A diketopyrrolopyrrole-based fluorescent probe for investigating mitochondrial zinc ions

Article abstract of DOI:10.1039/c7nj04940d

We demonstrate a strategy to asymmetrically introduce a Zn²⁺ chelator (MeO-DPEN) and a mitochondrial-targeting group (TPP) at the N-positions of both lactam moieties of diketopyrrolopyrrole (DPP) fluorophore. And two probes DPP-Mito and DPP-C2

Full text of DOI:10.1039/c7nj04940d

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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A diketopyrrolopyrrole-based fluorescent probe for investigating
mitochondrial zinc ions
Chenchen Du a,† Shibo Fu b,† Xiaolei Ren, a Xiaohua Wang, a Zhong Wang, b,∗ Juan
Zhou, b,* Hongyu Wang a,c,d*
We demonstrate a strategy to asymmetrically introduce Zn2+ chelator (MeOꢀDPEN)
and mitochondrialꢀtargeting group (TPP) at Nꢀpositions of both lactam moieties of
diketopyrrolopyrrole (DPP) fluorophore. And two probes DPP-Mito and DPP-C2
were reported for comparable studies. Both probes showed almost nonfluorescent due
to the photoꢀinduced electron transfer (PET) process from MeOꢀDPEN to DPP
fluorophore. Upon addition of Zn2+, fluorescence intensities of both probes increased
approximately 77ꢀfold. Both probes exhibited high dissociation constant, pH
insensitivity and high metal ion selectivity. Hela cell fluorescence imaging studies
demonstrated that both probes were low toxicity, and could monitor the changes in
intracellular Zn2+ concentrations. Confocal microscopy showed that DPP-Mito
specifically localized in mitochondria, while DPP-C2 was not organelleꢀspecific.
a
Department of Chemistry, College of Science, Shanghai University, 99 Shangda Road, Shanghai,
200444, P. R. China. Eꢀmail: wanghy@shu.edu.cn
b Department of Urology, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School
of Medicine, Shanghai, 200011, P. R. China. Eꢀmail: zhongwang2000@sina.com,
zhoujuan0002@163.com
c
Center for Supramolecular Chemistry and Catalysis, Shanghai University, 99 Shangda Road,
Shanghai, 200444, P. R. China
d Key Laboratory of Organosilicon Chemistry and Material Technology of Ministry of Education,
Hangzhou Normal University, Hangzhou 311121, People’s Republic of China
† The first two authors contributed equally to this study.

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1. Introduction
Zinc (II) (Zn2+) is a vital mineral that is integral to many enzymes and transcription
factors that regulate key cellular functions such as the response to oxidative stress,
metabolism, growth, proliferation, apoptosis, cell signaling and survival of cells.1
Imbalance of Zn2+ is associated to many diseases, such as Atherosclerosis, Alzheimer
diseases, diabetes and prostate disease.2 Due to the colorlessness and magnetic silence
of Zn2+, it is difficult to accurately understand the biological roles of Zn2+.3,4 However,
fluorescence imaging can provide direct information about the actions of Zn2+ through
vivid images. A variety of the fluorescent probes derived from quinolone,5,6
fluorescein,7,8
coumarin,9,10
naphthalene,11,12
boronꢀdipyrromethene,13,14
anthracene15,16 had been developed to understand the role of Zn2+ ions in physiology.
Recently, diketopyrrolopyrrole (DPP) derivatives have received intensive attention in
fluorescent probes due to their ease of synthesis, outstanding photoꢀphysical stability,
and high fluorescence quantum yields.17ꢀ19 DPP fluorescent probes have been
developed for chemical and biological detection, such as metal ions, anions, active
oxygen species, etc.20ꢀ24 So far, only a few DPPꢀbased Zn2+ fluorescent probes have
been reported. Wang, et al. introduced zinc chelator, N,Nꢀdi(pyridinꢀ2ꢀylmethyl)
amine (DPA), to the phenyl rings at positions 3 and 6 of DPP core, showing
fluorescence enhancement upon binding with Zn2+ through both photoꢀinduced
electron transfer (PET) and intraꢀmolecular charge transfer (ICT) mechanisms.25,26
Those fluorescent probes were successfully used for imaging Zn2+ in Hela cells.
However, it is wellꢀknown that 3,6ꢀdiphenyl DPP derivatives emit green color, which

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limits their biological applications because their absorption and emission maxima
appear below 600 nm.27,28 Moreover, those probes were not organelleꢀspecific, so
detection of the subcellular Zn2+ remains a significant challenge. Until now, most
reported molecular designing strategy is introducing the reporters to the aryl groups at
3,6ꢀpositions of DPP core, due to the easy synthesis through coupling reactions and
formation of effective conjugation between the DPP and reporters, and simultaneously
attaching side chains at Nꢀpositions of lactam moieties to increase the solubility. Few
attempts to functionalize DPP at the lactam Nꢀpositions have been made.29
Mitochondria are the principal organelles related to energy metabolism and adenosine
triphosphate (ATP) synthesis, and also the important apparatus for intracellular Zn2+
transportation and storage.30,31 So monitoring the change of mitochondrial Zn2+
concentration becomes the key to understanding the physiology of Zn2+. Herein, we
designed and synthesized a new 3,6ꢀdithienyl DPPꢀbased fluorescent probe
DPP-Mito for imaging mitochondrial zinc ions through asymmetrically introducing
Zn2+ chelator and mitochondrialꢀtargeting group at Nꢀpositions of both lactam
moieties (Scheme 1). The replacement of benzene with thiophene at positions 3 and 6
of DPPs decreased the dihedral angle from ca. 30 to 7 degrees,32,33 resulting in
significant bathochromic shift of both absorption and emission. A methoxy derivative
of N,Nꢀdiꢀ(2ꢀpicolyl)ethylenediamine (MeOꢀDPEN) was chosen as the Zn2+ chelator,
which is well suited for the detection of low zinc level in tissues, because it exhibited
high Zn2+ binding affinity and turnꢀon response.34ꢀ37 And triphenylphosphonium (TPP)
salt was employed as the mitochondrialꢀtargeting group.38,39 MeOꢀDPEN and TPP

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were attached on the opposite Nꢀpositions of lactam, and separated as far as possible
to minimize the interactions between them. A model compound DPP-C2, without the
chemical tag TPP, was also synthesized for comparison. Both probes exhibited high
dissociation constants with Zn2+, pH insensitivity and high metal ion selectivity. Hela
cell fluorescence imaging studies demonstrated that DPP-Mito was low toxicity,
mitochondrialꢀspecific localization, and could monitor the changes in intracellular
Zn2+ concentrations.
Scheme 1. Synthesis of the target DPP-C2 and DPP-Mito.
2. Results and discussion
2.1 Spectroscopic studies
The UVꢀvis and fluorescence spectra of DPP-C2 and DPP-Mito in different polarity
of solvents were showed in Figure 1, and the corresponding data was exhibited in
Table 1 and Table S1. According to the reported literatures, the absorption in the range

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of 400~600 nm and the fluorescence in 500~700 nm range were attributed to the DPP
fluorophore.29 The absorption and emission spectra of both probes showed almost
unchanged with increasing solvent polarity, which is due to the conjugation
interruption between the DPP fluorophore and MeOꢀDPEN. The utility of the
molecule can’t form a polar probe. In metalꢀfree buffer solution (50 mM HEPES, 100
mM KCl, 10 mM EGTA, pH 7.2, 1% DMSO), the maximum absorbance (λabsmax) and
emission (λflmax) wavelengths were 519 and 558 nm (ФF = 0.0036) for DPP-C2, and
515 and 559 nm (ФF = 0.0086) for DPP-Mito, respectively. Both probes showed
almost nonfluorescent due to the photoꢀinduced electron transfer (PET) process from
MeOꢀDPEN to DPP fluorophore.20,34ꢀ37 Upon addition of Zn2+, fluorescence
intensities of both probes increased dramatically with slightly affecting the absorption
spectra. It may be due to the decrease in the electronꢀdonating ability of the
MeOꢀDPEN upon complexation with Zn2+, thus blocking the PET process.
Consequently, the large “offꢀon” enhancement in the emission spectra were observed,
and the fluorescence enhancement factors [FEF = (Fmax ꢀ Fmin)/Fmin] of DPP-C2 and
DPP-Mito were both equal to 77 in the presence of excess of Zn2+. These results
demonstrated that the mitochondrial targeting group TPP exhibited negligible effect
on the optical properties of DPP fluorophore. The intensities of emission of
DPP-Mito/Zn2+ complex retained at 58% after exposure to natural light for one week
(Figure S8), showing a moderate degree of photostability. The precipitation of the
complex was observed during this period, which may be the main reason for the
decrease of fluorescence.

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Figure 1. Normalized absorption and emission spectra of DPP-C2 (a and b) and
DPP-Mito (c and d) with or without 10 mM Zn2+ in DCM, DMSO, ethanol, and
buffer (50 mM HEPES, 100 mM KCl, 10 mM EGTA, pH 7.2, 1% DMSO).
2.2 Binding affinity for Zn2+
The binding model of DPP-C2 and DPP-Mito with Zn2+ were determined by Job’s
plots, fluorescence titration curves and isothermal titration calorimetry (ITC) (Figure
2 and Figure S7). From the Job’s plots (Figure 2a), the value of the maximum
fluorescence appeared at 0.5 mole fraction, suggesting the formation of 1:1
probe/Zn2+ complex. We measured the MALDIꢀ TOFꢀMs spectra of the complexes
formed between the probes and Zn2+ (Figure S9 and S10). In the mass spectra of
DPP-C2/Zn2+ and DPP-Mito/Zn2+ complexes, ion peaks at 794 and 1083 were

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respectively observed, which were assigned as [DPP-C2 + Zn2+ ꢀ H+]+ and
{[DPP-Mito – Br¯]+ + Zn2+ ꢀ 2H+}+. To confirm the stoichiometry between the probes
and Zn2+, fluorescence titration experiment was conducted to evaluate the binding
behavior of the probes to Zn2+ in HEPES buffer solutions. The measured fluorescence
(
F
ꢀ
F
min)/ (
equation (Figure 2c), also indicating the 1:1 complex stoichiometry. And the
dissociation constants (
d) of DPP-C2 and DPP-Mito with Zn2+ in HEPES buffer
Fmax ꢀ F
min) as a function of [Zn2+]free fitted well to the BenesiꢀHildebrand
K
were found to be respective 3.08 and 2.04 nM with nanomolar detection limits,
suggesting that both probes have nanomolar affinity towards Zn2+ and are suitable for
the detection of low zinc levels in tissues and cells.34ꢀ37 Complexes formation between
both probes and Zn2+ involving 1:1 stoichiometry were further confirmed by Hill plots
(Figure 2d). The plots showed a linear regime in the [Zn2+]free concentration of 0.29 ~
47 nM. And the obtained Hill coefficients for DPP-C2 and DPP-Mito were 1.26 and
1.11, respectively.

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Figure 2. (a) Job’s plots for DPP-Mito binding Zn2+ in HEPES buffer (50 mM
HEPES, 100 mM KCl, pH 7.2, 1% DMSO). The total concentration of DPP-Mito and
Zn2+ was maintained at 1 ꢁM. (b) Emission spectra of DPP-Mito in HEPES buffer
(50 mM HEPES, 100 mM KCl, 10 mM EGTA, pH 7.2) with a series of [Zn2+]free (0 ~
127 nM). (c) The fluorescence titration curves of the complexation of DPP-Mito with
[Zn2+]free (0 ~ 127 nM). (d) Hill plots for the complexation of DPP-Mito with free
[Zn2+]free. The excitation wavelength was 500 nm.
Table 1. Spectroscopic properties of DPP-C2 and DPP-Mito, and their zinc
complexes in HEPES buffer solution. a
g
Ka
b
c
d
f
λabs
λfl
ФF
FEF e
Kd
(nM)
max
max
(× 108
Compound
(nm)
(nm)
Mꢀ1)

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523
524
520
524
558
558
560
560
0.0036
0.1330
0.0086
0.1562
DPP-C2
DPP-C2 + Zn2+
DPP-Mito
77
77
3.08
2.04
2.02
2.71
DPP-Mito +
Zn2+
a The probes were measured in HEPES buffer (50 mM HEPES, 100 mM KCl, 10 mM EGTA, pH
7.2, 1% DMSO) with or without 10 mM Zn2+. b The maximum absorption wavelength in HEPES
buffer. c The maximum emission wavelength in HEPES buffer. d Fluorescent quantum yields were
determined in reference to rhodamine 6G (ФF = 0.89, in CH2Cl2). e Fluorescence enhancement
factor FEF = (F ꢀ Fmin) /Fmin. f Apparent dissociation constants for Zn2+ measured by fluorescence
g
titration, and titration curves were fitted to BenesiꢀHildebrand equation. Association constants
measured by ITC in ethanol.
Further evidence for binding parameters of the interaction between the probes and
Zn2+ were determined by ITC. The stepwise injection of an ethanol solution of ZnCl2
(0.5 mM) into an ethanol solution of the probe (0.05 mM) at 298 K resulted in a
gradual release of heat as an exothermic binding process. Figure 3 shows a typical
ITC profile and the corrected heat values plotted as a function of the probe/Zn2+ molar
ratio. The binding parameters were obtained by fitting the binding curve with an
independent binding site nonlinear regression algorithm. The stoichiometry of the
binding between DPP-C2 and DPP-Mito with Zn2+ were both found to be 1,
indicating that one probe molecule is associated with one zinc ion, agreeing well with
the Job’s plots and fluorescence titration. The association constants (Ka) were
2.02×108 and 2.71×108 Mꢀ1 for DPP-C2/Zn2+ and DPP-Mito/Zn2+ complex,
respectively, also indicating the strong affinity between MeOꢀDPEN and Zn2+.

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Figure 3. ITC trace obtained from the titration of DPP-C2 (a) and DPP-Mito (b) with
o
Zn2+ in ethanol at 25 C. Integrated heats of binding plotted as a function of the
probe/Zn2+ molar ratio.
2.3 Effect of pH
In order to test the effect of pH on fluorescence intensities of DPP-C2 and DPP-Mito,
a series of HEPES buffer solutions of different pH (3.5 ~ 9) were prepared. DPP-C2
and DPP-Mito exhibited similar pH responsive trends (Figure 4a and Figure S11).
For Zn2+ absent buffer solutions, the fluorescence intensity increased gradually when
pH decreased from 6.5 to 3.5, indicating that the fluorescence of both probes were
partially recovered in this acidic condition. This may be attributed to partial
protonation of MeOꢀDPEN, thus resulting in partial PET process. In the pH range of
6.5 ~ 9, the fluorescence intensities of both DPP-C2 and DPP-Mito did not change

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significantly. However, upon addition Zn2+ to HEPES buffer of the probes, both
probe/Zn2+ complex showed insensitive to the pH changes in the whole testing range
of 3.5 ~ 9. This may be ascribed to stronger binding affinity with Zn2+ than the proton,
which indicates that both probes are able to detect Zn2+ without proton
interference.34ꢀ37
Figure 4. (a) The influence of pH on DPP-Mito with or without Zn2+ in HEPES buffer.
(b) Metal ion selectivity profiles of DPP-Mito (1 ꢁM) in HEPES buffer solutions (50
mM HEPES, 100 mM KCl, 10 mM EGTA, pH 7.2, 1% DMSO). Black bars represent
the relative fluorescence intensities (F/F0) of DPPꢀMito with various metal ions. Red
bars represent the relative fluorescence intensities (F/F0) of DPP-Mito in the presence
of the indicated metal ions, followed by Zn2+.
2.4 Metal ion selectivity
We further studied the selectivity of DPP-C2 and DPP-Mito in HEPES buffer (50
mM HEPES, 100 mM KCl, 10 mM EGTA, pH 7.2, 1% DMSO) through metal ion
competitive experiments. As shown in Figure 4b and Figure S8, the fluorescence
intensities of DPP-C2 and DPP-Mito kept unchanged with adding 10 mM Na+, Ca2+,

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Mg2+, and 5 mM Mn2+, Fe2+, Co2+, Ni2+, Cu2+ and Cd2+. Subsequently adding excess
Zn2+, the fluorescence intensities were recovered except Co2+ and Cu2+. These results
showed that both probes could detect Zn2+ with high selectivity over 10 mM Na+,
Ca2+, Mg2+, and 5 mM Mn2+, Fe2+, Cd2+, and modest selectivity over 5 mM Ni2+. Co2+
and Cu2+ could obviously quench the fluorescence due to metalꢀtoꢀligand electron
transfer upon excitation mechanism.40 Since Co2+ and Cu2+ are usually very low level
in living cells,41 their interference could be neglected. It is worth noting that both
probes exhibited small interference from Cd2+. Since Cd2+ is in the same group of the
periodic table and has similar properties with Zn2+. Therefore, most of the reported
probes showed a similar fluorescence changes including the change of intensity and
the shift of wavelengths when Zn2+ and Cd2+ are coordinated with fluorescent
sensors.42,43
2.5 Investigating mitochondrial zinc ions in Hela cells
Hela cells were used as a model system to evaluate the ability of our probes to target
mitochondria and respond to changes in intracellular zinc levels. Firstly, the
cytotoxicity of both probes was evaluated for Hela cells using the Cell Counting Kitꢀ8
(CCKꢀ8, Dojindo) cellꢀviability assay.44 Hela cells were incubated with the probes at
concentrations of 5 and 10 ꢁM for 24 h, respectively. The viabilities of Hela cells
were all higher than 90% under all these conditions (Figure S12), indicating low
toxicity of the probes.
To investigate the cellular location of the probes, coꢀlocalization experiments were
performed by coꢀstaining Hela cells with the probes and MitoTracker® Green FM, a

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wellꢀknown commercially available marker for mitochondria. Confocal images of the
probes with MitoTracker® Green FM were recorded in separate detection windows,
with minimum interference from each other (Figure 5). It was noticed that the
distribution of DPP-Mito is nearly identical to that of MitoTracker® Green FM, and
their fluorescent images overlapped very well with each other (Figure 5h).
Coꢀlocalizations were quantified using Pearson’s sample correlation factors (A),
which describes the correlation of the intensity distribution between the channels to
characterize the degree of overlap between images.45,46 The Pearson’s coꢀlocalization
coefficient of DPP-Mito and MitoTracker® Green FM was 0.83. However, DPP-C2
showed poor overlap with MitoTracker® Green FM (Figure 5d), with Pearson’s
coꢀlocalization coefficient of 0.12. To confirm the spatial localization of the probes,
3D images were reconstructed from images obtained at different values in Z axis.
Higherꢀmagnification 3D images clearly indicated overlapped localization of
DPP-Mito and MitoTracker® Green FM. These results demonstrated that DPP-Mito
specially targeted mitochondria.

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Figure 5. Confocal microscopy images of Hela cells coꢀlabeled with MitoTracker®
Green FM and the probes. (a, e) The cells’ nuclei (blue) were stained by DAPI. (b, f)
Stained by MitoTracker® Green FM. (c, g) Stained by DPP-C2 and DPP-Mito,
respectively. (d, h) Merged images.
We further investigated whether both probes could be used to monitor changes of Zn2+
in Hela cells. The Hela cells incubated with probes for 24 h showed strong red
fluorescence, apparently from the intracellular Zn2+ (Figure 6a). The fluorescence
signal decreased significantly when the probeꢀtreated cells were incubated with TPEN
(a membrane permeable zinc chelator)47 (Figure 6b). In contrast, when the
probeꢀtreated cells were again incubated with an exogenous zinc source (zinc
pyrithione),48 the fluorescence intensity increased obviously (Figure 6c). Both
DPP-C2 and DPP-Mito (Figure S13) showed similar results. These findings
demonstrate that both probes are capable of detecting the changes in intracellular Zn2+
concentrations.
Figure 6. Fluorescent microscopy images of Hela cells (a) the cells were incubated
with DPP-Mito (10 ꢁM) for 24 h; (b) the cells were incubated with DPP-Mito (10
ꢁM) for 24 h, followed by further incubation with TPEN (50 ꢁM) for 10 min; (c) the

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cells were incubated with DPP-Mito (10 ꢁM) for 24 h, followed by further incubation
with a mixture of 50 ꢁM zinc pyrithione for 10 min.
3. Conclusions
In conclusion, we developed a zincꢀresponsive and mitochondrialꢀtargetable probe
DPP-Mito. This compound is essentially nonfluorescent in the zincꢀfree state;
however, complexation with Zn2+ triggers fluorescence enhancement of
approximately 77ꢀfold. Spectroscopic studies demonstrated that the mitochondrial
targeting group TPP exhibited negligible effect on the optical properties of DPP
fluorophore. Job’s plots, fluorescence titration curves and ITC showed that the probe
and Zn2+ complexed in 1:1 stoichiometry with Kd = 2.04 nM. DPP-Mito exhibited
high selectivity for Zn2+, and DPP-Mito/Zn2+ complex showed pH insensitivity at pH
3.5 ~ 9. Hela cell imaging studies demonstrated that DPP-Mito was low toxicity,
mitochondrialꢀspecific localization, and could monitor the changes in intracellular
Zn2+ concentrations.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of
China (81402089, 61774099), Program for Outstanding Medical Academic Leader
and the Innovation Program of Shanghai Municipal Education Commission

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(15ZZ047).
4. Experimental section
Materials and instruments
All the commercial chemicals and solvents were used without further purified.
3,6ꢀDi(thiophenꢀ2ꢀyl)ꢀ2,5ꢀdihydropyrrolo[3,4ꢀc]pyrroleꢀ1,4ꢀdione 1 and MeOꢀDPEN
were synthesized according to our previous report.37,49,50 Deionized water was used
for all experiments.
The 1H NMR and 13C NMR spectra were recorded on a Brucker Avance III 500 MHz
NMR spectrometer. Mass spectroscopic data were obtained from the Shanghai
Institute of Organic Chemistry (SIOC) using an HP5973 mass spectrometer. Matrix
assisted
laser
desorption/ionization
timeꢀofꢀflight
mass
spectrometry
(MALDIꢀTOFꢀMS) was performed on
a
Brucker Autoflex Speed using
transꢀ2ꢀ[3ꢀ(4ꢀtertꢀbutylphenyl)ꢀ2ꢀmethylꢀ2ꢀ propenylidene] malononitrile (DCTB) as
the matrix. UVꢀvis spectra were recorded on a Shimadzu UVꢀ2501PC UVꢀvis
spectrophotometer. Fluorescence measurement was carried out on a PerkinꢀElmer
LSꢀ55 spectrofluorophotometer with a xenon lamp as a light source. Excitation
wavelength was provided at 500 nm. All UV and PL spectra were collected at room
temperature. A Mettler Toledo SevenCompact S220 PH meter was used for pH
measurement. ITC measurements were performed using a MicroCal VPꢀITC.
Absorption and fluorescence spectra
The UVꢀvis absorption and fluorescence spectra were measured in four polarity of

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solvents,
such
as
DCM,
ethanol,
DMSO,
and
HEPES
(4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid) buffer (50 mM HEPES, 100
mM KCl, 10 mM EGTA, pH 7.2, 1% DMSO). The probe was dissolved in solvent at a
concentration of 1.0
×
10ꢀ4 mol/L as the stock solution, which was further diluted to 1
ꢁM. Absorption and fluorescence spectra were determined at a concentration of 1 ꢁM
(3 mL) in a 1 cm standard quartz cell for zincꢀfree form (With the presence of 10 ꢁM
EDTA to remove residual metal ion in solution). For the zincꢀbound form of the probe,
1 ꢁM Zn2+ were added in DCM, ethanol and DMSO solution of the probe (1 ꢁM),
respectively, and 10 mM Zn2+ was added in HEPES buffer of the probe.
Determination of fluorescence quantum yields
Fluorescence quantum yields (ΦF) of DPP-C2 and DPP-Mito in solutions were
measured using the equation 1, where rhodamine 6G (ФF = 0.89 in CH2Cl2) was
chosen as the standard.51
Iunk
A
ηunk
std
Фunk = Фstd
⋅
⋅
Aunk Istd ηstd
Equation 1
Where Φ is the fluorescence quantum yield; I is integrated area under the corrected
emission spectrum; A is the absorbance at the excitation wavelength; η is the
refractive index of the solution (pure solvents were assumed); and the subscripts ‘unk’
and ‘std’ refer to the unknown and the standard, respectively.
Determination of apparent dissociation constants
A series of HEPES buffer solutions (50 mM HEPES, 100 mM KCl, 10 mM EGTA,
pH 7.2, 1% DMSO) containing various amounts of ZnSO4 (0 ~ 9.8 mM) were
app
prepared. The free zinc ion concentration ([Zn2+]free) were calculated from KZnꢀEGTA
,

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[EGTA]total, [Zn2+]total using the literature methods.52,53
[Zn2+]total (mM) 0.50 1.00 2.00 3.00 4.00 4.99 5.99 6.98 7.98 8.97 9.47 9.8
[Zn2+]free (nM) 0.14 0.29 0.65 1.1
1.8 2.6 3.9 6.1 10 23 47 127
To determine the apparent dissociation constants (Kd) for the probe/Zn2+ complex, the
fluorescence titration curves were obtained and the dissociation constants were
calculated by fitting to BenesiꢀHildebrand equation (Equation 2). The binding
stoichiometry and detection limit were obtained from the Hill plots (Equation 3).
F − Fmin
[Zn2+ ]free
Fmax − Fmin Kd +[Zn2+ ]free
=
Equation 2
Equation 3






F − Fmin
Fmax − F
log
= n log[Zn2+ ]free +logKa
Where F is the florescence intensity, Fmax is the maximum fluorescence intensity of
the probe in HEPES buffer with Zn2+ existence, and Fmin is the fluorescence intensity
of the probe in HEPES buffer without Zn2+ existence. Kd, Ka and n are the dissociation
constant, association constant and the Hill coefficient, respectively.
Job’s plot
A series of solutions were prepared containing different mole fractions of the probe
and Zn2+ in HEPES buffer (50 mM HEPES, 100 mM KCl, pH 7.2, 1% DMSO), and
the total concentration of the probe and Zn2+ was maintained to 1 ꢁM.
ITC titration
o
The ITC titration was carried out in absolute ethanol at 25 C, and the concentration
of the probe and Zn2+ were kept at 0.05 mM and 0.5 mM respectively. After the
titration was finished, the titration data was fitted to give the stoichiometry (n), the

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equilibrium association constant (Ka), the apparent association enthalpy (∆H) and
entropy (∆S).
Effect of pH
A series of HEPES buffer (50 mM HEPES, 100 mM KCl, 1% DMSO) of different pH
(3.5 ~ 9) were prepared. pH variations in the solutions were achieved by adding the
minimum volumes of KOH or HCl. The concentration of Zn2+ was kept at 0 ꢁM by
adding 10 mM EGTA. The effect of the pH on fluorescent intensity of 1 ꢁM HEPES
buffer in the absence and presence of Zn2+ were determined.
Zn2+ selectivity
Solutions of metal ions were prepared from NaCl, CaCl2, MgCl2·6H2O, Mn(OAc)2,
FeSO4, CoCl2·6H2O, NiCl2·6H2O, CuCl2, CdCl2, and ZnSO4·7H2O dissolved in water.
The fluorescence spectra of 1 ꢁM probe were recorded in HEPES buffer (50 mM
HEPES, 100 mM KCl, 10 mM EGTA, pH 7.2, 1% DMSO) solution after addition of
the metal stock solutions. The final concentrations of the metal ions were 10 mM for
Na+, Ca2+, Mg2+, and 5 mM for Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Cd2+. After acquisitions,
Zn2+ (10 or 6 ꢁM) was further added into relevant solutions, and the fluorescence of
competing samples were measured again.
Cell viability assay
Human cervical carcinoma cells (Hela) was purchased from American Type Culture
Collection (ATCC, USA). And Hela cells were cultured in highꢀglucose DMEM
(HyClone, CA) supplemented with 10% FBS (HyClone, CA) and 1%
penicillin/streptomycin. Cells were cultured at 37 °C with 5% CO2.

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Hela cells were plated in 96ꢀwell plates at 2000 cells per well in 100 ꢁL medium and
cultured for 24 hours. The cells were further incubated with DPP-C2/DPP-Mito (10
ꢁM) for another 24 hours. Then cell proliferation was analyzed by the Cell Counting
Kitꢀ8 assay (Dojindo, Japan) according to the manufacturer’s instructions. Briefly, 10
ꢁL of CCKꢀ8 solution was added to each well and cells were cultured at 37 °C for 2
hours. The absorbance at 450 nm was measured with a synergy H1 MultiꢀMode
Microplate Reader (Bio Tek, USA).
Cell culture and imaging
After incubated by DPP-C2/DPP-Mito for 24 hours, mitochondria were stained by
100 nM MitoTracker® Green FM (Invitrogen, USA) for 1 hour. Cell nuclei were
stained with blueꢀfluorescent DAPI. Hela cells were observed under a laser scanning
confocal microscope (Nikon A1, Japan) with 1000X magnification Coꢀlocalization
was measured with Image J as Pearson’s correlation value. 3D images were collected
using NISꢀElements Viewer 4 software. Cells shown are representative images from
replicate experiments (n = 5).
Hela Cells were plated in 6 well plates at 2×
105 per well and cultured for 24 hours.
Then cells were incubated with DPP-C2/DPP-Mito (10 ꢁM) for 24 hours, followed
by further incubation with zinc pyrithione (50 ꢁM) for 10 minutes or
N,N,N′,N′ꢀtetrakis(2ꢀpyridylmethyl)ethylenediamine (TPEN) (60 ꢁM) for 10 minutes.
The images were obtained under a 400× magnification fluorescence microscope
(Nikon TiꢀS, Japan).
Synthesis

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Synthesis of 2. A mixture of compound 1 (1 g, 3.33 mmol), tꢀbutyl bromoacetate
(0.78 g, 4 mmol), K2CO3 (0.92 g, 8.33 mmol) and catalytic amounts of NaI in 50 mL
o
DMF was stirred for 24 h at 55 C. The resulting mixture was extracted with
dichloromethane, dried with anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by column chromatography using a
mixture of ethyl acetate/petroleum ether (v/v = 1/2) as the eluent to give brown solid
(0.55 g, 38.25%). 1H NMR (500 MHz, CDCl3): δ 11.50 (s, 1H), 8.64 (d, J = 3.55, 1H),
8.26 (d, J = 3.55, 1H), 8.03 (t, J = 5.55, 2H), 7.30ꢀ7.42 (m, 2H), 4.75 (s, 2H), 1.40 (s,
9H). 13C NMR (125 MHz, CDCl3): δ 167.79, 162.38, 160.18, 138.43, 138.13, 134.17,
132.54, 132.49, 130.89, 129.96, 129.36, 129.08, 109.06, 106.65, 82.65, 44.11, 28.02.
Synthesis of 3. A mixture of compound 2 (0.1 g, 0.24 mmol), bromoethane (0.11 g, 1
mmol), K2CO3 (0.1 g, 0.72 mmol) and catalytic amounts of KI in 20 mL DMF was
o
stirred for 2 h at 80 C. Then the resulting mixture was extracted with
dichloromethane, dried with anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by column chromatography using a
mixture of dichloromethane/petroleum ether (v/v = 1/1) as the eluent to give brown
1
solid (0.064 g, 62.75%). H NMR (500 MHz, CDCl3): δ 8.88 (dd, J1 = 1.1 Hz, J2 =
3.95 Hz, 1H), 8.79 (dd, J1 = 1.00 Hz, J2 = 3.85 Hz, 1H) , 7.61ꢀ7.68 (m, 2H), 7.27ꢀ7.31
(m, 2H), 4.80 (s, 2H), 4.18 (q, J = 7.15, 2H), 1.45 (s, 9H ), 1.39 (t, J = 7.15, 3H). 13C
NMR (125 MHz, CDCl3): δ 167.31, 161.37, 160.87, 140.62, 139.25, 135.40, 134.68,
131.00, 130.36, 129.88, 129.54, 128.73, 128.68, 108.48, 106.91, 82.82, 44.12, 37.12,
27.97, 15.08.

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Synthesis of 4. A mixture of 1,4ꢀdibromobutane (2 g, 9.26 mmol), triphenyphosphine
(2.43 g, 9.26 mmol) in 30 mL toluene was refluxed for 12 h under nitrogen. Then the
resulting mixture was cooled to room temperature, the solid was filtrated and washed
o
with petroleum ether. The residue was dried under vacuum at 60 C to give white
1
solid (1.85 g, yield = 41.78%). H NMR (500 MHz, CDCl3): δ 7.42ꢀ7.58 (m, 15H),
3.54 (m, 2H), 3.24 (t, J = 6.2, 2H), 1.98 (m, 2H), 1.54 (m, 2H). 13C NMR (125 MHz,
CDCl3): 135.01, 134.98, 133.47, 133.39, 130.43, 130.33, 118.07, 117.38, 33.40, 32.02,
21.67, 20.72.
A mixture of compound 2 (0.2 g, 0.48 mmol), (4ꢀbromobutyl)triphenylphosphonium
bromide (0.23 g, 0.48 mmol), K2CO3 (0.2 g, 1.45 mmol) in 20 mL DMF was stirred
for 8 h at 30 oC. Then the resulting mixture was extracted with dichloromethane, dried
with anhydrous sodium sulfate and concentrated under reduced pressure. The crude
product was purified by column chromatography using
a
mixture of
dichloromethane/methanol (v/v = 15/1) as the eluent to give brown solid (0.23 g,
1
59%). H NMR (500 MHz, CDCl3): δ 8.78 (dd, J1 = 0.8 Hz, J2 = 3.85 Hz, 1H), 8.65
(dd, J1 = 0.85 Hz, J2 = 3.85 Hz, 1H) , 7.56ꢀ7.78 (m, 17H), 7.21ꢀ7.28 (m, 2H), 4.76 (s,
2H), 4.23 (t, J = 5.7, 2H), 3.85 (m, 2H), 2.21 (m, 2H), 1.65 (m, 2H), 1.46 (s, 9H ). 13C
NMR (125 MHz, CDCl3): δ 167.31, 161.73, 160.60, 140.39, 139.48, 135.60, 135.05,
135.03, 134.65, 133.73, 133.65, 131.91, 130.97, 130.48, 130.37, 129.78, 129.11,
128.75, 128.67, 118.27, 117.58, 107.71, 106.94, 82.95, 44.07, 39.85, 30.14, 28.01,
21.85, 19.29.
Synthesis of 5. A mixture of compound 3 (0.064 g, 0.17 mmol) in 5 mL TFA was

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stirred for 4 h at room temperature. Then the resulting mixture was concentrated
under reduced pressure. The resulting brown solid was used in the next reaction
without further purification.
Synthesis of 6. A mixture of compound 4 (0.23 g, 0.28 mmol) in 5 mL TFA was
stirred for 4 h at room temperature. Then the resulting mixture was concentrated
under reduced pressure. The resulting brown solid was used in the next reaction
without further purification.
Synthesis of DPP-C2. A mixture of compound 5 (100 mg, 0.26 mmol), TBTU (100
mg, 0.26 mmol), DIPEA (100 mg, 0.78 mmol) in 8 mL DMF was stirred for half an
hour
at
room
temperature.
To
this
mixture,
Nꢀ(4ꢀaminoꢀ2ꢀmethoxyphenyl)ꢀN’,N’ꢀ[bis(2ꢀpyridylmethyl)]ethylenediamine (141 mg,
0.39 mmol) was added and stirred for another 12 h at room temperature. The resulting
mixture was extracted with dichloromethane, dried with anhydrous sodium sulfate
and concentrated under reduced pressure. The crude product was purified by column
chromatography using a mixture of dichloromethane/methanol (v/v = 100/1) as the
eluent to give red solid (0.12 g, 63.16%). 1H NMR (500 MHz, DMSOꢀd6): δ 10.13 (s,
1H), 8.75 (t, J = 4, 2H), 8.48 (d, J = 4.2, 2H), 8.12 (d, J = 4.8, 1H), 8.05 (d, J = 4.9,
1H), 7.74 (m, 2H), 7.49 (d, J = 7.75, 2H), 7.40 (m, 2H), 7.23 (m, 2H), 7.15 (d, J = 1.8,
1H), 6.85 (dd, J1 = 1.8, J2 = 8.5 1H), 6.36 (d, J = 8.55, 1H), 4.83 (s, 2H), 4.09 (q, J =
7.05, 2H), 3.80 (s, 4H), 3.77 (s, 3H), 3.12 (t, J = 5.50, 2H), 2.72 (t, J = 5.30, 2H), 1.28
(t, J = 6.95, 3H). 13C NMR (125 MHz, DMSOꢀd6): δ 164.92, 160.91, 160.77, 159.53,
149.20, 146.56, 140.30, 139.73, 136.93, 135.22, 134.90, 134.69, 133.43, 133.26,

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130.01, 129.49, 129.22, 129.04, 128.38, 123.11, 122.59, 112.55, 109.51, 107.37,
106.74, 103.42, 60.04, 55.80, 52.55, 45.28, 41.01, 37.07, 15.31. ESIꢀMs calcd for
[C41H39N5O4S2], 731.23; found, 731.1 [M +].
Synthesis of DPP-Mito. A mixture of compound 6 (180 mg, 0.24 mmol), TBTU (77.1
mg, 0.24 mmol), DIPEA (93.1 mg, 0.72mmol) in 8 ml DMF was stirred for half an
hour
at
room
temperature.
To
this
mixture,
Nꢀ(4ꢀaminoꢀ2ꢀmethoxyphenyl)ꢀN’,N’ꢀ[bis(2ꢀpyridylmethyl)]ethylenediamine (130 mg,
0.36 mmol) was added and stirred for 12 h at room temperature. The resulting mixture
was extracted with dichloromethane, dried with anhydrous sodium sulfate and
concentrated under reduce pressure. The crude product was purified by column
chromatography using a mixture of dichloromethane/methanol (v/v = 10/1) as the
eluent to give red solid (0.16 g, 61.54%). 1H NMR (500 MHz, CDCl3): δ 10.8 (s, 1H),
8.61 (t, J = 3.8, 2H), 8.48 (d, J = 4.25, 2H), 7.43ꢀ7.69 (m, 22H), 7.21 (m, 2H), 7.11 (m,
3H), 6.87 (t, J = 4.15, 1H), 6.32 (d, J = 8.55, 1H), 5.11 (s, 2H), 4.06 (t, J = 5.0, 2H),
3.85 (s, 4H), 3.77 (br, 5H), 3.14 (t, J = 5.35, 2H), 2.86 (t, J = 5.55, 2H), 2.06 (t, J =
6.05, 2H), 1.65 (br, 2H). 13C NMR (125 MHz, CDCl3): δ 165.42, 161.33, 160.86,
159.14, 148.88, 146.76, 140.45, 139.22, 136.52, 135.04, 135.02, 134.46, 134.42,
133.54, 133.46, 131.53, 131.19, 130.45, 130.35, 130.10, 129.68, 129.29, 128.27,
128.26, 123.02, 122.13, 118.36, 117.68, 112.62, 111.06, 109.99, 107.21, 107.11,
103.39, 60.28, 55.61, 52.91, 45.80, 41.58, 39.93, 30.37, 21.84, 19.61. ESIꢀMs calcd
for [C59H55N7O4PS2 ] (loss of Br¯), 1020.35; found, 1020.2 [M +].
+

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