A selective turn-on fluorescent sensor for FeIII and application to bioimaging

Article abstract of DOI:10.1016/j.tetlet.2007.03.112

A novel compound FD1 was demonstrated as a turn-on fluorescent sensor for imaging of iron(III) ion in biological samples. Based on the spirolactam (nonfluorescence) to ring-open amide (fluorescence) equilibrium, FD1 exhibited high selectivity and sensitiv

Full text of DOI:10.1016/j.tetlet.2007.03.112

Tetrahedron Letters 48 (2007) 3709–3712
A selective turn-on fluorescent sensor for FeIII and application
to bioimaging
Meng Zhang,a Yanhong Gao,b Manyu Li,c,d Mengxiao Yu,a Fuyou Li,a,* Lei Li,a
a
a,
*
Minwei Zhu,e Jianping Zhang,c,d, Tao Yi and Chunhui Huang
*
aDepartment of Chemistry and Advanced Materials Laboratory, Fudan University, Shanghai 200433, PR China
bHospital of Xinhua, Medicine School of Shanghai Jiaotong University, Shanghai 200092, PR China
cState Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry,
Chinese Academy of Sciences, Beijing 1000872, PR China
dDepartment of Chemistry, Renmin University of China, Beijing 1000872, PR China
eHospital of Obstetrics and Gynecology, Fudan University, Shanghai 200433, PR China
Received 31 December 2006; revised 20 March 2007; accepted 21 March 2007
Available online 24 March 2007
Abstract—A novel compound FD1 was demonstrated as a turn-on fluorescent sensor for imaging of iron(III) ion in biological sam-
ples. Based on the spirolactam (nonfluorescence) to ring-open amide (fluorescence) equilibrium, FD1 exhibited high selectivity and
sensitivity for Fe3+ over other metal ions. Moreover, fluorescent microscopy experiments further established that FD1 could be used
for sensing Fe3+ within living cells.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The design and synthesis for selective and sensitive fluo-
rescent sensors for metal ions has tremendously gained
in importance,1 because metal ions involved in a variety
of fundamental physiological processes in organisms. As
an important physiologically relevant metal ion, FeIII
ion plays an indispensable role in many biochemical
processes at the cellular level,2 and both its deficiency
and excess can induce a variety of diseases3 with iron
trafficing, storage, and balance being tightly regulated
in an organism.4 As a consequence, intense research
efforts have been directed to develop sensitive and
Compared with the single-photon excited fluorescent
bioimaging technique, two-photon laser scanning
microscopy imaging (TPLSM) has the advantage of
high transmission at low incident intensity, reduced
photodamage, improved depth penetration, and reduced
background cellular autofluorescence, and has recently
attracted a great deal of interest since the pioneering
work of the Webb group on TPLSM.14 To image the
distribution of ions in cellular processes, suitable two-
photon chemosensors with turn-on fluorescence should
be developed. However, the cases of two-photon excited
fluorescence (TPEF) sensor for ions are still limited to
proton, fluoride anion, main group I and II metal ions,
and cysteine/homocysteine.15 Herein, we are interested
in developing a TPEF probe which could be utilized
for sensing Fe3+ in living cells. A major challenge to
achieve this goal is developing a fluorescent probe that
exhibits an increasing visible fluorescent emission upon
the addition of Fe3+ over physiologically relevant metal
ions. Based on the well-known spirolactam (nonfluores-
cence) to ring-open amide (fluorescence) equilibrium of
rhodamine,16 we described a significant emission-
increasing probe FD1 (Scheme 1) for Fe3+, and demon-
strated its utility in bioimaging.
selective sensors for Fe3+ 5–7 However, for the paramag-
.
netic nature of iron(III) ion, the fluorescent indication
for Fe3+ is mostly signaled by fluorescence quenching.6
Especially, the lack of suitable turn-on fluorescent iron
indicators7 is even more obvious when judged in terms
of application in bioimaging, although significant pro-
gress has been made in fluorescent molecular sensors
for intracellular imaging main group I and II metal
ions8,9 and transition metal ions (such as Zn2+
,
10
2+
Cu+,11 Pb2+
,
and Hg 13).
12
Keywords: Fluorescent sensor; Bioimaging; Fe3+
.
*
FD1 was synthesized by treatment of rhodamine B
hydrazide17 with acetone in a yield of ca. 40%.18 Single
Corresponding authors. Tel.: +86 21 55664185; fax: +86 21
55664621; e-mail: fyli@fudan.edu.cn
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.112

3710
M. Zhang et al. / Tetrahedron Letters 48 (2007) 3709–3712
O
O
O
O
N
1. POCl3
N-NH2
N
O
2. NH2NH2
N
O
N
N
O
N
N
O
N
1
3 (FD1)
2
Scheme 1. Synthetic route of FD1.
0.25
0.20
0.15
0.10
0.05
0.00
crystal of FD1 was obtained by slow evaporation of the
acetone solution. The single-crystal X-ray diffraction
study showed that the molecular structure of FD1 is
of spirocyclic form in the crystal (Fig. 1). The character-
istic peak of 66.2 ppm (9-carbon) in the 13C NMR spec-
trum of FD1 also supported this consideration.
0.15
0.12
0.09
0.06
0.03
0.00
Fe(III)
8 equiv.
0
0.0
0.2
0.4
0.6
XFe(III)
0.8
1.0
Absorption spectra of FD1 were performed in a CH3CN
solution diluted 20 times with HEPES buffer (20 mM,
pH 7). FeCl3 was used as Fe(III) source. A weak absorp-
tion peak at 561 nm was observed in FD1 solution
which appeared to be almost colorless. The adsorption
peak intensity increased dramatically with each addition
of Fe3+ (Fig. 2) and the color of solution changed to
red-orange, which can be attributed to the delocalized
xanthene moiety of the ring-open amide form of FD1.
The significant color change in FD1 solution upon addi-
tion of Fe3+ indicated that FD1 was a sensitive ‘naked-
eye’ indicator for Fe3+. Binding assays using the method
of continuous variations (Job’s plot) were consistent
with a 1:1 stoichiometry of the FD1–Fe3+ complex.
400
450
500
550
600
Wavelength (nm)
Figure 2. UV–vis absorption spectra of FD1 (10 lM) upon the
addition of Fe3+ (0–75 lM) in the CH3CN solution diluted 20 times
with HEPES buffer (20 mM pH 7) at 25 ꢁC. Each spectrum is obtained
after Fe3+ addition for 5 min. Inset: Job’ plot of FD1 and Fe3+. The
total concentration of FD1 and Fe3+ was kept at a fixed 10 lM. The
data are consistent with 1:1 Fe3+–FD1 complex.
The fluorescence enhancement effects of various
amounts of Fe3+ on FD1 were investigated under exci-
tation at kex = 510 nm (Fig. 3). No obvious fluorescent
emission was observed in FD1 solution with the absence
of Fe3+. When Fe3+ was introduced into a 10 lM FD1
solution, an obvious fluorescence peak was observed
100
500
Fe3+
a
400
300
200
100
0
80
60
40
20
0
and also enhanced upon further addition of Fe3+
,
whereas other metal ions displayed much weaker re-
sponse. The fluorescence intensity at 583 nm increased
by a factor of 112 when Fe3+ concentration increased
0
100
200
300
400
500
CFe(III) / 10-6 M
b
30
20
10
0
600
700
800
900
1000
1100
Wavelength (nm)
550
600
650
700
750
Wavelength (nm)
Figure 3. Fluorescence responses of FD1 (10 lM) upon the addition of
Fe3+ at 25 ꢁC. Each spectrum was obtained in the CH3CN solution
diluted 20 times with HEPES buffer (20 mM, pH 7) after Fe3+ addition
for 5 min. Inset: (a) the plot of fluorescent emission intensity at 583 nm
as a function of Fe3+ concentration (kex = 510 nm) and (b) two-photon
absorption spectrum of FD1 upon the addition of 80 lM Fe3+
.
from 0 to 80 lM. The stability constant of the complex
was calculated by the linear Benesi–Hildebrand
expression:20
1
1
1
1
Figure 1. The ORTEP drawing of FD1 (30% ellipsoid). H atoms were
omitted for clarity.19
¼
þ
½Fe3þꢀ
DI ½FD1ꢀD/ Kass½FD1ꢀD/

M. Zhang et al. / Tetrahedron Letters 48 (2007) 3709–3712
3711
where DI is the change in the fluorescence intensity at
583 nm; Kass is the stability constant; D/ (D/ =
complex ꢁ /FD1) is the difference of fluorescence quan-
tum yields between the complex and FD1; and [FD1]
and [Fe3+] are the concentrations of FD1 and Fe3+
/
,
respectively. On the basis of the plot of 1/DI and
1/[Fe3+], the stability constant was calculated to
be 2.3 · 104 Mꢁ1
.
Figure 5. Fluorescence and phase contrast images of live PIEC cells:
(a) fluorescence image of PIEC cells at 25 ꢁC; (b) fluorescence image of
PIEC cells incubated with 5 lM FD1 for 15 min at 25 ꢁC; (c)
brightfield image of live PIEC cells shown in panel b to confirm
viability.
Furthermore, the two-photon related photophysical
properties of FD1 were investigated in the absence and
presence of Fe3+, and the two-photon absorption
cross-sections were determined by the method of two-
photon induced fluorescence using Rhodamine B as a
standard with known two-photon absorption (TPA)
cross-section (d) (Supplementary data).21 A similar re-
sponse to the TPEF titration of Fe3+ with those of
FD1 in single-photon related photophysical properties
was observed. No obvious TPEF signal was recorded
under the excitation of fs laser from 650 to 1050 nm,
indicating that FD1 was not TPEF-active. However,
the TPEF intensity of FD1 (kex = 800 nm) increased
gradually upon addition of Fe3+ (Supplementary data).
As shown in the inset of Figure 3, FD1 exhibited two-
photon active properties with TPA cross-section of 31
GM in the presence of 80 lM Fe3+. This result indicated
However, only the addition of Fe3+ resulted in a prom-
inent enhancement (112-fold) of fluorescence emission at
583 nm, which indicated the high selectivity of FD1 to
Fe3+. The competition experiment was also carried out
by adding Fe3+ to FD1 solution in the presence of
other metal ions, as shown in Figure 4. The results
indicated that the recognition of Fe3+ by FD1 was not
significantly interfered by these commonly coexistent
ions.
To develop practical applicability of FD1 toward Fe3+
,
we next assessed the ability of FD1 to operate within liv-
ing pig iliac artery endothelium cells (PIEC) by fluores-
cence microscopy (Fig. 5). PIEC cells incubated with or
without 5 lM FD1 for 15 min at 25 ꢁC gave no intracel-
lular background fluorescence (Fig. 5a). Moreover, no
intracellular fluorescence was detected for PIEC cells
supplementing cells with 50 lM FeCl3 in the growth
medium for 5 h at 37 ꢁC. However, as shown in Figure
5b, bright intracellular fluorescence was observed for
supplementing cells with 50 lM FeCl3 in the growth
medium for 5 h at 37 ꢁC and then staining with FD1 un-
der the same loading conditions. It can be seen from the
brightfield transmission measurement after Fe3+ and
FD1 incubation (Fig. 5c) that the PIEC cells were viable
throughout the imaging experiments. These facts
implied that FD1 was membrane-permeable and could
sense intracellular Fe3+ in living cells.
that FD1 could be used as TPEF sensor for Fe3+
.
The selective coordination studies of FD1 were then
extended to related heavy, transition, and main group
metal ions by fluorescence spectroscopy. The representa-
tive chromogenic behavior of FD1 toward metal ions in
aqueous solution is shown in Figure 4. Upon the addi-
tion of 80 lM metal ions, very mild fluorescence
enhancement factor (FEF) in FD1 solution was ob-
served for Cu2+ (18-fold), while Na+, K+, Cu+, Ag+,
Ca2+, Cd2+, Co2+, Cr2+, Zn2+, Mg2+, Mn2+, Ni2+
,
Pb2+, and Fe2+ showed very weak response (Fig. 4).
140
120
100
80
In summary, we demonstrated a unique fluorescent
chemosensor FD1 for Fe3+ and its bioimaging applica-
tions. FD1 exhibited a turn-on fluorescent response for
detecting Fe3+ with excellent selectivity over other metal
ions. Moreover, fluorescent microscopy experiments
indicated that FD1 could be used as fluorescent probe
for sensing Fe3+ in living cells. We anticipate that the
turn-on fluorescent properties of this probe presage
many opportunities for studying the biological effect of
Fe3+ in future application.
60
40
20
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Figure 4. Fluorescence responses of FD1 (10 lM) to various metal
ions (80 lM, x-axis markers) in 20 mM HEPES, pH 7. Bars represent
the final integrated fluorescence response (Ff) over the initial integrated
emission (Fi). White bars represent the addition of an excess of the
appropriate metal ion (1 mM for Na+, K+, Mg2+, and Ca2+, 80 lM
for all other cations) to a 10 lM solution of FD1. Black bars represent
the addition of Fe3+ (80 lM) to the solution. (kex = 510 nm). 1, Na+; 2,
K+; 3, Cu+; 4, Ag+; 5, Ca2+; 6, Cd2+; 7, Co2+; 8, Cu2+; 9, Cr2+; 10,
Acknowledgements
The authors thank NSFC (20490210 and 20501006),
National High Technology Program of China
(2006AA03Z318), Huo Yingdong Education Founda-
tion (104012), and Shanghai Sci. Tech. Comm.
(05DJ14004 and 06QH14002) for the financial support.
Zn2+; 11, Mg2+; 12, Mn2+; 13, Ni2+; 14, Pb2+; 15, Fe3+
.

3712
M. Zhang et al. / Tetrahedron Letters 48 (2007) 3709–3712
Mittoo, S.; Bradley, M. Angew. Chem., Int. Ed. 2006, 45,
Supplementary data
5472.
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Chem. Biol. 2004, 8, 182; (b) Lim, N. C.; Freake, H. C.;
Synthetic and experimental details (PDF) are available.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2007.03.112.
Bruckner, C. Chem. Eur. J. 2005, 11, 38, and some
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18. Data for FD1: mp 210–213 ꢁC; 1H NMR (400 Hz, CDCl3):
d 7.89–7.92 (m, 1H), 7.42–7.47 (m, 2H), 7.09–7.13 (m, 1H),
6.53 (d, J = 8.8 Hz, 2H), 6.38 (d, J = 2.4 Hz, 2H), 6.26–
6.29 (dd, J = 8.8 Hz, J = 2.8 Hz, 2H), 3.30–3.35 (m, 8H),
1.96 (s, 3H), 1.81 (s, 3H), 1.16 (t, J = 7.2 Hz, 12H); 13C
NMR (100 Hz, CDCl3): d 12.6, 21.4, 25.5, 44.3, 66.2, 97.9,
106.5, 107.8, 122.8, 123.8, 128.0, 128.5, 130.8, 132.1, 148.6,
151.8, 153.6, 160.6, 174.2. MS (EI): m/z 496.5 (M+). Anal.
Calcd for C31H36N4O2: C, 74.97; H, 7.31; N, 11.28.
Found: C, 74.84; H, 7.17; N, 11.32.
19. Crystal data for C31H36N4O2: Fw = 496.65, Monoclinic
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˚
˚
(P2(1)/c),
a = 12.199(2) A,
b = 12.303(2) A,
c =
Buschel, M.; Tolmachev, A. I.; Daub, J.; Rurack, K.
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˚
˚
18.536(3) A, a = 90ꢁ, b = 107.501(3)ꢁ, c = 90ꢁ, V =
2653.3(8) A ,
3
q
calcd = 1.246 g cmꢁ3
,
Z = 4, l = 0.079
mmꢁ1, R1 [I > 2r(I)] = 0.0613, wR2 [I > 2r(I)] = 0.1401,
R1 (all data) = 0.1500, wR2 (all data) = 0.1788,
GOF = 1.046. CCDC deposition number: CCDC 605536.
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