Synthesis and evaluation of a new Rhodamine B and Di(2-picolyl)amine conjugate as a highly sensitive and selective chemosensor for Al3+ and its application in living-cell imaging

Article abstract of DOI:10.1016/j.bmc.2014.12.070

A new Rhodamine B derivative (RBDPA), namely, N¹-(2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)-N⁴,N⁴-bis(pyridin-2-ylmethyl)succinamide, was designed, synthesized and structurally characterized to develop a chemosensor. The studies show that RBDPA exhibits high sensitivity and selectivity toward Al³⁺ among many other metal cations in an ethanol/H₂O (1:1, v/v, pH = 7.2, HEPES buffer, 0.1 mM) solution. Fluorescence microscopy experiments further demonstrate that RBDPA can be used as a fluorescent probe to detect Al³⁺ in living cells.

Full text of DOI:10.1016/j.bmc.2014.12.070

Accepted Manuscript
Synthesis and evaluation of a new Rhodamine B and Di(2-picolyl)amine con-
3
+
jugate as a highly sensitive and selective chemosensor for Al and its application
in living-cell imaging
Xiaofeng Bao, Qiansheng Cao, Yazhou Xu, Yuanxue Gao, Yuan Xu, Xuemei
Nie, Baojin Zhou, Tao Pang, Jing Zhu
PII:
S0968-0896(15)00002-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.070
BMC 12002
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
20 November 2014
29 December 2014
30 December 2014
Please cite this article as: Bao, X., Cao, Q., Xu, Y., Gao, Y., Xu, Y., Nie, X., Zhou, B., Pang, T., Zhu, J., Synthesis
and evaluation of a new Rhodamine B and Di(2-picolyl)amine conjugate as a highly sensitive and selective
3
+
chemosensor for Al and its application in living-cell imaging, Bioorganic & Medicinal Chemistry (2015), doi:
http://dx.doi.org/10.1016/j.bmc.2014.12.070
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Synthesis and evaluation of a new Rhodamine B and Di(2-picolyl)amine
3
conjugate as a highly sensitive and selective chemosensor for Al and its
+
application in living-cell imaging
a,
Xiaofeng Bao *, Qiansheng Cao , Yazhou Xu , Yuanxue Gao , Yuan Xu , Xuemei
a
c
c
c
d
Nie , Baojin Zhou , Tao Pang , Jing Zhu *
d
c
b,
a
Department of Biochemical Engineering, Nanjing University of Science &
Technology, Chemical Engineering Building B308, 200 Xiaolinwei, Nanjing, 210094,
P.R.China.
b
Department of Pharmacy, Nanjing University of Traditional Chinese Medicine,
Nanjing 210046, P.R. China
c
Jiangsu Key Laboratory of Drug Screen, New Drug Screening Center, China
Pharmaceutical University, Nanjing, 210009, P.R. China.
d
Department of Chemistry, School of Chemical Engineering, Nanjing University of
Science & Technology, Nanjing, 210094,P.R.China
*Correspondence author: Dr. Xiaofeng Bao, baoxiaofeng@mail.njust.edu.cn; or Dr.
Jing Zhu, E-mail:zhujing1227@hotmail.com.

Abstract
A
new
Rhodamine
B
derivative
(RBDPA),
namely,
N1-(2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9-xanthen]-2-yl)ethyl)-N4,N4
bis(pyridin-2-ylmethyl)succinamide, was designed, synthesized and structurally
characterized to develop a chemosensor. The studies show that RBDPA exhibits high
-
3
+
sensitivity and selectivity toward Al among many other metal cations in an
ethanol/H O (1:1, v/v, pH=7.2, HEPES buffer, 0.1 mM) solution. Fluorescence
microscopy experiments further demonstrate that RBDPA can be used as a
2
3
fluorescent probe to detect Al in living cells.
+
3
Keywords: Rhodamine B derivative; Chemosensor; Al ; Fluorescent probe; Cell
+
imaging.

INTRODUCTION
Aluminum, which is the most abundant metallic element (8.3% by weight) and the
third most abundant of all elements (after oxygen and silicon) in the earth’s crust, is
[1]
widely used in our daily life and industrial fields, such as water treatment , food
[2]
[3,4]
additives , aluminum-based pharmaceuticals , aluminum containers and cooking
[
utensils . The vast majority of aluminum compounds, which include all
5]
aluminum-containing minerals and all commercially significant aluminum
3
compounds, exist in the form of Al in organisms and the environment . Although
+
[6]
[
aluminum and most of its derivatives exhibit low toxicity , it is well-known that Al
7]
3+
[8, 9]
plays important roles in cells and environmental food chains . For example, studies
have shown that the consumption of acidic foods or liquids with aluminum
significantly increases the accumulation of aluminum in the nervous and osseus
[10, 11]
tissues
[12]
. High doses of aluminum can also cause neurotoxicity
and are
[13]
associated with altered functions of the blood-brain barrier . Because aluminum
competes with calcium for absorption, increased amounts of dietary aluminum may
also contribute to the reduced skeletal mineralization (osteopenia), which is observed
[14]
in preterm infants and infants with growth retardation . Therefore, the design and
3
development of new probes for the simple and easy detection of Al in environmental
+
systems is of great interest to current researchers.
Fluorescent chemosensors are currently extensively used as powerful tools to detect
[15, 16, 17, 18]
various transition metal ions in environmental systems
. In comparison to
[19]
the known methods such as graphite furnace atomic absorption spectrometry and

[20]
inductively coupled plasma atomic emission spectrometry
,
fluorescent
chemosensors offer significant advantages because of their excellent properties of
[
21, 22]
high sensitivity, specificity, cost efficiency, and real-time monitoring
. Because
of their excellent photo-physical properties, such as a high fluorescence quantum
yield, large molar extinction coefficient, and visible wavelength excitation, rhodamine
B and its derivatives have an ideal skeleton structure to construct various fluorescent
[
23-26]
probes
. It is well known that there are rhodamine B as well as its derivatives in
[
27]
the non-fluorescent spirolactam form under approximate physiological conditions
,
which exhibits a weak fluorescence emission and UV absorption intensity; when they
bind to specific metal ions or protons, the ring-opened amides cause a strong
[28, 29]
fluorescence emission and UV absorption
. Based on this mechanism, a series of
rhodamine B derivatives have been designed and synthesized to use as fluorescence
3
+
2+
2+
2+
3+
chemosensors to detect metal ions, such as Fe , Hg , Cu , Sn , Cr , Sn and Pb
4+
2+
3+
[
30-36]
.
Although some fluorescence chemosensors have been reported to detect Al
using organic dyes, they have always been problematic because of the poor
[
37]
[38]
fluorescence intensity
characterization of a new Rhodamine B-derivatized fluorescent chemosensor based on
Di(2-picolyl)amine moiety, which is named RBDPA (Figure 1).
Di(2-picolyl)amine and its derivatives were widely reported to be receptors to
and selectivity
. Herein, we report the synthesis and
a
2
special-sized metal ions such as Cu , Zn and Cd
+
2+
2+[39-41]
. Therefore, we introduce it
onto the rhodamine B moiety as a host recognition site for metal ions. Our findings
3
+
show that RBDPA exhibits high selectivity and sensitivity toward Al ions in an

ethanol-HEPES buffer solution (1:1, v/v, 100 µM, pH 7.2). Fluorescence microscopy
experiments further demonstrate that RBDPA can also be used as a fluorescent probe
3
to detect Al in human liver (L-02) cells.
+
2. Experimental
2.1.Materials and general methods
All of the reagents and organic solvents that we used were of ACS grade or higher
and were used without further purification. Unless otherwise noted, all chemicals
were purchased from J&K Scientific (Shanghai, China) and used as received. All
solvents were of analytical grade, and double-distilled water was used in all
experiments. The salts that were used to prepare the metal-ion stock solutions were
3 3 3 2 2 2 2 2 3 2 2 3
AgNO , AlCl , Ba(NO ) , CaCl , CdCl2•2.5H O, CoCl •6H O, CrCl •6H O, Cs CO ,CuCl,
2 2 2 2 3 2 2 2 2 2 2 2
CuCl •2H O, FeCl •4H O, FeCl •6H O, HgCl , KCl, LiCl•H O, MgCl •6H O, MnCl •4H O,
2 2 2 2
NaCl, NiCl •6H O, PbCl , and ZnCl . The reactions were performed in an argon
atmosphere using standard Schlenk techniques. Thin-layer chromatography was
performed on a HAIYANG silica gel F254 plate, and the compounds were visualized

under UV light (λ = 254 nm). Column chromatography was performed using
HAIYANG silica gel (type: 200–300 mesh ZCX-2).
1
13
H (500 MHz) and C NMR (126 MHz) spectra were recorded on an Avance 500
spectrometer (Bruker; Billerica, MA, USA). The chemical shifts are reported in δ
units (ppm) downfield relative to the chemical shift of tetramethylsilane. The
abbreviations br, s, d, t and m denote “broad”, “singlet”, “doublet”, “triplet” and
“multiplet”, respectively. The mass spectra were obtained with a Finnigan TSQ
Quantum LC/MS Spectrometer. High-resolution mass spectra (HRMS) were acquired
under electron ionization conditions with a double-focusing high-resolution
instrument (Autospec; Micromass Inc.). The pH levels of the stock solutions were
measured using a PHS-25C Precision pH/mV Meter (Aolilong, Hangzhou, China).
UV-Vis and fluorescence spectra were obtained using a UV-3600 UV-VIS-NIR
spectrophotometer (Shimadzu, Japan) and an Edinburgh FLS920 fluorescence
spectrophotometer (Livingston, UK), respectively, at room temperature.
2.2 Synthesis of 2-(2-aminoethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-
xanthen]-3-one (1).
To a solution of Rhodamine B (1.91 g, 4.30 mmol) in 50 mL of ethanol,
ethane-1,2-diamine (2.60 mL, 40.00 mmol) was added. The mixture reaction was
refluxed overnight and subsequently dried in vacuum. The residue was dissolved in
CH
using anhydrous magnesium sulfate. After the solvent was removed, flash
chromatography (silica gel; MeOH/CH Cl ; 3:97; v/v) of the residue yielded 1 as a
2 2 2
Cl and subsequently washed with H O and brine. The organic layer was dried
2
2

1
pink solid (1.78 mg, 92%). H NMR (CDCl
3
), δ 7.90 (dd, J = 5.6, 3.0 Hz, 1H),
.61-7.38 (m, 2H), 7.09 (dd, J = 5.6, 2.9 Hz, 1H), 6.43 (d, J = 8.8 Hz, 2H), 6.37 (d, J =
.5 Hz, 2H), 6.27 (dd, J= 8.9, 2.6 Hz, 2H), 3.38-3.27 (m, 8H), 3.19 (t, J = 6.65 Hz, J
7
2
1
2
=
6.6 Hz, 2H), 2.41 (t, J
1
2 1 2
= 6.65 Hz, J =6.6 Hz,2H), 1.16 (t, J = 7.0 Hz,J = 7.1 Hz,
1
3
1
1
2
2H) ppm. C NMR (CDCl ), δ 168.59, 153.35, 148.79, 132.38, 131.20, 128.64,
3
28.02, 123.80, 122.71, 108.13, 105.65, 97, 64.91, 44.36, 44.06, 40.73, 12.56 ppm.
.3 Synthesis of 4-((2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthen]
-
2-yl)ethyl)amino)-4-oxobutanoic acid (2).
2 2
To a solution of Compound 1 (0.24 g, 0.50 mmol) in 10 mL of CH Cl , Succinic
anhydride (0.05 g, 0.50 mmol) was added. The reaction mixture was stirred at room
temperature for half an hour and dried in vacuum. Flash chromatography (silica gel;
1
MeOH/CH
NMR (CDCl
8.1 Hz, 1H), 6.45-6.34 (m, 4H), 6.28 (d, J = 8.7 Hz, 2H), 3.34 (dd, J = 14.1, 7.0 Hz,
0H), 3.05 (s, 2H), 2.63-2.57 (m, 2H), 2.53-2.43 (m, 2H), 1.17 (t, J = 7.0 Hz, J = 7.1
), δ 175.01, 172.63, 170.10, 153.68, 153.30, 149.00,
32.95, 130.29, 128.38, 123.92, 122.90, 108.32, 104.48, 97.81, 65.92, 44.35, 40.55,
2
Cl
2
; 1:99; v/v) of the residue yielded 2 as a pink solid (0.29 g, 100%). H
3
), δ 7.90 (d, J = 5.1 Hz, 1H), 7.53-7.43 (m, 2H), 7.37 (s, 1H), 7.10 (d, J
=
1
1
2
1
3
Hz,12H) ppm. C NMR (CDCl
3
1
3
+
9.91, 30.50, 12.55 ppm. HRMS (M +1) found, 585.3091; calculated for
+
C
34
H
41
N
4
O
5
, 585.3071.
1
.4 Synthesis of N -(2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'–xan
2
then]-2-yl)ethyl)-N4,N4-bis(pyridin-2-ylmethyl)succinamide (RBDPA).
To a solution of compound 2 (0.278 g, 0.475 mmol) in 10 mL of CH
2
2
Cl ,

N,N'-dicyclohexylcarbodiimide (0.124 g, 0.601 mmol), 1-Hydroxybenzotriazole
0.810 mg, 0.601 mmol) and N,N-Diisopropylethylamine (0.104 mL, 0.750 mmol)
(
were added. The reaction mixture was stirred at room temperature for half an hour;
then, Di(2-picolyl)amine (0.180 mL, 1 mmol) was added and stirred for additional 2
hours. The reaction mixture was dried in vacuum. The residue was dissolved in 20 mL
of dichloromethane and subsequently washed with brine three times. The organic
layer was dried with anhydrous magnesium sulfate and filtered through a pad of celite.
After the solvent was removed, flash chromatography (silica gel; MeOH/CH
2
Cl
2
; 1:99;
),
1
v/v) of the residue yielded RBDPA as a pink solid (0.310 g, 81%). H NMR (CDCl
3
δ 8.53 (d, J = 4.3 Hz, 1H), 8.46 (d, J = 4.3 Hz, 1H), 7.91-7.87 (m, 1H), 7.66-7.63 (m,
1
H), 7.61-7.58 (m, 1H), 7.46-7.43 (m, 2H), 7.29 (s, 1H), 7.23 (d, J = 7.8 Hz, 1H),
.19-7.10 (m, 2H), 7.10-7.05 (m, 1H), 6.78 (s, 1H), 6.44 (d, J = 8.9 Hz, 2H), 6.37 (d, J
7
=
2.5 Hz, 2H), 6.27 (d, J = 8.9, 2.5 Hz, 2H), 4.74 (d, J = 5.9 Hz, 4H), 3.31 (d, J = 11.8,
.3 Hz, 10H), 3.06 (s, 2H), 2.76 (t, J = 6.9 Hz, J = 6.8 Hz,2H), 2.50 (t, J = 6.8 Hz,J
= 7.0 Hz, J = 7.1 Hz,12H) ppm. C NMR (CDCl ), δ
6
1
2
1
2
1
3
=
5.45 Hz, 2H), 1.16 (t, J
1
2
3
172.88, 172.02, 169.54, 157.25, 156.44, 153.73, 153.23, 149.65, 148.87, 136.92,
132.66, 130.50, 128.28, 127.95,127.66, 123.81, 123.38,122.96, 122.48, 121.09,
108.24, 104.87, 97.76, 65.47, 52.86, 51.23, 44.28, 40.04, 31.19, 28.46, 12.55 ppm.
+
+
52 7 4
HRMS (M +1) found, 766.4072; calculated for C46H N O , 766.4075.
2.5 Stock solution preparation for spectral detection
-
3
+
3+
2+
2+
Stock solutions (10 M) of the chloride or nitrate salts of Ag , Al , Ba , Ca ,
2
+
2+
3+
2+
+
2+
2+
3+
2+
Cd , Co , Cr , Cs , Cu , Cu , Fe , Fe , Hg , K , Li , Mg , Mn , Na , Ni ,
+
+
2+
2+
+
2+

2
Pb and Zn in deionized water were prepared. The stock solution of RBDPA (10
+
2+
-3
M) was prepared in ethanol/HEPES (1:1; v/v; 100 µM, pH 7.2). Working solutions of
RBDPA were freshly prepared by diluting the highly concentrated stock solution to
the desired concentration prior to the spectroscopic measurements.
2.6 UV-Vis and fluorescence-spectral studies
All experiments were performed in an ethanol/HEPES (1:1; v/v; 100 µM, pH 7.2)
solution. In each titration experiment, a 10 µM solution of the probe RBDPA was
placed in a quartz optical cell with a 1-cm optical path length, and the appropriate
amount of ion stock solution was added to the quartz optical cell using a micropipette.
Spectral data were recorded 30 min after the ion addition. In the selectivity
experiments, the test samples were prepared by placing an appropriate amount of the
cation stock solution in 3 mL of the probe RBDPA solution (10 µM). In the
fluorescence measurements, excitation was provided at 560 nm, and emission was
collected from 565 to 680 nm.
2.7 Cell Studies
In vitro experiments were performed using human L-02 liver cells. L-02 cells were
cultured in RPMI-1640 medium, which was supplemented with 10% fetal bovine
serum (FBS) in an atmosphere of 5% CO2 at 37°C. The cytotoxicity of RBDPA was
determined using an MTT assay. Briefly, the cells were incubated with different
concentrations of RBDPA for 48 h. The cell viability was evaluated by incubating
with 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
for 4 h under 5% CO /95% air at 37°C. The media were replaced with 100 µL of
2

DMSO, and the absorbance was read at 570 nM. For fluorescence microscopy images,
one day before imaging, the cells were seeded in 6-well plates. Immediately before
3
the experiments, the cells were incubated with 0.1 mM AlCl in 50 mM HEPES
buffer for 30 min at room temperature, followed by incubation with 20 µM RBDPA
at 37°C under 5% CO2 for additional 30 min. The cells were washed with HEPES
three times and subsequently imaged. The fluorescence intracellular imaging was
observed under an Olympus inverted fluorescence microscope with a 20× objective
lens multiplied by 1.6 (excited with green light). The cells that were incubated with
the solvent DMSO were taken as a control.
3
. Results and discussion
.1 Synthesis of RBDPA
3
The synthesis procedure of RBDPA is illustrated in Scheme 1. Compound 1 was
prepared in a 92% yield from the commercially available Rhodamine B and ethane-1,
[
42]
2
-diamine, as previously described . Compound 2 was prepared in a 100 % yield
from the reaction of succinic anhydride with 1 in CH Cl at room temperature for 30
2
2

min, which was subsequently coupled with Di(2-picolyl)amine in the presence of
DCC, HOBt, and TEA in CH Cl at room temperature for 2 h to give RBDPA in an
1% yield. The structure of RBDPA was confirmed using H NMR, C NMR, and
HRMS (Supporting Materials).
2
2
1
13
8
3.2 Responses of RBDPA to pH
Initially, acid-base titration experiments were performed to evaluate the responses
of RBDPA to pH in different ethanol-H O (1:1, v/v) pH solutions. The fluorescence
2
intensities of RBDPA were measured at λex/em=560/582 nm in different pH solutions,
ranging from 2.0 to 11.0. As shown in Figure 2, RBDPA exhibits a notably low
fluorescence emission at 582 nm (λex=560 nm) in the pH range of 5.0 to 11.0, which
indicates that there is RBDPA in the spirolactam form (Scheme 2) in the pH range of
5.0 to 11.0. However, when the pH was adjusted below 5.0, because of the
ring-opening process of the spirocyclic moiety of Rhodamine B, the fluorescence
intensity at λex/em=560/582 nm drastically increased and reached its maximum value
when the pH was lower than 3.0, which indicates that there is RBDPA in the
open-ring form (Scheme 2). These results suggest that RBDPA is stable with a

notably low background fluorescence in the ethanol-H
.0-11.0. Therefore, further UV-Vis and fluorescence studies were performed in an
ethanol-H O (1:1, v/v, HEPES buffer, 100 µM, pH 7.2) solution.
2
O solution in the pH range of
5
2
2
Figure 2. Effect of pH on the fluorescence of RBDPA (10 µM) in ethanol-H O
solutions (1:1, v/v). The excitation and emission wavelengths were 560 nm and 582
nm, respectively.
3.3 Response time of RBDPA

3
+
Figure 3. Fluorescence spectra of RBDPA (10 µM) with Al (50 µM, 5 eq.) in an
ethanol-H O solution (1:1, v/v, HEPES buffer, 100 µM, pH 7.2). The excitation and
2
emission wavelengths were 560 and 582 nm, respectively. Inset: plot of the
fluorescence intensities at 582 nm over a period of 100 min.
3
To evaluate the sensitivity of RBDPA toward Al , the response time of the reaction
+
system was studied at room temperature. As shown in Figure 3, the fluorescence
3
+
intensity at 582 nm rapidly increased after Al (50 µM) was added to the RBDPA (10
µM) solution, reached its maximum value in 30 min and stabilized thereafter. These
3
+
results indicate that RBDPA is a sensitive sensor for the Al inanethanol-H
1:1, v/v, HEPES buffer, 100 µM, pH 7.2). Therefore, 30 min was set as the standard
for all fluorescence intensity measurements.
2
O solution
(
3.4 UV-vis spectral response of RBDPA

3
To evaluate the selectivity of RBDPA for Al , the sensing ability of RBDPA
+
toward different metal cations was studied using UV-Vis spectroscopy. As shown in
Figure 4, the UV-vis spectrum of RBDPA (10 µM) in an ethanol-H
2
O solution (1:1,
v/v, HEPES buffer, 100 µM, pH 7.2) exhibits notably weak absorbance at 560 nm.
3
+
The addition of 5 equiv. Al (50 µM) into the solution significantly enhances the
absorbance at 560 nm. Under the identical condition, no obvious response was
+
3+
2+
2+
2+
2+
3+
observed at 560 nm after other metal ions (Ag , Al , Ba , Ca , Cd , Co , Cr ,
2
+
+
2+
2+
3+
2+
+
+
2+
Cs , Cu , Cu , Fe , Fe , Hg , K , Li , Mg , Mn , Na , Ni , Pb and Zn ) were
2+
+
2+
2+
2+
added. The results demonstrate that RBDPA can serve as an excellent selective
3
chemosensor for Al in an ethanol-H
+
2
O solution (1:1, v/v, HEPES buffer, 100 µM,
pH 7.2).
Figure 4. UV-Vis absorbance spectra of RBDPA (10 µM) in an ethanol-HEPES
buffer solution (1:1; v/v; 100 µM; pH 7.2) in the absence and presence of 5
+
equivalents of various metal ions: Ag , Al , Ba , Ca , Cd , Co , Cr , Cs , Cu ,
3+
2+
2+
2+
2+
3+
2+
+

2
Cu , Fe , Fe , Hg , K , Li , Mg , Mn , Na , Ni , Pb and Zn . The absorption
+
2+
3+
2+
+
+
2+
2+
+
2+
2+
2+
wavelength for RBDPA was 560 nm.
3.5 Fluorescence titration of RBDPA with metal ions
3
To further understand the selectivity of RBDPA for Al , the changes in
+
fluorescence intensity after various metal ions were added under identical conditions
were also investigated. The fluorescence spectra of RBDPA (10 µM) in an
ethanol-H
2
O solution (1:1, v/v, HEPES buffer, 100 µM, pH 7.2) exhibits a notably
weak fluorescence at 582 nm (λex = 560 nm) in the absence of metal ions, which
3
+
indicates that the predominant form of RBDPA is the spirolactam form. When Al
(
50 µM, 5 eq.) was introduced to the RBDPA solution, a remarkable fluorescence
3
+
enhancement was observed (Figure 5), which indicates that the Al ions induce the
3
formation of the strongly fluorescent, ring-opened RBDPA-Al complex (Scheme 3).
+
3
+
The fluorescence enhancement of Al to RBDPA is as high as 180-fold. Other metal
ions showed no obvious fluorescence enhancement under the same conditions. These
results further demonstrate that RBDPA can function as a highly sensitive and
3
+
selective fluorescent chemosensor for Al over various other metal ions.

Figure 5. Fluorescence spectra of RBDPA (10 µM) in an ethanol-H
2
O solution (1:1,
v/v, HEPES buffer, 100 µM, pH 7.2) in the absence and presence of 5 equivalents of
+
3+
2+
2+
2+
2+
3+
2+
+
2+
2+
3+
various metal ions: Ag , Al , Ba , Ca , Cd , Co , Cr , Cs , Cu , Cu , Fe , Fe ,
2
+
+
+
2+
2+
+
2+
Hg , K , Li , Mg , Mn , Na , Ni , Pb and Zn . The excitation and emission
2+
2+
wavelengths were 560 nm and 582 nm, respectively.
3
+
3.6 Fluorescence titration of RBDPA with Al
3
+
3+
To gain further insight into the binding of RBDPA with Al , the Al titration
against RBDPA in an ethanol-H O solution (1:1, v/v, HEPES buffer, 100 µM, pH 7.2)
2
3
was monitored using fluorescence spectra. As shown in Figure 6, when no Al ion
+
was added to the RBDPA solutions, free RBDPA (10 µM) remained colorless and
3
+
exhibited notably weak fluorescence (λex =560 nm) at 582 nm. However, when Al

3
0-50 eq.) was added, the titration of Al ions with RBDPA significantly increased
+
(
the emission intensity at 582 nm, which resulted in a color change from colorless to
pink, which can be ascribed to the formation of the ring-opened amide form of
3
RBDPA upon Al ion binding (Scheme 3). The emission intensity reached its
+
3
maximum value after the addition of 10 equivalents of Al . A fluorescence
+
enhancement of over 500-fold was observed under saturation conditions.
2
Figure 6. Fluorescence spectra of RBDPA (10 µM) in an ethanol-H O solution (1:1,
3
+
v/v, HEPES buffer, 100 µM, pH 7.2) when different amounts of Al were added. The
excitation and emission wavelengths were 560 and 582 nm, respectively.
3.7 Job’s plot
3
To determine the binding stoichiometry of the RBDPA-Al complex, a Job’s
+
3
plot was generated by continuously varying the mole fraction of Al from 0 to 1 in a
+

3
+
solution of [Al ] + [RBDPA] with a total concentration of 50 µM. The Job’s plot
analysis revealed an approximate maximum at the 0.5 mole fraction, which indicates a
3
:1 stoichiometry for the RBDPA-Al complex (Figure 7).
+
1
Figure 7. Job’s plot of RBDPA in an ethanol-H
00 µM, pH 7.2) with a total concentration of [RBDPA] + Al = 50 µM. The
excitation and emission wavelengths were 560 and 582 nm, respectively.
2
O solution (1:1, v/v, HEPES buffer,
3
+
1
3.8 Calculations of the binding association constant
[
43]
The association constant was calculated using the Benesi-Hildebrand plot
:
1
1
1
= +
F-F0 K ×(F -F )×[Al ] Fmax -F0
based on a 1:1 stoichiometry, where F is the
3
+
a
max
0
obtained fluorescence intensity, F
0
is the fluorescence intensity of free RBDPA at the
emission wavelength, and Fmax is the saturated fluorescence intensity of the

3
RBDPA-Al complex. As shown in Figure 8, the association constant (Ka) was
+
3
+
graphically evaluated by plotting 1/[F-F
obtained, and the binding constant was calculated to be 3.84×10 M in an
ethanol-H O solution (1:1, v/v, HEPES buffer, 100 µM, pH 7.2) as inferred from the
0
] vs 1/[Al ]. A linear relationship was
4
-1
2
3
fluorescence titration curves of RBDPA with Al .
+
3
+
0
Figure 8. Benesi-Hildebrand plot (λem= 582 nm) of 1/(F - F ) vs. 1/[Al ] based on a
3
:1 association stoichiometry between RBDPA and Al .
+
1
3.9 Calculations of the Detection Limit
3
+
The detection limit of RBDPA for Al was also calculated based on the
fluorescence titration. The fluorescence emission spectrum of RBDPA was measured
0 times, and the standard deviation of the blank measurement was calculated. To
obtain the slope, the ratio of fluorescence intensity at 582 nm was plotted versus the
1
3
+
Al concentration (Figure 9). The detection limit was calculated using the following

[44]
equation : Detection limit = 3SD/S, where SD is the standard deviation of the blank
and S is the slope of the calibration curve. Thus, the calculated detection limit was
0.196 µM (Supporting Materials).
2
Figure 9. Fluorescence intensity at 582 nm of RBDPA (10 µM) in an ethanol-H O
3
+
solution (1:1, v/v, HEPES buffer, 100 µM, pH 7.2) with different amounts of Al . The
excitation wavelength is 560 nm.
3
.10 Competitive selectivity of RBDPA for Al with other metal ions
+
3
3
+
To further evaluate the selectivity of RBDPA for Al , the competitive selectivity of
3
RBDPA for Al in the presence of many other metal ions under identical conditions
+
was also investigated. As shown in Figure 10, no significant variation in emission of
3
the RBDPA-Al complex was observed compared with the results obtained with or
+
3
without other metal ions. These results indicate that the detection of Al is not
+
3
+
interfered by other metal ions and that RBDPA can be used as a selective Al
fluorescent chemosensor.

Figure 10. Metal-ion selectivity profiles of RBDPA (10 µM) in the presence of
various metal ions in an ethanol-H O solution (1:1, v/v, HEPES buffer, 100 µM, pH
.2). The black bars represent the fluorescence intensity (582 nm) of RBDPA in the
2
7
+
2+
2+
2+
2+
3+
2+
+
2+
2+
presence of 5 eq. of free ions: Ag , Ba , Ca , Cd , Co , Cr , Cs , Cu , Cu , Fe ,
3
+
2+
+
+
2+
2+
+
Fe , Hg , K , Li , Mg , Mn , Na , Ni , Pb , and Zn in the absence of Al . The
2+
2+
2+
3+
red bars represent the fluorescence intensity (582 nm) in the presence of various metal
3
ions after the addition of Al . The excitation wavelength is 560 nm.
+
1
.11 H NMR study of RBDPA with Al
3+
3
1 3+
The H NMR spectra of RBDPA in the presence and absence of Al were also
3
investigated to further elucidate the nature of the interaction between Al and
+
3
RBDPA. As shown in Figure 11, after 1.0 equivalent of Al was added to RBDPA in
+
a CD OD:D O (5:1, v/v) solution, the proton signals of H and H displayed apparent
5
3
2
4
downfield shifts from the single peaks, which were centered at 6.385 ppm and 6.431
ppm, to the broad peaks, which were centered at 6.672 ppm and 6.814 ppm,

respectively (∆δ = 0.287 ppm and 0.383 ppm, respectively). These shifts originate
3
+
from the Al -induced ring-opening process of the Rhodamine B spirocycle. In
addition, the proton signals of H10, H11, H13 and H14 also displayed significant shifts
from the triplet peaks centered at 3.225ppm, 2.396 ppm, 3.016 ppm and 2.747 ppm to
the triplet peaks centered at 3.207 ppm, 2.342 ppm, 2.921 ppm and 2.625 ppm,
respectively (∆δ = 0.018ppm, 0.054 ppm, 0.095 ppm and 0.121 ppm, respectively),
3
+
because of the coordination of RBDPA with Al . The single peaks of H15 and H20
proton adjacent to the pyridine ring also displayed significant shifts from the single
peaks at 4.804 ppm and 4.695 ppm to the single peaks at 5.042 ppm and 4.889 ppm,
respectively (∆δ = 0.238 ppm and 0.194 ppm, respectively). The obvious changes of
the signals from the protons on the pyridine ring were also observed. These results
3
indicate the coordination of RBDPA and Al , which was proposed in Scheme 3,
+
3
where Al may be coordinated with three oxygen atoms on the side chain of RBDPA.
+

1
4 2
Figure 11. H NMR spectra (500 MHz, 298K, methanol-d :D O (5:1, v/v)) of (a)
3
RBDPA and (b) RBDPA +1.0 equivalent Al .
+
Density functional theory (DFT) calculations
3
To better understand the nature of the coordination of Al with RBDPA, the
+
3
+
energy-optimized structures of RBDPA and RBDPA-Al (Figure 12) were obtained
using DFT calculations with the B3LYP method, where 6-31+G(d,p) was the basis set.
The spatial distributions and orbital energies of the HOMO and LUMO of RBDPA
3
+
and RBDPA-Al were also generated using DFT calculations (Figure 13). The
results indicate that in RBDPA, the LUMO was spread over the pyridine moiety,
whereas the HOMO was distributed on the spirolactam of the Rhodamine B moiety.
As a result, the C-N bond on the spirocycle of the Rhodamine B moiety breaks to
3
+
facilitate the binding of Al ions with the RBDPA carbonyl oxygen atoms. The π
3
electrons on the HOMO of the RBDPA-Al complex were also mainly located on the
+
on the spirocycle of the Rhodamine B moiety, whereas the LUMO was mostly located

3
on the guest Al ion. The energy gaps between the HOMO and LUMO in the probe
+
3
RBDPA and RBDPA-Al were calculated to be 97.51 kcal/mol and 30.29 kcal/mol,
+
3
respectively. The results show that the binding of Al to RBDPA decreases the
+
HOMO-LUMO energy gap of the complex and stabilizes the system.
3
+
Figure 12. Energy-minimized structures of (A) RBDPA and (B) the RBDPA-Al
complex.

3
+
Figure 13. HOMO and LUMO orbitals of (A) RBDPA and (B) the RBDPA-Al
complex.
3
+
Cell studies of RBDPA in the presence of Al
Because of its favorable molecular properties, RBDPA should be suitable for
fluorescence imaging in living cells. Therefore, we further evaluated the applicability
3
of RBDPA as a fluorescent probe for Al through in vitro cell studies. The direct
+
3
+
detection of Al in living cells using the probe RBDPA was examined using cultured
normal human liver cells (L-02). The cells were incubated with 0.1 mM AlCl in 50
mM HEPES buffer for 30 min at room temperature and subsequently incubated with
the probe RBDPA (20 µM) at 37°C under 5% CO for an additional 30 min.
3
2
Fluorescence microscopic studies show a lack of fluorescence for human L-02 liver
cells after they were treated with only RBDPA (Figure 14 D). After incubation with
3
AlCl , a bright fluorescence was observed in the L-02 cells (Figure 14 F). The
phase-contrast images in the bright fields of the cells show that the cells were viable
during the experiments, which indicates that RBDPA may be cell-permeable and
non-toxic to the cells.
To evaluate the cytotoxicity of RBDPA, the cell viability was determined using an
MTT assay in L-02 cells with RBDPA concentrations of 0-20 µM. When RBPY was
incubated with L-02 cells for 48 h, it showed no toxicity to the cells (Figure 15).
These results suggest that RBDPA may be suitable to be used as a potential probe to
3
detect Al in biological samples.
+

Figure 14. Fluorescence images of human L-02 hepatocytes incubated with RBDPA
3
+
3
and/or Al . The L-02 cells were incubated with 0.1 mM AlCl in 50 mM HEPES
buffer for 30 min at room temperature, followed by 20 µM RBDPA for additional 30
min (E: bright-field image; F: fluorescence image). The DMSO-treated cells were
taken as a control (A: bright-field image; B: fluorescence image), and only
RBDPA-treated cells were also taken as a control (C: bright-field image; D:
fluorescence image).

Figure 15. Cytotoxicity of RBDPA in human L-02 hepatocytes. The cells were
treated with different concentrations of RBDPA for 48 h, and the cell viability assay
was determined using an MTT assay. The data were expressed as the means ± SD.
Conclusion
In summary, a Rhodamine B derivative, RBDPA, was designed and synthesized as
3
a selective and sensitive chemosensor for Al ions in an ethanol/H
+
2
O (1:1, v/v,
pH=7.2, HEPES buffer, 0.1 mM) solution. The chemical structure of RBDPA was
1 13
analyzed using H NMR, C NMR and HRMS. The 1:1 coordination mode was
3
proposed based on a Job’s plot. The Al binding ability of RBDPA was further
+
3
demonstrated using DFT calculations, which suggest that the binding of Al to
+
RBDPA decreases the HOMO-LUMO energy gap of the complex. Cell studies further
3
demonstrate that RBDPA can be a potential probe to detect Al in human liver cells
+

(
L-02).
Acknowledgements
This research was supported by NSF China (81371616, 21402241), the Natural
Science Foundation of Jiangsu Province (BK20130653), the Fundamental Research
Funds for the Central Universities (30920130111024, JKZD2013006) and the
Research Fund for the Doctoral Program of Higher Education of China
(
20133219120020). We thank the NJUST Sensor Lab (Chief: Dr. Xueji Zhang, Co-PI:
Dr. Dan Shan and Dr. Jinming Kong) for providing the facilities for UV-Vis studies.
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