A highly selective fluorescence turn-on probe for Cu(II) based on reaction and its imaging in living cells

Article abstract of DOI:10.1021/ic401865e

A new oxidative C-O bond cleavage reaction-based probe FluHMPP was designed and prepared. FluHMPP displays excellent selective turn-on fluorescence response for Cu^II in aqueous solution under visible light excitation. The cleavage products are fully characterized. Fluorescein fragment is further oxidized to highly fluorescent MFME (3′-O-methylfluorescein methyl ester), and benzyl ether of imine fragment has been transformed to carboxyl. Confocal microscopy experiments have demonstrated that FluHMPP could also be used in live cells for the detection of Cu^II.

Full text of DOI:10.1021/ic401865e

Article
pubs.acs.org/IC
A Highly Selective Fluorescence “Turn-On” Probe for Cu(II) Based on
Reaction and Its Imaging in Living Cells
†
†
†
‡
†
†
†
Zhaohua Shi, Xiaoliang Tang, Xiaoyan Zhou, Ju Cheng, Qingxin Han, Ji-an Zhou, Bei Wang,
†
,†
,‡
Yanfang Yang, Weisheng Liu,* and Decheng Bai*
†Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied
Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of
China
‡
Key Laboratory of Preclinical Study for New Drugs of Gansu Province and Operative Surgery Institute, School of Basic Medical
Sciences, Lanzhou University, Lanzhou 730000, People’s Republic of China
*
S Supporting Information
ABSTRACT: A new oxidative C−O bond cleavage reaction-based probe FluHMPP was designed and prepared. FluHMPP
II
displays excellent selective turn-on fluorescence response for Cu in aqueous solution under visible light excitation. The cleavage
products are fully characterized. Fluorescein fragment is further oxidized to highly fluorescent MFME (3′-O-methylfluorescein
methyl ester), and benzyl ether of imine fragment has been transformed to carboxyl. Confocal microscopy experiments have
II
demonstrated that FluHMPP could also be used in live cells for the detection of Cu .
8
INTRODUCTION
applications recently, chemists still need to design novel
ones, which can overcome these limitations, including ultra-
violet excitation or operation at nonphysiological pH for
fluorescence imaging.
■
Copper, as the third most abundant transition metal in the
human body (after iron and zinc), plays a pivotal role involving
cellular energy generation, oxygen transport and activation, and
1
Turn-on fluorescence detection based on reaction is an
appealing strategy for paramagnetic metal ions. Furthermore,
X-ray diffraction analysis of reaction product can effectively
explain fluorescence enhancement mechanism. So far, most of
the reaction-based probes for Cu exploit metal-promoted
hydrolysis or desulfurization reaction, which may suffer from
However, there are only very few systems
using the redox activity of Cu .
Imine derivatives have strong coordination ability and can
modulate metal center electronic properties; they have been
widely used in catalytic oxidation, analytical chemistry, and
chemical simulation for biological systems. Recently, C−O
bond cleavage reaction-based systems were developed for turn-
on sensing of Cu and Co by Taki and Chang, respectively.
These results encouraged us to develop new systems for other
substances in biological studies. Herein, we present the
synthesis of FluHMPP (Scheme 1) in which a tridentate
signal transduction. Cells have developed sophisticated
regulatory mechanisms to maintain a critical balance for
2
ingestion of copper. However, copper is highly toxic to
organisms when its level exceeds cellular needs. For example, it
is a powerful catalyst of the Fenton reaction, which can
generate highly reactive hydroxyl radicals to interfere with
II
6b,9
3
competing ions.
cellular metabolism, and it can displace other metal ions and
II 8a,10
4
change enzyme-catalyzed reactions. Therefore, disruption of
copper homeostasis is related to some neurodegenerative
diseases such as Menkes syndrome, Wilson’s disease, and
5
Alzheimer’s disease. Despite the critical contributions of
11
copper homeostasis to physiology and pathology, the detection
of copper ions in living systems is still a challenge.
As one of the important means and techniques for detecting
copper, fluorescent probes display evident advantages in
selectivity and biological imaging. Because of the fast
I
II
12
13
6
II
electron/energy transfer involving intrinsic paramagnetic Cu
centers, most probes showed a “turn-off” response. Although
7
some “turn-on” or ratiometric sensors have been reported and a
Received: July 19, 2013
few of them have been used with success in biological
Published: October 11, 2013
©
2013 American Chemical Society
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Inorganic Chemistry
Article
Scheme 1. Synthesis of FluHMPP
NNO imine ligand, 6-(hydroxymethyl)-4-methyl-2-((2-
picolylimino)methyl)phenol (HMPP), is linked to the non-
conjugated form of a fluorescein platform through a benzyIlI
ether linkage. FluHMPP could selectively react with Cu
through imine complexation and exhibits turn-on fluorescence
response in aqueous media arising from oxidative cleavage. In
addition, confocal microscopy experiments have demonstrated
that FluHMPP is membrane permeable and can react with
Synthesis of FluHMPP. Preparation of 2-[3-(2-Allyloxy-3-
formyl-5-methyl-benzyloxy)-6-methoxy-9H-xanthen-9-yl]benzoic
Acid Methyl Ester (3). To a solution of 1 (1.81 g, 5.00 mmol) in
degassed CH CN (100 mL) were added 2 (1.03 g, 5.00 mmol) and
3
K CO (1.38 g, 10 mmol). The reaction mixture was refluxed under Ar
2
3
overnight. After removal of the insoluble materials by filtration, the
filtrate was evaporated. The residue was dissolved in AcOEt (100 mL),
and the organic layer was washed with water (100 mL × 2) and brine
(
100 mL), dried over MgSO , and concentrated. The crude residue
4
II
was purified by flash chromatography (SiO , AcOEt/petroleum ether
2
intracellular Cu .
1
:2, v/v) to give 3 as a white solid (2.47 g, 90%). Mp 64.5−65.3 °C.
Anal. Calcd for C H O : C, 74.17; H, 5.49. Found: C, 74.13; H, 5.43.
34
30
7
1
EXPERIMENTAL SECTION
H NMR (400 MHz, CDCl ): δ = 2.36 (s, 3H), 3.78 (s, 3H), 3.95 (s,
3
■
3H), 4.51 (d, J = 5.6 Hz, 2H), 5.06 (s, 2H), 5.26 (dd, J = 0.8, 10.4 Hz,
1H), 5.36 (dd, J = 1.2, 17.2 Hz, 1H), 5.99−6.09 (m, 1H), 6.22 (s, 1H),
6.53 (dd, J = 2.8, 8.8 Hz, 1H), 6.60 (dd, J = 2.4, 8.8 Hz, 1H), 6.65 (d, J
General Information and Materials. All solvents were of reagent
grade. CH CN was purified by distillation over CaH and transferred
3
2
under argon. Oxygen-sensitive reaction was carried out under an argon
atmosphere in a heat-dried flask. All reactions were monitored by thin-
layer chromatography (TLC) using UV light. 2-(3-Hydroxy-6-
=
2.4 Hz, 1H), 6.74 (d, J = 2.8 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 6.97
d, J = 8.8 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H),
7.32 (td, J = 1.2, 7.6 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.64 (d, 2.4 Hz,
(
12
methoxy-9H-xanthen-9-yl)benzoic acid methyl ester (1) and 2-
1
3
14
1H), 7.80 (dd, J = 1.2, 7.6 Hz, 1H), 10.34 (s, 1H). C NMR (100
MHz, CDCl ): δ = 20.7, 37.7, 52.3, 55.3, 64.7, 78.4, 101.0, 102.0,
10.2, 110.6, 116.8, 117.5, 119.0, 126.1, 129.0, 129.2, 129.3, 129.5,
allyloxy-3-chloromethyl-5-methylbenzaldehyde (2) were prepared
1
13
3
according to literature procedures. H NMR and C NMR spectra
1
1
1
5
were recorded in CDCl solution on a Bruker DRX400 spectrometer.
3
30.5, 130.7, 131.0, 131.7, 132.4, 132.5, 134.5, 136.9, 148.2, 151.3,
Chemical shifts (δ) were denoted in ppm, and calibrated by using
+
51.4, 157.9, 158.1, 159.2, 168.7, 189.9. ESI−MS m/z [(M + 1) ]:
residual undeuterated solvent (CHCl (7.27 ppm), or tetramethylsi-
3
51.2.
1
lane (0.00 ppm)) as internal reference for H NMR and the deuterated
Preparation of 2-[3-(2-Hydroxy-3-formyl-5-methyl-benzyloxy)-6-
solvent (CDCl (77.00 ppm) or tetramethylsilane (0.00 ppm)) as
3
methoxy-9H-xanthen-9-yl]benzoic Acid Methyl Ester (4). A stirred
mixture of 3 (1.71 g, 3.1 mmol), Pd(OAc) (22 mg, 0.1 mmol), PPh
13
internal standard for C NMR. Mass spectra and the high-resolution
mass spectrum (HRMS) were measured on a Bruker Esquire 6000
mass spectrometer and a Bruker maXis 4G mass spectrometer by
means of the electronic spray ionization (ESI) technique, respectively.
The melting points were measured on an X-6 melting point apparatus
without calibration (Beijing Fuka Keyi Science and Technology Co.,
Ltd.). Elemental analyses were conducted using an Elementar VarioEL
instrument. Tris−HCl solution (10 mM, pH 7.20) was prepared in
H O−CH CN (7:3, v/v). A starting solution (H O−CH CN) of 100
2
3
(
157 mg, 0.6 mmol), Et N (3.93 g, 38.8 mmol), and HCO H (1.79 g,
3
2
3
8.8 mmol) in 80% EtOH was refluxed for 30 min. After removal of
the solvent, the residue was partitioned between CH Cl and 0.1 M
2
2
HCl. The organic layer was dried (Na SO ) and concentrated. The
2
4
crude product was purified by flash chromatography (SiO , petroleum
2
ether/AcOEt 5:1, v/v) to afford 4 as a white solid (1.11 g, 70%). Mp
1
14.3−114.8 °C. Anal. Calcd for C H O : C, 72.93; H, 5.13. Found:
31
26
7
1
2
3
2
3
C, 72.73; H, 5.02. H NMR (400 MHz, CDCl ): δ = 2.35 (s, 3H), 3.80
3
mM NaOH (pH ≈ 13) was used for pH titrations. The pH values
were lowered to 4.0 by the addition of aqueous HCl (H O−CH CN).
(s, 3H), 3.97 (s, 3H), 5.13 (s, 2H), 6.22 (s, 1H), 6.54 (dd, J = 2.4, 8.8
Hz, 1H), 6.63−6.67 (m, 2H), 6.78 (d, J = 2.4 Hz, 1H), 6.96 (t, J = 8.8
Hz, 2H), 7.13 (d, J = 8.0 Hz, 1H), 7.20 (td, J = 1.2, 8.0 Hz, 1H), 7.30−
7.35 (m, 2H), 7.55 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 9.86 (s, 1H),
2
3
All pH measurements were made with a pH-10C digital pH meter.
Caution: Although no problems were encountered during the
preparation of perchlorate salts, suitable care should be taken when
handling such potentially hazardous compounds.
11.19 (s, 1H). 13C NMR (100 MHz, CDCl ): δ = 20.3, 37.7, 52.3,
3
55.3, 63.8, 101.0, 102.1, 110.2, 110.8, 116.8, 117.3, 120.1, 125.2, 126.0,
1
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Inorganic Chemistry
Article
1
1
1
29.1, 129.3, 129.5, 130.5, 130.6, 131.7, 132.4, 133.0, 136.9, 148.3,
temperature. The samples were directly measured using ESI-TOF.
51.3, 151.4, 156.8, 158.2, 159.2, 168.7, 196.6. ESI−MS m/z [(M −
For HPLC analysis, the above samples were analyzed with a reverse
−
18
) ]: 509.4.
phase HPLC (Varian-ProStar system, 4.6 × 250 mm, Diamonsil C
5
Preparation of FluHMPP. 2-Picolylamine (216 mg, 2 mmol)
μ) eluted with CH CN/H O. The retention time was compared to
3 2
dissolved in 2 mL of abs EtOH was added to 50 mL of abs EtOH
solution of 4 (1.02 g, 2 mmol), and the reaction mixture was stirred for
that of an authentic sample of 3′-O-methylfluorescein methyl ester
(MFME).
5
h at room temperature. After removal of the solvent, the crude
Cell Culture and Fluorescence Microscopic Imaging. The
Hela cell lines were provided by the Institute of Biochemistry and Cell
Biology (China). Cells were grown in Dulbecco’s modified eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum in an
product was subjected to flash chromatography (SiO , petroleum
ether/AcOEt 10:1, v/v) to afford FluHMPP as a yellow solid (1.08 g,
2
9
5
0%). Mp 80.0−81.1 °C. Anal. Calcd for C H N O : C, 73.98; H,
37
32
2
6
1
.37; N, 4.66. Found: C, 73.83; H, 5.18; N, 4.59. H NMR (400 MHz,
atmosphere of 5% CO and 95% air at 37 °C humidified air for 24 h.
2
CDCl ): δ = 2.31 (s, 3H), 3.79 (s, 3H), 3.96 (s, 3H), 4.95 (s, 2H),
One day before imaging, the cells were passaged and plated in phenol
red-free medium on 14 mm glass coverslips in 12-well plates. Copper
uptake was performed in the same medium with supplementation with
3
5
.15 (s, 2H), 6.21 (s, 1H), 6.54 (dd, J = 2.4, 8.8 Hz, 1H), 6.65−6.68
(
m, 2H), 6.81 (d, J = 2.4 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 7.08 (s,
1
H), 7.13 (d, J = 8.0 Hz, 1H), 7.17−7.22 (m, 2H), 7.30−7.38 (m,
H), 7.69 (td, J = 1.6, 7.6 Hz, 1H), 7.80 (dd, J = 0.8, 7.6 Hz, 1H), 8.51
CuCl at 100 μM for 1 h. It was washed twice with PBS buffer, and
2
3
then a solution of FluHMPP in CH CN (1 mM) was diluted into
3
13
(
s, 1H), 8.59 (d, J = 4.8 Hz, 1H), 13.55 (s, 1H). C NMR (100 MHz,
DMEM at 20 μM, added to the cells, and incubated for another 4 h.
Before analysis, the coverslips were removed from the 12-well plate
and plated on a glass slide. Next, fluorescence microscopic images were
acquired. Excitation of loaded cells at 480 nm was carried out with a
He−Ne laser. The confocal microscopic optical setup was in
multichannel mode. All confocal images were collected with a Zeiss
Leica inverted epifluorescence/reflectance laser scanning confocal
microscope.
CDCl ): δ = 20.4, 37.7, 52.2, 55.3, 64.6, 65.0, 101.0, 102.1, 110.1,
3
1
1
1
10.9, 116.9, 117.0, 118.2, 121.9, 122.3, 124.2, 126.0, 127.6, 129.3,
29.4, 130.5, 130.5, 131.3, 131.8, 132.4, 132.6, 136.8, 148.3, 149.4,
51.3, 151.4, 156.5, 157.9, 158.5, 159.1, 166.7, 168.7. ESI−MS m/z
+
[
(M + 1) ]: 601.4.
II
Synthesis of [Cu (MPP−NO )NO ] . A 10 mL methanol solution of
2
3 2
Cu(NO ) ·3H O (0.0121 g, 0.05 mmol) was added to a magnetically
3
2
2
stirred 10 mL acetonitrile solution of FluHMPP (0.0300 g, 0.05
mmol). The mixture was stirred in air for 1 day whereby a yellow-
green solution was formed. It was filtered and kept in air. Dark green
X-ray Diffraction Studies. Single-crystal X-ray diffraction
measurements were carried out on a Bruker APEX-II CCD
diffractometer operating at 50 KV and 30 mA using Mo Kα radiation
(λ = 0.71073 Å). Data collection and reduction were performed using
II
rhombic single crystals of [Cu (MPP−NO )NO ] suitable for X-ray
2
3 2
1
5
crystallography were obtained on slow evaporation of the filtrate
SMART and SAINT software. An empirical absorption correction
16
within 5 days. Anal. Calcd for C H Cu N O : C, 42.48; H, 3.06; N,
was applied using the SADABS program. The structure was solved
28
24
2
8
12
2
1
4.15. Found: C, 42.65; H, 3.18; N, 13.89. HRESI−MS m/z
by direct methods and refined by full-matrix least-squares on F using
II
−
+
17
[
Cu (MPP−NO ) ] : 333.0177.
the SHELXL-97 program package.
2
Determination of the Yield of the C−O Bond Cleavage Reaction.
Crystal data and details of the structure determination for
II
A 6 mL methanol solution of Cu(NO ) ·3H O (0.0121 g, 0.05 mmol)
[Cu (MPP−NO )NO ] are summarized in Supporting Information
3
2
2
2
3 2
was added to a magnetically stirred 5 mL acetonitrile solution of
FluHMPP (0.0300 g, 0.05 mmol). The mixture was stirred in dark for
Table S1. CCDC 910759 contains the supplementary crystallographic
data for this Article. These data can be obtained free of charge from
the Cambridge Crystallographic Data Centre via http://www.ccdc.
cam.ac.uk/cgi-bin/catreq.cgi.
2
h in air. After removal of the solvent, the crude product was
subjected to flash chromatography (SiO , CH Cl /CH OH 50:1, v/v)
2
2
2
3
to afford 3′-O-methylfluorescein methyl ester (MFME) as a yellow
1
solid (14.5 mg, 80.5%). H NMR (400 MHz, CDCl ): δ = 3.64 (s,
3
RESULTS AND DISCUSSION
3
1
H), 3.92 (s, 3H), 6.46 (d, J = 1.6 Hz, 1H), 6.54 (dd, J = 1.6, 9.6 Hz,
H), 6.74 (dd, J = 2.4, 9.2 Hz, 1H), 6.85 (d, J = 9.6 Hz, 1H), 6.89 (d, J
■
Synthesis of Complexes. The synthesis of FluHMPP is
shown in Scheme 1. 3 was synthesized via reaction of 2 with 1
=
9.2 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 7.31 (dd, J = 1.2, 7.6 Hz, 1H),
7.67 (td, J = 1.2, 8.0 Hz, 1H), 7.74 (td, J = 1.2, 7.6 Hz, 1H), 8.25 (dd, J
using anhydrous potassium carbonate in refluxing CH CN.
1.2, 8.0 Hz, 1H). 1 C NMR (100 MHz, CDCl ): δ = 52.5, 56.1,
3
3
=
3
Removal of allyl of 3 using palladium catalyst followed by
condensation with 2-picolylamine afforded FluHMPP. The
1
1
00.5, 105.9, 113.5, 115.0, 117.7, 129.0, 129.8, 130.0, 130.3, 130.5,
30.7, 131.3, 132.8, 134.8, 150.3, 154.4, 159.1, 164.2, 165.7, 185.8.
1
+
structure of FluHMPP was confirmed by elemental analysis, H
ESI−MS m/z [(M + 1) ]: 361.1.
13
NMR, C NMR spectroscopies, and ESI mass spectrometry. In
Fluorescence Spectroscopy. Fluorescence spectra were recorded
on a Hitachi F-4500 spectrofluorometer equipped with a xenon lamp
as the excitation source. To reduce the fluctuation in the excitation
intensity during measurement, the lamp was kept on for 1 h prior to
the experiment. The path length was 1 cm with a cell volume of 3.0
mL. Fluorescence responses of FluHMPP to various metal ions were
measured as follows. FluHMPP (final, 10 μM) was added to 1 mL of
Tris−HCl (10 mM, pH 7.20), H O−CH CN (7:3, v/v), then aqueous
II
addition, complex [Cu (MPP−NO )NO ] was also obtained.
2
3 2
Oxidation products were confirmed by ESI−MS, HPLC, and X-
ray diffraction measurement.
Fluorescence Response and Product Analysis. Our
II
research on a new Cu turn-on system is constructed of a
nonfluorescent nonconjugated fluorescein platform and a
receptor moiety (HMPP). Cu-mediated oxidative reaction
renews the conjugated form of fluorescein via removal of the
blocking receptor and thereby turns on the fluorescent signal of
the probe. All fluorescence properties of FluHMPP were
evaluated in Tris−HCl (10 mM, pH 7.20), H O−CH CN,
2
3
solution of transition metal ions (NaCl, KCl, MgCl , CaCl ,
2
2
Cr(ClO ) , Co(ClO ) , Ni(NO ) , FeCl , Cu(NO ) , ZnCl , Cd-
4
3
4
2
3
2
3
3
2
2
(
NO ) , Al(NO ) and Hg(ClO ) ) or [Cu(CH CN) ]BF in CH CN
3 2 3 3 4 2 3 4 4 3
were used to give a final concentration of 1 mM s- and 0.2 mM d-block
metal ions. In the same way, aqueous solutions of various copper salts
2
3
(
Cu(NO ) , Cu(ClO ) , CuSO , Cu(AcO) , Cu(CF SO ) , and
3 2 4 2 4 2 3 3 2
(7:3, v/v). Predictably, FluHMPP has negligible fluorescence
because the xanthene part of FluHMPP adopts a non-
conjugated form. We studied the fluorescent changes of 10
CuCl ) were used to detect the influence of the anions. The mixtures
2
were kept in the dark for 2 h. All fluorescence spectra were measured
with an excitation wavelength at 450 nm, and emission spectra were
collected from 470 to 700 nm.
II
μM FluHMPP by titration with various concentrations of Cu
in air (Supporting Information Figure S1). An obvious
Product Analysis. For ESI−MS analysis, a 0.2 mM solution of
enhancement of more than 30-fold in fluorescence intensity
Cu(NO ) in CH OH was added to a 10 μM FluHMPP solution in 1
3
3
II
mL of Tris−HCl (10 mM, pH 7.20), H O−CH CN (7:3, v/v) under
was observed with addition of 0.2 mM Cu within 2 h (Figure
2
3
aerobic conditions. The mixture was stirred for 2 h at room
1). HPLC (Supporting Information Figure S3) and ESI mass
1
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Inorganic Chemistry
Article
crystallizes in the monoclinic system, with space group P2 /c
1
from acetonitrile and methanol. The asymmetric unit contains
−
II
−
two (MPP−NO ) , two Cu ions, and two NO . A phenolic
2
3
II
−
oxygen atom bridges two Cu ions, and a NO also bridges
3
II
1
2
two Cu ions in μ -η :η mode. However, when Cu(ClO ) was
2
4 2
added to the reaction system instead of Cu(NO ) , we acquired
3
2
colorless crystals of [Cu(CH CN) ](ClO ) previously re-
3
4
4
18
I
ported. These results indicate the formation of Cu during
the reaction process. The possible mechanism we are
considering is that Cu coordinates and reacts with the NNO
receptor of FluHMPP, resulting in a transient Cu -complex
Supporting Information Figure S5), and then Cu catalyzes
II
I
19
I
(
II
benzyl ether C−O bond cleavage to form [Cu (MPP−
COOH)]+ and MFME with assistance of O through an
2
intramolecular mechanism similar to that reported in Taki’s and
1
2,20
Suzuki’s systems.
[
[
Elimination of the carboxyl group from
II
+
Cu (MPP−COOH)] followed by nitration affords
II +
Cu (MPP−NO )] . Supporting Information Figure S6
2
Figure 1. Fluorescence response of 10 μM FluHMPP before and after
shows a possible mechanism for the formation of
II
+
2
7
h of reaction with 0.2 mM Cu(NO ) in Tris−HCl (10 mM, pH
[Cu (MPP−NO )] . Cu(II) ions play a significant catalytic
3
2
2
21
.20), H O−CH CN, (7:3, v/v). (Inset) Time course of the
2
3
role in the decarboxylation and nitration procedures. The
yield of C−O bond cleavage was 80.5%. In the Cu system
reported by Taki, the fluorescence response had to be
performed in the reducing environment provided by GSH.
However, our system showed no fluorescence response for
fluorescence intensity change at 519 nm in the reaction of FluHMPP
(
10 μM) and Cu(NO ) (0.2 mM) under aerobic conditions. The
3
2
excitation wavelength was 450 nm.
II
I
both Cu and Cu in the presence of GSH within 2 h
spectrometry (Supporting Information Figure S4) analysis of
the reaction mixture revealed that there is a new product, 3′-O-
methylfluorescein methyl ester (MFME). This indicates that
the benzyl ether C−O bond of FluHMPP was cleaved, and the
resulting 2-(3-hydroxy-6-methoxy-9H-xanthen-9-yl)benzoic
acid methyl ester was easily air-oxidized to afford MFME.
Meanwhile, the ESI mass spectrum of the reaction mixture
revealed a peak at m/z 436.2 (Supporting Information Figure
(
Supporting Information Figures S7 and S8).
Metal-Ion Selectivity and Effect of pH. The fluorescence
selectivity of 10 μM FluHMPP was studied in the presence of 1
mM s- and 0.2 mM d-block metal ions after 2 h of mixing in air.
Physiologically important metal ions such as Na , K , Fe , and
Zn had no or negligible interference in the fluorescence of
+
+
3+
2+
FluHMPP (Figure 3). Because of the formation of [Cu-
II
S4) with isotope distribution pattern calculated for the Cu
II
complex having an oxidized ligand of HMPP, [Cu (MPP−
+
COO)(CH CN) ] + Na . This suggests that the NNO imine
3
2
part has been oxidized to the carboxylate-containing derivative,
HMPP−COOH.
II
Crystal Structure of [Cu (MPP−NO )NO ] and Mech-
2
3 2
II
anism Research. The structure of the Cu complex with
MPP−NO is shown in Figure 2. Selected bond lengths and
2
bond angles are given in Supporting Information Table S2. It
Figure 3. Metal ion selectivity of FluHMPP. The bars represent the
fluorescence intensity at 519 nm after 2 h of reaction of 10 μM
FluHMPP with each type of metal ion (1 mM s- and 0.2 mM d-block
metal ions) in Tris−HCl (10 mM, pH 7.20), H O−CH CN, (7:3, v/
2
3
v). The excitation wavelength was 450 nm.
+
I
II
(
CH CN) ] , Cu cannot be quickly oxidized to Cu , so it
3 4
showed a slightly enhanced fluorescence of FluHMPP in test
I
time. However, Cu could also induce observable fluorescence
enhancement if the reaction time was extended (Supporting
Information Figure S9). The anion responses to the detection
systems were further investigated. These results show that
II
Figure 2. Thermal ellipsoid (30% probability level) plot of
various kinds of anions Cu salts could cause fluorescence
II
[
Cu (MPP−NO )NO ] . All hydrogen atoms were omitted for clarity.
enhancement of FluHMPP (Scheme 2 and Supporting
2
3 2
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Scheme 2. Mechanism of Fluorescence Enhancement of
FluHMPP Induced by CuII
support for our speculation about debenzylation and that in
related literature. Ongoing efforts are underway in our group
12
to exploit the design strategy based on reaction to detect a
variety of other substances in biological studies.
ASSOCIATED CONTENT
Supporting Information
■
*
S
II
X-ray crystallographic data of [Cu (MPP−NO )NO ] in CIF
2
3 2
format, characterization, and additional spectroscopic data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Information Figure S10). In addition to excellent selectivity, it
is essential that the probe can be operated in the physiological
pH range for biological applications. Therefore, we investigated
the pH effect on the fluorescence response of FluHMPP in the
AUTHOR INFORMATION
Corresponding Author
Tel.: +86-931-8915151. Fax: +86-931-8912582. E-mail:
liuws@lzu.edu.cn.
■
*
II
presence of Cu . As was seen from Figure 4, FluHMPP was
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the NSFC (Grants 20931003,
1122007, 21001059), the Specialized Research Fund for the
■
9
Doctoral Program of Higher Education (Grant No.
0110211130002), and the Fundamental Research Funds for
2
the Central Universities (lzujbky-2012-189).
REFERENCES
■
(
1) (a) Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Chem.
Rev. 2006, 106, 1995. (b) Uauy, R.; Olivares, M.; Gonzalez, M. Am. J.
Figure 4. Effect of pH on the fluorescence intensity at 519 nm of
FluHMPP (●) and of FluHMPP (10 μM) reacted with Cu(NO3)2
Clin. Nutr. 1998, 67, 952S.
(
(
2) Zhou, B.; Gitschier, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7481.
3) (a) Halliwell, B.; Gutteridge, J. M. C. Biochem. J. 1984, 219, 1.
(
0.2 mM) after 2 h (○) in Tris−HCl (10 mM, pH 7.20), H O−
2
CH CN, (7:3, v/v) respectively. The excitation wavelength was 450
3
(
(
b) Linder, M. C.; Hazegh-Azam, M. Am. J. Clin. Nutr. 1996, 63, 797S.
c) Valko, M.; Morris, H.; Cronin, M. T. D. Curr. Med. Chem. 2005,
nm.
1
2, 1161. (d) Buettner, G. R.; Jurkiewicz, B. A. Radiat. Res. 1996, 145,
stable within the pH range of 4.0−9.5. On the other hand, it
532. (e) Chevion, M. Free Radical Biol. Med. 1988, 5, 27.
(4) (a) Harris, Z. L.; Gitlin, J. D. Am. J. Clin. Nutr. 1996, 63, 836S.
II
readily reacted with Cu within the biologically relevant pH
(b) Scheinberg, I. H.; Sternlieb, I. Am. J. Clin. Nutr. 1996, 63, 842S.
range (5.5−7.5), indicating that FluHMPP possesses high
sensing ability in living cells without interference from pH
effects.
(5) (a) Vulpe, C.; Levinson, B.; Whitney, S.; Packman, S.; Gitschier,
J. Nat. Genet. 1993, 3, 7. (b) Waggoner, D. J.; Bartnikas, T. B.; Gitlin, J.
D. Neurobiol. Dis. 1999, 6, 221. (c) Bull, P. C.; Thomas, G. R.;
Rommens, J. M.; Forbes, J. R.; Cox, D. W. Nat. Genet. 1993, 5, 327.
Fluorescence Images. Because of the favorable properties
of FluHMPP in vitro, we investigated a practical application of
(6) (a) Que, E. L.; Domaille, D. W.; Chang, C. J. Chem. Rev. 2008,
II
FluHMPP, and bioimaging for Cu was examined in living cells
108, 1517. (b) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J.
Chem. Rev. 2012, 112, 1910. (c) Du, J.; Hu, M.; Fan, J.; Peng, X. Chem.
Soc. Rev. 2012, 41, 4511.
using confocal fluorescence microscopy (Supporting Informa-
tion Figure S11). HeLa cells treated with 20 μM FluHMPP
alone in the growth medium for 4 h at 37 °C exhibited no
fluorescence (Supporting Information Figure S11a and S11b).
(7) (a) Bergonzi, R.; Fabbrizzi, L.; Licchelli, M.; Mangano, C. Coord.
Chem. Rev. 1998, 170, 31. (b) Tang, B.; Niu, J.; Yu, C.; Zhuo, L.; Ge, J.
Chem. Commun. 2005, 41, 4184. (c) Park, S. M.; Kim, M. H.; Choe, J.-
I.; No, K. T.; Chang, S.-K. J. Org. Chem. 2007, 72, 3550. (d) Jung, J.
H.; Lee, M. H.; Kim, H. J.; Jung, H. S.; Lee, S. Y.; Shin, N. R.; No, K.;
Kim, J. S. Tetrahedron Lett. 2009, 50, 2013. (e) Jung, H. S.; Kwon, P.
S.; Lee, J. W.; Kim, J. I.; Hong, C. S.; Kim, J. W.; Yan, S.; Lee, J. Y.;
Lee, J. H.; Joo, T.; Kim, J. S. J. Am. Chem. Soc. 2009, 131, 2008. (f) Xie,
However, supplementing the cells with 100 μM of CuCl in the
2
growth medium for 1 h, followed by washing twice with PBS
buffer prior to FluHMPP staining, results in a significant
increase in the observed intracellular fluorescence (Supporting
Information Figure S11d and S11e). Obvious changes indicated
that FluHMPP was cell membrane permeable and capable of
J.; Menand, M.; Maisonneuve, S.; Metivier, R. J. Org. Chem. 2007, 72,
́
́
II
detecting Cu ions in the living cells.
5980.
8) (a) Lin, W.; Long, L.; Chen, B.; Tan, W.; Gao, W. Chem.
(
CONCLUSIONS
We have synthesized and evaluated FluHMPP, a novel
Commun. 2010, 46, 1311. (b) Ciesienski, K. L.; Hyman, L. M.;
Derisavifard, S.; Franz, K. J. Inorg. Chem. 2010, 49, 6808. (c) Goswami,
S.; Sen, D.; Das, N. K. Org. Lett. 2010, 12, 856. (d) Shao, N.; Jin, J. Y.;
Wang, H.; Zhang, Y.; Yang, R. H.; Chan, W. H. Anal. Chem. 2008, 80,
■
reaction-based probe for selective turn-on fluorescence
II
detection of Cu in both aqueous media and living cells
3
466. (e) Xu, Z.; Qian, X.; Cui, J. Org. Lett. 2005, 7, 3029. (f) Zhang,
under visible light excitation. The results obviously suggested
X.; Shiraishi, Y.; Hirai, T. Org. Lett. 2007, 9, 5039. (g) Xu, Z.; Xiao, Y.;
Qian, X.; Cui, J.; Cui, D. Org. Lett. 2005, 7, 889. (h) Royzen, M.; Dai,
Z.; Canary, J. W. J. Am. Chem. Soc. 2005, 127, 1612. (i) Wen, Z.-C.;
Yang, R.; He, H.; Jiang, Y.-B. Chem. Commun. 2006, 42, 106. (j) You,
II
that only Cu could cause oxidative benzyl ether C−O bond
cleavage via metal binding assisted by O . Moreover, single-
2
crystal X-ray crystallographic analysis further provided strong
1
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Article
Y.; Han, Y.; Lee, Y.-M.; Park, S. Y.; Nam, W.; Lippard, S. J. J. Am.
Chem. Soc. 2011, 133, 11488.
(
9) Quang, D. T.; Kim, J. S. Chem. Rev. 2010, 110, 6280.
(
10) Jo, J.; Lee, H. Y.; Liu, W.; Olasz, A.; Chen, C.-H.; Lee, D. J. Am.
Chem. Soc. 2012, 134, 16000.
11) (a) Canali, L.; Sherrington, D. Chem. Soc. Rev. 1999, 28, 85.
b) Bai, S.; Li, B.; Peng, J.; Zhang, X.; Yang, Q.; Li, C. Chem. Sci. 2012,
, 2864. (c) El-Gamel, N. E. A. RSC Adv. 2012, 2, 5870. (d) Peyralans,
(
(
3
J. J. P.; Otto, S. Curr. Opin. Chem. Biol. 2009, 13, 705. (e) Zhou, X.;
Yu, B.; Guo, Y.; Tang, X.; Zhang, H.; Liu, W. Inorg. Chem. 2010, 49,
4
002.
12) Taki, M.; Iyoshi, S.; Ojida, A.; Hamachi, I.; Yamamoto, Y. J. Am.
Chem. Soc. 2010, 132, 5938.
13) Au-Yeung, H. Y.; New, E. J.; Chang, C. J. Chem. Commun. 2012,
8, 5268.
14) Amato, M. E.; Ballistreri, F. P.; Pappalardo, A.; Sciotto, D.;
Tomaselli, G. A.; Toscano, R. M. Tetrahedron 2007, 63, 9751.
15) (a) SMART, 5.05 ed.; Bruker AXS, Inc.: Madison, WI, 1998.
b) Bruker AdVanced X-ray Solutions SAINT, version 6.45; Bruker
AXS, Inc.: Madison, WI, 1997−2003.
16) Sheldrick, G. M. SADABS: Area-Detector Absorption Correction;
University of Gottingen: Gottingen, Germany, 1996.
17) Heldrick, G. M. SHELXL-97, Program for the Refinement of
Crystal Structures; University of Gottingen: Gottingen, Germany, 1997.
18) Hao, H.-G.; Zheng, X.-D.; Lu, T.-B. Angew. Chem., Int. Ed. 2010,
9, 8148.
(
(
4
(
(
(
(
̈
̈
(
̈
̈
(
4
(
(
19) Cao, H.; Shi, W.; Xie, J.; Huang, Y. Anal. Methods 2011, 3, 2102.
20) (a) Cho, J.; Furutachi, H.; Fujinami, S.; Suzuki, M. Angew.
Chem., Int. Ed. 2004, 43, 3300. (b) Mizuno, M.; Honda, K.; Cho, J.;
Furutachi, H.; Tosha, T.; Matsumoto, T.; Fujinami, S.; Kitagawa, T.;
Suzuki, M. Angew. Chem., Int. Ed. 2006, 45, 6911.
(
21) Zhang, X.-M. Coord. Chem. Rev. 2005, 249, 1201.
1
2673
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