Mono-and bithiophene-substituted diarylethene photoswitches with emissive open or closed forms

Article abstract of DOI:10.3762/bjoc.15.227

We present a new series of photochromic 1,2-bis(2-ethylbenzo[b]thiophen-3-yl)perfluorocyclopentenes with an oxidized benzothiophene core (O) or a nonoxidized one, decorated with mono-(Th₁) and bithiophene (Th₂) units attached to positions 6 and 6′ (Sy = symmetric) or only to position 6 (As = asymmetric). Oxidized compounds have highly fluorescent closed forms emitting in the visible region (yellow to red). The dyes with nonoxidized benzothiophenes possess fluorescent open forms with rather low emission efficiency. The photoswitching kinetics was studied at several wavelengths with UV and visible light. New diarylethenes underwent ring-closure reactions by irradiation with UV light (365 nm, 405 nm), and the reversible ring-opening by irradiation with visible light (470 nm, 530 nm). The on-switching of fluorescence due to the ring-closure reaction was observed also with visible light of 470 nm (to an extent of 10% for compound SyOTh₁) and attributed to the Urbach tail effect. Due to a high degree of fluorescence modulation (>270), good fatigue resistance and large fluorescence quantum yield, compound SyOTh₁ emerged as a candidate for single-molecule based super-resolution fluorescence microscopy.

Full text of DOI:10.3762/bjoc.15.227

Mono- and bithiophene-substituted diarylethene
photoswitches with emissive open or closed forms
A. Lennart Schleper1,2, Mariano L. Bossi3, Vladimir N. Belov*1 and Stefan W. Hell1,3
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 2344–2354.
1Department of NanoBiophotonics, Max Planck Institute for
doi:10.3762/bjoc.15.227
Biophysical Chemistry, 37077 Göttingen, Germany, 2Department of
Organic Chemistry, Heidelberg University, Im Neuenheimer Feld 270,
69120 Heidelberg, Germany and 3Department of Optical Nanoscopy,
Max Planck Institute for Medical Research, Jahnstrasse 29, 69120
Heidelberg, Germany
Received: 31 May 2019
Accepted: 10 September 2019
Published: 01 October 2019
This article is part of the thematic issue "Dyes in modern organic
chemistry".
Email:
Vladimir N. Belov* - vbelov@mpibpc.mpg.de
Guest Editor: H. Ihmels
* Corresponding author
© 2019 Schleper et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
diarylethenes; dyes; fluorescence; organic synthesis; photochromism;
photoswitching
Abstract
We present a new series of photochromic 1,2-bis(2-ethylbenzo[b]thiophen-3-yl)perfluorocyclopentenes with an oxidized benzothio-
phene core (O) or a nonoxidized one, decorated with mono- (Th1) and bithiophene (Th2) units attached to positions 6 and 6′
(Sy = symmetric) or only to position 6 (As = asymmetric). “Oxidized” compounds have highly fluorescent closed forms emitting in
the visible region (yellow to red). The dyes with nonoxidized benzothiophenes possess fluorescent open forms with rather low
emission efficiency. The photoswitching kinetics was studied at several wavelengths with UV and visible light. New diarylethenes
underwent ring-closure reactions by irradiation with UV light (365 nm, 405 nm), and the reversible ring-opening by irradiation with
visible light (470 nm, 530 nm). The on-switching of fluorescence due to the ring-closure reaction was observed also with visible
light of 470 nm (to an extent of 10% for compound SyOTh1) and attributed to the Urbach tail effect. Due to a high degree of fluo-
rescence modulation (>270), good fatigue resistance and large fluorescence quantum yield, compound SyOTh1 emerged as a candi-
date for single-molecule based super-resolution fluorescence microscopy.
Introduction
Reversibly photoswitchable diarylethenes (DAEs) with highly diarylethenes with fluorescent open forms are rare, while photo-
fluorescent “closed” forms combine photochromic and fluores- switchable compounds with highly fluorescent “open” and
cent entities in one molecule [1] and contain a perfluorocyclo- “closed” forms represent a yet unknown and unique class of
pentene bridge linking two 2-alkyl-1-benzothiophene-1,1-oxide ratiometric fluorophores.
residues with a C=C bond via C-3 and C-3′ atoms [2,3]. The
“open” form of the DAE core (see graphical abstract and Recently, we demonstrated that asymmetric DAEs with
Table 1) is only weakly fluorescent (φFl ≈ 0.01) [4]. In general, oxidized 2-ethylbenzo[b]thiophene-3-yl units have high cyclo-
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reversion quantum yields (a desirable feature for super-resolu- SyTh2, AsOTh1, AsOTh2, SyOTh1, and SyOTh2 prepared and
tion RESOLFT microscopy), large Stokes shifts and acceptable studied in this work are given in Figure 1.
absorptivity [5]. Yet unknown asymmetric “thiophenylated”
DAEs may have unique properties. Oligothiophenes are highly Methyl esters of the thiophene carboxylic acids were chosen as
fluorescent [6,7], and therefore, we reasoned that their incorpo- model building blocks, because they possess optical properties
ration into DAEs with oxidized 2-alkylbenzo[b]thiophene units similar to the free acids. The future promising candidates which
might produce fluorescent open forms in addition to the were expected to emerge in the course of synthesis and
intrinsic fluorescence of the closed forms. In particular, we ex- screening could be eventually transformed to the corresponding
pected that the prolonged conjugation path, even at “one side” carboxylic acids possessing higher solubility in aqueous buffers
of the molecule (in asymmetric compounds) would enable and reactive groups required for bioconjugation.
switching with focusable light of 375 nm and above. To test this
Results and Discussion
hypothesis, we designed a series of DAEs with mono- and
bithiophene substituents attached to positions 6 and 6′ of an Synthetic procedures
oxidized or a nonoxidized 2-alkylbenzo[b]thiophene core. The To enable a facile synthesis of oxidized and nonoxidized 1,2-
structures of “thiophenylated” DAEs AsTh1, AsTh2, SyTh1, bis(2-ethylbenzo[b]thiophen-3-yl)perfluorocylopentenes
Figure 1: Structures of “thiophenylated” DAEs prepared and studied in this work.
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decorated with thiophene units, mono- and diiodinated core The transformations leading to diarylethene cores 3 and 6 were
structures 4, 5, 7, and 8 (Scheme 1), as well as mono- carried out on a 10–100 mmol scale (Scheme 1). The iodina-
and bithiopheneboronic esters 9 and 12 (Scheme 2) were pre- tion at positions 6 and 6' of benzo[b]thiophene residues has
pared.
been reported [5,8]. Following those protocols, diiodides 5 and
Scheme 1: Synthesis routes towards mono- and diiodinated core structures 4, 5, 7, and 8.
Scheme 2: Synthesis of thiophene- and bithiopheneboronic esters 9 and 12 (bpy – 4,4’-di-tert-butyl-2,2’-dipyridine; COD – cycloocta-1,5-diene;
NBS – N-bromosuccinimide, DCM – dichloromethane).
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8 were obtained in fairly good yields by treatment of 3 with 12 (see Scheme 3). Throughout the text, abbreviations “As” and
elemental iodine and periodic acid in aqueous acetic acid with “Sy” denote asymmetric and symmetric substitution patterns,
addition of sulfuric acid, and by treatment of 6 with elemental respectively; “O” indicates oxidized benzothiophene units, and
iodine and periodic acid in concentrated sulfuric acid, respec- “Th1”/ “Th2” specify the number of thiophene units in the side
tively. The conditions for monoiodination of 6 have been chain. The coupling products were isolated via preparative
published recently [5]. We observed that the reproducible HPLC in yields ranging from 21% to 75%. While in all cases
monoiodination of DAE 3 to form compound 4 was possible the open forms of the diarylethenes were isolated, small
under similar conditions and in low yield (14%) by using amounts of SyOTh1 in its closed form (formed by handling in
smaller amounts of periodic acid (0.34 equiv) and iodine the lab not fully protected from light) were detected by HPLC
(0.68 equiv) than the amounts of these reagents used for diiodi- (Figure S33 in Supporting Information File 1). The constitution,
nation (see Experimental for details). The progress of the reac- structures, and purities of the final products were confirmed by
tion leading to monoiodide 4 was monitored by HPLC. Despite HRMS, 1H and 19F NMR spectroscopy, as well as analytical
incomplete conversion, the formation of diiodide 5 could not be HPLC.
fully suppressed. The Rf values of 3 (TLC [SiO2, hexane]:
Rf = 0.17), monoiodide 4 (Rf = 0.19), and diiodide 5 (Rf = 0.20) Saponification of the ester groups was performed only for
were very similar, and DAE 4 could not be isolated by column nonoxidized benzothiophenes, as shown for compound SyTh2
chromatography on regular silica gel. Instead, monoiodide 4 in Scheme 4. Diarylethenes with “oxidized” benzothiophene
was obtained in 14% yield as colorless solid after preparative units and perfluorocyclopentene rings react with aqueous or
HPLC on a reversed phase (C18) column and lyophilization. alcoholic solutions of strong bases [5,10,15]. Under these
The constitution and structure of 4 was confirmed by HRMS, conditions, substitution of fluorine and/or the addition of
1H and 19F NMR spectroscopy (see Figure S1 in Supporting nucleophiles to the electron-poor double bond take place.
Information File 1). These results show that “desymmetriza- Therefore, tert-butyl protection is required for carboxylates
tion” of DAEs still represents a real synthetic challenge. present in diarylethenes with “oxidized” benzothiophene units
Diiodides 5 and 8 provide the possibility [9,10] to obtain sym- and perfluorocyclopentene rings. Photoswitches containing the
metric DAEs, and monoiodides 4 and 7 allow for a short and free carboxylic acid residues benefit from solubility in aqueous
straightforward approach towards asymmetric DAEs.
buffers and the presence of a reactive group required for further
modifications. The methyl groups were cleaved using 1 M aq
The thiophene- and bithiopheneboronic esters 9 and 12 were NaOH, and the product SyTh2-H was isolated by preparative
prepared as shown in Scheme 2. C–H activation of methyl thio- HPLC in 84% yield (see Scheme 4). As shown in Figure S2
phene-2-carboxylate in position 5 was achieved by using a (see Supporting Information File 1), tetraester SyTh2 displayed
bis(1,5-cyclooctadiene)di-μ-methoxydiiridium(I) catalyst with six 1H NMR signals between 3.70 and 4.00 ppm, belonging to
4,4’-di-tert-butyl-2,2’-dipyridyl as ligands. The reaction with two inequivalent methyl ester groups and the methylene group
pinacolborane (HB(pin)) gave ester 9 in 97% yield [11]. of the parallel and antiparallel isomers. After the reaction, four
Another ester (10) was synthesized by bromination of 3-thio- of the six signals vanished, and only two methylene signals
pheneacetic acid with NBS (applying an ultrasound bath) fol- (two isomers) were detected, confirming cleavage of the methyl
lowed by esterification of the crude product with methanol in esters.
the presence of sulfuric acid [12]. Other regioisomeric bro-
mides were also formed, but they were separated by column Photophysical properties
chromatography on the next step. Bithiophene 11 was prepared The absorption and emission properties as well as the photo-
in 74% yield by using a Suzuki–Miyaura coupling (catalyzed by chromic properties of eight “thiophenylated” DAEs were
PEPPSI-IPr) between boronic ester 9 and bromide 10 [13,14]. studied in acetonitrile solutions (Table 1). The properties of the
Boronation of bithiophene 11 was achieved under conditions OFs (open forms) were obtained from diluted solutions at a
similar to the preparation of thiophene 9 and afforded ester 12 known concentration prior to irradiation. Solutions were irradi-
in 55% yield (79% crude yield with 70% purity). However, in ated in a home-made setup [5] with 355 nm light until the
this case, larger amounts of catalyst and pinacolborane were re- photostationary state (PSS365 nm). Figure 2 shows changes of
quired for good conversions.
absorption and emission spectra in the course of the switching
process for compound SyTh2. Irradiated solutions in the
Finally, the photoswitchable DAEs AsTh1, SyTh1, AsTh2, PSS365 nm were analyzed by HPLC at the isosbestic point. Con-
SyTh2, AsOTh1, SyOTh1, AsOTh2, and SyOTh2 (Figure 1) version to the CF (closed form) αPSS = [CF]/([CF] + [OF]) was
were obtained using a standard procedure for a Suzuki–Miyaura calculated from the proportion of the area corresponding to the
coupling of iodides 4, 5, 7, and 8 with boronic acid esters 9 and CF (areaCF/[areaCF + areaOF]). The absorption spectrum of the
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Beilstein J. Org. Chem. 2019, 15, 2344–2354.
Scheme 3: Photoswitchable diarylethenes AsTh1, SyTh1, AsTh2, SyTh2, AsOTh1, SyOTh1, AsOTh2, and SyOTh2 synthesized via a
Suzuki–Miyaura coupling. Conditions: 60 °C, argon atmosphere, an emulsion of the starting compounds, Pd2(dba)3, P(Cy)3 stock solution, and
aqueous K2CO3 in THF (see Supporting Information File 1 for details).
Scheme 4: Saponification of methyl ester groups in tetraester SyTh2 leading to tetracarboxylic acid SyTh2-H.
CF was calculated from the absorption spectrum at the We first analyzed the absorption spectra of the open forms (see
PSS365 nm and the absorption spectrum of the OF, using the ob- Figure 3A) in more detail. Addition of thiophene rings to the
tained conversion. The obtained spectra for all CF’s matched, core structures 3 and 6 induced a red-shift in the OF (Table 1)
within experimental errors, the absorption recorded on the diode [4,16]. Their absorption maxima were located between 327 nm
array detector of the HPLC, at the center of the elution peak of and 385 nm, and present interesting correlations. First, the posi-
the corresponding isomer (see for example, Figures S26 and tions of the main absorption bands of the corresponding sym-
S38 in Supporting Information File 1). Emission properties of metric and asymmetric DAEs were very similar. The symmet-
the CF’s were obtained from solutions at the PSS365 nm, with ric DAEs have higher absorption coefficients than their asym-
excitation at a wavelength at which the OF’s do not absorb metric counterparts (by a factor of 1.3–3.2). Considering the
(only the CF absorbs). The data on switching kinetics is given very similar band positions, this indicates that in the OF each
in Table 2 and discussed in the next section.
benzothiophene unit (with aligning thiophene group(s)) contrib-
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Figure 2: Absorption (A) and emission (B) spectra of SyTh2 in acetonitrile in the course of the cyclization reaction (irradiation at 365 nm). Emission
was not fully depleted due to incomplete conversion in the photostationary state (PSS, α = 71%).
Table 1: Optical properties of the photochromic DAEs (in acetonitrile solution).
Absorption
CFa
λabs ε/103
[nm] [M−1cm−1] [nm] [M−1cm−1]
Emission
Stokes shift [nm]
(cm−1)
OFa
λabs ε/103
Separationb
[nm]
(cm-1)
OF
φFld
CF
φFld
λemc
(λex)
[nm]
τFle
λemc
(λex)
[nm]
τFle
[ns]
[ns]
AsTh1
SyTh1
AsTh2
SyTh2
327 25.8
328 50.6
363 20.0
363 63.1
562 13.5
584 21.3
566 12.1
591 32.5
447 37.1
478 53.5
480 58.6
524 63.8
536 9.6
235
410
0.01
0.01
0.09
0.08
–
0.20
0.23
0.24
0.24
–
–
–
–
–
–
–
83
(12800)
(320)
(6190)
256
(13400)
203
(9880)
228
(10600)
91
(5720)
123
(7250)
95
(5140)
140
(6960)
278
(20100)
410
(340)
462
(360)
469
(410)
–
–
–
82
(6100)
99
(5900)
106
(6230)
103
(4190)
82
(3060)
176
(5590)
146
(4160)
–
–
–
–
–
AsOTh1 356 15.5
SyOTh1 355 31.4
AsOTh2 385 36.4
SyOTh2 384 47.9
550
(460)
560
(470)
656
(460)
670
(530)
0.36
0.48
0.16
0.18
–
1.63
1.91
1.28
1.16
–
–
–
–
–
–
–
–
–
–
3f [16]
6 [4]
258 16.5
310 5.3
–
–
–
–
412 23.7
102
(7990)
464
0.01
0.26g 509
5.04h
0.06
0.51
154i
(10700)i
97j
(4630)j
aWavelength and extinction coefficient at the absorption maxima. bSeparation between absorption maxima of OF and CF. cPosition of the emission
maxima (excitation wavelength). dFluorescence quantum yield. eFluorescence lifetime. fIn hexane solution. gParallel isomer. hAntiparallel isomer. iOF
(open form). jCF (closed form).
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Figure 3: Absorption spectra of the OFs (A) and CFs (B) of AsTh1 (a), SyTh1 (b), AsTh2 (c), SyTh2 (d), AsOTh1 (e), SyOTh1 (f), AsOTh2 (g), and
SyOTh2 (h). The absorption of the OFs in (A) above 460 nm is due to the presence of low amounts of the corresponding CFs (formed during manipu-
lation of the solids and/or preparation of the solutions).
utes into absorption independently. It was confirmed that asym- feature, as both compounds consist of the same parts: two
metric compounds with “compact” structures can be trans- oxidized benzothiophenes and, in total, two thiophenes as sub-
formed to the closed form with the same light as bulkier stituents. It appears that the sequence of the building blocks is
analogs (the latter featuring slower ring-opening reactions; see less important than their nature and quantity. The same regu-
Table 2). Second, a bithiophene residue induces a red-shift of larity holds true (with less precision) for the most red-shifted
30–35 nm in the absorption of the OF with respect to a mono- absorption bands of SyTh1 and AsTh2 (CFs have 18 nm differ-
thiophene ring. The oxidized DAEs (OF) absorbed at higher ence in band positions); both DAEs consisting of same build-
wavelengths than the corresponding nonoxidized analogs (by ing blocks, again!
ca. 20–30 nm), and a splitting of the band is observed with a
shoulder appearing at the blue edge. In general, substitution in If we consider the positions of the absorption bands of the OF
the OF with a bithiophene side chain induced a slightly larger and CF of each oxidized DAE, we observe that they are separat-
red-shift than oxidation of benzothiophene units.
ed “only” by 90–140 nm. On the other hand, in nonoxidized
DAEs, these bands are separated by 200–260 nm. This large
The absorption maxima of the CFs were located between difference is justified by the fact that the oxidation red-shifts the
447 nm and 591 nm; they present remarkably larger differences band positions of the OF, and blue-shifts the band positions of
than the corresponding OFs (see Figure 3B). Compared with the the CF. A conversion of the separation between the absorption
parent compounds 3 and 6 [4,16], the CFs of all new DAEs bands from wavelengths to wavenumbers allows the evaluation
displayed, again, red-shifted absorption bands. Unlike for the of the relative energy gaps between the HOMO and LUMO. We
OFs (Figure 3A), oxidation produces a strong blue shift of the can see that the energy gap between HOMO and LUMO in
absorption bands of the CF (compare the curves a–d and e–h in nonoxidized DAEs is decreased to a higher extent upon cycliza-
Figure 3B). This shift is stepwise reduced from 124 nm in the tion than in oxidized DAEs. Furthermore, we noted that asym-
unsubstituted compounds (3→6), to 65 nm in the presence of metric DAEs have a smaller separation between absorption
four thiophene residues (SyTh2→SyOTh2). It is worth noting maxima of OF and CF than their symmetric counterparts. We
that for asymmetric compounds (AsTh2→AsOTh2), the shift observed that nonoxidized DAEs exhibited a higher extinction
(20 nm) is smaller than for symmetric DAEs having the same coefficient in the OF than in the CF (by a factor of 1.7–2.4);
total number of thiophene rings (SyTh1→SyOTh1). All CFs oxidized DAEs revealed an opposite trend (higher absorption in
showed several absorption bands, while nonoxidized DAEs the CF by a factor of 1.3–2.4).
have two distinct absorption bands in the visible range. The data
in Figure 3 indicate that, contrary to the OF, both benzo[b]thio- New DAEs have very different emission properties (see
phene moieties (with side rings) interact and determine the loca- Figure 4 and Table 1). the oxidized DAEs strongly emit in their
tion of the absorption band in the CF. If we compare the absorp- CFs, and the nonoxidized DAEs emit in their OFs, but rather
tion spectra of SyOTh1 and AsOTh2, we see that the most red- weakly. Interestingly, unsubstituted DAEs 3 and 6 (Scheme 1)
shifted bands have very similar positions. This is a remarkable were either nonemissive at all (3), or very poorly emissive in
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Beilstein J. Org. Chem. 2019, 15, 2344–2354.
They exceeded lifetimes measured for the fluorescence of the
unsubstituted DAE 6 by a factor of 4, and the fluorescence
quantum yield of the CF of 6 was surpassed by the oxidized
DAEs with thiophene side rings by a factor of 3–8 [4]. The
open forms of the substituted oxidized compounds were
nonemissive.
The Stokes shift of the OFs of nonoxidized DAEs was always
ca. 6000 cm−1, independent of the substitution pattern. For
comparison, this value was much lower than the Stokes shift of
the OF of compound 6 (10,700 cm−1) [4]. The Stokes shift of
the CFs of oxidized DAEs varied between 3000 cm−1 and
5600 cm−1, which is comparable with the Stokes shift observed
for the CF of 6 (4600 cm−1) [4]. In the case of oxidized DAEs,
asymmetric compounds possessed higher Stokes shifts by
ca. 1000 cm−1 than symmetric DAEs. Only compound AsOTh2
has a higher Stokes shift than the CF of unsubstituted DAE 6.
Two important observations were made in this part of the work.
First, the OFs of oxidized DAEs absorb at higher wavelengths,
and their CFs absorb at lower wavelengths than the correspond-
ing isomers of the nonoxidized counterparts. Second, the CFs of
nonoxidized DAEs were found to be nonemissive. This impor-
tant property will be taken into account in the design of the
future photoswitchable compounds with ratiometric fluores-
cence modulation.
Figure 4: Solutions of compounds AsTh1 (a), SyTh1 (b), AsTh2 (c),
SyTh2 (d), AsOTh1 (e), SyOTh1 (f), AsOTh2 (g), and SyOTh2 (h) in
CDCl3 as mixtures of OFs and CFs under irradiation with 366 nm (top),
and the corresponding emission spectra in MeCN (bottom). Emission
of the compounds covers the range from UV to the blue and red
regions. Sharp peaks at ca. 390 nm (a and b) are Raman bands of the
solvent.
both forms (6) [4,16]. The new DAEs – when nonoxidized – Switching kinetics
possessed blue emission with maxima between 410 nm and Table 2 contains an overview of the most important parameters
470 nm. In this case, the difference in emission between asym- related to the switching kinetics.
metric and symmetric substitution patterns was negligible. This
feature can be explained by the fact that due to the twisted con- All compounds, except AsOTh2 and SyOTh2, were reversibly
formation of the open form, the emission transition involves the switchable between open and closed forms by irradiation with
isolated benzothiophene unit (including the side ring). As ex- 365 nm (cyclization) and 470 nm (cycloreversion) light. For
pected, in MeCN quantum yields of nonoxidized compounds compounds AsOTh2 and SyOTh2, irreversible photobleaching
with a bithiophene side chain were higher (ca. 8% compared to was the main reaction path observed after irradiation with
1% for a monothiophene derivative), but still moderate. Fluo- visible (470–530 nm) light (see Figures S55 and S61 in Sup-
rescence lifetimes were short (0.2 ns), in accordance with mod- porting Information File 1). Considering the trend for other
erate quantum yields.
DAEs [1,5,9,10], we expect the cycloreversion quantum yields
of compounds AsOTh2 and SyOTh2 to be <10−6–10−7, which
The oxidized compounds were strongly fluorescent in their means that the bleaching quantum yields are on the order of
closed forms, with emission maxima in the range between >10−5–10−6. The nonoxidized DAEs were also switchable by ir-
550 nm and 670 nm. The difference between the positions of radiation of the OF at 530 nm. In general, nonoxidized DAEs
emission bands of the structurally related symmetric and asym- switched faster in both directions than the oxidized DAEs (by
metric compounds was small, compared to the change caused 1–4 orders of magnitude). Furthermore, the nonoxidized com-
by an additional thiophene ring to the benzothiophene core. pounds (apart from SyTh1) revealed higher cycloreversion
Oxidized DAEs with monothiophene units as side chains exhib- quantum yields (φCF→OF) than cyclization quantum yields
ited higher fluorescence quantum yields than the structurally (φOF→CF); the oxidized compounds showed an opposite trend.
related DAEs with bithiophenes (>35% compared to <20%). As a result, the degree of conversion in the photostationary state
Longer fluorescent lifetimes (1.3–1.9 ns) of the oxidized DAEs found for nonoxidized compounds was moderate <70% (ca.
(CFs) correspond to the better fluorescence quantum yields. 30% for asymmetric and 70% for symmetric dyes), but the
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Beilstein J. Org. Chem. 2019, 15, 2344–2354.
Table 2: Switching kinetics of the photochromic compounds (in acetonitrile solution).
αa [%]
φOF→CFb
φCF→OFc
φon-switching/φoff-switching
Nphd
AsTh1
SyTh1
AsTh2
SyTh2
AsOTh1
SyOTh1
AsOTh2
SyOTh2
3h [16]
6 [4]
36
71
28
71
>98
>98
>98
>99
53i
31
0.13
0.16
0.03
0.04
9.0 · 10−2
2.4 · 10−2
7.6 · 10−5
1.9 · 10−4
0.39i
0.14
0.07
0.15
0.06
1.6 · 10−3
8.8 · 10−5
–g
–g
0.35j
0.25k
1.1e
0.4e
5.0e
1.5e
56f
273f
–
–
–
–
<1
<1
3
2
225
5230
–
–
–
0.24i
–
aConversion into the CF in the PSS (365 nm). bIrradiation at 365 nm. cIrradiation at 530 nm. dThe number of average emitted photons before off-
switching is proportional to the ratio of fluorescence quantum yield and off-switching quantum yield; the off-switching quantum yield is φOF→CF for
nonoxidized and φCF→OF for oxidized compounds. eRatio of cycloreversion and cyclization. fRatio of cyclization and cycloreversion. gIrreversible
bleaching overcame the cycloreversion reaction. hIn hexane. iIrradiation at 313 nm. jIrradiation at 517 nm. kIrradiation at 405 nm.
oxidized analogs show complete conversions (>98%). Interest- solution, extrapolation of the first 160 switching cycles pre-
ingly, decoration of the core structures 3 and 6 (Scheme 1) with dicted that >3500 switching cycles may be expected before a
thiophene rings lowered the switching quantum yields by 50% signal loss will occur. The high fatigue resistance of com-
several orders of magnitude. It became obvious that the rate of pound SyTh1 indicated that nonoxidized DAEs may, in general,
cyclization and cycloreversion reactions decreased when the be more photostable than oxidized DAEs. However, due to poor
conjugation path became longer (transition from the unsubsti- emission, SyTh1 was not considered as a fluorescence marker.
tuted core to monothiophene and then to bithiophene deriva- Another promising compound for applications in super-resolu-
tives). In all cases, asymmetric substitution allowed faster cy- tion microscopy was DAE AsOTh1. A high emission effi-
cloreversion than symmetric substitution, which is a positive ciency and a relatively fast cycloreversion, makes it a good
effect regarding potential application of these dyes as revers- candidate for a photoswitch applicable in RESOLFT microsco-
ible fluorescent markers in RESOLFT microscopy [5].
py.
Two processes – fluorescence and off-switching – compete for Urbach tail effect
the depopulation of the same excited state [10]. Hence, the ratio Under irradiation with visible light, most DAEs show complete
of fluorescence quantum yield and off-switching quantum yield conversion into the OF, because OFs do not absorb at wave-
is proportional to the number of emitted photons (Nph) per cycle lengths corresponding to the absorption band of the CF. How-
or per incursion into the fluorescent state (i.e., before off- ever, compound SyOTh1 presented a remarkably “low” value
switching takes place). Due to its high fluorescence quantum of αCF→OF [470 nm] = 0.9 in MeCN. A similar behavior has
yield (φfl) and low cycloreversion quantum yield (φCF→OF), been recently reported and ascribed to the so-called Urbach tail
compound SyOTh1 is expected to emit more than 5200 photons effect [19,20], i.e., the ring-closing photoreaction is induced by
per cycle on average. Furthermore, compound SyOTh1 proved absorbing very weak hot bands of the OF, in the visible range.
to be highly fatigue resistant. As shown in Figure 5, it endured Neither nonoxidized DAEs, nor the asymmetric compound
>160 switching cycles in acetonitrile solution while losing only AsOTh1 show this effect at observable levels; the other
ca. 15% of its absorption. The extrapolation of the change in oxidized compounds are “irreversible”. To further investigate
absorption yielded about 340 switching cycles, until a 50% this phenomenon, SyOTh1 was irradiated with visible light of
signal loss occurs. Thus, this compound is a good candidate for even longer wavelength (530 nm). Remarkably, conversion
a marker applicable in STORM/PALM and MINFLUX super- from the CF to the OF was practically complete (Figure 6).
resolution techniques [17,18]. It even showed superior Thus, this photochromic DAE can be switched “on” with
switching kinetics (regarding the ratio of fluorescence quantum 470 nm light to an extent of 10%, and switched “off” with
yield and cycloreversion quantum yield) compared to DAEs 530 nm light. In Figure 6C we present ten switching cycles per-
which were already applied in super-resolution microscopy formed with visible light, alternating between blue and green ir-
[10,15]. The nonoxidized analog of SyOTh1, compound radiation (no UV light was used for switching!). The emission
SyTh1, showed even higher fatigue resistance. In acetonitrile signal (not shown) presents the same on/off behavior.
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Beilstein J. Org. Chem. 2019, 15, 2344–2354.
Figure 5: Fatigue resistances of compounds SyOTh1 (A and B) and SyTh1 (C). Parts A and C show the absorbance at certain wavelengths after
cyclization (top values) and cycloreversion (bottom values). Part B shows the change in absorbance at 478 nm for each full switching cycle
(CF–OF–CF) for compound SyOTh1. Dots: the measured values; blue line: an exponential fit. Part D shows the structures of both compounds.
Figure 6: (A) Absorption spectra of compound SyOTh1 in MeCN at the photostationary states under irradiation with 365 nm light (blue/black lines),
470 nm light (red line), and 530 nm light (purple line). Part B, irradiation kinetics (same color code). Part C, ten complete on/off switching cycles per-
formed with visible light.
Conclusion
high cycloreversion quantum yields. Due to a high on/off ratio
We prepared eight 1,2-bis[(2-ethylbenzo[b]thiophene)-3- of >270, good fatigue resistance and large fluorescence quan-
yl]perfluorocyclopentenes with oxidized and nonoxidized cores tum yield, compound SyOTh1 may be an excellent marker for
and decorated them with mono- and bithiophene rings in sym- single-molecule based super-resolution fluorescence microsco-
metric and asymmetric fashions. By studying the optical proper- py, e.g., MINFLUX and STORM. In addition, SyOTh1 presents
ties of the new compounds, we found that DAEs with nonoxi- a remarkable on-switching of 10% of its maximal fluorescence
dized benzothiophene units possess fluorescent open forms with signal with 470 nm light, and the ring-closure reaction was fully
low emission efficiencies. The new DAEs are relatively fast reversible by irradiation with longer wavelengths. This offers
switches, and, in particular, the nonoxidized DAEs exhibited the possibility of reversible switching a DAE only with visible,
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Beilstein J. Org. Chem. 2019, 15, 2344–2354.
10.Roubinet, B.; Weber, M.; Shojaei, H.; Bates, M.; Bossi, M. L.;
Belov, V. N.; Irie, M.; Hell, S. W. J. Am. Chem. Soc. 2017, 139,
6611–6620. doi:10.1021/jacs.7b00274
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avoiding harmful (cytotoxic) UV irradiation. In view of the
forthcoming solubilizing strategies applicable to fluorescent
DAEs and intended for the crucial improvement of their fatigue
resistance in aqueous solutions [21], the switching core of com-
pound AsOTh1 is expected to provide a promising marker for
RESOLFT microscopy (after attaching the reactive group).
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Supporting Information
Supporting Information File 1
Experimental part and additional spectra of synthesized
compounds.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-227-S1.pdf]
doi:10.1002/anie.201607940
Angew. Chem., 2016, 128, 15655–15659.
doi:10.1002/ange.201607940
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Biopolymers 2011, 95, 322–331. doi:10.1002/bip.21586
18.Balzarotti, F.; Eilers, Y.; Gwosch, K. C.; Gynnå, A. H.; Westphal, V.;
Stefani, F. D.; Elf, J.; Hell, S. W. Science 2017, 355, 606–612.
doi:10.1126/science.aak9913
Acknowledgements
Jürgen Bienert, Jens Schimpfhauser, Jan Seikowki (Facility für
synthetische Chemie, Max-Planck-Institut für biophysikalische
Chemie, Göttingen), Dr. Holm Frauendorf, and the central
analytics’ team (Institut für organische und biomolekulare
Chemie der Georg-August Universität Göttingen) are cordially
acknowledged for recording NMR and mass spectra. ALS was
supported by a Cusanuswerk scholarship.
19.Kashihara, R.; Morimoto, M.; Ito, S.; Miyasaka, H.; Irie, M.
J. Am. Chem. Soc. 2017, 139, 16498–16501.
doi:10.1021/jacs.7b10697
ORCID® iDs
20.Arai, Y.; Ito, S.; Fujita, H.; Yoneda, Y.; Kaji, T.; Takei, S.; Kashihara, R.;
Morimoto, M.; Irie, M.; Miyasaka, H. Chem. Commun. 2017, 53,
4066–4069. doi:10.1039/c6cc10073b
Mariano L. Bossi - https://orcid.org/0000-0001-6755-8074
Vladimir N. Belov - https://orcid.org/0000-0002-7741-4653
21.Uno, K.; Bossi, M. L.; Irie, M.; Belov, V. N.; Hell, S. W.
J. Am. Chem. Soc. 2019, 141. doi:10.1021/jacs.9b08748
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