Fluorescence imaging mitochondrial copper(II) via photocontrollable fluorogenic probe in live cells

Article abstract of DOI:10.1016/j.cclet.2017.07.016

Monitoring mitochondrial derived copper(II) in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper(II). Herein, we have reported the design, synthesis and characterization of photocontrollable f

Full text of DOI:10.1016/j.cclet.2017.07.016

Accepted Manuscript
Title: Fluorescence imaging mitochondrial copper(II) via
photocontrollable fluorogenic probe in live cells
Authors: Liulin Wang, Buxiang Chen, Pingping Peng, Wenbo
Hu, Zhipeng Liu, Xiaohua Pei, Weihong Zhao, Chengwu
Zhang, Lin Li, Wei Huang
PII:
S1001-8417(17)30261-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.07.016
CCLET 4134
To appear in:
Chinese Chemical Letters
Please cite this article as: Liulin Wang, Buxiang Chen, Pingping Peng, Wenbo Hu,
Zhipeng Liu, Xiaohua Pei, Weihong Zhao, Chengwu Zhang, Lin Li, Wei Huang,
Fluorescence imaging mitochondrial copper(II) via photocontrollable fluorogenic probe
in live cells, Chinese Chemical Lettershttp://dx.doi.org/10.1016/j.cclet.2017.07.016
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Communication
Fluorescence imaging mitochondrial copper(II) via photocontrollable fluorogenic
probe in live cells
Liulin Wanga, Buxiang Chena, Pingping Penga, Wenbo Hua, Zhipeng Liua, Xiaohua Peib, Weihong Zhaob,
Chengwu Zhanga, Lin Lia, , Wei Huanga, c,*
a Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced
Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, China
b Division of Nephrology, Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210009, China
c
Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Nanjing University of Posts &
Telecommunications, Nanjing 210023, China
Graphical abstract
Aberrant homeostasis of mitochondrial copper(II) is implicated in the pathogenesis of various diseases. Monitoring
mitochondrial derived copper(II) in live cells is highly demanded, but accurately detecting is unmet due to the interference with
cytoplasmic copper(II). Herein, we present a photo-controlled fluorogenic probe, MCu-3, which could perfectly avoid this
interference.
ABSTRACT
Monitoring mitochondrial derived copper(II) in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic
copper(II). Herein, we have reported the design, synthesis and characterization of photocontrollable fluorogenic probe, MCu-3, which is equipped with a photo-
labile group (nitrobenzyl group) and mitochondria targeting unit (triphenylphosphonium salt). This novel probe showed an intense fluorescence enhancement in
response to copper(II) without interference from other metal cations in the biological condition (pH 6-9). The detection limit is 1.7 ×10-7 mol/L in HEPES
———
 Corresponding authors.

buffer. The confocal fluorescence imaging results demonstrated MCu-3 can visualize mitochondrial copper(II) in live mammalian cells. The clear advantage of
our photocontrollable method is successful to avoid the influence of cytoplasmic copper(II) during mitochondria specific detection
Keywords:
Mitochondria
Photocontrollable
Fluorogenic Probe
Fluorescence imaging
Copper(II)
Mitochondria are organelles with critical roles in key processes within eukaryotic cells, and summarily described as the “power
factory” of life, supplying energy which our bodies depend on all the time in every biochemical reaction and action of lives [1-3].
Dysfunction of mitochondria underlies the pathogenesis of variety of diseases, such as Parkinson’s disease (PD), Alzheimer’s disease
(AD), atherosclerosis, acute and chronic degenerative cardiac myocyte death, neuronal death (ischemic and hemorrhagic stroke) and
cancer [4-7]. Therefore, mitochondrial damage factors have been one of the top priority scientific topics [8-10]. In mitochondria,
copper(II), an essential trace element, is an inalienable part of many important enzymes involved in a range of vital physiological
processes such as transportation and catalysis in living cells [11-13]. Copper(II)-induced toxicity is the result of ionic imbalance
affecting on mitochondrial respiratory chain, apparently causing transition metals-induced reactive oxygen species (ROS) formation,
mitochondrial membrane depolarization, lipid peroxidation, and cytochrome c release which ignite cell apoptosis [12]. Disorder of
copper(II)/copper(I) homeostasis plays a leading role in the pathogenetic mechanisms of many neurodegenerative diseases [14,15].
Copper interacting with the proteins is also the signs of neurodegenerative disorders, such as PD and AD [16,17]. In consequence,
detection of mitochondrial copper(II) is significant to study its pathogenetic mechanism in neurodegenerative diseases and monitor its
homeostasis. Regretfully, there is no available tools which could precisely detect mitochondrial copper(II) in live cells without
interfered by cytoplasmic copper.
Given the pivotal role of mitochondria, development of mitochondria specific probe to detect the elements insider it attracts
increasing attention. For example, in the past decade, various reactive mitochondria-specific fluorescent probes have been developed
for the detection of reactive oxygen species (ROS) [18-22], reactive sulphur species (RSS) [23-25], metal ions [26-29], enzymes [30,31]
and other targets [32-36] inside mitochondria. Nevertheless, most of these fluorescent probes have the limitation that they hardly
overcome the interference of the targeted species inside cytoplasm. For detection of mitochondrial copper(II) or other species, it is
necessary to design more appropriate strategies to conquer this long-standing problem.
Herein, with this aim in mind, we have designed a novel mitochondrial specific fluorogenic probe MCu-3 (Scheme 1) to accurately
visualize copper(II) in mitochondria of live cells. In our approach, alkyl-linked fluorescein hydrazide provided a fluorescence-
responsive for copper(II) [37-39]. Its photocaged version with 2-nitrobenzyloxy unit, efficiently photolyzed by ultraviolet (UV) light
———
E-mail addresses: iamlli@njtech.edu.cn (L. Li), iamwhuang@njtech.edu.cn (W. Huang).

illumination, not only increases the cell permeability of the probe, but also at the same time provides additional spatial and temporal
controls over imaging mitochondrial copper(II) [40-42]. In the current study, we chose a simple photolabile 2-nitrobenzyloxy group as
the caging molecule for synthetic convenience, but it may be readily replaced with two-photon caging molecules [43-45].
Triphenylphosphonium salt (TPP) group serves as the mitochondrial-targeting moiety according to previous literature report [46-50].
The overall “piggyback” strategy of the probe MCu-3 is shown in the right side of Scheme 1. As shown, fluorescein hydrazide
hydrolysed by cytoplasmic copper would normally be blocked due to the existence of caged unit after cellular uptake (Step 1), which
facilitated MCu-3 to accumulate completely with no fluorescence. The TPP would be internalized to mitochondria (Step 2). Subsequent
UV irradiation (Step 3) will force photocaged group to leave to release the uncaged probe MCu-2 (still conjugated to the TPP), which
would then be hydrolyzed by mitochondrial copper(II), generating the specific localized and highly fluorescent MCu-4 (Step 4).
Based on this design, the fluorogenic probe MCu-3 and two reference probes MCu-1/2 were synthesized following the revised
literature procedures [28,51-53], as shown in Scheme 1. The copper-recognizing part, fluorescein hydrazide (2), was synthesized in one
step with the overall yield of 90% from the commercially available fluorescein and hydrazine hydrate. Alkylation reactions were
carried out with 1,4-dibromobutane in 70%-78% yield to generate 1/3/5 from fluorescein/2/4, respectively. The photocaged group was
synthesized with bromination reaction of 1-ethyl-2-nitrobenzene (80% yield, Supporting information). The nitrobenzyl protected
fluorescein hydrazide (4) was prepared by condensation between 2 and 1-(1-bromoethyl)-2-nitrobenzene in 52% yield in the presence
of Cs2CO3, while bisnitrobenzyl protected fluorescein hydrazide (6) was obtained in 85% yield as shown in Supporting information.
The mitochondria-targeted group-containing MCu-1/2/3 were synthesized under high temperature reflux of toluene (60%-71% yield).
The structures of all compounds were fully characterized by 1H NMR, 13C NMR and HRMS analysis (see Supporting information).
To unequivocally establish that our newly developed MCu-3/2/4 probe group was indeed a good fluorogenic system, capable of "keep
silence" and emitting strong fluorescence during MCu-3→2 and MCu-2→4 conversion, respectively, we first investigated their
photophysical properties (Fig. S1 in Supporting information). As shown, under physiological conditions (HEPES buffer at pH 7.5),
MCu-1, ester version reference of MCu-4, had maximum absorption at 450 nm (Fig. S1A), and emission at 525 nm (Fig. S1B, Φ = 0.20),
while there is nearly no absorption and emission for MCu-2/3/3+UV (Φ = 0.001~0.0015) around the same wavelength. Consequently,
the caged probe has a very low fluorescence background and high S/N ratio in vitro assay and live cell imaging. As we expected,
incubated with copper(II), MCu-3+UV has very similar absorption and emission properties with MCu-1 in HEPES buffer (Fig. S1A and
B, pH 7.5). As shown in Fig. 1A, the photolabile nitrobenzyl group was released after exposure to UV light (500 μJ/cm2 for 5 min), and
hydrolysis reaction could be finished in 60 min. Experiments were monitored by HPLC-MS, and peaks were unambiguously assigned
based on their molecular weights. At the meantime, we also demonstrated MCu-3 will not coordinate with copper(II) in physiological
conditions by using HPLC-MS, which may prevent it from bringing cytoplasmic copper into mitochondria. As expected, there is
almost no fluorescent signal even in the presence of copper(II) before UV illumination, indicating that the caged probe is very stable
and does not be hydrolyzed to MCu-4 in the presence of copper(II) in the dark. We then proceeded to investigate the optimal response

time of MCu-3 toward copper(II) upon exposure to UV light for 5 min. A largest fluorescence enhancement (Φ = 0.17) at 525 nm was
observed after 60 min and no longer change over time (Fig. S2 in Supporting information), which is consistent with the HPLC-MS
analysis.
We further examined the sensing response of MCu-3 to copper(II). Upon photolysis, introduction of an increasing concentration of
copper(II) to the solution of MCu-3 (2 μmol/L) induced a gradual increase in the emission around 525 nm (Fig. 1B). When the amount
of copper(II) was more than 10 equiv., the spectra saturation was reached, which is similar with MCu-2 (Fig. S4 in Supporting
information) and 6 (Fig. S5 in Supporting information). In this case, a linear calibration graph of the fluorescent intensities toward
copper(II) concentrations was plotted. Fluorescence intensity also exhibited a good linearity against the copper(II) concentrations as
shown in Fig. S3 (R2 = 0.994, Supporting information). A linear regression curve was then fitted to these normalized fluorescent
intensity data, and the point at which this line crossed the axis was considered as the detection limit as 1.7 ×10-7 mol/L (Supporting
information).
The pH of cytoplasma was between 6.8 and 7.4, however the pH of the mitochondrial matrix was around 8.2-7.5 [49,51]. Therefore,
we determined whether the probe can emit fluorescence and be stable in the scope. Hence, the effect of pH on the fluorescence
response of MCu-3 to copper(II) was tested. A quantitative analysis of the fluorescence intensity at 525 nm vs a wide pH range from 3
to 12 (Fig. S6 in Supporting information) indicated that MCu-3 can act as a detector in physiological pH conditions (pH, 6-9) (Fig. 1C).
Thus, MCu-3 could work properly in mitochondria. Next, we also tested the selectivity of MCu-3 (2 μmol/L) toward a series of metal
ions (10 equiv.) upon photolysis for 5 min, including Zn2+, Co2+, Mn2+, Ni2+, K+, Na+, Ca2+, Ba2+, Fe3+, Cr3+, Al3+, Cd2+, Mg2+, Pd2+, to
evaluate the selectivity of the probe for copper(II). As shown in Fig. 1D and E, addition of copper(II) to MCu-3 upon photolysis resulted
in a significant enhancement of the fluorescence intensity, other species have a negligible fluorescent intensity and little interference,
which indicated MCu-3 has a good selectivity and anti-interference ability for copper(II).
We next investigated that our newly developed MCu-1/2/3 fluorogenic system was indeed useful for bioimaging application to detect
mitochondrial copper(II). The cytotoxicity activity assays were shown there is no obvious cell toxicity at concentrations up to 20
μmol/L for all three probes in the human hepatocellular carcinoma (HepG2) cells (Fig. S7 in Supporting information), making them
suitable for living cell imaging. Indeed, when live HepG2 cancer cells were treated with MCu-1, followed by one-photon fluorescence
microscopy, we observed fluorescent staining exclusively within the cells (Fig. 2). As shown in Fig. 2(4), the green fluorescence signal
of the reference probe MCu-1 was well-overlapped with the red fluorescence signal from the Mito-Tracker with a high contrast ratio. In
contrast, fluorescein, a control free dye without TPP, only can stain whole cells without any specific localization. Prolonged incubation
of the cells with MCu-1 was comparable with known Mito-Tracker without leading to any diffusion, confirming MCu-1 has good cell
permeability and mitochondrial specificity.
Encouraged by these results, we next determined whether MCu-3 works as a mitochondria specific probe for imaging copper(II). As
shown in Fig. 3, HepG2 cells were incubated with MCu-3 and MCu-2 in the presence of copper(II). Cells treated with MCu-3, if without

UV-irradiation, showed no fluorescence, indicating the temporal control of our "caging" strategy (Fig. 3(2)). However, the significant
fluorescent enhancement of MCu-2-treated cells have been observed in the same way (Fig. 3(7)). Upon UV irradiation, a gradual
increase in green fluorescence was observed both in MCu-3 (Fig. 3(3)) and MCu-2-treated cells (Fig. 3(8)). However, there is no
fluorescence if MCu-3-treated cells were only exposed to UV light without copper(II) as shown in Fig. S8 (Supporting information).
Furthermore, cell membrane-impermeable copper(II) chelator (EDTA) was used to treat cells before and after probe incubation
together with UV irradiation. The images (Fig. S8(3)) showed EDTA had almost no effect on fluorescence because the reaction
between MCu-3 and copper(II) is irreversible. And, a faint fluorescence signal in Fig. S8(4) indicated pre-treated EDTA did not totally
blocked the reaction. The cells were simultaneously treated with commercially available Mito-tracker to independently verify the
localization results. Both MCu-3 and MCu-2-treated cells showed similar mitochondria-specific images by comparing the colocalization
analysis results between Fig. 3(5) and (10), indicating the strategy is amenable to the original design principle. This suggested that the
probes could accurately detect mitochondrial copper(II).
In summary, we have described a photocontrollable probe can detect mitochondrial copper(II) over cytoplasmic via fluorescence
imaging. The probe would be very useful for study pathogenic mechanism of mitochondrial copper(II) involved diseases. This novel
probe shows a 15-fold fluorescence enhancement in response to copper(II) without interference from other metal ions in the biological
condition. The clear advantage of our method is successful to avoid the influence of chemical species inside cytoplasm during
mitochondrial specific detection. Of course, the method maybe also play an instruction role in mitochondria-specific drug delivery and
control release, not only to the study of targeting mitochondrial fluorescence probe. A series of probes, with detection limit in the
femtomolar range, which could distinguish the endogenous copper(II) in different organelles would be good compensation to our probe.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (Nos. 81672508, 61505076), Natural
Science Foundation of Jiangsu Province (No. BK20140951), Key University Science Research Project of Jiangsu Province (No.
16KJA180004) and SICAM Fellowship & Scholarship by Jiangsu National Synergetic Innovation Center for Advanced Materials.

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(A)
(B)
(C)
5k
4k
3k
2k
1k
0
MCu-4
Cu2+, 37 oC, 60 min
M
-3
Cu
3k
2k
1k
0
2+
M
M
-3+UV+Cu
-1
20 μmol/L
Cu
Cu
UV, 5 min
UV, 3 min
0
UV, 2 min
UV, 1 min
0 min
MCu-2
MCu-3
5
6
7
8
9
500
600
Wavelength (nm)
700
pH value
0
2.5
5.0
7.5
(D)
700
Cu2+()
Cu2+(+)
2+
2+
2+
2+
8. Ba
1. Zn
2. Co
3+
9. Fe
(E)
3+
10. Cr
11. Al
3. Mn
3+
2+
2+
4. Ni
5. Pd
2+
+
12. Cd
13. Mg
2+
6. Na
7. Ca
+
2+
14. K
2+
15. Cu
0
0
5
10
15
n
20
25
30
Fig. 1. (A) MCu-3→MCu-2 conversion by UV irradiation (5 min), followed by MCu-2→MCu-4 hydrolysed by mitochondrial Cu2+ (60 min). Reactions were carried
out at room temperature in HEPES buffer (pH 7.5; probe concentration: 2.0 mol/L). Cu2+/probe concentration ratio = 10:1. (B) One-photon fluorescence
emission spectra of 5 min UV-assisted uncaged MCu-3 (2.0 mol/L) after addition of Cu2+ (0→10 equiv.) in HEPES buffer after 60 min incubation at room
temperature. (C) The fluorescence intensities at 525 nm of MCu-1 (2 mol/L), MCu-3 (2 mol/L) and uncaged MCu-3 (2 mol/L)/Cu2+ (10 equiv.) at various pH
values at room temperature. The excitation wavelength was 455 nm. (D) Effect of the analytes (10 equiv.) to uncaged MCu-3 with/without Cu2+ in HEPES buffer,
red bars represent the fluorescence intensity of uncaged MCu-3 (2.0 mol/L) in the presence of various analytes (10 equiv.) before (1-14) and after (16-29)
adding Cu2+ (10 equiv.) at room temperature, respectively. (E) Photos of the fluorescence of effect of the analytes (10 equiv.) to uncaged MCu-3 in HEPES buffer
after 60 min incubation at room temperature.
DIC
488 nm
Mito-tracer
(3)
Merged
(1)
(2)
(6)
(4)
(8)
MCu-1
(5)
(9)
(7)
Fluorescein
DMSO
(10)
(11)
(12)
Fig. 2. One-photon fluorescence imaging of live HepG2 cells by control probe MCu-1 (5 μmol/L), fluorescein (5 μmol/L) and DMSO. Live cells were incubated
o
with the probes for 1 h at 37 C, followed addition of a commercial available Mito-tracker Red CMXRos dye (200 nmol/L), then further incubated for 30 min,
before being imaged. The excitation wavelength were 488 and 543 nm, while the PMT range were 500-550 and 570-650 nm for control probes and Mito-tracker,
respectively. Insert in (4) is the colocalization analysis of merged-image of (2)/(3) (R = 0.85). All images were acquired in the same way. DIC = Differential
interference contrast image. Scale bar = 10 µm

488 nm
(3)
DIC
Mito-Tracker
(4)
Merged
－UV
＋UV
(1)
(2)
(5)
MCu-3
(7)
(8)
(9)
(10)
(6)
MCu-2
Fig. 3. One-photon fluorescence imaging of live HepG2 cells by probes MCu-3/2 (5 μmol/L). Live cells were incubated with the probe for 1 h at 37 oC, followed
o
by UV(+) or UV(−) irradiation (2 min) at room temperature and addition of with Cu2+ (10 equiv.) for another 30 min at 37 C. After that, cells were incubated
with the tracker (200 nmol/L) for 30 min, before being imaged. The excitation wavelength and PMT range were 488 and 500-550 nm for both of two probes,
respectively. Insets are DIC images in (3)/(8), and colocalization analysis of merged-image (3)/(4) (R = 0.81) and (8)/(9) (R = 0.78) in (5) and (10), respectively.
All images were acquired in the same way. Scale bar = 10 µm.
Scheme 1. The synthetic route and overall strategies of fluorogenic probes for accurately visualizing copper(II) ion in mitochondria in live cells. (a) Ethanol,
H2SO4, reflux, quantitative; (b) 1,4-Dibromobutane, Cs2CO3, DMF, 70%-78%; (c) Triphenylphosphine, toluene, reflux, 60%-71%; (d) Hydrazine hydrate,
MeOH, reflux, 91%; (e) 1-(1-Bromoethyl)-2-nitrobenzene, Cs2CO3, DMF, 52%.