Development of a near-infrared ratiometric fluorescent probe for glutathione using an intramolecular charge transfer signaling mechanism and its bioimaging application in living cells

Article abstract of DOI:10.1039/c8tb02864h

A novel near-infrared (NIR) ratiometric fluorescent probe HBT-GSH derived from conjugated benzothiazole was developed for the selective detection of glutathione (GSH) over cysteine (Cys) and homocysteine (Hcy). The probe was sophisticatedly designed based

Full text of DOI:10.1039/c8tb02864h

Journal of
Materials Chemistry B
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Development of a near-infrared ratiometric
fluorescent probe for glutathione using an
intramolecular charge transfer signaling
mechanism and its bioimaging application
in living cells†
Cite this:DOI: 10.1039/c8tb02864h
a
a
ab
a
Yong Zhou, Li Zhang, Xuan Zhang
*
and Zhi-Jia Zhu
A novel near-infrared (NIR) ratiometric fluorescent probe HBT-GSH derived from conjugated benzo-
thiazole was developed for the selective detection of glutathione (GSH) over cysteine (Cys) and
homocysteine (Hcy). The probe was sophisticatedly designed based on the GSH selectively induced
enhancement of intramolecular charge transfer (ICT) fluorescence. It was synthesized by masking the
active phenol group of 2,6-bis(2-vinylbenzothiazolyl)-4-fluorophenol through an acetyl group that acts
both as a trigger of the ICT fluorescence and as a recognition moiety for GSH. On its own, the probe
HBT-GSH exhibited strong blue fluorescence emission at 426 nm and weak NIR fluorescence emission
at 665 nm in aqueous solution, whereas the NIR fluorescence was significantly enhanced and the short
emission decreased upon the addition of GSH. Thus an NIR ratiometric fluorescent probe for GSH was
developed based on the GSH-selective removal of the acetyl group, therefore switching on the ICT in
HBT-GSH. The fluorescence intensity ratio (I665 nm/I426 nm) showed a linear relationship with a GSH
concentration of 0–100 mM with a detection limit of 0.35 mM. Moreover, the fluorescent probe was
successfully used for the ratiometric fluorescence bioimaging of GSH in living cells.
Received 30th October 2018,
Accepted 22nd December 2018
DOI: 10.1039/c8tb02864h
rsc.li/materials-b
The design and synthesis of small molecule-based fluorescent
probes is one of the most promising methods for intracellular GSH
Introduction
Glutathione (GSH) is one of the most important intracellular detection due to the many merits of fluorescence analysis, such
biothiols that play crucial roles in many physiological processes, as high sensitivity and selectivity, bioimaging in vivo, and non-
9
–27
such as the maintenance of redox homeostasis, intracellular invasiveness.
However, it is still a challenge to discriminate
1–3
signal transduction and gene regulation. As the most abundant GSH from cysteine (Cys) and homocysteine (Hcy) due to their
2
8–35
non-protein thiol, the normal GSH concentration ranges from similar molecular structures and reactivity.
Furthermore,
to 15 mM in various cells, while abnormal GSH levels could probes with near-infrared (NIR, 650–900 nm) fluorescence
be related to some diseases, such as cancer, Alzheimer’s, and emission have attracted increasing attention due to their char-
4
1
5
–8
lung and cardiac diseases. Thus the development of a novel acteristic advantages, such as deeper tissue penetration and
strategy for quantitatively monitoring the intracellular GSH minimum interference from the indigenous auto-fluorescence
level will be very helpful for the early diagnosis and treatment of the biosystem background.
2
3–27,36
Additionally, ratiometric
of related diseases.
fluorescent probes could provide an inherent reliability originat-
ing from their advantage of effective self-calibration by monitor-
3
7
ing two well-resolved emissions. To date, some ratiometric
3
8–46
a
fluorescent probes for GSH have been reported,
but only a
Key Laboratory of Science and Technology of Eco-Textiles, Ministry of Education,
few of them have shown fluorescence emission in the NIR
College of Chemistry, Chemical Engineering and Biotechnology, Donghua
University, Shanghai 201620, China. E-mail: xzhang@dhu.edu.cn
4
5,46
region.
Therefore, the development of an NIR ratiometric
b
State Key Laboratory of Fine Chemicals, Dalian University of Technology,
fluorescent probe for intracellular GSH-selective detection is a
more promising but still challenging task.
Recently, we have found that p-conjugated extended benzo-
thiazole derivatives exhibit NIR emission in a polar solvent,
originating from the deprotonation of the phenol group
Dalian 116024, China
†
Electronic supplementary information (ESI) available: Absorption titration
1
13
spectra, H NMR, C NMR, and MALDI-TOF-MS spectra of related compounds,
and mass spectra of the probe HBT-GSH in the absence and presence of GSH.
See DOI: 10.1039/c8tb02864h
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Scheme 1 Synthesis of HBT-GSH and the proposed signaling mechanism
for GSH detection.
Fig. 1 Fluorescence (a) and absorption (b) spectra of HBT-GSH in the
absence and presence of 10 equiv. of various amino acids and biologically
relevant species in aqueous PBS buffer (pH = 7.4). Insets are the fluores-
4
7
switching on an intramolecular charge transfer (ICT) process.
In this work, we have developed a novel NIR ratiometric
fluorescent probe HBT-GSH (Scheme 1) based on a conjugated cence (a) and solution (b) color change of the probe in the presence of
GSH, Cys, Hcy and other species tested in this work, where the fluorescence
benzothiazole framework, where the active phenol group was
masked with an acetyl group that acts both as a trigger of the
ICT fluorescence and as a recognition moiety for GSH. The probe
in (a) was obtained by exposure to a UV lamp at 365 nm. The excitation
wavelength is 350 nm for fluorescence measurement.
showed a strong short-wavelength fluorescence emission at 426 nm
that decreased and an ICT fluorescence emission at 665 nm
that increased upon addition of GSH, making NIR ratiometric
fluorescence analysis available. Furthermore, the probe HBT-GSH
could distinguish GSH well from Cys/Hcy in aqueous solution and
be successfully applied in imaging of living cells.
noticeable change in NIR fluorescence emission at 665 nm.
A fluorescence color change from blue to red was clearly observed
in the presence of GSH (Fig. 1a). This indicates that the probe
HBT-GSH exhibited a high selectivity toward GSH in aqueous
solution. Meanwhile, the absorption intensity at 516 nm showed a
large increase in the presence of GSH accompanied by a color
change from colorless to red (Fig. 1b), suggesting the availability
of a naked-eye detection of GSH. In addition, the ratios of two
emission intensities (I665 nm/I426 nm) clearly showed significant
Results and discussion
Design and synthesis
p-Conjugated extended benzothiazole derivatives exhibit ICT enhancement in the presence of GSH over Cys/Hcy and other
fluorescence emission in the NIR range with a high quantum species, and the existence of these species did not cause sub-
yield and a large Stokes shift, making them promising for the stantial interference to GSH detection (Fig. S2, ESI†).
development of NIR fluorescent probes by masking their active
The time-dependent fluorescence response of the probe
phenol group. Benzothiazole derivative 1 was firstly synthesized HBT-GSH in the presence of 10 equiv. of GSH and Cys/Hcy
47
4
7
by following a similar procedure to that established previously,
was also performed, respectively. As shown in Fig. 2a, the NIR
and then the probe HBT-GSH was easily afforded by treating 1 with fluorescence intensity at 665 nm increased continuously and
acetyl chloride in acetone, as shown in Scheme 1. The chemical reached equilibrium at about 120 min for GSH, but slower
1
13
structure of the probe HBT-GSH was confirmed by H NMR,
C
reaction kinetics were observed for Cys/Hcy, demonstrating the
NMR, and MALDI-TOF-MS, elemental analysis, and the synthesis high selectivity of the probe HBT-GSH toward GSH. The probe
details are presented in the Experimental section.
HBT-GSH displayed no noticeable changes in NIR fluorescence
in the absence of GSH, suggesting that the probe is stable
enough under experimental conditions.
Selectivity toward GSH
Compound 1 showed a short-wavelength emission at 426 nm in
CHCl but an NIR emission at 688 nm in DMSO (Fig. S1, ESI†),
3
Ratiometric fluorescence determination of GSH
due to the switching on of an ICT process in the latter polar The change in fluorescence character of the probe HBT-GSH
4
7
solvent. This allowed conjugated benzothiazole derivative 1 to means it is possible to develop it as a ratiometric probe for GSH.
be a suitable platform to construct the NIR fluorescent probe Firstly, the influence of pH on the fluorescence of the probe in
HBT-GSH. To examine the selectivity of probe HBT-GSH toward the absence and presence of GSH were investigated over the
GSH, the fluorescence and absorption spectra were measured range of pH 2–10. Without GSH, the fluorescence intensities
in the presence of 10 equiv. of various species such as common ratio displayed negligible changes at pH o 8 but increased
amino acids (GSH, Cys, Hcy, Asp, Asn, Ser, Pro, Ala, Gly, Val,
Leu, lle, Thr, Arg, Glu, Gln, Tyr, His, Met, Phe, Trp, Lys, Tau),
+
+
2+
2+
2À
À
2À
À
cations (Na , K , Ca , Mg ), anions (SO
4
, NO
3
, CO
3
, Cl ),
Na S and glucose, respectively (Fig. 1). As shown in Fig. 1a, the
2
probe HBT-GSH only showed a short wavelength fluorescence
at 426 nm, but the addition of GSH strongly enhanced the NIR
fluorescence at 665 nm accompanied by a decrease in blue
emission at 426 nm. The fluorescence quantum yields were
measured to be 0.138 and 0.245 in the absence and presence of
GSH, respectively. While Cys and Hcy induced a moderate
Fig. 2 Effect of time and pH on fluorescence intensity of HBT-GSH in the
increase in NIR fluorescence, other species did not make a absence and presence of various biothiols.
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obviously after that (Fig. 2b), revealing that partial hydrolysis of
the probe could occur under the condition pH 4 8. In the
presence of GSH, while the I665 nm/I426 nm ratio showed almost no
change at pH o 6, a noticeable increase appeared at pH 4 6 and
the ratio was significantly enhanced under pH 4 8 (Fig. 2b),
suggesting that the GSH-induced removal of the acetyl group in
the probe HBT-GSH was facilitated under basic conditions. Then
the ratiometric fluorescence titrations were performed at the
physiological pH 7.4 in the phosphate buffer solution (10 mM,
5
0% DMSO). As shown in Fig. 3a, the fluorescence emission
Fig. 4 LUMO and HOMO orbitals of HBT-GSH and anion of 1 in the
ground state.
gradually decreased at 426 nm and increased at 665 nm with
increasing amounts of GSH up to 180 mM. The I665 nm/I426 nm
intensity ratios were found to follow a good linear relationship
2
(
R = 0.9968) with a GSH concentration ranging from 0 to 100 mM and the terminal COOH groups in these biothiols. This sug-
Fig. 3b). The detection limit was estimated to be 0.35 mM gested that such possible hydrogen bonding interactions might
(
according to S/N = 3. Hence, the probe HBT-GSH could detect therefore promote the cleavage reaction rate of GSH toward the
GSH quantitatively by a ratiometric fluorescence method with acetyl moiety in probe HBT-GSH. A DFT calculation based on
excellent sensitivity. In addition, the absorption titration showed the Gaussian 09 program was further performed to gain better
that the peak at 516 nm gradually increased upon the addition of insights into the NIR fluorescence and signaling mechanism.
GSH and the solution color became red (Fig. S3, ESI†).
Fig. 4 presents the optimized ground state structures of both
the probe HBT-GSH and the anion of 1. It revealed that the p
electrons of the probe HBT-GSH were well delocalized over the
whole molecular skeleton on both the lowest unoccupied
molecular orbital (LUMO) and the highest occupied molecular
orbital (HOMO). However, the p electrons of the 1 anion were
mainly localized on the phenolate group on the HOMO, and
delocalized over the whole molecular skeleton on the LUMO,
suggesting the occurrence of an ICT process from the phenolate
donor to the benzothiazole acceptor. The phenol moiety has
been known to be a latent electron donor but acts as a strong
electron donor when it is transformed into a phenolate, and
Sensing mechanism
To confirm that probe HBT-GSH has been transformed into 1 in
the presence of GSH, as shown in Scheme 1, MALDI-TOF-MS
mass analysis of a mixed solution of the probe with 10 equiv. of
GSH was conducted. A prominent peak at m/z = 453.1 corres-
+
ponding to [1 + Na] was observed, providing solid evidence for
the fact that GSH induced the cleavage of the acetyl moiety in
probe HBT-GSH (Fig. S4, ESI†). The high selectivity toward GSH
over Cys/Hcy has usually been considered from the point of
view of their structural differences, where GSH has a longer
flexible backbone that could provide the additional chance to
form some intermolecular interactions with the probe, such as
4
7,48
could therefore switch on an ICT emission.
Based on the
TD-DFT calculation on the excited state, the fluorescence
emission wavelengths were predicted to be 420 nm and
1
8,23,26
hydrogen bonds and electrostatic interactions.
Accord-
670 nm for the probe HBT-GSH and 1 anion, respectively.
ingly, we speculated that the benzothiazole units in the probe
HBT-GSH could be involved in hydrogen bond interactions with
carboxylic acid groups in biothiols. Only GSH has a much better
match distance between HS and the terminal COOH groups
The good reproduction of the experimental results implied the
reliability of the present theoretical calculation level. Thus the
signaling mechanism could be rationalized by GSH-induced
removal of the acetyl group and switching-on of ICT, as shown
in Scheme 1.
(the HS–COOH separation for Cys is 3 bonds, that for Hcy is
4
bonds, and that for GSH is 6/8 bonds) and two carboxylic acid
groups that were favourable to the formation of hydrogen
bonds. The reaction kinetics showed the order of GSH 4 Hcy 4
Cys (Fig. 2a) that matched well with the distance between HS
Fluorescence imaging in living cells
To evaluate the potential practical applications of probe HBT-GSH,
the fluorescence imaging of GSH in living HeLa cells was also
performed. Firstly, the cytotoxicity of probe HBT-GSH was
evaluated by a CCK-8 assay. The results showed that high cell
viability remained even after treatment with 40 mM of probe
at 37 1C for 24 h (Fig. S5, ESI†), which suggested the low
cytotoxicity of the probe HBT-GSH and its potential in the
imaging of live cells. Thus 20 mM of the probe HBT-GSH was
used for bioimaging of GSH in living HeLa cells with a Leica
TCS SP8 microscope, where dual blue and red channels were
monitored upon excitation at 405 nm and 552 nm respectively
Fig. 3 Change in fluorescence spectra of HBT-GSH (10 mM) with the
addition of various amounts of GSH (0–180 mM) (a). The corresponding
(Fig. 5). When HeLa cells were incubated with the probe HBT-GSH
(20 mM) for 60 min at 37 1C, both blue and red fluorescence
linear relationship between the fluorescence intensity ratio (I665 nm/I426 nm
)
and the concentration of GSH (0–100 mM) (b).
emissions were observed in the two channels (Fig. 5b and c),
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through an acetyl group that acts both as a trigger of ICT
fluorescence and as a recognition moiety for GSH. Based on mass
analysis and DFT calculations, the signaling mechanism of the
GSH-induced removal of the acetyl group and the switching on of
the ICT fluorescence was proposed. The fluorescence intensity ratio
(I665 nm/I426 nm) showed a linear relationship with a GSH concen-
tration of 0–100 mM with a detection limit of 0.35 mM. Moreover,
the probe had low cytotoxicity and was successfully used for the
ratiometric fluorescence bioimaging of GSH in living cells.
Experimental
Materials and methods
All the chemicals are analytical grade and purchased from
Sinopharm Chemical Reagents Corp. (Shanghai, China). Phosphate
buffered saline (PBS, pH = 7.4) was prepared from K
and KH PO (0.1 M). A stock solution of the probe HBT-GSH was
prepared in DMSO and all the other species solutions were
2 4
HPO (0.1 M)
2
4
1
13
prepared in deionized water. H NMR and C NMR spectra
were recorded on a Bruker AVANCE III 400 MHz spectrometer.
Mass spectra were obtained on an AB Sciex MALDI-TOF/TOFt
MS. Fluorescence spectra were measured on an Edinburgh FS5
spectrofluorometer with Ex/Em slit widths of 5 nm. Absorption
spectra were obtained on a SHIMADZU UV-1800 spectrophoto-
meter. Confocal fluorescence imaging experiments in living
HeLa cells were carried out with a Leica TCS SP8 microscope.
Theoretical calculations were performed based on the
Fig. 5 Confocal fluorescence images of living HeLa cells incubated with
the probe HBT-GSH (20 mM) for 60 min at 37 1C: cells without treatment
49
Gaussian 09 package. The ground state and the first singlet
excited state geometries of the compounds were optimized in
the gas phase using density functional theory (DFT) and time-
(
a–d), cells with pre-treatment by NEM (1 mM) (e–h), and cells with pre-
treatment by NEM (1 mM) and the further addition of 200 mM GSH (i–l).
a, e and i) are the bright-field images; (b, f and g) and (c, g and k) are the
(
fluorescence images at blue and red channels, respectively; (d, h and l) are dependent density functional theory (TDDFT) at the B3LYP/
the overlap of the fluorescence and bright-field images.
6-31+G(d) level, respectively. The fluorescence emission properties
were calculated using TDDFT based on the optimized first singlet
excited state geometries, respectively.
where the NIR emission resulted from the intracellular GSH-induced
removal of the acetyl group in the probe. In the control experiment,
HeLa cells were pretreated with N-ethylmaleimide (NEM, a known
Synthesis
Synthesis of 2,6-diformyl-4-fluorophenol. The available pro-
scavenger for GSH, 1 mM for 60 min), and thereafter incubated cedures were followed in a similar way to previous work.47,50
with probe HBT-GSH (20 mM) for another 60 min. As shown in Briefly, under an N atmosphere, 4-fluorophenol (5 mmol) and
2
Fig. 5f and g, while the blue fluorescence remained, there was no hexamethylenetetramine (15 mmol) were dissolved in TFA (8 mL)
fluorescence in the red channel. Then the NEM-pretreated HeLa and refluxed at 110 1C for 90 h. The mixture was then cooled
cells were further sequentially incubated with GSH (200 mM) down to room temperature and poured into a 0.8 M HCl solution
for 30 min, and the probe HBT-GSH (20 mM) for 60 min at 37 1C. (60 mL), and the 2,6-diformyl-4-fluorophenol was obtained by
1
A bright fluorescence in the red channel was again observed filtration. Yield: 48%. H NMR (400 MHz, CDCl ), d (ppm): 11.38
3
1
3
inside the cells (Fig. 5k) accompanied by a weak fluorescence in (s, 1H), 10.22 (s, 2H), 7.69 (d, J = 4 Hz, 2H). C NMR (100 MHz,
the blue channel (Fig. 5j). The above results suggested that probe DMSO-d ), d (ppm): 190.87, 190.85, 158.46, 158.45, 156.17,
6
HBT-GSH can be used as a promising fluorescent probe for GSH 153.78, 125.06, 125.01, 122.43, 122.20.
imaging in living cells.
Synthesis of 1. Under an N2 atmosphere, 2,6-diformyl-4-
fluorophenol (1 mmol) and 2-methylbenzothiazole (4 mmol)
were refluxed in acetic anhydride (2 mL) for 48 h. After cooling
to room temperature, the solid was obtained by filtration and
Conclusions
In summary, a novel NIR ratiometric fluorescent probe HBT-GSH thoroughly washed with water. The obtained solid was further
was developed for the selective detection of GSH over Cys and Hcy dissolved in pyridine (8 mL) and refluxed at 115 1C for 1 h, then
in aqueous solution. The probe was synthesized by masking the water (8 mL) was added and it was stirred at 100 1C for another
active phenol group of 2,6-bis(2-vinylbenzothiazolyl)-4-fluorophenol 4 h. After cooling down to room temperature, the crude product
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was collected by filtration and purified by washing with CH
to give 1 as a yellow solid. Yield: 33%. H NMR (400 MHz, the absorbance of the control group (i.e., HeLa cells without
2
Cl
2
calculated with the equation: VR = A/A
0
 100%, where A
0
is
1
DMSO-d ), d (ppm): 9.98 (s, 1H), 8.13 (d, J = 8 Hz, 2H), 8.00 the probe) and A is the absorbance of the experimental group
6
(
t, J = 8 Hz, 4H), 7.77 (d, J = 8 Hz, 2H), 7.71 (d, J = 16 Hz, 2H), (i.e., HeLa cells treated with the probe). The cell survival rate
13
7.56 (t, J = 8 Hz, 2H), 7.48 (t, J = 8 Hz, 2H); C NMR (100 MHz, from the control group was considered to be 100%.
DMSO-d ), d (ppm): 166.47, 153.48, 150.11, 134.11, 131.20, 126.57,
6
1
25.51, 123.21, 122.61, 122.21, 114.07, 113.83. MALDI-TOF-MS: m/z
+
Conflicts of interest
2 2
calcd for C24H15FN OS , 430.53; found 431.01 [M + H] .
Synthesis of HBT-GSH. Compound 1 (0.1 mmol) was dis-
solved in acetone (100 mL) and acetyl chloride (0.15 mmol
dissolved in 5 mL of acetone) was slowly added dropwise under
There are no conflicts to declare.
stirring at 0 1C in the presence of Et N (0.2 mmol). After stirring Acknowledgements
3
at this temperature for 2 h, the mixture was warmed to room
temperature and stirred for another 14 h. The reaction mixture
was concentrated in a vacuum and the residue was dissolved in
This work was financially supported by the State Key Laboratory
of Fine Chemicals, Dalian University of Technology (KF 1715)
and the Shanghai Municipal Natural Science Foundation
CH
MgSO
2
Cl
2
and then washed with water and brine, dried with
; then the product was obtained by evaporation to
(16ZR1401700). We would like to thank Dr Xiao-Juan Huang
4
1
for her kind assistance with cell imaging experiments.
dryness under reduced pressure. Yield: 79%. H NMR (400 MHz,
CDCl ), d (ppm): 8.04 (d, J = 8 Hz, 2H), 7.89 (d, J = 8 Hz, 2H),
3
1
3
7
.52–7.37 (m, 5H), 2.54 (s, 3H). C NMR (100 MHz, CDCl ), d
3
Notes and references
(ppm): 169.00, 165.68, 161.79, 159.35, 153.86, 143.08, 134.52,
1
1
C
31.41, 131.33, 129.31, 129.29, 126.61, 126.02, 125.88, 123.40,
1 H. Chen, L. Zhou, C.-Y. Lin, M. C. Beattie, J. Liu and
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21.60, 114.07, 113.83, 20.62. MALDI-TOF-MS: m/z calcd for
+
26
H17FN
2
O
2
S
2
, 472.56; found 473.04 [M + H] . Elemental
analysis: calcd for C26
N 5.71%; found: C 63.90%, H 3.57%, N 5.59%.
H
2 2 2 2
17FN O S + H O, C 63.65%, H 3.90%,
3
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Cell imaging
HeLa cells were cultured in Dulbecco’s Modified Eagle Medium
(
DMEM) supplemented with 10% fetal bovine serum at 37 1C in
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a 95% humidity atmosphere under 5% CO environment. Then
the cells were seeded in confocal microscope culture dishes
with a density of 2 Â 10 cells per well. The cells were then
2
5
incubated with probe HBT-GSH (20 mM) for 60 min at 37 1C,
and washed with PBS buffer (10 mM) three times to remove free
probe. In the control experiments, the cells were pretreated
with NEM (1 mM) for 60 min at 37 1C, followed by washing with
PBS three times, and incubated with probe HBT-GSH (20 mM)
for 60 min at 37 1C. In another control experiment, the cells
were pretreated with NEM (1 mM) for 60 min at 37 1C, followed
by washing with PBS three times, then incubated with GSH
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5
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