Synthesis and evaluation of a novel fluorescent chemosensor for glutathione based on a rhodamine B and N-[4-(carbonyl) phenyl]maleimide conjugate and its application in living cell imaging

Article abstract of DOI:10.1016/j.dyepig.2016.08.063

A novel rhodamine B spirolactam derivative bearing an N-[4-(carbonyl)phenyl] maleimide moiety (L1) was designed, synthesized and structurally characterized to develop a chemosensor. The interactions of L1 with amino acids and metal ions were studied by UV–vis absorption and fluorescence spectroscopy. L1 exhibited a highly sensitive and selective turn-on fluorescence response toward glutathione (GSH) over other biological species in EtOH/HEPES (3:2, v/v, 0.1?mM, pH 7.34) solution. The detection limit of GSH by L1 was 0.219?μM. Intracellular imaging applications demonstrated that L1 can be used as a fluorescent probe for the detection of GSH in HepG-2 and HUVEC cells.

Full text of DOI:10.1016/j.dyepig.2016.08.063

Accepted Manuscript
Synthesis and evaluation of a novel fluorescent chemosensor for glutathione based
on a rhodamine B and N-[4-(carbonyl) phenyl]maleimide conjugate and its application
in living cell imaging
Hai Shu, Xiaolei Wu, Baojing Zhou, Yingbin Han, Mingjie Jin, Jing Zhu, Xiaofeng Bao
PII:
S0143-7208(16)30596-4
10.1016/j.dyepig.2016.08.063
DYPI 5443
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 21 May 2016
Revised Date: 29 August 2016
Accepted Date: 30 August 2016
Please cite this article as: Shu H, Wu X, Zhou B, Han Y, Jin M, Zhu J, Bao X, Synthesis and evaluation
of a novel fluorescent chemosensor for glutathione based on a rhodamine B and N-[4-(carbonyl)
phenyl]maleimide conjugate and its application in living cell imaging, Dyes and Pigments (2016), doi:
10.1016/j.dyepig.2016.08.063.
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ACCEPTED MANUSCRIPT

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Synthesis and evaluation of a novel fluorescent chemosensor for glutathione
based on a rhodamine B and N-[4-(carbonyl) phenyl]maleimide conjugate and its
application in living cell imaging
Hai Shua, Xiaolei Wua, Baojing Zhouc, Yingbin Hand, Mingjie Jina, Jing Zhub,*,
Xiaofeng Baoa,*
a School of Environmental and Biological Engineering, Nanjing University of Science
and Technology, 200 Xiaolinwei, Nanjing 210094, P.R. China. E-mail:
baoxiaofeng@mail.njust.edu.cn
b
Department of Pharmacy, Nanjing University of Chinese Medicine, 138
XianlinDadao, 210023, P.R. China. E-mail: zhujing1227@hotmail.com
c School of Chemical Engineering, Nanjing University of Science and Technology,
Nanjing 210094, P.R. China

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Keywords: Rhodamine B; Colorimetric fluorescent probe; GSH; Cell imaging
Abstract
A novel rhodamine B spirolactam derivative bearing an N-[4-(carbonyl)phenyl]
maleimide moiety (L1) was designed, synthesized and structurally characterized to
develop a chemosensor. The interactions of L1 with amino acids and metal ions were
studied by UV-vis absorption and fluorescence spectroscopy. L1 exhibited a highly
sensitive and selective turn-on fluorescence response toward glutathione (GSH) over
other biological species in EtOH/HEPES (3:2, v/v, 0.1 mM, pH 7.34) solution. The
detection limit of GSH by L1 was 0.219 µM. Intracellular imaging applications
demonstrated that L1 can be used as a fluorescent probe for the detection of GSH in
HepG-2 and HUVEC cells.

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1. Introduction
Biological thiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione
(GSH), play vital and distinct roles in a number of biological systems.[1] Glutathione
(GSH) is the most abundant non-protein thiol species in mammalian cells, and it plays
an important role in various biological processes, including regulating redox
homeostasis, detoxification, and metabolism.[2-4] Abnormal levels of GSH are related
to a variety of diseases, such as cancer, AIDS, liver damage and neurodegenerative
diseases.[5-7] Accurate quantification of thiols in biological systems may contribute to
the early diagnosis and therapy of these diseases.[8] Therefore, the development of
simple, rapid, sensitive methods to monitor the variation in biological systems is of
great importance.
Several methods have been developed for thiol measurement, for instance,
high-performance liquid chromatography (HPLC),[9,10] capillary electrophoresis,[11]
UV-vis spectroscopy,[12,13] mass spectrometry,[14,15] electrochemical assays,[16] and
fluorescence techniques.[17] Among these techniques, fluorescence assays are the most
attractive detection methods due to several desirable features, such as low cost,
simplicity, rapid detection, non-destructive nature and potential for in vivo
imaging.[18-22] To date, significant efforts have been devoted to the construction of
fluorescent probes for intracellular thiols. These probes are usually designed based on
various specific reactions between the probe and thiols, such as Michael addition,[23]
cleavage of GSH-sensitive bonds (e.g., sulfonamide and sulfonate ester,[24] selenium–

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nitrogen bonds,[25,26] disulfide bonds[27,28]), metal complex-displacement
coordination,[29] and metal complex oxidation-reduction.[30] However, these reported
probes sometimes suffer from the limitations of high background fluorescence,[31,32]
bad selective performance[33] or working in high pH aqueous solutions.[34] Thus, there
is great interest to develop a thiol probe based on the Michael addition reaction with
more desirable features.
Herein, we designed a novel rhodamine B-derivatized fluorescent chemosensor
with an N-[4-(carbonyl) phenyl] maleimide moiety, namely L1. The maleimide
moiety and its derivatives have been used as receptors for thiols. Therefore, we
conjugated an N-[4-(carbonyl) phenyl] maleimide moiety to the rhodamine B moiety
as a host recognition site for thiols. Our findings demonstrate that L1 exhibits high
selectivity and sensitivity toward GSH in EtOH/H2O (3:2, v/v, pH 7.34, HEPES
buffer, 0.1 mM) solution. The fluorescence microscopy studies show that L1 can be
used as a fluorescent probe for the detection of GSH in living cells.
2. Experimental
2.1. Materials and instruments
All reagents and organic solvents were of ACS grade or higher and were used
without further purification. Unless otherwise noted, all chemicals were purchased
from J&K Scientific (Shanghai, China) and were used as received. All solvents were
of analytical grade, and double-distilled water was used in all experiments. The
reactions were performed under an argon atmosphere using standard Schlenk
techniques. Thin-layer chromatography was performed on a HAIYANG silica gel

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F254 plate, and compounds were visualized under UV light (λ= 254 nm). Column
chromatography was performed using HAIYANG silica gel (type: 200–300 mesh
ZCX-2).
13
1H (500 MHz) and C NMR (126 MHz) spectra were recorded on an Avance 500
spectrometer (Bruker; Billerica, MA, USA). The chemical shifts are reported in δ
units (ppm) downfield relative to the chemical shift of tetramethylsilane. The
abbreviations br, s, d, t and m denote broad, singlet, doublet, triplet and multiplet,
respectively. Mass spectra were obtained with a Finnigan TSQ Quantum LC/MS
spectrometer. High-resolution mass spectra (HRMS) were acquired under electron
ionization conditions with a double-focusing high-resolution instrument (Autospec;
Micromass Inc.). The pH levels of stock solutions were measured using a PHS-25C
Precision pH/mV meter (Aolilong, Hangzhou, China). UV-vis and fluorescence
spectra were obtained on a UV-3600 UV-vis-NIR spectrophotometer (Shimadzu,
Japan) and an Edinburgh FLS920 fluorescence spectrophotometer (Livingston, UK),
respectively, at room temperature. Cell imaging was performed with an inverted
fluorescence microscope (OLYMPUS, IX83).
2.2. Synthesis of 2-(2-aminoethyl)-30,60-bis(diethylamino)spiro-[isoindoline-1,90-
xanthen]-3-one (6)
Ethane-1, 2-diamine (4.46 mL, 10 equiv.) was added to a solution of rhodamine B
(3.2 g, 6.68 mmol) in ethanol (80 mL). The mixture was refluxed for 12 h and then
evaporated to dryness under a vacuum. The crude solid was dissolved in CH2Cl2 and
then washed with H2O and brine. The organic layer was dried with MgSO4. After

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removal of the solvent, flash chromatography (silica gel; MeOH/CH2Cl2, 3:97 v/v) of
the residue yielded 6 as a pink solid (3.1 g, 95%). Mp. 217-219 ℃. IR(KBr, cm-1):
υNH, 3417.5;
υasCH, 2907.1, 2927.6;
υC=O, 1685.6;
υ
C=C, 1616.2, 1513.9; C-N, 1394.4;
υ
1
υ
asC-O-C, 1222.7, 1118.6. H NMR (CDCl3): δ 8.12–7.78 (m, 1H), 7.64–7.34 (m, 2H),
7.19–6.96 (m, 1H), 6.43 (d, 2H, J = 8.9 Hz), 6.37 (d, J = 2.5 Hz, 2H), 6.27 (dd, J1 =
2.6, J2 = 2.5 Hz, 2H), 3.66–3.24 (m, 8H), 3.28–2.91(m, 2H), 2.41 (t, J1 = 6.7 Hz, J2 =
13
6.6 Hz, 2H), 1.40 (s, 2H), 1.16 (t, J1 = 7.0 Hz, J2 = 7.1 Hz, 12H) ppm. C NMR
(CDCl3): δ 168.59, 153.43, 153.26, 148.79, 132.37, 131.20, 128.64, 128.01, 123.79,
122.70, 108.13, 105.64, 97.71, 77.38, 77.13, 76.88, 64.91, 44.31, 43.80, 40.73, 12.56
ppm.
2.3. Synthesis of N-[4-(chlorocarbonyl) phenyl] maleimide (4)
p-Aminophenyl maleamic acid (200 mg, 1.46 mmol) was added to a mixture of
maleic anhydride (215 mg, 2.19 mmol) in ethyl acetate (20 mL). The resulting
mixture was stirred for 4 h at room temperature. The precipitate was filtered, washed
with ethyl acetate and dried to give 4-(2,5-dioxo-2H-pyrrol-1(5H)-yl)benzoic acid
(305 mg, 88%) as a white solid.
A mixture of the crude (Z)-4-(4-carboxy-2-oxobut-3-en-1-yl)benzoic acid (220 mg,
0.94 mmol), acetic anhydride (10 mL) and sodium acetate (25 mg, 0.30 mmol) was
heated at 80 °C and stirred for 6 h. The mixture was poured into ice water, and the
solid was isolated by filtration, washed with water to neutral pH and dried in vacuum
to afford a pale yellow solid. The residue was purified by column chromatography
using
0–4%
MeOH/DCM
as
an
eluent
to
give

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4-(2,5-dioxo-2H-pyrrol-1(5H)-yl)benzoic acid (3) as a pale yellow solid (75 mg, 37%).
Mp. 225-228 ℃ . IR(KBr, cm-1):
υOH, 3417.5;
υ=C-H, 3105.1;
υ
C=O, 1712.6.6; υC-N
,
1394.4. 1H NMR (500 MHz, DMSO-d6): δ 7.23 (s, 2H), 7.50 (d, J = 7.5 Hz, 2H), 8.05
(d, J = 8.5 Hz, 2H), 13.08 (s, 1H). 13C NMR (500 MHz, DMSO-d6): δ 169.51, 166.69,
-
135.50, 134.82, 129.89, 129.58, 126.10 ppm. ESI–MS m/z: calcd for C11H6NO4
[M-H]-, 216.0375; found, 216.0293.
A mixture of 4-(2,5-dioxo-2H-pyrrol-1(5H)-yl)benzoic acid (30 mg, 0.128 mmol)
and thionyl chloride (10 mL) was refluxed overnight at 80 °C. Unreacted thionyl
chloride was evaporated to yield the residual product N-[4-(chlorocarbonyl) phenyl]
maleimide (4) which was used directly.
2.4. Synthesis of compound L1
The obtained N-[4-(chlorocarbonyl) phenyl] maleimide (40 mg, 0.170 mmol) was
suspended in dry CH2Cl2 (15 mL). Thereafter, compound 6 (124 mg, 0.254 mmol)
and Et3N (38 µL) were added. The mixture was stirred at room temperature for 4.5 h,
and the solvent was removed in a vacuum. Dichloromethane (30 mL) was then added
to the residue, and the organic phase was extracted with saturated brine (10 mL) three
times. The organic phase was dried with anhydrous magnesium sulfate. After the
crude mixture was filtered through a pad of celite, the solvent was removed in a
vacuum to obtain the crude solid. The residue was purified by column
chromatography using 0–1% MeOH/DCM as an eluent to give L1 as an orange solid
(64 mg, 55%). Mp. 196-198 ℃. IR(KBr, cm-1):
υNH, 3421.4, 3132.1;
υasCH, 2970.1,
2929.6; C=O, 1702.3; C=C, 1620.0;
υ
υ
υ
C-N, 1396.3. 1H NMR (500 MHz, CDCl3): δ 8.41

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(s, 1H), 8.03 (d, 2H), 7.19–6.96 (m, 1H), 7.95–7.93 (m, 1H), 7.51–7.46 (m, 4H),
7.11–7.09 (m, 1H), 6.89 (s, 2H), 6.45 (d, J = 9 Hz, 2H), 6.39 (d, J = 3 Hz, 2H), 6.27
(dd, J1 = 3, J2 = 9 Hz, 2H), 3.45 (t, J1 = 5 Hz, J2 = 10.5 Hz, 2H), 3.36–3.32 (m, 8H),
13
3.19 (t, J1 = 3 Hz, J2 = 6 Hz, 2H), 1.17 (t, J1 = 7 Hz, J2 = 14 Hz, 12H) ppm. C
NMR (500 MHz, CDCl3): δ 170.03, 168.71, 165.59, 153.48, 152.87, 133.91, 133.39,
132.97, 132.48, 129.86, 127.95, 127.70, 125.08, 123.48, 122.53, 107.95, 104.11,
97.38, 65.58, 43.93, 41.54, 39.46, 31.48, 31.06, 29.73, 29.24, 28.91, 24.41, 22.25,
13.69, 12.17 ppm. ESI–MS m/z: calcd for C41H42N5O5 + [M+H]+, 684.3108; found,
684.3230.
2.5. Stock solution preparation for spectral detection
The stock solution of L1 (1 mM) was prepared in EtOH/HEPES (3:2, v/v, 0.1 mM,
pH 7.34) solution. Stock solutions (5 mM) of Pro, Val, His, Lys, Thr, Leu, Arg, Met,
Ala, Ser, Glu, Hcy, GSH, Cys and metal chloride salts of Na+, K+, Mg2+ and Zn2+
were dissolved in double-distilled water. Working solutions of L1 were freshly
prepared by diluting the highly concentrated stock solution to the desired
concentration prior to the spectroscopic measurements.
2.6. UV-vis and fluorescence spectral studies
All experiments were performed in EtOH/H2O solution (3:2, v/v, pH 7.4, HEPES
buffer, 0.1 mM). In the selectivity experiments, the test samples were prepared by
adding the cation stock or amino acid solution (200 mM) to a 3 mL solution of the
probe L1 (20 mM). In the titration experiment, a 20 mM solution of the probe L1 was
prepared at room temperature with an appropriate amount of the ion stock solution or

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GSH solution, which was added to the quartz optical cell using a micropipette.
Spectral data were recorded 100 min after the addition of the amino acids or metal
ions. In the fluorescence measurements, excitation was performed at 563 nm, and
emission was collected from 568 to 702 nm (slit: 3/3 nm).
2.7. Determination of the detection limit
The detection limit was calculated based on the fluorescence titration. The
fluorescence emission spectrum of probe L1 was measured 10 times, and the standard
deviation of the blank measurement was determined. To obtain the slope, the
fluorescence emission intensity (586 nm) was plotted as a function of the
concentration of GSH. The detection limit was calculated using the following
equation: Detection limit = 3δ/S
Where δ is the standard deviation of the blank measurement, and S is the slope of the
fluorescence intensity at 586 nm versus the GSH concentration.
2.8. Cell incubation and fluorescence imaging
HUVEC and HepG-2 cells were cultured in Dulbecco’s modified Eagle’s medium
(high glucose) supplemented with 10% FBS (fetal bovine serum), 100 units/mL
penicillin, and 100 units/mL streptomycin in a 5% CO2/95% air incubator at 37 °C.
HUVEC and HepG-2 cells were seeded at 1 × 105 cells per well in 24-well
flat-bottomed plates in an atmosphere of 5% CO2/95% air at 37 °C and incubated for
2 h before treatment. For living cell imaging, cells were incubated with 20 µM L1 in
culture medium for 5 hours in an air incubator at 37 °C. In the control experiment,
cells were pretreated with 200 µM GSH for 2 hours and then incubated with 20 µM

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L1 in culture medium for an additional 5 hours. Fluorescence imaging was performed
after washing the cells with PBS 3 times and was recorded using the red channel
under an inverted fluorescence microscope.
2.9. L1 cytotoxicity determination by an MTT assay
The
cytotoxicity
of
L1
was
evaluated
by
an
MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay using
HUVEC cells. Cytotoxicity was determined by an MTT colorimetric method using
four parallel wells at each time point. HUVEC cells were cultured in 96-well plates at
a density of 9 × 103 cells per well and were incubated at 37 °C in 5% CO2/95% air for
12 h. Then, cells were incubated with different concentrations of L1 (0–100 µM) for
24 h, followed by further incubation with 5 mg/mL of MTT for 4 h at 37 °C. The
supernatant was then discarded, and DMSO (150 µL/well) was added to dissolve the
formazan. Finally, the absorbance was measured at 550 nm with a microplate reader
(Infinite M200 Pro).
3. Results and discussion
3.1. Synthesis of L1
The synthesis of L1 is illustrated in Scheme 1. Compound 4 was prepared
according to a previously reported procedure.[35] Compound 6 was prepared in 95%
yield by refluxing commercially available rhodamine B with ethane-1,2-diamine in
EtOH, which was followed by coupling with 4 in the presence of Et3N in CH2Cl2 at
room temperature for 4.5 h to give L1 in a 55% yield. The structure of L1 was
confirmed by 1H NMR, 13C NMR and HRMS (Supporting Materials).

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[Scheme 1]
3.2. pH and time-dependent response of L1
The pH response of L1 in EtOH/H2O (3:2, v/v) solution was investigated.
Acid-base titration experiments were performed in different EtOH/H2O (3:2, v/v) pH
solutions. The fluorescence intensity of L1 at 586 nm in different pH solutions was
recorded. As shown in Figure 1a, the probe L1 displayed almost no fluorescence in
the pH range of 6.0–13.0, which indicated that the spirolactam form of L1 was
dominant. In contrast, the fluorescence intensity drastically increased at pH less than
6.0 due to the ring-opening of the spirocyclic moiety of rhodamine B and reached its
maximum value when the pH was less than 3.0. These results suggested that L1 was
insensitive to pH from 6.0 to 13.0 and could work well in approximate physiological
conditions with low background fluorescence. Therefore, further UV-vis and
fluorescence studies were performed in EtOH/H2O (3:2, v/v, pH 7.34, HEPES buffer,
0.1 mM) solution. The response time of L1 was studied at room temperature to
evaluate the sensitivity of L1 toward GSH. As shown in Figure 1b, the fluorescent
intensity of L1 (20 µm) at 586 nm rapidly increased after GSH (200 µM) was added,
reached its maximum in approximately 100 min and stabilized thereafter. These
results indicated that L1 is a sensitive probe for GSH in EtOH/H2O (3:2, v/v, pH 7.34,
HEPES buffer, 0.1 mM) solution. Therefore, a 100 min reaction time was selected for
the following experiments.

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[Figure 1]
3.3. Fluorescence response of probe L1 toward GSH
The change in fluorescence intensity upon the addition of various amounts of GSH
was also investigated at room temperature. As shown in Figure 2, the free probe L1
remained colorless and emitted almost negligible fluorescence at 586 nm. However,
upon addition of GSH (0–90 equiv.) to the solution of probe L1, the fluorescence
intensity of probe L1 at 586 nm significantly increased, resulting in a color change
from colorless to pink, which was attributed to the reaction of GSH with the
carbon-carbon double bond of the maleimide moiety of probe L1. When 1000 µM
GSH was added to the L1 solution, a greater than 200-fold fluorescence enhancement
was observed. The fluorescence intensity of probe L1 also displayed good linearity
with the GSH concentration in the range of 2 to 26 µM (Figure S9). The detection
limit of probe L1 for GSH was calculated to be 0.219 µM. The dramatic fluorescence
enhancement together with the low detection limit suggested that probe L1 is highly
sensitive to GSH.
[Figure 2]
3.4. Selectivity and competitiveness of the performance of probe L1
To investigate the selectivity of probe L1 for biological thiols, we studied the
fluorescence performance of probe L1 (20 µM) toward other biologically relevant
species, including amino acids (Pro, Val, His, Lys, Thr, Leu, Arg, Met, Ala, Ser, Glu,
Hcy, GSH, Cys) and metal ions (K+, Na+, Mg2+, Zn2+) under identical conditions. As

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shown in Figure 3a, the only prominent fluorescence enhancement (158-fold)
appeared when the GSH (200 µM) was added. Other species caused negligible
fluorescence response under identical conditions, except for Glu, which induced a
slight fluorescence intensity change (blue line). Moreover, the visual fluorescence
response of probe L1 toward various species further suggests that the probe L1 can be
applied as a direct visualization fluorescence indicator for GSH. The UV-vis
absorption spectrum of probe L1 for various species was also recorded under identical
conditions (Figure S10). In accordance with the results of the fluorescence
experiment, only GSH caused a strong absorbance at 563 nm for probe L1.
The possible interference of other species was evaluated by competitive assays. As
shown in Figure 3b, no obvious variation in the fluorescence intensity of the L1-GSH
complex was observed compared with the results with or without other competitive
species, indicating that the co-existence of other species had no visible effect on the
probe L1 detection of GSH. These results indicated that probe L1 had good selectivity
toward GSH, even in the presence of other physiologically relevant species.
[Figure 3]
3.5. Fluorescence spectroscopy studies of the L1-GSH complex with various anions
As mentioned above, L1 could selectively bind with GSH to form an L1-GSH
complex, leading to a dramatic change in the spectral properties of L1. To further
study the effects of different anions on the rupture of the L1-GSH complex to
regenerate L1. Fluorescence spectra of the L1-GSH complex were recorded in the
-
-
2-
2-
-
presence of different anions, such as Cl-, Br-, I-, NO3 , H2PO4 , SO4 , CO3 , BrO3 ，

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-
2-
3-
IO3 , S2O8 and PO4 . As shown in Figure S11, no anions caused discernible spectral
changes in the L1-GSH complex, which indicated the L1-GSH complex is quite
stable in the presence of these anions.
3.6. Proposed sensing mechanism
Based on the chemistry of the Michael addition reaction and the optical changes of
compound L1 with thiols, a plausible reaction mechanism was proposed (Scheme 2).
The fluorescence of probe L1 was extremely weakened by the attachment of the
maleimide ring. When the electron-deficient maleimide group became saturated
through the Michael addition of thiols, one of the carboxyl groups on GSH ionized the
hydrogen ion. The distance between the spirocyclic moiety of rhodamine B and the
negatively charged carboxylate group decreased, which generated a highly acidic
microenvironment. Therefore, the spirocyclic moiety of rhodamine B may be
transformed into the fluorescent open-ring formation by the turn-on of the parent
rhodamine backbone fluorescence.
[Scheme 2]
To further confirm the proposed sensing mechanism, a Job plot was produced by
continuously varying the mole fraction of GSH from 0 to 1 in a solution of
[GSH]+[L1] at a total concentration of 50 µM. The Job plot analysis revealed an
approximate maximum at a mole fraction of 0.5, which indicated compound L1
reacted with GSH in a 1:1 molar ratio (Figure 4). The mass spectra of L1 in the
presence and absence of GSH were also acquired. When 5 equiv. of GSH was added
to the L1 solution, a new peak appeared at m/z 991.4035, which was assigned to [L1–

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GSH + H]+ (Figure 5). This result indicated that compound L1, with an m/z of
684.3230 for [L1 +H]+, can be converted into [L1-GSH+H]+ in the presence of GSH.
These results confirmed the proposed reaction mechanism.
[Figure 4]
[Figure 5]
3.7. Theoretical calculation
To better understand the optical response of probe L1 to thiols, energy-optimized
structures of L1 and the L1-GSH complex (Figure S12) were obtained using DFT
calculations with the B3LYP method using the 6-31+G(d,p) basis set. The spatial
distributions and orbital energies of the HOMO and LUMO of L1 and L1-GSH were
also generated using DFT calculations (Figure S13). The results indicated that in L1,
the HOMO is distributed on the spirolactam of the rhodamine B moiety, whereas the
LUMO is spread over the maleimide group. The π electrons on the HOMO of the
L1-GSH adduct are mainly located on the xanthene part, whereas the LUMO is
mostly located on N-[4-(carbonyl) phenyl] maleimide. The energy gaps between the
HOMO and LUMO in probe L1 and the L1-GSH complex were calculated 48.57
kcal/mol and 86.66 kcal/mol, respectively. The results indicated that the binding of
GSH to L1 increased the HOMO–LUMO energy gap of the complex and stabilized
the system.
3.8. Fluorescence imaging in living cells
To investigate the permeability of cells and to clarify whether L1 was sufficiently

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sensitive to detect biothiols in living cells, the cytotoxicity of L1 was first evaluated
using MTT assays. As shown in Figure 6, more than 83% of HUVEC cells remained
alive after internalizing 20 µM L1 for 24 h. Therefore, L1 has low cytotoxicity and
good biocompatibility under the experimental conditions.
[Figure 6]
Next, cell-imaging experiments with L1 were performed and observed under an
inverted fluorescence microscope. As shown in Figure 7, HepG-2 cells without L1
did not show fluorescence, whereas after incubating with L1 for 5 h, a weak red
fluorescence was observed. The HUVEC cells displayed no intracellular background
fluorescence (Figure 8). However, after incubating with probe L1 for 5 h, the
HUVEC cells displayed visible fluorescence. These results proved that L1 was
capable of permeating cells and reacting with intracellular GSH.
[Figure 7]
To further study the response of probe L1 to changes in the intracellular GSH level,
HepG-2 cells and HUVEC cells were separately treated with GSH (200 µM) and then
incubated with L1 for an additional 5 h (Figure 7 and Figure 8). Strong red
fluorescence was observed. These results indicated that probe L1 was capable of
detecting differences in GSH concentrations in living cells. The bright-field images
confirmed that the cells were viable throughout the imaging experiments.
[Figure 8]

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4. Conclusions
In summary, a rhodamine B derivative L1 was applied as a selective fluorescent
and colorimetric chemosensor to specifically recognize GSH in EtOH/H2O (3:2, v/v,
pH 7.34, HEPES buffer, 0.1 mM) solution through its UV-vis and fluorescence
1
13
response. The chemical structure of L1 was analyzed by H NMR, C NMR and
HRMS. Highly sensitive and selective detection of GSH was feasible with compound
L1, with a detection limit of 0.219 µM. Preliminary biological experiments
demonstrated that this probe has the potential to detect and image GSH in biological
systems.
Acknowledgements
This research was supported by the National Natural Science Foundation of China
(81371616, 21606132), a Specially Appointed Professor Grant by Jiangsu Province
(2015, Prof. Jing Zhu), Natural Science Foundation of Jiangsu Province
(BK20160823) and the Research Fund for the Doctoral Program of Higher Education
of China (20133219120020). We thank the sensor Lab of NUST for providing the
facilities for the UV-vis studies.
Notes and references
[1] Lun S, Qian S, Nan W, Chen ZY, Zhang WB and Qian JH. A fluorescent probe
for the discrimination between Cys and GSH. Anal Methods 2015; 7: 10371-5.

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Figure captions
Scheme 1. Synthesis of L1
Figure 1. (a) The effect of pH on the fluorescence of L1 (20 µM) in EtOH/H2O
solution (3:2, v/v). (b) Fluorescence spectra of L1 (20 µM) with GSH (200 µM) in
EtOH/H2O (3:2, v/v, pH 7.34, HEPES buffer, 0.1 mM) solution. Inset: plot of the
fluorescence intensity at 586 nm over a period of 180 min. The excitation and
emission wavelengths were 563 and 586 nm.
Figure 2. Fluorescence spectrum of L1 (20 µM) in EtOH/H2O (3:2, v/v, pH 7.34,
HEPES buffer, 0.1 mM) solution with various equiv. of GSH (0–90 equiv.). The
excitation and emission wavelengths were 563 and 586 nm. Inset: the figure on the
right displays the fluorescence intensity vs. GSH concentration.
Figure 3. (a) Fluorescence spectra of L1 (20 µM) in EtOH/H2O (3:2, v/v, pH 7.34,
HEPES buffer, 0.1 mM) solution upon the addition of 10 equiv. of various amino
acids and metal ions with excitation at 563 nm. (b) Fluorescence intensity (at 586 nm)
response of L1 (20 µM) towards various species (200 µM) in the absence (black bar)
and presence (red bar) of GSH (200 µM) in EtOH/H2O (3:2, v/v, pH 7.34, HEPES
buffer, 0.1 mM) solution with excitation at 563 nm.
Scheme 2. Proposed mechanism for the response of compound L1 to GSH.
Figure 4. Job plot of L1 in EtOH/H2O (3:2, v/v, pH 7.34, HEPES buffer, 0.1 mM)
solution with a total concentration of [L1]+[GSH]=50 µM. The emission wavelength

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was 586 nm (λex= 563 nm).
Figure 5. HRMS spectrum of the L1-GSH complex.
Figure 6. Cytotoxicity of probe L1 in HUVEC cells. Cells were treated with different
concentrations of probe L1 for 24 h, and the cell viability was determined by the MTT
assay. Data are expressed as the mean ± SD.
Figure 7. Fluorescence images of HepG-2 cells. (A): Cells treated with no probe L1
or GSH; (B): cells treated with 20 µM probe L1 for an additional 5 h; (C): cells
incubated with 200 µM GSH for 2 h, followed by treatment with 20 µM probe L1 for
an additional 5 h. (1): Bright field; (2): red channel; (3): merged images of (1) and (2).
Conditions: 5% CO2/95% air atmosphere at 37 °C.
Figure 8. Fluorescence images of HUVEC cells. (A): Cells treated with no probe L1
or GSH; (B): cells treated with only 20 µM probe L1 for an additional 5 h; (C): cells
incubated with 200 µM GSH for 2 h, followed by treatment with 20 µM probe L1 for
an additional 5 h. (1): Bright field; (2): red channel; (3): merged images of (1) and (2).
Condition: f 5% CO2/95% air atmosphere at 37 °C.

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