A fluorescent probe for the Fe3+ ion pool in endoplasmic reticulum in liver cells

Article abstract of DOI:10.1016/j.dyepig.2016.03.032

We have presented rhodamine-derived Schiff base probes (1-6) selective of the Fe³⁺ ion in biological systems. In the presence of Fe³⁺ ions, the probes display new absorption and emission bands at 526 nm and 551 nm, respectively. Conf

Full text of DOI:10.1016/j.dyepig.2016.03.032

Accepted Manuscript
3+
A Fluorescent Probe for the Fe Ion Pool in Endoplasmic Reticulum in Liver Cells
Min Hee Lee, Hoyeon Lee, Min Jung Chang, Hyeong Seok Kim, Chulhun Kang, Jong
Seung Kim
PII:
S0143-7208(16)30092-4
10.1016/j.dyepig.2016.03.032
DYPI 5157
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 20 February 2016
Revised Date: 17 March 2016
Accepted Date: 18 March 2016
Please cite this article as: Lee MH, Lee H, Chang MJ, Kim HS, Kang C, Kim JS, A Fluorescent Probe
3+
for the Fe Ion Pool in Endoplasmic Reticulum in Liver Cells, Dyes and Pigments (2016), doi: 10.1016/
j.dyepig.2016.03.032.
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A Fluorescent Probe for the Fe Ion Pool in
Endoplasmic Reticulum in Liver Cells
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a,*
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Min Hee Lee , Hoyeon Lee , Min Jung Chang , Hyeong Seok Kim ,
b,*
c,*
Chulhun Kang , and Jong Seung Kim
a
Department of Chemistry, Sookmyung Women’s University, Seoul 140-742, Korea
b
The School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea
c
Department of Chemistry, Korea University, Seoul 136-701, Korea
*Corresponding authors: minheelee@sookmyung.ac.kr (M. H. Lee); kangch@khu.ac.kr (C. Kang);
jongskim@korea.ac.kr (J. S. Kim)
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Abstract
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We have presented rhodamine-derived Schiff base probes (1–6) selective of the Fe ion in biological
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systems. In the presence of Fe ions, the probes display new absorption and emission bands at 526 nm
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and 551 nm, respectively. Confocal microscopic analysis using the HepG2 cell line showed that probes 1–
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6 provide fluorescence Off–On changes caused by cellular Fe ions, and demonstrated a largely
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enhanced fluorescence upon iron overloading of the cells. We also found that the Fe ion-mediated
fluorescence changes of probes 1–6 are mainly localized in the ER (Endoplasmic Reticulum) in HepG2
cells. Among them, probe 5 exhibits the highest selectivity for ER localization over the other organelles.
Key Words
Iron detection, fluorescent probe, rhodamine, cell imaging
1
. Introduction
Iron is an essential trace element in human cells. It exists as a crucial cofactor for proteins that are
involved in processes such as oxygen delivery, the electron transfer chain of mitochondria, and
oxidation/reduction of biological substrates [1–3]. However, unbound Fe ions are potentially toxic species
due to their high propensity to induce, through the Fenton reaction, reactive oxygen species that are
responsible for oxidative cell damage [4]. In fact, the release of Fe ions from iron-containing proteins
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induces iron (Fe )-mediated cytotoxicity that has been strongly implicated in many neurodegenerative
diseases, including Alzheimer’s disease and Parkinson’s disease [5,6]. However, how the Fe ions cause
the cytotoxicity is still unclear. Therefore, detecting Fe ions in live cells would be essential for
understanding its toxicity and the relationship with human diseases.
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Currently, several fluorescent probes are being exploited for the selective detection of Fe ions in
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human cells [7–11]. These probes have provided fluorescent images for cellular Fe ions; however,
investigations at the organelle level in cells are rare. Previously, we reported a rhodamine-based probe
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that gives a fluorescent enhancement for cellular Fe ions through a Fe -mediated hydrolysis of the
Schiff base and the spirolactam ring opening of the rhodamine moiety, as shown in Scheme 1 [11]. Based
on this, we have synthesized rhodamine-based Schiff base derivatives (1–6), and assessed their abilities
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for Fe -sensing at the organelle level. Herein, we present rhodamine-based probes that allow for Fe
imaging, mainly localized in the ER (Endoplasmic Reticulum) of HepG2 cells.
Scheme 1.
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2
. Materials and Methods
2
.1. Materials and instrumentation
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All fluorescence and UV/Vis absorption data were collected using RF-5301PC (Shimadzu Corporation,
Kyoto, Kyoto Prefecture, Japan) and S-3100 (Betatek Inc., Toronto, Ontario, Canada) spectrophotometers,
respectively. NMR was recorded using a Varian INOVA spectrometer (400 MHz). All reagents and
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cationic compounds such as chloride salts of Na , K , Li , Ag , Hg , Cu , Pb , Cd , Co , Ni , Ba ,
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Ca , Mg , Zn , Mn , Fe , and Fe were purchased from Aldrich (St. Louis, MO, USA). CH CN for
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spectra detection was an HPLC grade reagent without fluorescent impurity, and deionized water was used.
2.2. UV/Vis and fluorescence spectroscopic methods
Stock solutions of metal chloride salts were prepared in deionized water. Stock solutions of synthetic
probes were prepared in CH CN. All spectra were recorded in aqueous solution (CH CN:H O = 5:95).
3
3
2
Excitation was carried out at 528 nm with all excitation and emission slit widths at 3 nm.
2.3. Cell culture and confocal microscopic methods
A human hepatoma cell line, HepG2, was grown in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 100 unit/mL penicillin, and 100 g/mL of streptomycin at 37 C under 95% humidified air
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containing 5% CO . A total of 1 × 10 cells were cultured and stabilized in a confocal dish and the
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medium was replaced with fresh medium containing 500 μM of ferric citrate. After 24 h of incubation,
the medium was removed and the cells were briefly washed with 1 mL of PBS (phosphate buffered
saline). Then, 1 mL of media containing each of the probes, 1–6 (final concentration was 1 μM), was
added and incubated. The images were obtained using a confocal microscope from Carl Zeiss (LSM 510
META model; Oberkochen, Germany). Probes 1–6 were excited at 488 nm using an argon laser and the
emission image was acquired using a band-path filter (505–550 nm). All images were collected under the
same experimental parameters to minimize possible variations in fluorescence intensity.
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2.4. Synthetic methods
Compounds 2 and 7 were prepared according to a previously described procedure [11] and compounds 1
and 3–6 were newly synthesized in this study by modifying the procedure (Scheme 2a).
2
.4.1. Compound 1
Rhodamine-ethylene diamine (7) (0.3 g, 0.66 mmol) was stirred with salicylaldehyde (0.08 g, 0.66 mmol)
in methanol for 26 h at room temperature. The precipitates were collected from the methanol. Finally,
+
0
.25 g of probe 1 (69% yield) was obtained as a yellow solid. ESI-MS m/z (M ) calcd 560.3, found 561.2.
1
H NMR (CDCl , 400 MHz): δ 13.00 (s, 1H); 8.01 (s, 1H); 7.95 (d, 1H, J = 6.40 Hz); 7.45 (t, 2H, J = 3.60
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Hz); 7.26–7.22 (m, 1H); 7.11 (d, 1H, J = 7.60 Hz); 7.05 (d, 1H, J = 7.20 Hz); 6.89 (d, 1H, J = 8.40 Hz);
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.80 (t, 1H, J = 7.60 Hz); 6.36 (s, 2H); 6.21 (s, 2H); 3.46–3.42 (m, 4H); 3.35 (t, 2H, J = 7.20 Hz); 3.15–
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.8 (m, 4H); 1.84 (s, 6H), 1.32 (t, 6H, J = 7.20 Hz). C NMR (CDCl 100 MHz): 168.5, 165.9, 161.2,
3,
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53.8, 151.9, 147.6, 132.7, 132.1, 131.3, 131.2, 128.6, 128.2, 124.0, 123.0, 118.9, 118.4, 118.1, 117.1,
106.1, 96.7, 65.1, 57.3, 41.1, 38.5, 16.8, 14.9 ppm.
2
.4.2. Compound 3
Rhodamine-ethylene diamine (7) (0.2 g, 0.44 mmol) was stirred with 2-hydroxy-3,5-dinitrobenzaldehyde
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0.09 g, 0.44 mmol) in methanol for 15 h at room temperature. The precipitates were collected from
+
methanol. Finally, 0.24 g of 3 (83% yield) was obtained as a yellow solid. ESI-MS m/z (M ) calcd 650.2,
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found 651.2. H NMR (CDCl , 400 MHz): δ 8.93 (s, 1H); 8.23 (s, 1H); 7.94–7.92 (m, 1H); 7.83 (s, 1H);
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.53–7.48 (m, 2H); 7.08–7.06 (m, 1H); 6.35 (s, 2H); 6.13 (s, 2H); 3.56–3.52 (m, 6H); 3.23–3.18 (m, 4H);
13
.84 (s, 6H); 1.33 (t, 6H, J = 6.80 Hz). C NMR (CDCl 100 MHz): 170.6, 169.1, 166.8, 153.1, 152.0,
3,
47.9, 135.9, 133.4, 130.6, 128.7, 128.5, 128.2, 124.2, 123.3, 118.5, 116.7, 105.5, 96.7, 65.4, 50.2, 39.5,
8.5, 16.8, 14.7 ppm.
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.4.3. Compound 4
Rhodamine-ethylene diamine (7) (0.2 g, 0.44 mmol) was stirred with 3-nitrobenzaldehyde (0.07 g, 0.44
6

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mmol) in methanol for 14 h at room temperature. The precipitates were collected from methanol. Finally,
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.20 g of probe 4 (78% yield) was obtained as a yellow solid. FAB-MS m/z (M ) calcd 589.3, found
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90.2. H NMR (CDCl , 400 MHz): δ 8.33 (s, 1H); 8.21 (m, 1H); 7.95–7.92 (m, 3H); 7.52 (t, 1H, J = 8.01
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Hz); 7.46–7.42 (m, 2H); 7.05–7.02 (m, 1H); 6.35 (s, 2H); 6.21 (s, 2H); 3.49–3.47 (m, 6H); 3.21–3.18 (m,
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4H); 1.83 (s, 6H); 1.32 (t, 6H, J = 7.20 Hz). C NMR (CDCl , 100 MHz): 168.7, 159.5, 153.9, 151.9,
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48.6, 147.5, 138.0, 133.5, 132.7, 131.1, 129.5, 128.7, 128.2, 124.9, 124.0, 123.1, 123.0, 118.0, 106.3,
6.8, 65.2, 59.2, 41.2, 38.5, 16.8, 14.9 ppm.
2
.4.4. Compound 5
Rhodamine-ethylene diamine (7) (0.16 g, 0.35 mmol) was stirred benzaldehyde (0.04 g, 0.35 mmol) in
methanol for 20 h at room temperature. The precipitates were collected from methanol. Finally, 0.07 g of
+
1
probe 5 (38% yield) was obtained as a pink solid. FAB-MS m/z (M ) calcd 544.3, found 545.3. H NMR
(
CDCl , 400 MHz): δ 8.00 (s, 1H); 7.94–7.92 (m, 1H); 7.58–7.56 (m, 2H); 7.45–7.42 (m, 2H); 7.36–7.31
3
(
m, 3H); 7.05–7.03 (m, 1H); 6.35 (s, 2H); 6.23 (s, 2H); 3.47–3.39 (m, 6H); 3.21–3.20 (m, 4H); 1.85 (s,
1
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6
H); 1.32 (t, 6H, J = 7.20 Hz). C NMR (CDCl , 400 MHz): 196.3, 168.5, 162.5, 156.4, 154.0, 151.9,
3
147.5, 136.3, 132.6, 131.2, 130.6, 128.7, 128.5, 128.2, 128.1, 123.9, 122.9, 118.0, 106.2, 105.2, 96.8, 77.7,
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5.2, 59.1, 41.4, 38.5, 16.8, 14.9 ppm.
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.4.5. Compound 6
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.08 g of probe 6 (28% yield) was obtained as an orange solid. FAB-MS m/z (M ) calcd 634.3, found
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635.2. H NMR (CDCl , 400 MHz): δ 8.81 (d, 1H, J = 1.60 Hz); 8.45–8.42 (m, 1H); 8.24–8.22 (d, 2H, J
3
=
9.21 Hz); 7.93–7.91 (m, 1H); 7.45 (t, 2H, J = 3.20 Hz); 7.05–7.03 (m, 1H); 6.33 (s, 2H); 6.18 (s, 2H);
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.55 (t, 2H, J = 6.80 Hz); 3.48–3.45 (m, 4H); 3.22–3.16 (m, 4H); 1.82 (s, 6H); 1.33 (t, 6H, J = 6.80 Hz).
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C NMR (CDCl , 400 MHz): 196.3, 168.5, 162.5, 156.4, 154.0, 151.9, 147.5, 136.3, 132.6, 131.2, 130.6,
3
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ppm.
3
. Results and Discussion
3+
3
.1. Absorption and fluorescence spectra of probes 1–6 responding to Fe ions
The synthetic routes for the iron probes 1–6 and their chemical structures are displayed in Scheme 2.
Among the probes presented here, probe 2 was previously reported by our group and probes 1 and 3–6
were newly synthesized by modifying the synthetic procedure for probe 2 [11]. All chemical structures of
1
13
the probes were confirmed by H NMR, C NMR, and ESI−MS (Figs. S1–S15). The probes 1 and 4 were
also confirmed by X-ray crystallographic analysis as displayed in Fig. 1 (see also Tables S1–S10).
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Scheme 2.
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Fig. 1.
3+
For the Fe -sensing mechanism of compound 2, we previously demonstrated that the irreversible
3
+
binding of Fe with probe 2 resulted in the ring opening of spirolactam in the rhodamine moiety followed
1
by the hydrolysis of the Schiff base linkage (Scheme 1) [11]. This was confirmed by H-NMR and ESI-
MS spectroscopy, and X-ray crystallography. Similarly, in ESI-MS analysis experiments, the mass peaks
3+
for the probes 1 and 3–6 disappeared upon the addition of Fe ions, whereas rhodamine-ethylene diamine
(
7) was mainly detected (Figs. S16–S21). These results tell us that probes 1–6 provide a fluorescence
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change due to the addition of the Fe ions through a Fe -mediated hydrolysis of the Schiff base, which
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3+
We tested the selectivity of the probes (1–6) for Fe ions over other metal ions in aqueous solution. As
1
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seen in Figs. 2a and 2b, probe 5 alone displayed no distinct absorption and emission bands, whereas upon
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the addition of Fe ions to a solution of probe 5, new absorption and emission bands appeared at 526 nm
2+
and 551 nm, respectively. Upon the addition of Hg , similar fluorescence change was observed. However,
+
+
+
+
2+
2+
2+
2+
2+
2+
2+
2+
in the case of other metal ions, including Na , K , Li , Ag , Cu , Pb , Cd , Co , Ni , Ba , Ca , Mg ,
2+
2+
2+
Zn , Mn , and Fe , the changes were relatively negligible. In addition, the absorption and emission
3+
bands at 526 nm and 551 nm were gradually enhanced with an increasing concentration of Fe ions (Fig.
3). Similar photophysical changes were also seen in the case of the other probes (Figs. S22–S33). These
3+
results imply that the probes 1–6 are predominantly selective for Fe over the other metal ions, including
2+
Fe ions.
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Fig. 3.
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+
Fluorescence changes caused by a Fe -induced ring opening of the rhodamine part in probes 1–6
3
+
were also monitored as a function of time. As seen in Fig. 4, upon the addition of 100 equiv. of Fe ions
to a solution of probe 5, the emission band at 552 nm was gradually increased over the course of 60 min
3+
(
Fig. 4 inset). Similar changes were also monitored upon the addition of Fe ions to other probes (Figs.
S35–S39).
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Fig. 4.
In order to investigate the interference of other biologically active metals, the fluorescence changes of
+
+
2+
2+
probes 1–6 were tested in the presence of various essential metal ions, including K , Na , Ca , Fe ,
2+
2+
3+
Mg , and Mn . In Fig. 5, the Fe -mediated fluorescence enhancements at 552 nm were readily observed
with probes 1–6 in the presence of high concentrations of other biologically relevant metal ions such as
+
+
2+
2+
2+
2+
K , Na , Ca , Fe , Mg , and Mn (5 mM each). The levels of enhanced fluorescence intensities were
3+
also similar to those of probes 1–6 recorded upon the addition of 50 μM of Fe ions. This indicates that
3
+
probes 1–6 can be employed to detect Fe in biological systems without interference from other
competitive metal ions.
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Fig. 5.
3+
3
.2. Sensing abilities of probes 1–6 to the Fe ions in live cells
3+
The Fe -sensing abilities of probes 1–6 were tested in iron-overloaded HepG2 cells where the iron
overloading was achieved by treatment with ferric citrate. It is reported that the hepatocytes are important
in iron metabolism, and ferric citrate is the major iron carrier in the blood system to the liver cells in iron
overload and related diseases [12]. Therefore, in the presence of ferric citrate, the fluorescent intensity of
the probe in the cells was anticipated to be increased. Indeed, upon incubation with probe 5, ferric citrate-
treated cells display a stronger fluorescence than the cells in the absence of ferric citrate (Fig. 6). The
fluorescence images were also monitored as a function of incubation time upon the addition of 500 µM of
ferric citrate (Fig. S40). Thus, we demonstrated that probe 5 provides a fluorescence Off–On image for
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Cytotoxicity tests using probes 1–6 were also carried out to determine their cell compatibilities. As
inferred from an MTT assay (Fig. S41), the probes 1–6 displayed negligible cytotoxicity for the cellular
3+
Fe imaging in HepG2 cells.
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Fig. 6.
To identify the cellular location for the fluorogenic reactions of 1–6, co-localization experiments were
performed using selective fluorescent dyes for the ER (Endoplasmic Reticulum), lysosome (Lyso), and
mitochondria (Mito), in HepG2 cells. As revealed in Figs. 7 and S42–S46, the green images of the probes
1–6 are found to be more co-localized with the red image of the ER tracker than those of Mito and Lyso
trackers. The colocalization analysis for each probe was performed using the Pearson coefficient (Rr
value) [13, 14]. We found that the values of 1-6 for ER are 0.6718, 0.5294, 0.5425, 0.5527, 0.7854, and
0
.6587, respectively (Figs. S42–S46), suggesting that the variable substituents (-H, -OH, or -NO ) on the
2
phenyl group of Schiff base linkage of probes 1-6 affect the ER-accumulation ability of the probe without
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+
major changes of the Fe ion selectivity or reactivity. Interestingly, the fluorescence image of probe 5 is
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highly overlapped with that of the ER. The Pearson coefficients of probe 5 for ER, Lyso, and Mito
trackers were also determined to be 0.7854, 0.4709, and 0.2034, respectively (Fig. 7). From this, we could
3+
infer that probes 1–6 are mainly localized in the ER and give a fluorescence Off–On change with Fe
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ions. In addition, probe 5 was highly selective for the ER-localized Fe ions. This would be useful for the
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+
imaging of Fe involved in the ER in iron-overloaded HepG2 cells.
Fig. 7.
4
. Conclusions
3+
In summary, we have presented rhodamine-derived Schiff base probes (1–6) that allow for Fe imaging
in HepG2 cells. Probes 1–6 alone displayed no distinct absorption and emission bands, whereas upon the
3+
addition of Fe ions to the solutions of probes 1–6, new absorption and emission bands appeared at 526
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nm and 551 nm, respectively. The Fe ions irreversibly bind to the probes and induce the ring opening of
spirolactam in the rhodamine part. However, in the case of other biologically relevant metal ions,
+
+
2+
2+
2+
2+
2+
2+
2+
including Na , K , Fe , Cu , Co , Ca , Mg , Zn , and Mn , a negligible change was observed under
1
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3+
similar conditions. These results indicate that probes 1–6 can be used for the detection of Fe in
biological systems without interference from other competitive metal ions. In confocal microscopic
analysis using the HepG2 cell line, we demonstrated that the probes 1–6 provide a fluorescence Off–On
3+
image for cellular Fe ions and it could be used to discriminate between normal and iron-overloaded cells.
3
+
We also found that probes 1–6 give rise to the fluorescence changes, due to the Fe ions, mainly
localized in the ER in HepG2 cells. Particularly, probe 5 exhibits a highly selective fluorescent image for
3
+
the ER-localized Fe ions over other organelles. We thus believe that probe 5 would be further useful for
3
+
the imaging of Fe involved in the ER of iron-overloaded HepG2 cells.
Acknowledgments
This research was supported by the NRF (2015R1C1A2A01054496, MHL), the CRI project of Korea (No.
2009-0081566, JSK) and the NRF (No. 2015R1A5A1037656, CK).
Supplementary data
Supplementary data related to this article can be found http:////////////
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References
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2
.
.
Ganz T. Molecular control of iron transport. J Am Soc Nephrol 2007;18:394.
Eisenstein RS. Iron regulatory proteins and the molecular control of mammalian iron metabolism.
Annu Rev Nutr 2000;20:627.
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Aisen P, Wessling-Resnick M, Leibold EA. Iron metabolism. Curr Opin Chem Biol 1999;3:200.
4. Koppenol WH. The centennial of the Fenton reaction. Free Radical Biol Med 1993;15:645.
5. Deugnier Y, Turlin B. Pathology of hepatic iron overload. World J Gastroenterol 2007; 13: 4755.
6. Zecca L, Youdim MB, Riederer P, Connor JR, Crichton RR. Iron, brain aging and
neurodegenerative disorders. Nat Rev Neurosci 2004;5:863.
7. Sahoo SK, Sharma D, Bera RK, Crisponic G, Callan JF. Iron(III) selective molecular and
supramolecular fluorescent probes. Chem Soc Rev 2012;41:7195.
8. Li CY, Zou CX, Li YF, Tang JL, Weng C. A new rhodamine-based fluorescent chemosensor for
3+
Fe and its application in living cell imaging. Dyes Pigments 2014;104:110.
9. Ji S, Meng X, Ye W, Feng Y, Sheng H, Cai Y, Liu J, Zhuc X, Guo Q. A rhodamine-based "turn-on"
3
+
fluorescent probe for Fe in aqueous solution. Dalton Trans 2014;43:1583.
1
1
0. Ma S, Yang Z, She M, Sun W, Yin B, Liu P, Zhang S, Li J. Design and synthesis of functionalized
3
+
rhodamine based probes for specific intracellular fluorescence imaging of Fe . Dyes Pigments
015;115:120.
2
1. Lee MH, Giap TV, Kim SH, Lee YH, Kang C, Kim JS. A novel strategy to selectively detect Fe(III)
in aqueous media driven by hydrolysis of a rhodamine 6G Schiff base. Chem Commun
2
010;46:1407.
1
1
1
2. Brissot P, Ropert M, Le Lan C, Loréal O. Non-transferrin bound iron: A key role in iron overload
and iron toxicity. BBA-Gen Subjects 2012;1820:403.
3. Dunn KW, Kamocka MM, McDonald JH. A practical guide to evaluating colocalization in
biological microscopy. Am J Physiol Cell Physiol 2011;300:C723.
4. Škerget K, Taler-Verčič A, Bavdek A, Hodnik V, Čeru S, Tušek-Žnidarič M, Kumm T, Pitsi D,
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Pompe-Novak M, Palumaa P, Soriano S, Kopitar-Jerala N, Turk V, Anderluh G, Žerovnik E.
Interaction between oligomers of stefin B and amyloid-beta in vitro and in cells. J Bio Chem
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010;285:3201.
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Supporting Information
3+
A Fluorescent Probe for the Fe Ion Pool in
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Endoplasmic Reticulum in Liver Cells
1
,*
2
1
3
Min Hee Lee , Ho Hyun Lee , Min Jung Chang , Hyeong Seok Kim ,
2
,*
3,*
Chulhun Kang , and Jong Seung Kim
1
Department of Chemistry, Sookmyung Women’s University, Seoul 140-742, Korea
2
The School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea
3
Department of Chemistry, Korea University, Seoul 136-701, Korea
*
Corresponding authors: minheelee@sookmyung.ac.kr (M. H. Lee); kangch@khu.ac.kr (C.
Kang); jongskim@korea.ac.kr (J. S. Kim)
Contents
1
2
3
4
. Materials and Instrumentation
. UV/Vis and Fluorescence Spectroscopic Methods
. Cell culture and Confocal Microscopic Methods
. Experimental Section
1
13
5. H / C NMR and FAB-MS Analysis
6
7
. X-ray Crystallographic Analysis
. Confocal Microscopic Analysis
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1
. Materials and instrumentation
All fluorescence and UV/Vis absorption spectra were recorded in RF-5301PC and S-3100
spectrophotometer, respectively. NMR was recorded at Varian instrument (400 MHz). All reagents and
+
+
+
+
2+
2+
2+
2+
2+
2+
2+
cationic compounds such as chloride salts of Na , K , Li , Ag , Hg , Cu , Pb , Cd , Co , Ni , Ba ,
2+
2+
2+
2+
2+
3+
Ca , Mg , Zn , Mn Fe , and Fe were purchased from Aldrich and used as received. All solvents
1
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6
6
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6
6
were analytical reagents. CH CN for spectra detection was HPLC reagent without fluorescent impurity
3
and water was deionized water.
2
. UV/Vis and Fluorescence Spectroscopic Methods
Stock solutions of metal chloride salts were prepared in deionized water. Stock solutions of synthetic
probes were prepared in CH CN. All spectra were recorded in aqueous solution (CH CN:H O = 5:95).
3
3
2
Excitation was carried out at 528 nm with all excitation and emission slit widths 3 nm.
3
. Cell Culture and Confocal Microscopic Methods
A human hepatoma cell line, HepG2, was grown in RPMI 1640 medium supplemented with 10 % fetal
bovine serum, 100 unit/ml penicillin and 100 g/ml of streptomycin at 37 C under 95 % humidified air
5
containing 5% CO
. The 1 x 10 cells were located and stabilized in a confocal dish and the medium was
2
replaced with a new one containing 500 M of ferric citrate. After 24 h of incubation at the incubator, the
medium was removed and the cells were briefly washed with 1 mL of phosphate buffered saline (1×PBS).
And 1 mL of probes 1-6 containing medium (the final concentration was 1 M) was placed and incubated
at the incubator. Then, the images were obtained using a confocal microscope from Carl Zeiss (LSM 510
META model). Probes 1-6 were excited at 488 nm using an argon laser and emission image was acquired
using a band-path filter (505-550 nm). All images were taken under the same experimental parameters to
minimize possible variations in fluorescence intensity.
2
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. Experimental Section
1
Compound 2 was prepared according to the literature procedure and compounds 1 and
-6 were newly synthesized in this study by modifying the procedure.
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Compound 1
Rhodamine-ethylene diamine (7) (0.3 g, 0.66 mmol) was stirred with salicylaldehyde (0.08 g, 0.66 mmol)
in methanol for 26 h at room temperature. The precipitates were collected from methanol, finally 0.25 g
+
1
of 1 (69% yield) was obtained as a yellow solid. ESI-MS m/z (M ) calcd 560.3, found 561.2. H NMR
(
CDCl , 400 MHz): δ 13.00 (s, 1H); 8.01 (s, 1H); 7.95 (d, 1H, J = 6.40 Hz); 7.45 (t, 2H, J = 3.60 Hz);
3
7
1
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.26~7.22 (m, 1H); 7.11 (d, 1H, J = 7.60 Hz); 7.05 (d, 1H, J = 7.20 Hz); 6.89 (d, 1H, J = 8.40 Hz); 6.80 (t,
H, J = 7.60 Hz); 6.36 (s, 2H); 6.21 (s, 2H); 3.46~3.42 (m, 4H); 3.35 (t, 2H, J = 7.20 Hz); 3.15~2.8 (m,
13
H); 1.84 (s, 6H), 1.32 (t, 6H, J = 7.20 Hz). C NMR (CDCl 100 MHz): 168.5, 165.9, 161.2, 153.8,
3,
151.9, 147.6, 132.7, 132.1, 131.3, 131.2, 128.6, 128.2, 124.0, 123.0, 118.9, 118.4, 118.1, 117.1, 106.1,
6.7, 65.1, 57.3, 41.1, 38.5, 16.8, 14.9 ppm.
9
Compound 3
Rhodamine-ethylene diamine (7) (0.2 g, 0.44 mmol) was stirred with 2-hydroxy-3,5-dinitrobenzaldehyde
(0.09 g, 0.44 mmol) in methanol for 15 h at room temperature. The precipitates were collected from
+
methanol, finally 0.24 g of 3 (83% yield) was obtained as a yellow solid. ESI-MS m/z (M ) calcd 650.2,
1
found 651.2. H NMR (CDCl , 400 MHz): δ 8.93 (s, 1H); 8.23 (s, 1H); 7.94~7.92 (m, 1H); 7.83 (s, 1H);
3
7
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.53~7.48 (m, 2H); 7.08~7.06 (m, 1H); 6.35 (s, 2H); 6.13 (s, 2H); 3.56~3.52 (m, 6H); 3.23~3.18 (m, 4H);
13
.84 (s, 6H); 1.33 (t, 6H, J = 6.80 Hz). C NMR (CDCl 100 MHz): 170.6, 169.1, 166.8, 153.1, 152.0,
3,
147.9, 135.9, 133.4, 130.6, 128.7, 128.5, 128.2, 124.2, 123.3, 118.5, 116.7, 105.5, 96.7, 65.4, 50.2, 39.5,
8.5, 16.8, 14.7 ppm.
3
1. M. H. Lee, T. V. Giap, S. H. Kim, Y. H. Lee, C. Kang and J. S. Kim, Chem. Commun. 2010, 46,
1407.
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Compound 4
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mmol) in methanol for 14 h at room temperature. The precipitates were collected from methanol, finally
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0
.20 g of 4 (78% yield) was obtained as a yellow solid. FAB-MS m/z (M ) calcd 589.3, found 590.2. H
NMR (CDCl , 400 MHz): δ 8.33 (s, 1H); 8.21 (m, 1H); 7.95~7.92 (m, 3H); 7.52 (t, 1H, J = 8.01 Hz);
3
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.46~7.42 (m, 2H); 7.05~7.02 (m, 1H); 6.35 (s, 2H); 6.21 (s, 2H); 3.49~3.47 (m, 6H); 3.21~3.18 (m, 4H);
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.83 (s, 6H); 1.32 (t, 6H, J = 7.20 Hz). C NMR (CDCl , 100 MHz): 168.7, 159.5, 153.9, 151.9, 148.6,
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47.5, 138.0, 133.5, 132.7, 131.1, 129.5, 128.7, 128.2, 124.9, 124.0, 123.1, 123.0, 118.0, 106.3, 96.8, 65.2,
9.2, 41.2, 38.5, 16.8, 14.9 ppm.
Compound 5
Rhodamine-ethylene diamine (7) (0.16 g, 0.35 mmol) was stirred benzaldehyde (0.04 g, 0.35 mmol) in
methanol for 20 h at room temperature. The precipitates were collected from methanol, finally 0.07 g of 5
+
1
(
38% yield) was obtained as a pink solid. FAB-MS m/z (M ) calcd 544.3, found 545.3. H NMR (CDCl ,
3
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00 MHz): δ 8.00 (s, 1H); 7.94~7.92 (m, 1H); 7.58~7.56 (m, 2H); 7.45~7.42 (m, 2H); 7.36~7.31 (m, 3H);
7.05~7.03 (m, 1H); 6.35 (s, 2H); 6.23 (s, 2H); 3.47~3.39 (m, 6H); 3.21~3.20 (m, 4H); 1.85 (s, 6H); 1.32 (t,
1
3
6
1
4
H, J = 7.20 Hz). C NMR (CDCl , 400 MHz): 196.3, 168.5, 162.5, 156.4, 154.0, 151.9, 147.5, 136.3,
3
32.6, 131.2, 130.6, 128.7, 128.5, 128.2, 128.1, 123.9, 122.9, 118.0, 106.2, 105.2, 96.8, 77.7, 65.2, 59.1,
1.4, 38.5, 16.8, 14.9 ppm.
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Compound 6
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.08 g of 6 (28% yield) was obtained as an orange solid. FAB-MS m/z (M ) calcd 634.3, found 635.2. H
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H, J = 6.80 Hz); 3.48~3.45 (m, 4H); 3.22~3.16 (m, 4H); 1.82 (s, 6H); 1.33 (t, 6H, J = 6.80 Hz).
C
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H / C NMR and FAB-MS Analysis
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Figure S1. H-NMR spectrum of 1 in CDCl .
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Figure S2. C-NMR spectrum of 1 in CDCl .
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Figure S3. ESI-MS spectrum of 1.
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Figure S4. H-NMR spectrum of 3 in CDCl .
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Figure S5. C-NMR spectrum of 3 in CDCl .
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Figure S6. ESI-MS spectrum of 3.
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Figure S7. H-NMR spectrum of 4 in CDCl .
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Figure S8. C-NMR spectrum of 4 in CDCl .
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