A Novel Ratiometric Fluorescent Probe for Highly Sensitive and Selective Detection of β-Galactosidase in Living Cells

Article abstract of DOI:10.1002/cjoc.201800539

β-Galactosidase, a glycoside hydrolase enzyme, has been proved to be an important biomarker of cell senescence and primary ovarian cancer. Effective detection of β-galactosidase has attracted wide attention. Herein, one ratiometric fluorescent probe has been successfully synthesized for detecting the β-galactosidase in living cells. The as-prepared probe exhibits two emission peaks at 490 nm and 530 nm, respectively, and the ratio of fluorescence intensities from the two emission peaks could be utilized to monitor the β-galactosidase. This present ratiometric fluorescent probe is, therefore, very promising for effective, sensitive, and selective detection of the β-galactosidase in living cells.

Full text of DOI:10.1002/cjoc.201800539

Accepted Article
Title: A novel ratiometric fluorescent probe for highly sensitive and selective
detection of β-galactosidase in living cells
Authors: Min Chena, Lixuan Mu,* Xingxing Cao, Guangwei Shea, Wensheng Shi*
This manuscript has been accepted and appears as an Accepted Article online.
This work may now be cited as: Chin. J. Chem. 2019, 37, 10.1002/cjoc.201800539.
The final Version of Record (VoR) of it with formal page numbers will soon be
published online in Early View: http://dx.doi.org/10.1002/cjoc.201800539.
ISSN 1001-604X • CN 31-1547/O6
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A novel ratiometric fluorescent probe for highly sensitive and selective
detection of β-galactosidase in living cells
Min Chena,b, Lixuan Mu*,a, Xingxing Caoa,b, Guangwei Shea, Wensheng Shi*,a
a Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
Beijing 100190, China
b University of Chinese Academy of Sciences, Beijing 100049, China
Cite this paper: Chin. J. Chem. 2019, 37, XXX—XXX. DOI: 10.1002/cjoc.201900XXX
Summary of main observation and conclusion β-galactosidase, a glycoside hydrolase enzyme, has been proved to be an important biomarker of cell
senescence and primary ovarian cancer. Effective detection of β-galactosidase has attracted widely attention. Herein, one ratiometric fluorescent probe
has been successfully synthesized for detecting the β-galactosidase in living cells. The as-prepared probe exhibits two emission peaks at 490 nm and 530
nm, respectively, and the ratio of fluorescence intensities from the two emission peaks could be utilized to monitor the β-galactosidase. This present
ratiometric fluorescent probe is, therefore, very promising for effective, sensitive, and selective detection of the β-galactosidase in living cells.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/cjoc.201800539
*E-mail: Prof. Wensheng Shi, E-mail: shiws@mail.ipc.ac.cn
Dr. Lixuan Mu, E-mail: mulixuan@mail.ipc.ac.cn
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In
the
present
work,
we
designed
an
Introduction
intramolecular-charge-transfer (ICT) based ratiometric probe
(AM-RP-G) composed of 4-amino-1, 8-napthalic anhydride
[7,
14]
As a lysosomal exoglycosidase, β-galactosidase (β-Gal) plays a
vital role in the human body for removing galactose residues from
various substrates, such as gangliosides, sphingolipids,
glycoproteins, and keratin sulfate [1]. A variety of diseases (e.g.
ovarian cancer, β-galactosialidosis, and morquio B syndrome) was
implicated in the alteration of the β-galactosidase concentration
[2]. At the cellular level, the β-galactosidase can also be employed
as a reporter to examine the transcription and transfection
derivative
as
the
fluorophore
,
esterified
α-D-galactopyranosyl as the β-galactosidase hydrolytic site, and
4-hydroxybenzaldehyde as the linkage group, respectively, for
β-galactosidase detection. The hydrolysis by the β-galactosidase
would make the emission wavelength of AM-RP-G shift from 490
nm to 530 nm. Moreover, as the increase of the β-galactosidase
concentration, the emission intensity of unhydrolyzed probe at
490 nm would be decreased. Meanwhile, the emission intensity
of the hydrolyzed probe at 530 nm would be increased
simultaneously. So the ratio of the fluorescence intensity at 530
nm and 490 nm would sensitively depend on the concentration of
β-galactosidases. It was found that the fluorescence intensity
ratio of the probe showed good linear dependence on the
concentration of the β-galactosidase in the range of 0-0.07 U/ml,
and a detection limit of 1.4 × 10-3 U/mL was achieved. Moreover,
this probe can also be applied to monitor the β-galactosidase
activity in cells, which opens opportunities for biomedical
research, containing researches for cell aging, cells or tissues
efficiencies, or as a marker for targeted the expression of gene or
destruction in cells [3]. It is also a vital biomarker for cell aging
.
[4]
So, monitoring the β-galactosidase activity within living cells or
tissues has aroused considerable interests. Some methods have
been explored for detecting the β-galactosidase, such as magnetic
resonance, positron emission tomography, and single
photoemission computed tomography et al [5]. However, these
methods are hampered by the high cost, laborious manipulation,
modest sensitivity, and invalid monitor of β-galactosidase in living
cells [6]. Due to the advantage of fluorescent probes, such as
advanced sensitivity, convenient handling procedures, less
expensive instruments, and strong talent of bioimaging, the
imaging even targeted tumor imaging [15]
.
fluorescent probes have received considerable attentions for Results and Discussion
monitoring the β-galactosidase activity in living cells [7]
.
Owing to the high photo stability and large Stokes shifts of the
polycyclic aromatic skeleton, the fluorophore 4-amino-1,
8-napthalic anhydride derivative was chosen as the fluorophore
As the enzymatic hydrolysis could produce a blue dimerized
chromophore for fluorescent observation, the
[14, 15,
20]. Then α-D-galactopyranosyl was used as identifying
component and 4-hydroxybenzaldehyde was selected as the
linker. For improving the accumulation of the probe in cells, the
α-D-galactopyranosyl as a part of the probe was esterified. All the
three parts make up the probe AM-RP-G. Once the probe
AM-RP-G entered into the cells, the ester moiety of the probe
would react with the ubiquitous intracellular esterases and the
RP-G was resultantly cleaved from AM-RP-G [10]. Then, the
identifying component α-D-galactopyranosyl of the RP-G was
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) has
been used as one common fluorescent probe for β-galactosidase
[8]. However, if the X-gal was used to detect the β-galactosidase in
cell, the cells must be fixed and usually the detection efficacy of
[9]
β-galactosidase is manually counted
. The fluorescein
di-β-D-galactopyranoside (FDG) as another common fluorescent
probe exhibits a turn-on fluorescent response after it was
hydrolyzed [10]. However, its poor ability to be loaded in cell would
restrict the FDG for the observation of the cellular β-galactosidase
recognized
β-galactosidase-mediated
by
the
target
hydrolysis
β-galactosidase.
would induce
The
a
[11]. On the other hand, the fluorescent intensity of these probes
self-immolative reaction in RP-G. Consequently, the amino group
was freed [7]. The amino group as a strong electron donor could
enhance the ICT in the fluorophore. As a result, the wavelength of
maximal emissions from fluorophore would shift from 490 nm to
530 nm (SI, Figure. S1). The mechanism was illuminated in
scheme 1.
[12]
were directly utilized for detecting the β-galactosidase
.
However, the measurement for fluorescence with this strategy
would be influenced by many factors, such as various
environment conditions (pH, polarity, temperature, etc), the
localization of the probes in cells, collection efficiency of emission,
and excitation intensity [13]. To eliminate the influence of these
factors, the emission ratiometric probe could provide a selective
[14,
measurement mechanism for the detection of β-galactosidase
.
19]
Scheme 1. The principle of AM-RP-G for β-galactosidase
detection.
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2.8×10-5 D-galactose with time after addition of 0.06 U/L
β-galactosidase in 4.5×10-5 M AM-RP-G solution in 50 mM PBS
buffer ( pH 7.4, 9 U/L esterase, 1% DMSO, 37 °C); (b) The
fluorescence ratio I530/I490 of AM-RP-G with time in Figure.1 and
Figure.2a (red line and black line represent with and without
D-galactose, respectively).
The response of the AM-RP-G to β-galactosidase with various
concentrations was investigated for qualitatively detection of
β-galactosidase. Different concentrations of β-galactosidase (0
U/mL to 0.09 U/mL) in 4.5×10-5 M AM-RP-G solution in 50 mM
PBS buffer ( containing 9 U/mL esterase, 1% DMSO, pH 7.4, 37 °C)
were incubated. As shown in Figure. 3a, the emission at 490 nm
was gradually decreased as the concentration of β-galactosidase
was increased, while, the emission at 530 nm was enhanced
gradually.
To investigate the response of the AM-RP-G to
β-galactosidase in aqueous solution, the AM-RP-G was firstly
incubated with esterase to produce RP-G. The stock solution of
the AM-RP-G was diluted with 0.05M PBS to form a solution of
4.5×10-5 M AM-RP-G (the system containing 9 U/mL esterase, pH
7.4, 1% DMSO, 37 ℃). As shown in SI, Figure.S2, insignificant
change was observed from the fluorescence when the AM-RP-G
was treated with the esterase of 9 U/mL for 0.5 h. However, when
the β-galactosidase was subsequently added into the system, the
fluorescence intensities at 530 nm gradually increased
accompanying the gradual decrease of the fluorescence
intensities at 490 nm (Figure. 1), indicating the AM-RP-G has an
effective response to β-galactosidase.
Figure.3. (a) The fluorescence emission spectra of AM-RP-G at
different concentrations of β-galactosidase after reaction for 110
min. The inset plots in (a) shows the ratio of I530/I490 at different
concentrations of β-galactosidase after reaction for 110 min. (b)
The linear dependence of the ratio of I530/I490 on β-galactosidase.
The probe concentration is 4.5х10-5 M, in PBS buffer (0.05 M, pH
7.4, containing 9 U/L esterase, 1% DMSO, 37 °C).
In addition, the intensity ratio of I530/I490 was improved almost
four times when the concentration of the β-galactosidase
increased from 0 U/mL to 0.09 U/mL. The reaction nearly reached
equilibrium with 0.07 U/mL β-galactosidase (Figure. 3a inset). A
linear dependence of I530/I490 on the concentrations of
β-galactosidase from 0 U/mL to 0.07 U/mL can be obtained. A
detection limit of 1.4×10-3 U/mL was achieved according to LOD =
3σ/s, where σ was the standard deviation of ten blank signals and
s was the slope value [17], as shown in Figure. 3b.
Next, the ability of AM-RP-G to monitor the β-galactosidase in
real time was examined. The change of I530/I490 with the
incubation time was analyzed at various β-galactosidase
concentrations from 0 U/mL to 0.09 U/mL. The fluorescent
intensities ratios were measured with the time interval of 10 min
and the results were shown in Figure. 4.
Figure.1. Fluorescence spectra of AM-RP-G with time after
addition of 0.06 U/L β-galactosidase in 4.5×10-5 M AM-RP-G
solution in 50 mM PBS buffer ( pH 7.4, containing, 9 U/L esterase,,
1% DMSO , 37 °C).
In order to further verify that the β-galactosidase indeed
caused the change of the emission wavelength of the AM-RP-G,
the inhibitor experiments were conducted. The D-galactose, a
[16]
competitive inhibitor for β-galactosidase
was selected, with
the β-galactosidase were added into the 4.5×10-5 M AM-RP-G
solution in 50 mM PBS buffer ( pH 7.4, containing 9 U/L esterase,
1% DMSO, 37 °C), incubated for 110 min. The results are
showed in Figure.2. From Figure.2a, it can be found little change
of the emission spectra was observed from AM-RP-G after adding
D-galactose and β-galactosidase (Figure. 2a). But the emission
spectra from AM-RP-G increased gradually at 530 nm with
incidental decrease at 490 nm after adding only β-galactosidase
(Figure.1). Figure.2b indicated the ratio of the fluorescence at 530
nm and 490 nm from Figure.1 and Figure.2a. Obviously, the ratio
change of the fluorescence with D-galactose could be ignored and
the ratio change of the fluorescence without D-galactose was
significant. These results illuminated that the change of the
emission from the AM-RP-G were caused by the β-galactosidase.
Figure.4. Fluorescence ratio I530/I490 of AM-RP-G recorded
every 10 min under varying β-galactosidase concentrations. The
probe concentration is 4.5х10-5 M, in PBS buffer (0.05 M, pH 7.4,
containing 9 U/L esterase, 1% DMSO, 37 °C).
It can be found that the ratio of I530/I490 gradually increased
with either the increase of the β-galactosidase concentrations or
Figure.2. (a).The fluorescence spectra of AM-RP-G with
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duration of incubation time, which are in accord with the
enzymatic reaction kinetics.
excitation of 405 nm laser. As shown in Figure. 7a, the HeLa cells
only exhibited blue fluorescence emission in 0 min. However,
when the cells were incubated with β-galactosidase for 1 h
（Figure.7b-merged）and 2 h（Figure.7c-merged）, the blue
emission from HeLa cells was gradually weakening (Figure.
7b-blue and c-blue) and the HeLa cells simultaneously exhibited
remarkable green (Figure. 7b-green and c-green) emission. At the
same time, the florescence changes of HeLa cells along the yellow
arrows in Figure. 7 (a, b, c) were collected in different incubation
times of 0 min, 60 min and 120 min. (Figure. 7d, e, f). It can be
observed from Figure.7e-f that the intensities of green emission
were gradually enhanced.
The selectivity of the probe to β-galactosidase was further
investigated. Various co-existing enzymes in cells were added into
the 4.5×10-5 M AM-RP-G system (0.05 M PBS, containing 9 U/L
esterase, 1% DMSO, pH=7.4, 37 ℃) and incubated for 110 min.
The emission wavelengths of the systems showed little change
except the β-galactosidase. (Figure.5a).
Figure.5. (a) The fluorescence emission spectra of 4.5×10-5
M
AM-RP-G incubated with different enzymes. The fluorescence
ratio I530/I490 of AM-RP-G after incubated with different enzymes.
The concentration of esterase is 9 U/mL, ALP is 3U/mL, Thrombin
is 3 U/mL, Pepsin is 3U/mL, GOD (Glucose Oxidase) is 3 U/mL,
Tryspin is 3 U/mL, β-galactosidase is 0. 35 U/mL. ( 0.05 M PBS , 1%
DMSO , 37℃, 110 min).
As shown in Figure.5b, the ratio of I530/I490 almost unchanged
with the addition of other enzymes. These results indicated that
the present AM-RP-G had favorable selectivity to β-galactosidase.
Moreover, to study the stability of AM-RP-G for detection of
β-galactosidase at various pH, the AM-RP-G was dissolved in
solutions with different pH at 37 ℃ for 10 min and then the
fluorescence of the AM-RP-G was monitored. Varying the pH from
3 to 10, the ratio of I530/I490 almost exhibited little change (Figure.
6a), indicating the AM-RP-G owns intrinsic stability under various
pH conditions. The stability and performance of AM-RP-G in a
complex system was also detected. The AM-RP-G was dissolved in
a solution containing 10% BSA incubated at 37 ℃ for 120 min.
The ratio of I530/I490 almost exhibited little change (Figure. 6b)
in the BSA solution at 120 min. Then 0.05U β-galactosidase was
added and the ratio changed obviously after the addition of
β-galactosidase for 120 min. This result manifested that the
AM-RP-G can be used to detect β-galactosidase activity at
physiological environment.
Figure.7. Confocal fluorescence microscopy images of HeLa
cells. HeLa cells sequentially treated with 0.005 U/ml
-galactosidase for 0 min (a), 60 min (b), 120 min (c), (d, e, f) the
fluorescence intensity profile within the regions, yellow arrows of
(a, b, c). The scale bar is 20 μm. The green line indicates the green
emission and the blue line indicates the blue emission. Excited
at 405 nm; blue channel: 430 nm-490 nm; green channel: 510
nm-580 nm.
The change of fluorescence emission intensity in HeLa cells at
different times were calculated, as shown in Figure.S4, the ratio
of green emission and blue emission had an obvious change. And
the change of fluorescence emission from HeLa cells was
accordant with the change of the fluorescence spectra shown in
Figure. 1. All results confirmed that AM-RP-G could be used as a
novel ratiometric fluorescence probe for an effective detection of
β-galactosidase in cells.
To apply this probe to detect endogenous β-galactosidase,
live SKOV3 cells in which the concentration of β-galactosidase is
high than normal cells were incubated with AM-RP-G for 8 mins in
cell culture medium and subsequently washed with PBS solution
for three times to remove the extracellular AM-RP-G, before the
fluorescence images were captured. The fluorescence images of
the SKOV3 cells were captured with a Nikon A1 confocal
microscopy with an oil 60× objective lens and excitation of 405
nm laser. As shown in Figure. 8a, the SKOV3 cells only exhibited
blue fluorescence emission in 0 min. However, when the cells
were incubated with cells for 1 h（Figure.8b-merged）and 2 h
（Figure.8c-merged）, the blue emission from SKOV3 cells was
gradually weakening (Figure. 8b-blue and c-blue) and the SKOV3
cells simultaneously exhibited remarkable green (Figure. 8b-green
and c-green) emission. At the same time, the florescence changes
of SKOV3 cells along the yellow arrows in Figure. 8 (a, b, c) were
collected in different incubation times of 0 min, 60 min and 120
min. (Figure. 8d, e, f). It can be observed from Figure.8e-f that the
intensities of green emission were gradually enhanced. And the
change of fluorescence intensity in SKOV3 cells at different times
were calculated, as shown in Figure.S5 the ratio of green emission
and blue emission had an obvious change. For verify that the
fluorescence change was caused by the β-galactosidase in live
SKOV3 cells, another dish full of SKOV3 cells was incubated with
inhibitor (D-galactose) before the AM-RP-G was dropped in, the
Figure.6. (a) The fluorescence ratio of I530/I490 in different pH
reaction environment. (b) The fluorescence ratio of I530/I490 in 10%
BSA solution. The probe concentration is 4.5х10-5 M, 1% DMSO,
37 °C.
For further application of AM-RP-G in cells, the cytotoxicity of
AM-RP-G was investigated with the MTT method and the results
were shown in Figure S3. The viability of HeLa cells was 80% after
incubation for 12h with 1.0 10-6 M AM-RP-G. Then, the AM-RP-G
was used to detect exogenous β-galactosidase in live HeLa cells.
Before taking the fluorescence image, the cells were incubated
with AM-RP-G for 5 min in cell culture medium and subsequently
washed with PBS solution for five times to remove the
extracellular AM-RP-G. Then the exogenous β-galactosidase were
added to the dishes and incubated for different times. The
fluorescence images of the HeLa cells were captured with a Nikon
A1 confocal microscopy with an oil 100× objective lens and
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fluorescence change had almost no change in 120 min, Figure.S6.
The results confirmed that AM-RP-G could be used as a
ratiometric fluorescence probe for an effective detection and
imaging of β-galactosidase in cells.
spectra were got from a Hitachi 4600 spectrophotometer with
excitation of 420 nm. Absorption spectra were got from a Hitachi,
U-3900 spectrophotometer. Samples for fluorescence emission
measurements were contained in 1 cm × 1 cm quartz cuvette.
Fluorescent images of the cells were got from a confocal laser
Figure.8. Confocal fluorescence microscopy images of SKOV3
scanning microscopy Nikon A1 with a 100× oil-immersion
objective lens, excited at 405 nm, collected in blue and green
channels.
cells. SKOV3 cells sequentially treated with 5.5 10-6 M AM-RP-G
Cell culture
Before taking the fluorescence image, Hela cells and SKOV3
cells were cultured in confocal microscope dishes in Dulbecco's
Modified Eagle Medium (DMEM) media at 5% CO2 air atmosphere
at 37 °C in a humidified incubator of Panasonic MCO-5AC for 48 h
for cell attachment.
Synthesis of AM-RP-G
The synthetic routes for the AM-RP-G were outlined in
scheme 2.
for 0 min (a), 60 min (b), 120 min (c), (d, e, f) the fluorescence
intensity profile within the regions, yellow arrows of (a, b, c). The
scale bar is 20 μm. The green line indicates the green emission
and the blue line indicates the blue emission. Excited at 405 nm;
blue channel: 430 nm-490 nm; green channel: 510 nm-580 nm
Scheme 2. Synthesis of AM-RP-G.
Conclusions
In summary, we have designed and fabricated the AM-RP-G
composed of 4-amino-1, 8-napthalic anhydride derivative as
fluorescence group and α-D-galactopyranosyl as the identifying
component for accurate detection of the β-galactosidase in
At first, the molecule 1 and 2 were synthesized following a
previous work in supporting information [16]. And then, molecule 3
was synthesized as follow: 4-amino-1, 8-napthalic anhydride (150
mg, 0.7 mmol) was dissolved in 25 mL ultra-dry ethanol ethanol
under nitrogen atmosphere and heated to 90 ℃ in dark. Then,
n-butylamine (0.3 mL, 3 mmol) was added into the ethanol and
refluxed for 20 hours [17]. After cooling, the solvent was removed
under reduced pressure, and the crude materials was purified by
chromatography on silica gel (20:1 DCM: EtAc) to obtain the
compound 3 as a yellow solid in yield of 93.5 %.1H NMR (400 MHz,
CDCl3) δ 8.61 (d, J = 7.4 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H),
7.52(s,2H ),7.49 – 7.38 (m, 1H), 7.00 (d, 1H), 6.89 (d, J = 8.3 Hz,
1H), 4.15 (t, J = 7.4 Hz, 2H), 1.71 – 1.67 (m,2H), 1.67 – 1.09 (m, 2H),
0.97 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3) δ, 164.6, 164.1, 148.9,
aqueous solution and cells. The probe showed
a linear
dependence on the concentration of the β-galactosidase between
0-0.07 U/ml with a detection limit of 1.4 × 10-3 U/mL. Moreover,
the probe also exhibited high selectivity toward the
β-galactosidase. Present probe may have a potential for the
monitor of the β-galactosidase in living cells to evaluate the
efficacy of cancer chemotherapy.
Experimental
Material and apparatus
133.7, 129.8, 125.0, 123.3, 120.2, 112.4, 109.6, 40.1, 30.3, 20.4,
4-hydroxybenzaldehyde, 4-amino-1, 8-napthalic anhydride
and N-butylamine were purchased from Sigma-Aldrich. 2, 3, 4,
6-Tetra-O-acetyl-α-D-galactopyranosylbromide and triphosgene
were purchased from Aladdin. Other reagents and solvents were
purchased from Beijing Chemical Regent Co. Hela cells were
purchased from the center of cells, Peking Union Medical College.
All the reagents and solvents were used without further
purification.
-
13.9. ESI-HRMS (m/z): calculated for C16H16N2O2 [M -H]
267.1279, found: 267.1141.
:
Next, a solution of triphosgene (61 mg, 0.2 mmol) in toluene
(3 mL) was added dropwise to the mixture of compound 3 (53.6
mg, 0.2 mmol) and DMAP（4-dimethylaminopyridine）(74 mg, 0.6
mmol) in toluene (4 mL). The solution was heated at 100 ℃ for
3.5 hours. After the solution was cooled to room temperature,
the reaction mixture was diluted and filtered with
dichloromethane (6 mL) and the filtrate was obtained without
further purification. Then, the compound 2 (90.8 mg, 0.2 mmol)
was added into above filtrate and the solution was stirred at
room temperature for 4 hours [18]. After that, the solution was
concentrated and purified by column chromatography (silica gel,
1:10 EtOAc/CH2Cl2) and obtained AM-RP-G as yellow solid (49.7
1H NMR spectra were recorded on a Bruker Advance 400
spectrometer with tetramethylsilane as an internal standard.
Electrosprayionization high resolution mass spectra (ESI-HRMS)
were recorded on a Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry (FTICR-MS, Bruker Apex IV). 13C NMR (100 Hz)
spectra were obtained on a Bruker Avance 400. Fluorescence
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mg, 0.07 mmol, 35% yield). 1H NMR (400 MHz, CDCl3) δ 8.62
– 8.47 (m, 2H), 8.32 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H),
7.69 (t, J = 7.9 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.02 – 6.88 (m,
2H), 5.48 – 5.38 (s, 2H), 5.28 (d, 1H), 5.18 (s, 1H), 5.02 (dd, 8.2
Hz, 2H), 4.3（t, 1H）, 4.17(t, 2H), 4.15(t, 1H), 4.10(d, 2H), 2.15(s, 3H)
2.02 (s, 3H), 1.98 (s, 3H), 1.95 (s, 3H), 1.68-1.62 (m, 2H), 1.43-1.32
(m, 2H), 0.90 (dd, 3H). 13C NMR (CDCl3) δ, 170.3, 169.5, 164.2,
163.8, 157.4, 153.1, 138.9, 132.6, 131.4, 130.6, 130.4, 129.1,
126.8, 125.8, 123.7, 123.0, 118.2, 117.1, 99.7, 77.4, 71.3, 70.9,
68.8, 67.6, 70.0, 61.4, 40.4, 30.4, 29.8, 20.8, 20.7, 20.5, 14.0.
ESI-HRMS (m/z): calculated for C38H40N2O14 [M+Na]+ 771.25,
found 771.2373. The stock solution of the AM-RP-G (4.5 × 10-3M)
were prepared in dimethyl sulphoxide (DMSO).
Alpha-peptide of Beta-galactosidase. Analyst. 1998, 123, 1309-1314.
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Cytotoxicity assay of AM-RP-G
HeLa cells were firstly seeded to a 96-well plate with the
density of 5 × 103 per well and cultured in the Dulbecco’s
modified eagle medium (DMEM) at 37 °C for 24 h. Then,
AM-RP-G with different concentrations were added to each well,
which were incubated with the cells for 12 h. After the incubation,
the cells were washed thrice with PBS. Then, 10% volume ratio
(v/v) MTT dye solution (10 mg/mL) were added to obtain the
value of cell viability. All experiments were performed in
triplicate.
Acknowledgement
[5] Chauvin, T.; Durand, P.; Bernier, M.; Meudal, H.; Doan, B. T.; Noury,
F.; Badet, B.; Beloeil, J. C.; Toth, E. Detection of Enzymatic Activity by
PARACEST MRI: a General Approach to Target a Large Variety of
Enzymes. Angew. Chem. Int. Edit. 2008, 47, 4370-4372.
This work was supported by National Key R&D Program of
China (2016YFA0200800); NSFC (51672284); Beijing Natural
Science Foundation (2181002); Chinese Academy of Sciences
(1A1111KYSB20180017, QYZDJ-SSW-JSC032, XDB17000000)
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(The following will be filled in by the editorial staff)
Manuscript received: XXXX, 2019
Manuscript revised: XXXX, 2019
Manuscript accepted: XXXX, 2019
Accepted manuscript online: XXXX, 2019
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Entry for the Table of Contents
Page No.
A Novel Ratiometric Fluorescent Probe for Highly
Sensitive
and
Selective
Detection
of
β-galactosidase in Living Cells
Min Chena,b
, ,
Lixuan Mu*,a, Xingxing Caoa,b
A novel ratiometric fluorescent probe for β-galactosidase detection in living cells was
designed and synthesized.
Guangwei Shea, Wensheng Shi*,a
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