BODIPY based realtime, reversible and targeted fluorescent probes for biothiol imaging in living cells

Article abstract of DOI:10.1039/d0cc06313d

Real-time live cell imaging and quantification of biothiol dynamics are important for understanding pathophysiological processes. However, the design and synthesis of rational probes that have reversible and real-time capabilities is still challenging. In

Full text of DOI:10.1039/d0cc06313d

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BODIPY Based Realtime, Reversible and Targeted Fluorescent
Probes for Biothiols Imaging in Living Cells
a, b
a
a
a
a
Rongkun He, Yichuan Zhang, Suresh Madhu, Quan Gao, Qianjin Lian, Sriram Srinivasa
Received 00th January 20xx,
Accepted 00th January 20xx
c
a,
Raghavan and Jin Geng *
DOI: 10.1039/x0xx00000x
Real-time live cell imaging and quantification of biothiols dynamics
are important for understanding the pathophysiological process.
However, it is still challenging in the design and synthesis of rational
probes that have reversible and real-time capabilities. In this work,
we have prepared Boron-dipyrrolemethene (BODIPY) based
fluorescent molecules as ratiometric probes that allow the real-
time biothiols dynamics in living cells. The Michael reaction
between α-formyl-BODIPY (BOD-JQ) and GSH exhibited a reversible
fluorogenic mechanism with fluorescent emission shifting from 592
nm to 544 nm with t1/2 = 16 ms. In particular, we showed that the
probes with targeting agents are capable of detecting biothiols in
mitochondria and endoplasmic reticulum (ER) with high temporal
resolution.
Figure 1. (a) Time course of the response of BOD-JQ to GSH. Kinetic studies were
Biothiols play crucial roles in maintaining biological redox performed using a stopped-flow spectrophotometer at 25 ˚C under pseudo-first-order
homeostasis in biological systems.1,2,3,4,5,6Alternations of the conditions (5 μM BOD-JQ and 1 mM GSH). (b) Effect of pH on the fluorescence
cellular thiols levels have been found to relate a number of responses of BOD-JQ (5μM) to GSH (500 μM), R
0
is the ratio of I544 nm / I592nm of BOD-
7
,8,9,10
cancer,11,12,13 AIDS,12 JQ, R is the ratio of I544 nm / I592nm of BOD-JQ in the presence of GSH. (c) Fluorescence
16, 17, 18, 19,
diseases, such as leucocyte loss,
1
4, 15
5
Alzheimer’s disease,
psoriasis and liver damage.
emission spectra (λex = 510 nm) of BOD-JQ (5 μM) upon the addition of GSH. (d)
2
0, 21
Such fundamental biological roles explain the considerable Fluorescence intensity ratio (I544 nm / I592 nm) of BOD-JQ (5 μM) toward substance to be
contemporary effort devoted to the development of efficient detected (500 μM) Reaction solvent: DMSO : H O : PBS = 14 : 5 :1 (v : v : v), PBS: 0.2
2
methods for the detection and quantification of biothiols.
M potassium phosphate buffer, pH 7.4.
Many efforts have been made to develop fluorescent probes
for detecting biothiols. However, many of them showed slow
response and thus cannot be used for real-time detection. To date,
only a limited number of real-time fluorescent probes for biothiol
detections have been reported, of which the equilibrium can be
reached within one minute. 17, 22 However drawbacks such as
complicated synthesis route21 and narrow emission shift (≤ 39 nm)
Scheme 1. Reversible reaction of BOD-JQ with thiols.
resulted in high background interferences22 and the response
mechanism based on turn-on fluorescence response17 (see
Supplementary Table 1).
a. Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced
BODIPY and its diversities is one of the most exploited
fluorophores and has played a pivotal role in fluorescent probe
developments.16 However, there has not been any BODIPY
based biothiol probes reported for biothiol detections with
reversible and fast reaction kinetics23, 24 (see Supplementary
Technology, Chinese Academy of Sciences, Shenzhen, China.
College of Chemistry and Chemical Engineering, Xiamen University, Xiamen,
b.
China.
c.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Electronic Supplementary Information (ESI) available. See
†
DOI: 10.1039/x0xx00000x
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Journal Name
Table 1). Therefore, it is of great significance to develop a
BODIPY-based probe to overcome the drawbacks discussed
above allowing real-time detection of biothiols in living cells.
Herein, we introduce a robust BODIPY-based molecular
fluorescent probe that enabled a real-time sensing, ratiometric
and continuous monitoring of biothiols in living cells. The probe
was designed to rapidly and reversibly react with biothiols
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DOI: 10.1039/D0CC06313D
(Scheme 1). We observed that these probes can react with
biothiols, including GSH, Cys, Hcy, resulting in a shifted
emission for about 50 nm from 592 nm to 544 nm. The
reversibility of the reaction was successfully demonstrated using
the GSH and N-ethylmaleimide (NEM) in aqueous solutions and
living cells (Supplementary Movie S1 and S2). In addition,
BOD-JQ was reacted with Cys or Hcy and then NEM to
demonstrate their reversibility with biothiols. Supplementary
Movie 3 and 4 showed strong evidence that BOD-JQ react with
Cys and Hcy in a reversible manner, too. In particular, we have
shown that the probes with targeting moieties are capable of
Figure 2. Electron density distributions in the HOMO and LUMO. (a) Scheme of
measuring biothiols dynamics in mitochondria and endoplasmic reversible reaction of BOD-JQ with EtSH and the possible product. (b) Electron density
reticulum (ER) with a high temporal resolution.
distributions in the HOMO and LUMO of BOD-JQ, BOD-EtSH1 and BOD-EtSH2. Color
To detect biothiols in biological systems, we have designed code: C (gray), H (white), O (red), N (blue), F (light green), B (pink), S (yellow).
and synthesized an α-formyl-BODIPY dye with a carbon-carbon
double bond for Michael addition with biothiols (BOD-JQ,
Figure 1a). To accelerate the reaction rate of Michael addition,
electron-withdrawing groups were introduced to the α-position
as reported,26 i.e. probe 1 (with an ester group), 6 (with a cyano
adding GSH (100 equiv.) to a solution of BOD-JQ (5 µM) in the
presence of amino acids or ascorbic acid (100 equiv.). The results
revealed that the reaction of BOD-JQ with biothiols was not
significantly interfered (Supplementary Figure S50).
group) and 7 (with an aldehyde group) in Supplementary Table
The reversibility of the reaction between BOD-JQ and GSH was
demonstrated by the addition of N-ethylmaleimide (NEM) (1
equiv. to GSH, 1 mM) to the reaction mixture. The reaction was
monitored by measuring the absorbance at 570 nm of the reaction
solution after the addition of GSH and NEM. The absorbance of
the BOD-JQ at 570 nm decreased immediately after the addition
of GSH, and the subsequent addition of NEM resulted in a rapid
and complete recovery of the intensity, even after three cycles,
confirming the reversibility of the reaction (Supplementary
Figure S51). The colour changes of the solution can be directly
visualized by naked eyes as shown in Supplementary Figure S52
and Supplementary Movie 1).
Due to the reversibility of the Michael addition between
BOD-JQ and GSH, the dissociation of GSH from BOD-JQ can
be evaluated without a scavenger. As “Dilution” favors the
dissociation and reforming of BOD-JQ. Thus, we have further
confirmed the reversibility of the reaction, because the
absorbance of solution C (570 nm) was more than half of B, and
has the same intensity as that of A (Supplementary Figure S53).
Molecular descriptors of the BODIPY dye were calculated
using Density functional theory (DFT) calculations. Ethanethiol
(EtSH) was used to react with BOD-JQ giving two possible
products BOD-EtSH1 and BOD-EtSH2 as shown in Figure 2a.
The energy difference between highest occupied molecular
orbital (HOMO) and lowest unoccupied molecular orbital
1
. It is worth noting that some electron withdrawing groups,
such as nitro groups, reduced the reaction rates.27 In this work,
we have chosen aldehydes to modify the BODIPY core. We
found that the obtained probe, BOD-JQ, reacted with GSH
within 0.2 s in an aqueous solution (PBS/DMSO, 3/7). Figure 1a
showed the time-dependent response of BOD-JQ to GSH in a
pseudo-first-order reaction manner (5 μM BOD-JQ with 1 mM
GSH). The observed rate constant (κobs) at pH 7.4 and 25 ˚C was
found 42.4 s (t1/2 = 16 ms), indicating BOD-JQ can rapidly react
with GSH under such experimental conditions.
We then investigated the influences of pH on the reaction of
BOD-JQ with GSH. The most significant fluorescence intensity
change upon reacting with GSH was observed at pH 7.4, as
-1
shown in Figure 1b (R / R
0 0
= 240.7, where R and R are I544 nm /
I592 nm in the absence and presence of GSH, respectively). BOD-
JQ showed ratiometric fluorescence responses with a wide
dynamic range and exhibited a maximum fluorescence intensity
at 592 nm and 544 nm before and after the reaction with GSH,
respectively (Figure1c and Supplementary Figure S27). The
fluorescence intensity ratio (I544 nm / I592 nm) was found to be
linearly related to the GSH concentration in the range of 4 μM -
2
7
0 μM with R = 0.997 (Supplementary Figure S47).
Selectivity is an important parameter in evaluating the
performance of a probe. Commonly existed biomolecules in
living systems, including amino acids and ascorbic acid, were
used to evaluate the reaction selectivity of BOD-JQ with
biothiols. We observed that the addition of biothiols gave a
notable change in fluorescence intensities, while all other
reagents gave neglectable changes upon biothiol additions
(LUMO) has been used as an easiest indicator for the kinetic
stability.21 A large HOMO-LUMO energy gap implied the high
kinetic stability or low chemical reactivity of the molecules,
because the favorability of the addition or extraction of electrons
(Figure 1d). The competition experiments were carried out by
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Figure 3. Quantification of biothiols in several cell lines. (a) Confocal fluorescence
images of B16F10, A375 and HEKa Cells incubated with BOD-JQ (2 μM) for 30 min:
green channel at 500 – 550 nm (BOD-JQ-SH), λex = 488 nm, red channel at 570 – 620 nm
(
BOD-JQ), λex = 561 nm. (b) Calibration curve of GSH concentration against the intensity
Figure 4. Targeting fluorescent probes. (a) BOD-PPh3 with targeting group for
mitochondrial. (b) Confocal fluorescence images of B16F10, A375 and HEK-A cells
incubated with BOD-PPh3 (2 μM) for 30 min. followed by containing with MBF for 5 min,
from left to right: blue 430 – 470 nm, λex = 405 nm (MBF); green 500 – 550 nm, λex = 488
nm (BOD-PPh3-SH); merged images of blue and green channels (c) BOD-Cl with targeting
group for ER. (d) Confocal fluorescence images of B16F10, A375 and HEK-A Cells
incubated with probe BOD-Cl (2 μM) for 30 min followed by containing with ERT for 5
min, from left to right: blue channel at 430 – 470 nm, λex = 405 nm (ERT); green channel
at 500 – 550 nm, λex = 488 nm (BOD-Cl-SH); merged images of blue and green channels.
ratio of green and red channels. The plots were fitted with a quadratic equation, and the
calculated fitting equation is given in the graph as R = 0.02797 + 0.01265c + 0.0011c2,
where R represents for intensity ratio of green and red channels, and c represents for GSH
concentration. (c) Box plots of biothiols concentration in each cell line calculated via the
equation showing in (b). Values (mM) above each box plot represent mean ± s.d. (n is the
number of cells). Scale bar = 400 μm.(d) Calculated real time biothiols concentration in
living B16F10 cells. Error bars represent standard deviation (n = 19 cells).
from a low-lying HOMO indicated by this energy gap. The
simulation studies gave the energy gap of BOD-EtSH2 as
against biothiols concentrations were fitted well in a quadratic
equation (Figure 3b). Therefore, we were able to calculate the
intracellular biothiols concentrations based on this equation i.e.,
3
.112eV and BOD-EtSH1 as 2.873eV, suggesting the formation
of BOD-EtSH2 was more favorable and stable than BOD-EtSH1.
This result is in well agreement with previous studies.12, 27, 28
As discussed, the probe exhibited high sensitivity, selectivity
and ratiometric fluorescent responses to biothiols. We therefore
continued to investigate the reaction of the probes with biothiols
in living cells. We incubated BOD-JQ with several cell lines,
including B16F10 (mouse melanoma cells), HEK-A (human
epidermal keratinocyte) and A375 cells (malignant melanoma
cells). 2 µM was chosen as the concentration of BOD-JQ for
furtehr studies because of the compatibility of the probe (> 85%
viability at this concentration) (Supplementary Figure S54 –
S62).
1
1.3 ± 1.7 mM for A375 cells, 13.8 ± 1.2 mM for B16F10 cells,
and 13.8 ± 3.1 mM for HEK-A (Figure 3c). The biothiols
concentrations in these cells determined by the BOD-JQ probes
were in agreement with previously reported values,15, 28 indicating
the potential applications of our probes and methodologies in
quantifying intracellular biothiol concentrations.
The significant biological roles of biothiols has explained the
considerable contemporary effort devoted to the development
of efficient methods for the detection and quantification of
biothiols in different organelles. In this work, we have prepared
two organelle-targeting probes, BOD-PPh3 and BOD-Cl,
functionalized with mitochondria targeting group and ER
targeting group as shown in Figure 4a and 4c. We incubated
B16F10 cells with the probes under the same conditions as
discussed above. Colocalization experiments were carried out
using two commonly employed mitochondrial tracker and ER
tracker. We observed that the fluorescence of Mito-tracker was
well colocalized with that of reacted BOD-PPh3 in cells, with
Pearson’s correlation coefficients (Rr) determined as 0.85 for
B16F10 cells, 0.88 for A375 cells, and 0.82 for HEK-A (Figure
All the cell lines (B16F10, HEK-A and HEK-A) were
incubated with BOD-JQ (2 μM) for 30 min. We observed that
BOD-JQ-SH was shown in green and BOD-JQ was shown in red
(Figure 3a), confirming sufficient cellular permeability of the
probe and the Michael addition reactions happened in cells as
expected. The fluorescent signal ratios varied between cells lines,
implying the differences in biothiols level in these cells. The
fluorescence intensity ratio between BOD-JQ-SH and BOD-JQ
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J. Name., 2013, 00, 1-3 | 3
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b). The similar level of colocalization was observed for ER
targeting probe, BOD-Cl (Figure 4d), where we determined the
Pearson’s correlation coefficients were 0.86 for B16F10 cells,
Journal Name
6
7
8
9
1
. Y. Yue, C. Yin, F. Huo, J. Chao, Y. Zhang, SensV.ieAwcAtrutiacletoOrnslinBe
Chem. 2016, 223, 496-500.
4
DOI: 10.1039/D0CC06313D
. D. M. Townsend, K. D. Tew and H. Tapiero, Biomed.
Pharmacother., 2003, 57, 145-155.
. J. M. Estrela, A. Ortega and E. Obrador, Crit. Rev. Clin. Lab. Sci.,
2006, 43, 143-181.
. X. Yang, Y. Guo and R. M. Strongin, Angew. Chem., Int. Ed.,
0
.86 for A375 cells, and 0.89 for HEK-A. In the contrast, when
using probe, BOD-JQ, without any targeting agents, the
Pearson’s correlation coefficients were found as poor as 0.21 for
both mitochondria and ER in B16F10, a375 and HEK-A cells
2
011, 50, 10690-10693.
0. S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
R. B. D'Agostino, P. W. F. Wilson and P. A. Wolf, N. Engl. J.
Med., 2002, 346, 476-483.
(supplementary Figure S63).
We have successively demonstrated that BOD-JQ can reflect
the changes of the intracellular biothiol concentrations. The 11. L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung and Q.-Z.
Yang, J. Am. Chem. Soc., 2012, 134, 18928-18931.
2. L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem., Int.
Ed., 2009, 48, 4034-4037.
3. H. Jiang and H. Ju, Anal. Chem., 2007, 79, 6690-6696.
4. K. Amarnath, V. Amarnath, K. Amarnath, H. L. Valentine and
W. M. Valentine, Talanta, 2003, 60, 1229-1238.
5. F. Wang, L. Zhou, C. Zhao, R. Wang, Q. Fei, S. Luo, Z. Guo, H.
Tian and W.-H. Zhu, Chem. Sci., 2015, 6, 2584-2589.
6. T. Matsumoto, Y. Urano, T. Shoda, H. Kojima and T. Nagano,
Org. Lett., 2007, 9, 3375-3377.
cellular biothiol level was manipulated by the addition of NEM
and the cell-permeable glutathione monoethyl ester (a GSH
precursor). Initially, we incubated BOD-JQ (2 μM) with B16F10
cells for 30 min. After the addition of NEM (500 μM), the
fluorescence intensity of BOD-JQ-SH decreased rapidly and
reached a plateau within 30s. This was due to the disassociation
of BOD-JQ and the re-formation of BOD-JQ. We observed that
the fluorescence intensity was unchanged without further
treatments. After 5 min, GSH monoethyl ester (5 mM) was added
and the fluorescence intensity was gradually restored, indicating
the detection of intracellular biothiols changes in real time by
BOD-JQ (Supplementary Figure S64 and Figure 3d).
In summary, we have rationally designed and synthesized a
highly sensitive BODIPY-based fluorescence probe which
exhibited tremendous fast reaction kinetics with thiols, thus it
enabled the real-time monitoring of biothiols concentrations in
living cells. We demonstrated the probe for quantifying
intracellular GSH concentration by measuring GSH levels in
various cell lines and by successfully monitoring the GSH
dynamics in living cells. The results presented here support our
view that these probes are valuable tools for the investigation of
how thiols dynamics are regulated in a physiological context, due
to their capability for real-time quantification of biothiols with
high temporal resolution. We envision that these new probes will
enable opportunities to study biothiols dynamics and
transportation and expand our understanding of the physiological
1
1
1
1
1
1
1
1
2
2
2
2
2
7. B. K. McMahon and T. Gunnlaugsson, J. Am. Chem. Soc., 2012,
134, 10725-10728.
8. X. Xie, M. Li, F. Tang, Y. Li, L. Zhang, X. Jiao, X. Wang and B.
Tang, Anal. Chem., 2017, 89, 3015-3020.
9. Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52,
7
11– 760
0. Z. Lu, Y. Lu, C. Fan, X. Sun, M. Zhang and Y. Lu, J. Mater. Chem.
B, 2018, 6, 8221-8227.
1. Z. Cai, X. Liu, X. Wang, Z. Chen, Z. Song, Y. Xu, H. Tao, Y. Li, R.
Chen, L. Guo and F. Fu, Anal. Chim. Acta, 2019, 1091, 127-134.
2. K. Umezawa, M. Yoshida, M. Kamiya, T. Yamasoba and Y.
Urano, Nat. Chem., 2017, 9, 279-286.
3. J. Zhang, N. Wang, X. Ji, Y. Tao, J. Wang and W. Zhao, Chem. -
Eur. J., 2020, 26, 4172-4192.
4. X. Jiang, J. Chen, A. Bajić, C. Zhang, X. Song, S. L. Carroll, Z.-L.
Cai, M. Tang, M. Xue, N. Cheng, C. P. Schaaf, F. Li, K. R.
MacKenzie, A. C. M. Ferreon, F. Xia, M. C. Wang, M. Maletić-
Savatić and J. Wang, Nat. Commun., 2017, 8, 16087.
5. E. H. Krenske, R. C. Petter and K. N. Houk, J. Org. Chem., 2016,
81, 11726-11733.
6. H. Hino, M. Kamiya, K. Kitano, K. Mizuno, S. Tanaka, N.
Nishiyama, K. Kataoka, Y. Urano and J. Nakajima, Transl.
Oncol., 2016, 9, 203-210.
2
2
and pathological roles of biothiols in living cells.
This work was completed with support by National Natural
Science Foundation of China (22071263), Hundred-Talent Program
2
2
7. Y. Urano, M. Sakabe, N. Kosaka, M. Ogawa, M. Mitsunaga, D.
Asanuma, M. Kamiya, M. R. Young, T. Nagano, P. L. Choyke
and H. Kobayashi, Sci. Transl. Med., 2011, 3, 110-119.
8. S.-Y. Lim, K.-H. Hong, D. I. Kim, H. Kwon and H.-J. Kim, J. Am.
Chem. Soc., 2014, 136, 7018-7025.
(Chinese Academy of Sciences) and Guangdong Natural Science
Foundation (2020A1515010994).
Conflicts of interest
There are no conflicts to declare
Notes and references
1
2
3
4
5
. J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J.-H. Ryu and J.
Yoon, J. Am. Chem. Soc., 2014, 136, 5351-5358.
. B. Helbling, J. Von Overbeck and B. H. Lauterburg, Eur. J. Clin.
Invest., 1996, 26, 38-44.
. X.-F. Yang, Q. Huang, Y. Zhong, Z. Li, H. Li, M. Lowry, J. O.
Escobedo and R. M. Strongin, Chem. Sci., 2014, 5, 2177-2183.
. Y.-F. Kang, L.-Y. Niu and Q.-Z. Yang, Chin. Chem. Lett., 2019,
3
0, 1791-1798.
. Y. Yue, F. Huo, F. Cheng, X. Zhu, T. Mafireyi, R. M. Strongin and
C. Yin, Chem. Soc. Rev., 2019, 48, 4155-4177.
4
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