Rational Design of a Red-Emissive Fluorophore with AIE and ESIPT Characteristics and Its Application in Light-Up Sensing of Esterase

Article abstract of DOI:10.1021/acs.analchem.6b04974

The development of red fluorophores with efficient solid-state emission is still challenging. Herein, a red fluorophore 1 with aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) characteristics is rationally design

Full text of DOI:10.1021/acs.analchem.6b04974

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Article
Rational design of a red-emissive fluorophore with AIE and ESIPT
characteristics and its application in light-up sensing of esterase
Lu Peng, Shidang Xu, Xiaokun Zheng, Xiamin Cheng, Ruoyu Zhang, Jie Liu, Bin Liu, and Aijun Tong
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04974 • Publication Date (Web): 03 Feb 2017
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Analytical Chemistry
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Rational design of a red-emissive fluorophore with AIE and ESIPT
characteristics and its application in light-up sensing of esterase
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Lu Peng,†,‡ Shidang Xu,‡ Xiaokun Zheng,† Xiamin Cheng,‡ Ruoyu Zhang,‡ Jie Liu,‡ Bin Liu*,‡,§
and Aijun Tong*,†
†
Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Key Laboratory of Bioorꢀ
ganic Phosphorus Chemistry and Chemical Biology, Tsinghua University, Beijing 100084, People’ s Republic of China. Fax:
+86ꢀ10ꢀ62787682. Email: tongaj@mail.tsinghua.edu.cn.
‡
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singaꢀ
pore 117585, Singapore. Fax: +65ꢀ67791936. Eꢀmail: cheliub@nus.edu.sg.
§
Institute of Materials Research and Engineering (A*STAR), 3 Research Link, Singapore 117602, Singapore
ABSTRACT: The development of red fluorophores with efficient solidꢀstate emission is still challenging. Herein, a red fluoroꢀ
phore 1 with aggregationꢀinduced emission (AIE) and excitedꢀstate intramolecular proton transfer (ESIPT) characteristics is rationꢀ
ally designed and facilely synthesized by attaching an electron donor diethylamine and an electron acceptor maleonitrile group to
salicyladazine. In contrast to many red fluorophores which undergo serious aggregationꢀcaused quenching (ACQ), compound 1
emits bright red fluorescence (λem = 650 nm, ΦF = 24.3%) in the solid state with a large Stokes shift of 174 nm. Interestingly, conꢀ
trol compounds 2 and 3, which have similar structures as 1, exhibit obvious aggregationꢀcaused quenching (ACQ) characteristics.
The difference in the crystal structures of 1, 2 and 3 reveals that the interplanar spacing among molecules plays a decisive role in
realizing the AIE characteristics of 1. Moreover, when the hydroxyl group of 1 was substituted by an esterase reactive acetoxyl, a
fluorescence lightꢀup probe 4 was developed for sensing of esterase based on the selective reaction between 4 and esterase to genꢀ
erate the AIE and ESIPT active molecule 1. The linear range for in vitro quantification of esterase is 0.01ꢀ0.15 U/mL with a detecꢀ
tion limit of 0.005 U/mL. Probe 4 was also successfully applied to image esterase in mitochondria of living cells.
Fluorescence detection of various analytes has received much
research interest because of its advantages such as low cost,
simplicity, good sensitivity, and capability of realꢀtime and
nonꢀdestructive bioꢀimaging1ꢀ8. Among all the luminescent
molecules, red fluorophores play a crucial role in fluorescence
detection and bioꢀimaging because they could minimize phoꢀ
toꢀdamage to living cells, enable deep tissue penetration and
overcome the interference from biological background fluoꢀ
rescence9ꢀ12. However, most fluorophores have bright emission
in the solution state, but their luminescence declines obviously
in the aggregated state, which is known as aggregationꢀcaused
quenching (ACQ)13, 14. The situation is even worse for red
fluorophores because most of them contain large aromatic
rings which could form strong intermolecular πꢀπ interacꢀ
tions15, 16, to cause nonꢀradiative decay in the aggregated state.
Fluorophores with aggregationꢀinduced emission (AIE)17ꢀ22
properties are an emerging class of molecules displaying
strong fluorescence in their solid or aggregate states. So far,
many AIE fluorophores based on hydrocarbons, heteroatoms
or organometallics have been constructed as fluorescent mateꢀ
rials for biomedical applications23ꢀ27. Although fluorophores
with both red fluorescence and AIE characteristics are more
preferable, unfortunately, limited red AIE fluorophores have
been reported28ꢀ33. The scarcity of red AIE fluorophore is
mainly due to the difficulty of molecular design and structural
modifications. To design red AIE fluorophores, one has to
consider either increasing the molecular conjugation length, or
selecting a proper combination of an electron donor and an
acceptor to redꢀshift the emission. Researchers often encounter
a dilemma because this strategy may usually lead to ACQ
characteristics34, 35. In addition, in many cases, twisted molecuꢀ
lar structures are needed to acquire AIE properties, which
makes the design and synthesis of red AIE fluorophores even
more difficult.
Previously, we have developed one class of AIEꢀactive
fluorophores36, 37 which is based on the excited state intramoꢀ
39
lecular proton transfer (ESIPT)38,
mechanism. For these
fluorophores, twisted structure is not necessary to exhibit AIE
characteristics, while the hydroxyl groups are responsible for
ESIPT and essential for their AIE characteristics in aggregate
and solid states. Once the hydroxyl group is blocked, the fluoꢀ
rescence of the molecule will be quenched or changed to a
different wavelength. This brings the advantages of facile synꢀ
thesis and ease of chemical modifications for the development
of various lightꢀup probes40, 41. However, the limitations of the
ESIPT dyes include the short absorption (λabs < 400 nm) and
emission wavelengths (λem < 600 nm), which does not match
well with the excitation of confocal microscope (≥ 405 nm).
Herein, we rationally developed a fluorophore 1 with both red
fluorescence and AIE characteristics based on salicyladazine
(Scheme 1a). Red fluorescence is achieved by attaching an
electron donor diethylamine and an electron acceptor maleꢀ
onitrile group to salicyladazine. Meanwhile, 1 maintains the
hydroxyl group to keep AIE and ESIPT characteristics. More
importantly, the proper crystal packing pattern of 1, which
makes the intermolecular πꢀπ interactions very weak, prevents
the nonꢀradiative decay of excited electrons and allows 1 to
show good red emission property (λem = 650 nm, ΦF = 24.3%)
in the solid state. Apart from the red fluorescence, 1 possesses
appropriate absorption band (λabs = 437 nm in solution state
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and 476 nm in solid state) for confocal imaging with a large (diethylamino)salicylaldehyde (1.93 g, 10 mmol) and diaꢀ
1
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Stokes shift, which greatly reduces selfꢀquenching of fluoresꢀ
cence.
minomaleonitrile (0.50 g, 5 mmol) were added. The mixed
solution was refluxed for 3 days, the product was precipitated
as black solid. The product was filtrated under vacuum,
washed by ethanol and dried under vacuum to afford pure
A lightꢀup probe 4 for esterase based on 1 was also develꢀ
oped through blocking the hydroxyl group of 1 with an esterꢀ
1
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ase selective acetoxyl group42, (Sheme 1b). Esterase was
product of 2 (1.71 g, 3.7 mmol, 75% yield). H NMR (400
MHz, d6ꢀDMSO):
δ = 8.57 (s, 2H), 7.57 (d, 2H, J = 11.4 Hz),
selected as the model target because esterase is an important
drug target44, 45 and prodrug activator46. After 4 was reacted
with esterase, 1 was expected to be regenerated to show both
AIE and ESIPT characteristics. Although several fluorescent
probes42, 43, 47, 48 have been developed for esterase, they are not
able to selectively image esterase in a specific organelle of
cells. Moreover, they suffer from disadvantages such as ACQ
when accumlated in cells and small Stokes shifts (< 40 nm)42,
43. Previously, we have reported a lightꢀup probe49 based on
AIE and ESIPT characteristics for specific detection of lysoꢀ
somal esterase, which emits green fluorescence (λem = 532
nm). Considering that a red fluorescent probe with AIE and
ESIPT characteristics would be more suitable for bioꢀimaging,
probe 4 was developed in this work. 4 showed red lightꢀup
fluorescence and high specificity to esterase. Moreover, it was
found to be accumulated in mitochondria of living cells and
emit bright red fluorescence even in the aggregated state. Beꢀ
cause mitochondria plays critical roles in many vital cellular
processes, such as ATP production, central metabolism and
apoptosis50, 51, probe 4 might find promising potential in the
biological study of esterase.
6.44 (d, 2H, J = 11.0 Hz), 6.16 (s, 2H), 4.47 (t, 8H), 1.15 (t,
12H). Solidꢀstate 13C NMR (600 MHz): 166.64, 165.72,
163.42, 161.82, 156.30, 154.00, 137.53 (2C), 123.06, 121.52,
111.57 (2C), 110.26 (2C), 107.43 (2C), 98.62, 97.24, 48.91
(2C), 47.38 (2C), 15.89 (2C), 14.29 (2C).
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Synthesis of 3. In 50 mL of ethanol, 4ꢀ
(dimethylamino)salicylaldehyde (1.93 g, 10 mmol) and diaꢀ
minomaleonitrile (1.08 g, 10 mmol) were added. The mixed
solution was stired at r.t. for 7 days, the product was precipiꢀ
tated as black solid. The product was filtrated under vacuum,
washed by ethanol and dried under vacuum to afford pure
1
product of 3 (1.66 g, 6.5 mmol, 65% yield). H NMR (400
MHz, d6ꢀDMSO):
δ =10.58 (s, 1H), 8.37 (s, 1H),7.63 (d, 1H, J
= 11.9 Hz),7.38 (s, 2H), 6.33 (d, 1H, J = 11.8 Hz), 6.11 (d, 1H,
J = 3.2 Hz), 3.03 (q, 6H); 13C NMR (400 MHz, d6ꢀDMSO):
160.82, 155.94, 154.65, 132.57, 123.35, 115.76, 114.85,
110.09, 105.46, 105.18, 97.84, 44.04 (2C). Mass spectrometry:
calc. for C13H12N5O [MꢀH]ꢀ 254.1, found 254.1 (ESI).
Synthesis of 4. In 20 mL of acetonitrile, 4ꢀ
(diethylamino)salicylaldehyde (0.725 g, 3.75 mmol), acetic
anhydride (0.7 mL, 7.5 mmol) and cesium carbonate (1.225 g,
3.725 mmol) were added. The mixed solution was refluxed
overnight and extracted three times using 30 mL of ethyl aceꢀ
tate, washed with brine solution, dried over anhydrous Na2SO4
and the solvent was evaporated to afford crude product of 5
(0.63 g, 2.7 mmol, 72% yield). 5 (0.62 g, 2.63 mmol) was
further reacted with diaminomaleonitrile (0.284 g, 2.63 mmol)
in 20 mL ethanol at r.t. for 5 days. The resulting precipitates
were filtrated and washed three times with 30 mL of ethanol to
yield 4 as brown powder (0.318 mg, 0.97 mmol, 37% yield).
Scheme 1. (a) Chemical structures of compound 1, 2, 3 and
corresponding photographs of their solids without or with a
UV lamp at 365 nm. (b) The fluorescent light-up probe 4 for
sensing of esterase.
1H NMR (400 MHz, CDCl3):
δ = 8.32 (s, 1H), 7.88 (d, 1H, J =
12.36 Hz), 6.59 (d, 1H, J = 9.6 Hz), 6.33 (d, 1H, J = 2.32 Hz),
5.00 (s, 2H), 3.43 (t, 4H), 2.37 (t, 3H), 1.22 (t, 6H); 13C NMR
(400 MHz, CDCl3): 169.75, 153.48 (2C), 152.01, 130.12,
122.17, 114.39, 114.22, 112.82, 110.19, 109.78, 104.56, 44.93
(2C), 21.11, 12.66 (2C). Mass spectrometry: calc. for
C17H18N5O2 [MꢀH]ꢀ 324.1, found 324.1 (ESI).
Analytical procedures. The stock solution (1.0×10−2 M)
of 4 was prepared in DMSO. Stock solutions of esterase
(100 U/mL) and other analytes (CaCl2, Mg(NO3)2,
Zn(NO3)2, Fe(NO3)3, Cu(NO3)2, pepsin, lysozyme, cysteine,
vitamin C, alanine, BSA) were prepared in distilled
deionized water, respectively. In a typical detection, 10 ꢁL
of 4 stock solution was added into a test tube, which was
diluted to 1.0 mL with PBS buffer (10 mM, pH 7.4).
Subsequently, a proper amount of esterase stock solution
was added. The fluorescence spectra were recorded before
and after the addition of esterase at different time.
EXPERIMENTAL SECTION
Synthesis of 1. In 50 mL of ethanol, 4ꢀ
(diethylamino)salicylaldehyde (1.93 g, 10 mmol) and diaꢀ
minomaleonitrile (1.08 g, 10 mmol) were added. After being
stirred at r.t. for 7 days, the product was precipitated as brown
solid. The product was filtrated under vacuum, washed by
ethanol and dried under vacuum to afford pure product of 1
(2.31 g, 8.2 mmol, 82% yield). 1H NMR (400 MHz, d6ꢀ
DMSO):
δ = 10.55(s, 1H), 8.35(s, 1H), 7.60 (d, 1H, J = 12.0
Cell imaging. MCFꢀ7 and HeLa cells were seeded at a
density of 2×104/well in the chamber (LABꢀTEK,
Chambered Cover glass System) and grown for 18 h at 37
℃ in a humidified 5% CO2 incubator. The cells were
treated without or with the esterase inhibitor solution
(AIEBSF, 10 mM) in PBS buffer (10 mM, pH 7.4) for 20
min at 37 ℃. After washing with PBS buffer to remove the
remaining inhibitor, the cells were further incubated with
Hz), 7.33(s, 2H), 6.21 (dd, 1H, J1 = 8.0 Hz, J2 = 8.0 Hz), 6.10
(d, 1H, J = 4 Hz), 3.39 (dd, 4H, J1 = 16.0 Hz, J2 = 16.0 Hz),
1.12 (t, 6H); 13C NMR (400 MHz, d6ꢀDMSO): 161.12, 155.92,
152.29, 132.88, 123.00, 115.71, 114.76, 109.55, 105.31,
105.03, 97.14, 44.45, 31.13, 13.02 (2C). Mass spectrometry:
calc. for C15H17N5NaO [M+Na]+ 306.1, found 306.1 (ESI).
Synthesis of 2. In 50 mL of ethanol, 4ꢀ
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the solution of 4 (50 ꢁM, DMSO:H2O=5:995 v/v) for 10 and S8). The fluorescence change of 1 was investigated in
1
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min. The solution of 4 was prepared by adding 5 ꢁL of its
DMSO stock solution (10 mM) into a microtube, subsequently
995 ꢁL Dulbecco’s modified Eagle medium (DMEM) was
added with vigorous mixing. The fluorescence imaging of
the cells was collected using the red channel (λex=405 nm,
547ꢀ647 nm). For coꢀlocalization imaging, HeLa cells were
preꢀstained with nucleus staining dye Hochest (10 mg/mL)
or MitoꢀTracker Green (100 nM) for 20 min and then
incubated with 4 (50 ꢁM) for 10 min. The images were
collected using blue channel (λex = 405 nm, λem = 420ꢀ480
nm), green channel (λex = 488 nm, λem = 500ꢀ540 nm) and
red channel (λex = 405 nm, λem = 550ꢀ650 nm), respectively.
water/DMSO coꢀsolvents with different water volume fracꢀ
tions (fw = 0ꢀ90 vol%), and the results are shown in Figure 2.
In a good solvent (fw = 0 vol%), 1 disperses well and exhibits
green fluorescence. Nevertheless, in a poor solvent (fw = 90
vol%), an intense red fluorescence can be observed, suggesting
1 with AIE characteristics. The AIE effect of 1 occurs when
the water volume fraction in the coꢀsolvent is more than 70%.
The intensity ratio (I619/I513) is enhanced at a higher water volꢀ
ume fraction, which correlates well with the increased aggreꢀ
gates formation in poorer solvents. Moreover, in the poor solꢀ
vent (fw = 90 vol%), a levelingꢀoff in the visible region of its
absorption spectra (commonly observed in nanoꢀaggregate
suspensions52) strongly suggests the formation of a poorlyꢀ
soluble “aggregate” state (Figure S1a). The dynamic light
scattering (DLS) analysis results of 1 indicate that the average
particle diameter is 540.4 nm (Figure S2a).
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Scheme 2. Synthesis of compound (a) 1 , (b) 2, (c) 3 and (d)
4.
NC
CN
a)
NC
N
CN
H2
N
NH2
OH
OH
(1.0 eq.)
NH2
N
N
CHO
EtOH, r.t.
1
NC
H2
CN
b)
NC
OH
CN
N
N
NH2
HO
OH
CHO
(2.0 eq.)
N
N
N
N
EtOH, reflux
NC
2
CN
c)
NC
CN
H2
(1.0 eq.)
N
NH2
OH
OH
N
NH2
N
N
CHO
EtOH, r.t.
3
d)
O
O
O
OH
O
O
Figure 2. Effect of water volume fraction on the (a) fluorescence
spectra and (b) ratio of intensity (I619/I513) of 1 (100 ꢁM) in waꢀ
ter/DMSO containing 10 mM PBS buffer at pH 7.4. Excitation
was set at 437 nm. (c) The corresponding photographs of 1 with a
UV lamp at 365 nm.
N
CHO
N
CHO
CH3CN, reflux
5
NC
CN
O
NC
CN
NH2
O
H2
N
NH2
(1.0 eq.)
N
N
To further confirm that the green fluorescence of 1 was
originated from the solution state while red fluorescence was
from the aggregate state, the effects of concentration and pH
on its fluorescence were investigated. As shown in Figure S3,
1 emits green fluorescence at low concentrations (0ꢀ2.0 mM),
but red fluorescence at high concentrations (5.0ꢀ100.0 mM),
suggesting that the green and red fluorescence are attributed to
the solution and aggregate state, respectively. Moreover, 1
exhibits a significant fluorescence color change from red to
green with an intensity ratio (I547/I619) enhanced when the pH
is increased from 3.0 to 13.0 in aqueous solution (Figure S4).
This accords with the fact that 1 forms aggregates in acidic
condition because of poor solubility in water while it stays in
solution state under alkaline condition because the deprotonaꢀ
tion process can improve its solubility. In addition, the fluoꢀ
rescence lifetime of 1 (Figure S5) in a poor solvent (fw = 90
vol%, τ = 2.6 ns) is comparable to that in solid state (τ = 3.2
ns), indicating the red fluorescence of 1 in the poor solvent is
attributed to the aggregate state. On the other hand, the short
fluorescence lifetime in a good solvent (fw = 0 vol%, τ = 0.2
ns) is resulted from the nonꢀradiative decay of excited elecꢀ
trons due to frequent intermolecular collision53.
EtOH, r.t.
4
RESULTS AND DISCUSSION
Optical Properties. The solid state fluorescence of 1, 2 and
3 was studied first to test our design principle. As shown in
Scheme 1a and Figure 1a, powders of 1 emit strong red fluoꢀ
rescence (λem = 650 nm, ΦF = 24.3%), while the powders of 2
and 3 display low fluorescence (ΦF < 1.0%). This accords well
with the absorption spectrum in solid state (Figure 1b) that 2
and 3 have strong absorption in visible region, which could
cause serious selfꢀquenching of their fluorescence.
Figure 1. (a) Fluorescence and (b) absorption spectra of comꢀ
pound 1, 2 and 3 in solid state. Excitation was set at 500 nm.
The redꢀshifted emission of fluorophores could arise from
intramolecular charge transfer (ICT)54, 55, which lowers the πꢀ
π* energy gap. To evaluate whether the red emission of 1 is
caused by ICT or not, the effect of solvent polarity is studied
Furthermore, we studied the effect of water volume fraction
on the fluorescence of 1, 2 and 3, respectively (Figures 2, S7
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because solvatochromism is usually a strong evidence of the
molecules form effective Jꢀaggregates. The ethyl groups result
existence of ICT56, 57. However, as shown in Figure S6 and
Table S1, the absorption and emission spectra do not change
much with the increase of solvent polarity, confirming that
ICT has little contribution to the redꢀshifted emission of 1.
in increased steric hindrance between two adjacent molecules.
The distance between the central benzene planes of adjacent
molecules is 4.01 Å, which is greater than 3.80 Å and could
impede intermolecular πꢀπ interactions and thus avoid the
quenching of fluorescence in either the solid state or aggregate
state. Contrarily, in the crystals of 2, molecules stack up in
disorder. The molecular planes were twisted into an archꢀlike
shape. The interꢀplanar spacing between two adjacent moleꢀ
cules is 3.25 Å, within the range of intermolecular πꢀπ stackꢀ
ing. Although the ethyl groups are far from each other due to
steric hindrance, the molecular plane is bent and the central
benzene planes of adjacent molecules are much closer than
those of 1, thus making the intermolecular πꢀπ interactions
strong enough to quench the fluorescence in the solid and agꢀ
gregate state. As for 3, molecules adopt Jꢀaggregate packing
pattern, but the steric hindrance of methyl groups is too weak
to prevent πꢀπ interactions with an interꢀplanar spacing of 3.32
Å, resulting in its ACQ characteristics.
1
2
3
4
5
6
7
8
Interestingly, 2 and 3 were found to be with ACQ characterꢀ
istics (Figures S7 and S8). 2 and 3 both dissolve well in
DMSO but are not soluble in water. In DMSO (fw = 0 vol%), 2
and 3 disperse well and exhibit red and green fluorescence,
respectively. However, in a poor solvent (fw = 90 vol%), little
fluorescence could be observed. The fluorescence intensity
was found to decrease at a higher water volume fraction,
which correlates well with the increased aggregates formation
in poorer solvents. The fluorescence intensity decrease of 2 in
DMSO with its concentration increase also suggests its ACQ
characteristics (Figure S9).
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Crystal Analysis. To investigate the decisive factor that
makes 1 AIE while 2 and 3 ACQ, we further studied the single
crystal structure of 1, 2 and 3 (Figure 3). In the crystals of 1,
Figure 3. (a) Photographs of the single crystal of 1 without or with a UV lamp at 365 nm. (b) Confocal micrographs of the crystal of 1.
The excitation was set at 405 nm. “R”, “L”: red and bright channels, respectively. Red channel: 547ꢀ647 nm. (cꢀe) Single crystal analyses
and packing patterns of 1, 2 and 3, respectively.
of acetoxyl group. Upon addition of esterase, the fluorescence
of 4 enhanced gradually until red fluorescence was observed.
The timeꢀdependent fluorescence intensity change of 4 was
recorded without and with the addition of esterase (Figure 4b),
showing that the fluorescence lightꢀup effect only occurs in
the presence of esterase. As shown in Figures 4c and 4d, a
Light-up sensing of esterase. Compound 1 was further deꢀ
veloped into a red lightꢀup fluorescent probe 4 for sensing of
esterase. The fluorescence of 4 in the absence and presence of
esterase was studied to prove our design principle above
(Scheme 1b). The blocked compound 4 displayed very weak
fluorescence (Figure 4a), suggesting successful blocking effect
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good linearity was found in the esterase concentration range of
0.01ꢀ0.15 U/mL. The detection limit was calculated as 0.005
U/mL based on the definition by IUPAC (CDL = 3Sb/m) from
10 blank solutions, which is similar to the previously reported
probes48.
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Figure 5. Imaging esterase in living MCFꢀ7 cells. Cells (a) withꢀ
out or (b) with incubation of 4 (50 ꢁM) for 10 min; (c) cells were
preꢀincubated with 10 mM inhibitor for 20 min and then treated
with 4 (50 ꢁM) for 10 min; “L”: bright field; “R”: red fluoresꢀ
cence image of 4 (λex = 405 nm, λem = 547ꢀ647 nm), respectively.
Figure 4. (a) Fluorescence spectra of 4 (100 ꢁM) in the presence
of various concentrations of esterase (0ꢀ1.0 U/mL) in 10 mM PBS
buffer solution at pH 7.4, 37
℃. Insets from left to right: photoꢀ
graphs of 4 (100 mM) without or with esterase (1.0 U mL) under
UV light (365 nm). (b) The corresponding fluorescence intensity
(I580) change over time and (c) concentration. (d) Calibration
curve of the fluorescence intensities (I580) versus esterase concenꢀ
trations. The measurements in (c) and (d) were performed after
the addition of esterase for 20 min.
It has been reported by Nagano’s group42 and others43 that
acetoxyl can be cleaved by esterase to release hydroxyl group.
To verify this, the fluorescent product after reaction between
probe 4 and the esterase was isolated by filtration and subseꢀ
quently characterized. The results in Figure S10 clearly sugꢀ
gest the formation of 1 from 4, supporting the hypothesis in
our design (Scheme 1b). The selectivity of probe 4 towards
esterase was also studied (Figure S11). No change was obꢀ
served with some commonly used inorganic salts or biomoleꢀ
cules. Thus, probe 4 shows good selectivity to esterase. To
confirm that the fluorescence enhancement was caused by
Figure 6. Coꢀlocalization imaging of the HeLa cells. Cells were
preꢀstained with (a) nucleus staining dye Hochest (10 mg/mL ) or
(b) MitoꢀTracker Green (100 nM) for 20 min and then incubated
with 4 (50 ꢁM) for 10 min. “L”: bright field; “B”: blue fluoresꢀ
cence image of Hochest (λex = 405 nm, λem = 420ꢀ480 nm); “G”:
green fluorescence image of MitoꢀTracker Green (λex=488 nm,
λem = 500ꢀ540 nm); “R”: red fluorescence image of 4 (λex = 405
nm, λem = 550ꢀ650 nm), respectively.
esterase,
the
inhibition
effect
of
4ꢀ(2ꢀ
aminoethyl)benzenesulfonyl fluoride (AIEBSF)42 on esterase
activity has been examined. As shown in Figure S12, the fluoꢀ
rescence intensity was weakened by the addition of AIEBSF,
suggesting the mechanism of fluorescence enhancement by the
enzymatic reaction between 4 and esterase.
Then coꢀlocalization experiments were performed to exꢀ
amine which subcellular location the probe stains. HeLa cells
were firstly stained with Hochest (a nucleus stain, Figure 6a)
or MitoꢀTracker Green (a mitochondrial stain, Figure 6b), and
then treated with the probe 4 (50 ꢁM). Confocal microscopy
analysis shows that the red fluorescence signal of probe 4 is
well overlapped with the green fluorescence signal from the
MitoꢀTracker. Then the Pearson’s correlation coefficient, used
to quantify the overlap between the emission of 4 and Mitoꢀ
Tracker, is further calculated as 0.86 (Figure S14), indicating
that the probe shows good selectivity to mitochondria. The
specifity is probably because some molecules of 4 (containing
amino group) might undergo ionization to generate positive
charges, which could bind to the mitochondrial membrane of
Imaging esterase in living cells. To further demonstrate the
potential of probe 4 to image esterase in living matrices, we
carried out the experiments in living cells. After the cells were
incubated with 4 (50 ꢁM) for 10 min, strong fluorescence was
exhibited (Figures 5b and S13). To demonstrate the specificity
of 4 to esterase, a control experiment was undertaken (Figure
5c). Cells were pretreated with the inhibitor AIEBSF (10
mM), which was expected to inhibit the activity of esterase,
and then incubated with probe 4, a remarkable decrease in
fluorescence intensity was observed. This confirms the speciꢀ
ficity of probe 4 towards esterase over other analytes in living
cells.
negative potential58ꢀ60
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6. Kim, H. N; Ren, W. X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2012,
CONCLUSION
41, 3210ꢀ3244.
1
2
3
4
5
6
7
8
A new red fluorescent molecule 1 with AIE and ESIPT
characteristics was designed and synthesized. To prolong the
emission wavelength of the AIE molecule salicyladazine, 1
incorporates diethylamino group as the electron donor and
maleonitrile as the electron acceptor, thus making it exhibiting
bright red fluorescence (λem = 650 nm, ΦF = 24.3%) in the
solid state. Interestingly, control compound 2 and 3, which
have similar structures as 1, show obviously contrary characꢀ
teristics (ACQ). The crystal analysis reveals that the interꢀ
planar spacing plays a decisive role for realizing the AIE charꢀ
acteristics of 1. Moreover, a lightꢀup fluorescent probe 4 was
further developed for esterase activity detection by blocking
the hydroxyl group of 1 with an esterase selective acetoxyl
group. Upon the addition of esterase, the hydroxyl group of 4
was recovered, resulting in 1 with ESIPT and strong fluoresꢀ
cence in the aggregate states. The reaction product of 4 with
esterase well accumulated in mitochondria of living cells and
displayed strong emission due to its AIE characteristics, makꢀ
ing 4 a good contrast agent for imaging mitochondrial esterꢀ
ase.
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ASSOCIATED CONTENT
Supporting Information
Materials and instrumentation; absorption and fluoꢀ
rescence spectroscopic data of 1 in different organic
solvents; effect of concentration and pH on the fluoꢀ
rescence of 1, 2 and 3; confirmation of the formation
of 1 from 4 and esterase; NMR and MS results of
compound 1, 2, 3, 4, coꢀlocalization imaging of
HeLa cells; Z potential analysis of 4; DLS analysis
of 4 in the presence of esterase
Cif files of compound 1, 2 and 3
AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail: cheliub@nus.edu.sg. Fax: +65ꢀ67791936.
* Email: tongaj@mail.tsinghua.edu.cn. Fax: +86ꢀ10ꢀ
62787682.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are very grateful for the financial support from the Naꢀ
tional Natural Science Foundation of China (Nos. 21375074,
21390410 and 21621003), Singapore National Research
Foundation (Rꢀ279ꢀ000ꢀ444ꢀ281, R279ꢀ000ꢀ483ꢀ281), Naꢀ
tional University of Singapore (R279ꢀ000ꢀ482ꢀ133), the Instiꢀ
tute of Materials Research and Engineering (IMRE/14ꢀ
8P1110) and
China Postdoctoral Science Foundation
(2016M601000).
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