Lysosome targeting fluorescence probe for imaging intracellular thiols

Article abstract of DOI:10.1039/c5ob00889a

A BODIPY-based fluorescence turn-on probe, exhibiting high selectivity and sensitivity towards intracellular thiols with excellent lysosomal localization is reported. The probe displayed fast response towards biothiols in aqueous solution. Localization of the probe in lysosome was demonstrated by intracellular colocalization studies with the aid of LysoSensor Green.

Full text of DOI:10.1039/c5ob00889a

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Lysosome targeting fluorescence probe for imaging intracellular
thiols
Dnyaneshwar Kand,a† Tanmoy Saha,a† Mayurika Lahiri,b and Pinaki Talukdara*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
lysosomal thiols it is important to develop thiol selective fluoresent
probes capable of targeting lysosomes.
A BODIPY-based fluorescence turn-on probe, exhibiting high selectivity
and sensitivity towards intracellular thiols with excellent lysosomal
localization is reported. Probe displayed fast response towards biothiols in
aqueous solution. Localization of the probe in lysosome was demonstrated
Herein, the design, synthesis and biothiol sensing properties of
lysosome targeting fluorescence turnꢀon probe 1 are reported (Fig.
1). To obtain a photostable water soluble fluorescent thiol probe with
excitation and emission wavelengths in the visible region boronꢀ
dipyrromethene (BODIPY) was selected as the fluorophore.33 The
necessary molecular decorations for thiol recognition and lysosome
targeting were incorporated via 2,4ꢀdinitrobenzenesulfonyl (DNs)
group and morpholine ring,34 respectively. As BODIPYꢀbased
chemosensors operate by perturbing the reduction potential of the
mesoꢀsubstituent,35 the DNs group was attached to aryl group at
mesoꢀposition.36 Moreover, a phenyl ring at 5ꢀposition provided
extended conjugation resulting in excitation at longer wavelengths
while maintaining high quantum yield.37
by intracellular colocalization studies with the aid of LysoSensor Green
.
Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are three
important low molecular weight thiol biomolecules (biothiols) which
perform various physiological functions essential for survival.1
Alteration in intracellular and plasma levels of biothiols are
associated with various diseases and disorders2 e.g. abnormal levels
of Cys results in hair depigmentation, edema, liver damage, slow
growth in children etc.3 Hcy is known as a risk factor for
cardiovascular and Alzheimer’s diseases.4 GSH, a tripeptide is most
abundant intracellular nonprotein thiol which plays a critical role in
controlling oxidative stress in order to maintain the redox
homeostasis,5 crucial for cell growth and function.6 Although,
numerous fluorescent probes have been developed for selective
detection of biothiols7ꢀ9 and cell imaging applications,7, 10ꢀ13 probes
specific for locating in particular subcellular organelles are rare.14ꢀ26
Fluorescent probes for detection of biothiols in mitochondria are
reported.27, 28
Lysosome is an important cell organelle that contains
approximately 50 different degradative enzymes which are active at
the acidic pH (pH = 4ꢀ6) of the compartment.29 The lysosomal
membrane constitutes a physiological barrier between the lysosome
matrix and the surrounding cytoplasm. The membrane’s
impermeability ensures the retention of both the lysosomal enzymes
and their substrates within the lysosomes.30 It is believed that GSH
may be involved in stabilizing lysosome membranes.31 Thiols
facilitate intralysosomal proteolysis by reducing disulphide bonds.32
For example, Cys is an effective stimulator of albumin degradation
in liver lysosomes.31 For better understanding of the role of
Synthesis of probe 1 was carried out from salicylaldehyde in five
steps (Scheme 1). Dipyrromethane
3 was synthesized from
salicylaldehyde 2 and pyrrole in presence of catalytic CF3COOH in
64% yield. Compound 3 was dibrominated with two equivalents of
Nꢀbromosuccinimide in tetrahydrofuran at ꢀ78 °C and oxidized with
DDQ in dichloromethane followed by addition of BF3·Et2O and
Et3N afforded the dibromoꢀBODIPY 4 in 60% yield. Reaction of 4
with morpholine resulted in BODIPY derivative 5 in 80% yield
which on subsequent Suzuki coupling reaction with phenyl boronic
acid provided compound 6 in 79% yield. Compound 6 upon
treatment with 2,4ꢀdinitrobenzenesulfonyl chloride provided probe 1
in 91% yield. Apart from the spectroscopic characterization of all
compounds, single crystal Xꢀray diffraction structures for compound
5 and probe 1 were also recorded. Crystal structure analysis of probe
O2N
NO2
Perturbation
of r eduction
potential
O
S
O
Thiol sensing group
O
Extended
conjugat ion
Fluorophore
a.Department of Chemistry, Indian Institute of Science Education and Research
Pune, Pune 411008, India. Email: ptalukdar@iiserpune.ac.in
b.Department of Biology, Indian Institute of Science Education and Research Pune,
Pune 411008, India.
N
N
B
N
F
F
Lysosomal directing group
† D.K. and T.S. contributed equally to this work.
O
Electronic Supplementary Information (ESI) available: Experimental procedures,
crystal structure parameters, analytical and spectroscopic data. See
DOI: 10.1039/x0xx00000x
1
Fig. 1 Structure of the lysosomal targeting thiol probe 1.
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i) NBS, THF, 78 oC, 2 h
ii) DDQ, rt, 2 h
OH
Pyrrole, TFA (cat.),
CHO
Morpholine, Et3N,
CH3CN, rt, 6 h
iii) BF3 Et2O, Et3N,
OH
CH2Cl2, rt, 10 h
CH2Cl2, rt, 3 h
OH
OH
64%
60%
80%
N
N
B
N
N
Br
N
F
F
B
NH HN
Br
Br
F
F
O
2
3
4
5
Phenyl boronic acid,
Pd(PPh3)4, 1M Na2CO3
OH
/
2,4-Dinitrobenzenesulfonyl chloride,
Et3N, CH2Cl2, rt, 2 h
toluene (1:1), reflux, 12 h
1
N
N
79%
91%
B
N
O
F
F
6
Crystal structure of 5
Crystal structure of 1
Scheme 1 Synthesis of probe 1.a
1 was useful to confirm the relative spatial arrangement between
BODIPY and DNs moieties (Fig. S2).
A
B
24
18
12
6
C
40 ꢀM
20
20
M
ꢀ
To validate the fluorescence turnꢀon nature of sensing, the
photophysical properties of compounds 1 and 6 were investigated in
aqueous HEPES buffer (10 mM, pH 7.4, 1 mM CTAB) solution. The
absorption spectrum of probe 1 exhibited λmax = 510 nm with a molar
extinction coefficient ε = 18600 M−1 cm−1 (see Fig. S3). The probe
displayed very weak fluorescence (λex = 510 nm) and quantum yield,
Φ = 0.0026 (standard: Rhodamine G, Φ = 0.76 in water).38
Compound 6 exhibited an absorption band centred at λmax = 515 nm
with a molar extinction coefficient ε = 23966 M−1 cm−1. The
fluorescence spectrum acquired for 6 indicate a strong fluorescence
emission centred at λem = 584 nm (λex = 510 nm) and Φ = 0.17
(standard: Rhodamine G, Φ = 0.76 in water). This photophysical
data satisfy the criteria of probe 1 to act as an efficient fluorescent
turnꢀon probe. Reactivity of the probe 1 (10 ꢁM) towards nꢀBuNH2,
Cys, GSH and Hcy (100 ꢁM) were determined by fluorescence
emission kinetics in HEPES buffer (10 mM, 1 mM CTAB, pH =
7.4). In each experiment, emission intensity at λ = 585 nm (λex = 510
nm) was recorded with time (Fig. 2A). Addition of nꢀBuNH2 to
probe 1 did not indicate formation of fluorescent species during the
reaction. A pronounced fluorescent intensity increase up to ~ 64ꢀfold
was obtained within 1 min after the addition of Cys (t1/2 = 6.4 s and
pseudo first order rate constant, k = 0.108 s−1), Hcy (t1/2 = 14.49 s
and k = 0.0478 s−1) and GSH (t1/2 = 7.47 s and k = 0.0928 s−1).
Quantitative turnꢀon response of probe 1 towards Cys was
examined by fluorometric titrations. Sharp enhancements in
fluorescence intensity (at λem = 585 nm) were observed (Fig. 2B)
when titrations were carried out by addition of increasing
10 ꢀM
8
M
ꢀ
6 ꢀM
4 ꢀM
2 ꢀM
0 ꢀM
15
10
D
1
1+BuNH 2
1+Cys
1+Hcy
5
0
1+GSH
0
520
0
100
t (s)
200
620
720
λ
(nm)
Fig. 2 (A) Fluorescence kinetics of probe 1 (10 ꢁM in 10 mM HEPES buffer, 1 mM
CTAB, pH = 7.4) with various analytes (100 ꢀM). All data were recorded at λem = 585
nm (λex = 510 nm). (B) Fluorescence spectra of 1 (10 ꢁM) in presence of Cys (0 to 40
ꢀM) with λex = 510 nm. Changes in visible color (C) and fluorescence (D) for 1 (10 ꢁM)
upon addition of Cys (100 ꢀM).
concentrations of Cys (0, 2, 4, 6, 8, 10, 20 and 40 ꢁM) to the probe 1
(10 ꢀM) in HEPES buffer (10 mM, 1 mM CTAB, pH = 7.4). An
equilibration time of 1 min was given after each addition of Cys to
ensure the completion of reaction. When fluorescence intensities at
585 nm were plotted against concentrations of Cys, good linear
correlation (Regression factor, R = 0.9686) was observed up to one
equivalent of the Cys added (Fig. S6A). A detection limit of 8.2 nM
was calculated for the probe 1, based on signal to noise ratio, S/N =
3. Sensing of Cys by 1 was also associated with the change in color
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A
by fluorometric titrations. Significant enhancements in fluorescence
intensity (at λem = 585 nm) were observed when titrations were
carried out by adding increasing concentrations of Cys (0, 2, 4, 6, 8,
10, 20, 40 and 100 ꢁM) in the probe 1 (10 ꢀM) at this pH (Fig. 3C).
An equilibration time of 8 min was given after each addition of Cys
to ensure the completion of reaction. When fluorescence intensities
at 585 nm were plotted against concentrations of Cys, a linear
correlation (Regression factor, R2 = 0.9787) was observed up to one
equivalent of the Cys added (Fig. S6B). A detection limit of 95.7 nM
was calculated for the probe 1, based on signal to noise ratio, S/N =
3.
25
20
15
10
5
0
pH 4
pH 5
pH 6
= 1 + Cys
pH 7
pH 8
= 1
= 6
B
C
To prove the formation of compound 6 during the thiol sensing,
HPLC studies were carried out under a gradient method using
CH3CN and H2O eluent (for details information see SI). HPLC
20
20
100
40
20
M
M
M
M
16
12
8
10
chromatograph of pure probe 1 provided the retention time, tR
=
15
10
5
8
6
4
2
0
M
M
17.56 min while, tR = 16.34 min was obtained for compound 6 (Fig.
4). Compound 1 upon treatment with Cys (0.5 and 1.0 equiv) clearly
showed consumption of the probe and formation of 6 (Fig. 4).
MALDIꢀTOF analysis of the isolated compound corresponding to tR
= 16.34 min showed m/z = 445.13 confirming the formation of
compound 6 (Fig. S9).
M
M
M
1
4
In next stage, the selectivity of probe 1 towards biological thiols
1+Cys
0
0
0
200
t (s)
600
400
520
720
620
(nm)
Fig. 3 Fluorescence intensities at 585 nm for probe 1 (blue), probe 1+Cys (red) and
compound 6 (green) at pH 4, 5, 6, 7 and 8, respectively in phosphate buffer (10 mM, 1
mM CTAB) (A). Fluorescence kinetics measurements of probe 1 (10 ꢁM) with and
without addition of Cys (100 ꢁM) recorded at 585 nm (λex = 510 nm) nm in phosphate
buffer (10 mM, 1 mM CTAB, pH = 5) (B). Fluorescence spectra of 1 (10 ꢁM) in
presence of Cys (0 to 100 ꢀM) with λex = 510 nm in phosphate buffer (10 mM, 1 mM
CTAB, pH = 5) (C).
from orchid to hot pink under the ambient light (Fig. 2C) and
excitation under hand held UVꢀlamp (λex = 365 nm) resulted in the
appearance of orange fluorescence (Fig. 2D).
We have already stated that the lysosomal pH ranges between 4ꢀ
6.39 As a lysosome targeting probe for biothiols, the molecule should
also respond to biothiols in the pH range of lysosome. To verify this,
fluorescence intensities (at 585 nm) of probe 1 (10 ꢁM) and
compound 6 (10 ꢁM) were individually recorded in phosphate buffer
(10 mM, 1 mM CTAB) at pH values 4, 5, 6, 7 and 8 (Fig. 3A).
Simultaneously the sensing activity of 1 (10 ꢀM) was monitored at
pH = 4ꢀ8 by treating with Cys (100 ꢀM) in phosphate buffer (10
mM, 1 mM CTAB) for 10 min. From this study, response of the
probe towards Cys at pH lower than the physiological pH was
observed, and fluorescence enhancements in the pH range of 4ꢀ8
were also comparable. Encouraged by this results, reactivity of the
probe 1 (10 ꢀM) towards Cys (100 ꢀM) at pH 5 was monitored in
phosphate buffer (10 mM, 1 mM CTAB, pH = 5). For free probe, no
fluorescence intensity enhancement λ = 585 nm (λex = 510 nm) was
observed indicating the stability of the compound under acidic pH
(Fig. 3B). However, a sharp enhancement of fluorescent intensity up
to ~ 95ꢀfold was obtained within 8 min after the addition of Cys.
From this reaction kinetics, k = 0.00553 s−1 and t1/2 = 125.3 s were
calculated. The slower rate in comparison with pH 7.4 can be
rationalized with the lower nucleophillicity of the thiol group of Cys.
Moreover, quantitative turnꢀon response of probe 1 towards Cys in
phosphate buffer (10 mM, 1 mM CTAB, pH = 5) was also examined
Fig. 4 HPLC chromatographs of probe 1 (10 ꢁM) upon reaction with Cys (0.5 and 1.0
equivalent) recorded in a gradient solvent system of CH3CN and H2O.
Fig. 5 Relative fluorescence intensity enhancements (I/I0) at 585 nm for probe 1 (10 ꢁM)
towards Ala, Arg, His, NaCl, Na2SO4, NaSCN, NaNO3, BuNH2, Ser, GSH, Cys and Hcy
(100 ꢁM each) in HEPES buffer. Front row: changes in intensities in the presence of
nonꢀthiol based analytes (100 ꢁM); First nine bars of back row: changes in intensities
upon addition of Cys (100 ꢁM) to the resulting solutions of nonꢀthiol addition. Last three
bars of back row: changes in intensities upon addition of GSH, Cys and Hcy (100 ꢁM
each) to probe 1 (10 ꢁM).
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was examined under physiological pH. In each case, probe 1 (10
ꢀM) was treated separately with 100 ꢀM of each analyte (either of
Ala, Arg, His, NaCl, Na2SO4, NaSCN, NaNO3, BuNH2, Ser, GSH,
Cys and Hcy) in HEPES buffer (10 mM, 1 mM CTAB, pH = 7.4)
and fluorescence spectra (λex = 510 nm) were recorded after 5
minutes at room temperature. No significant fluorescence
enhancement was observed for analytes nonꢀthiol analytes (Fig. 5).
Treatment of probe 1 with GSH, Cys and Hcy under identical
conditions provided strong fluorescence enhancements in the range
of 54 − 63 fold. Comparable fluorescence enhancements were
observed upon addition of Cys to the solutions preꢀtreated with nonꢀ
thiol analytes. This study demonstrates the effectiveness of
compound 1 as a selective probe for biothiols under competitive
environments.
Fig. 6 DIC image (A), overlay of fluorescence and DIC images (B), of HeLa cells
incubated with of probe 1 (5.0 ꢁM) for 10 min. Overlay image of fluorescence and DIC
of cells preꢀincubated with Nꢀphenylmleimide (5 mM) for 30 min followed by
incubation with probe 1 (5 ꢁM) for 10 min(C). Fluorescence image of cells preꢀ
incubated with probe 1 (5.0 ꢁM) for 10 min followed by incubation with LysoSensor
Green (1.0 ꢁM) for 10 min, green channel (D), red channel (E) and overlay image of
images D and E. (F).
Based on the aforestated outcome, localization of probe 1 in the
lysosome and its ability to sense biological thiols in living cells were
examined. First, the cell permeability and intracellular thiol sensing
ability of probe 1 was evaluated by liveꢀcell imaging of Human
cervical cancer cell line (HeLa). Strong fluorescence was observed
when HeLa cells were incubated with probe 1 (10 ꢀM in 1:100
o
DMSOꢀDNEM v/v, pH = 7.4) at 37 C for 10 min (Fig. 6B and E).
These cells then incubated with commercially available lysosome
specific dyeꢀLysoSensor Green (1.0 ꢁM) showed green fluorescence
(Fig. 6D). Colocalization image of green and red channels shows the
localization of probe 1 in lysosomes (Fig. 6F). In control experiment,
cells were preꢀtreated with an excess (5 mM) of the thiolꢀreactive Nꢀ
phenylmaleimide and then incubated with the probe 1. The confocal
microscopic studies did not show fluorescence signal (Fig. 6C). This
confirms the specificity of probe 1 for thiols over other analytes in
living cells.
Next, the colocalization experiments were performed by coꢀ
staining HeLa cells with LysoSensor Green and probe 1 to determine
the location of fluorescence emission. When Hela cells were
incubated with probe 1 (5 ꢀM in 1:100 DMSOꢀDNEM v/v, pH =
7.4) at 37 oC for 10 min, strong red fluorescence was observed (Fig.
7A). These cells were then incubated with LysoSensor Green (1 ꢀM
in 1:100 DMSOꢀ DNEM v/v, pH = 7.4) at 37 oC for 10 min showed
green fluorescence (Fig. 7B). As seen in Fig. 7C the fluorescence
image of probe 1 was mainly overlapped with that of LysoSensor
Green indicates ability of probe 1 to target lysosomes. The intensity
profiles of the linear regions of interest (ROI) across HeLa cells
stained with probe 1 and LysoSensor Green vary in close synchrony
(Fig. 7D). Pearson’s coefficient and overlap coefficient are 0.963
and 0.984, respectively; evaluated using the conventional dye
overlay method. Overlap coefficients k1 and k2 found to be 0.9 and
1.1 respectively. Colocalization coefficients (Manders' Coefficients)
M1 = 0.77 (fraction of LysoSensor Green overlapping probe) and M2
Fig. 7 Colocalization experiments using probe 1 to lysosomes in HeLa cells. HeLa cells
were stained with (A) probe 1 (5.0 ꢁM) for 5 min at 37 °C and (B) LysoSensor Green
(1.0 ꢁM) (C) Overlay of (A) and (B). (D) Intensity profile of regions of interest (ROI)
across HeLa cells. (E) Intensity correlation plot of stain probe 1 and LysoSensor Green.
= 0.818 (fraction of probe overlapping LysoSensor Green) also ICA plots of (F) stain LysoSensor Green and (G) probe 1.
confirms an excellent overlap. An intensity correlation analysis
(ICA) is employed to assess the intensity distribution of the two coꢀ
existing dyes. The pixel intensity of the LysoSensor Green was
plotted against that of the probe 1 (Fig. 7E). The dependent staining
results in a highly correlated plot, and the ICA plots for the two
stains generate an unsymmetrical hourglassꢀshaped scatter plot that
are markedly skewed toward positive values (Fig. 7F, G). Li’s
intensity correlation quotient (ICQ) for the two dyes is 0.459, very
close to 0.5, suggesting that the staining intensities are dependent on
each other.
Various concentrations of probe 1 (5, 10, 20 and 50 ꢁM) were used
to determine toxicity level of probe towards HeLa cell. The result
revealed that cells were not affected by incubation with probe 1 (up
to 10 ꢁM) for 2 h as about 95% cell viability was determined at 10
ꢀM concentration of probe 1 (Fig. S8).
Conclusions
In summary, we have developed lysosome targeting BODIPYꢀbased
fluorescence turnꢀon probe for rapid, selective and sensitive
detection of biothiols. At pH 7.4, the probe displayed very fast
The cytotoxicity of probe 1 was determined by MTT assay.
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reaction with biothiols such as Cys, Hcy and GSH. Reaction of the
probe with Cys provided pseudo first order rate constant, k = 0.108
s−1 and t1/2 = 6.4 s. The reaction also provided ~ 64ꢀfold fluorescence
enhancement within 1 min of reaction. The probe was also reactive
towards the biothiol at pH 5 however, with lower value of k =
0.00553 s−1 and longer t1/2 = 125.3 s. At this pH, a sharp
enhancement of fluorescent intensity up to ~ 95ꢀfold was obtained
after 8 min of Cys addition. Liveꢀcell imaging studies of HeLa cells
confirmed the cell permeability, lysosome specificity and
intracellular biothiol detection ability of the probe. MTT assay
disclosed about 95% cell viability at 10 ꢁM concentration of the
probe.
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We acknowledge IISER Pune and DSTꢀSERB (Grant No.
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