A mitochondria-targeting fluorescent probe for the selective detection of glutathione in living cells

Article abstract of DOI:10.1039/c6ob02407f

We report a fluorescent probe for the selective detection of mitochondrial glutathione (GSH). The probe, containing triphenylphosphine as a mitochondrial targeting group, exhibited ratiometric and selective detection of GSH over Cys/Hcy. The probe was used for imaging mitochondrial GSH in living HeLa cells.

Full text of DOI:10.1039/c6ob02407f

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: X. Liu, L. Niu, Y.
Chen, M. Zheng, Y. yang and Q. Yang, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB02407F.
This is an Accepted Manuscript, which has been through the
Volume 14 Number
1 7 January 2016 Pages 1–372
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 4
View Article Online
DOI: 10.1039/C6OB02407F
Journal Name
COMMUNICATION
A mitochondria-targeting fluorescent probe for the selective
detection of glutathione in living cells
Xue-Liang Liu a, Li-Ya Niu,b,* ,Yu-Zhe Chenc, Mei-Ling Zhengc, Yunxu Yang a,*, and Qing-Zheng Yangb,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report a fluorescent probe for the selective detection of different roles in biological system, the development of
mitochondrial glutathione (GSH). The probe, containing fluorescent probes which can distinguish Cys/Hcy/GSH from
a
triphenylphosphine as mitochondrial targeting group, exhibited each other has emerged as an active topic in recent years7.
ratiometric and selective detection of GSH over Cys/Hcy. The However, few probes have been developed to selectively
probe was used for imaging mitochondrial GSH in living HeLa cells. detect mitochondrial GSH8. Herein, we reported a fluorescent
probe for the selective detection of GSH in mitochondria of the
Mitochondrion is a double membrane-bound organelle in
eukaryotic cells, utilizing oxygen to generate adenosine
triphosphate (ATP) as the source of biochemical energy, thus
has been described as the powerhouse of the cells1. In
addition, they are involved in many biological processes, such
as signaling, cellular differentiation, cell death, and
maintaining control of the cell cycle and cell growth2.
Mitochondria are recognized as a significant source of reactive
oxygen species (ROS), which can damage mitochondrial
membrane and hinder intracellular oxidative phosphorylation.
living cells. The probe was composed of chlorinated BODIPY as
GSH recognition group and a typical mitochondrial-targeting
moiety of triphenylphosphonium cation. It showed
a
ratiometric fluorescent response towards GSH, while Cys/Hcy
induced on-off fluorescence signals. More importantly, it
successfully detected mitochondrial GSH in living HeLa cells.
In our previous work 7b,9, we developed a BODIPY-based
fluorescent probe for the selective detection of GSH over
Cys/Hcy. The probe bearing a chlorine at 3-position as a
leaving group reacted with thiols by nucleophilic aromatic
substitution (SNAr) to afford thioether. The amino groups of
Cys/Hcy but not GSH further went through an intramolecular
rearrangement to yield the amino derivatives. The distinct
photophysical properties of thioether- and amino- substituted
BODIPY enabled the selective detection of GSH. On the other
hand, a functional group can be introduced at 5-position
through click reaction (e.g., a triethyleneglycol group to
improve the water-solubility of the probe). In order to develop
a fluorescent probe processing both GSH detection and
3
The oxidative damage to the mitochondria is responsible for
cell signaling and cell death which cause many
neurodegenerative diseases and pathologies, such as
Alzheimer’s disease, Parkinson’s disease, neurodegenerative
disorders, etc4. As the most abundant biothiols in cells, GSH
plays a key role in maintaining the redox environment in
mitochondria to avoid or repair oxidative damage5. Therefore,
it is of great importance to monitor mitochondrial GSH in living
cells.
Fluorescence probes are considered to be one of the most
efficient molecular tools monitor and visualize analytes in
living cells and tissues due to their high sensitivity and
selectivity, high spatiotemporal resolution, and real-time
imaging6. Given that biothiols, including GSH, Cys and Hcy, play
mitochondrial-targeting ability,
a
mitochondrial-targeted
functional group is necessary. Positively charged groups,
especially triphenylphosphonium moiety, are commonly used
as mitochondrial delivery vehicles10. Because the
mitochondrial membrane spans across a negative potential,
probes with these positive charge take advantage of
electrostatic forces to deliver the probe to the mitochondria 11
.
Therefore, our fluorescent probe was designed by integrating
triphenylphosphonium moiety into the chlorinated BODIPY.
BODIPY-PPh3 was synthesized from 3,5-dichlorinated
BODIPY as a precursor. First, a trimethylsilylacetylene was
attached to the 3-position through Sonogashira coupling. Then
the triphenylphosphonium group was introduced by the one
pot reaction of the desilylation and click reaction (Scheme 1).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 4
View Article Online
DOI: 10.1039/C6OB02407F
COMMUNICATION
Journal Name
Compound BODIPY-PPh3 was well characterized by 1H NMR
and 13C NMR spectroscopy and high-resolution mass
spectrometry (HRMS).
shifted absorption band at 500 nm was observed. Meanwhile,
the fluorescence at 557 nm turned off gradually. In the case of
Hcy, with the decrease of the absorption at 540 nm, an
absorption band centred at 570 nm emerged and increased at
the initial 40 min. Then the absorption at 570 nm decreased
with the appearance of a blue-shifted absorption band at 500
nm. The emission intensity at 557 nm gradually decreased,
with the simultaneous enhancement of the fluorescence at
588 nm initially. As the reaction progressed, the fluorescence
intensity at 588 nm decreased. The absorption and emission
spectra of BODIPY-PPh3 in the presence of GSH, Cys and Hcy
were shown in Fig. 2, indicating BODIPY-PPh3 selectively
detected GSH over Cys/Hcy.
N3
P+
Br-
Si
N
N
TBAF, TERN, CuI, CH2Cl2
Pd(PPh3)2Cl2, CuI
toluene, Et3
N
N
B
N
N
B
B
N
F
F
Cl
F
F
Cl
Cl
F
F
Cl
N
N
BODIPY-Cl2
Si
BODIPY-tri
N
P+
Br-
BODIPY-PPh3
Scheme 1 Synthesis of BODIPY-PPh3
At first, we performed the time-dependent absorption and
fluorescence response of BODIPY-PPh3 in the presence of GSH
in HEPES buffer. As shown in Fig. 1, in the presence of GSH, the
absorption band of free BODIPY-PPh3 centred at 540 nm
gradually decreased with concomitant growth of a new band
at 570 nm. In the fluorescence spectra, the free BODIPY-PPh3
exhibited an emission band at 557 nm. Upon addition of GSH,
the original emission at 557 nm decreased, and a new
emission band centred at 588 nm increased significantly when
excited at the isosbestic point of 550 nm. The ratio of
Fig. 2 (a) Absorption and (b) fluorescence spectra of BODIPY-PPh3 (10 μM) upon
addition of 1mM GSH, Cys and Hcy in acetonitrile/ HEPES buffer (5:95, v/v, 20
mM, pH 7.4) after 3 h addition at 37 °C. λex = 550 nm.
fluorescence intensities at 588 nm and 557 nm (I588/I557
)
showed good linear relationship with the GSH concentrations
(0-80 μM, R2 = 0.993), and the detection limit was determined
to be 1.1 μM (S/N = 3).
n=1 fast
n=2 slow
HOOC
H2N
n
SH
N
N
N
N
B
2
B
COOH
n SH
NH2
F
F
HN
S
F
R
F
R
n
COOH
Cys n=1
Hcy n=2
1
R =
N
F
N
B
GSH
F
R
Cl
N
N
N
P+
Br-
BODIPY-PPh3
N
F
N
B
3
SG
F
R
Scheme 2 Reaction mechanism for BODIPY-PPh3 with Cys, GSH and Hcy.
The spectroscopic properties of BODIPY-PPh3 were in good
agreement with our previous studies. Based on the above
observation and previous explorations, we proposed the
reaction mechanism, as illustrated in Scheme 2. Initially, the
chloro leaving group is replaced by the thiols through the thiol-
Fig. 1 Time-dependent (a) absorption and (b) emission spectra of BODIPY-PPh3
(10 μM) in the presence of 1 mM GSH. (c) Fluorescence spectra of BODIPY-PPh3
(10 μM) after 1h upon addition of increasing concentrations of GSH. (d) The
fluorescence ratio of 588 nm and 557 nm (I588/I557) as a function of the GSH
concentration (0 to 80 μM). λex = 550 nm. Each spectrum was obtained in
acetonitrile/ HEPES buffer (5:95, v/v, 20 mM, pH 7.4) at 37 °C.
halogen SNAr nucleophilic substitution to produce compound
and For Cys/Hcy, once is formed, the following
rearrangement reaction take place by a five- or six-membered
cyclic transition state to yield compound with more
1
3
.
1
2
The spectroscopic responses of BODIPY-PPh3 to Cys and Hcy
were also investigated (Fig. S1 and S2). Upon addition of Cys,
the absorption at 540 nm decreased gradually, and a blue-
thermodynamically stable C-N bond than C-S bond. In addition,
the rearrangement reaction for Hcy is much slower than Cys
due to the less favourite six-membered cyclic transition state
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 3 of 4
View Article Online
DOI: 10.1039/C6OB02407F
Journal Name
COMMUNICATION
than the five-membered one. On the other hand, in the case of To examine the mitochondrial-locating performance of
GSH, after the thiol-halogen SNAr nucleophilic substitution, the BODIPY-PPh3, colocalization experiments were conducted by
intramolecular rearrangement is difficult to happen due to the costaining HeLa cells with a commercial mitochondrial tracker
unstable macrocyclic transition state, thus it result in the rhodamine 123 and BODIPY-PPh3. The fluorescence of
stable product
3
.
rhodamine 123 (Fig. 4a) from the costained cells in the
presence of GSH overlaps well with that of BODIPY-PPh3 (Fig.
4b), as shown in the merged yellow image (Fig. 4c). Obviously,
the changes in the intensity profiles of the linear region of
interest (ROI) 1 across the living HeLa cell are synchronous in
the two channels (Fig. 4d). More importantly, a high Pearson's
coefficient of 0.96 and an overlap coefficient of 0.95 are
obtained from the intensity correlation plots (Fig. 4e).
Next, we evaluated the selectivity of BODIPY-PPh3 toward
other amino acids. As shown in Fig. 3, only GSH triggered
significant fluorescence ratio changes of 16.99. Cys and Hcy
showed certain fluorescence responses but their fluorescence
ratio was smaller compared to GSH (0.69 and 2.02 for Cys and
Hcy, respectively). Other amino acids did not promote
noticeable fluorescence variations. We further check the
detection of GSH in the presence of other amino acids and
Meanwhile, in order to further approve the location of
metal ions (Fig. S3). The existence of other interferent did not BODIPY-PPh3 in mitochondria, we studied the colocation
affect the detection of GSH. The results confirmed the high between the probe BODIPY-PPh3 and rhodamine 123 in
selectivity of probe BODIPY-PPh3 for GSH.
multiple cell levels. Fig. S4 show that BODIPY-PPh3 and
rhodamine 123 can colocate in a plurality of living cells in the
mitochondria, and high Pearson’s colocalization coefficient
0.95 and overlap coefficient 0.95 indicate that the staining of
BODIPY-PPh3 fits well with that of mitochondrial tracker
rhodamine 123, The intensity profile of ROI 1 across HeLa cells
in the two channels also varies in close synchrony. Besides, a
high correlationship is found in the intensity scatter plot of
green and red channels, suggesting BODIPY-PPh3 exists
predominantly for multiple cells in mitochondria. The results
further confirmed that the probe can accurately locate and in
the mitochondria of the cell.
Fig. 3 Fluorescence intensity ratio (I588/I557) responses of BODIPY-PPh3 (10 μM)
upon addition of various amino acids (1mM) in acetonitrile/HEPES buffer (5:95
v/v, 20 mM, pH = 7.4) after 3 h addition at 37 °C. λex = 550 nm.
Fig. 5 Confocal fluorescence images of living HeLa cells incubated with GSH (1
mM) for 20 min, and then incubated with BODIPY-PPh3 (2μM) for 15 min: (a) red
channel (λex 561 nm; λem 570−620 nm); (b) green channel (λex 488 nm; λem
500−550 nm); (c) bright-field transmission image;(d) ratio image generated from
(a) and (b). Confocal fluorescence images of living HeLa cells incubated with NEM
(1 mM) for 20 min, and then incubated with the BODIPY-PPh3 (2μM) for 15 min:
(e) red channel (λex 561 nm; λem 570−620 nm); (f) green channel (λex 488 nm; λem
500−550 nm); (g) bright-field transmission image; (h) ratio image generated from
(e) and (f).
The selective detections of GSH in the mitochondria of the
living HeLa cells were carried out by using confocal
fluorescence imaging. As shown in Fig. 5, when incubation with
GSH for 20 min at 37℃, and then incubation with BODIPY-
PPh3 for 15 min, fluorescence from both green (500-550 nm)
and red channels (570-620 nm) was observed (Fig. 5a and 5b).
The fluorescence ratio of red/green channels was ~5 (Fig. 5d).
By contrast, the incubation of n-ethylmaleimide (NEM) to trap
intracellular thiols prior to the addition of BODIPY-PPh3
resulted in relatively weak emission in red channel and strong
Fig. 4. Single cell colocalization of rhodamine 123 and BODIPY-PPh3 in
mitochondria of HeLa cells. HeLa cells were pretreated with GSH (1mM) for
20min, and then costained with (a) rhodamine 123 (2 μM, Channel 1 (Ch1), λex
488 nm; λem 500−550 nm) and (b) BODIPY-PPh3 (2 μM, Channel 2 (Ch2), λex 561
nm; λem 570−620 nm). (c) Merged image of (a) and (b). (d) Intensity profile of
region of interest 1 (ROI 1) cross the HeLa cell costained with Rh 123 and
BODIPY-PPh3. (e) Intensity scatter plot of Ch1 and Ch2.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 4
View Article Online
DOI: 10.1039/C6OB02407F
COMMUNICATION
Journal Name
emission in green channel (Fig. 5e and f), and the fluorescence
ratio decreased to ~2. (Fig.5h). It indicated that BODIPY-PPh3 is
capable of monitoring GSH in the mitochondria of living cells.
Tang, Chem. Commun. ,2014, 50, 15439-15442; (h) F. Y. Wang, L.
Zhou, C. C. Zhao, R. Wang, Q. Fei, S. H. Luo, Z. Q. Guo, H. Tian and
W. H. Zhu, Chem. Sci., 2015, 6, 2584-2589; (i) X. Yang, Y. Guo and
R. M. Strongin, Angew. Chem. Int. Ed., 2011, 50, 10690-10693; (j)
J. Zhang, J. Wang, J. Liu, L. Ning, X. Zhu, B. Yu, X. Liu, X. Yao and H.
Zhang, Anal. Chem., 2015, 87, 4856-4863. (k) H. Chen, Y. Tang, M.
Ren and W. Lin, Chem. Sci., 2016, 7, 896-1903; (l) J. Y. Wang, Z.-R.
Liu, M. Ren, X. Kong and W. Lin, J. Mater. Chem. B, 2017, DOI:
10.1039/C6TB02610A; (l) L. He, Q. Xu, Y. Liu, H. Wei, Y. Tang and
W. Lin, ACS Appl. Mater. Interfaces, 2015, 7, 12809–12813
8. (a) H. Zhang, C. X. Wang, K. Wang, X.P. Xuan, Q. Z. Lv and K. Jiang
, Biosens. Bioelectron., 2016, 85, 96-102; (b) J. Zhang, X. L. Bao, J.
L. Zhou, F. F. Peng, H. Ren, X. C. Dong and W. L. Zhao, Biosens.
Bioelectron., 2016, 85, 164-170; (c) S. Y. Lim, K. H. Hong , D. I.
Kim, H. Kwon and H. J. Kim, J. Am, J. Am. Chem. Soc., 2014, 136,
7018-7025; (d) X. Zheng and X. Lin, Chem. Commun.,2015, 52,
1094-1119.
In conclusion, we have developed a mitochondrial-targeting
fluorescent probe BODIPY-PPh3 for the selective detection of
GSH. The probe contained
a triphenylphosphine as a
mitochondrial targeting group and showed ratiometric
response and selectivity for GSH over Cys/Hcy with high
sensitivity. Colocalization experiments demonstrated that the
probe specially located mitochondrial in living cells. Confocal
fluorescence microscopy experiments confirmed its
applicability to detect GSH in mitochondria of the living cells.
We anticipate the probe can be used for exploring biological
effects of mitochondrial GSH.
9. L.-Y. Niu, Y.-Shi. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tunga and Q.-Z.
Yang, Chem. Commun., 2013, 49, 1294-1296.
We thank Prof. Z. Niu (TIPC) for providing HeLa cells. This
work was financially supported by the 973 program
(2013CB933800), National Natural Science Foundation of
China (21525206, 21402216, 21272243 and 61475164), the
Fundamental Research Funds for the Central Universities and
Beijing Municipal Commission of Education.
10. (a) S.I. Nigam, B. P. Burke and L. H. Davies, Chem. Commun.,
2016, 52, 7114-7117; (b) H. B. Yu, X. F. Zhang, Y. Xiao, W.Zou,
L.P. Wang and L. J. Jin, Anal. Chem., 2013, 85, 7076-7084; (c) J.
Zhou, L. H. Li, W. Shi, X. H. Gao, X. H. Li and H. M. Ma, Chem.
Sci., 2015, 6, 4884-4888; (d) J. Xu , Y. Zhang, H. Yu, X. D.Gao and
S. J.Shao, Anal. Chem., 2016, 88, 1455-1461; (e) P. Ning, J.C.
Jiang , L. C. Li, S. X. Wang, H. Z. Yu, Y. Feng, M. Z. Zhu, B. C.
Zhang, H. Yin, Q. X. Guo and X. M. Meng, Biosens. Bioelectron.,
2016,77, 921–927; (f) X. Y. Gong, X. F.Yang, Y. Y. Zhong, Y. H.
Chen and Z. Li, Dyes and Pigments., 2016, 131, 24-32; (g) B.N.
Sui, S. Tang, A. W. Woodward, B. Kim and K. D. Belfield, Eur. J.
Org. Chem., 2016, 2851-2857.
Notes and references
1. K.Henze and W. Martin, Nature. ,2003, 426, 127-128.
2.(a) T.Finkel, M.Serrano and M. A.Blasco, Nature., 2007, 448, 767-
774; (b) H.M. Mcbride, M. Neuspiel and S. Wasiak, Curr. Biol.,
2006, 16, 551-660; (c) A. T.Hoye, J. E.Davoren , P.Wipf , M. P.Fink
and V. E.Kagan, Accounts. Chem. Res., 2008, 41, 87-97; (d)
V.Gogvadze , S.Orrenius and B.Zhivotovsky , Semin. Cancer. Biol,
2009, 19, 57-66.
11. M. P. Murphy and R. A. J. Smith, Annu. Rev. Pharmacol. Toxicol.,
2007, 47, 629-656.
3. Y. Y. Wang, S. M.Chen and H. Li , Acta Pharma. Sinica., 2010, 31,
160-164.
4. (a) A. M.Strohecker and E. White, Cancer Discov., 2014, 4, 766;
(b) R E. E. amsay, P. J.Hogg and P. J.Dilda, Pharm. Res., 2011, 28,
2731; (c) P.W.Szlosarek , S. Lee and P. J.Pollard , Mol. Cell. ,2014,
56, 343.
5. (a) H. J.Forman, H.Zhang and A.Rinna, Mol. Aspects. Med. ,2009,
30, 1-12; (b) R.A.Smith, R.C.Hartly and M.P.Murphy, Antioxid.
Redox. Signal., 2011, 15:3021-3038.
6.(a) M. Vendrell, D. Zhai, J. C. Er and Y.T. Chang, Chem. Rev., 2012,
112, 4391-4420; (b) H. Kobayashi, M. Ogawa, R. Alford, P. L.
Choyke and Y. Urano, Chem. Rev., 2010, 110, 2620-2640; (c) R.
W.Sinkeldam, N. J. Greco and Y. Tor, Chem. Rev., 2010, 110,
2579-2619;(d) Y. M. Yang, Q. Zhao, W.Feng , F. Y.Li , Chem. Rev.,
2013, 113, 192-270. (e) H. Chen, Y. Tang and W. Lin, TRAC-Trend.
Anal. Chem., 2016, 76, 166-181.
7. (a) X.-L. Liu, L.-Y. Niu, Y.-Z. Chen, Y. Yang and Q.-Z. Yang, Biosens.
Bioelectron. 2016, doi: 10.1016/j.bios.2016.06.076; (b) 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; (c) L.-Y. Niu, Y.-Z. Chen, H.-
R. Zheng, L.-Z. Wu, C.-H. Tung and Q.-Z.Yang, Chem. Soc. Rev. ,
2015, 44, 6143-6160; (d) L.-Y. Niu, Q.-Q. Yang, H.-R. Zheng, Y.-Z.
Chen, L.-Z. Wu, C.-H. Tung and Q.-Z. Yang. , RSC Adv., 2015, 5,
3959-3964; (e) M.-Y. Jia, L.-Y. Niu, Y. Zhang , Q.-Z. Yang, C.-H.
Tung, Y.-F. Guan and L. Feng, ACS Appl. Mater. Interfaces., 2015,
7, 5907-5914; (f) H. Li, J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P.
Sun and X. Peng, Chem. Commun., 2009, 39, 5904-5906; (g) X.
Wang, J. Lv, X. Yao, Y. Li, F. Huang, M. Li, J. Yang, X. Ruan and B.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins