A fluorogenic probe with aggregation-induced emission characteristics for carboxylesterase assay through formation of supramolecular microfibers

Article abstract of DOI:10.1002/asia.201301326

Herein, we report a novel fluorescent "light-up" probe useful for carboxylesterase assay that is based on a tetraphenylethylene derivative containing carboxylic ester groups. The specific cleavage of the carboxylic ester bonds by carboxylesterase results

Full text of DOI:10.1002/asia.201301326

FULL PAPER
DOI: 10.1002/asia.201301326
A Fluorogenic Probe with Aggregation-Induced Emission Characteristics for
Carboxylesterase Assay through Formation of Supramolecular Microfibers
Xiaojing Wang, Huan Liu, Jingwen Li, Kaiguo Ding, Zhonglin Lv, Yonggang Yang,
Hong Chen,* and Xinming Li*[a]
Abstract: Herein, we report a novel fluorescent “light-up” probe useful for carbox-
ylesterase assay that is based on a tetraphenylethylene derivative containing car-
boxylic ester groups. The specific cleavage of the carboxylic ester bonds by carbox-
ylesterase results in the generation of a relatively hydrophobic moiety that self-as-
sembles into supramolecular microfibers, thus giving rise to “turn-on” fluorescent
signals. A high sensitivity towards carboxylesterase was achieved with a detection
limit as low as 29 pm, which is much lower than the corresponding assays based on
other fluorescent approaches.
Keywords: aggregation-induced
emission · carboxylesterase · fluo-
rescence · self-assembly · tetraphe-
nylethylene
Introduction
Unlike conventional fluorophores that are easily subject
to a fluorescence quenching effect at high concentrations or
in the solid state due to intra- or intermolecular interac-
tions,[13] tetraphenylethene (TPE) contains four propeller-
shaped conjugated phenyl rings and shows an unusual fluo-
rescent behavior; it is almost non-emissive when dissolved
in solution but turns to be highly fluorescent in the aggre-
gated form.[14] Because of this unique property, TPE-based
molecules with aggregation-induced emission (AIE) proper-
ties have emerged as powerful and versatile building
blocks[15] for the design of synthetically readily accessible
and environmentally stable probes for the detection of vari-
ous analytes.[16] Despite their remarkable advantages, a limit-
ed number of examples have so far been reported to apply
AIE-type molecules in the design of fluorescence “light-up”
probes for different enzymatic assays.[17] To the best of our
knowledge, the development of new biomolecular probes
for carboxylesterase detection based on the AIE feature has
not been reported so far. Thus, we explored the potential of
TPE conjugated with a carboxylic ester as a novel fluoro-
genic probe for carboxylesterase detection.
In this work, based on the tetraphenylethylene motif, we
designed and synthesized a novel tetraphenylethylene deriv-
ative (1) that contains four carboxylic ester groups subject
to carboxylesterase recognition and catalytic hydrolysis
(Scheme 1). The design rationale and fluorescent sensing
mechanism of 1 toward carboxylesterase are illustrated in
Scheme 1 and are explained as follows: 1) 1 exhibits an ex-
cellent solubility in water due to the presence of four car-
boxylic acid groups in its molecular structure before enzy-
matic catalysis and displays very low fluorescence (emission
quantum yield FF =0.02) in aqueous solution at pH 7.4;
2) after catalytic hydrolysis of the carboxylic ester bonds,
1 is enzymatically converted into 2, a relatively more hydro-
phobic entity, thus resulting in the self-assembly of the AIE
Carboxylesterases are a group of isoenzymes commonly
found in mammalian organs,[1] and play an important role in
catalyzing the hydrolysis of carboxyl esters[2] for detoxifica-
tion of narcotics or chemical toxin clearance.[3] Due to their
broad substrate specificity and high enantioselectivity, car-
boxylesterases constitute attractive biocatalysts for the pro-
duction of optically pure compounds in fine chemical syn-
thesis[4] and represent important drug candidates for pro-
tein-based therapeutics or drug targets for chemotherapeutic
prodrug activation.[5] Therefore, carboxylesterase assays
would benefit various biochemical studies for understanding
its mechanism of action and role in drug metabolism. A
number of strategies have been established for carboxyles-
terase detection with the application of chromatography,[6]
mass spectrometry,[7] chemiluminescence,[8] and fluores-
cence.[9] Among them, biomolecular probes based on differ-
ent fluorogenic substrates have received much attention due
to the simplicity, speed, and sensitivity of their assays.[10] Al-
though these probes have been proven successful in carbox-
ylesterase activity assays, the development of water-solu-
ble[11] and synthetically readily accessible fluorogenic
probes[12] with improved assay sensitivity and selectivity is
still desirable.
[a] X. Wang, H. Liu, J. Li, K. Ding, Z. Lv, Prof. Y. Yang, Prof. H. Chen,
Prof. X. Li
College of Chemistry, Chemical Engineering and Materials Science
Soochow University
Suzhou, 215123 (China)
Tel : (+86)0512-65882917
E-mail: xinmingli@suda.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201301326.
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Scheme 1. Illustration of the fluorometric detection of carboxylesterase through the formation of supramolecular microfibers by carboxylic ester hydroly-
sis of the AIE probe.
residues in aqueous solution with enhanced fluorescent sig-
nals. In this way, 1 can be used for the fluorescence turn-on
detection of carboxylesterase with a detection limit as low
as 29 pm. This result clearly demonstrates that 1 could not
only be applied to the real-time monitoring of carboxylester-
ase activity but also to the screening of potential carboxyles-
terase inhibitors.
structures of these new products were verified by NMR
spectroscopy and electrospray ionization mass spectrometry
(ESI-MS; see Figure S1 in the Supporting Information). As
expected, 1 was soluble in water, and an aqueous solution of
1 (300 mm) in 10 mm Tris-HCl buffer (pH 7.4) was prepared
for the following studies.
Before the addition of carboxylesterase, the solution of
1 was completely transparent, colorless, and non-fluorescent
under illumination with visible light or UV light (365 nm;
Figure 1). However, the addition of carboxylesterase at
a final concentration of 3.2 nm transformed the solution of
1 into a suspension owing to the generation of insoluble 2
(Figures S2 and S3 in the Supporting Information), which
was emissive under irradiation with UV light. The obvious
transition from a clear solution of 1 to a suspension of 2 trig-
gered by carboxylesterase could be observed with the naked
eye. From the fluorescence spectroscopic analysis shown in
Figure 1C, we observed that the solution of 1 was weakly
fluorescent at 475 nm (FF =0.02), which is consistent with
the non-emissive character of AIE fluorogens in the soluble
state. However, an emission band at 475 nm appeared and
increased with time after the addition of carboxylesterase,
and the enhancements were attributed to the generation
Results and Discussion
Scheme 2 shows the synthetic route for the preparation of
fluorogenic probe 1. By following the reaction conditions
and procedures described by Tang et al. in a previous
report,[18] we synthesized 4 from 4, 4’-dihydroxybenzophe-
none as the starting material through a McMurry coupling
reaction. The next step consisted of a condensation reaction
with tert-butyl bromoacetate, and the subsequent treatment
with TFA afforded 6 in a yield of 42% over two steps.
Upon activation by N-hydroxysuccinimide (NHS), 6 reacted
with 2-aminoethanol to afford 2 in 63% yield. Further reac-
tion with succinic anhydride in the presence of N,N’-diiso-
propylethylamine led to 1 in a yield of 83%. The chemical
Scheme 2. The synthetic routes for the preparation of fluorogenic probe 1 and enzymatic generation of 2 through carboxylesterase hydrolysis. Reagents
and conditions: a) TiCl4, Zn powder, reflux, 12 h; b) tert-butyl bromoacetate, K2CO3, reflux, 72 h; c) TFA, RT, 12 h; d) DIC, NHS, 2-aminoethanol, RT,
12 h; e) succinic anhydride, DIEA, RT, 12 h.
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Figure 2. Bright field (A) and fluorescence (B) microscopy images of mo-
lecular aggregates observed after incubation of a solution of 1 (300 mm,
pH 7.4) with carboxylesterase (3.2 nm) for 120 min.
Figure 1. Optical images of solutions of 1 (300 mm, pH 7.4) in the absence
(left) or presence (right) of carboxylesterase (3.2 nm) under A) visible
and B) UV light (365 nm). C) Time evolution of fluorescence emission
spectra (lex =375 nm) of the solution of 1 (300 mm, pH 7.4) upon addition
of carboxylesterase (3.2 nm).
and accumulation of insoluble 2 (Figures S2 and S3 in the
Supporting Information), which aggregated and self-assem-
bled in water.
As demonstrated in Figure 1, such fluorescence genera-
tion and enhancement may be ascribed to the formation of
molecular aggregates of 2 that were produced from the reac-
tion of 1 with carboxylesterase. We used fluorescence mi-
croscopy to identify the aggregates self-assembled from mol-
ecules of 2 moieties in its suspension. As displayed in Fig-
ure 2A, fluorescence microscopy analysis revealed that the
aggregates were one-dimensional microfibers with length of
more than 50 mm. The microfibers were highly fluorescent,
emitting a bright blue light upon photo-excitation (lex =
365 nm) (Figure 2B). By comparison, almost no fluorescent
aggregates could be observed in the solution of 1 before the
addition of carboxylesterase (Figure S4A and S4B in the
Supporting Information), thereby indicating that carboxyles-
terase catalyzed the transformation of 1 to 2 for the forma-
tion of supramolecular microfibers and turn-on of fluores-
cence (Figure S7 in the Supporting Information).
Due to the capability of self-assembly, 2 was packed in
a one-dimensional fashion to afford crystalline microfibers
through enzymatic catalysis (Figure S7 in the Supporting In-
formation). Figure 3A shows a representative scanning elec-
tron microscopy (SEM) image of the microfibers, which are
several micrometers in width and up to hundreds of micro-
meters in length. In comparison, for the solution of 1 before
Figure 3. A) SEM image and B) XRD pattern of microfibers observed
after incubation of a solution of 1 (300 mm, pH 7.4) with carboxylesterase
(3.2 nm) for 120 min.
incubation with carboxylesterase, only highly dispersed mi-
cellar microstructures with an average diameter of around
10 mm were observed by SEM (Figure S4C in the Supporting
Information). From the X-ray diffraction (XRD) pattern
displayed in Figure 3B, we observed multiple strong and
sharp diffraction peaks at 16.768, 20.658, 29.628, and 34.168,
implying both a polymorphous and microcrystalline nature
of the microfibers[19] self-assembled from 2.
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To address the selectivity of the probe to carboxylester-
ase, we conducted control experiments by treating 1 with
other nonspecific proteins, such as pepsin, trypsin, lysozyme,
human serum albumin (HSA), alkaline phosphatase, and
exonuclease I under the same conditions. As shown in
Figure 4, only the solution containing carboxylesterase led
to an intense fluorescence signal at 475 nm that was about 9
times higher than that for the other proteins. These data
Figure 5. A) Time evolution of the fluorescence intensities of a solution
of 1 (300 mm, pH 7.4) at 475 nm upon addition of different concentrations
of carboxylesterase. B) Linear plot of the relative fluorescence intensity
(F/F0, lem =475 nm) of a solution of 1 as a function of the concentration
of carboxylesterase (0.05–3.2 nm). The fluorescence intensities were mea-
sured after incubation for 100 min at 378C.
Figure 4. Fluorescence intensity changes (lem =475 nm) of solutions of
1 in 10 mm Tris-HCl buffer (pH 7.4) after incubation with different pro-
teins (3.2 nm each) for 100 min at 378C.
mined. The Km and Vmax values were estimated to be
55.3 mm and 0.15 mmminÀ1, respectively (Figure S5 in the
Supporting Information).
confirmed that these proteins cannot recognize 1 as a sub-
strate for carboxylic ester hydrolysis. Moreover, we also
tested the response of 1 to acetylcholinesterase, one kind of
commercially available esterase that hydrolyses the carbox-
ylic ester bond of acetylcholine.[20] The fact that we did not
observe an obvious increase in fluorescence emission under
the same conditions provides further support of the selectiv-
ity of 1 towards carboxylesterase (Figure 4).
It is well known that the hydrolytic activity of carboxyles-
terase is greatly reduced in the presence of inhibitors. In
order to examine the possibility to use our probe for inhibi-
tor screening assays, we selected two common carboxylester-
ase inhibitors, 4-chlorobenzenesulfonamide and 2-thenoyltri-
fluoroacetone, to investigate their inhibitory effects on the
hydrolysis of 1. We found that the enhancement of fluores-
cence intensity by our probe was inhibited in a dose-depen-
dent manner. As shown in Figure 6, on the basis of the plot
of the relative activity of enzyme as a function of the con-
centration of inhibitors, the IC50 values were calculated to
be 8.21 mm for 4-chlorobenzenesulfonamide and 0.73 mm for
Furthermore, we examined the kinetics of the carboxyles-
terase-catalyzed hydrolysis of 1 using different amounts of
carboxylesterase. Figure 5A and Figure S6 in the Supporting
Information show an increase in fluorescence intensity at
475 nm with increasing carboxylesterase concentration (0–
3.2 nm) over 4 hours. Obviously, at higher concentrations of
carboxylesterase, a greater fluorescence enhancement was
observed, and the fluorescence intensity increased more
quickly when the probe was incubated with higher concen-
trations of enzyme. These results indicate that increasing the
enzyme concentration gave rise to a higher cleavage reac-
tion rate so that less time was required to complete the hy-
drolysis process. Furthermore, on the basis of the plot of the
relative fluorescence intensity (F/F0) of 1 in solution at
475 nm as a function of the concentration of carboxylester-
ase ranging from 0.05 to 3.2 nm, the detection limit was cal-
culated to be 29 pm under these conditions (Figure 5B),
which was much lower than the previously reported data for
carboxylesterase assays.[21] In addition, the kinetic parame-
ters of carboxylesterase-catalysed hydrolysis of 1 were deter-
Figure 6. Relative activity of carboxylesterase (3.2 nm) as a function of
different concentrations of carboxylesterase inhibitors in 10 mm Tris-HCl
buffer (pH 7.4) at 378C.
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lected and concentrated under reduced pressure. The crude product was
further purified by silica gel column chromatography (MeOH/CH2Cl2
1:16) to give
2-thenoyltrifluoroacetone, which are in good agreement
with previously reported values.[22] These results clearly
demonstrated that our system can be used not only for the
real-time monitoring of carboxylesterase activity but also
for the screening of potential carboxylesterase inhibitors.
4 as a
white solid (1.5 g, 78%). 1H NMR (400 MHz,
[D6]DMSO): d=9.23 (s, 4H), 7.26–7.35 (d, 8H), 6.54–6.65 ppm (d, 8H);
MS (m/z) calcd for C26H20O4: 395.14 [MÀH]À; found: 395.20.
Synthesis of 5: Briefly, under a nitrogen atmosphere, tert-butyl bromoace-
tate (7.88 g, 40.3 mmol) was added dropwise to dry acetone (80 mL) con-
taining tetrakis(4-hydroxyphenyl)ethane (TPE-OH4; 2.0 g, 5.04 mmol)
and K2CO3 (11.2 g, 80.9 mmol), and the reaction mixture was heated at
reflux for 72 h. After filtration and removal of the solvent, the residue
was purified by silica gel column chromatography (EtOAc/n-hexane
1:15) to afford 5 as a yellow powder (2.3 g, 54.7%). 1H NMR (400 MHz,
[D6]DMSO): d=7.46–7.57 (m, 8H), 7.12–7.02 (m, 8H), 4.66–4.74 (m,
8H), 4.52–4.63 ppm (m, 36H); MS (m/z) calcd for C50H60O12: 851.41
[MÀH]À; found: 851.30.
Conclusions
In summary, we have demonstrated the development of
a novel, simple, and sensitive carboxylesterase assay system
relying on the aggregation-induced emission characteristics
of tetraphenylethylene luminogens for the first time. Upon
the addition of carboxylesterase, which induced the specific
cleavage of the carboxylic ester bond in a tetraphenylethy-
lene derivative, 1 was enzymatically converted into 2, a rela-
tively more hydrophobic entity, to result in the supramole-
cualr self-assembly and aggregation of the AIE residues in
aqueous solutions and enhancement of the fluorescent sig-
nals at 475 nm. Under these conditions, a high sensitivity for
carboxylesterase detection was achieved with a detection
limit as low as 29 pm, which was much lower than those of
previously reported assays.[21] Moreover, probe 1 also exhib-
ited the potential for carboxylesterase inhibitor screenings.
Synthesis of 6: To 30 mL of 10% trifluoroacetic acid in dry CH2Cl2, TPE-
COOtBu4 (1.3 g, 1.5 mmol) was added. The reaction mixture was stirred
at room temperature overnight and then concentrated in vacuo. The re-
sulting residue was purified by silica gel column chromatography
(EtOAc/n-hexane 5:1) to afford 6 as a white powder (684 mg, 73.7%).
1H NMR (400 MHz, [D6]DMSO): d=6.87–6.90 (m, 8H), 6.61–6.63 (m,
8H), 4.49–4.53 ppm (m, 8H). MS (m/z) calcd for C34H28O12: 627.16
[MÀH]À; found: 627.20.
Synthesis of 2: Compund 6 (400 mg, 0.64 mmol), N,N’-diisopropylcarbo-
diimide (710 mg, 5.63 mmol), and N-hydroxysuccinimide (589 mg,
5.12 mmol) were added to dry CH2Cl2 (20 mL), and the reaction mixture
was stirred at room temperature for 6 h. The crude product was then
used in the next reaction without purification. Ethanolamine (313 mg,
5.12 mmol) was dissolved in deionized water (10 mL), and the aforemen-
tioned solution containing activated compound 6 was added dropwise.
The reaction mixture was stirred overnight at room temperature. Subse-
quently, the solvent was evaporated, and the residue was purified by
silica gel chromatography (CHCl3/MeOH 12:1) to afford 2 as a pale
Experimental Section
1
yellow powder (322 mg, 63%). H NMR (400 MHz, [D6]DMSO): d=8.00
General Experimental Details
(s, 4H), 6.86–6.88 (d, 4H), 6.73–6.76 (d, 4H), 4.74 (s, 4H), 4.40 (s, 8H),
3.41 (s, 8H), 3.20 ppm (s, 8H); MS (m/z) calcd for C42H48N4O12: 845.33
[M+HCOOHÀH]À; found: 845.30.
Materials
Esterase from porcine liver was obtained from Sigma–Aldrich (unit size:
218 unitsmgÀ1 protein, 0.163 mL, 28.1 mgmLÀ1 (biuret)). Alkaline phos-
phatase was purchased from Fermentas (unit size: 300 units,
1.0 unitmLÀ1). Exonuclease I was obtained from Shanghai Shifeng Biolog-
ical Technology Company (unit size: 750 units, 5 unitsmLÀ1). All other
starting materials were obtained from Sigma and J&K Chemical. Com-
pound 6 was synthesized by following the reported procedures.[18] Other
commercially available reagents were used without further purification,
unless noted otherwise.
Synthesis of 1: Compound 2 (400 mg, 0.50 mmol), succinic anhydride
(600 mg, 6 mmol), and N,N-diisopropylethylamine (DIPEA, 517 mg,
4 mmol) were dissolved in DMF 25 mL, and the mixture was stirred over-
night at room temperature. After the reaction was completed, the solvent
was removed under reduced pressure, and the solid residue was washed
with diluted HCl (1 mm), dried, and then purified by HPLC to yield 1 as
a pale yellow powder (996 mg, 83%). 1H NMR (400 MHz, [D6]DMSO):
d=12.19 (s, 4H), 8.13 (s, 4H), 6.83–6.86 (d, 8H), 6.69–6.72 (d, 8H), 4.37
(s, 8H), 4.00 (s, 8H), 3.30 (s, 8H), 2.48 (s, 8H), 2.45 ppm (s, 8H); MS (m/
z) calcd for C58H64N4O24: 1199.39 [MÀH]À; found: 1199.4.
Measurements
1H NMR spectra were obtained on a Varian Unity Inova 400 spectrome-
ter by using [D6]DMSO as the solvent. LC-MS analyses were performed
on Agilent 6220 Quadrupole LC/MS system with an ESI resource. HPLC
purification and analysis were carried out on a Waters 600E Multi-sol-
vent Delivery System using a YMC C18 RP column with CH3CN (0.1 v%
of TFA) and water (0.1 v% of TFA) as the eluents. SEM images were re-
corded on a Hitachi S-4800 scanning electron microscope. X-ray diffrac-
tion experiments were performed on a PANalytical X’Pert PRO MRD
diffractometer using Cu Ka radiation operated at 40 kV and 20 mA and
an X’celerator detector. Fluorescence spectroscopy measurements were
taken on a FluoroMax-4 spectrofluorometer and a Thermo Scientific Var-
ioskan Flash spectral scanning multimode reader. Fluorescence microsco-
py images were recorded on an Olympus IX71 fluorescence microscope.
Kinetic Studies of the Transformation of 1 to 2 by Carboxylesterase
For the studies of the hydrolysis reaction kinetics, different concentra-
tions of probe 1 (20, 50, 100, 200, and 300 mm) in 10 mm Tris-HCl buffer
were hydrolyzed by carboxylesterase (3 nm). The reaction was monitored
by measuring the fluorescence change at 475 nm (excitation at 375 nm)
at 378C. The kinetic parameters for the enzymatic hydrolysis reaction of
probe 1 were determined by using the Lineweaver–Burk analysis, thus
yielding values of Km =55.3 mm and Vmax =0.15 mmminÀ1
.
Determination of the Detection Limit of Carboxylesterase with Probe 1
To assess the sensitivity of the fluorescent assays, we prepared a series of
solutions in 10 mm Tris-HCl buffer (pH 7.4) containing 300 mm of 1 and
different concentrations of carboxylesterase (0, 0.05, 0.2, 0.8, 1.6, and
3.2 nm), and incubated them at 378C. The fluorescence intensities of the
mixtures were collected at various time points (0, 20, 40, 60, 80, 100, 120,
140, 160, 180, 200, 220, and 240 min), and the relationships between
known concentrations of carboxylesterase and the fluorescent response
at different times were analyzed by using a calibration curve. A good
linear relationship between the carboxylesterase concentration and the
relative fluorescence intensity (F/F0) was obtained in the range between
Fluorogenic Probe Synthesis
Synthesis of 4: 4, 4’-Dihydroxybenzophenone (2.1 g, 10 mmol) and Zn
power (1.44 g, 22 mmol) were dissolved in dry THF under a nitrogen at-
mosphere. The solution was cooled to 08C, followed by the dropwise ad-
dition of TiCl4 (1.3 mL, 12 mmol). After refluxing overnight, the solution
was cooled to room temperature, treated with 40 mL HCl solution
(1 molLÀ1), and then extracted with CH2Cl2. The organic layer was col-
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0.05 and 3.2 nm at 100 min. Its regression equation is Y=2.0928X+
1.2099 (where Y is the relative fluorescence intensity and X is the con-
centration of carboxylesterase in nm) with a correlation coefficient of
0.9986. The limit of detection was calculated from the standard deviation
of the response (SD=0.0184) and the slope of the calibration curve (S=
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We thank the support of this work by the National Science Fund for Dis-
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