A mitochondria-targeted turn-on fluorescent probe based on a rhodol platform for the detection of copper(i)

Article abstract of DOI:10.1039/c4ob00527a

A new spirocyclized rhodol-based fluorescent probe has been developed for detecting mitochondrial Cu⁺. Alkylation of the hydroxy group of a xanthene moiety with a tris(2-pyridylmethyl)amine-based ligand induced the formation of a non-fluorescent spirocyclic structure. The reaction with Cu ⁺ in the presence of submillimolar concentrations of glutathione at physiological pH resulted in the elimination of the ligand together with an increase in the fluorescence of the rhodol fluorophore. This probe was used to visualize mitochondrial Cu⁺ in copper supplemented cells. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00527a

Organic &
Biomolecular Chemistry
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A mitochondria-targeted turn-on fluorescent
probe based on a rhodol platform for the
detection of copper(I)†
Cite this: Org. Biomol. Chem., 2014,
12, 4999
Masayasu Taki,*a,b Kazushi Akaoka,b Koji Mitsuib and Yukio Yamamotob
A new spirocyclized rhodol-based fluorescent probe has been developed for detecting mitochondrial
Cu+. Alkylation of the hydroxy group of a xanthene moiety with a tris(2-pyridylmethyl)amine-based ligand
induced the formation of a non-fluorescent spirocyclic structure. The reaction with Cu+ in the presence
of submillimolar concentrations of glutathione at physiological pH resulted in the elimination of the ligand
together with an increase in the fluorescence of the rhodol fluorophore. This probe was used to visualize
mitochondrial Cu+ in copper supplemented cells.
Received 10th March 2014,
Accepted 12th May 2014
DOI: 10.1039/c4ob00527a
www.rsc.org/obc
Fluorescent probes that can effectively visualize the distri-
bution of copper within discrete subcellular organelles rep-
Introduction
Mitochondria are membrane-bound organelles that account resent powerful tools for investigating copper homeostasis at
for as much as 20% of the total cell volume. The main func- the cellular level.4 Chang et al. reported a mitochondria-
tion of the mitochondrion is the generation of ATP via the res- targeted fluorescent probe for Cu+ (Mito-CS1) based on a
piratory reduction of oxygen to water with the simultaneous photo-induced electron transfer mechanism.5 This probe con-
oxidation of NADH and FADH2. Copper is an essential cofactor tained a cationic triphenylphosphonium (TPP) group that
within the mitochondrial matrices of cytochrome c oxidase selectively accumulated in the mitochondria and allowed for
(CcO) and superoxide dismutase (SOD1) enzymes, which play changes in mitochondrial Cu+ concentrations to be monitored
critical roles in the production of cellular energy and the in living samples. The use of this probe was limited, however,
execution of apoptotic events, respectively.1 In contrast, copper because the small fluorescence on/off ratio (ca. 10-fold) orig-
is also potentially toxic because of its chemical redox potential inating in high background fluorescence of the metal-
and ability to produce hydroxyl radicals via the Fenton reac- unbound form may often obscure the detection of the desired
tion. The uptake, efflux, and subcellular distribution of copper signals. We have previously reported the development of a
are therefore strictly regulated through a system of protein reaction-based Cu+ fluorescent probe (FluTPAs) and its practi-
transporters and copper-chaperones.2 Within the mitochon- cal application in intracellular Cu+ imaging.6 A reduced fluor-
drial intermembrane space (IMS), CcO and SOD are metallated escein platform of FluTPAs is essentially non-fluorescent.
by pathways consisting of several IMS copper metallochaper- When copper ions are present in the intracellular reducing
ones, such as COX17 and CCS, which are supplied with Cu+ by environment, C–O bond cleavage of the benzyl ether group fol-
a small copper–ligand complex in the mitochondrial matrix lowed by air-oxidation results in the formation of a strongly
(MM).3 The basic biological knowledge of copper concen- fluorescent compound (Fig. 1a). Although we have succeeded
trations within the IMS and MM, however, as well as the in monitoring intracellular Cu+ levels in living systems with
general level of understanding of the mechanisms underlying FluTPA (R = Me), this probe could not be used to visualize Cu+
mitochondrial copper homeostasis, remains poor.
stored in organelles because of the diffusion of not only the
probe but also the fluorescent product into the cytosol. To
address this issue, we have designed a new fluorogenic Cu+-
selective fluorescent probe bearing an aminoethyl group at the
xanthene moiety capable of being modified with an organelle
targeting functional group. Hydroxymethyl rhodol, which was
recently developed by Urano et al.,7 was employed as the
fluorophore. When the phenolic hydroxy group of rhodol is
alkylated, the equilibrium between the non-fluorescent spiro-
cyclic and fluorescent ring-opened forms should shift to the
aInstitute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa,
Nagoya 464-8602, Japan. E-mail: taki@itbm.nagoya-u.ac.jp
bGraduate School of Human & Environmental Studies, Kyoto University, Yoshida,
Sakyo-ku, Kyoto 606-8501, Japan
†Electronic supplementary information (ESI) available: NMR spectra of the new
compounds, optical spectra, product analyses, and localization analyses of the
probe to cell organelles. See DOI: 10.1039/c4ob00527a
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additional oxidation step (Fig. 1b). Herein, we describe the syn-
thesis of the TPP-linked Cu+-selective fluorescent probe
RdlTPA-TPP and demonstrate the use of this probe for optical
imaging of mitochondrial Cu+.
Results and discussion
To evaluate the fundamental fluorescence properties of the
rhodol-based Cu+ probe, we initially prepared RdlTPA-Et2 by
the coupling reaction of N,N-diethyl rhodol bearing a hydroxy-
methyl group (HMDER)7 with the 6-chloromethyl derivative of
TPA (6-TPA-Cl),8 as shown in Scheme 1a. Any fluorescent
impurities were removed by recycling gel permeation chromato-
graphy (rec-GPC) prior to its use. All of the spectroscopic
measurements of RdlTPA-Et2 were conducted in an aqueous
buffer solution (50 mM HEPES, pH 7.20) containing 2 mM
GSH, which is the most abundant cellular thiol compound
(0.5–10 mM in cells), to simulate the intracellular environ-
ment.6 RdlTPA-Et2 did not exhibit absorption in the visible
region (Fig. S1; see ESI†) and had negligible fluorescence
(dotted line in Fig. 2), indicating that the spirocyclic form is
the dominant species at neutral pH. When 20 μM of Cu+ was
added to the solution of RdlTPA-Et2 at ambient temperature,
Fig. 1 Fluorogenic probes for Cu+ based on (a) reduced fluorescein
and (b) rhodol platforms.
former of the two forms at the physiological pH of 7.4. Accord-
ing to this fluorescence control mechanism, very little fluo-
rescence should be observed when the phenolic hydroxy group
is alkylated with the TPA ligand. Once the TPA moiety has
been eliminated by the reaction with Cu+, as was found in the
FluTPA system, a strong fluorescence of the ring-opened form
of the rhodol fluorophore should be observed without an
Scheme 1 Synthesis of RdlTPA-Et2 and RdlTPA-TPP.
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would have little influence on intracellular imaging of Cu+
because of the existing low level of Co2+ in vivo. In cells, the
oxidation state of the copper ion pool is maintained at +1 with
defined redox buffers of glutathione.10 In fact, no increase was
observed in the fluorescence when Cu2+ was added in the
absence of GSH, which strongly supported the notion that
Cu2+ is rapidly reduced to Cu+ under these experimental con-
ditions. Mitochondrial GSH represents approximately 10–15%
of the total cellular GSH and the concentration can be as high
as 10 mM.11 We confirmed that RdlTPA-Et2 exhibited detect-
able fluorescence at concentrations of up to 1 μM of copper
ions even in the presence of such high concentrations of GSH
(Fig. S4; see ESI†). These results demonstrate that the mito-
chondria-targeting probe RdlTPA-TPP could be applied to
monitor the levels of mitochondrial Cu+ in cells.
The synthesis of RdlTPA-TPP is outlined in Scheme 1b. The
benzophenone derivative 1 was synthesized by the decompo-
sition of fluorescein under reflux in a KOH solution.12 A sub-
sequent Friedel–Crafts reaction between 1 and aminophenol
213 in boiling TFA afforded the rhodol platform 3. The de-
protection of the N-acetyl group in a boiling HBr solution fol-
lowed by esterification with methanolic sulphuric acid yielded
ester 4, which was then reduced with LiAlH4. Subsequent oxi-
dation with p-chloranil afforded the rhodol fluorophore
5 bearing a hydroxymethyl group. The coupling reaction
between 5 and TPP-carboxylic acid 6 afforded Rdl-TPP and it is
the envisaged fluorescent product generated by the reaction of
RdlTPA-TPP with Cu+. The final desired fluorescence probe
was obtained following the alkylation of the phenolic OH
group with 6-TPA-Cl and the purification of the resulting crude
product by HPLC (ODS, CH3CN–H2O).14
Prior to the imaging experiments, the fluorescence of the
RdlTPA-TPP towards Cu+ was confirmed to be comparable to
that of RdlTPA-Et2 (Fig. S5; see ESI†). HeLa cells loaded with
10 μM of RdlTPA-TPP exhibited a punctate staining pattern
(Fig. 4a and 4e). To elucidate the stained subcellular localiz-
ation of the probe, the cells were coincubated with tetramethyl
rosamine (TMR) or LysoTracker Red (Fig. 4b and 4f), which are
commercially available fluorescent dyes that can be used for
labelling mitochondria or acidic cellular compartments, such
as lysosome, respectively. Surprisingly, the micrograph of
intracellular RdlTPA-TPP did not colocalize with that of the
TMR (Fig. 4c), although it did colocalize well with the
LysoTracker Red (Fig. 4g). The spirocyclized structure of
RdlTPA-TPP became the ring-opened fluorescent dye at an
acidic pH value of ≤6 (Fig. S6; see ESI†). The probe would
therefore exist predominantly in the ring-opened form in the
acidic cellular compartments. In contrast, the pH of the MM is
7.8.15 At this pH, the probe would exist in the spirocyclic form
and is consequently unable to reveal any fluorescence. For this
reason, the presence of the probe in the mitochondria was
confirmed using m-chlorophenylhydrazone (CCCP), which
causes mitochondrial depolarization and uncoupling of respir-
ation. Because the treatment of cells with CCCP induces
redistribution of mitochondrially targeted dyes to lysosomal
compartments,16 the lysosomal concentration of RdlTPA-TPP
Fig. 2 Fluorescence spectral changes observed upon addition of 20 μM
[CuI(CH3CN)4]PF6 to the solution of 1 μM RdlTPA-Et2 in 50 mM HEPES
(pH 7.20) containing 2 mM GSH. The spectra were measured every
15 min with the excitation wavelength at 525 nm. Inset: time course of
the fluorescence intensity change at 542 nm.
a 100-fold increase in the fluorescence was observed (solid line
in Fig. 2). Analysis of the reaction mixture by HPLC and electro-
spray ionization mass spectroscopy (Fig. S2 and S3, respect-
ively; see ESI†) reveals that HMDER (Φ = 0.37) is generated in
almost quantitative yield (>99%) following a 3 h reaction
period. This result indicates that the C–O bond cleavage reac-
tion induced by Cu+ recognition as demonstrated for FluTPAs
proceeds even in the case of the spirocyclized rhodol scaffold.
The fluorescence response of RdlTPA-Et2 was specific for
copper ions (i.e., Cu+ and Cu2+). Although Co2+ caused a rela-
tively small enhancement in the fluorescence (Fig. 3),9 this
Fig. 3 Fluorescence response of RdlTPA-Et2 (1 μM) at 542 nm as a
function of various added metal cations (5 mM for Na+, K+, Ca2+, and
Mg2+, 20 μM for all other cations) in 50 mM HEPES (pH 7.20). 1, no
metal; 2, Cu+; 3, Cu2+; 4, Na+; 5, K+; 6, Mg2+; 7, Ca2+; 8, Mn2+; 9, Fe3+
;
10, Co2+; 11, Ni2+; 12, Zn2+; 13, Cd2+; 14, Hg2+; 15, Cu+ + Na+; 16, Cu+
K+; 17, Cu+ + Mg2+; 18, Cu+ + Ca2+
+
.
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Fig. 4 Localization of RdlTPA-TPP in HeLa cells grown in the basal media. (a–d) Confocal images of the cells loaded with 10 μM RdlTPA-TPP and 75
nM tetramethyl rosamine. (a) RdlTPA-TPP; (b) TMR; (c) overlay of (a) and (b); (d) overlay of bright field image with (c). (e–f) Confocal images of the
cells loaded with 10 μM RdlTPA-TPP and 50 nM LysoTracker Red. (e) RdlTPA-TPP; (f) LysoTracker Red; (g) overlay of (e) and (f); (h) overlay of bright
field image with (f).
should increase, resulting in an increase of fluorescence pattern to those described above, it was clear that the probe
signals of the ring-opened form. As predicted, an enhance- had not reacted in the lysosome to emit fluorescence.
ment in the fluorescence of RdlTPA-TPP was observed,
although the intensity remained unchanged for LysoTracker
Red (Fig. S7; see ESI†). This result strongly supports that the
non-fluorescent spirocyclized probe is certainly localized to
Conclusions
We have developed a new mitochondria-targeted fluorescent
probe, RdlTPA-TPP, for detecting Cu+. This probe is essentially
non-fluorescent at neutral pH because of the spirocyclization
of its hydroxymethyl group. The reaction of the probe with Cu+
under reduction conditions induced the C–O bond cleavage of
the benzyl ether, with the resulting cleavage product being
simultaneously converted into its fluorescent ring-opened
form. Because RdlTPA-TPP needs a long incubation time to
generate sufficient fluorescence for detection, this probe is
suitable for imaging mitochondrial Cu+ pools in fluorescence
with a high S/N ratio, rather than for the spatiotemporal deter-
mination of Cu+ in living biological samples. The most
notable feature of this probe design is the ease with which the
subcellular localization of probes can be modulated via the
introduction of the different organelle-targeting group instead
of the TPP tag. Such organelle specific Cu+ probes could be
used as powerful tools for elucidating the distribution of
copper as well as metabolism pathways at the cellular level.
mitochondria. Finally, imaging experiments were then per-
formed with Cu-supplemented HeLa cells, which were grown
in DMEM containing 100 μM of CuCl2 for 6 h at 37 °C. As
shown in Fig. 5a, much brighter fluorescence signals com-
pared to those in panels (a) and (e) in Fig. 4 were observed in
the cells following 3 h of incubation with 5 μM RdlTPA-TPP. A
co-staining experiment with TMR provided a fully merged
image that was orange in appearance (Fig. 5b), suggesting that
the Cu+ induced C–O bond cleavage reaction of the probe pro-
ceeded to afford a fluorescent product (Rdl-TPP) in the mito-
chondria. It is noteworthy that a punctate staining pattern
derived from the acidic organelle was still visible in Fig. 5b
(depicted as green). Given that cells prepared separately that
were loaded with Rdl-TPP did not show a similar staining
Experimental
Synthesis
General. All chemicals used in this study were commercial
products of the highest available purity. 2-(2,4-Dihydroxy-
benzoyl)benzoic acid (1),12 3-hydroxy-N-(2-acetamidoethyl)-N-
methylaniline (2),13 HMDER,7 and 6-TPA-Cl8 were prepared
according to the reported procedures. NMR spectra were
Fig. 5 Detection of mitochondrial copper ions with RdlTPA-TPP. Cu-
supplemented HeLa cells were coincubated with 5 μM RdlTPA-TPP and
TMR for 3 h at 37 °C. (a) Fluorescence image of RdlTPA-TPP; (b) merged
image of (a) and TMR; (c) bright field image.
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recorded on a JNM-ECX-500 (at 500 MHz to 1H, 125 MHz to of the solvent, the residue was dissolved in MeOH (50 mL), fol-
13C). Chemical shifts are given in ppm relative to tetramethyl- lowed by addition of conc. H2SO4 (0.5 mL). The solution was
silane (Si(CH3)4). HRMS were recorded on a Bruker micrOTOF refluxed for 2 days. After neutralizing with NaHCO3, insoluble
II (ESI). TLC analyses were performed on silica gel 60-F254 inorganic materials were removed by filtration and the filtrate
(Merck). Flash chromatography was performed on silica gel was concentrated in vacuo to yield the ester 4. The crude was
(Merck Silica Gel 60). ODS column chromatography was per- purified by ODS column chromatography eluted with H2O–
formed on a YFLC system (Yamazen Co., Osaka, Japan). Re- CH3CN (containing 0.1% TFA) to give an orange powder
1
cycling preparative HPLC was performed on a LC-9110NEXT (58 mg, 44% for 2 steps). H NMR (500 MHz, CD3OD): δ = 3.28
system (Japan Analytical Industry Co., Japan) connected to a (2H, t, brt, overlapped with solvent peaks), 3.36 (3H, s), 3.57
UV detector. Reverse phase HPLC was performed on a Waters (3H, s), 4.01 (2H, brt), 7.02 (1H, dd, J = 9.0, 2.5 Hz), 7.20 (1H,
Delta 600 system (10 × 250 mm, YMC-Triart C18).
d, J = 2.5 Hz), 7.25–7.35 (4H, m), 7.43 (1H, d, J = 6.5 Hz),
RdlTPA-Et2. To a solution of HMDER (281 mg, 0.75 mmol), 7.81–7.86 (2H, m), 8.32 (1H, d, J = 6.5 Hz); 13C NMR (500 MHz,
Cs2CO3 (688 mg, 2.25 mmol), and KI (125 mg, 0.75 mmol) in CD3OD): δ = 36.3, 38.9, 49.8, 51.7, 96.9, 102.1, 115.7, 115.8,
dry CH3CN (50 mL) was slowly added 6-TPA-Cl (254 mg, 116.3, 118.0, 130.1, 130.7, 131.1, 131.6, 132.0, 133.0, 133.4,
0.75 mmol), and the reaction mixture was refluxed overnight 157.8, 159.0, 159.1, 163.7, 165.5, 168.9; MS (ESI pos.) m/z calcd
under an Ar atmosphere. After removal of the insoluble for C24H22N2O4 ([M + H]+) 403.17, found 403.17; calcd for
materials by filtration, the filtrate was evaporated. The residue C24H22N2O4 ([M + Na]+) 425.15, found 425.15.
was dissolved in CH2Cl2 (100 mL), and the organic layer was
Compound 5. To a solution of compound 4 (50 mg,
washed with water (100 mL × 2) and brine (100 mL), dried over 0.12 mmol) in dry THF (20 mL) was slowly added LiAlH4 in
MgSO4, and concentrated. The crude residue was purified by small portions at 0 °C until the starting materials were con-
Al2O3 column chromatography (CH2Cl2 –MeOH = 40 : 1) to give sumed. The mixture was warmed to room temperature and
the title compound as a pale yellow oil (410 mg, 81%). For stirred overnight. 2 M HCl aq. was added to the solution until
spectroscopic analyses, the compound was further purified by the pH became neutral to quench the reaction. The resulting
preparative HPLC with a linear gradient from 70% solvent A insoluble material was filtered through a pad of Celite and the
(0.1% TFA in water) and 30% B (0.1% TFA in acetonitrile) to filtrate was evaporated in vacuo. The residue was dissolved in
10% A and 90% B and the collected fraction was lyophilized. dry MeOH (10 mL), followed by addition of p-chloranil (89 mg,
1H NMR (500 MHz, CDCl3): δ 1.15 (6H, t, J = 6.8 Hz), 3.34 (4H, 0.36 mmol). After stirring for 2 h at room temperature, the
q, J = 6.8 Hz), 3.91 (2H, s), 3.92 (4H, s), 5.19 (2H, s), 5.23 (2H, solvent was removed and the crude was purified by ODS
d, J = 3.0 Hz), 6.36–6.40 (2H, m), 6.66 (1H, dd, J = 8.8, 2.4 Hz), column chromatography eluted with H2O–CH3CN (containing
6.73 (1H, d, J = 8.8 Hz), 6.78, (1H, d, J = 2.4 Hz), 6.82 (1H, d, J = 0.1% TFA) to give an orange powder (28 mg, 63%).
8.8 Hz), 6.93 (1H, d, J = 8.0 Hz), 7.13 (2H, m), 7.24–7.27 (2H,
Rdl-TPP. To a solution of amine 4 (5 mg, 10 μmol) and
m), 7.33–7.36 (3H, m), 7.51 (1H, d, J = 7.5 Hz), 7.59 (2H, d, J = (3-carboxypropyl)triphenylphosphonium iodide (4.6 mg,
7.5 Hz), 7.65 (2H, td, J = 7.5, 1.5 Hz), 7.67 (1H, t, J = 7.5 Hz), 10 μmol) in dry DMF (3 mL) were added EDC (3.8 mg,
8.54 (2H, ddd, J = 5.0, 2.0, 0.5 Hz); 13C-NMR (126 MHz, CDCl3) 20 μmol), DIEA (10 mg, 80 μmol) and HOBt (3.0 mg, 20 μmol).
δ 12.7, 44.6, 60.3, 60.4, 70.9, 71.6, 84.0, 97.8, 101.9, 111.2, After stirring overnight at room temperature, the solvent was
111.6, 118.1, 120.0, 120.7, 121.9, 122.2, 123.2, 124.2, 128.0, removed and the crude was purified by ODS column chromato-
128.3, 129.7, 130.1, 136.6, 137.4, 139.8, 145.0, 148.8, 149.2, graphy eluted with H2O–CH3CN (containing 0.1% TFA) to give
152.1, 152.1, 156.5, 159.0, 159.2, 159.5; MS (ESI pos.) m/z calcd an orange powder (2.5 mg, 26%). Because the available
for C43H42N5O3 ([M + H]+) 676.33, found 676.31; calcd for amount of this compound was too small for NMR analyses,
C43H41N5O3Na1 ([M + Na]+) 698.31, found 698.29.
mass analysis was performed to identify the product. MS (ESI
Compound 3. A mixture of benzophenone 1 (8 g, 31 mmol) pos.) m/z calcd for C45H42N2O4P ([M]+) 705.28, found 705.28;
and aminophenol 2 (6.5 g, 31 mmol) in TFA (120 mL) was C45H43N2O4P ([M + H]2+) 353.15, found 353.14. The overall
stirred for 3 h at 95 °C. After cooling to room temperature, TFA purity of this compound was also confirmed by HPLC analysis
was removed by evaporation. The residue was directly purified (Fig. S11, see ESI†).
by SiO2 column chromatography (CHCl3–MeOH = 9 : 1) to give
RdlTPA-TPP. This compound was prepared according to the
rhodol 3 as a red oil (140 mg, 18%). 1H NMR (500 MHz, synthetic procedure for RdlTPA-Et2 by using Rdl-TPP instead
CD3OD): δ = 1.83 (3H, s), 3.25 (3H, s), 3.46 (2H, brt), 3.73 (2H, of HMDER. The final product was purified by ODS column
m), 6.86 (1H, dd, J = 9.0, 2.5 Hz), 6.98–7.09 (2H, m), 7.07–7.09 chromatography eluted with H2O–CH3CN (containing 0.1%
(2H, m), 7.36 (1H, d, J = 7.5 Hz), 7.79 (1H, t, J = 7.5 Hz), 7.84 TFA) to give an orange powder (4.7 mg, 80%). HRMS (ESI pos.)
(1H, t, J = 7.5 Hz), 8.28 (1H, d, J = 7.5 Hz); 13C NMR (500 MHz, m/z calcd for C45H42N2O4P ([M]+) 1007.441, found 1007.417.
CD3OD): δ = 22.5, 37.9, 39.8, 52.8, 79.4, 98.1, 103.4, 114.7, The overall purity of this compound was also confirmed by
115.5, 115.9, 117.7, 129.9, 131.0, 131.5, 132.0, 132.1, 134.4, HPLC analysis (Fig. S12, see ESI†).
139.2, 157.7, 158.6, 158.8, 167.9, 169.0, 174.0; MS (ESI pos.)
Steady-state absorption and fluorescence spectroscopy
m/z calcd for C25H23N2O5 ([M + H]+) 431.16, found 431.16.
Compound 4. A solution of rhodol 3 (140 mg, 0.33 mmol) The UV absorption spectra were recorded on a Hewlett-Packard
in 1.5 M HCl aq. (10 mL) was refluxed overnight. After removal 8453 spectrometer. Fluorescence spectra were recorded using a
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Hitachi F-2500 spectrometer with a slit width of 2.5 nm. The RdlTPA-TPP in DMSO was diluted to 5 μM in colorless DMEM
photomultiplier voltage was 700 V. To reduce the fluctuation (without phenol red, Invitrogen). The cells were then loaded
in the excitation intensity during measurements, the lamp was with RdlTPA-TPP (5 μM) and TMR (75 nM) by incubation for
kept on for 1 h prior to the experiment. The path length was 30 min at 37 °C, rinsed three times with colorless DMEM, and
1 cm with a cell volume of 3.0 mL. Fluorescence responses of imaged under the same conditions as those described above.
RdlTPA-Et2 to various metal ions were measured as follows.
RdlTPA-Et2 (final, 1 μM) was added to 1 mL of HEPES (50 mM,
pH 7.20) in the presence of 2 mM GSH. Aqueous solution of
transition metal ions (MnCl2, CoCl2, NiCl2, FeCl3, and ZnCl2)
Acknowledgements
or [Cu(CH3CN)4]PF6 in CH3CN was added to give a final con-
This work was financially supported by a Grant-in-Aid for
centration of 20 μM. The mixtures were vortexed and kept in
Young Scientists (A) from JSPS (grant no. 23685039 for M.T.)
the dark at room temperature for 3 h. All fluorescence spectra
and the Naito Foundation (M.T.).
were measured with an excitation wavelength at 525 nm.
Product analysis
To a 1 μM solution of RdlTPA-Et2 in 50 mM HEPES buffer (pH
7.20) containing 2 mM GSH was added a 20 μM solution of
Notes and references
[CuI(CH3CN)4]PF6 in CH3CN under aerobic conditions. The
mixture was stirred for 3 h at room temperature and analyzed
with a reverse phase HPLC (Waters Delta 600 system, 4.6 ×
150 mm, YMC-Triart C18) eluted with CH3CN–H2O (0.1%
TFA). The retention time was compared with that of an auth-
entic sample of HMDER. The yield of the production of
HMDER was calculated from the integrated areas of the emis-
sion for HMDER to be >99%. For ESI-MS analysis, a 10 μM
solution of [CuI(CH3CN)4]PF6 in CH3CN in water was added to
a 2 μM solution of RdlTPA-Et2 in 10 mM ammonium acetate
containing 100 μM GSH under aerobic conditions. The
mixture was stirred for 3 h at room temperature. The samples
were directly measured using a micrOTOF II focus (Bruker).
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Cell culture experiments
Preparation of cells. HeLa cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Sigma) containing 10%
fetal bovine serum (FBS, Gibco) and 1% Antibiotic–Antimyco-
tic (Gibco) at 37 °C in a 5% CO2/95% air incubator. Two days
before imaging, cells (5 × 104) were plated in a 35 mm dia-
meter glass-bottomed dish and cultured in DMEM. Copper
uptake experiments were performed in the same medium
except the overnight supplementation of 100 μM CuCl2 at the
end of the incubation period noted above.
Intracellular localization analysis. A 5 mM stock solution of
RdlTPA-TPP in DMSO was diluted to 10 μM in colorless DMEM
(without phenol red, Invitrogen). HeLa cells (no copper sup-
plementation) were coincubated with 10 μM RdlTPA-TPP and
75 nM N,N′-tetramethyl rosamine (TMR) or 50 nM LysoTracker
Red for 30 min at 37 °C. The cells were rinsed three times with
colorless DMEM to remove the excess probe, and imaged with
a FV10i-DOC confocal laser-scanning microscope (OLYMPUS).
The fluorescence images were obtained using the corres-
ponding lasers (RdlTPA-TPP: λex = 473 nm, λem = 490–540 nm,
TMR and LysoTracker Red: λex = 559 nm, λem = 574–674 nm).
Fluorescence imaging of Cu+ in cells. Immediately before
the staining experiments, Cu-supplemented cells were washed
twice with PBS buffer containing 200 μM EDTA to remove
extracellular heavy metal ions. A 5 mM stock solution of
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5004 | Org. Biomol. Chem., 2014, 12, 4999–5005
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Organic & Biomolecular Chemistry
Paper
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1H/13C NMR analyses, the overall purity of the product was
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