Reversibly Photoswitchable Fluorescent Diarylethenes Resistant against Photobleaching in Aqueous Solutions

Article abstract of DOI:10.1021/jacs.9b08748

Low photostability in aqueous solutions is the main drawback of synthetic photochromic dyes, which limits their switching performance and utility in biology, medicine, and life sciences. Even the most promising photochromes - reversibly photoswitchable di

Full text of DOI:10.1021/jacs.9b08748

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Article
Reversibly Photoswitchable Fluorescent Diarylethenes
Resistant against Photobleaching in Aqueous Solutions
Kakishi Uno, Mariano L Bossi, Masahiro Irie, Vladimir N Belov, and Stefan W. Hell
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b08748 • Publication Date (Web): 22 Sep 2019
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Reversibly Photoswitchable Fluorescent Diarylethenes Resistant
against Photobleaching in Aqueous Solutions
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Kakishi Uno†, Mariano L. Bossi‡, Masahiro Irie§*, Vladimir N. Belov†*, and Stefan W. Hell†‡*
†Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen,
Germany
‡Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg,
Germany
§Research Center for Smart Molecules, Rikkyo University, Tokyo, Japan
ABSTRACT: Low photostability in aqueous solutions is the main drawback of synthetic photochromic dyes, which limits
their switching performance and utility in biology, medicine and life sciences. Even the most promising photochromes -
reversibly photoswitchable diarylethenes (DAEs) with fluorescent “closed” forms - were known to undergo only several tens
(typically 20-30) switching cycles in aqueous solutions. In this work, we introduce water-soluble and highly photostable 1,2-
[bis-(2-ethyl/isobutyl-1-benzothiophene-1,1-dioxide-6-phenyl-3-yl)]perfluorocyclopentenes decorated with four
-
CONHC(CH2R)3 residues capped with 12 carboxylic acid groups (R = CH2CO2H or O(CH2)2CO2H). Under irradiation with
UV- (365 nm) and visible light (470 nm), they provide several hundred (the “rapid” DAEs with isobutyl groups – up to
thousand) full switching cycles in aqueous solutions without exclusion of air oxygen (outperforming the photoswitchable
and fluorescent protein Dreiklang). The new branching approach based on secondary carboxamides with N-tert-alkyl
residues decorated with polar groups may serve as a blueprint for the design of highly fatigue-resistant and reversibly
photoswitchable fluorescent probes applicable in life sciences as aqueous solutions.
■INTRODUCTION
much more difficult approach to the bulky fluorescent and
photochromic dyads10-12 turned to be less attractive. A
particularly challenging problem in the design of fluorescent
diarylethenes (DAEs) relates to water-solubility and switching
performance in aqueous buffers.6-8,11 We previously reported a
series of polycarboxylated DAEs (DAE-n×COOH; Scheme 1,
compounds 1a-c), demonstrated their switching in aqueous
solutions and first applications in superresolution
microscopy.6-8 We also designed an automated set-up and
developed the uniform methodology for evaluation of the
switching performance of DAEs.8 These advances enable to
screen various DAEs and compare their fatigue response in
different solvents and environments. Several thousand of
cycles have been observed in air-saturated organic solvents
(e.g., acetonitrile)8 without exclusion of air oxygen.14
However, in aqueous solutions, DAE-n×COOH endured only
several tens of cycles, and their performance as fluorescent
probes applicable in life sciences was limited by
photobleaching.6-8 Thus, it was critical to improve the fatigue
resistance of photoswitches in aqueous environments. In the
present work, we introduce new structures (Figure 1c) of the
reversibly photoswitchable DAEs with unprecedented fatigue
resistance in aqueous solutions.
Organic photochromic dyes have been applied as molecular
switches1 in photo- and supramolecular chemistry, material
science, optogenetics and bioimaging. They have been used
for construction of optical memory media,2 as solar energy
storage materials,3 molecular machines and actuators,4
artificial photoreceptors,5 and probes for biological
microscopy.6-8 The ultimate sensitivity of the fluorescence
readout (down to single molecules)9 enables to apply the
organic photochromes in fluorescence nanoscopies.7 If the
switching unit is non-fluorescent, it has to be combined with
a fluorescent reporter and incorporated in a dyad-type
assembly.10-12 The photochromic parts of these dyads are
represented by azobenzenes, spiropyrans, spirooxazines and
diarylethenes (DAEs).13 DAEs with perfluorocyclopentene
rings undergo 1.4×104 switching cycle in methyl cyclohexane14
and 3×104 cycle in the crystalline state.15 Both of their states are
thermally stable and feature a large separation between
absorption bands of the two isomers, enabling a reliable
control of the switching efficiencies.2 Due to multistep
synthesis, the structural diversity and switching performance
of the complex photochromic-fluorescent dyads is limited.10-13
Recently, a new type of turn-on mode photo-switchable
molecule has been developed.16-18 After the new “oxidized” 1,2-
bis(2-alkyl-6-aryl-1-benzothiophen-3-
Scheme 1: (a) Photochromism of sulfone derivatives of
DAEs with fluorescent closed-ring isomers emitting
visible light:16,17 b) tetra- and octa-acids;6,7 c) extended
yl)perfluorocyclopentenes were introduced and shown to
have emission efficiencies of up to 90%,17 the earlier and
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(6-R1-R2) and compact (7-R1-R2) dodeca-acids with N-
(tert-alkyl)amido groups (DAE-12×COOH; this work).
This kind of transformation may occur up to 8 times, reducing
1
2
3
4
5
6
the number of carboxylic acid groups and giving products with
longer retention times and lower polarity. The photoproducts
can aggregate and precipitate from aqueous solutions, and
this phenomenon can account for the drastic loss of the
switching capacity (i.e. number of cycles).
F
F
F F
(a)
F
F
F
F
F
F
F
F
UV light
R1
R1
R1
R1
X
X
X
X
S
S
S
S
visible light
O
O
O
O
O
O
O O
On the other hand, the presence of a tertial alkyl group
attached to the nitrogen atom in fluorescent dyes was recently
shown to provide an efficient protection against
photooxidative N-dealkylation.19 Therefore, in a search for
water-soluble and fatigue resistant DAEs, we decided to keep
the aromatic bis-amide residue, but replace N,N-diacetic acid
groups with N-tert-alkyl substituents. Thus, we kept the
central core in DAE-4×COOH 1a (Scheme 1B) that provides
reliable switching and a highly fluorescent CF (see Table 1),
and decorated it with four functionally substituted N-(tert-
alkyl) groups, so that each branching point leads to three
carboxylic acid groups, and the whole number of carboxylates
is 12 (DAE-12×COOH in Scheme 1c).
7
8
9
"open" form (OF)
fluorescent "closed" form (CF)
(b) Previous work
O
O
COOH
COOH
"Tetra-acid"
"Octa-acids"
X =
OH
N
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X =
R2
OH
R2
1
2
1
2
COOH
1a
: R = Et, R = H
1b
: R = Et, R = H
N
1
2
1c
: R = Et, R = OMe
O
O
COOH
COOH
(c) This work
HOOC
O
COOH
COOH
"Dodeca-acids"
long
"Dodeca-acids"
short
O
COOH
O
O
NH
R2
N
H
R2
O
X =
X =
COOH
COOH
1
2
1
2
6
7
-R -R
-R -R
NH
NH
O
O
COOH
In addition to the expected photostability against C-C bond
cleavage, 12 carboxylic acid groups in DAE-12×COOH
(Scheme 1c) make these compounds more polar than tetra-
(1a) or octa-acids (1b,c). Numerous carboxylic residues
provide lower mass-to-charge ratio, better solubility in
aqueous solutions at physiological pH and prevent
aggregation more efficiently. In particular, we expected that
due to lower mass-to-charge ratio, compounds with shorter
linkers (7-R1-R2) will be more hydrophilic than analogs with
longer linkers (6-R1-R2). The methoxy groups attached to C-4
and C-4′ of the phenyl rings are expected to slow down the
ring-opening reactions, compared with unsubstituted
compounds (R2 = H).7,20 On the other hand, the bulky isobutyl
O
O
6
6
7
7
7
7
-Et-H
-Et-H
O
-Et-OMe
-Et-OMe
-iBu-H
COOH
COOH
6-
iBu-H
HOOC
6-
iBu-OMe
-iBu-OMe
COOH
These fluorescent molecular switches accommodate hundreds
(up to one thousand) of switching cycles in purely aqueous
solutions without exclusion of air oxygen and under
alternating irradiations with UV and visible light. Remarkably,
some of the new DAEs were found to be more photoresistant
in aqueous solution than in acetonitrile. The new synthetic
fluorescent switchers can now be seriously compared with the
reversibly photoswitchable fluorescent proteins (rsFPs)
Dreiklang and EGFP2.
groups attached to positions
2 and 2′ in oxidized
benzo[b]thiophenes (R1) are expected to accelerate the ring-
opening reactions (in comparison with the relatively small
ethyl or methyl groups).18 By varying the residues R1 and R2,
we planned to learn if there is any correlation between the
isomerization quantum yields, in particular, the quantum
yields of the ring-opening reaction, and the fatigue resistance
(i.e. the number of switching cycles). In principle, we expected
to observe fewer switching cycles for compounds with lower
isomerization quantum yields, in particular for DAEs with low
ring-opening quantum yields. Indeed, if the ring-opening
quantum yield is low, a large number of excitation cycles of
the CF is required to complete the transformation to the open-
form (OF), and the longer irradiation times increase the
probability of side reactions from the excited state. Therefore,
we expected that the number of switching cycles will decrease
in the row: iBu-H – iBu-OMe – Et-H – Et-OMe (Table 1). On
the other hand, in the series of highly asymmetric DAEs with
“push-pull” substituents there was no clear correlation
between cycloreversion quantum yield and fatigue resistance
(at least for the structurally divers DAEs).8 Therefore, as new
targets, we have chosen compounds 6-R1-R2 and 7-R1-R2 (R1 =
Et, iBu, R2 = H, OMe) and compared them (in respect of
photoswitching properties) with available tetra-acid 1a and
octa-acids 1b,c.6,7 In addition, we have chosen Dreiklang as an
example of a reversibly photoswitchable fluorescent protein
with spectroscopic properties similar to DAE-12×COOH, and
compared their switching properties. Dreiklang is a reversibly
photoswitchable variant of GFP, and represents a genetically
■ RESULTS AND DISCUSSION
Analysis of photoproducts and design of new structures.
In our previous studies we found that DAE-n×COOH
dissolved in aprotic solvents (MeCN) endure much more
switching cycles than the same compounds “cycled” in protic
solvents (alcohols) or water.6-8,
HPLC analyses of the
11
photoirradiated solutions showed the presence of several
photoswitchable by-products with lower polarity. For
example, photocyclization and ring-opening reactions
(Scheme 1A) of aromatic carboxamides with N,N-
bis(carboxymethyl) groups (octa-acids 1b,c in Scheme 1)
gradually produced new photochromic compounds with
absorption and emission spectra almost identical to the initial
dyes (see Figures S23 and S25 in Supporting Information).
When the degree of conversion was moderate (octa-acid 1b
with M = 1348), the LCMS analysis displayed a series of
resolved peaks with lower molecular masses, higher retention
times and the constant mass increment of 30 Da (M+H): 1349
(starting material) - 1319 - 1289 - 1259 - 1229 (Figures S28 and
S29). This pattern is consistent with the stepwise extrusion of
formaldehyde from N-carboxymethyl groups:
Ar-CON(CH2COOH)2 --> Ar-CON(CH2COOH)CHO + H2CO
(m = 30).
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encoded fluorescent switch already established as a marker for
Miyaura reaction with the catalytic Pd(dpa)2/Sphos system
was employed to couple diiodide sulfones Et-Ox-2I and iBu-
Ox-2I with boronic esters 3-R2 and 5-R2. The intermediate
dodeca-t-butyl esters were isolated by flash chromatography
and readily deprotected by treating with TFA in
dichloromethane to afford the target DAE-12×COOH (6- R1-R2
and 7-R1-R2 in Schemes 1 and 2) in good yields (see SI for
detailed experimental procedures and analytical data).
RESOLFT fluorescence nanoscopy.21
1
2
3
4
5
6
7
8
Synthesis. For the synthesis of all dodeca-acids, we used 5-
bromoisophthalic and 5-bromo-2-methoxyisophthalic acids 1-
R2,7 as well as commercially available aminotriesters A and B
(Scheme 2). The building blocks 2-R2 and 4-R2 were obtained
by generation of acid chlorides from compounds 1-R2 (using
oxalyl chloride) followed by the reaction with aminotriesters
(A and B). The protocol of Myaura-borylation was employed
to prepare esters 3-R2 and 5-R2 in good yields. A Suzuki-
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Scheme 2. Synthesis of dodeca-acids 6-R1-R2 and 7-R1-R2 (R1 = Et, iBu, R2 = H, OMe).
ButO2C
ButO2C
O
CO2But
CO2But
OH
O
O
Br
2
O
O
1
1
-R
O
O
R2
OH
1
2
-H
N
H
R2
N
H
R2
6
-R -R
O
O
O
b
c
d, e
1-
OMe
Br
B
CO2But
CO2But
CO2But
CO2But
O
O
6
6
-Et-H (47%)
A
-Et-OMe (42%)
NH
NH
a
O
O
O
O
6-iBu-H (57%)
6-
iBu-OMe (65%)
2
2
2
3
-R
-R
O
O
O
O
2
3
O
-H (77%)
-H (75%)
CO2But
CO2But
2
3
-OMe (82%)
-OMe (85%)
Cl
R2
ButO2C
ButO2C
Br
Cl
O
ButO2C
ButO2C
O
CO2But
CO2But
CO2But
CO2But
O
1
2
7
-R -R
N
H
R2
N
O
b
c
d, e
H
Br
B
7
7
7
7
-Et-H (44%)
O
R2
e
-Et-OM (59%)
B
-iBu-H (37%)
NH
NH
CO2But
CO2But
O
O
-iBu-OMe (58%)
2
2
4
4
4
5
5
5
-R
-R
-H (52%)
-OMe (57%)
-H (66%)
CO2But
CO2But
ButO2C
ButO2C
-OMe (81%)
CO2But
CO2But
F
F
F
CO2But
ButO2C
F
ButO2C
F
F
O
O
R1
R1
NH2
Aminotriester (
CO2But
I
I
O
S
O
S
B
)
NH2
O
O O
A
Aminotriester (
)
R1 = Et:
Et-Ox-2I
Bu-Ox-2I
R1 = iBu: i
Reagents and conditions: a) oxalyl chloride, cat. DMF, CH2Cl2, r.t., 6 h; b) Et3N, CH2Cl2; c) (BPin)2, AcOK, Pd(dppf)Cl2, 1,4-dioxane, 90 °C,
overnight; d) Pd(dba)2, Sphos, K2CO3, aq. THF, reflux, 30 min; e) CF3CO2H, CH2Cl2, r.t., 1 h.
fluorescent proteins (rsFPs) endure several hundreds or even
thousands (rsEGFP2) of switching cycles;21,22 they may be
Photophysical properties. The photophysical properties of
fused with a protein of interest and enable to observe living
cells and tissues via transfection protocols (often with optical
superresolution; e. g., in RESOLFT nanoscopy).21-24 We have
chosen Dreiklang and rsEGFP2, because their spectral and
switching properties are similar to those of compounds 1a,b
and new DAEs (see Table 1). For all compounds in Table 1
(including Dreiklang, but not rsEGFP2), photocyclization
(conversion into the fluorescent state) was induced by
irradiation with 365 nm light, while cycloreversion reactions
(for all DAEs, excluding Dreiklang) occurred under irradiation
with 470 nm light (488 nm for rsEGFP2).22 At pH 7.5 (near its
pKa value of 7.2), Dreiklang has two absorption maxima of the
fluorescent form (at 412 and 511 nm),21 and for its back-
switching into the non-fluorescent state we applied 405 nm
light.
the newly synthesized dodeca-acids DAE-12×COOH (Table 1)
have been measured in acetonitrile and aqueous solutions at
pH = 7 (phosphate buffer, 100 mM) in an automated setup
under conditions reported earlier.8 As references, we used the
previously published tetra-acid 1a and octa-acids 1b and 1c.6,7
Compounds 6-Et-H, 6-iBu-H, 7-Et-H, and 7-iBu-H have the
same chromophore (core switching unit), as compounds 1a,b,
and compounds 6-Et-OMe, 6-iBu-OMe, 7-Et-OMe, and 7-iBu-
OMe – the same, as compound 1c. Thus, absorption spectra of
the open forms are very similar within each group. The same
is true for the absorption spectra of the closed forms; they
feature small red shifts of ca. 5 nm for the CFs with bulky iBu
groups in the central positions, with respect to ethyl
substituted DAEs (see Figure S31 in Supporting Information).
For comparison with our synthetic switchers, we also used the
reversibly photoswitchable fluorescent proteins (rsFPs)
Dreiklang21 and EGFP2.22 Reversibly photoswitchable
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Table 1. Photophysical properties in aqueous solutions of photoswitchable DAEs (phosphate buffer 100 mM, pH
= 7), Dreiklang (DK in 100 mM aqueous Tris-HCl with150 mM NaCl at pH 7.5), as well as rsEGFP2.
1
2
3
4
5
6
7
8
9
max
[nm]
substitution
pattern
maxabs [nm]
em
e
b
PSS
365nm
(±0.03)
N1/2
fl
isom
/ ×10–3 [M-1cm-1]a
fl/
CFOF
linke
OFCFc
CFOFd
CO2H
r
OF
CF
CF
CF
buffer
MeCN
1af
1bg
4
---
---
340/12.4
337/16.5
334/12.5
333/14.4
334/13.3
334/14.2
345/17.1
338/15.2
450/35
447/45.6
450/36.1
455/38.9
451/36.5
456/38.6
457/37.3
458/43.1
534
522
525
526
528
529
582
579
0.48
0.57
0.44
0.46
0.46
0.46
0.45
0.30
1.9×10-1
2.3×10-1
5.2×10-1
5.0×10-1
4.1×10-1
4.5×10-1
1.5×10-3
9.5×10-3
1.2×10-3
2.0×10-3
1.9×10-3
4.2×10-2
1.6×10-3
3.3×10-2
1.8×10-4
9.8×10-5
---
---
400
290
235
11
171
37
769
429
405
1150
305
679
88
8h
12i
12i
12j
12j
8h
12i
6-Et-H
6-iBu-H
7-Et-H
7-iBu-H
1cg
long
long
short
short
---
0,97
0,90
0,99
0,93
---
301
609
1084
806
17
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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45
46
47
48
49
50
51
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53
54
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57
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59
60
290
14
2500
3052
6-Et-OMe
6-iBu-
OMe
7-Et-OMe
7-iBu-
OMe
long
0,96
305
420
12i
12j
12j
long
short
short
339/16.1
338/15.7
339/16.3
462/41.8
461/44.5
465/44.6
581
575
582
0.34
0.33
0,34
5.7×10-3
5.0×10-3
3.4×10-3
2.5×10-3
7.6×10-5
2.1×10-3
(3,4×10-3)
405nm
n.d. (480
– 500
0,78
0,95
0,75
137
4337
159
841
450
756
1949
109
347
412l
(2.4×10-2)
365nm
(0,92)
405nm
DK k
---
---
---
---
340
408
529
503
0.41
0.3
---
---
260
n.d.
n.d.
511l/83
n.d (405
nm)
rsEGFP2m
478/61
n.d.
2100
nm)
a) lowest energy absorption peak; b) fluorescence quantum yield; c) measured at 365 nm; d) measured at 470 nm (see Supporting
Information for details); e) number of cycles providing half of the initial amount of the closed form left in the PSS (365 nm); see SI for
details; f) data from ref. 6; g) data from ref. 7; h) CON(CH2CO2H)2; i) CONHC[CH2O(CH2)2CO2H]3; j) CONHC[(CH2)2CO2H]3; k) data from
ref. 21: “on”- and “off”-switching with 365 nm and 405 nm light, respectively; l) two absorption bands of the fluorescent form were observed
for the neutral and anionic states at pH 7.5; excitation with 515 nm light; m) data from ref. 22.
Figure 1. Comparative photoswitching kinetics (A: ON-switching with 365 nm, B: OFF-switching with 470 nm irradiation) of 5 μM
solutions (phosphate buffer, 100 mM, pH = 7) of compounds 1a (orange circles), 1b (gray circles), 6-Et-H (black circles), 6-iBu-H
(red circles), 7-Et-H (green circles), 7-iBu-H (blue circles), 1c (gray triangles), 6-Et-OMe (black triangles), 6-iBu-OMe (red
triangles), 7-Et-OMe (green triangles), and 7-iBu-OMe (blue triangles). The absorption at the maximum of the CF (450-460 nm)
is plotted vs. the irradiation time.
spectra. HPLC measurements show full conversion (PSS-
> 0,95) to the CF (fluorescent form) for all DAEs with
The photoconversion reactions were monitored by UV-VIS
and fluorescence spectroscopy, as well as HPLC analysis with
a diode array detector (for DAEs). The isomers (OF and CF,
Scheme 1A) have different retention times and optical
365nm
ethyl groups in the central positions. Their replacement with
bulky and isobutyl groups accelerates the ring-opening
reaction18 to values of the same order of magnitude as for the
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ring-closing reaction. We isolated the closed-ring isomer of
switching quantum yield of compound 7-iBu-H is 3.3%. At
the other extreme, there are very slowly off-switching
compounds having electron donating methoxy groups at
positions 4/4′ of the phenyl rings and ethyl groups in the
central positions (1c, 6-Et-OMe and 7-Et-OMe). The
intermediate group includes compound 1b, 6-Et-H, 7-Et-H,
6-iBu-H 6-iBu-OMe, and 7-iBu-OMe. It is interesting to note
that the “accelerating” effect of isobutyl groups attached to
the central positions compensates the effect of the electron
donating OMe groups in positions 4/4′ of the phenyl rings.
In respect of cycloreversion quantum yields, primary amides
are slower than secondary amides having the similar
substitution pattern (Table 1 and Figure 1). The fatigue
resistance of DAEs (Table 1, Figure 2) was evaluated as
number of cycles required for bleaching half of the initial
amount of the closed form.
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compound 7-iBu-OMe and showed that it remained
unchanged in the dark for at least 36 h at 50°C (in aqueous
acetonitrile). This result indicates that in spite of the high
steric strain, the closed-ring isomers of isobutyl-substituted
DAEs are thermally stable. Introduction of methoxy groups
at 4,4′-positions of phenyl rings decreased both cyclization
and cycloreversion quantum yields. The decrease in the
yields is ascribed to intramolecular donor (methoxy) –
acceptor (SO2) interactions. In respect of cyclization
quantum yields, primary amides are generally faster (up to
3-fold) than secondary amides, particularly in the case of
slowly ON-switching dyes. Regarding the OFF-switching
rates, three groups of compounds were identified (Figure
1B). The first one contains only one compound (7-iBu-H)
having isobutyl groups at the central core (R1) and hydrogen
atoms at positions 4/4′ of the phenyl rings (R2). The off-
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Figure 2. (A) Fatigue resistance (N ) in aqueous solutions; (B) the ratio (R) of switching cycles achieved in aqueous buffer and
½
MeCN. See ref. 8 and Table 1 the definition and values of N , and text for discussion.(N1/2) detected in the photostationary state
½
(PSS) at 365 nm (PSS365).8
This parameter can be evaluated by measuring the absorption
or emission intensity in the course of switching. The values of
N1/2 in Table 1 were obtained from changes in absorption.
Changes in emission provide similar values; however, the
more diluted samples are required, and this results in “noisier”
data. The products of photodecomposition were detected by
HPLC in their CFs for several reasons: CFs produce narrower
peaks than the OF, the separation is more efficient, the
absorption coefficients are higher (see Table 1), and there are
fewer overlapping with artefact peaks, compared with the
detection in the UV region (for HPLC traces, see Supporting
Information). The striking differences in photostabilities were
observed for structurally similar DAEs having the same type
of the central core but various types of amide groups. The
secondary amides ArCON(CH2CO2H)2 (1b,c) turned out to be
far less photoresistant than the structural analogs with
primary amides. In aqueous buffered solutions, primary
amides ArCONHC(X-COOH)3 show a 10- to 50-fold increase
in fatigue resistance, with respect to compounds 1b,c. For
example, the fatigue resistance of a OMe/OMe compound has
been improved by a factor of 49 (cf. 6-iBu-OMe and 1c).
However, the new structural feature – primary amides with
truncated N-tert-alkyl groups capped with carboxylates – has
a less significant effect for H/H compounds. Having the aim
to create DAEs with high photoswitching capacities in
aqueous media, we introduced and compared the ratio R in of
the switching cycles achieved in acetonitrile and aqueous
solutions (Table 1 and Figure 2B). The ratios observed for
compounds 1b,c are in agreement with our previous
measurements performed with other DAEs, where a steep
decrease in fatigue resistance was observed in aqueous
solutions (even for compounds demonstrating excellent
photostabilities in acetonitrile). The introduction of primary
amides with long linkers (ArCONHC(CH2O(CH2)2CO2H)3)
brings the parameter R nearer to 1, due to the increased
number of polar and charged carboxylate groups (from 8 to
12). However, compounds 6-Et-H, 6-iBu-H, 6-Et-OMe, and 6-
iBu-OMe still show poorer performance in water (Table 1 and
Figure 2B). On the contrary, “short-linker” compounds have a
remarkably improved fatigue resistance in water,
demonstrating up to 4-fold increase in photostability, with
respect to acetonitrile. To our knowledge, this is the first
example of fluorescent photoswitchable DAEs having better
performance in aqueous media than in organic solvents. In
particular, compound 7-Et-H accommodated over 1000 cycles
(Figure 3) in a buffered aqueous solution; this is an
outstanding value for DAE, especially taking into account that
oxygen was not excluded (all test were performed without
exclusion of air oxygen). Compounds with high fatigue
resistance are extremely valuable labels for RESOLFT
nanoscopy.6,8 For these applications, a faster response is also
desired. On the other hand, switchers with a high fl/onoff
ratio (Table 1) are expected to provide a large number of
photons emitted (on average) per incursion into the “bright”
state, making them promising candidates for single molecule-
based stochastic nanoscopies,7 or for the molecular-scale
opto-electronic devices.9 While high fatigue-resistance may
be not critical for single molecule-based stochastic
nanoscopies, it is advantageous for prolonged imaging
applications. It is remarkable that compounds with methoxy
substituents (6-Et-OMe and 7-Et-OMe) demonstrated an
improved photon statistics (3000 and 4300 photons per cycle
are predicted) with respect to dye 1c (2500 photons), in
addition to an improved fatigue resistance of over one order
of magnitude (in aqueous buffer). Tetra-acid 1a (without any
amide groups)6 has an intermediate photoresistance (N1/2
=
171), probably due to insufficient protection against
aggregation (aggregates are likely to bleach faster). Numerous
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details). These compounds demonstrate a “conventional”
1
or expected fatigue, where bleaching results in the “dark”
photoproducts. Therefore, the better performance of
compounds with short linkers, in the pairs with
compounds having long linkers, must be due to their lower
mass-to-charge ratio (higher solubilizing capacity of the
polar groups). The reversibly photoswitchable FPs
Dreiklang and EGFP2 underwent ca. 160 and 2100
switching cycles, respectively, before the fluorescence was
reduced by 50%.21,22 In cuvette experiments we observed
260 full switching cycles, – a reasonably good agreement
with the reported value for Dreiklang obtained in living E.
coli expressing this protein.21 Under the similar switching
conditions, all rationally designed and synthetic DAEs 6-
R1-R2 and 7-R1-R2 can be favorably compared with
Dreiklang, which has been identified in the course of several
consecutive screenings among 70 000 clones of the natural FP
Citrine.21 The extinction coefficients of Dreiklang (83 000 M-1
cm-1) and rsEGFP2 (61 300 M-1 cm-1) are larger than the molar
absorptivities of the synthetic photoswitches (35 000 – 46 000
M-1 cm-1), but the molecular masses of Dreiklang (28 510 Da)21
and rsEGFP2 (26 900 Da)22 are much higher than these of
compounds 6-R1-R2 and 7-R1-R2 (ca. 2200 Da). Therefore, the
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“normalized brightness”
– product of the fluorescence
quantum yield and extinction coefficient calculated on a unit
of the molecular mass – is much higher for synthetic switches.
Additional and not always favorable feature of Dreiklang is its
acid-sensitivity; the pKa value of the “on-state” is 7.2. The
values of the fluorescence quantum yield and extinction
coefficient of Dreiklang (Table 1) were measured at pH 7.5.
Two absorption bands of the fluorescent form (maxima at 412
nm and 511 nm) were observed for the neutral and anionic
states, respectively, at pH nearby the pKa value (7.2).21 Under
acidic conditions and in the fluorescent state, this protein
features only one absorption band (412 nm), and excitation
with 515 nm light becomes impossible. The pKa of the rsEGFP2
chromophore in the thermal equilibrium state is ~5.8.22 The
switching and fluorescent properties of synthetic dyes 6-R1-R2
and 7-R1-R2 as well as rsEGFP2 are less dependent on pH than
these of Dreiklang. Dreiklang, rsEGFP2, DAEs 1a,b, and
compounds unsubstituted at C-4/4′ (R2 = H in Scheme 1; first
part of Table 1), are relatively fast switches: their cyclization
and cycloreversion quantum yields are higher than the
corresponding parameters of the slower switches 1c and the
methoxy-substituted compounds (R2 = OMe in Scheme 1;
second part of Table 1). For dyes 6-Et-OMe and 7-Et-OMe, one
full cycle required irradiation for more than 20 min, and the
whole series lasted for about 5-10 days (until 50% bleaching
occurred). Compounds 6-iBu-OMe and 7-iBu-OMe occupy
intermediate positions between slow and fast switchers. The
data in Table 1 allow to compare photostabilities of the
methoxy-substituted compounds (R2 = OMe in Scheme 1) and
their unsubstituted analogs (R2 = H in Scheme 1). We can
conclude that the lower cycloreversion quantum yields are not
always associated with higher photobleaching rates. However,
in the series of structurally very similar DAEs with the same
aromatic systems (6-Et-H/6-iBu-H, 6-Et-OMe/6-iBu-OMe
and 7-Et-OMe/7-iBu-OMe), the values of N1/2 generally
increase with increasing of cycloreversion quantum yields.
However, the pair of compounds 7-Et-H/7-iBu-H represents
an exception from this rule: in aqueous solutions the “slower”
dye 7-Et-H underwent more switching cycles (more than 1000;
Table 1). As shown in Table 1 and Figure 2, the fatigue
carboxylic residues in dyes 6-R1-R2 and 7-R1-R2 provide lower
mass-to-charge ratios, better solubility in aqueous solutions at
physiological pH and prevent aggregation more efficiently.
The comparison with the relatively photostable “tetra-
acid” 1a demonstrates the detrimental effect of the
secondary amide residues on photostability, even if N,N-
bis(carboxymethyl) groups have a remarkable solubilizing
effect. To reveal the differences in degradation paths found
for compounds 1b,c, on one hand, and for the new DAEs,
on the other hand, we analyzed solutions of all compounds
(HPLC and mass-spectroscopy), after performing 20 full
cycles. All new compounds (12×COOH,
Figure 3. Fatigue resistance of compound 7-Et-H in MeCN
(red symbols) and in phosphate buffer (black symbols).
The difference in absorption (normalized) at 450 nm (the
maximum of the CF) was measured between the PSSs
achieved with 365 nm and 470 nm irradiations, and plotted
vs. the cycle number (A). The full set of absorption (B) and
emission (C) spectra for the first 100 cycles in aqueous
phosphate buffer (0,1 M, pH = 7). Insets show changes in
absorption and emission maxima for the CF, for each half
cycle (PSSs under 365 nm and 470 nm irradiation).
primary amides) displayed only one main peak of the
colored CF in HPLC analyses of photoreaction mixtures,
with only minor peaks of photoproducts gradually
appearing in the course of numerous transitions between
open- and closed-forms (see Supporting Information for
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resistance in acetonitrile solutions is not always better, though
We are grateful to Max-Planck-Gesellschaft for the generous
financial support. We thank J. Bienert (MPIBPC), Dr. H.
Frauendorf, Dr. M. John and co-workers (Institut für
Organische und Biomolekulare Chemie, Georg-August-
Universität, Göttingen, Germany) for recording mass- and
NMR spectra.
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in the previous studies the higher switching velocity in aprotic
solvents always associated with higher photoresistance.6-8
■ CONCLUSION AND OUTLOOK
5
The high photostability of the GFP-like proteins is believed to
be determined by enclosure of the chromophore
(autocatalytically formed from Gly65-Tyr66-Gly67 tripeptide)
into an alpha-helical segment surrounded by an 11-stranded
beta-barrel.21 This feature is unavailable for synthetic dyes.
However, the numerous polar groups “embracing” the central
core in compounds 6-R1-R2 and 7-R1-R2 probably have a
protecting effect, in addition to imparting water solubility.
Therefore, the switching fatigue in all solvents and especially
in water improves, due to “isolation” of the switching unit
from the harmful contact with nucleophiles. In a similar
manner, the amino acid barrel protects the photochemically
unstable fluorophore of all FPs. An important structural
element introduced in this report is the presence of four
secondary amides with N-tert-alkyl residues capped with 12
carboxylic acid groups. Under alternating irradiation with UV-
(365 nm) and visible light (470 nm), the new water-soluble
fDEAs having this structural unit undergo several hundred
(the “rapid” DAEs with isobutyl groups – up to thousand) full
switching cycles in aqueous solutions without exclusion of air
oxygen. The new branching approach based on secondary
carboxamides and N-tert-alkyl residues may serve for
designing of various photo-switchable and fluorescent dyes
with further improved photoresistance against bleaching in
protic solvents. However, the rapid and endurable switching
demonstrated for the free dyes in aqueous solutions does not
necessarily mean the same performance for the conjugates of
the dyes with proteins. In future, we are going to prepare
bioconjugates of the new DAEs introduced in this study and
study their imaging capabilities in fluorescence microscopy
and nanoscopy. In addition, it may turn out that further
optimizations in respect of photostability and switching
kinetics will be necessary, in particular, for DAEs undergoing
the ring-closure reaction under irradiation with 405 nm light.
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■ REFERENCES
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■ ASSOCIATED CONTENT
(12) Xiong, Y.; Rivera- Fuentes, P.; Sezgin, E.; Vargas Jentzsch, A.;
Eggeling, C.; Anderson, H. L. Photoswitchable Spiropyran Dyads for
Biological Imaging. Org. Lett. 2016, 18, 3666–3669.
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: . It contains complete
description of the synthesis (with copies of NMR spectra of all
new compounds), details of photoswitching and
photodecomposition experiments.
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Switches Based on Photochromic Compounds. Eur. J. Org. Chem.
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(17) Uno, K.; Niikura, H.; Morimoto, M.; Ishibashi, Y.; Miyasaka, H.;
■ AUTHOR INFORMATION
Corresponding Authors
*vbelov@gwdg.de
*iriem@rikkyo.ac.jp
*shell@gwdg.de
ORCID
Kakishi Uno: 0000-0002-3747-2656
Vladimir N. Belov: 0000-0002-7741-4653
Stefan W. Hell: 0000-0002-9638-5077
Notes
The authors declare no conflict of interests.
■ ACKNOWLEDGMENTS
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Irie, M. In Situ Preparation of Highly Fluorescent Dyes upon
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