Mitochondrial thioredoxin-responding off-on fluorescent probe

Article abstract of DOI:10.1021/ja308446y

We synthesized a new probe, Mito-Naph, to visualize mitochondrial thioredoxin (Trx) activity in cells. A fluorescence off-on change is induced by disulfide cleavage of the probe, resulting from a reaction with Trx and subsequent intramolecular cyclization by the released thiolate to give a fluorescent product. By measuring the fluorescence at 540 nm, Trx activity can be detected at nanomolar concentrations (down to 50 nM) well below its physiological levels. The in vitro and in vivo Trx preference of Mito-Naph was demonstrated by fluorometric and confocal microscopic experiments. In vitro kinetic analysis of the disulfide bond cleavage revealed that the second-order rate constant for Trx is (4.04 ± 0.26) × 10³ (M s) ^-1, approximately 5000 times faster than that for GSH. The inhibition experiments involving PX-12, a selective inhibitor of Trx, also revealed that the emission from Mito-Naph significantly decreased in PX-12 dose-dependent manners, both in living cells and in cellular protein extracts. The Trx preference was further supported by an observation that the fluorescence intensity of rat liver extract was decreased according to the Trx depletion by immunoprecipitation. On the basis of these results, it is concluded that Mito-Naph preferentially reacts with Trx, compared with other biological thiols containing amino acids in vitro and in vivo.

Full text of DOI:10.1021/ja308446y

Article
pubs.acs.org/JACS
Mitochondrial Thioredoxin-Responding Off−On Fluorescent Probe
Min Hee Lee,†,∥ Ji Hye Han,‡,∥ Jae-Hong Lee,† Hyo Gil Choi,§ Chulhun Kang,*,‡ and Jong Seung Kim*,†
†Department of Chemistry, Korea University, Seoul, 136-701, Korea
‡The School of East-West Medical Science, Kyung Hee University, Yongin, 446-701, Korea
§Protected Horticulture Research Station, National Institute of Horticultural and Herbal Science, Rural Development Administration,
Busan, 618-800, Korea
S
* Supporting Information
ABSTRACT: We synthesized a new probe, Mito-Naph, to visualize mitochondrial
thioredoxin (Trx) activity in cells. A fluorescence off−on change is induced by disulfide
cleavage of the probe, resulting from a reaction with Trx and subsequent intramolecular
cyclization by the released thiolate to give a fluorescent product. By measuring the
fluorescence at 540 nm, Trx activity can be detected at nanomolar concentrations (down to
50 nM) well below its physiological levels. The in vitro and in vivo Trx preference of Mito-
Naph was demonstrated by fluorometric and confocal microscopic experiments. In vitro
kinetic analysis of the disulfide bond cleavage revealed that the second-order rate constant for
Trx is (4.04 0.26) × 103 (M s)−1, approximately 5000 times faster than that for GSH. The
inhibition experiments involving PX-12, a selective inhibitor of Trx, also revealed that the emission from Mito-Naph significantly
decreased in PX-12 dose-dependent manners, both in living cells and in cellular protein extracts. The Trx preference was further
supported by an observation that the fluorescence intensity of rat liver extract was decreased according to the Trx depletion by
immunoprecipitation. On the basis of these results, it is concluded that Mito-Naph preferentially reacts with Trx, compared with
other biological thiols containing amino acids in vitro and in vivo.
discussing what is the true target in thiol sensing, the
abundance of each thiol and the reactivity for the probe need
to be carefully evaluated, which has never been demonstrated in
the literatures dealing with thiol sensing so far.
The goal of the present study is to develop a probe able to
detect the thiols in mitochondria and to characterize its
selectivity on the basis of comparison of the reactivity of Trx
with that of GSH, considering their abundances. In particular,
the mitochondrial Trx level would be informative in
investigating cellular functions related to cancer. This method-
ology has never been exploited, to the best of our knowledge,
and promises to enable researchers to design new drugs in the
future.
Here, we report the synthesis and biological application of a
fluorescent naphthalimide-based probe (Mito-Naph). It has a
mitochondrial guiding moiety and disulfide bond to bring it to
the mitochondria and undergo fluorogenic changes upon thiol-
catalyzed disulfide bond cleavage.27,29 The naphthalimide unit
provides a suitable fluorescent scaffold owing to its excellent
spectroscopic characteristics such as outstanding internal charge
transfer (ICT) efficiency, high quantum yield, and structural
flexibility for introducing various modifications.27,30 The
triphenylphosphonium headgroup guides it to the mitochon-
dria and also improves its water solubility.29 The combination
of these variations is believed to make in vitro mitochondrial
INTRODUCTION
■
Intracellular thiols play essential roles in redox regulation of cell
growth, apoptosis inhibition, DNA synthesis, and angio-
genesis.1−3 The major thiols of the redox system are
glutathione (GSH) and thioredoxin (Trx).4,5 Trx is primarily
involved in the reducing process of protein disulfides such as
ribonucleotide reductase,6 methionine sulfoxide reductases,7
and peroxiredoxins.8 Furthermore, the overexpression of Trx in
intracellular and extracellular compartments has been impli-
cated in several types of cancers, cardiovascular diseases,
etc.9−12 In addition, mitochondrial Trx is essential in
scavenging oxidatively damaged mitochondrial proteins,13
regulating mitochondrial permeability transition,14−16 and
participating against apoptosis.17−19 Moreover, inappropriate
levels of mitochondrial Trx are directly associated with
mitochondria dysfunctions, resulting in increased reactive
oxygen species, higher apoptosis rate, and lower growth.16,20−23
Developing sensitive and specific probes toward a thiol is of
critical importance for understanding its roles in disease. On
discussion of thiol-sensing in biological samples, GSH is always
at the center of attention because it is the most abundant thiol
in cells.24 On the other hand, Trx has been mostly overlooked
in vivo or in vitro thiol sensing simply because its cellular level
seems to be negligible compared to that of GSH (approx-
imately 0.1% of the level of GSH).25 However, it should be
reminded that Trx can catalyze disulfide reduction much faster
than dithiothreitol or GSH.26−28 So, it may be a hasty
conclusion that GSH be the most predominant species in the
thiol sensing, simply based on its abundance. Accordingly,
Received: August 25, 2012
© XXXX American Chemical Society
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thiol imaging possible in biological and sensory applica-
tions.27,29
RESULTS AND DISCUSSION
■
Mito-Naph was prepared by the synthetic route outlined in
Scheme 1. The naphthalimide derivatives 131 and 232 were
Scheme 1. Synthetic Route to Mito-Naph
Figure 1. Photophysical changes of Mito-Naph during its reaction with
Trx. (a) UV/vis absorption and (b) fluorescence spectra of Mito-Naph
(5.0 μM) in the absence (black) and presence (red) of Trx (5.0 μM).
(c) Fluorescence changes of Mito-Naph (1.0 μM) with increasing
concentrations of Trx (0−5.0 μM). (d) Linear correlation between the
fluorescence changes at 540 nm and the Trx concentrations (0−1.0
μM). All spectra were acquired 30 min after Trx addition at 37 °C in
PBS buffer (pH 7.4), with excitation λ = 428 nm.
synthesized by adapting published procedures. For the
synthesis of 4, 2 was reacted with 333 in the presence of 1-
ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI)/4-
(N,N-dimethylamino)pyridine (DMAP) in DMF. Compound
4 was then reacted with phosgene, followed by treatment with
2,2′-dithiodiethanol, to produce Mito-Naph in 63% yield. To
verify the mitochondrial-targeting role of the triphenylphos-
phonium unit in Mito-Naph, a structural analogue 9 without
triphenylphosphonium was also prepared (Supporting In-
formation, Scheme S1). The overall chemical structures of 4
1
and Mito-Naph were confirmed by H NMR, 13C NMR, HR-
FAB, and ESI-MS (Supporting Information, Figures S9−S17).
Mito-Naph absorbs and fluoresces at the maxima of 376 and
472 nm, respectively. Upon its addition to a Trx-containing
solution under physiological conditions (at 37 °C in PBS buffer,
pH 7.4), a new absorption band centered at 428 nm (yellow)
and a green emission band at 540 nm appeared concomitantly
(Figure 1a,b). The fluorescence spectra at various Trx
concentrations are also shown in Figure 1c. With an increasing
Trx concentration (0−5.0 μM), the fluorescence intensity at
472 nm decreased, along with the appearance of a new emission
band at 540 nm. The spectral change was initiated by disulfide
bond cleavage of Mito-Naph by Trx, which in turn was induced
by the released thiolate that mounted an intramolecular attack
on its amide carbonyl carbon to yield a five-membered
carbamate ring and the fluorescent product 4 (Figure 4). We
also found a linear correlation between the fluorescence
intensity at 540 nm and the Trx concentration (Figure 1d)
such that Trx can be detected at nanomolar concentrations
(down to 50 nM), below the physiological levels (5−10 μM) of
Trx in live cells.25,34
Before we attempted to detect Trx in cells with Mito-Naph,
interference from other biologically relevant analytes was
evaluated under simulated physiological conditions (at 37 °C
in PBS buffer of pH 7.4). The fluorescence intensity of Mito-
Naph was measured in the presence of amino acids, essential
metal ions, or hydrogen peroxide, and the results are depicted
in Figure 2. Upon reaction with Trx, the fluorescence of Mito-
Naph was enhanced at 540 nm by up to 14 times. In contrast,
Figure 2. Reactivity of Mito-Naph with Trx in the presence of other
putative nonthiol interferants. Fluorescence responses of Mito-Naph
(5.0 μM) toward (a) 5.0 mM of nonthiol amino acids (1, only probe;
2, Val; 3, Tyr; 4, Thr; 5, Tau; 6, Ser; 7, Pro; 8, Phe; 9, Met; 10, Lys; 11,
Leu; 12, Ile; 13, His; 14, Gly; 15, Gluc; 16, Glu; 17, Gln; 18, Asp; 19,
Asn; 20, Arg; 21, Ala; 22, Trp) and 5.0 μM of Trx (23) and (b) 1.0
mM of monovalent metal ions [24, K(I); 25, Na(I)] and 100 μM of
di- and trivalent metal ions [26, Ca(II); 27, Cu(II); 28, Fe(II); 29,
Fe(III); 30, Mg(II); 31, Zn(II)], and 5.0 mM of H2O2 (32). The bars
represent the fluorescence intensity at 540 nm. All fluorescence
changes were acquired 30 min after the addition of the analytes at 37
°C in PBS buffer (pH 7.4), with an excitation λ = 428 nm.
only an insignificant increase in the emission intensity with
other analytes was detected. Furthermore, it should be noted
that a new band at 428 nm was apparent in the absorption
spectrum only in the presence of Trx (Supporting Information,
Figure S1). Together with the data presented in Figure 2, these
results demonstrate clearly that reaction of the Mito-Naph with
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Trx is highly favored over other thiols and the accompanying
fluorescence enhancement is sufficient to detect cellular Trx
pools with negligible interference from other biologically
relevant analytes.
In consideration of the preference of Trx for catalyzing
disulfide cleavage reactions with Mito-Naph, fluorescence
experiments with other biological thiols, such as GSH, cysteine
(Cys), and homocysteine (Hcy), were also implemented. The
GSH and Trx are known to exist in concentrations of ∼10 mM
and ∼10 μM, respectively, in the human body.25,34 However,
since 10 μM of Trx can be easily oxidized in vitro due to its
high activity, the fluorescence changes of Mito-Naph were
investigated with 1.0 μM of Trx and 1.0 mM of GSH. As shown
in Figures 3 and S2 (Supporting Information), the initial
The dramatic Trx preference of Mito-Naph versus other
thiols (Table 1) seems quite unusual considering the fact that
the disulfide bond cleavage reaction has been widely used in
thiol-mediated sensors26−28,35−39 or drug delivery systems,40−42
where it is frequently presumed to occur with GSH or other
metabolic thiols such as Cys or Hcy. In contrast, to the best of
our knowledge, Trx-mediated disulfide bond cleavage has never
been reported in any sensory application yet. To extend our
understanding of the basis for the Trx preference in disulfide
bond cleavage, a structural analogue possessing a disulfide bond
with an ammonium (7)26,35 instead of a hydroxyl end group
was also prepared (Scheme S2 and see also Figures S18−S23,
Supporting Information), and its kinetic properties were
characterized (Supporting Information, Figures S5 and S6).
As shown in Table S1 (Supporting Information), similar to the
results with Mito-Naph, analogue 7 also shows a much higher
rate constant for Trx than for other thiols. Therefore, we firmly
conclude that regardless of the end group species, the disulfide
bond is preferentially cleaved by Trx, proposing that Trx could
contribute to the drug release through the disulfide-mediated
drug delivery systems.
To investigate the role of the Cys residues of Trx, the
products were analyzed by MALDI-TOF mass spectroscopy.
After the reaction with Trx, a peak at m/z 572.0, corresponding
to 4, was predominantly detected (Supporting Information,
Figure S8). We also obtained m/z values of 11 611.7 (Trx) and
11 610.3 (Trx-S2) before and after the reaction, respectively
(Supporting Information, Figures S7 and S8). These results
confirm that upon reaction with Mito-Naph, Trx was oxidized
to yield Trx-S2 and compound 4 is concomitantly produced
(Figure 4). We speculate that the oxidized Trx-S2 could be
reduced by a Trx reductase in cells.
Figure 3. Comparison of the fluorescence enhancement of Mito-Naph
in reactions with Trx, GSH, Cys, and Hcy. Fluorescence emission
intensities of Mito-Naph (1.0 μM) in the presence of 1.0 μM Trx, 1.0
mM GSH, or 100 μM Cys or Hcy. The bars represent the initial slope
of the change in fluorescence intensity at 540 nm as a function of time
(ΔF/Δt). All fluorescence changes were measured at 37 °C in PBS
buffer (pH 7.4), with an excitation λ = 428 nm.
reaction rate (ΔF/Δt) in the presence of GSH (1.4 × 10−2) is
slower than that with Trx (7.3 × 10−2), although GSH (1.0
mM) is more abundant (1000 times) than Trx (1.0 μM).
Moreover, only weak or no detectable changes in fluorescence
in the presence of 100 μM Cys (2.2 × 10−3) or Hcy (1.5 ×
10−4) were observed. From these findings, we infer that Mito-
Naph shows a selective response to Trx over other thiols, such
as GSH, Cys, and Hcy.
To gain insight into the preference of Trx for other thiols, the
reaction rate constants were calculated from kinetic analyses of
reactions between Mito-Naph and Trx, GSH, Cys, Hcy, or
dithiothreitol (DTT). The results are summarized in Table 1,
Table 1. Second-Order Rate Constants for the Reaction of
Mito-Naph with GSH, Trx, Cys, Hcy, and DTT
Figure 4. Proposed mechanism of the reaction of Mito-Naph with Trx.
a
b
thiols
k
k/kGSH
GSH
Trx
0.79 0.06
1.00
(4.04 0.26) × 103
1.06 0.06
5.12 × 103
1.06
To investigate the contribution of Trx to fluorescence
changes generated by Mito-Naph reaction in cells, confocal
microscopic observations were performed in the presence of
PX-12, a well-known inhibitor of Trx.43,44 The results (Figures
5a−c) indicate that the fluorescence intensity significantly
decreased in a dose (PX-12)-dependent manner (see also
Supporting Information, Figure S24). These results also imply
that the fluorescence intensity is mainly associated with a Trx-
mediated reaction and not with GSH or other metabolic thiols.
Moreover, the fluorescence intensity of Mito-Naph in crude
cytosolic protein extracts of HepG2 cells reciprocally depends
on the concentration of PX-12 (Figure 5d). The removal of Trx
by immunoprecipitation (IP) also significantly reduced
fluorescence intensity (Figure 5e). From these results, we
Cys
Hcy
DTT
0.58 0.02
0.73
2.28 0.07
2.88
a
b
The second order rate constants are expressed as (M s)−1. The
relative ratio of the rate constants to that of GSH.
and more detailed calculations and plots are shown in Figures
S3 and S4 (Supporting Information). We found that the
reaction kinetics with Trx, k = (4.04 0.26) × 103 (M s)−1, was
approximately 5000 times higher than with GSH. Moreover,
the k value for DTT, a strong reducing agent, was much lower
[k = 2.28 0.07 (M s)−1].
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of the MitoTracker over other Lyso- or ER Trackers, indicating
that Mito-Naph is localized to the mitochondria. In contrast,
this type of image overlap was not observed in the case of 9,
where mitochondria-targeting triphenylphosphine is not
present (Supporting Information, Figure S25), which is
strongly supportive that Mito-Naph reacts mainly with Trx in
mitochondria to emit fluorescence.
CONCLUSIONS
■
In conclusion, a new probe bearing naphthalimide, triphenyl-
phosphine, and disulfide moieties has been synthesized, and its
photophysical properties and biological application to sensing
Trx in a biological environment were examined. We found that
the probe can preferentially visualize the activity of Trx in
mitochondria, where fluorescence emission is induced by the
cleavage of a disulfide bond by the Trx. The colocalization
experiments using Mito-Naph and MitoTracker Red demon-
strated that Mito-Naph localized to the mitochondria. The in
vitro kinetic analysis of disulfide bond cleavage showed that the
reaction rate constant for Trx is (4.04 0.26) × 103 (M s)−1,
which means approximately 5000 times faster cleavage than
that for the more abundant GSH. The in vivo preference of
Mito-Naph for Trx was evident from the confocal microscopic
and fluorometric experiments, which revealed that the use of an
inhibitor of Trx (PX-12) markedly decreases the intracellular
fluorescence in the HepG2 cells and in cell extracts. The
preference for Trx was further supported by a concomitant
decrease of fluorescence intensity in cell extracts after Trx was
depleted by immunoprecipitation. Therefore, Mito-Naph is a
mitochondrial Trx probe in cells and could play indispensable
roles for understanding the mitochondrial Trx system in vitro
and in vivo.
Figure 5. Fluorescence changes of Mito-Naph (5.0 μM) in HepG2
cells and in cytosolic protein extracts. The cells were incubated with
media containing PX-12 at (a) 0, (b) 2.0, and (c) 10.0 μM at 37 °C.
After 24 h, Mito-Naph was added to each well and incubated for 20
min. Phase contrast (bottom) and fluorescence (top) images were
acquired using confocal microscopy at an excitation wavelength of 458
nm and a long path (>505 nm) emission filter. (d) Inhibition of
fluorescence intensity of Mito-Naph by PX-12 (0−50 μM) in HepG2
cytosolic protein (1.0 mg/mL, 10 mM of PBS buffer, pH 7.4). (e)
Effect of removing Trx by IP on the fluorescence of Mito-Naph (10.0
μM) before (cytosol) and after (IP supernatant). The immunor-
eactivity of cytosol and IP-S are shown in a Western blot. The
excitation and emission wavelengths were 450 and 535 nm,
respectively.
EXPERIMENTAL SECTION
■
firmly conclude herein that the disulfide bond cleavage reaction
of Mito-Naph is undergone by Trx in cells.
We also performed the colocalization experiments using
Mito-Naph and organelle trackers (Mito-, Lyso-, ER Tracker
Red) to determine the location of fluorescence emission. As
seen in Figures 6 and S26 (Supporting Information), the
fluorescence image of Mito-Naph mainly overlapped with that
Compounds 1,31 2,32 3,33 5,26 and 827 were prepared by adapting
published procedures.
Synthesis of 4. A mixture of 2 (1.1 g, 3.8 mmol), EDCI (0.74 g,
3.8 mmol), and DMAP (0.47 g, 3.8 mmol) in anhydrous DMF was
stirred under nitrogen for 30 min at room temperature. 3 (1.49 g, 3.8
mmol) was then added to the mixture. The reaction mixture was
stirred overnight. After removal of solvent, the crude product was
purified over silica gel using CH2Cl2/MeOH (v/v, 8:2) as the eluent to
yield 4 as a orange solid (1.0 g, 68%). ESI-MS: m/z (M+) calcd 572.2,
found 572.3 (M+). HR-FAB: m/z (M+) calcd 572.2103, found
572.2103. 1H NMR (CD3OD, 400 MHz): δ 8.41 (d, 1H, J = 7.3 Hz),
8.33 (d, 1H, J = 7.6 Hz), 8.10 (d, 1H, J = 8.1 Hz), 7.89−7.71 (m,
15H), 7.50 (t, 1H, J = 8.0 Hz), 6.74 (d, 1H, J = 8.2 Hz), 4.33 (t, 2H, J
= 7.0 Hz), 3.72−3.47 (m, 4H), 2.48 (t, 2H, J = 7.0 Hz). 13C NMR
(CD3OD, 100 MHz): 173.2, 164.9, 164.2, 153.4, 136.4, 135.2, 133.7,
131.3, 130.3, 129.1, 124.0, 122.1, 119.8, 118.7, 108.4, 108.0, 36.6, 34.6,
33.2, 22.0, 21.5 ppm.
Synthesis of 6. To a mixture of 4 (0.2 g, 0.3 mmol) and phosgene
(0.9 mL, 1.7 mmol) in 20 mL of CH2Cl2 was added N,N-
diisopropylethylamine (DIPEA) (0.4 mL, 2.4 mmol) dropwise. The
resulting solution was stirred for 3 h under nitrogen gas. The reaction
mixture was then flushed with nitrogen gas. After unreacted phosgene
gas was removed and neutralization occurred in an NaOH bath,
synthetic 5 (0.4 g, 1.4 mmol) in CH2Cl2 was added to the mixture.
The reaction mixture was stirred overnight. The solvent was
evaporated off, at which point CH2Cl2 (100 mL) and water (100
mL) were added, and the organic layer was collected. The CH2Cl2
layer was dried over anhydrous MgSO4. After removal of the solvents,
the crude product was purified over silica gel using DCM/MeOH (v/
v, 25:2) as the eluent to yield 6 as a yellow solid (0.2 g, 68%). ESI-MS:
m/z (M+) calcd 851.2, found 851.4 (M+). HR-FAB: m/z (M+) calcd
Figure 6. Colocalization experiments using Mito-Naph and Mito-
Tracker Red in HeLa cells. The cells were incubated with 5.0 μM of
Mito-Naph for 20 min at 37 °C, and the medium was replaced with
fresh medium containing 0.05 μM of MitoTracker Red and incubated
for 10 min. Images for Mito-Naph and MitoTracker Red were then
obtained using excitation at 458 and 633 nm and band path at (a)
470−600 nm and (b) 650−700 nm, respectively. Panel c is a merged
image of panels a and b. Panel d shows phase-contrast image.
1
851.2702, found 851.2701. H NMR (CDCl3, 400 MHz): δ 9.18 (s,
D
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1H), 9.12 (s, 1H), 8.53 (d, 1H, J = 8.5 Hz), 8.25 (d, 1H, J = 7.6 Hz),
8.04 (d, 1H, J = 8.2 Hz), 7.83−7.69 (m, 15H), 7.46 (t, 1H, J = 7.7 Hz),
5.34 (br s, 1H), 4.50 (t, 2H, J = 6.4 Hz), 4.38 (t, 2H, J = 6.9 Hz),
3.81−3.61 (m, 4H), 3.53−3.43 (m, 2H), 3.04 (t, 2H, J = 6.0 Hz), 2.84
(t, 2H, J = 6.4 Hz), 2.65 (t, 2H, J = 6.8 Hz), 1.41 (s, 9H). 13C NMR
(CDCl3, 100 MHz): 172.4, 164.1, 163.6, 156.2, 154.1, 140.3, 135.6,
133.6, 131.9, 131.0, 130.9, 128.8, 126.1, 123.7, 122.5, 118.4, 118.0,
117.5, 79.7, 63.2, 53.7, 38.2, 37.5, 36.9, 34.2, 33.2, 34.2, 33.2, 29.7,
28.6, 22.8, 22.3 ppm.
Tracker Red is purchased from Molecular Probes (Invitrogen). All
solvents were HPLC reagent grade, and triple-deionized water was
used throughout the analytical experiments. Silica gel 60 (Merck,
0.063−0.2 mm) was used for column chromatography. Analytical thin
layer chromatography was performed using Merck 60 F254 silica gel
1
(precoated sheets, 0.25 mm thick). H and 13C NMR spectra were
collected in CDCl3 or CD3OD (Cambridge Isotope Laboratories,
Cambridge, MA) on Varian 300 and 400 MHz spectrometers. ESI
mass spectrometric analyses were carried out using an LC/MS-2020
Series (Shimadzu) instrument. HR-FAB mass spectral analyses were
carried out at Korea Basic Science Institute.
Synthesis of 7. A solution of TFA/DCM (v/v, 2:1) was added to
compound 6 (0.2 g, 0.2 mmol). After 2 h of stirring, the solution was
removed under reduced pressure. Diethyl ether was poured in the
crude product dissolved in DCM to yield 7 as a yellow solid (0.1 g,
68%). ESI-MS: m/z (M+) calcd 752.2, found 751.3 (M − H+). HR-
FAB: m/z (M − H+) calcd 751.2178, found 751.2178. 1H NMR
(CDCl3 with 10% CD3OD, 400 MHz): δ 8.66 (br s, 1H), 8.49 (d, 1H,
J = 9.2 Hz), 8.27 (d, 1H, J = 7.0 Hz), 8.18 (d, 1H, J = 8.3 Hz), 8.05 (d,
1H, J = 8.0 Hz), 7.86−7.66 (m, 15H), 7.49 (t, 1H, J = 7.4 Hz), 4.47 (t,
2H, J = 5.6 Hz), 4.34 (t, 2H, J = 5.8 Hz), 3.58−3.43 (m, 4H), 3.37−
3.27 (m, 2H), 3.13−3.01 (m, 4H), 2.57−2.47(m, 2H). 13C NMR
(CDCl3 with 10% CD3OD, 100 MHz): 172.8, 164.4, 163.9, 153.9,
140.5, 135.6, 133.5, 132.2, 131.2, 128.7, 126.3, 123.8, 122.2, 118.3,
117.4, 116.2, 63.2, 38.9, 37.7, 37.1, 34.7, 34.5, 33.5, 22.3, 21.4 ppm.
Synthesis of 9. To a mixture of 8 (0.2 g, 0.7 mmol) and phosgene
(0.4 g, 1.4 mmol) in 20 mL of CH2Cl2 was added DIPEA (0.5 mL, 2.5
mmol) dropwise. The resulting solution was stirred for 3 h under
nitrogen gas. The reaction mixture was flushed with nitrogen gas. After
removal of unreacted phosgene gas and neutralization in an NaOH
bath, a solution of 2,2′-dithiodiethanol (0.6 g, 3.5 mmol) in CH2Cl2/
THF (v/v, 1:1) was added to the mixture. The reaction mixture was
stirred overnight. The solvent was evaporated off, at which point
CH2Cl2 (100 mL) and water (100 mL) were added, and the organic
layer was collected. The CH2Cl2 layer was dried over anhydrous
MgSO4. After removal of the solvents, the crude product was purified
over silica gel using ethyl acetate/hexane (v/v, 3:2) as the eluent to
yield 9 as a yellow solid (0.2 g, 60%). ESI-MS: m/z (M+) calcd 448.1,
found 447.1 (M − H+). HR-FAB: m/z (M + H+) calcd 449.1205,
found 449.1204. 1H NMR (CDCl3, 400 MHz): δ 8.61−8.58 (m, 2 H),
8.34 (d, 1H, J = 8.2 Hz), 8.25 (d, 1H, J = 8.5 Hz), 7.62 (br, 1H), 7.75
(t, 1H, J = 8.0 Hz), 4.56 (t, 2H, J = 6.3 Hz), 4.16 (d, 2H, J = 7.5 Hz),
3.96 (t, 2H, J = 5.6 Hz), 3.06 (t, 2H, J = 6.3 Hz), 2.96 (t, 2H, J = 5.8
Hz), 2.40 (br, 1H), 1.74−1.67 (m, 2H), 1.47−1.39 (m, 2H), 0.97 (t,
3H, J = 7.3 Hz). 13C NMR (CDCl3, 100 MHz): 164.3, 163.8, 153.2,
139.0, 132.6, 131.4, 129.1, 126.8, 126.3, 123.6, 123.2, 118.2, 117.2,
64.1, 60.8, 41.7, 40.5, 37.7, 30.4, 20.6, 14.1 ppm.
UV/Vis and Fluorescence Spectroscopy. Stock solutions of
Mito-Naph, 7, and biologically relevant analytes, including Trx, GSH,
Cys, Hcy, Val, Tyr, Thr, Tau, Ser, Pro, Phe, Met, Lys, Leu, Ile, His,
Gly, Gluc, Glu, Gln, Asp, Asn, Arg, Ala, Na(I), K(I), Zn(II), Mg(II),
Fe(III), Fe(II), Cu(II) and Ca(II), were prepared in triple-distilled
water. Absorption spectra were recorded on an S-3100 (Scinco)
spectrophotometer, and fluorescence spectra were recorded using an
RF-5301 PC spectrofluorometer (Shimadzu) equipped with a xenon
lamp. Samples for absorption and emission measurements were
contained in quartz cuvettes (3 mL volume). Excitation was provided
at 428 nm with excitation and emission slit widths at 3 nm. All
spectroscopic measurements were performed under physiological
conditions (at 37 °C in PBS buffer, pH 7.4).
Kinetic Data. Kinetic measurements were performed using a
Shimadzu RF-5301PC spectrometer in single-mixing mode by mixing
the two reactants. The time dependences of the response of Mito-
Naph (1.0 μM) or 7 (1.0 μM) to thiols (GSH, Trx, Cys, Hcy, and
DTT) were determined at 540 nm, with a time interval of 0.16 s. In
the case of Trx, reduced Trx was prepared by incubating 50 μM Trx
with 250 μM DTT for 1 h at 37 °C. Excitation was performed at 428
nm, with all excitation and emission slit widths at 3 nm. All kinetic
measurements were performed under physiological conditions (at 37
°C in PBS buffer, pH 7.4).
Cell Culture. A human hepatoma cell line (HepG2) and a human
cervical cancer cell line (HeLa) were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal calf serum,
1% penicillin, and 10 000 units/mL of streptomycin at 37 °C, under
humidified air containing 5% CO2; 1 ×105 cells per well were plated
on a 24-well plate. Cell images were obtained using a confocal
microscope from Carl Zeiss (LSM 510 META model). Other
information is available in the figure captions.
Immunoprecipitation. Cells were homogenized in homogeniza-
tion buffer (320 mM sucrose, 4.0 mM HEPES, 1.0 mM EDTA, and
100 mM DTT, pH 7.4), and homogenates were centrifuged at 10 000g
for 10 min at 4 °C. The pellet was removed, and the crude cytosol was
collected. The collected crude cytosol was treated with anti-Trx
antibody (Abcam) overnight at 4 °C and further incubated with anti-
antibody Protein G PLUS-Agarose (Santa Cruz Biotechnology)
overnight. The agarose beads were pelleted by centrifugation (1000g
for 5 min), and the Trx-depleted supernatant was collected and
immediately used for Trx activity assay.
Western Blot. Thirty mircograms of protein per sample was
loaded per well on 12% polyacrylamide gels. After electrophoresis was
performed, the separated proteins were transferred onto nitrocellulose
membranes (Bio-Rad, Hercules, CA) in transfer buffer [39 mM
glycine, 48 mM Tris-base, pH 8.3, 0.37% (w/v) SDS, 20% (v/v)
methanol]. Membranes were blocked with 5% nonfat dried milk in
TBS-T buffer [20 mM Trix-base, pH 7.6, 137 mM NaCl, 0.1% (v/v)
Tween-20] for 1 h at room temperature. Membranes were then
incubated with anti-Trx antibody (Abcam) overnight at 4 °C and
further incubated with HRP-conjugated anti-antibody (Santa Cruz
Biotechnology) for 1 h. Anti-β-actin (Sigma-Aldrich) was used to
establish the internal standard. The blots were washed three times with
TBS-T buffer, and the immune-reactive protein bands were visualized
by application of ECL substrate solution (INTRON Biotechnology).
Synthesis of Mito-Naph. To a mixture of 4 (0.7 g, 1.3 mmol) and
phosgene (2.8 mL, 6.5 mmol) in 20 mL of CH2Cl2 was added DIPEA
(1.4 mL, 9.1 mmol) dropwise. The resulting solution was stirred for 3
h under nitrogen gas. The reaction mixture was then flushed with
nitrogen gas. After removal of unreacted phosgene gas and
neutralization in an NaOH bath, a solution of 2,2′-dithiodiethanol
(0.8 g, 6.5 mmol) in CH2Cl2/THF (v/v = 1:1) was added to the
mixture. The reaction mixture was stirred overnight. After removal of
the solvent, the crude product was purified over silica gel using ethyl
CH2Cl2/MeOH (v/v, 9:1) as the eluent to yield Mito-Naph as a
yellow solid (0.6 g, 63%). ESI-MS: m/z (M+) calcd 752.2, found 753.5
1
(M + H+). HR-FAB: m/z (M+) calcd 752.2018, found 752.2022. H
NMR (CDCl3, 400 MHz): δ 9.49 (s, 1H), 9.26 (br t, 1H), 8.50 (d, 1H,
J = 7.3 Hz), 8.13 (d, 1H, J = 7.6 Hz), 8.07 (d, 1H, J = 8.0 Hz), 7.99 (d,
1H, J = 8.0 Hz), 7.86−7.69 (m, 15H), 7.28 (d, 1H, J = 7.6 Hz), 4.68 (t,
1H, J = 6.0 Hz), 4.50 (t, 2H, J = 6.1 Hz), 4.35 (t, 2H, J = 6.5 Hz),
3.93−3.89 (m, 2H), 3.80−3.75 (m, 2H), 3.69−3.99 (m, 2H), 3.10 (t,
2H, J = 6.2 Hz), 2.99 (t, 2H, J = 6.3 Hz), 2.73 (t, 2H, J = 7.1 Hz). 13C
NMR (CDCl3, 100 MHz): 172.7, 164.1, 163.6, 154.2, 140.6, 135.6,
133.6, 131.9, 130.9, 130.8, 128.8, 128.4, 126.0, 123.4, 122.0, 118.4,
117.6, 116.9, 63.6, 60.6, 41.7, 38.0, 37.1, 34.4, 33.4, 22.6, 22.9 ppm.
Synthetic Materials and Methods. All reactions were carried out
under nitrogen atmosphere. All reagents, including thioredoxin (from
Escherichia coli), amino acids, metal ions, thiols, and other chemicals
for synthesis, were purchased from Aldrich and used as received. Mito
E
dx.doi.org/10.1021/ja308446y | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(21) Zhou, R.; Tardivel, A.; Thorens, B.; Choi, I.; Tschopp, J. Nat.
Immunol. 2010, 11, 136−140.
ASSOCIATED CONTENT
* Supporting Information
Additional spectra (UV/vis absorption, fluorescence, NMR,
ESI-MS) and imaging data and full reference information. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
kangch@khu.ac.kr; jongskim@korea.ac.kr
■
Author Contributions
∥These authors contributed equally to the work.
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the CRI project (20120000243) of
the National Research Foundation of Korea (J.S.K.) and the
Cooperative Research Program for Agriculture Science &
Technology Development (Project No. PJ008959), Rural
Development Administration, Republic of Korea (C.K.).
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