Photoswitchable Turn-on Mode Fluorescent Diarylethenes: Strategies for Controlling the Switching Response

Article abstract of DOI:10.1246/bcsj.20170365

A new type of photoswitchable fluorescent diarylethenes, which have no fluorophore unit but emit strong fluorescence (ˉ_f³ 0.9) in the closed-ring isomers, has been developed. They are sulfone derivatives of 1,2-bis(2-alkyl-4-methyl-5-phenyl-3-thienyl)perfluorocyclopentenes and 1,2-bis(2-alkyl-1-benzothiophen-3-yl)perfluorocyclopentenes. By chemical modifications of the structures their switching response was tuned to meet the requirements for super-resolution fluorescence microscopies. The water-soluble derivatives have been successfully applied to acquire super-resolution bioimages using a single-wavelength visible beam.

Full text of DOI:10.1246/bcsj.20170365

Advance Publication Cover Page
Photoswitchable Turn-on Mode Fluorescent Diarylethenes:
Strategies for Controlling the Switching Response
Masahiro Irie* and Masakazu Morimoto
Advance Publication on the web December 19, 2017
doi:10.1246/bcsj.20170365
© 2017 The Chemical Society of Japan

Photoswitchable Turn-on Mode Fluorescent Diarylethenes: Strategies for
Controlling the Switching Response
Masahiro Irie* and Masakazu Morimoto
Department of Chemistry and Research Center for Smart Molecules
Rikkyo University
Nishi-Ikebukuro 3-34-1, Toshima-ku, Tokyo, 171-8501, Japan
Abstract:
A new type of photoswitchable fluorescent diarylethenes, which have no fluorophore unit but
emit strong fluorescence (Φf ~ 0.9) in the closed-ring isomers, has been developed. They are
sulfone derivatives of 1,2-bis(2-alkyl-4-methyl-5-phenyl-3-thienyl)perfluorocyclopentenes and
1,2-bis(2-alkyl-1-benzothiophen-3-yl)perfluorocyclopentenes. By chemical modifications of the
structures their switching response was tuned to meet the requirements for super-resolution
fluorescence microscopies. The water-soluble derivatives have been successfully applied to
acquire super-resolution bioimages using a single-wavelength visible beam.
1

Fluorescence is the most convenient tool to detect small amounts of molecules.1 Even single
molecules can be detected using the fluorescence owing to remarkable progress of optical
-
4
detection techniques.2
Various types of highly fluorescent chromophores have been
synthesized and widely applied in materials and life sciences, such as microanalysis and
bioimaging.5 - 9 Their application is expected to be further extended when an additional
switching property is provided to the chromophores.10 The changes in fluorescence spectra
and/or intensity by external stimuli, such as chemicals, electrons (or holes) or photons, are
ingeniously adopted for the analysis and the imaging. When the fluorescence is photoactivated
or switched on and off upon photoirradiation, the fluorescence modulation offers the
opportunities to monitor diffusion processes in real time,11 – 13 store optical information in
memory media,14 - 20 and increase the resolution in imaging.10, 20 - 23
There are two types of fluorescence switching molecules, photoactivatable and
photoswitchable ones. The photoactivatable molecules irreversibly switch from non-fluorescent
to fluorescent states, while photoswitchable ones undergo reversible switching between
fluorescent and non-fluorescent states. The reversibly photoswitchable molecules can be
designed and constructed by integrating both photochromic and fluorescent chromophores in a
molecule.14 - 20 Based on this strategy numerous photoswitchable fluorescent molecules have
been synthesized. Even limited to molecules having a photochromic diarylethene derivative as a
photoswitching unit, a number of derivatives having a fluorescent unit, such as triphenyl
imidazole,24 anthracene,14, 15, 25, 26 perylenebisimide,16 – 19, 27 or indacene,28 have been prepared.
The molecules are initially fluorescent, while the fluorescence is quenched by an energy or an
electron transfer mechanism when the diarylethene unit converts from the open- to the
closed-ring isomer upon UV irradiation. The fluorescence switching from fluorescent to
non-fluorescent states can be applied to optical memory media but is hardly applicable to
super-resolution bioimaging, which visualizes and tracks microstructures and molecular events
in cells.29
Recent progress of fluorescent microscopies has revolutionized the optical imaging,
especially spatial resolution.30, 31 The diffraction limit no longer restricts the spatial resolution in
optical microscopy; it was overcome by using photoswitching of fluorescent probes between
dark and emitting states. The super-resolution techniques are usually grouped into two
categories, coordinate-targeted and coordinate-stochastic methods. In coordinate-targeted
methods (stimulated emission depression (STED)32 - 34 and reversible saturable (switchable)
optical linear fluorescence transition (RESOLFT) microscopies35, 36), the position of the on and
off states is determined by a doughnut-shaped pattern of light inducing a turn-on or a turn-off
transition in the sample, except at the central position with vanishing intensity. In
coordinate-stochastic super-resolution microscopies (photoactivated localization microscopy
2

(PALM)37, 38 and stochastic optical reconstruction microscopy (STORM)39, 40), fluorescence off–
on events take place molecules by molecules, with the molecules located at random positions.
PALM and STORM are based on the technique to detect single fluorescent molecules.
The reversibly photoswitchable fluorescent molecules can be applied to both RESOLFT
microscopy and PALM/STORM, while the irreversibly photoactivatable molecules can be used
only for PALM/STORM. The application of turn-off mode photoswitchable fluorescent
molecules to PALM/STORM is limited, because the fluorescence quenching, in many cases, is
imperfect and the imaging technique requires a dark background to detect single fluorescent
molecules. For the application, it is strongly desired to develop turn-on mode photoswitchable
fluorescent molecules, which can efficiently and instantaneously activated upon
photoirradiation.
Although several turn-on mode photoactivatable fluorophores, such as dihydrofuran,41
coumarine,42 fluorescein,43 rhodamine44 and anthracene,45 have been reported, their
photoactivation quantum yields are relatively low and the reactions are irreversible. Recently, a
new type of turn-on mode photoswitchable fluorescent molecules have been developed. They
are sulfone derivatives of 1,2-bis(2-alkyl-4-methyl-5-phenyl-3-thienyl)perfluorocyclopentenes46
and 1,2-bis(2-alkyl-1-benzothiophen-3-yl)perfluorocyclopentenes.47 Although they are initially
non-luminous under excitation with visible light, they emit strong fluorescence upon irradiation
with UV light.
In this account we describe how to improve the photochemical and photophysical properties
of the sulfone derivatives by chemical modifications to meet the requirements for
super-resolution fluorescence microscopies. New molecular photoswitches with improved and
outstanding properties will surely contribute to the progress of super-resolution fluorescence
bioimaging.
1. Photoswitchable highly fluorescent diarylethenes
Sulfone derivatives of 1,2-bis(2-alkyl-4-methyl-5-phenyl-3-thienyl)perfluorocyclopentenes.
1,2-Bis(2,4-dimethyl-5-phenyl-3-thienyl)perfluorocyclopentene (1) is one of typical
photochromic diarylethenes.48 - 50 The open-ring 1a isomer converts to the closed-ring 1b isomer
upon irradiation with UV light. The colorless n-hexane solution containing 1a (λmax = 268 nm,
ε = 2.8 × 104 M−1 cm−1) turns to blue due to the formation of 1b (λmax = 562 nm, ε = 1.1 × 104
M−1 cm−1). Upon irradiation with visible light (λ > 450 nm) 1b returns back to 1a and the blue
color disappears. The cyclization and cycloreversion quantum yields were determined to be 0.45
and 0.018, respectively. Although 1 exhibits an excellent color change, both isomers are
non-fluorescent.
During the course of study on the fatigue property of diarylethene derivatives Ahn and
3

co-workers51 - 55 found that oxidation of sulfur atoms to sulfones is effective to provide a
fluorescent property to the derivatives. Following up the research the synthesis of a
dithienylperfluorocyclopentene derivative having thiophene 1,1-dioxide groups was examined.52,
56
As
expected
the
derivative,
1,2-bis(2,4-dimethyl-5-phenylthiophene-1,1-dioxide-3-yl)perfluorocyclopentene (2), switched
to a fluorescent dye upon UV irradiation. Upon irradiation with UV light the open-ring 2a
isomer converted to the closed-ring 2b isomer and the photogenerated 2b emitted fluorescence
under excitation with 423 nm light. The fluorescence quantum yield was, however, as low as
0.03 in 2-methylteterahydrofuran. Although 2 exhibits the turn-on mode fluorescence property,
the low fluorescence quantum yield prevents its practical use. To improve the fluorescence
performance chemical modifications were made and it was found that introduction of short alkyl
chains at 2- and 2’-positions of the thiophene 1,1-dioxide groups is effective to increase the
fluorescence quantum yield.46
Scheme 1 shows the structures of derivatives prepared. Figure 1 shows the absorption and
fluorescence spectra of 3-6 in 1,4-dioxane. The 1,4-dioxane solutions of 3a-6a are colorless and
have no absorption in the visible-wavelength region, while upon irradiation with UV light 3a-6a
converted to 3b-6b and the solutions turned yellow. The closed-ring 3b-6b isomers emit green
fluorescence around 530 nm upon excitation with 430 nm light. Subsequent irradiation with
visible light (λ > 450 nm) caused the cycloreversion reactions from 3b-6b to 3a-6a and the
absorption and fluorescence spectra decreased. Thus, 3-6 undergo cyclization/cycloreversion
reactions and fluorescence switching upon alternate irradiation with UV and visible light.
[Scheme 1]
[Figure 1]
Table 1 summarizes photophysical and photochemical properties of diarylethenes 2-6 in
1,4-dioxane. Alkyl substitution at 2- and 2’-positions markedly affected the fluorescence
quantum yield and the cycloreversion reaction. When methyl substituents were replaced with
ethyl, n-propyl, isopropyl and isobutyl substituents, the fluorescence quantum yield increased
dramatically from 0.07 to 0.42 – 0.50. The fluorescence quantum yield was significantly
improved by replacing the methyl substituents with longer or branched alkyl ones.
Photocyclization and photocycloreversion quantum yields are also shown in Table 1. The
cyclization quantum yields of 2a-6a are moderate and almost in the range of 0.1-0.2. The alkyl
substituents scarcely affected the photocyclization reactivity. The cycloreversion quantum yields
of 2b-5b are in the order of 10−5, which are much lower than the yield of non-oxidized
diarylethene 1b (0.018).50 When sulfur atoms were oxidized to sulfones, the cycloreversion
reaction was strongly suppressed. It is worth to note that the cycloreversion quantum yield of 6b,
which has isobutyl substituents at 2- and 2’-positions, increases to 2.1 × 10−3, which is 81 times
4

larger than that of 5b. Although the origin of the effect of isobutyl substitution is not clear at
present, this result indicates that introduction of isobutyl substituents at the reactive carbons is
useful to increase the cycloreversion quantum yield.
[Table 1]
Figure 2 shows the relationship between the quantum yields and the dielectric constants of
the solvents. Methyl substituted derivative 2b shows relatively high fluorescence quantum yield
in non-polar n-hexane, while the yield dramatically decreases to 0.07 in 1,4-dioxane and further
decreases to 0.02 in ethanol. In contrast, diarylethenes 3b-6b keep relatively high yields even in
ethanol. In ethanol the yields of ethyl and n-propyl substituted derivatives (3b and 4b) are 0.18
and 0.19, respectively. The derivatives having branched alkyl substituents (5b and 6b) show
higher values (5b: 0.32, 6b: 0.27). These results indicate that introduction of branched alkyl
substituents into the reactive carbon atoms is effective to improve the fluorescent performance
in polar solvents.
[Figure 2]
Sulfone derivatives of 1,2-bis(2-alkyl-6-aryl-1-benzothiophen-3-yl)perfluorocyclopentenes.
1,2-Bis(2-methyl-1-benzothiophen-3-yl)perfluoropentene (7) is also one of typical
photochromic diarylethenes, which undergo thermally irreversible and fatigue resistant
photochromic reactions.48, 50, 57, 58 The open-ring 7a isomer converts to the closed-ring 7b isomer
upon irradiation with UV light. The colorless n-hexane solution containing 7a (λmax = 258 nm,
ε = 1.5 × 104 M−1 cm−1) turns red due to the formation of 7b (λmax = 516 nm, ε = 1.0 × 104 M−1
cm−1). Upon irradiation with visible light (λ > 450 nm) 7b turns back to 7a and the red color
disappears. The cyclization and cycloreversion quantum yields were determined to be 0.31 and
0.29, respectively. In contrast to dithienylethene 1 both open- and closed-ring isomers of 7
exhibit weak fluorescence. The quantum yields of the open- and closed-ring isomers are
reported to be 0.012 and ~0.0001, respectively.53, 59
Oxidation of the sulfur atoms to sulfones improved the fluorescent property. Figure 3 shows
the
absorption
and
fluorescence
spectra
of
1,2-bis(2-methyl-1-benzothiophen-1,1-dioxide-3-yl)perfluorocyclopentene (8) in ethyl acetate.51
The absorption spectrum of the open-ring 8a isomer is in the UV region and the ethyl acetate
solution is colorless, while the closed-ring 8b isomer generated by irradiation with UV light has
absorption at 398 nm and the solution is pale yellow. The absorption band shows a significant
hypsochromic shift in comparison with that of non-oxidized 7b. Upon excitation with 400 nm
light 8b exhibits fluorescence. The fluorescence quantum yields of the open- and closed-ring
isomers are reported to be 0.025 and 0.011, respectively.53
[Figure 3]
5

Although both non-oxidized derivative 7 and oxidized sulfone derivative 8 possess the
turn-on mode fluorescence switching property, their fluorescence quantum yields are too low to
be applied to super-resolution fluorescence microscopies. For the application it is required to
improve the fluorescence performance: (i) increase in the absorption coefficient, (ii)
bathochromic shift of the absorption and fluorescence spectra, and (iii) increase in the
fluorescence quantum yield.
Just as examined in sulfone derivatives of 1,2-bisthienylethenes linear or branched alkyl
chains were substituted at 2- and 2’-positions of the benzothiophene 1,1-oxide groups.78, 60 Aryl
substituents were also introduced at 6- and 6’-positions. Scheme 2 shows the structures of the
modified sulfone derivatives. Both alkyl and aryl substituents markedly influenced the
photochromic performance and the fluorescence property. Figure 4 shows the absorption and
fluorescence spectra of 9 having methyl substituents at 2- and 2’-positions and 12 having
n-butyl substituents in 1,4-dioxane. The open-ring 9a and 12a isomers have no absorption in the
visible wavelength region (λ > 400 nm) and convert to the closed-ring 9b and 12b isomers
having absorption in the visible wavelength region upon irradiation with UV (330 nm) light.
The absorption maxima of 9a and 12a are observed at 296 nm and 298 nm, respectively, while
those of 9b and 12b show the maxima at 443 nm and 456 nm, respectively. The absorption
maxima of the closed-ring isomers of these aryl substituted derivatives showed a significant
bathochromic shift in comparison with that of non-substituted derivative 8b. In addition, the
absorption coefficients increased two or three times. Both closed-ring isomers exhibited brilliant
green fluorescence at around 540 nm. The fluorescence quantum yield was found to increase
from 0.011 up to 0.89 in 1,4-dioxane by introducing n-propyl substituents at 2- and 2’-positions
and phenyl substituents at 6- and 6’-positions.
[Scheme 2]
[Figure 4]
Table 2 summarizes photophysical and photochemical properties of the derivatives having
various alkyl substituents. As can be seen from Table 2 the absorption maxima of the open-ring
isomers are similar, but the absorption maxima of the closed-ring isomers show significant
bathochromic shifts when substituents are altered from methyl to ethyl, n-propyl, n-butyl or
isobutyl substituents.
[Table 2]
Solvent polarity dependence of the fluorescence quantum yields of 9b-13b was examined as
shown in Figure 5. The quantum yield of 9b having methyl substituents dramatically decreases
with increase in the solvent polarity, while the decrease is moderate for ethyl, n-propyl, n-butyl
and isobutyl substituted derivatives 10b-13b. The ethyl-substituted derivative 10b keeps the
quantum yield of 0.70 even in polar acetonitrile, while the methyl-substituted derivative 9b
6

decreases the yield to 0.15. The different behavior can be explained as follows. In polar solvents,
intermolecular interactions between the sulfone moieties and polar solvent molecules are
considered to accelerate the non-radiative decay process. When bulky alkyl substituents are
introduced at the connecting carbon atoms, they can defend the sulfone moieties from the attack
of polar solvent molecules and suppress the non-radiative decay process. Thus, the derivatives
having bulky alkyl substituents keep the relatively high fluorescence quantum yield even in
polar solvents.
[Figure 5]
Figure 6 shows the absorption and fluorescence spectra of 14 in 1,4-dioxane.48 Replacement
of phenyl substituents at 6- and 6’-positions with thiophene rings shifts the absorption
maximum of the closed-ring 14b isomer to 506 nm (ε = 5.8 × 104 M−1 cm−1). The closed-ring
isomer can be excited by irradiation with 532 nm light and emits brilliant orange fluorescence.
The fluorescence quantum yield was measured to be 0.78 in 1,4-dioxane. 14b is thermally as
well as photochemically stable and hardly return back to the open-ring 14a isomer by irradiation
with visible light (the cycloreversion quantum yield < 10−5). Figure 7 shows photographs of the
fluorescence switching of 1,4-dioxane solutions containing 10 and 14 upon irradiation with 365
nm light under excitation with 488 nm (left-side solution containing 10) and 532 nm light
(right-side solution containing 14). Before UV irradiation both solutions are non-luminous,
while brilliant green and orange fluorescence emissions instantaneously appear upon UV
irradiation. The emissions are attributable to the formation of fluorescent 10b and 14b isomers.
[Figure 6]
[Figure 7]
2. Control of Cycloreversion Quantum Yields
Coordinate-targeted super-resolution fluorescence microscopy, such as RESOLFT microscopy
claims easy photoswitching of probe molecules from on-states to off-states upon irradiation with
36, 61
a doughnut-shaped low-power laser beam.35,
Therefore, it is required to develop
photoswitchable fluorescent molecules with high switching-off (cycloreversion) efficiency.
According to numerical simulations,62 the resolution of images increases with increasing the
switching-off quantum yield. However, if the quantum yield is too high, the irradiation that is
required to probe the residual fluorescence will also deplete the fluorescence emission. To avoid
the reduction in the fluorescence intensity during the probe phase, the quantum yield less than
10−2 is preferable. Taking into consideration of these factors, the optimal switching-off quantum
yield is concluded to be in the order of 10−2 - 10−3.
To obtain the desirable cycloreversion quantum yield for the RESOLFT microscopy, various
substituents were introduced at 6- and 6’-positions of the sulfone derivatives of
7

1,2-bis(2-ethyl-1-benzothiophen-3-yl)perfluorocyclopentene.63 The structures of the substituted
derivatives are shown in Scheme 3. Although the cycloreversion quantum yield of unsubstituted
sulfone derivative 15b is 0.18,47 introduction of phenyl substituents at 6- and 6’-positions, such
as 10b, decreases the yield to 5.9 × 10−4. This result suggests that the cycloreversion quantum
yield can be controlled by changing the aryl substituents, which affect the π-conjugation length
and the coplanarity of the closed-ring isomers. In other words, the dihedral angle between the
phenyl ring at 6-position (or 6’-position) and the oxidized benzothiophene is considered to
influence the cycloreversion quantum yield.
[Scheme 3]
The absorption maxima of the open-ring isomers 15a, 16a, 17a and 18a were observed at
275 nm and 310 nm (15a), 293 nm and 328 nm (16a), 289 nm and 326 nm (17a), and 285 nm
and 320 nm (18a), while those of the closed-ring isomers 15b, 16b, 17b, and 18b were observed
at 414 nm, 445 nm, 441 nm, and 430 nm, respectively. The absorption maxima of the
closed-ring isomers having ortho-substituted phenyl rings, 16b, 17b, and 18b, showed a small
but significant hypsochromic shift in comparison with that of 10b (456 nm). The fluorescence
quantum yields of 10b, 16b, 17b, and 18b were determined to be 0.87, 0.79, 0.84 and 0.79 in
1,4-dioxane, respectively. The yields are very similar and the ortho-substitution scarcely affects
the fluorescence process.
Table 3 shows the cyclization and cycloreversion quantum yields of the derivatives having
ortho-substituted phenyl rings. As shown in Tables 2 and 3, the cyclization quantum yields of
the four derivatives, 10a, 16a, 17a, and 18a, are similar each other. On the other hand, the
cycloreversion quantum yields are strongly dependent on the ortho-substitution. As described
the previous section, the yield of 10b is as low as 5.9 × 10−4, while the yield increases to 2.8 ×
10−3 (16b), 3.2 × 10−3 (17b), and 1.8 × 10−2 (18b) by introducing fluorine or methyl group(s) at
ortho-position(s) of the phenyl rings. By replacing ortho-hydrogen with fluorine the yield
increased by 5 times. The introduction of ortho-dimethyl substituents increased the yield by as
large as 30 times. Ortho-substitution is very effective to increase the cycloreversion quantum
yield to 10−2 – 10−3.
[Table 3]
Ortho-substitution is considered to decrease the coplanarity of the closed-ring isomers.
Table 3 also shows the dihedral angles between the phenyl rings and the oxidized
benzothiophene, which were measured by X-ray crystallographic analysis. When
ortho-hydrogen is replaced with fluorine atom, such as 16b, the dihedral angle increases from
30.2° to 35.9°. It further increases to 61.0° by introducing a methyl group. The dihedral angle of
the derivative having ortho-dimethyl substituted phenyl ring is as large as 65.7°. The increase in
the dihedral angle loses the coplanarity of the closed-ring isomer and decreases the extension of
8

π-conjugation length, resulting in the hypsochromic shift of the absorption spectrum and the
increase in the cycloreversion quantum yield.
Figure 8 shows the correlation between absorption maxima of the closed-ring isomers and
cyclization/cycloreversion quantum yields in less polar 1,4-dioxane and polar ethanol.
Fluorescence quantum yields of the closed-ring isomers are also shown. Although the shift of
the absorption maxima of the closed-ring isomers by ortho-substitutions scarcely affects the
fluorescence quantum yields as well as cyclization quantum yields, it strongly increases the
cycloreversion quantum yields by as large as 30 times. The photogenerated closed-ring isomers,
16b, 17b, and 18b, exhibit brilliant green fluorescence with quantum yields higher than 0.79
and their cyclization quantum yields are higher than 0.58 in 1,4-dioxane. In addition, the
cycloreversion quantum yields are in the range of 2.8 × 10−3 – 1.8 × 10−2. These compounds
fulfill the necessary requirements for RESOLFT super-resolution microscopy.
[Figure 8]
Another strategy to increase the cycloreversion quantum yield is to introduce isobutyl
substituents at reactive carbon atoms. As shown in Tables 1 and 2, the cyclization quantum
yields of 6b and 13b having isobutyl substituents at 2- and 2’-positions of thiophene 1,1-dioxide
or benzothiophene 1,1-dioxide are larger than the values of the derivative having ethyl
substituents. The yields of 6b and 13b are more than one order of magnitude (19 – 55 times)
larger than those of 3b and 10b. The cycloreversion quantum yields of these derivatives in the
range of 10−2 – 10−3 are favorable for the application to RESOLFT microscopy.
3. Reversible Fluorescence Photoswitching by Irradiation with Single-Wavelength Visible
Light 64
As described in Introduction, localization super-resolution fluorescence microscopies, such as
PALM and STORM, are based on single-molecule fluorescence detection. The large majority of
probe molecules must be in the off states, except for a very sparse population that is in the on
states. Both techniques require a dark background to detect single fluorescent molecules and
need to turn on and off independently the emission of fluorophores positioned within the same
diffraction limit. By adjusting the irradiation conditions on intensities of photoactivation as well
as photobleaching only a sparse population of probe molecules is maintained in the emissive
state during the image reconstruction sequence. Each localized probe at the single-molecular
level is visualized in the form of a diffraction pattern and the center of the pattern can be
approximated as the center of the molecule. The coordinate of each localized probe can then be
stored and accumulated. This sequence of events, activation, localization and bleaching, is
reiterated multiple times, until enough coordinates become available to reconstruct a complete
image of the sample.
9

The diarylethenes so far synthesized, except several specially designed derivatives,65 - 67
cannot be activated with visible light (> 400 nm). The visible light responsive property is
favorable for the application to biological systems. During the course of study of
photoswitchable diarylethenes we have discovered unprecedented photoreactions upon
irradiation with single-wavelength visible light. Upon irradiation with the visible light both
cyclization and cycloreversion reactions simultaneously take place and a very sparse population
of molecules is reversibly activated to on states.
Upon irradiation with UV light (365 nm light) the open-ring 10a isomers readily converted
to the closed-ring 10b isomers, which have absorption at 456 nm and give fluorescence at
around 550 nm under excitation with 450 nm light. The photogenerated 10b isomers were
bleached upon irradiation with 450 nm light and reverted back to the open-ring 10a isomers.
Although both absorption and fluorescence spectra considerably decreased, noticeable intensity
of fluorescence ascribed to 10b remained even after prolonged irradiation with 450 nm light.
10a has no apparent absorption at 450 nm. This means that all closed 10b isomers are expected
to completely revert back to the open-ring 10a isomers by irradiation with 450 nm light. But, a
small amount of 10b survived. This result indicates that 450 nm light induces the cyclization
reaction of 10a in addition to the cycloreversion reaction.
We assumed that the reversible photoisomerizations are induced by irradiation with
single-wavelength (λ) light.
Open-ring isomer 10a
Closed-ring isomer 10b
(1)
(2)
ꢀꢁ
B
ꢄ
ꢇ
ꢃ ABAꢅA ꢆ BABꢅB ꢈ
ꢀꢂ
ꢌꢍ
ꢎ ꢏ ꢎ ꢐꢑ
A
B B
ꢕ
A
ꢈ ꢃ ꢉꢊꢋ
ꢓ ꢉꢔ ꢖ
ꢗ
 ꢁ ꢒ ꢁ
A B B
ꢘ
A
where ΦAB, ΦBA, εA, εB, CA, CB, d, and I0 are the cyclization quantum yield, the cycloreversion
quantum yield, the absorption coefficient of 10a at λ, the absorption coefficient of 10b at λ, the
concentration of 10a (mol/L), the concentration of 1b (mol/L), the cell length (cm), and the light
intensity (einstein/cm2·s), respectively. When εACA + εBCB << 1, the formation of the
closed-ring isomers is expressed as follows, where CO is the total concentration of 10a and 10b.
ꢕ


A
AB
ꢄ
ꢇ

B
 ꢒ
A
ꢅOꢍ1 ꢆ ꢚꢊ 

B
ꢓꢉꢔ ꢖ ꢂꢐ
(3)
AB
BA
ꢗ
ꢅꢙ ꢃ 
 ꢒ
AB
A
BA
In the photostationary state,
ꢁꢛ
ꢁꢗ
ABA
ꢃ 
(4)
ABAꢒBA B

Figure 9 shows the formation of 10b from pure 10a in n-hexane and CCl4 upon irradiation
10

with 450 nm light. To detect a very small amount of 10b, less than 10−7 M, fluorescence was
used to measure the concentration. The formation curves were analyzed according to eq. (3). It
is possible to determine the very weak absorption coefficients of 10a at 450 nm from the
concentration ratios of 10a and 10b in the photostationary state from eq. (4). Based on
numerical simulations of the formation process of 10b from 10a the absorption coefficients at
450 nm in n-hexane and CCl4 were estimated to be 0.084 M−1 cm−1 and 0.19 M−1 cm−1,
respectively. Although the absorption coefficients were very low, the formation of 10b was
clearly observed upon irradiation with 450 nm light. This is attributed to very low
cycloreversion quantum yields of 10b in n-hexane and CCl4.
[Figure 9]
According to Wondrazek et al.68 and Kinoshita et al.69 the absorption spectra (or the
emission excitation spectra) of fluorescent dyes, such as coumarin 7 and rhodamine 6G, show
an exponential frequency dependence in the off-resonance region of the absorption tails at room
temperature. The tails have been reported to obey the Urbach rule known in the absorption
spectra of solid materials, as expressed by the following formula.68 - 72
ꢄ
ꢇ
ꢞ ꢟ ꢊꢟ
ꢗꢗ
ꢄ ꢇ
ꢄ
ꢇ
ε ꢜ ∝ ε ꢜꢔꢔ exp ꢝꢆ
ꢢ
(5)
ꢠ ꢡ
B
where ε, kB , T, and σ are the absorption coefficient, the Boltzmann constant, the absolute
temperature of the sample and the steepness parameter, respectively.
The absorption coefficients estimated from eq. (4) were plotted in Figure 10 along with the
absorption tails of the open-ring 10a isomer. As can be seen in Figure 10 the coefficients obey
the exponential frequency dependence at the wavelengths longer than ~ 415 nm (~ 24000 cm−1).
The exponential frequency dependence indicates that the photoreactions with the visible light in
the far-resonance region of the absorption edge are caused by the absorption of hot bands or
Urbach tails. The open-ring 10a isomers in the vibrationally excited states undergo optical
transition into electronically excited states by absorbing the very weak hot bands and the
cyclization reaction from 10a to 10b takes place. The reversible fluorescence photoswitching
with single-wavelength visible light is advantageously applicable to super-resolution
microscopies, as shown in the next section.
[Figure 10]
4. Application to Super-resolution Bioimaging 73 - 76
As already being discussed in the previous sections, properly designed sulfone derivatives of
bisthienylethenes and bisbenzothienylethenes fulfill the requirements for the application to
super-resolution fluorescence microscopies. For the application to bioimaging another
indispensable property is water solubility. Several approaches to provide water solubility to
11

diarylethenes have been reported.77 In order to give both the water solubility and bioconjugation
ability we introduced several carboxylic acid groups to the fluorescent diarylethenes. The
presence of eight caroboxylates provided a fairly good solubility at pH > 5 and inhibited
aggregation even after photocyclization. One of the carboxylates can be conveniently
transformed into any reactive group for bioconjugation. Remarkably, the bioconjugates retain
the photoswitching and fluorescent properties of the derivatives. The caroboxylated diarylethene
19 was applied to RESOLFT bioimaging.
To demonstrate the applicability of 19 for RESOLFT microscopy, fixed immunolabeled
Vero cells were imaged using a modified IC RESOLFT QUAD Scanning microscope (Abberior
Instruments, Göttingen). The imaging was performed by applying the following pulse sequence:
switching the diarylethenes to their closed-ring isomers with 355 nm light (130 W cm−2) for 50
μs, 200 μs delay, application of the doughnut-shaped 488 nm beam (36 kW cm−2) for 1.2 ms to
switch the diarylethenes in the periphery of the focal spot to their non-fluorescent open-ring
isomers. Finally, the remaining area with the fluorescent isomers was probed for 80 μs with a
Gaussian shaped 488 nm light beam (9.7 kW cm−2). The confocal image was obtained by the
same pulse sequence without the off-switching. Using this imaging scheme, it was possible to
acquire RESOLFT images with a resolution of 74 nm FWHM (full width at half minimum) and
confocal images of 175 nm FWHM. Figure 11 shows the RESOLFT and confocal images.
[Figure 11]
The success of the use of the diarylethene for RESOLFT is attributed to the relatively large
cycloreversion quantum yield. The yield of 19b was measured to be 2.0 × 10−3 ± 3 × 10−4. 19b
was switched off in 1.5 ms. The resolution achieved in RESOLFT is limited mainly by the
photobleaching rate and not by the residual fluorescence after off-switching. RESOLFT
microscopy requires the cycloreversion quantum yield in the order of 10−2 – 10−3.
On the contrary, coordinate-stochastic super-resolution fluorescence microscopies, such as
PALM and STORM, require a low cycloreversion quantum yield, because the resolution of
these techniques depends on the localization process. Two important requirements for the probes
are (i) a high number of photons detected during the on state to ensure a high localization
accuracy, and (ii) the possibility to achieve a high [off]/[on] ratio to allow for a high labeling
density. The second requirement is inherent to diarylethenes, because a complete conversion to
open-ring isomers is achieved when the irradiation wavelength is properly selected to avoid
excitation of hot bands or Urbach tails. The first requirement is dependent on both the ratio of
emission efficiency (Φf) and the cycloreversion quantum yield (ΦBA). 19b used in the above
RESOLFT microscopy presents Φf/ΦBA < 300. This means that only 30 photons are expected to
be detected during the on state in average, because a photon collection efficiency is around 10%.
12

To increase the localization accuracy, it is required to increase Φf/ΦBA one order of magnitude.
The preferable cycloreversion quantum yield is in the order of 10−4 or less. A sulfone derivative
20 having methoxy substituents and eight carboxylates in both sides was prepared and used for
STORM. The cycloreversion and the fluorescence yields of 20b were measured to be 1.8 × 10−4
and 0.45, respectively. Φf/ΦBA increased to 2500.
The standard labeling protocol provided a reproducible control of the degree of labeling.
The obtained bioconjugate was stable and showed no precipitation or aggregation after several
months. The diarylethene retained the photoswitching ability even after bioconjugation with
secondary antibodies and presented no reduction in the emission efficiency of the closed-ring
isomer. Figure 12 shows the demonstration of wide-field STORM images. Photoactivation was
carried out with 405 or 375 nm laser. Remarkably, it was possible to acquire up to - 200000
frames before any additional photoactivation with UV light. In other words, the on and off
fluorescence switching of a sparse population of probe molecules can be attained by irradiation
with only 488 nm laser. This imaging technique, which uses a single-wavelength visible beam
without harmful UV light, is favorable for the application to biological systems. As described in
the previous section, the activation in the absence of UV light is ascribed to the optical
transition induced by the absorption of very weak hot bands or Urbach tails. Images acquired
without and with UV photoactivation laser (375 nm) were identical within experimental error, as
shown in Figure 12(b).
[Figure 12]
Here, super-resolution fluorescence imaging using photoswitchable fluorescent
diarylethenes was demonstrated. By properly tuning the structures the derivatives were
successfully applied to RESOLFT microscopy and STORM.
5. Summary and Future Prospect
A new type of turn-on mode fluorescent diarylethenes, which have no fluorophore unit but emit
strong fluorescence (Φf ~ 0.9) in the closed-ring isomers, has been synthesized and successfully
applied to super-resolution fluorescence imaging, such as RESOLFT microscopy and
PALM/STORM.
Oxidation
of
sulfur
atoms
of
1,2-bis(2-alkyl-4-methyl-5-phenyl-3-thienyl)perfluorocyclopentenes
and
1,2-bis(2-alkyl-1-benzothiophen-3-yl)perfluorocyclopentenes was effective to provide a highly
fluorescent property to the closed-ring isomers. By chemical modifications of the structures the
physical and chemical properties of the diarylethenes were tuned to meet the requirements for
the microcopies. RESOLFT microcopy requires the cycloreversion quantum yield in the order
of 10−2 – 10−3, while the yield in the order of 10−4 or less is claimed in PALM/STORM imaging.
The cycloreversion as well as fluorescence quantum yields were optimized by introducing
13

appropriate alkyl and aryl substituents to thiophene 1,1-dioxide and benzothiophene 1,1-dioxide
groups. The water-soluble derivatives have been applied to acquire super-resolution bioimages
using a single-wavelength visible beam.
The super-resolution microscopies using photoswitchable diarylethenes can be applied not
only to biological systems but also to materials science. It is capable of resolving apolar
nano-structures in cylindrical micelles formed by amphiphilic block copolymers by using
super-resolution PALM.74 The progress of super-resolution microscopy technique is continuing.
Recently, Hell and co-workers78 reported a new nanometer-resolution imaging method named
MINFLUX. The method can image individual molecules separated by just 1 nm. The present
photoswitchable diarylethenes have a potential to be used in the MINFLUX microscopy.
Acknowledgments
This work was supported by MEXT-Supported Program for Scientific Research Foundation at
Private Universities. We are grateful to Prof. K.-H. Ahn (Kyung Hee University) for early
discussion on the synthesis of sulfone derivatives. We also wish to express our thanks to Prof. S.
W. Hell and Dr. V. N. Belov (Max Planck Institute for Biophysical Chemistry) for their
guidance and collaboration to use the compounds for super-resolution fluorescence
microscopies.
14

References
1. J. R. Lakowicz, Principles of fluorescent spectroscopy, 3rd ed. Springer, New York, 2010.
2. W. E. Moerner, L. Kador, Phys. Rev. Lett. 1989, 62, 2535.
3. E. Betzig, R. J. Chichester, Science 1993, 262, 1422.
4. R. Rigler, M. Oritt, T. Basché, Single molecule spectroscopy. Nobel conference lectures
Springer-Verlag, Berlin, 2001.
5. S. W. Thomas, G. D. Joly, T. M. Swager, Chem. Rev. 2007, 107, 1339.
6. X. Chen, T. Pradhan, F. Wang, J. S. Kim, J. Yoon, Chem. Rev. 2012, 112, 1910.
7. X. F. Wang, B. Herman, Fluorescence Imaging Spectroscopy and Microscopy John Wiley
& Sons, New York, 1996.
8. R. Ando, H. Mizuno, A. Miyawaki, Science 2004, 306, 1370.
9. A. S. Stender, K. Marchuk, C. Liu, S. Sander, M. W. Meyer, E. A. Smith, B. Neupane, G.
Wang, J. Li, J-. X. Cheng, B. Huang, N. Fang, Chem. Rev. 2013, 113, 2469.
10. F. M. Raymo, Phys. Chem. Chem. Phys. 2013, 15, 14840.
11. T. J. Johnson, D. Ross, M. Gaitan, L. E. Locascio, Anal. Chem. 2001, 73, 3656.
12. J. Patrick, D. T. Chiu, Anal. Chem. 2003, 75, 1387.
13. G. Zheng, Y-M. Guo, W-H. Li, J. Am. Chem. Soc. 2007, 129, 10616.
14. M. Irie, T. Fukaminato, T. Sasaki, N. Tamai, T. Kawai, Nature 2002, 420, 759.
15. T. Fukaminato, T. Sasaki, T. Kawai, N. Tamai, M. Irie, J. Am. Chem. Soc. 2004, 126,
14843.
16. T. Fukaminato, T. Umemoto, Y. Iwata, S. Yokojima, M. Yoneyama, S. Nakamura, M. Irie,
J. Am. Chem. Soc. 2007, 129, 5932.
17. T. Fukaminato, T. Doi, N. Tamaoki, K. Okuno, Y. Ishibashi, H. Miyasaka, M. Irie, J. Am.
Chem. Soc. 2011, 133, 4984.
18. M. Berberich, A.-M. Krause, M. Orlandi, F. Scandola, F. Würthner, Angew. Chem. Int. Ed.
2008, 47, 6616.
19. M. Berberich, M. Natali, O. Spenst, C. Chiorboli, F. Scandola, F. Würthner, Chem. Eur. J.
2012, 18, 13651.
20. T. Fukaminato, J. Photochem. Photobiol. C. 2011, 12, 177.
21. M. Heilmann, P. Dedecker, J. Hofkens, M. Sauer, Laser Photon. Rev. 2009, 3, 180.
22. A. Fürstenberg, M. Heilemann, Phys. Chem. Chem. Phys. 2013, 15, 14919.
23. J. Kwon, J. Hwang, J. Park, G. R. Han, K. Y. Han, S. K. Kim, Sci. Rep. 2014, 5, 17804.
24. K. Yagi, C. F. Soong, M. Irie M, J. Org. Chem., 2001, 66, 5419.
25. A. de Meijere, L. Zhao, V. N. Belov, M. L. Bossi, M. Noltemeyer, S. W. Hell, Chem. Eur.
J. 2007, 13, 2503.
26. H. Ohara, M. Morimoto, M. Irie, Photochem. Photobiol. Sci. 2010, 9, 1079.
15

27. M. Pärs, C. C. Hofmann, K. Willinger, P. Bauer, M. Thelakkat, J. Köhler, Angew. Chem.
Int. Ed. 2011, 50, 11405.
28. T. A. Golovkova, D. V. Kozlov, D. C. Neckers, J. Org. Chem. 2005, 70, 5545.
29. P. Sengupta, S. B. van Engelenburg, J. Lippincott-Schwartz, Chem. Rev. 2014, 114, 3189.
30. S. W. Hell, Science 2007, 316, 1153.
31. S. W. Hell, Nat. Methods 2009, 6, 24.
32. S. W. Hell, J. Wichmann, J. Opt. Lett. 1994, 19, 780.
33. V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, Science 2008,
320, 246.
34. S. Berning, K. I. Willing, H. Steffens, P. Dibai, S. W. Hell, Science 2012, 335, 551.
35. S. W. Hell, S. Jakobs, L. Kastrup, Appl. Phys. A 2003, 77, 859.
36. M. Hoffmann, C. Eggeling, S. Jakobs, S. W. Hell, Proc. Nat. Acad. Sci. USA 2005, 102,
17565.
37. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M.
W. Davidson, J. Lippincott-Schwartz, H. F. Hess, Science 2006, 313, 1642.
38. S. T. Hess, T. P. K. Girirajan, M. D. Mason, Biophys. J. 2006, 91, 4258.
39. M. J. Rust, M. Bates, X. Zhuang, Nat. Methods 2006, 3, 793.
40. M. Bates, B. Huang, G. T. Dempsey, X. W. Zhuang, Science 2007, 317, 1749.
41. S. J. Lord, N. R. Conley, H. D. Lee, R. Samuel, N. Liu, R. J. Twieg, W. E. Moerner, J. Am.
Chem. Soc. 2008, 130, 9204.
42. E. Deniz, M. Tomasulo, J. Cusido, I. Yildiz, M. Petriella, M. L. Bossi, S. Sortino, F. M.
Raymo, J. Phys. Chem. C. 2012, 116, 6058.
43. T. Kobayashi, Y. Urano, M. Kamiya, T. Ueno, H. Kojima, T. Nagano, J. Am. Chem. Soc.
2007, 129, 6696.
44. J. Fölling, V. N. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. L.
Bossi, S. W. Hell, Angew, Chem. Int. Ed. 2007, 46, 6266.
45. E. R. Thapaliya, B. Captain, F. M. Raymo, J. Org. Chem. 2014, 79, 3973.
46. M. Morimoto, T. Sumi, M. Irie, Materials 2017, 10, 1021.
47. K. Uno, H. Niikura, M. Morimoto, Y. Ishibashi, H. Miyasaka, M. Irie, J. Am. Chem. Soc.
2011, 133, 13558.
48. M. Irie, T. Fukaminato, K. Matsuda, S. Kobatake, Chem. Rev. 2014, 114, 12174.
49. M. Irie, K. Sakemura, M. Okinaka, K. Uchida, J. Org. Chem. 1995, 60, 8305
50. T. Sumi, Y. Takagi, A. Yagi, M. Morimoto, M. Irie, Chem. Commun. 2014, 50, 3928.
51. Y-C. Jeong, S. I. Yang, K-h. Ahn, E. Kim, Chem. Commun. 2005, 2503.
52. Y-C. Jeong, E. Kim, K-H. Ahn, S. I. Yang, Bull. Korean Chem. Soc. 2005, 26, 1675.
53. Y-C. Jeong, S. I. Yang, E. Kim, K-H. Ahn, Tetrahedron 2006, 62, 5855.
16

54. Y-C. Jeong, D. G. Park, E. Kim, K-H. Ahn, S. I. Yang, Chem. Commun. 2006, 1881.
55. Y-C. Jeong, D. G. Park, I. S. Lee, S. I. Yang, K-H. Ahn, J. Mater. Chem. 2009, 19, 97.
56. M. Taguchi, T. Nakagawa, T. Nakashima, T. Kawai, J. Mater. Chem. 2011, 21, 17425.
57. M. Hanazawa, R. Sumiya, Y. Horikawa, M. Irie, J. Chem. Soc., Chem. Commun. 1992, 206.
58. K. Uchida, E. Tsuchida, Y. Aoi, S. Nakamura, M. Irie, Chem. Lett. 1999, 28, 63.
59. S. Shim, T. Joo, S. C. Bae, k. S. Kim, E. Kim, J. Phys. Chem. A 2003, 107, 8106.
60. Y. Takagi, T. Kunishi, T. Katayama, Y. Ishibashi, H. Miayasaka, M. Morimoto, M. Irie,
Photochem. Photobiol. Sci. 2012, 11, 1661.
61. T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I.
Willig, C. Eggelling, S. Jakobs, S. W. Hell, Nature 2011, 478, 204.
62. P. Dedecker, J-i Hotta, C. Flors, M. Sliwa, H. Uji-i, M. B. J. Roeffaers, R. Ando, H.
Mizuno, A. Miyawaki, J. Hofkens, J. Am. Chem. Soc. 2007, 129, 16132.
63. Y. Takagi, M. Morimoto, R. Kashihara, S. Fujinami, S. Ito, H. Miayasaka, M. Irie,
Tetrahedron 2017, 73, 4918.
64. R. Kashihara, M. Morimoto, S. Ito, H. Miyasaka, M. Irie, J. Am. Chem. Soc. 2017, 139,
16498.
65. T. Sumi, T. Kaburaki, M. Morimoto, K. Une, H. Sotome, S. Ito, H. Miyasaka, M.
Irie, Org. Lett. 2015, 17, 4802.
66. T. Fukaminato, T. Hirose, T. Doi, M. Hazama, K. Matsuda, M. Irie, J. Am. Chem. Soc.
2014, 136, 17145.
67. S. Fredrich, R. Göstl, M. Herder, L. Grubert, S. Hecht, Angew. Chem. Int. Ed. 2016, 55,
1208.
68. F. Wondrazek, A. Seilmeier, W. Kaiser, Chem. Phys. Lett. 1984, 104, 121.
69. S. Kinoshita, N. Nishi, A. Saitoh, T. Kushida, J. Phys. Soc. Jpn. 1987, 56, 4162.
70. K. Mizuno, A. Matsui, G. J. Sloan, J. Phys. Soc. Jpn. 1984, 53, 2799.
71. F. Urbach, Phys. Rev. 1953, 92, 1324.
72. J. B. Birks, Photopysics of Aromatic Molecules; John Wiley & Sons Ltd: London, 1970.
73. B. Roubinet, M. L. Bossi, P. Alt, M. Leutenegger, H. Shojaei, S. Schnorrenberg, S. Nizamov,
M. Irie, V. N. Belov, S. W. Hell. S. Angew. Chem. Int. Ed. 2016, 55, 15429.
74. O. Nevskyi, D. Sysoiev, A. Oppermann, T. Huhn, D. Wöll, D. Angew. Chem. Int. Ed. 2016,
55, 12698.
75. Y. Arai, S. Ito, H. Fujita, Y. Yoneda, T. Kaji, S. Takei, R. Kashihara, M. Morimoto, M. Irie,
H. Miyasaka, Chem. Commun. 2017, 53, 4066.
76. B. Roubinet, M. Weber, H. Shojaei, M. Bates, M. L. Bossi, V. N. Belov, M. Irie, S. W. Hell.
J. Am. Chem. Soc. 2017, 139, 6611.
77. Y. Shoji, A. Yagi, M. Horiuchi, M. Morimoto, M. Irie, Isr. J. Chem. 2013, 53, 303.
17

78. F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, S. W.
Hell, Science 2017, 355, 606.
18

Scheme 1. Photoisomerization of diarylethenes 1–6.
19

Scheme 2. Photoisomerization of diarylethenes 7–14.
20

Scheme 3. Photoisomerization of diarylethenes 15–18.
21

Table 1. Photophysical and photochemical properties of 2‒6 in 1,4-dioxane. λmax: absorption maximum, ε:
molar absorption coefficient, Φoc: cyclization quantum yield under irradiation with 313 nm light, Φco:
cycloreversion quantum yield under irradiation with 450 nm light, Φf: fluorescence quantum yield under
irradiation at the absorption maximum.
Open-ring isomer, a
Closed-ring isomer, b
λ
max / nm
λmax / nm
(ε / 104 M−1 cm−1)
Φoc
Φco
Φf
(ε / 104 M−1 cm−1)
2
3
4
5
6
296 (0.79)
297 (0.79)
298 (0.80)
301 (0.79)
299 (0.83)
0.14
0.09
0.10
0.21
0.12
424 (2.4)
3.5 × 10−5
3.8 × 10−5
7.1 × 10−5
2.6 × 10−5
2.1 × 10−3
0.07
0.42
0.42
0.42
0.50
433 (2.2)
434 (2.1)
427 (1.8)
438 (2.1)
22

Table 2. Photophysical and photochemical properties of 8‒14. λmax: absorption maximum, ε: molar
absorption coefficient, Φoc: cyclization quantum yield, Φco: cycloreversion quantum yield, Φf: fluorescence
quantum yield. Ethyl acetate and 1,4-dioxane were used as solvents for 8 and 9‒14, respectively.
Open-ring isomer, a
Closed-ring isomer, b
λ
max / nm
λmax / nm
(ε / 104 M−1 cm−1)
Φoc
Φco
Φf
(ε / 104 M−1cm−1)
8
276 (0.37), 308 (0.41)
0.22 a
0.61 b,c,g
0.62 b,c,g
0.58 c,g
0.56 c,g
0.62 b
398 (2.10)
0.061 d
0.011
0.64
0.87
0.89
0.85
0.80
0.78
9
296 (1.8), 335 (1.4)
298 (1.9), 336 (1.5)
297 (2.0), 336 (1.6)
298 (2.0), 336 (1.6)
300 (1.6), 336 (1.4)
330 (2.4), 374 (2.5)
443 (5.1)
456 (4.6)
456 (4.5)
456 (4.5)
460 (3.7)
506 (5.8)
1.2 × 10−3 e,g
5.9 × 10−4 e,g
9.0 × 10−4 e,g
1.2 × 10−3 e,g
1.1 × 10−2 e
10
11
12
13
14
0.23 c,g
< 1.0 × 10−5 e,f,g
a Under irradiation with 312 nm light.
b Under irradiation with 313 nm light.
c Under irradiation with 330 nm light.
d Under irradiation at λmax of closed-ring isomer.
e Under irradiation with 450 nm light.
f Under irradiation with 488 nm light.
g
Re-examined using a photoreaction quantum yield measuring system (Shimadzu, QYM-01) with a
corrected power meter.
23

Table 3. Photophysical photochemical properties of 15–18 in 1,4-dioxane. λmax: absorption maximum, ε: absorption
coefficient, Φoc: cyclization quantum yield, Φco: cycloreversion quantum yield, Φf: fluorescence quantum yield, τf:
fluorescence lifetime. ω: averaged dihedral angle between the phenyl ring and the oxidized benzothiophene observed by
X-ray crystallographic analysis.
Open-ring isomer, a
Closed-ring isomer, b
ω / °
λ
max / nm
λmax / nm
(ε / 104 M−1 cm−1)
Φoc
Φco
Φf
τf / ns
(ε / 104 M−1cm−1)
15
16
17
18
275 (0.52), 310 (0.52)
293 (1.7), 328 (1.4)
289 (1.2), 326 (1.0)
285 (0.97), 320 (0.82)
0.28 a
0.58 b
0.63 b
0.59 b
414 (1.8)
0.18 c
0.22
0.79
0.84
0.79
1.2
3.0
3.1
3.2
–
445 (4.2)
441 (3.7)
430 (3.5)
2.8 × 10−3 d
3.2 × 10−3 d
1.8 × 10−2 d
35.9
61.0
65.7
a Under irradiation with 313 nm light.
b Under irradiation with 330 nm light.
c Under irradiation with 405 nm light.
d Under irradiation with 450 nm light.
24

Figure 1. Absorption and fluorescence spectra of 3 (a), 4 (b), 5 (c), and 6 (d) in
1,4-dioxane (3.0 × 10−5 M). Black dashed lines: absorption spectra of open-ring isomers
3a–6a, black solid lines: absorption spectra of closed-ring isomers 3b–6b, green solid
lines: fluorescence spectra (uncorrected) of closed-ring isomers 3b–6b.
25

Figure 2. Relationship between fluorescence quantum yields (Φf) of closed-ring
isomers 2b‒6b and the relative dielectric constants (εr) of the solvents. The solvents
used are n-hexane (εr = 1.89), 1,4-dioxane (εr = 2.22), 2-methyltetrahydrofuran (εr =
6.97), 2-propanol (εr = 20.2), and ethanol (εr = 25.3).
26

Figure 3. (a) Absorption spectra of 8a (solid line) and 8b (dashed line) in ethyl acetate
(1.0 × 10−5 M). (b) Fluorescence spectra of 8a (solid line) and 8b (dashed line) in ethyl
acetate (1.0 × 10−5 M) under photo-excitation at the absorption maxima. Reproduced
from ref. 51 with permission from The Royal Society of Chemistry.
27

Figure 4. Absorption spectra of open-ring isomers (black dot-dashed line), closed-ring
isomers (black solid line) and photostationary states under irradiation with 330 nm light
(black dotted line) and fluorescence spectra of closed-ring isomers under irradiation
with 450 nm light (green solid line, uncorrected) in 1,4-dioxane (2.0 × 10−5 M). (a) 9,
(b) 12. Reproduced from ref. 60 with permission of The Royal Society of Chemistry
(RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the
RSC.
28

Figure 5. Relationship between fluorescence quantum yields (f) and relative dielectric
constants of the solvents (εr, hexane: 1.89, 1,4-dioxane: 2.22, dichloromethane: 8.93,
ethanol: 25.3, acetonitrile: 36.64). Red circles: 9b, blue squares: 10b, green triangles:
11b, black circles: 12b, orange squares: 13b. Adapted from ref. 60 with permission of
The Royal Society of Chemistry (RSC) on behalf of the Centre National de la
Recherche Scientifique (CNRS) and the RSC.
29