A naked-eye rhodamine-based fluorescent probe for Fe(III) and its application in living cells

Article abstract of DOI:10.1016/j.tetlet.2011.03.104

A naked-eye turn-on fluorescent Fe³⁺ probe (RQ6) was developed by linking a new conjugated quinoline fluorescent group to the rhodamine platform. The probe can detect Fe³⁺ with high selectivity over other metal ions. Bioimaging studies indicated that RQ6 was cell permeable and suitable for detecting Fe³⁺ in the living cells by confocal microscopy.

Full text of DOI:10.1016/j.tetlet.2011.03.104

Tetrahedron Letters 52 (2011) 2840–2843
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A naked-eye rhodamine-based fluorescent probe for Fe(III) and its application
in living cells
⇑
⇑
Shuxin Wang, Xiangming Meng , Manzhou Zhu
Department of Chemistry, Anhui University, Hefei 230039, China
a r t i c l e i n f o
a b s t r a c t
A naked-eye turn-on fluorescent Fe3+ probe (RQ6) was developed by linking a new conjugated quinoline
fluorescent group to the rhodamine platform. The probe can detect Fe3+ with high selectivity over other
metal ions. Bioimaging studies indicated that RQ6 was cell permeable and suitable for detecting Fe3+ in
the living cells by confocal microscopy.
Article history:
Received 1 February 2011
Revised 12 March 2011
Accepted 22 March 2011
Available online 30 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Rhodamine
Fe3+ probe
Fluorescent probe
Bioimaging
FRET
Fe3+ plays an important role in many biochemical processes at
the cellular level.1 The change of the amount of Fe3+ is associated
with many diseases, including certain cancers and dysfunction of
organs.2 Thus, real-time detecting of trace Fe3+ in living cells is of
great importance. Fluorescent probes have become powerful
detecting tools in environmental, biological, and chemical aspects
for its real-time analysis during the last decades.3,4 However, due
to the paramagnetic nature of Fe3+, the fluorescent indication for
Fe3+ is mostly signaled by fluorescence quenching, which may limit
the application of probes.5 Thus, developing a turn-on fluorescent
probe for Fe3+ has attracted increasing research efforts in recent
years.6
Rhodamine-based fluorescent probes have become popular for
its particular structure. The equilibrium between spirocyclic
(nonfluorescent) and ring-open forms (high fluorescent) makes
rhodamine an ideal platform for fluorogenic probe design.7 But a
Scheme 1. Proposed mechanism of Fe3+-selective probe RQ6.
⇑
Corresponding authors. Tel.: +86 551 5108970; fax: +86 551 5107342.
E-mail addresses: mengxm@ahu.edu.cn (X. Meng), zmz@ahu.edu.cn (M. Zhu).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.104

S. Wang et al. / Tetrahedron Letters 52 (2011) 2840–2843
2841
Br
MeO
Br
a
b
N
N
2
NH2
1
N
O
N
MeO
c
d
N
N
O
N
O
N
O
RQ6
3
Scheme 2. Reagents and conditions: (a) crotonaldehyde, 6 N HCl, 105 °C, 75.6%; (b)
1-methoxy-4-vinylbenzene, PdCl2(PPh3)2, K2CO3, DMF, 160 °C, 24 h, 89%; (c) SeO2,
dioxane, 80 °C, 2.5 h, 90.9%; (d) hydrazine rhodamine, EtOH, 60 °C, 61%.
Figure 2. Fluorescence spectra of 25
l
M RQ6 in ethanol/H2O (1:2, pH 7.15) upon
addition of Fe3+ (0–2 equiv). kex = 378 nm. Inset: (A) Job’s plot of RQ6 and Fe3+. The
total concentration of RQ6 and Fe3+ are 10
lM in ethanol/H2O (1:2, pH 7.15),
kex = 378 nm, kem = 584 nm. (B) Amplification picture around 482 nm.
energy transfer (FRET) would be a choice for its convenience to
modify the excitation source.10
Here we report a turn-on fluorescent probe (RQ6) for Fe3+ based
on the rhodamine platform. A conjugated quinoline fluorescent
group, which could be excited by about 400 nm wavelength of
light, was linked to the rhodamine platform. The emission of con-
jugated quinoline was partly located in the absorption of rhoda-
mine. Comparison of the rhodamine derivatives with the
appended quinoline unit, which has already been reported for
the detection of Cr3+ 7b,10a
we extended the conjugated system to
,
make a red shift of quinoline in fluorescence emission, and re-
moved the hydroxy group at 8-position to reach a different coordi-
nation mode choice on the Fe3+ ions.6b The ring-open rhodamine of
RQ6 could be excited by the emission of the conjugated quinoline
via FRET process, eliminating the excitation influence during the
detection of ions (Scheme 1).
The RQ6 was easily synthesized via four steps from readily
available initial materials with an overall yield of 37% (Scheme
2). The structures of RQ6 and the intermediates were all confirmed
by 1H NMR and 13C NMR.11
Absorption and fluorescence spectra were performed in etha-
nol/H2O solvent system (1:2, pH 7.15). As shown in Figure 1, the
maximum absorption was obtained at 378 nm for RQ6, which
was attributed to the quinoline group. Upon addition of Fe3+, the
absorption spectrum was shifted to 550 nm gradually, with a color
change of the solution from colorless to pink (Fig. 3). This indicated
Figure 1. UV–vis spectra of 25 lM RQ6 in ethanol/H2O (1:2, pH 7.15) upon addition
of Fe3+ (0–2 equiv).
problem of the rhodamine-based fluorescent probe is the small
Stock’s shift of the spectrum. The emission was disturbed by exci-
tation, which may reduce the detection accuracy.
On the other hand, from the point of bioimaging, two-photon
laser scanning has lots of advantages over one-photon fluores-
cence.8 At present, the most popular excitation wavelength for
two-photon laser scanning is near 800 nm (i.e., near 400 nm for
single-photon excitation), which is the best out-put of Ti-sapphire
laser.9 Thus, it has become a challenge to tune the excitation wave-
length of the rhodamine-based probe so as to make it more suit-
able for bioimaging. To achieve this aim, fluorescence resonance
Figure 3. Top: color of RQ6 and RQ6 with different metal ions. Bottom: fluorescence (kex = 365 nm) change upon addition of different metal ions.

2842
S. Wang et al. / Tetrahedron Letters 52 (2011) 2840–2843
binding model between Fe3+ and RQ6 (Fig. 2 inset). The stability
constant was calculated to be 8.75 Â 103 MÀ1 by the reported
method.12
Ion selectivity study was performed in HEPES buffer. Due to the
abundance of Na+, K+, and Ca2+ in biological systems, these ions
were tested at the higher concentration (40 times of Fe3+). As
shown in Figure 4, no fluorescence emission changed after adding
Na+, K+, and Ca2+, which means that the probe can be used under
biological conditions. The data also show that Co2+, Cd2+, Mn2+
,
Hg2+, Fe2+, Ni2+, and Cu2+ caused negligible response to the fluores-
cence of RQ6, while Cr3+ has a slight effect. Photographs of the
aqueous solutions of RQ6 with the addition of different ions clearly
show that Fe3+ changed the color of the solution dramatically,
while other ions caused no perceivable changes (Fig. 3).
Figure 4. Fluorescence responses of RQ6 upon addition of various metal ions
(green: RQ6 with other metals, red: RQ6 with other metals and Fe3+). Experimental
conditions: 25
l
M RQ6 in ethanol/H2O (1:2, pH 7.15), 1.0 mM for K+(1), Na+(2),
l
Ca2+(3), and 25
M for Co2+(4), Mn2+(5), Ni2+(6), Mg2+(7), Fe2+(8), Cu+(9), Cd2+(11)
Furthermore, the pH effect on fluorescence of RQ6 was investi-
gated in water. When pH <5, orange fluorescence appeared due to
the formation of the open-ring form. While in the range of 5–9,
RQ6 shows very stable fluorescence, indicating that the probe
could be used under physiological condition.
With the above data in hand, we then applied RQ6 in the cell
imaging of Fe3+ by laser scanning confocal microscopy. HeLa cell
was chosen as the sample. And the common 800 nm two-photon
laser was used for excitation. Incubation for 30 min at 37 °C with
Hg2+(10), Cr3+(12) and Fe3+(13). (kex = 378 nm. kem = 584 nm).
that the spriolactam structure RQ6 was induced to form open-ring
structure by the addition of Fe3+. The dramatical color change en-
sured the sensor as a sensitive ‘naked-eye’ probe for Fe3+
.
Interestingly, with the addition of Fe3+, the emission at 584 nm
was enhanced dramatically, and the emission at 482 nm also chan-
ged (Fig. 2, details see Supplementary data), along with the fluores-
cence color changing from pale green to light orange (Fig. 3). This
could be explained by the FRET from conjugated quinoline to rho-
damine. Thus, the excitation interference of the new rhodamine-
based probe was eliminated as a result of efficient FRET. Fluores-
cence intensity at 584 nm was increased by 186 times with the
addition of Fe3+, which indicated that the new sensor could be used
as a highly sensitive Fe3+ probe. The Job’s plot indicated the 1:2
10
l
M
RQ6, no intracellular fluorescence was detected
(Fig. 5A,B). Then when the cells were treated with 30
l
M Fe3+ for
2.5 h at 37 °C, red-orange fluorescence appeared (Fig 5 C,D). The re-
sults indicate that the RQ6 is cell permeable and can be used for
imaging of Fe3+ in living cells.
To ascertain the cytotoxic effect of RQ6, the MTT (5-dimethylthi-
azol-2-yl-2,5-diphenyltetrazolium bromide) assay was performed
Figure 5. Confocal fluorescence. (A) Two-photon fluorescence (TPF) image of Hela cells labeled with 10
lM RQ6 after 30 min of incubation, washed with PBS buffer.
kex = 800 nm. (B) Brightfield of HeLa cells. (C) TPF image of RQ6 labeled HeLa cells after 2.5 h treated with 30
lM FeCl3 (emission collected from 565 nm to 615 nm). (D) the
overlay of B and C.

S. Wang et al. / Tetrahedron Letters 52 (2011) 2840–2843
2843
References and notes
1. (a) Aisen, P.; Wessling-Resnick, M.; Leibold, E. A. Curr. Opin. Chem. Biol. 1999, 3,
200; (b) Eisenstein, R. S. Annu. Rev. Nutr. 2000, 20, 627.
2. (a) Halliwell, B. J. J. Neurochem. 1992, 59, 1609; (b) Swaminathan, S.; Fonseca, A.
V.; Alam, G. M.; Shah, V. S. Diabetes Care 2007, 30, 1926; (c) laris, D.; Skiada, V.;
Barbouti, A. Cancer Lett. 2008, 266, 21.
3. For reviews, see: (a) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443; (b)
Beija, M.; Afonso, C. A. M.; Martinho, J. M. G. Chem. Soc. Rev. 2009, 38, 2410; (c)
Xu, Z. C.; Yoon, J. Y.; Spring, D. R. Chem. Soc. Rev. 2010, 39, 1996.
4. For example, see: (a) Kiyose, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem.
Soc. 2006, 128, 6548; (b) Qian, F.; Zhang, C.; Zhang, Y.; He, W.; Gao, X.; Hu, P.;
Guo, Z. J. Am. Chem. Soc. 2009, 131, 1460; (c) Lee, D. N.; Kim, G. J.; Kim, H. J.
Tetrahedron Lett. 2009, 50, 4766; (d) Dodani, S. C.; He, Q.; Chang, C. J. J. Am.
Chem. Soc. 2009, 131, 18020; (e) Wang, J.; Qian, X. H.; Cui, J. J. Org. Chem. 2006,
71, 4308; (f) Meng, X. M.; Liu, L.; Hu, H. Y.; Zhu, M. Z.; Guo, Q. X. Tetrahedron
Lett. 2006, 47, 7961; (g) Meng, X. M.; Zhu, M. Z.; Liu, L.; Guo, Q. X. Tetrahedron
Lett. 2006, 47, 1559.
Figure 6. Cell viability was quantified by the MTT assay (mean SD).
5. (a) Weizman, H.; Ardon, O.; Mester, B.; Libman, J.; Dwir, O.; Hadar, Y.; Chen, Y.;
Shanzer, A. J. Am. Chem. Soc. 1996, 118, 12386; (b) Wolf, C.; Mei, X. F.; Rokadia,
H. K. Tetrahedron Lett. 2004, 45, 7867; (c) Ma, Y.; Luo, W.; Quinn, P. J.; Liu, Z.;
Hider, R. C. J. Med. Chem. 2004, 47, 6349; (d) Ouchetto, H.; Dias, M.; Mornet, R.;
Lesuissee, E.; Camadro, J. M. Bioorg. Med. Chem. 2005, 13, 1799.
6. (a) Xiang, Y.; Tong, A. J. Org. Lett. 2006, 8, 1549; (b) Mao, J.; Wang, L. N.; Dou,
W.; Tang, X. L.; Yan, Y.; Liu, W. S. Org. Lett. 2007, 9, 4567.
7. (a) Soh, J. H.; Swamy, K. M. K.; Kim, S. K.; Kim, S.; Lee, S.-H.; Yoon, J. Tetrahedron
Lett. 2007, 47, 5966; (b) Huang, K. W.; Yang, H.; Zhou, Z. G.; Yu, M. G.; Li, F. Y.;
Gao, X.; Yi, T.; Huang, C. H. Org. Lett. 2008, 10, 2557; (c) Zhang, X.; Shiraishi, Y.;
Hirai, T. Tetrahedron Lett. 2008, 49, 4178; (d) Zhang, W.; Tang, B.; Liu, X.; Liu, Y.;
Xu, K.; Ma, J.; Tong, L.; Yang, G. Analyst 2009, 134, 367; (e) Lee, M. H.; Giap, T. V.;
Kim, S. H.; Lee, Y. H.; Kang, C.; Kim, J. S. Chem. Commun. 2010, 46, 1407; (f) Hu,
Z. Q.; Wang, X. M.; Feng, Y. C.; Ding, L.; Li, M.; Lin, C. S. Chem. Commun. 2011, 47,
1622.
8. (a) Pond, S. J. K.; Rumi, M.; Levin, M. D.; Parker, T. C.; Beljonne, D.; Day, M. W.;
Brédas, J. L.; Marder, S. R.; Perry, J. W. J. Phys. Chem. A 2002, 104, 11470; (b) Kim,
H. M.; Kim, B. R.; Hong, J. H.; Park, J. S.; Lee, K. J.; Cho, B. R. Angew. Chem., Int. Ed.
2007, 46, 7445; (c) Pawlicki, M.; Collins, H. A.; Denning, R. G.; Anderson, H. L.
Angew. Chem., Int. Ed. 2009, 48, 3244; (d) Kim, H. M.; Cho, B. R. Acc. Chem. Res.
2009, 42, 863.
9. He, G. S.; Tan, L. S.; Zheng, Q.; Prasad, P. N. Chem. Rev. 2008, 108, 1245.
10. (a) Zhou, Z. G.; Yu, M. X.; Yang, H.; Huang, K. W.; Li, F. Y.; Yi, T.; Huang, C. H.
Chem. Commun. 2008, 3387; (b) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.;
Yoon, J. Y. Chem. Soc. Rev. 2008, 37, 1465; (c) Zhang, X.; Xiao, Y.; Qian, X. Angew.
Chem., Int. Ed. 2008, 47, 8025; (d) Xu, M. Y.; Wu, S. Z.; Zeng, F.; Yu, C. M.
Langmuir 2010, 26, 4529.
according to the reported method.13 HeLa cells were treated with 0,
10, 30, and 50
Figure 6. The cell viability remains 98% under the treatment of
10 M RQ6, which indicated that the new probe is low cytotoxic
lM RQ6 for 24 h. The results were illustrated in
l
to cells and suitable for bioimaging (Fig. 6).
In conclusion, a high selective rhodamine-based turn-on probe
for Fe3+ was synthesized. According to the efficient intramolecular
FRET, the probe could be excited by near 400 nm wavelength of
light (or 800 nm two-photon laser source), which is suitable for
bioimaging by confocal microscopy. The Two-photon fluorescence
(TPF) image confirmed that RQ6 can be used for monitoring intra-
cellular Fe3+ in living cells with low cytotoxicity. We anticipate that
the new probe can be useful in studying the bioactivity of Fe3+ in
biological systems.
Acknowledgments
We acknowledge financial support by NSFC (20871112 and
21072001), Anhui Province Natural Science Foundation
(090416231), Natural Science Foundation of Education Depart-
ment of Anhui Province (KJ2008B159 and KJ2010A028), and 211
Project of Anhui University for supporting the research.
11. Data for RQ6: 1H NMR (400 MHz, CDCl3, ppm): d 1.14–1.17 (12H, t, J = 6.88 Hz),
3.31–3.33 (8H, d, J = 6.76 Hz), 3.83 (3H, s), 6.50 (2H, s), 6.57–6.57 (2H, d,
J = 7.91 Hz), 6.90–6.92 (2H, d, J = 8.54 Hz), 7.06–7.20 (3H, m), 7.47–7.53 (4H,
dd, J = 7.70, 18.15), 7.70 (1H, s), 7.85–7.87 (1H, d, J = 8.76 Hz), 7.95–8.07 (4H,
m), 8.74 (1H, s). 13C NMR (100 MHz, CDCl3, ppm): d 12.65, 44.34, 55.30, 66.22,
98.30, 105.88, 108.10, 114.22, 118.64, 123.66, 123.85, 125.25, 125.71, 127.15,
127.72, 127.95, 128.41, 128.45, 128.59, 129.44, 129.73, 129.79, 146.97, 147.39,
149.01, 152.27, 153.18, 154.47, 159.59, 165.41. MS (ESI): m/z 728.3 (M+). The
detail experiment and the data of other intermediates are in the
Supplementary data.
Supplementary data
12. Connors, K. A. Binding Constants; John Wiley & Sons: New York, 1987. Chapter
4.
13. Maszewska, M.; Leclaire, J.; Cieslak, M.; Nawrot, B.; Okruszek, A.; Caminade, A.
M.; Majoral, J. P. Oligonucleotides 2003, 13, 193.
Supplementary data (synthetic and experimental details) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2011.03.104.