (WAKO).
N-(4-{[4-(Dimethylamino)phenyl]azo}phenyl)acetamide (8): Com-
pound 2 (0.10 g, 0.42 mmol) in CH2Cl2 (1.4 mL) and triethylamine
(0.1 mL) solution was added dropwise under nitrogen to acetyl
chloride (33 mg, 0.42 mmol, WAKO) prepared as a CH2Cl2 solu-
tion (6% v/v, 0.8 mL). The product was added to CH2Cl2 (20 mL),
the solution was filtered, and the organic layer was washed with
saturated aqueous solutions (20 mL) of NaHCO3 (twice) and NaCl
(twice) and then dried with anhydrous Na2SO4. The solution was
concentrated and dried in vacuo. Silica gel column chromatography
(CH2Cl2/MeOH, 95:5) provided 8 (0.10 g, 85% yield). 1H NMR
[300 MHz, [D6]DMSO (TMS)]: δ = 10.17 (s, 1 H, NHCO), 7.77–
6.81 (m, 8 H, aromatic protons of azobenzene), 3.08 [s, 6 H,
N(CH3)2], 2.08 (s, 3 H, CH3) ppm. Rf = 0.44 (CH2Cl2/MeOH,
95:5).
3-[Bis(4-methoxyphenyl)(phenyl)methoxy]-N-{4-[(4-dimethylamino)-
phenylazo]phenyl}-2-(hydroxymethyl)-2-methylpropionamide (4): A
dry pyridine (15 mL) solution of 3 (1.97 g, 5.53 mmol) and 4-(di-
Reversed-Phase HPLC Purification of 6: The sample solution was
filtered (Millex, 0.45 µm, Millipore) before application to the col-
umn (Symmetry C18, 5 µm, 100 Å, Waters) for reversed-phase
HPLC purification (JASCO). The developing solvent flowed for
50 min at a rate of 1 mLmin–1 and was composed of a linear gradi-
ent from 5:95% to 45:55% acetonitrile/water (50 m ammonium
formate) at room temperature. The first major peak was observed
at ca. 17 min, and the second peak was observed ca. 40 s later after
correction for the dead volume. The monitoring wavelength was
260 nm. The results reported in this paper were obtained by use of
the fraction with the longer retention time, although two distinct
spectral profiles corresponding to the (E) and (Z) forms of 4-
DMAzo-tethered 6 were also observed in DMSO with the fraction
of shorter retention time. Furthermore, the time constant for recov-
ery in aqueous solution after flash photolysis was 0.14Ϯ0.1 ms for
the fraction with the shorter retention time, similar to that obtained
for that with the longer retention time.
methylamino)pyridine
(DMAP,
Tokyo
Kasei,
5.66 mg,
0.276 mmol) was cooled under nitrogen with ice, and 4,4Ј-dimeth-
oxytrityl chloride (DMT-Cl, Wako, 2.25 g, 6.63 mmol) in CH2Cl2
(4.5 mL) was added dropwise. After 19 h, CH2Cl2 (60 mL) was
poured into the mixture, and the solution was filtered. After having
been washed with saturated aqueous solutions (60 mL) of NaHCO3
(twice) and NaCl (once), the organic layer was dried with anhy-
drous Na2SO4, the solution was concentrated and dried in vacuo,
and silica gel column chromatography (CH2Cl2/iPrOH/Et3N,
97.5:2.5:1) provided 4 (1.93 g, 53.0% yield). 1H NMR [300 MHz,
CDCl3(TMS)]: δ = 9.45 (s, 1 H, NHCO), 7.87–6.74 (m, 21 H, aro-
matic protons of azobenzene and DMT), 3.79–3.64 (s, 6 H, OCH3,
m, 2 H, CH2OH), 3.44–3.36 (d, 2 H, DMT-OCH2), 3.07 [s, 6 H,
N(CH3)2] 1.35 (s, 3 H, CH3) ppm. Rf = 0.25 (CH2Cl2/iPrOH/Et3N,
97.5:2.5:1). ESI MS: m/z = 659.3 [M + H]+ (calcd. 659.3).
2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-3-(4-{[4-(dimeth-
ylamino)phenyl]azo}phenylamino)-2-methyl-3-oxopropyl 2-Cyano-
ethyl diisopropylphosphoramidite (5): In a dry acetonitrile (10 mL)
solution, 2 (0.296 g, 0.45 mmol) and 2-cyanoethyl N,N,NЈ,NЈ-tetra-
ESI and MALDI-TOF Mass Measurements: Compounds 3–5 were
identified by NMR spectroscopy and ESI mass spectrometry (posi-
tive mode, MeOH solvent; ABI). The synthesized oligonucleotide 6
was identified by MALDI-TOF mass spectrometry (negative mode,
THAP matrix; ABI).
isopropylphosphorodiamidite
(Sigma–Aldrich,
0.16 mL,
0.50 mmol) were treated with 1H-tetrazole (Tokyo Kasei, 35 mg,
0.50 mmol). After 1 h, the product was added to ethyl acetate
(100 mL), and the organic solution was filtered and washed with
100 mL portions of saturated aqueous solutions of NaHCO3 and
NaCl, dried with Na2SO4, and desalted by filtration. Finally, the
solvent was removed in vacuo. The resultant product was purified
by silica gel column chromatography (hexane/AcOEt, 2:1) and
dried in vacuo to provide 0.14 g (36.2% yield). 1H NMR
[300 MHz, CDCl3(TMS)]: δ = 9.16 (s, 1 H, NHCO), 7.86–6.74 (m,
21 H, aromatic protons of azobenzene and DMT), 3.85–3.71 (m,
10 H, OCH3, CH2OP, CH2-OP), 3.54–3.46 [m, 2 H, CH(CH3)2],
3.42–3.37 (m, 2 H, DMT-OCH2), 3.08 [s, 6 H, N(CH3)2], 2.56–2.47
(m, 2 H, CH2CN), 1.28–1.07 [m, 15 H, CH(CH3)2, CH3] ppm. Rf
= 0.28 (hexane/AcOEt, 2:1). ESI MS: m/z = 881.5 [M + Na]+
(calcd. 881.4).
UV/Vis Spectroscopy: Spectra of the synthesized oligonucleotide,
4-DMAzo and its derivatives were measured with a DU800 spec-
trophotometer (Beckman). Prior to the (Z)/(E) transition measure-
ments with the spectrophotometer, the sample solutions were irra-
diated with light. The sample cell (quartz) was irradiated with UV
(350 nm) or visible (420 or 550 nm) light from a 150 W Xenon lamp
through a 5 nm slit for 25 min or longer at 20.5Ϯ1 °C by placing
a quartz cuvette containing the sample in the light path of a fluo-
rescence spectrometer (RF-5300PC, Shimadzu). Before each ex-
periment we confirmed that the absorbance of the sample at
425 nm did not change over a 15–30 min period in the dark; this
is an indication that the majority of the 4-DMAzo moieties were
originally in the (E) form, as was also confirmed by NMR analysis
of 8 (data not shown). All procedures were performed in a dark
room.
Synthesis of Photoresponsive Oligonucleotide 6: To prepare the pho-
toresponsive DNA oligonucleotide with an automated synthesizer
(DNA/RNA synthesizer 392/394, ABI), the phosphoramidite
monomer 5 was diluted to 0.1  in dry acetonitrile. Monomer 5
was coupled with a conventional adenine-phosphoramidite mono-
Kinetic Measurement in DMSO: The rate constants of thermal (Z)-
to-(E) isomerization were estimated by first-order analysis of the
absorbance at λ = 425 nm for at least three measurements. The R
values were Ͼ0.99 for all compounds. The mixtures of solute (6 or
the 4-DMAzo derivatives) and DMSO were dried thoroughly by
incubation with molecular sieves for more than 8 h before each
measurement in a dark place. The concentration of each compound
was as follows: 6 (3.9–8.3 µ), 2 (7.1–8.7 µ), 7 (8.3–8.7 µ), and
mer to synthesize the photoresponsive oligonucleotide on
a
0.2 µmol scale. The synthesized oligonucleotide was adsorbed on a
CPG column (500 Å pore size) and eluted by ammonia treatment
for cleavage. The resultant crude product for HPLC purification
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1846–1853

Photoresponsivity of 4-(Dimethylamino)azobenzene in Single-Stranded DNA
FULL PAPER
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ture. The sample solution was irradiated in
a
cuvette
(10ϫ10ϫ16 mm) at λ = 532 nm with a laser (average power
375 mW; pulse width 3–5 ns). The beam size was ca. 3 mm and was
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and was detected on the other side of the cuvette. Traces after 30
flashes made at 1 s intervals were integrated, and the rate constants
for thermal (Z)-to-(E) isomerization were obtained by first-order
kinetic analysis of the traces. The control oligonucleotide, 5Ј-
AAAAAAAA-3Ј (HPLC-purification grade), was purchased from
Hokkaido System Science.
Acknowledgments
This work was supported by the AIST Upbringing of Talent in
Nanobiotechnology Course, Promotion Budget for Science and
Technology, from the Ministry of Education, Culture, Sports, and
Technology of Japan.
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Received: October 26, 2006
Published Online: February 23, 2007
Eur. J. Org. Chem. 2007, 1846–1853
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1853