A mitochondria-specific fluorescent probe for visualizing endogenous hydrogen cyanide fluctuations in neurons

Article abstract of DOI:10.1021/jacs.7b12545

An ability to visualize HCN in mitochondria in real time may permit additional insights into the critical toxicological and physiological roles this classic toxin plays in living organisms. Herein, we report a mitochondria-specific coumarin pyrrolidinium-derived fluorescence probe (MRP1) that permits the real-time ratiometric imaging of HCN in living cells. The response is specific, sensitive (detection limit is ca. 65.6 nM), rapid (within 1 s), and reversible. Probe MRP1 contains a benzyl chloride subunit designed to enhance retention within the mitochondria under conditions where the mitochondria membrane potential is eliminated. It has proved effective in visualizing different concentrations of exogenous HCN in the mitochondria of HepG2 cells, as well as the imaging of endogenous HCN in the mitochondria of PC12 cells and within neurons. Fluctuations in HCN levels arising from the intracellular generation of HCN could be readily detected.

Full text of DOI:10.1021/jacs.7b12545

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Article
A Mitochondria-Specific Fluorescent Probe for Visualizing
Endogenous Hydrogen Cyanide Fluctuations in Neurons
Lingliang Long, Meiyu Huang, Ning Wang, yanjun wu, kun
wang, Aihua Gong, zhijian zhang, and Jonathan L. Sessler
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12545 • Publication Date (Web): 16 Jan 2018
Downloaded from http://pubs.acs.org on January 16, 2018
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A Mitochondria-Specific Fluorescent Probe for Visualizing
Endogenous Hydrogen Cyanide Fluctuations in Neurons
†
,‡
‡
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‡
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Lingliang Long, Meiyu Huang, Ning Wang, Yanjun Wu, Kun Wang, Aihua Gong, Zhijian
§
*,†
Zhang, Jonathan L. Sessler
†Department of Chemistry, The University of Texas at Austin, 105 East 24th Street, Stop A5300, Austin, Texas 78712-1224,
United States
‡
2
Scientific Research Academy & School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu
12013, P. R. China.
§
School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P. R. China
Supporting Information Placeholder
drogen cyanide (HCN) due to its pKa value of 9.3. In leukocytes,
HCN is produced by the myeloperoxidase-mediated oxidative
chlorination of glycine or N-terminal glycyl peptides during
phagocytosis, and is used as a defense against bacteria, fungi, and
ABSTRACT: An ability to visualize HCN in mitochondria in
real time may permit additional insights into the critical toxicolog-
ical and physiological roles this classic toxin plays in living or-
ganisms. Herein, we report a mitochondria-specific coumarin
pyrrolidinium-derived fluorescence probe (MRP1) that permits
the real time ratiometric imaging of HCN in living cells. The re-
sponse is specific, sensitive (detection limit is ca. 65.6 nM), rapid
7
other pathogens. It has also been suggested that HCN is generat-
ed within neurons upon the activation of opioid receptors by en-
8
dogenous or exogenous opioids. The HCN generated in this way
has been proposed as a novel neuromodulator within the central
and peripheral nervous system in loose analogy to nitric monoxide
(
within 1 s), and reversible. Probe MRP1 contains a benzyl chlo-
ride subunit designed to enhance retention within the mitochon-
dria under conditions where the mitochondria membrane potential
is eliminated. It has proved effective in visualizing different con-
centrations of exogenous HCN in the mitochondria of HepG2
cells, as well as the imaging of endogenous HCN in the mito-
chondria of PC12 cells and within neurons. Fluctuations in HCN
levels arising from the intracellular generation of HCN could be
readily detected.
9
(
NO), carbon monoxide (CO), and hydrogen sulfide (H
2
S). Un-
fortunately, experimental support for the putative role of HCN in
neurons remains elusive. Mitochondria have been suggested as
being the main locus for the production of HCN within the neu-
8
c
rons; however, this is far from established. Effective HCN
probes that operate within mitochondria may allow deeper in-
sights into the critical toxicological and physiological roles of
HCN within living organisms. As detailed below, we have now
prepared a probe that appears promising in this regard.
Fluorescent probes have received considerable attention due to
the high sensitivity, fast response time, technical simplicity, and
INTRODUCTION
1
0
applicability to biological milieus. In combination with laser
scanning confocal microscope (LSCM), appropriately designed
fluorescent probes can be used to sense important species in sub-
–
1
The cyanide anion (CN ) is a recognized toxin. For humans
blood cyanide levels higher than 20 µM are considered toxic.
However, cyanide is widespread in the modern world, and cya-
nide exposure from gold mining, electroplating, metallurgy,
2
1
1
cellular organelles in a non-invasive manner. Currently, a large
number of fluorescent probes for cyanide are known. As a general
rule, these systems rely on the unique nucleophilic reactivity of
3
a
3
b
3c
consumption of certainꢀfoods, bacterial cyanogenesis, uncon-
12
3
d
3e
3f
the cyanide anion to trigger a change in the fluorescent response.
trolled fires, cigarette smoke, vehicle exhaust, and from
3g
pharmaceutical by-products, are potential concerns. At the sub-
Fluorescent probes for the cyanide anion that are based on its
1
3
+ 14
15
interactions with metal ions, H , and boronic acid derivatives
are also known. To date, cyanide anion probes have been used to
cellular level, the mitochondria are recognized as being the prima-
ry site of cyanide toxicity. This is because cyanide inactivates
mitochondrial cytochrome C oxidase and halts cellular respira-
13a, 16
sense endogenous cyanide in plant samples.
However, to our
4
knowledge, no cyanide anion probe has been used successfully to
monitor endogenous HCN levels in mitochondria.
tion. It also induces production of reactive oxygen species, in-
5
cluding hydroxyl radicals (·OH) within mitochondria. This leads
to the oxidative damage to mitochondrial DNA and further results
in mitochondrial dysfunction. This dysfunction is linked to a vari-
To be effective at the mitochondrial level, we suggest that a
fluorescent HCN probe needs to meet the following prerequisites:
(a) It should display a fast response upon exposure to HCN. (b)
Give rise to a so-called ratiometric output so as to reduce potential
artifacts associated with, e.g., photobleaching, probe distribution,
or changes in excitation intensity. (c) The induced changes
should be reversible so as to allow fluctuations in HCN levels in
cells and organelles to be monitored in real time. (d) Possess a
6a
ety of diseases, including Alzheimer’s disease, Huntington’s
6
b
6c
6d
6e
disease, Parkinson’s disease, Diabetes, and cancer.
On the other hand, despite its high toxicity, growing evidence
suggests that the cyanide anion is generated in specific cells and
1
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-9
plays important physiological roles within the human body. At
the physiological pH of 7.4, cyanide exists predominantly as hy-
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high inherent fluorescence quantum yield (Φ ) and be effective at
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low concentrations (25-500 nM). (e) Be retained in the mitochon-
dria when the mitochondria membrane potential is eliminated.
The latter requirement is particularly germane to the design of an
HCN probe. The negative mitochondrial membrane potential (Δψ)
typically allows the targeting of positively charged species to the
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mitochondria. However, high concentrations of HCN can lead to
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a loss in the mitochondrial membrane potential, leading to diffu-
sion of erstwhile targeted species into the cytoplasm. In such in-
stances, the further study of mitochondria-related changes in HCN
concentrations would be precluded.
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RESULTS AND DISCUSSION
Rational design of the Probe
Scheme 1. (a) Components making up the present series of HCN
probes. Note that different substituent are present at the 4-position
of the phenyl group; (b) proposed basis for the sensing response
of MRP1 and related HCN probes.
Bearing the above considerations in mind, a ratiometric fluo-
rescent probe for visualizing HCN within the mitochondria of
living cells was designed as shown in Scheme 1a. Full synthetic
details are provided in the Supporting Information. A 7-
diethylamino-coumarin dye (green color) was selected as the
fluorophore while a 1-methylenepyrrolidinium subunit (pink color)
was used as the reactivity-based recognition site for HCN. In pre-
liminary studies, we found that the 1-methylenepyrrolidinium
group can rapidly, specifically, and reversibly react with HCN. By
Upon excitation at 521 nm, compound 1a displayed a fluores-
cence emission band centered at 602 nm with an associated quan-
tum yield (Φ
fluorescence quantum yields of 1d (Φ
0
f
) of 0.184 (Fig. S1b, Table S1). Compared to 1a, the
= 0.166) and 1e (Φ
.062) were reduced. In contrast, compound 1b, had a higher
f
f
=
f
fluorescence quantum yield (Φ = 0.285). On the other hand,
tethering
a
7-diethylamino-coumarin moiety to the 1-
compound 1c displayed little appreciable fluorescence intensity
= 0.002).
methylenepyrrolidinium subunit through an ethylene linker, we
expected to have a system that would give rise to a ratiometric
fluorescence response to HCN as the result of modulating the
(Φ
f
To obtain insights into the optical properties of compounds 1a-
19
1e, time-dependent density functional theory (TD-DFT) calcula-
tions were carried out. The electron densities in the HOMO and
LUMO of these compounds in the ground state and first excited
singlet state are shown in Fig. S2. On the basis, it is concluded
that the HOMO→ LUMO transitions for compound 1a, 1b, 1d,
and 1e involve electron density redistributions from the EDG to
EWG. In other words, ICT is predicated upon excitation. In stark
contrast, in the first excited singlet state of compound 1c, the
HOMO→ LUMO transition involves an electron density redistri-
bution from the 4-diethylamino-phenyl group to the 7-
diethylaminocoumarin moiety, which acts as an electron acceptor,
not donor.
intramolecular charge transfer (ICT) interactions between the
electron donating coumarin and electron withdrawing pyrrolidini-
um groups (referred to as EDG and EWG, respectively). Perturb-
ing the ICT interactions between linked EDG and EWGs can have
2
0,21
a significant effect on the fluorescence emission.
In an effort
to study this effect in the present instance and optimize the fluo-
rescence quantum yield of the probe, a 4-substituted phenyl group
(
blue color) was tethered to the electron deficient 1-
methylenepyrrolidinium group. We hypothesized that changing
the substituents at the 4-position of the phenyl group might allow
the extent of the ICT interaction to be fine-tuned. Thus com-
pounds 1a-1e with different electron donating or electron with-
drawing substituents at the 4-position of the appended phenyl
group were designed and synthesized (Scheme 1a). Predicative
studies of these HCN probes, discussed below, then allowed for
the preparation of a mitochondrial probe, MRP1, suitable for use
as in vitro.
The dihedral angles between the 7-diethylamino-coumarin ring
and the 4-substituted phenyl ring of compound 1a-1e in the opti-
mized geometry are given in Table S2 and Fig. S3. In the ground
state (S
0
), the dihedral angles between the coumarin ring and the
phenyl ring in compounds 1a, 1b, 1d, and 1e were approximately
o
6
0 . In the first excited singlet state (S
1
) these dihedral angles
As a first step towards understanding the HCN response func-
tion of compounds 1a-1e, their optical properties were investigat-
ed (Fig. S1, Table S1). Compound 1a exhibited a dominant ab-
sorption feature centered at 521 nm, which is ascribed to an ICT
transition. In the case of the systems bearing electron withdrawing
substituents (Cl, CN) at the 4-position of the phenyl group, i.e., 1d
and 1e, slightly red-shift absorption bands were observed at 524
and 530 nm, respectively. In contrast, more substantial blue shifts
were seen upon the introduction of electron donating groups (i.e.,
OH, diethylamino); in fact, absorption features at 515 nm and 513
nm were seen for compounds 1b and 1c, respectively.
o
decrease to around 50 . The dihedral angle of compound 1c in its
ground (S
b, 1d, and 1e. Whereas in the S
dral angle for 1c increases to 88 . It was also found that the fluo-
rescence intensity of 1c increases with increasing solvent viscosi-
ty (Fig S4). Taken in concert, these findings lead us to suggest
that the fluorescence quenching of compound 1c is best ascribed
o
0
) state is 57 , which is similar to that calculated for 1a,
state, the corresponding dihe-
1
1
o
22
to a twisted intramolecular charge transfer (TICT) from the 4-
diethylamino phenyl group to the 7-diethylamino-coumarin.
The charge transfer process underlying the presumed ICT may
be described in terms of the charge transfer parameter fCT
=
Δq
D A D A
−Δq , where Δq and Δq denote the changes in the charge
of the donor and the acceptor, respectively, upon excitation from
2
3
the ground state to the lowest excited state. As shown in Table
S3, in the S state, compound 1a has fCT around 0.45. By varying
1
the substituents, the fCT value could be increased to 0.57 in the
case of 1e. Likewise, it decreases to about 0.39 in 1b. Thus, the
ICT strength can be fine-tuned by varying the substituents at the
4
-position of the phenyl group.
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Compound 1b displayed the highest fluorescence quantum
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yield within the series 1a-1e. The key para-oxygen atom was thus
retained in designing a putative ratiometric fluorescent probe for
HCN, namely MRP1 (Scheme 1a). Probe MRP1 differs from 1b
in that an additional benzyl chloride group (orange color) is in-
corporated into the framework. This group can react with free
thiol groups present in mitochondrial proteins and thus serve po-
tentially to immobilize the probe within mitochondria, even in
instances when the mitochondria membrane potential is eliminat-
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Spectral response of probe MRP1 to HCN
The ability of probe MRP1 to react with HCN was evaluated in
2
0 mM potassium phosphate buffer/acetonitrile (pH 7.4, 1:3 v/v)
at room temperature. In its free form, probe MRP1 exhibited a
fluorescence emission feature centered at 599 nm (Φ = 0.261)
f
under these conditions. Upon addition of cyanide (as its KCN
salt), the fluorescence emission of MRP1 at 599 nm gradually
decreased while a new emission feature at 490 nm was seen to
grow in (Fig. 1a). An isoemission point was clearly visible at 552
nm during this addition process. The emission ratio (I490/I599), an
indicator of a ratiometric response, was found to be linearly pro-
portional to the concentration of cyanide anion over the 0-35 µM
Fig. 1 (a) Changes in the fluorescence emission spectra of MRP1
(1 µM) seen upon the addition of increasing quantities cyanide (0-
60 µM as its KCN salt) at room temperature (λex = 467 nm). (b)
Time dependent changes in the fluorescence emission intensities
recorded at 490 nm and 599 observed upon treatment MRP1 (1
µM) with cyanide (60 µM). (c) Changes in the fluorescence inten-
sity of MRP1 (1 µM) seen upon the sequential addition of first
-1
concentration range (Fig. S5). Based on a signal-to-noise (S N )
cyanide (60 µM) and then AgNO
599 nm); (■) MRP1, (▲) MRP1 + CN , (●) MRP1 + CN + Ag-
NO . (d) Ratiometric fluorescence response of MRP1 (1 µM)
3
(60 µM) (λex = 467 nm, λem =
-
-
ratio of 3, a detection limit of 65.6 nM was calculated; this is
much lower than the concentration of HCN typically found in the
3
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b
brain of healthy individuals (2-10 µM). The absorption maxi-
seen upon exposure to 60 µM of various putative analytes. Inset:
Photos of MRP1 (1 µM) taken in the presence of various species
(60 µM) under the illumination of a handheld UV lamp. All stud-
ies were carried out in 20 mM potassium phosphate buff-
er/acetonitrile, pH 7.4, 1:3 v/v.
mum of probe MRP1 was also found to undergo a blue shift
(
from 516 nm to 411 nm) upon treatment with cyanide (Fig S6).
The ratiometric fluorescence response of MRP1 to cyanide was
also evaluated at different pH values. On the basis of the results
obtained (cf. Fig. S7) it is concluded that MRP1 can be used to
detect cyanide over the pH 5–8 range. The stability of MRP1 in
aqueous solution was tested by means of fluorescence spectral
analyses. In various mixtures of acetonitrile and buffer, no change
in the fluorescence signature was seen over the course of 24 h,
leading us to infer that MRP1 is stable over at least such a time
The response of MRP1 towards other putative analytes was al-
so explored. When MRP1 (1 µM) was treated with either of other
–
–
–
–
–
physiologically relevant anions (e.g., F , Cl , Br , I , CH
3
COO ,
–
–
–
–
+
+
+
3 3 4 3
HSO , NO , ClO , and N ; studied as their Na , Li , or K
salts) or typical biological molecules (Glucose, Gly and Cys; all at
60 µM), no significant change was observed in the(I490/I599) emis-
sion ratio (Fig. 1d). Only when cyanide was added was a noticea-
ble response observed. This response toward cyanide was retained
in the presence of other relevant species, most of which produced
no appreciable or only minimal interference (Fig. S10). This se-
lectivity was apparent to the naked eye. Under conditions of illu-
mination using a handheld UV lamp cyanide produced a distinc-
tive color signature with MRP1 not seen in the case of other puta-
tive analytes (Fig. 1d).
3
scale (Fig. S8). The effect of the relative concentration of CH CN
was also investigated (Fig. S9); on this basis, it was determined
that MRP1 can be used to sense cyanide even in aqueous solu-
tions containing only 30% acetonitrile.
Time-dependent studies of the fluorescence changes produced
when MRP1 is treated with cyanide revealed a response that oc-
curs within ≤1 s (Fig. 1b). The fast response time is considered
beneficial for a probe designed to monitor HCN concentrations in
real time. After reacting with cyanide, probe MRP1 was treated
3
with AgNO (Fig. 1c). Sequential additions of cyanide and sil-
Compound 1a displayed a similar ratiometric fluorescence re-
sponse to cyanide (Fig S11). However, its structure is much sim-
pler than that of MRP1. Therefore, for clarity, compound 1a was
used to investigate the sensing reaction mechanism. The reaction
product of 1a with cyanide was isolated and characterized. As
ver(I) led to a turning off and on of the 599 nm fluorescent fea-
ture. On this basis, we conclude that MRP1 may allow for the
reversible monitoring of HCN fluctuations in living cells.
1
13
inferred from H NMR and C NMR spectral studies and mass
spectrometric analyses (Fig. S12 - Fig. S14), the optical response
seen in the case of 1a and by extension MRP1 reflects the nucle-
ophilic
addition
of
cyanide
anion
to
the
1-
methylenepyrrolidinium moiety present in these two congeneric
probes. This addition interferes with an efficient ICT from the
EDG to the EWG, which, in turn, results in a blue shift in both the
emission and absorption spectral features (Scheme 1b). Support
for this conclusion came from TD-DFT calculations (Figs. S15
and S16).
Fluorescence imaging of HCN in the mitochondria
of living cells
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To assess the mitochondrial staining ability of MRP1, co-
localization experiments were carried out using Mitotracker Deep
Prior to testing MRP1 as a sensor for endogenous cyanide, its
ability to function as ratiometric fluorescence probe for exoge-
nous HCN was examined. It was found that HepG2 cells loaded
with MRP1 alone showed negligible fluorescence in the green
channel but a strong fluorescence in the red channel (Fig. 4a-4c).
On this basis, it is concluded that negligible quantities of HCN are
present in HepG2 cells. After the addition of 15 µM KCN, which
is largely transformed to HCN under physiological conditions, the
fluorescence seen in the red channel decreased, while that in the
green channel increased (Fig. 4d-4f). In the presence of 40 µM
cyanide, the fluorescence in the red channel was almost fully
quenched, while that of the green channel became commensurate-
ly brighter (Fig. 4g-4i). The subsequent addition of cyanide, lead
to an increase in green-to-red fluorescence ratio (Fgreen / Fred) (Fig
4m). The increase was found to be linearly proportional to the
cyanide concentration, leading us to suggest that probe MRP1 is
potentially suitable for quantitative studies (Fig. 4n).
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Red (MTDR), a commercially available mitochondrial dye. In
these studies, HepG2 cells were co-incubated with MRP1 (0.5
µM) and MTDR (0.5 µM) at 37 °C for 15 min. As can be seen by
inspection of Fig. 2c, a clear mitochondria profile with strong
near-infrared (NIR) fluorescence was observed in the NIR chan-
nel, which was ascribed to MTDR. A similar mitochondria profile
was observed in the red channel, which is largely reflective of
probe MRP1 (Fig. 2b). The MRP1 response coincides well with
that of MTDR (Fig. 2d) as reflected in a Pearson’s co-localization
coefficient of 0.94. Compound 1b was also studied and likewise
found to display mitochondria-specific staining (Fig. 2e-2h). Con-
sidered in concert, these results provide support for the notion that
both MRP1 and 1b act as organelle specific probes that target the
mitochondria.
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To verify that the fluorescence response was due to HCN, the
HepG2 cells loaded with cyanide were treated with methemoglo-
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7
bin (MHb; a scavenger of cyanide) to reduce the intracellular
levels of HCN. In the presence of MHb, the fluorescence response
was significantly inhibited (Fig 4j-4l), as expected for a fluores-
cence response that is directly correlated to the HCN levels.
Fig. 2 Confocal image of HepG2 cells co-stained with either
MRP1 and MTDR or 1b and MTDR. (a)-(c) Bright field, red
(λem = 575-650 nm) and NIR (λem = 660-740 nm) images of
HepG2 cells co-stained with MRP1 and MTDR. (d) Intensity
profile of regions of interest (ROIs) in the co-stained HepG2 cells
as indicated by the white arrows in (b) and (c). (e)-(g) Bright
field, red and NIR images of HepG2 cells co-stained with 1b and
MTDR. (h) Intensity profile of the ROIs indicated in (f) and (g).
To test whether MRP1 would remain in the mitochondria after
the mitochondrial membrane potential was eliminated, the HepG2
cells were stained with MRP1 and then further incubated with
CCCP (carbonyl cyanide m-chlorophenyl hydrazone). CCCP is a
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reagent that can collapse the mitochondrial membrane potential.
Fig. 4 Confocal images of live HepG2 cells stained with MRP1
As inferred from the results summarized in Fig 3, MRP1 is re-
tained in the mitochondria after co-incubating with CCCP for 80
min. However, after treatment with CCCP, compound 1b was
found to diffuse from the mitochondria into the cytoplasm. The
difference between these two dye systems is ascribed to the pres-
ence of the benzyl chloride moiety in MRP1 that, per the design
expectations, acts as an immobilizing group.
(
0.5 µM) in the presence of different concentrations of exogenous
cyanide. (a)-(c) Images recorded over the green (λem = 460-530
nm) and red (λem = 575-650 nm) channels, and the corresponding
(Fgreen / Fred) ratio for HepG2 cells stained with MRP1 cells and
incubated with 0 µM cyanide. (d)-(f) Green and red channel and
ratiometric imaging of HepG2 cells stained with MRP1 and incu-
bated with 15 µM cyanide. (g)-(i) Green and red channel and rati-
ometric imaging of MRP1 stained HepG2 cells incubated with 40
µM cyanide. (j)-(l) Green and red channel and ratiometric imaging
of MRP1 stained HepG2 cells pretreated with 40 µ M MHb, and
then incubated with 40 µ M cyanide. (m) Average intensity ratios
from images (c), (f), (i), and (l). (n) Linear relationship between
the average intensity ratio and cyanide concentration. Error bars
represent the standard deviation.
Next, the potential utility of MRP1 as a probe for of endoge-
nously generated HCN was evaluated. This was done using rat
pheochromocytoma cells (PC12 cells), neuron-like cells that are
Fig. 3 Confocal images of HepG2 cells stained with MRP1 (0.5
µM) or 1b (0.5 µM) in the presence of CCCP (10 µM) and rec-
orded at different time intervals. The images were produced by
monitoring the red channel (λem = 575-650 nm).
8
a
known to release HCN. Upon incubation with MRP1 for 15
min, a strong fluorescence in the green channel (Fig. 5a) and weak
fluorescence in the red channel (Fig. 5b) was seen. The fluores-
cence ratio (Fig. 5c) was enhanced in the case of the PC12 cells as
compared to what was seen with HepG2 cells (cf. Fig. 4c). Based
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Journal of the American Chemical Society
on the fluorescence ratio and the equation given in Fig 4n, the
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response in the PC12 cells was equivalent to that seen when the
HepG2 cells were treated with 8.1 µM cyanide.
Hydromorphine (HYDROM) is a type of µ-opiate agonist that
8
a
can stimulate nerve cells to produce HCN. After treatment with
0 µM HYDROM for 15 min, the fluorescence ratio seen in the
1
case of the PC12 cells was further enhanced. In this case, the en-
dogenous HCN concentration was calculated to be 23.0 µM. On
the other hand, pretreatment of the PC12 cells with 40 µM MHb
(
to remove the endogenous HCN) followed by incubatation with
MRP1 for 15 min led to little if any fluorescence being observed
in the green channel (Fig. 5g). However, under these conditions a
strong fluorescence response was seen in the red channel (Fig.
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Fig. 6 Confocal image of endogenous mitochondrial HCN in neu-
rons. (a) Bright field image. (b) Ratiometric (Fgreen / Fred) imaging
of the neuron cells after incubation with MRP1 (0.5 µ M) for 10
min. (c) Ratiometric (Fgreen / Fred) imaging of the cells in pane (b)
and then further stimulated with HYDROM (10 µ M) for 10 min.
(d) Ratiometric imaging of the cells in pane (c) that were then
treated with MHb (40 µ M).
5
h). The fluorescence ratio was reduced significantly (Fig. 5i, 5j),
and the HCN concentration was calculated to be only 0.2 µM.
CONCLUSION
In summary, we have prepared and characterized a mitochon-
dria-specific ratiometric fluorescence probe for HCN. This probe
relies on 7-diethylamino-coumarin as the fluorophore, a 1-
methylenepyrrolidinium group as the reactive cyanide recognition
and mitochondria targeting subunit, and a benzyl chloride group
to provide immobilization within mitochondria. Photophysical
and spectral characterization studies revealed that a lead probe
(
MRP1) reacts rapidly (within 1 s) and responds reversibly to
HCN with both high selectivity and sensitivity (the detection limit
is 65.6 nM). Notably, probe MRP1 is retained in the mitochondria
even when the mitochondria membrane potential is decreased. It
can be used to visualize different concentration of exogenous
HCN within the mitochondria of HepG2 cells and proved effec-
tive in monitoring fluctuations in endogenous HCN concentra-
tions within the mitochondria of PC12 Cells and neurons. It is
thus believed that probe MRP1 and related systems could emerge
as useful tools that might allow the critical toxicological and
physiological roles of HCN in human body to be understood in
greater detail.
Fig. 5 Confocal image of endogenous mitochondrial HCN in
PC12 cells. (a)-(c) Green (λem = 460-530 nm), red (λem = 575-650
nm) and ratiometric (Fgreen / Fred) imaging of PC12 cells incubated
with MRP1 (0.5 µ M). (d)-(f) Green, red, and ratiometric imaging
of PC12 cells incubated with MRP1 (0.5 µ M), and then incubat-
ed with 10 µ M HYDROM. (g)-(i) Green, red, and ratiometric
imaging of PC12 cells pretreated with 40 µM MHb and then incu-
bated with MRP1 (0.5 µ M). (j) Average intensity ratios from
images (c), (f), and (i). Error bars represent the standard devia-
tions.
The above results provide support for the suggestion that probe
MRP1 may be used to detect different concentrations of endoge-
nous HCN in PC12 cells. Due to the fast response time seen in the
initial predicative studies discussed above, a further effort was
made to test whether MRP1 could be applied to visualize fluctua-
tions in HCN levels in neurons. The neurons were differentiated
from the neural stem cells as detailed in the Supporting Infor-
mation. After incubating the neurons with MRP1 for 15 min, the
fluorescence ratio (Fgreen / Fred) was determined to be 0.89 (Fig.
ASSOCIATED CONTENT
Supporting Information: Synthetic details, the method of
cell culture and fluorescence imaging, and other spectroscopic
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
6
b). On this basis, the concentration of the endogenous HCN was
calculated to be 8.4 µM. The cells were also stimulated with
HYDROM (10 µM) for 15 min. Under these conditions, the fluo-
rescence ratio increased to 2.09 (Fig. 6c). This value corresponds
to an HCN concentration of 21.7 µM. When the HCN scavenger
MHb (40 µM) was added to these cells, the fluorescence ratio
immediately decreased to 0.18 (Fig. 6d). Thus, we suggest that
MRP1 can be used to monitor in real time variations in endoge-
nous HCN concentrations within the mitochondria of neurons. To
our knowledge this represents the first instance wherein a fluores-
cent probe is used to visualize endogenous HCN in nerve cells.
*sessler@cm.utexas.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The work in Austin was supported by the National Institutes of
Health (RGM 103790A to J.L.S.) and the Robert A. Welch Foun-
dation (F-0018). L. Long gratefully acknowledges the program of
China Scholarships Council (201608320096 to L.L.) and the Na-
tional Sciences Foundation of China (21202063 to L.L.) for finan-
cial support.
The cytotoxicity of MRP1 in HepG2 cells was determined us-
ing a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoli-
um bromide (MTT) assay. On the basis of the results obtained, we
infer that the low cytotoxicity of MRP1 in live cells is relatively
low (≥95% cell viability at a concentration of 5 µM; cf. Fig. S17).
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Mishra, S.; Mishra, S. K.; Suresh, E.; Das, S.; Das, A. Org. Lett. 2010, 12,
406. (c) Sharma, S.; Hundal, M. S.; Hundal, G. Org. Biomol. Chem.
2013, 11, 654.
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Graphical Abstract
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