The mechanodonor-acceptor coupling (MDAC) approach for unidirectional multi-state fluorochromism

Article abstract of DOI:10.1007/s11426-020-9874-6

Uni-directional multi-state fluorochromic scaffolds are valuable photofunctional molecules and yet scarce. We report a general approach for their design, i.e., mechanodonor-acceptor coupling (MDAC). A photochromic molecule is a mechanodonor, due to its capability to convert photonic energy into mechanical force. Upon proper coupling, it can be used to drive a mechanochromic molecule for uni-directional multi-state fluorochromism. The embodiment of this approach is a rhodamine-dithienylethylene hydride (RDH), which has been successfully employed in super-resolution localization microscopy[Figure not available: see fulltext.]

Full text of DOI:10.1007/s11426-020-9874-6

SCIENCE CHINA
Chemistry
•ARTICLES•
February 2021 Vol.64 No.2: 253–262
https://doi.org/10.1007/s11426-020-9874-6
The mechanodonor-acceptor coupling (MDAC) approach for uni-
directional multi-state fluorochromism
Luyan Gu1†, Lujia Zhang2†, Xiao Luo4*, Ying Zheng6, Zhiwei Ye6, Meng Lv3, Jinquan Chen3,
Chunlai Chen2*, Yi Xiao6, Weihong Zhu1,5, Xuhong Qian1 & Youjun Yang1*
1State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University
of Science and Technology, Shanghai 200237, China;
2School of Life Sciences; Beijing Advanced Innovation Center for Structural Biology; Beijing Frontier Research Center for Biological
Structure, Tsinghua University, Beijing 100084, China;
3State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200062, China;
4School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China;
5School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China;
6State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
Received August 17, 2020; accepted September 14, 2020; published online December 28, 2020
Uni-directional multi-state fluorochromic scaffolds are valuable photofunctional molecules and yet scarce. We report a general
approach for their design, i.e., mechanodonor-acceptor coupling (MDAC). A photochromic molecule is a mechanodonor, due to
its capability to convert photonic energy into mechanical force. Upon proper coupling, it can be used to drive a mechanochromic
molecule for uni-directional multi-state fluorochromism. The embodiment of this approach is a rhodamine-dithienylethylene
hydride (RDH), which has been successfully employed in super-resolution localization microscopy
mechanodonor-acceptor coupling, fluorochromism, diarylethene-rhodamine hybrid, single-molecule imaging
Citation:
Gu L, Zhang L, Luo X, Zheng Y, Ye Z, Lv M, Chen J, Chen C, Xiao Y, Zhu W, Qian X, Yang Y. The mechanodonor-acceptor coupling (MDAC)
approach for uni-directional multi-state fluorochromism. Sci China Chem, 2021, 64: 253–262, https://doi.org/10.1007/s11426-020-9874-6
1 Introduction
novel binary photochromic scaffolds, such as the acylhy-
drazones [8], azo-BF2 switches [9], NHC-chelate dimesi-
tylboron [10], a NIR-light operated dihydropyrene [11],
imidazolyl based systems [12], dibenzobarrlenes [13], het-
erodiazocines [14], Stenhouse adducts [15], hydroazulenes
[16], hemithioindigo [17], and AIEgen systems [18]. All
these aforementioned ones are two-state photochromes
(A→B→A). Recently, a paradigm shift from these binary to
ternary or even higher-order molecular systems is ongoing
because of their capability to exponentially expand the at-
tainable information space in molecular logic gates and
functional complexities in optomechanical systems. Though
multi-state fluorochromes are not rare feats, design of uni-
directional multi-state systems remains challenging.
Photochromic materials [1] exhibiting photo-triggered
switching between two or more spectrally distinct states are
the cornerstones of a wide-variety of cutting-edge applica-
tions, e.g., spatiotemporally controlled drug-release [2],
diffraction-unlimited super-resolution microscopy [3], pho-
topharmacology [4], molecular logic gates [5], and mole-
cular motors/machines [6]. Classic examples are
azobenzene, diarylethene, spiropyran, fulgide, and chromene
[7]. Recent years have also witnessed the emergence of many
†These authors contributed equally to this work.
*Corresponding authors (email: youjunyang@ecust.edu.cn; chunlai@tsinghua.edu.cn;
xluo@chem.ecnu.edu.cn)
© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020
chem.scichina.com link.springer.com

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
Multi-state fluorochromic systems have been routinely
constructed with two or more (n≥2) binary photochromic
molecules [19]. Typically, the photochromic units are or-
thogonal and therefore can be switched independently. For
applications in optomechanics, unidirectional switching of a
multi-state system, e.g., a four-state system operating in way
of A→B→C→D→A, is required [20]. This could theoreti-
cally be constructed with two engaging photochromic scaf-
folds, i.e., the photoswitching of one enabling/disabling the
switching of the other. Engaging mechanism can be elec-
tronic [3a], or steric [5g] in the existing systems. It is our
interest to develop a rational approach allowing systematic
design of such unidirectional multistate systems, via a me-
chanic engaging mechanism. We argue that a photo-
Figure 1 The MDAC to access unidirectional multi-state fluorochromism
(color online).
toluenesulfonate (5.48 g, 3 equiv., 29.43 mmol) and NaH
(259 mg, 1.1 equiv., 10.79 mmol) was added. The mixture
was stirred at 80 °C for 10 h before 1 mL H2O was added
into the solution. The dissolvent was removed under reduced
pressure. A saturated solution of NH4Cl (50 mL) was added
and the resulting mixture was extracted repeatedly with di-
chloromethane. The organic layer was combined, dried with
MgSO4 and filtered. The solvent was removed by evapora-
tion and the residue was purified by silica gel column
chromatography (petroleum ether and EtOAc (50:1, v/v)) to
give 1.65 g of compound 3 in a 63% yield as a white solid.
1H NMR (400 MHz, CDCl3): δ 3.13 (s, 3H).
chromophore can be regarded as
a
photo-induced
mechanodonor, because photochromism inevitably triggers a
concomitant structural alteration, which has been elegantly
exploited as the mechanistic basis of molecular machines
and photopharmacology (Figure 1). Upon proper mechano-
donor-acceptor coupling (MDAC), such a mechanodonor
could be harnessed as a trigger of a second mechanoacceptor
to furnish multi-state fluorochromes. The mechanodonor is a
fluorochrome responsible for converting the light energy into
molecular motion or intramolecular strain, which is then
exploited to activate the mechanochemistry [21] of a second
mechanochromphore, i.e., mechanoacceptor.
2.2.2 Synthesis of (2,5-dimethylthiophen-3-yl)boronic acid
(4 [23])
3-Bromo-2,5-dimethylthiophene (2 g, 1 equiv., 10.47 mmol)
was dissolved in 40 mL anhydrous tetrahydrofuran (THF). n-
Butyllithium (2.5 M in hexane, 6.28 mL, 1.1 equiv.,
11.51 mmol) was slowly added at −78 °C, and the reaction
solution was stirred at the same temperature for 15 min. A
solution of tri-n-butyl borate (3.09 mL, 1.1 equiv.,
11.51 mmol) in THF (10 mL) was added over a period of
10 min. The reaction solution was slowly heated to room
temperature and stirred for 8 h. Then 9 mL 2 M HCl was
added and the mixture was stirred for another 10 h. After the
reaction was completed, the excess solvent was removed and
NaOH solution was added to neutralize. The resulting mix-
ture was extracted with dichloromethane and water for three
times before the combined organic phases were dried over
MgSO4 and the solvent was removed to obtain a crude
product. The crude product was dissolved in 15 mL of di-
chloromethane and washed three times with 2 M NaOH
solution. The resulting aqueous phases were combined and
10 mL concentrated hydrochloric acid (12 M) was added
dropwise to commence the precipitation of 4 in a 73% yield
In this work, we report a novel multi-state fluorochrome
(RDH) following the MDAC approach. Structurally, RDH is
a hybrid of a dithienylethene and a rhodamine lactone, which
are engaged to each other via a five-membered spiro-lactone
ring. Photo-triggered unidirectional four-state fluorochromic
properties were evaluated. Colorimetrically, these four forms
adopt either colorless or magenta-colored states. Fluorome-
trically, they may adopt one of the three different states, i.e.,
dark, weak and strong. Excited-state dynamics were studied
and their three-state fluorescence signals were confirmed by
single-molecule spectroscopy. Potentials for localization
microscopy were verified.
2 Experimental
2.1 Materials and instruments
All the starting materials were obtained from commercial
suppliers and used as received. Details are in the Supporting
Information online.
1
as a white powder (1.2 g). H NMR (400 MHz, CDCl3): δ
7.07 (s, 1H), 2.82 (s, 3H), 2.46 (s, 3H).
2.2 Synthesis and characterization
2.2.3 Synthesis of 3,4-bis(2,5-dimethylthiophen-3-yl)-1-
met-hyl-1H-pyrrole-2,5-dione (5 [24])
2.2.1 Synthesis of 3,4-dibromo-1-methyl-1H-pyrrole-2,5-
dione (3 [22])
3,4-Dibromo-1H-pyrrole-2,5-dione (2.5 g,
9.81 mmol) was dissolved in 30 mL of MeCN, and methyl p-
Compound 3 (600 mg, 1 equiv., 2.23 mmol) and compound 4
(765.84 mg, 2.2 equiv., 4.91 mmol) were dissolved in diox-
ane (50 mL), and then CsF (257.86 mg, 4 equiv., 8.93 mmol)
1
equiv.,

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
was added. This solution was bubbled with Ar for 15 min.
613.2559; found 613.2558.
Then Pd(PPh3)4 (1.67 g, 0.1 equiv., 0.22 mmol) was added.
The solution was once again bubbled with Ar for 15 min,
heated to 90 °C and stirred for 10 h before cooled to room
temperature. The dissolvent was removed under reduced
pressure. A saturated solution of Na2CO3 (30 mL) was added
to the solution and the mixture was extracted with di-
chloromethane for three times. The organic layer was com-
bined, dried over MgSO4 and filtered. The solvent was
removed by evaporation and the residue was purified by
silica gel column chromatography (petroleum ether and
EtOAc (40:1, v/v)) to give 325 mg of compound 5 in a 44%
yield as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 6.71
(s, 2H), 3.11 (s, 3H), 2.41 (s, 6H), 1.86 (s, 6H).
2.2.6 Synthesis of 3,5-dibromo-2-methylthiophene (7 [26])
2-Methylthiophene (5 g, 1 equiv., 50.93 mmol) was dis-
solved in 50 mL glacial acetic acid solution and then N-
bromosuccinimide (NBS) (18.13 g, 2 equiv., 101.87 mmol)
was slowly added to the solution at room temperature. After
stirring for 1.5 h, the reaction solution was poured into ice
water and saturated Na2CO3 solution was added to neu-
tralize. The resulting mixture was extracted with di-
chloromethane and the organic layer was dried over MgSO4
and concentrated. The obtained crude product was separated
by silica gel column chromatography (petroleum ether) to
give 11.50 g of compound 7 in an 88% yield as a colorless
liquid. 1H NMR (400 MHz, CDCl3): δ 6.85 (s, 1H), 2.34 (s,
3H).
2.2.4 Synthesis of 3,4-bis(2,5-dimethylthiophen-3-yl)fur-
an-2,5-dione (1 [25])
Compound 5 (225 mg, 1 equiv., 0.679 mmol) was dissolved
in 10% KOH aqueous solution (20 mL). The reaction mix-
ture was heated to 100 °C and maintained for 10 h before
cooled to room temperature. The product was then obtained
by adding 2 M HCl to commence the precipitation of 3,4-bis
(2,5-dimethylthiophen-3-yl)furan-2,5-dione (1, 201 mg) in a
2.2.7 Synthesis of 3-bromo-5-(4-methoxyphenyl)-2-me-
thyl-thiophene (8 [26])
Pd(PPh3)4 (316 mg, 0.01 equiv., 0.273 mmol) and (4-meth-
oxyphenyl)boronic acid (3.27 g, 1.1 equiv., 21.49 mmol) was
added to a stirred THF solution (120 mL) of 7 (5 g, 1 equiv.,
19.53 mmol). 2 M aqueous Na2CO3 (60 mL) was delivered
to the reaction mixture, which was heated at 80 °C under an
argon atmosphere. After 10 h, the solution was cooled down
to room temperature and poured into water. The organic layer
was extracted with dichloromethane, dried over anhydrous
MgSO4 and concentrated. The residue was purified by silica
gel column chromatography (dichloromethane and methanol
(50:1, v/v)), to give 4.2 g of compound 8 in a 76% yield as a
1
93% yield as a light yellow powder. H NMR (400 MHz,
CDCl3): δ 6.75 (d, J=0.9 Hz, 2H), 2.42 (s, 6H), 1.92 (s, 6H).
2.2.5 Synthesis of 3′,6′-bis(diethylamino)-3,4-bis(2,5-di-
methyl-thiophen-3-yl)-5H-spiro[furan-2,9′-xanthen]-5-one
(RDH)
3,3′-Oxybis(4-bromo-N,N-diethylaniline) (589.28 mg, 1.5
equiv., 1.25 mmol) was dissolved in 40 mL anhydrous THF.
Then n-butyllithium (2.5 M in hexane, 1 mL, 3 equiv.,
2.51 mmol) was slowly added at −78 °C, and the reaction
solution was stirred at the same temperature for 15 min to
yielded 6, A solution of compound 1 (266 mg, 1 equiv.,
0.83 mmol) in anhydrous THF (10 mL) was added over a
period of 10 min. The reaction solution was slowly heated to
room temperature and stirred for 8 h. Saturated NH4Cl so-
lution was added to the reaction flask, and the mixture was
extracted with dichloromethane for three times. The organic
layer was combined, dried over MgSO4 and filtered. The
solvent was removed by evaporation and the residue was
purified by silica gel column chromatography (petroleum
ether and EtOAc (20:1, v/v)) to give 262 mg of RDH in a
51% yield as a light red solid. 1H NMR (400 MHz, CDCl3): δ
7.10 (d, J=8.9 Hz, 2H), 6.80 (d, J=0.9 Hz, 1H), 6.45 (dd,
J=8.9, 2.6 Hz, 2H), 6.36 (d, J=2.5 Hz, 2H), 5.71 (d, J=
0.9 Hz, 1H), 3.37 (dd, J=7.1, 2.2 Hz, 8H), 2.42 (s, 3H), 2.13
(s, 3H), 1.97 (s, 3H), 1.73 (s, 3H), 1.18 (t, J=7.1 Hz, 12H).
13C NMR (151 MHz, CDCl3): δ 172.2, 158.4, 152.9, 149.6,
138.2, 136.6, 136.3, 136.2, 129.3, 127.8, 127.7, 126.7, 124.2,
124.0, 108.3, 104.1, 98.1, 44.5, 15.4, 15.3, 14.6, 14.5, 12.7.
ESI-HRMS (m/z): [M+H]+ calcd. for C36H41N2O3S2,
1
beige solid. H NMR (400 MHz, CDCl3): δ 7.43 (d, J=
8.9 Hz, 2H), 6.98 (s, 1H), 6.90 (d, J=8.9 Hz, 2H), 3.83 (s,
3H), 2.40 (s, 3H).
2.2.8 Synthesis of (5-(4-methoxyphenyl)-2-methylthio-
phen-3-yl)boronic acid (9 [27])
Compound 8 (4.2 g, 1 equiv., 14.83 mmol) was dissolved in
40 mL anhydrous THF. n-Butyllithium (2.5 M in hexane,
8.9 mL, 1.5 equiv., 22.25 mmol) was slowly added at
−78 °C, and the reaction solution was stirred at the same
temperature. After 15 min, a solution of tri-n-butyl borate
(6 mL, 1.5 equiv., 22.25 mmol) in THF (10 mL) was added
over a period of 10 min. The reaction solution was slowly
heated to room temperature and stirred for 8 h. Then 9 mL 2
M HCl was added and the reaction solution was stirred for
another 10 h. After the reaction is complete, saturated NaOH
solution was added, and the resulting mixture was extracted
with dichloromethane and water. After three extractions, the
combined organic phases are dried over anhydrous MgSO4
and the solvent was removed to obtain a crude product. The
crude product was dissolved in 15 mL of dichloromethane
and washed three times with 2 M NaOH solution. The re-
sulting aqueous phases were combined and 10 mL con-

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
centrated hydrochloric acid (12 M) was added dropwise to
commence the precipitation of 9 (3.0 g) in an 82% yield as a
white solid. 1H NMR (400 MHz, (CD3)2SO): δ 7.46–7.43 (m,
3H), 6.96 (d, J=8.8 Hz, 2H), 3.77 (s, 3H), 2.58 (s, 3H).
NH4Cl solution was added to the reaction flask, and the
mixture was extracted with dichloromethane for three times.
The organic layer was combined, dried over MgSO4 and
filtered. The solvent was removed by evaporation and the
residue was purified by silica gel column chromatography
(petroleum ether and EtOAc (5:1, v/v)) to give 150 mg of
2.2.9 Synthesis of 3,4-bis(5-(4-methoxyphenyl)-2-methyl-
thiop-hen-3-yl)-1-methyl-1H-pyrrole-2,5-dione (10)
1
compound 12 in a 47% yield as a light blue solid. H NMR
Compound 3 (600 mg, 1 equiv., 2.23 mmol) and compound 9
(1.38 g, 2.5 equiv., 5.58 mmol) were dissolved in dioxane
(50 mL), and CsF (1.36 g, 4 equiv., 8.93 mmol) was added.
This solution was bubbled with Ar for 15 min. Then
Pd(PPh3)4 (257.86 mg, 0.1 equiv., 0.22 mmol) was added.
The solution was once again bubbled with Ar for 15 min,
heated to 90 °C and stirred for 10 h before cooled to room
temperature. The dissolvent was removed under reduced
pressure. A saturated solution of Na2CO3 (30 mL) was added
to the solution and the mixture was extracted with di-
chloromethane for three times. The organic layer was com-
bined, dried over MgSO4 and filtered. The solvent was
removed by evaporation and the residue was purified by
silica gel column chromatography (petroleum ether and
EtOAc (15:1, v/v)) to give 410 mg of compound 10 in a 34%
yield as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 7.47
(d, J=8.8 Hz, 4H), 7.19 (s, 2H), 6.90 (d, J=8.8 Hz, 4H), 3.82
(s, 6H), 3.17 (s, 3H), 2.01 (s, 6H). 13C NMR (151 MHz,
CDCl3): δ 171.0, 159.4, 141.3, 140.2, 133.2, 127.8, 127.0,
126.6, 123.2, 114.4, 55.4, 24.4, 15.1. ESI-HRMS (m/z): [M
+Na]+ calcd. for C29H25NO4S2Na, 538.1123; found
538.1122.
(600 MHz, CDCl3): δ 7.48 (d, J=8.8 Hz, 2H), 7.24 (s, 1H),
7.17 (d, J=8.9 Hz, 2H), 7.13 (d, J=8.8 Hz, 2H), 6.89 (d, J=
8.8 Hz, 2H), 6.76 (d, J=8.8 Hz, 2H), 6.51 (dd, J=8.9, 2.6 Hz,
2H), 6.37 (d, J=2.5 Hz, 2H), 6.08 (s, 1H), 3.82 (s, 3H), 3.78
(s, 3H), 3.38 (dd, J=9.3, 7.2 Hz, 8H), 2.10 (s, 3H), 1.81 (s,
3H), 1.20 (t, J=7.1 Hz, 12H). 13C NMR (151 MHz, CDCl3): δ
172.3, 159.5, 159.2, 159.1, 153.0, 149.6, 140.8, 140.5, 139.1,
137.7, 130.3, 128.7, 127.7, 127.2, 127.0, 126.9, 126.6, 123.6,
123.2, 121.2, 114.3, 114.2, 108.4, 103.7, 98.2, 84.1, 55.5,
55.5, 44.6, 14.9, 14.5, 12.7. ESI-HRMS (m/z): [M+H]+ calcd.
for C48H49N2O5S2, 797.3083; found 797.3082.
2.2.12 Synthesis of 3′,6′-bis(diethylamino)-3,4-bis(5-(4-
methoxyphenyl)-2-methylthiophen-3-yl)-5H-spiro[furan-
2,9′-xanthen]-5-one (13)
Under nitrogen atmosphere, BBr3 (1 M in CH2Cl2, 0.88 mL,
5 equiv., 0.88 mmol) was added dropwise to a stirred solu-
tion of compound 12 (140 mg, 0.18 mmol) in anhydrous
dichloromethane (20 mL) at 0 °C. After that, the reaction
mixture was stirred for 5 h at room temperature. Then, the
mixture was poured into ice water and filtered to give a light
purple solid 13 (45 mg, 33.3%). The compound was used
without further purification. ESI-HRMS (m/z): [M+H]+
calcd. for C46H45N2O5S2, 769.2770; found 769.2759.
2.2.10 Synthesis of 3,4-bis(5-(4-methoxyphenyl)-2-me-
thylthiophen-3-yl)furan-2,5-dione (11)
Compound 10 (200 mg, 1 equiv., 0.387 mmol) was dissolved
in 10% KOH aqueous solution (20 mL). The reaction mix-
ture was heated to 100 °C, maintained for 10 h before cooled
to room temperature. The product was then obtained by
adding 2 M HCl to commence the precipitation of compound
2.2.13 Synthesis of 3′,6′-bis(diethylamino)-3,4-bis(2-me-
thyl-5-(4-(prop-2-yn-1-yloxy)phenyl)thiophen-3-yl)-5H-
spiro[furan-2,9′-xanthen]-5-one (14)
Compound 13 (80 mg, 1 equiv., 0.1 mmol) was dissolved in
30 mL of acetone, and 3-bromoprop-1-yne (62 mg, 5 equiv.,
0.52 mmol), Cs2CO3 (80 mg, 4 equiv., 0.41 mmol) were
added. The mixture was stirred for 9 h at 60 °C. The dis-
solvent was removed under reduced pressure. A saturated
solution of NH4Cl (50 mL) was added and the resulting
mixture was extracted repeatedly with dichloromethane. The
organic layer was combined, dried over MgSO4 and filtered.
The solvent was removed by evaporation and the residue was
purified on silica gel using dichloromethane as the eluent to
give 38 mg of compound 16 in a 44% yield as a dark blue
1
11 (175 mg) in a 90% yield as a blackish green powder. H
NMR (600 MHz, CDCl3): δ 7.47–7.45 (m, 4H), 7.20 (s, 2H),
6.92–6.90 (m, 4H), 3.83 (s, 6H), 2.07 (s, 6H).
13C NMR (151 MHz, CDCl3): δ 165.0, 159.7, 142.2, 142.1,
134.6, 127.1, 126.6, 126.2, 122.6, 114.5, 55.5, 15.3. ESI-
HRMS (m/z): [M+Na]+ calcd. for C28H22O5S2Na, 525.0806;
found 525.0805.
2.2.11 Synthesis of 3′,6′-bis(diethylamino)-3,4-bis(5-(4-
metho-xyphenyl)-2-methylthiophen-3-yl)-5H-spiro[furan-
2,9′-xanthen]-5-one (12)
Compound 6 was prepared in 40 mL anhydrous THF and
cooled downed to −78 °C. A solution of compound 11
(200 mg, 1 equiv., 0.40 mmol) in THF (10 mL) was added
over a period of 10 min. The reaction solution was slowly
heated to room temperature and stirred for 8 h. Saturated
1
solid. H NMR (400 MHz, CDCl3): δ 7.51–7.46 (m, 2H),
7.24 (s, 1H), 7.17 (d, J=8.9 Hz, 2H), 7.15–7.11 (m, 2H),
6.98–6.94 (m, 2H), 6.84 (d, J=8.8 Hz, 2H), 6.50 (dd, J=8.9,
2.5 Hz, 2H), 6.37 (d, J=2.5 Hz, 2H), 6.08 (s, 1H), 4.68 (dd,
J=18.9, 2.4 Hz, 4H), 3.38 (dd, J=6.9, 5.4 Hz, 8H), 2.52 (d, J=
10.8 Hz, 2H), 2.10 (s, 3H), 1.80 (s, 3H), 1.19 (t, J=7.0 Hz,
12H). 13C NMR (151 MHz, CDCl3): δ 172.2, 159.4, 156.9,

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
156.8, 152.8, 149.4, 140.5, 140.1, 139.2, 137.8, 130.2, 128.6,
128.0, 127.6, 127.6, 126.8, 126.4, 123.7, 123.0, 121.3, 115.3,
115.2, 108.3, 103.6, 98.1, 84.0, 55.9, 55.9, 44.5, 14.8, 14.4,
12.6. ESI-HRMS (m/z): [M+H]+ calcd. for C52H49N2O5S2,
845.3083; found 845.3082.
magenta color, which is the signature of the rhodamine core,
and yet a weak fluorescence because the close-form of the
DTE is a known fluorescence quencher via photo-induced
electron transfer (PET) [30]. Further green-light triggered
ring-opening of the B-ring yields RDHoo, whose fluores-
cence is now fully turned-on. The steric repulsion of the two
thiophene units of RDHoo further promotes the ring closure
of the cyclolactone to complete the unidirectional four-state
cycle.
2.3 Single molecule imaging
The fluorophore was covalently attached to a surface-
immobilized double stranded DNA using a click reaction
between azide and alkyne. The single-molecule imaging
experiments were performed at 25 °C using a home-built
objective-type. Details of the total internal reflection fluor-
escent (TIRF) microscope were described before [28] (Fig-
ure S14, Supporting Information online).
3.2 Spectral studies
The photochromic behavior of RDH was evaluated in a
series of solvents with varying polarity. Irradiation of the
CH2Cl2, THF, acetone, MeCN, or DMF solution of RDH
with LED light of 375 nm (30 mW/cm2) induced an emer-
gence of a broad absorption band covering a broad range of
500–600 nm with a maximum of 550 nm (Figure S4). The
quantum yields of the UV light-promoted ring-closing from
RDH to RDHcc, and green light-promoted ring-opening
from RDHcc-RDH were determined to be 48% and 8% re-
spectively in CH3CN following the reported methods [31].
Apparently, cyclization of the DTE moiety of RDH to
RDHcc occurred. However, further ring-opening of RDHcc
was not observed due to the magenta color of rhodamine core
did not appear, suggesting that the carboxylate was not a
sufficiently good leaving group in these non-protic solvents.
Therefore, the solvent was switched to MeOH, which is
protic and expected to further stabilize the carboxylate via
hydrogen-bonding and promote the ring-opening (Figure 4).
Upon UV-irradiation of a RDH solution in MeOH, the
magenta color corresponding to the absorption band at
552 nm instantaneously appeared and reached a plateau in
2.33 min (Figure 4(a)). We note that a lower-intensity peak at
355 nm, corresponding to the ring-closed form of the di-
thiopheneethylene unit, rose concomitantly (the white oval
circled region, Figure 4(a1)). The co-existence of the ab-
sorption bands at 552 and 355 nm confirmed the generation
of RDHoc (Figure 4(a)). The fluorescence emission studies
provided extra evidence for the formation of RDHoc in
MeOH upon UV irradiation. Excitation of the solution at
555 nm yielded an emission band at 583 nm with a weak
fluorescence quantum yield of 0.006 (Figure 4(b)). This is
also in agreement with our expectation that RDHoc is not
supposed to be highly fluorescent due to PET quenching.
Then, the solution was irradiated with a green LED light
(50 mW/cm2) to convert RDHoc to the highly fluorescent
RDHoo. This was indeed the case that the fluorescence
emission intensity at 590 nm increased by more than 17 fold
(Figure 4(b2)). This phenomenon explicitly proved the for-
mation of RDHcc, whose fluorescence quantum yield was
estimated to be 0.35. While the emission at 583 nm gradually
enhanced (Figure 4(a2)), the absorbance at 552 nm de-
2.4 Super-resolution imaging (PALM)
HeLa cells were stained with 1 μM RDH (10% DMSO) for
5 min and washed with PBS for three times. Imaging was
performed in normal culture medium without any additives
under 2,000 W/cm2, 532 nm irradiation and reconstructed
from 3,000 frames collected in 30 s. Super-resolution ima-
ging analysis was performed in a Thunder-Storm plugin of
Image. Briefly, the raw frames were filtered with difference-
of-Gaussians filter to select the single-molecule candidates.
Then the point spread function (PSF) of single molecule was
fitted with an integrated form of symmetric 2D Gaussian
function (Fitting radius: 3.0 pixel) following Maximum
likelihood method to estimate the precise location and sin-
gle-molecule intensity. The localization precision was cal-
culated according to the Thompson formula.
3 Results and discussion
3.1 Design rationale and synthesis
RDH was synthesized via a 5-step cascade starting from 3,4-
dibromo-N-methylmaleimide (3) (Figure 2). Suzuki-
Miyaura coupling of 3 with a dimethylthiophene boronic
acid (4) afforded 5 in a 44% yield. Further hydrolysis with
10% KOH at 90 °C, followed by an acid-promoted con-
densation, gave the anhydride (1) in a 93% yield. Treatment
of 1 with dilithium reagent (6) at −78 °C furnished RDH in a
51% yield, following a recent xanthene preparation [29].
The design rationale of RDH is shown in Figure 3(c).
Expectedly, UV-light irradiation of RDH (the resting state)
potentially promotes the formation of the B-ring of RDHcc
via an electrocyclic reaction of the dithiopheneetheylene
(DTE) moiety. During this process, the two thiophene units
are pulled together, which results in an internal strain in the
ring A of RDHcc, and further promot their ring-opening to
generate RDHoc. RDHoc is expected to exhibit an intense

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
Figure 2 Synthesis of (A) RDH and (B) a terminal alkyne substituted RDH analogue (14). (a) Pd(PPh3)4, CsF, dioxane, 100 °C, 44%; (b) 1) 10% KOH,
90 °C, 2) 2 M HCl, 93%; (c) THF, −78 °C, 51%; (d) Pd(PPh3)4, Na2CO3, THF/H2O, 90 °C, 76%; (e) 1) n-BuLi, 2) (nBuO3)B, 3) 12 M HCl, 82%; (f)
Pd(PPh3)4, CsF, dioxane, 100 oC, 34%; (g) 1) 10% KOH, 90 oC, 2) 2 M HCl 90%; (h) 6, n-BuLi, THF, −78 °C, 47%; (i) BBr3, 0 °C, 33%; (j) 3-bromoprop-1-
yne, Cs2CO3, acetone 60 °C, 44%.
Figure 3 Design rationale of RDH. (a) Photo-induced cyclization of dithienylethylene promoted strain in the five-membered anhydride ring. (b) The
mechanochemistry of rhodamine lactam. (c) The structure of RDH and its photo-triggered unidirectional switching between the four potential forms (color
online).
creased (Figure 4(b2)).
MeOH/H2O, with the spectral results in 50% MeOH in-
cluded in Supporting Information online (Figures S2, S3).
The reversible photoswitching of RDH was studied (Fig-
ure 5). The solution of RDH in MeOH was irradiated se-
quentially with 375 and 520 nm for multiple cycles and the
This suggested that the ring-closing of RDHoo back to
RDH occurred, which concluded the unidirectional four-
state switching of RDH. We further showcased that the
switching cycle can also be carried out in a mixture of

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
Figure 4 The UV-Vis absorption spectral changes (a) and the fluorescence emission spectral changes (b) of a RDH solution (5 μM, in MeOH). (a1) The
contour plot of the UV-Vis absorption spectra of a MeOH solution of RDH (5 μM) upon UV-irradiation of 375 nm from 0–2.33 min and then green-
irradiation of 520 nm from 2.33–22.33 min. (a2) The changes of the absorbance at 355 and 552 nm respectively with respect to the irradiation time. (a3) The
UV-Vis absorption spectrum of the solution at 2.33 min. (b1) The contour plot of the fluorescence emission spectra (λex=583 nm) of a solution of RDH
(5 μM) upon UV-irradiation of 375 nm from 0–2.33 min and then green-irradiation of 520 nm from 2.33–22.33 min. (b2) The changes of the emission
intensity at 583 nm respectively with respect to the irradiation time. (b3) The fluorescence emission spectrum of the solution at 2.33 min (color online).
fluctuation of both UV-Vis absorbance at 552 nm and the
emission intensity at 583 nm was monitored. The general
trend was that the extend of the absorbance/emission turn-on
at 552 and 583 nm respectively gradually decreased and a
background absorbance/emission gradually increases, with
respect to the number of cycles (Figures 5(a, b)). This phe-
nomenon suggested a build-up of a previously unexpected
substance exhibiting both an absorption at ca. 552 nm and a
notable emission at ca. 583 nm. This substance is not
RHDoo because it was stable under this condition and not
able to spontaneously convert to RDH upon sitting. The
dithienylethene can go through photo-induced cis-trans
Figure 5 (a) The reversible photoswitching of RDH monitored by UV-
Vis absorbance at 552 nm. (b) The reversible photoswitching of RDH
monitored by fluorescence emission intensity at 583 nm with excitation at
550 nm. (c) The cis-trans isomerization of dithienylethylene accounting for
the reduction of reversibility upon photoirradiation (color online).
isomerization if the two thiophene units are not locked in the
cis-conformation (Figure 5(c)) [32]. Therefore, we propose
that this unknown substance is trans-RDHoo. From rever-
sibility point of view, the formation of trans-RDHoo is un-
desired. However, this actually further enriches the already
facile photochemistry of RDH and can be proved a useful
feature in future applications.
of the rhodamine core. Using a TIRF microscope, we cap-
tured fluorescence signals produced by individual fluor-
ophore molecules over time. First of all, three-state
fluorescence properties were unambiguously observed,
which further supported the proposed switching mechanism
of 14 (Figure 6(h)). Statistically, the fluorophore stayed in its
emissive states for ~1 s, both in aqueous and 50% methanol
solutions (Figure S15). And during this period, ca. (1.8
−2.3)×105 photons were detected, comparable to the photon
flux by two bright fluorophores, i.e., Cy3 and TMR (Figures
6(b–g)). Second, we note that different molecules of 14 ex-
hibited interesting heterogeneity under our experimental
conditions. While some (ca. 33%) events showed multiple
3.3 Single molecular imaging of RDH analogue 14
The three-state fluorescence properties, i.e. dark-weak-
strong, of RDH were rare in the literature and therefore, we
synthesized a RDH analogue 14 which contained alkynyl
groups and resorted to single-molecule spectroscopy for
confirmation (Figure 6). The fluorophore was covalently
attached to a surface-immobilized double stranded DNAwith
click chemistry (Figure S10). The 405 nm laser-line was
used for activation of 14, and 532 nm was used for excitation

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
Figure 6 (a) The immobilization approach of 14 onto the surface of PEG-modified glass. (b–d) The histogram of the photon counts per second from each
activation event of 14, Cy3 and TMR, in aqueous phosphate buffer. (e–g) The histogram of the photon counts per second from each activation event of 14,
Cy3 and TMR, in aqueous phosphate buffer containing 50% MeOH. (h) Typical single-molecule fluorescence trajectories of events displayed multiple
emission cycles with variable intensities within each emission cycle (blue); of events displayed multiple emission cycles with stable intensities (orange); of
events displayed a single emission cycle with variable intensities (red); and of events displayed a single emission cycle with a stable intensity (green) (color
online).
off-on cycles, other events were switched on once and was
then probably bleached due to the high intensity of laser used
in this study. While some exhibited the expected dark-weak-
strong profile (Figure 6(h), blue and red), a large fraction
exhibited two-state, i.e., OFF-ON emission profile (Figure 6
(h), orange and green). However, this did not necessarily
contradict our proposed mechanism since the molecule could
spend flitting time on either 14oc or 14oo state and render the
corresponding “weak” or “strong” state undetected. Also, it
was seen that the molecule under investigation was turned on
to the “strong” state first and then transited back to the
“weak” state, suggesting the UV-triggered conversion of
14oo to the 14oc was also viable.
scopy [33]. Basically, the two fluorophores in close proxi-
mity were stochastically photo-activated to allow
independent localization of each fluorophore. Therefore,
photo-triggered reversible switching or irreversible activa-
tion is a prerequisite for a fluorophore to be feasible for
localization based microscopy. The off-on switching of the
fluorescence properties of RDH renders it feasible for photo-
activated localization microscopy. The biocompatibility of
RDH was verified by a cytotoxicity study with CCK8 kit and
the cell viability was found to be higher than 85% (Figure
S17). In a proof-of-concept microscopic study, photo-
activation of RDH in HeLa cells was tested. Prior to acti-
vation, the cells were dark as expected (Figure 7(a)). Upon
irradiation of cells with 365 nm (2.4 mW/cm2) laser line for
60 s, fluorescence of the rhodamine dye at 590 nm upon
excitation at 559 nm was observed (Figure 7(b)). This deli-
neated the potentials of RDH for PALM imaging applica-
tions. Colocalization studies confirmed that RDH was mito-
specific (Figure S16). Therefore, diffraction-unlimited mi-
tochondrial images were obtained, with a localization pre-
cision of 22.7 nm. A side-by-side comparison with the
3.4 Potentials of RDH in PALM imaging
Traditionally, the resolution of light microscopy is diffrac-
tion-limited [3b]. Namely, the two fluorophores within the
distance of ca. λ/2 cannot be distinguished under epi-
illumination. In 2006, super-resolution microscopic methods
were reported based on single-molecule localization micro-

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Gu et al. Sci China Chem February (2021) Vol.64 No.2
photochromic scaffolds with the bright fluorescence of
classic fluorochromic scaffolds.
Acknowledgements This work was supported by the National Natural
Science Foundation of China (21822805, 21922704, 21877069, 21908065,
22078098), China Postdoctoral Science Foundation (2019M651427,
2020T130197) and the Commission of Science and Technology of Shanghai
Municipality (18430711000).
Conflict of interest The authors declare no conflict of interest.
Supporting information The supporting information is available online at
http://chem.scichina.com and http://link.springer.com/journal/11426. The
supporting materials are published as submitted, without typesetting or
editing. The responsibility for scientific accuracy and content remains en-
tirely with the authors.
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