Highly Sensitive Bioluminescent Probe for Thiol Detection in Living Cells

Article abstract of DOI:10.1002/asia.201701774

The sensitive detection of thiols including glutathione and cysteine is desirable owing to their roles as indispensable biomolecules in maintaining intracellular biological redox homeostasis. Herein, we report the design and synthesis of SEluc-1 (sulfinate ester luciferin), a chemoselective probe exhibiting a ratiometric and turn-on response towards thiols selectively in fluorescence and bioluminescence, respectively. The probe, which was designed based on the “caged” luciferin strategy, displays excellent selectivity, high signal/noise ratio (>240 in the case of bioluminescence), and a biologically relevant limit of detection (LOD, 80 nm for cysteine), which are all desirable traits for a sensitive bioluminescent sensor. SEluc-1 was further applied to fluorescence imaging of thiol activity in living human cervical cancer HeLa cell cultures, and was successfully able to detect fluctuations in thiol concentrations induced by oxidative stress in a bioluminescent assay utilizing African green monkey fibroblast COS-7 cells and human breast adenocarcinoma MCF-7 cells.

Full text of DOI:10.1002/asia.201701774

CHEMISTRY
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Accepted Article
Title: Highly sensitive bioluminescent probe for thiol detection in living
cells
Authors: Mayu Hemmi, Yuma Ikeda, Yutaka Shindo, Takahiro
Nakajima, Shigeru Nishiyama, Kotaro Oka, Moritoshi Sato,
Yuki Hiruta, Daniel Citterio, and Koji Suzuki
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10.1002/asia.201701774
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Highly sensitive bioluminescent probe for thiol detection in living
cells
Mayu Hemmi,[a] Yuma Ikeda,[a] Yutaka Shindo,[a] Takahiro Nakajima,[b] Shigeru Nishiyama,[a] Kotaro
Oka,[a] Moritoshi Sato,[b] Yuki Hiruta,[a] Daniel Citterio,*[a] Koji Suzuki*[a]
Dedicated to Dr. Hidemasa Hidaka
Abstract: The sensitive detection of thiols including glutathione and
cysteine is desirable due to their roles as indispensable biomolecules
in maintaining intracellular biological redox homeostasis. Herein, we
report the design and synthesis of SEluc-1 (Sulfinate Ester Luciferin),
a chemoselective probe exhibiting ratiometric and turn-on response
towards thiols selectively in fluorescence and bioluminescence,
respectively. The probe, which was designed based on the “caged”
luciferin strategy, displays excellent selectivity, high S/N ratio (＞240
in the case of bioluminescence), and a biologically relevant limit of
detection (LOD, 80 nM for cysteine), which are all desirable traits for
a sensitive bioluminescent sensor. SEluc-1 was further applied to
fluorescence imaging of thiol activity in living human cervical cancer
HeLa cell cultures, and was successfully able to detect fluctuations in
thiol concentrations induced by oxidative stress in a bioluminescent
assay utilizing African green monkey fibroblast COS-7 cells and
human breast adenocarcinoma MCF-7 cells.
produced, cells are detoxified through the glutathione-ascorbate
cycle; GSH is oxidized to glutathione disulfide (GSSG),
subsequently followed by reduction to GSH catalyzed by
glutathione reductase.[6] Thus, the level of reduced GSH
concentration in cells serves as an indicator for oxidative stress-
related diseases including cancer and Alzheimer’s disease.[7]
Cysteine (Cys) is a sulfhydryl group-containing amino acid which
is also biologically relevant in cellular functions. It is the rate-
limiting substrate of GSH synthesis, since L-glutamate and L-
cysteine are catalyzed by glutamate cysteine ligase to form ɤ-
glutamyl-L-cysteine,
sensitive and selective detection of these thiols in cells is of great
importance in both clinical and research practice.
a
precursor of GSH.[8] Therefore, the
Recently, various efforts for the development of intracellular
thiol-selective probes have been made, most of which utilize
fluorescence as detection signal.[9] Fluorescent probes enable
simple and noninvasive detection of thiols, and therefore have
great potential for bioimaging. Nevertheless, the need of
excitation light contributes to a high background signal and
possible photobleaching, which may lead to limitations in
biological applications.[10]
Introduction
Thiols, including glutathione and cysteine, are low-molecular
weight compounds containing the most reactive and reduced form
of sulfur (-SH).[1] Glutathione (L-ɤ-glutamyl-L-cysteinylglycine,
GSH) is the most abundant non-protein biothiol and can be found
in mM levels intracellularly.[2] It plays a direct or indirect role in
multiple biological phenomena including enzyme activity,
metabolism, and cell protection.[3] Importantly, it exists in a redox
equilibrium established at a given electrical potential between the
reduced (sulfhydryl, -SH) and oxidized (disulfide, -SS-) form and
is therefore essential in maintaining the reducing environment in
cells.[4] In healthy cells and tissues, GSH is highly reducing, with
more than 90% of the total GSH content existing in the reduced
form.[5] When excess amounts of reactive oxygen species (ROS)
such as hydrogen peroxide (H2O2) and lipid peroxides are
To achieve large signal-to-noise (S/N) ratios for increased
sensitivity, we selected bioluminescence as a signaling technique.
In particular, the firefly luciferin-luciferase pair is the brightest
known bioluminescence system and has been widely studied and
applied.[11] Because there are virtually no endogenous
luminescent phenomena in human living cells, bioluminescent
assays can achieve comparatively high S/N contrast ratio.[12] The
light generated by the enzymatic reaction is applicable towards
highly sensitive detection of analytes in living cells due to the
favorable properties of the substrate such as rapid molecular
diffusion and excellent biocompatibility.[13] In 2006, electrophilic
aromatic substituted luciferins were reported as bioluminescent
probes for glutathione S-transferase (GST) assays, and was later
commercialized as a GSH quantifying reagent in cell lysates.[14]
However, because the probe cannot react directly with GSH, it
requires the addition of GST as a catalysis which places
limitations in live cell studies. For practical applications, the
bioluminescent probe should contain a ligand attached by a self-
immolative linker, allowing the direct detection of alterations in
reduced thiol concentration in living cells. Therefore, we sought to
develop a highly sensitive and selective probe that meet these
requirements employing the firefly luciferin-luciferase system.
Herein, two bioluminescent probes for the sensitive and
selective detection of thiols in living cells were designed and
synthesized. Sulfinate Ester Luciferin-1 (SEluc-1) and SEluc-2
are small-molecule probes based on the firefly luciferin, in which
a sulfonate ester serving as a thiol-selective ligand is connected
[a]
M. Hemmi, Y. Ikeda, Dr. Y. Shindo, Prof. Emeritus S. Nishiyama,
Prof. K. Oka, Dr. Y. Hiruta, Prof. D. Citterio, Prof. Emeritus K. Suzuki
Faculty of Science and Technology
Keio University
3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, Japan
citterio@applc.keio.jp
[b]
Dr. T. Nakajima, Prof. M. Sato
Graduate School of Arts and Sciences, College of Arts and
Sciences
The University of Tokyo
3-8-1 Komaba, Meguro, Tokyo, Japan
Supporting information for this article is given via a link at the end of
the document.
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to the phenolic position of the luciferin either through a self-
immolative linker (SEluc-1) or by direction conjugation (SEluc-2).
We established that SEluc-1 in particular exhibits excellent
selectivity to biothiols over other relevant species, notable S/N
ratio, and sufficient cell permeability for the monitoring of thiol
activity in living cells.
influence of enzyme activity and co-factors including ATP and
Mg2+. 50 µM of each probe was incubated with 100 µM cysteine
at 37 °C, and changes in fluorescence spectra and intensity were
monitored. In the case of SEluc-1, only the emission peak of the
probe itself (428 nm) was notable in the absence of cysteine. No
visible variations in fluorescence intensities was detected
(Supporting Figure S1a), suggesting the stability of the probe
under the assay conditions. The conjugation of the ligand to the
benzothiazole moiety decreases the electron density on the
hydroxyl oxygen at the 6th position and presumably leads to the
blue-shift of the probes' emission wavelength compared with that
of D-luciferin.[19] A new emission peak at 533 nm upon addition of
cysteine, as shown in Fig. 1a, confirmed the release of luciferin
by a cysteine-mediated cleavage process. The intensity of this
peak increased until reaching a plateau after 30 min of incubation
(Fig. 1b). On the other hand, incubation of SEluc-2 at 37 °C
resulted in the gradual generation of D-luciferin, hence suggesting
the vulnerability of the probe to decomposition in aqueous media
(Supporting Figure S1b). SEluc-2 exhibited larger enhancement
in fluorescence intensity at 533 nm when incubated with cysteine
compared with SEluc-1, and the fluorescence intensity saturated
after 25 min (Supporting Figure S2).
Scheme 1. (a) Chemical structures of SEluc-1, SEluc-2, and D-luciferin; (b)
Bioluminescence detection mechanism of novel probes, with SEluc-1 as an
example. (c) Synthesis scheme of SEluc-1 and SEluc-2.
Results and Discussion
We focused on the “caged” luciferin, a concept well-established
as an effective and versatile strategy for the development of a
highly sensitive bioluminescent sensor.[15] Modification of the
hydroxyl group of D-luciferin with a caging group prohibits
recognition by the enzyme, and luminescence is prevented.
Therefore, we envisioned a bioluminescent probe in which the
caged firefly luciferin is selectively unmasked by thiols to generate
D-luciferin, and subsequently oxidized by luciferase to emit
light.[16] Because the intensity of the light generated directly
correlates to the amount of liberated D-luciferin, it would serve as
an indicator of thiol activity. The tert-butyl sulfinate ester was
selected as the caging group, considering that its selective
cleavage by thiols owing to its thiophilicity had been previously
reported.[17]
By combining the two components, we designed SEluc-1 and
SEluc-2 as shown in Scheme 1. Both the caging group of SEluc-
1 and D-luciferin were synthesized as previously reported.[18]
Initial attempts to synthesize SEluc-1 by attaching the ligand to 2-
cyano-6-hydroxybenzothiazole, followed by a coupling reaction
with D-cysteine, proved futile as the addition of D-cysteine
resulted in the immediate cleavage of the ligand. Instead, SEluc-
1 was successfully synthesized after an SN2 reaction between the
ligand and D-luciferin with optimized reaction conditions. In a
similar manner, SEluc-2 was successfully synthesized from
commercially available tert-Butylsulfinyl chloride (Scheme 1c).
Preliminary experiments to analyze the reactivity of SEluc-1
and SEluc-2 were performed by fluorescence titration, to omit the
Figure 1. (a) Fluorescence spectral changes of 50 µM SEluc-1 alone (dotted)
or after incubation with 100 µM cysteine (solid) in 25 mM phosphate buffer (pH
7.4) at 37 °C for 30 min. λex = 370 nm; (b) Time course of fluorescence intensity
at 533 nm (D-luciferin). Red line indicates fluorescence saturation.
In order to quantitatively compare the sensitivity of the two
probes, the ratios in fluorescence enhancement before and after
incubation with 10 equivalents of thiols including cysteine, GSH,
and homocysteine were calculated (Supporting Figure S3, Table
S1). Although SEluc-2 showed higher absolute signal intensity
after reaction with thiols, SEluc-1 marked higher ratios in
fluorescence enhancement (33-fold in case of cysteine) due to
lower background signal compared with that of SEluc-2. Overall,
SEluc-1 was determined to be the most potent probe for further
investigation and application to cell imaging for its stability and
superior sensitivity towards thiols in aqueous solution.
Having established that SEluc-1 is more desirable as an optical
reporter for thiols, its ability to quantitatively detect thiols in
solution was examined. 50 µM SEluc-1 was incubated with
increasing concentrations of cysteine (0-100 µM) for 30 min and
the changes in fluorescence intensity was recorded. A linear
calibration curve was established for the cysteine concentration-
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dependent fluorescence emission ratio values of the probe and
liberated D-luciferin, with a detection limit of 0.33 µM (S/N = 3)
(Supporting Figure S4). We concluded that SEluc-1 functions
efficiently as a ratiometric fluorescent probe to quantitatively
measure the concentration of cysteine within its intracellular
concentration (~100 µM).[20]
We next sought to examine the response of SEluc-1 to cysteine
concentration increments in a bioluminescent assay. Incubation
of SEluc-1 with 0 to 200 µM of cysteine for 30 min, followed by
the addition of firefly luciferase (Fluc), ATP, and Mg2+ resulted in
a novel peak at 564 nm, confirming the generation of D-luciferin
by the cleavage reaction (Fig. 2a). Bioluminescence emitted from
the liberated luciferin was linearly dependent on the cysteine
concentration between 0-40 µM, and a lower detection limit was
achieved than in the case of fluorescence (Fig. 2b, 80 nM).
Similarly, calibration curves were obtained under the same
experimental conditions in the case of GSH and homocysteine
(Supporting Figure S5). These results suggest the practicability of
SEluc-1 towards highly sensitive thiol detection.
heavily dependent on the deprotonation degree of the sulfhydryl
group. Nevertheless, we were successfully able to obtain stable
and reproducible results with 25 mM phosphate buffer solutions
adjusted to a specific pH. It is especially important to note that the
high selectivity of SEluc-1 over H2O2 and oxidized thiols would
make this probe suitable for the study of reduced thiol activity
during oxidative stress within cells.
Figure 3. Peak luminescence intensities after incubation with 10 µM SEluc-1
alone or with 100 µM of various biomolecules or inorganic nucleophiles in 25
mM phosphate buffer (pH 7.4) at 37 °C for 30 min, followed by addition of 0.05
mg/mL Fluc, 4 mM ATP, and 8 mM MgSO4.
Encouraged by the desirable luminogenic traits of SEluc-1
including its excellent selectivity and physiologically relevant
detection limit towards thiols in aqueous solution, we sought to
verify whether it can be further translated to cell cultures. Firstly,
standard MTT assays of SEluc-1 were performed to investigate
its cell toxicity, which showed that 20 µM of the probe did not
exhibit cytotoxic effects on cultured live human cervical cancer
HeLa cells after incubation for up to 8 h (Supporting Figure S7).
Therefore, further experiments were performed with 20 µM
SEluc-1. Next, in order to determine whether SEluc-1 can
permeate cells to allow visualization of thiols in living cells,
changes in fluorescence intensity of the emission peak of D-
luciferin was observed with a fluorescence microscope. HeLa
cells incubated with SEluc-1 for 30 min exhibited bright
fluorescence, presumably owing to the liberation of D-luciferin by
the endogenous thiol-mediated cleavage reaction within the cell.
Furthermore, the manipulation of intracellular thiol level by
incubation with N-ethylmaleimide (NEM), a well-known thiol
scavenger prior to probe addition, elicited a sharp decrease in
fluorescence (Fig. 4, Supporting Figure S8).[21] Overall, the results
suggest that SEluc-1 is a cell-permeable molecule capable of
selectively visualizing intracellular thiols in a uniform manner.
Figure 2. (a) Concentration-dependent bioluminescence spectral changes of 50
µM SEluc-1 with 0-200 µM cysteine incubated in 25 mM phosphate buffer (pH
7.4) at 37 °C for 30 min, followed by addition of 1.0 mg/mL Fluc, 4 mM ATP, and
8 mM MgSO4; (b) Peak luminescence intensities plotted against cysteine
concentration (0-40 µM). 10 µM SEluc-1 was incubated with 0-40 µM in 25 mM
phosphate buffer (pH 7.4) at 37 °C for 30 min, followed by the addition of 0.04
mg/mL Fluc, 4 mM ATP, and 8 mM MgSO4.
Following the investigation of its fluorescence and
bioluminescence properties, we evaluated the selectivity of
SELuc-1 towards thiols over other biomolecules in
a
bioluminescent assay. SEluc-1 was incubated with a panel of
thiols, amino acids, and other nucleophiles (10 equivalents) for 30
min, prior to subsequent addition of luciferase, ATP, and Mg2+.
Robust enhancement was observed after the addition of thiols
including cysteine, GSH, and homocysteine, with over 240-fold
increase in bioluminescence in the case of cysteine (Fig. 3). On
the other hand, little to no increase in signal was found when
exposed to nucleophiles such as H2S and H2O2, esterase (porcine
liver), GSSG, various amino acids not bearing free sulfhydryl
groups, and inorganic nucleophiles. This confirmed that a free
sulfhydryl group is necessary to trigger the cleavage response. To
verify that a thiolate serves as the nucleophile during the cleavage
process, the response of the probe to cysteine under various
buffer conditions within the physiological pH range (pH 6-8) was
evaluated by HPLC analysis (Supporting Figure S6). Indeed, the
probe was found to be the most reactive under basic conditions
(Supporting Table S2), signifying that the reactivity of the thiol is
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reflects the generation of hydrogen peroxide and successive thiol
consumption. In contrast, MCF-7 cells deprived of glucose for
merely 5 min displayed a sharp decrease in thiol concentration,
followed by bioluminescence recovery with further incubation time
(Fig. 5f, Supporting Figure S12). This is in close agreement with
previous works suggesting that in human tumor cell lines, steady
state levels of intracellular prooxidant increase immediately after
introduction of glucose-free medium, and that cells appear to
compensate by increasing the synthesis of glutathione.[26]
Altogether, the data highlights the potential of SEluc-1 to monitor
oxidative stress-related fluctuations of endogenous thiol
concentrations in long-term studies within living cells.
Figure 4. (a) Live cell imaging carried out with HeLa cells. (A) Incubated with
20 µM SEluc-1 (B) Incubated with 1 mM NEM prior to addition of 20 µM SEluc-
1; (b) Quantitative comparison of fluorescence intensity between (A) and (B).
Finally, SEluc-1 was applied to the monitoring of alterations in
thiol concentration by bioluminescence. Because NEM is known
to inhibit the sulfhydryl groups of firefly luciferase,[22] thiol
concentrations were manipulated by inducing oxidative stress.
Firstly, cultured live African green monkey fibroblasts COS-7 cells
transfected with Fluc were incubated with 0, 100, or 500 µM of
H2O2, a typical reactive oxygen species, to promote the oxidation
of GSH to GSSG,[23] followed by incubation with 20 µM SEluc-1.
Marked decrease in bioluminescence intensity was observed in
an H2O2-concentration dependent manner. Moreover, successive
addition of 200 µM cysteine solution following H2O2 incubation
resulted in bioluminescence recovery (Fig. 5a, b). A similar trend
was observed in the case of human breast adenocarcinoma MCF-
7 cells (Fig. 5c, d).
Previous reports have indicated that depletion of glucose
induces oxidative stress, especially in cancerous cells. Glucose
metabolism results in the formation of pyruvate and NAPDH,
which are believed to function in the cellular detoxification of
hydrogen peroxide that is generated during mitochondrial
metabolism. Studies have suggested that glucose deprivation in
cancer cells may compromise the cell’s ability to detoxify the
ROS.[24] Umezawa et al. demonstrated that the resulting oxidative
stress and subsequent decrease of thiol concentration occur
within minutes after the introduction of glucose-free medium to the
cells.[25] We sought to determine whether the same observation
can be made utilizing SEluc-1. Specifically, COS-7 cells were
incubated with either high glucose (25 mM) or glucose-free (0
mM) medium for 5, 15, 30, or 60 min, followed by the addition of
20 µM SEluc-1. Indeed, with longer incubation time, we noted
further suppression in bioluminescence (Fig. 5e, Supporting
Figure S9). Because the bioluminescence of D-luciferin was
unaffected by the presence of the glucose-free medium and
hydrogen peroxide did not inhibit the luciferin-luciferase reaction
(Supporting Figure S10 and S11), we believe that this decrease
Figure 5. Live cell assays carried out in bioluminescence. (a) Total
bioluminescence intensity integrated for 6000 s and (b) time course of changes
in bioluminescence after wells with COS-7 cells were incubated with 0, 100, or
500 µM H2O2 for 15 min at 37 °C prior to addition of 20 µM SEluc-1. The bar on
the right represents cells further incubated with 200 µM L-cysteine prior to probe
addition; (c) Total bioluminescence intensity integrated for 6000 s and (d) time
course of changes in bioluminescence after MCF-7 cells were incubated with 0,
100, or 500 µM H2O2 for 15 min at 37 °C prior to addition of 20 µM SEluc-1. The
bar on the right represents cells further incubated with 200 µM L-cysteine prior
to probe addition; (e) Total bioluminescence intensity integrated for 6000 s after
COS-7 cells were incubated with 25 mM or 0 mM glucose medium for 5, 15, 30,
or 60 min at 37 °C prior to addition of 20 µM SEluc-1; (f) Total bioluminescence
intensity integrated for 6000 s after wells with MCF-7 cells were incubated with
25 mM or 0 mM glucose medium for 5, 15, 30, or 60 min at 37 °C prior to addition
of 20 µM SEluc-1.
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color. To this solution, 3 (268 mg, 1.04 mmol) in ethanol (5.8 mL) was
added dropwise at 0 °C and the reaction mixture was stirred at room
temperature for 3.5 hours. The reaction was quenched with 3 drops of 1 M
HCl, and water was evaporated under reduced pressure. The crude
material was purified by preparative layer chromatography
(methanol/chloroform = 1 : 3), and finally reverse-phase preparative layer
chromatography (water/methanol = 1 : 3) to obtain SEluc-1 as a yellow
solid. Yield = 11.7 mg, 12%. Rf = 0.32 (methanol/chloroform = 1 : 3). Rf =
0.49 (reverse-phase chromatography water/methanol = 1 : 3); 1H NMR
(400 MHz, CD3OD): δ (ppm) = 7.95 (d, J = 9.2 Hz, 1H), 7.62 (d, J = 2.8
Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 3.2 Hz, 1H), 7.20 (d, J =
8.4 Hz, 2H), 5.24 (t, J = 9.2 Hz, 1H), 5.16 (s, 2H), 3.73 (m, 2H), 1.34 (s,
9H); 13C NMR (100 MHz, CD3OD): δ (ppm) = 176.52, 165.72, 160.50,
159.65, 155.61, 148.99, 138.96, 135.66, 130.54, 125.68, 121.39, 118.56,
106.31, 82.41, 70.88, 59.61, 37.02, 21.77; HR-ESI-MS: calcd for
C22H22O5S3 [M+H]+ ꢀ/ꢁ 491.0769, found 491.0764.
Conclusions
We developed two bioluminescent sensors applying the “caged”
luciferin strategy for the sensitive and selective monitoring of
intracellular thiols. Based on preliminary evaluation by
fluorescence, SEluc-1 was selected for further investigations
owing to its superior stability and S/N ratio. Incubation of SEluc-1
with cysteine resulted in the liberation of D-luciferin and the
successive red-shifting of fluorescence emission wavelength from
428 nm to 533 nm. By calculating the ratio of fluorescence
intensity after incubation with increasing cysteine concentrations,
a calibration curve within its intracellular concentrations was
successfully obtained. Furthermore, when SEluc-1 was applied
to bioluminescence assays, highly sensitive and selective
detection of thiols was achieved, with a notable S/N ratio (>240)
and satisfactory LOD (80 nM). SEluc-1 enabled fluorescence
imaging of intracellular thiols, and was proved to be sensitive
enough to detect oxidative stress-mediated variations in thiol
concentration. We believe that this bioluminescent probe will be
helpful in further elucidating the role of the potent biomolecules
within their cellular environment.
Synthesis of SEluc-2. To a solution of D-luciferin (15 mg, 0.054 mmol) in
water (1.5 mL, not solubilized), 0.5 M NaOH (400 µL) was added at 0 ℃.
D-luciferin solubilized immediately, and the solution turned yellow-green in
color. To this solution, tert-Butylsulfinyl chloride (40 mg, 0.27 mmol) was
added dropwise at 0 °C and the reaction mixture was stirred at room
temperature for 2 hours. The solvent was evaporated under reduced
pressure. The crude material was purified by preparative layer
chromatography (methanol/chloroform = 1 : 3), and finally reverse-phase
preparative layer chromatography (water/methanol = 3 : 5) to obtain
Experimental Section
SEluc-2 as
a yellow solid. Yield = 8.0 mg, 40%. Rf = 0.28
(methanol/chloroform = 1 : 3). Rf = 0.29 (reverse-phase chromatography
water/methanol = 3 : 5); 1H NMR (500 MHz, CD3OD): δ (ppm) = 8.08 (d,
J = 8.0 Hz, 1H), 7.89 (t, J = 2.0 Hz, 1H), 7.39 (dt, J = 8.0, 2.0 Hz, 1H),
5.28 (t, J = 9.0 Hz, 1H), 3.75 (d, J = 7.0 Hz, 2H), 1.383 (s, 9H); 13C NMR
(100 MHz, CD3OD): δ (ppm) = 176.27, 165.57, 163.32, 154.80, 151.98,
138.59, 126.20, 121.48, 114.50, 82.40, 60.00, 37.07, 21.78; HR-ESI-MS:
calcd for C15H16N2O4S3 [M+H]+ ꢀ/ꢁ 385.0350, found 385.0328.
Materials and Instruments. All chemicals were purchased from
commercially available sources (Sigma-Aldrich, Tokyo Chemical
Industries, and Wako Pure Chemical) and used as received unless stated
otherwise. All reactions were conducted in nitrogen atmosphere. Column
chromatography was performed using Silica gel 60 (63-200 μm) from
Merck KGaA (Darmstadt, Germany) as stationary phase. All preparative
and analytical TLC silica gel plates (Kieselgel 60 F254 plates) were
purchased from Merck KGaA (Darmstadt, Germany). 1H and 13C MR
spectra were recorded on JNM ECA-500 and JNΜ-α400 from JEOL
(Tokyo, Japan) at 25 ℃. Chemical shifts are relative to an internal standard
of tetramethylsilane (δ = 0.0 ppm) and coupling constants (J) are given in
Hz. High-resolution mass spectra (HRMS) were obtained using LCT
Premier XE mass spectrometer from Waters (Milford, MA, U.S.). High
Measurement of fluorescence: Stock solutions of 1 mM probe in DMSO:
ultrapure water (distributed by Organo Corporation from Tokyo, Japan) =
1 : 1 (v/v) and 1 mM thiol (GSH or L-cysteine) in ultrapure water were
prepared. Fluorescence spectra and intensity were measured in 25 mM
phosphate buffer (pH 7.4) containing probe (50 µM), thiol (0-100 µM), with
DMSO as a co-solvent (2%). Each experiment was repeated 3 times.
performance liquid chromatography (HPLC) was conducted on
a
Prominence-i LC-2030C HPLC from Shimadzu (Kyoto, Japan). Absorption
spectra were recorded on a UV-1800 Spectrophotometer from Shimadzu
(Kyoto, Japan). Fluorescence spectra were recorded on a VarioskanTM
Flash multi spectra microplate reader from Thermo Fisher Scientific
(Waltham, MA, U.S.). Absorption spectra for MTT assays were measured
on a FluoroskanTM Ascent FL Microplate Fluorometer from Thermo Fisher
Scientific (Waltham, MA, U.S.). Bioluminescence intensity and kinetics
were measured with a TriStar2 LB 942L multimode microplate reader from
Berthold Technologies (Bad Wildbad, Germany). Bioluminescence spectra
were collected on an integrating sphere-based multichannel spectrometer
equipped with a liquid N2-cooled CCD detector as described previously.[27]
Cell imaging data was collected with an Eclipse TE300 inverted
microscope from Nikon (Tokyo, Japan). Bioluminescence measurements
for cell assays were obtained with a Centro XS3 LB 960 high sensitivity
microplate luminometer from Berthold Technologies (Bad Wildbad,
Germany). D-luciferin and 6 were prepared as previously reported.[18]
Details of synthesis procedures and NMR charts are shown in Supporting
information.
Measurement of bioluminescence: Stock solutions of 1 mM SEluc-1 in
DMSO : ultrapure water = 1 : 1 (v/v) and 1 mM thiol (GSH or L-cysteine) in
ultrapure water were prepared. Bioluminescence intensity was measured
in 25 mM phosphate buffer (pH 7.4) so that the final concentrations of each
component were as follows: SEluc-1 (50 µM), L-cysteine (0, 5, 20, 30, 40
µM), 0.05 mg/mL recombinant Photinus pyralis luciferase (Fluc) (promega,
E1701) (0.05 mg/mL), ATP-2Na (4 mM), MgSO4 7H2O (8 mM) and DMSO
as a co-solvent (2%). The solution containing ATP and MgSO4 7H2O was
injected automatically before each measurement. To obtain the
bioluminescence spectra, SEluc-1 (50 µM) was incubated with L-cysteine
(0, 2, 5, 10, 15, 20, 30, 50, 75, 100, 200 µM) in 25 mM phosphate buffer
(pH 7.4) and DMSO as a co-solvent (2%). The absolute spectral sensitivity
was calibrated with a spectral irradiance standard lamp. Bioluminescence
reactions were carried out at ambient temperature with a cocktail
containing 10 μL reaction mixture and 10 µL Fluc (1.0 mg/mL. Reaction
was initiated by injection of 90 μL of ATP-Mg2+ solution (3 mM ATP-2Na,
8 mM MgSO4 7H2O in 0.1 M phosphate buffer (pH 7.4)). Total light
emissions were measured for 120 s (spectra) and 60 s (intensity),
respectively. Each experiment was repeated 4 times.
Synthesis of SEluc-1. To a solution of D-luciferin (58.3 mg, 0.21 mmol) in
water (2.3 mL, not solubilized), 0.5 M NaOH (1740 µL) was added at 0 °C.
D-luciferin solubilized immediately, and the solution turned yellow-green in
Measurement of selectivity: Stock solutions of 1 mM probe in DMSO :
ultrapure water = 1 : 1 (v/v) and 1 mM analyte in ultrapure water were
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prepared. Bioluminescence intensity was measured in 25 mM phosphate
buffer (pH 7.4) containing probe (10 µM) and each analyte (GSH, L-
cysteine, homocysteine, NaHS, H2O2, oxidized GSH, porcine liver
esterase, arginine, L-tyrosine methyl ester HCl, serine, alanine, glutamine,
glycine, L-threonine, L-methionine, L-tryptophan, L-aspartic acid, L-
histidine, leucine, L(-)-phenylalanine, NaSCN, NaNO2, NH4Cl, NaF,
NaNO3, Na2S2O3, Na2SO3・5H2O, NaHCO3, 100 µM) and DMSO as a co-
solvent (2%) incubated for 30 min, followed by addition of recombinant
Photinus pyralis luciferase (Fluc) (promega, E1701) (0.05 mg/mL), ATP-
2Na (4 mM), and MgSO4 7H2O (8 mM). The solution containing ATP and
MgSO4 was injected automatically before each measurement. Each
experiment was repeated at least 4 times. Counting time for each well: 0.1
s, total measurement time: 6000 s.
or 0 mM glucose DMEM for 0, 5, 15, 30, and 60 min at 37 °C. After washing
twice with HBSS (for 25 mM glucose wells) or PBS buffer (for 0 mM
glucose wells), 100 µLSEluc-1 (20 µM, diluted with PBS buffer) was added
to each well, and the luminescence was measured. Counting time for each
well: 0.1 s, total measurement time: 6000 s.
Acknowledgements
This study was supported by Grant-in Aid for Scientific
Research (S) (Grant No. 24225001) to K.S. from Japan Society
for the Promotion of Science (JSPS).
Cell preparation for fluorescence imaging: HeLa cells (RIKEN BRC
CELL BANK) were cultured in Dulbecco’s modified eagle medium (DMEM,
Invitrogen) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin
and 100 µg/mL streptomycin (Nacalai tesque, Japan) at 37 °C under 5%
CO2. For fluorescence imaging, cells were plated on glass bottom dish and
allowed to adhere for 24 h.
Keywords: thiol• bioluminescence • fluorescence • luciferin •
imaging agents
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Abstract
Mayu Hemmi,[a] Yuma Ikeda,[a] Yutaka
Shindo,[a] Takahiro Nakajima,[b]
Shigeru Nishiyama,[a] Kotaro Oka,[a]
Moritoshi Sato,[b] Yuki Hiruta,[a] Daniel
Citterio,*[a] Koji Suzuki[a]
Introduction
Results and Discussion
Conclusions
Experimental Section
Supporting Information
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Highly sensitive bioluminescent
probe for thiol detection in living
cells
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Author(s), Corresponding Author(s)*
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Title
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