Two novel rhodamine-perylenediimide fluorescent probes: Synthesis, photophysical properties, and cell imaging

Article abstract of DOI:10.1016/j.cclet.2016.01.039

In this study, two novel dual-switch fluorescent chemosensors based on rhodamine-peryleneiimide have been designed and synthesized. The dual-switching behaviors of the sensors were based on the structural transformations of rhodamine and an intramolecular photoinduced electron transfer (PET) process from rhodamine to perylenediimide. These probes exhibited excellent sensitivity to protons with enhanced fluorescence emission from 500 nm to 580 nm. The fluorescence changes of probes were reversible within a wide range of pH values from 2.0 to 11.0. Moreover, the sensors exhibited high selectivity, short response time, and long lifetime toward protons. The possible mechanism was investigated by the DFT calculation and ¹H NMR. According to the experiment of confocal laser scanning microscopy, these probes could be used to detect the acidic pH variations in living cells.

Full text of DOI:10.1016/j.cclet.2016.01.039

G Model
CCLET 3567 1–8
Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
3
Two novel rhodamine-perylenediimide fluorescent probes:
Synthesis, photophysical properties, and cell imaging
*
Q1 Huan-Ren Cheng, Ying Qian
4
5
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
A R T I C L E I N F O
A B S T R A C T
Article history:
In this study, two novel dual-switch fluorescent chemosensors based on rhodamine-peryleneiimide
have been designed and synthesized. The dual-switching behaviors of the sensors were based on the
structural transformations of rhodamine and an intramolecular photoinduced electron transfer (PET)
process from rhodamine to perylenediimide. These probes exhibited excellent sensitivity to protons
with enhanced fluorescence emission from 500 to 580 nm. The fluorescence changes of probes were
reversible within a wide range of pH values from 2.0 to 11.0. Moreover, the sensors exhibited high
selectivity, short response time, and long lifetime toward protons. The possible mechanism was
investigated by the DFT calculation and 1H NMR. According to the experiment of confocal laser scanning
microscopy, these probes could be used to detect the acidic pH variations in living cells.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 30 June 2015
Received in revised form 14 September 2015
Accepted 14 January 2016
Available online xxx
Keywords:
Perylenediimide
Rhodamine
Photoinduced electron transfer
Dual-switch
6
7
1. Introduction
derivatives are suitable to monitor the pH value in acidic 28
environment with enhanced fluorescence signals. This type of 29
8
9
As common cations, protons play key roles in both of
metabolism and cellular events, such as cell growth, calcium
regulation chemotaxis, and cell adhesion [1]. Many detection
methods in the determination of pH have resulted in the research
of pH sensors [2–11]. Among these methods, fluorescent probes
are regarded as outstanding detection systems due to their terms
of high sensitivity, selectivity, and more convenient operation in
many applications [12–14]. So far, strenuous efforts have been
made to the development of efficient fluorescent chemosensors for
the detection of pH [15–19]. Rhodamine derivatives have been
widely used for detection of various metal ions due to their high
photostability, high quantum yield, and special structure [20–27].
The sensing mechanism is based on fluorescence enhancement
caused by spirolactam ring-opening of rhodamine derivatives
[28]. The spirolactam structure is also sensitive to the pH value
of the environment. Under neutral or basic conditions, the
spirolactam remains closed and the rhodamine derivatives
are non-fluorescent, while under acidic conditions, proton leads
to spirolactam ring-opening and the rhodamine derivatives
exhibit strong fluorescence emission [29–31]. Thus, rhodamine
rhodamine-based pH probes have been prepared and attained 30
considerable effects [30–34], while pH-sensitive rhodamine 31
derivatives as pH fluorescence probes are less common. Among 32
these rhodamine-based probes, dual-switch pH sensors that emit 33
fluorescence with dual emission wavelengths and enable a built in 34
correction for the undesired environmental effects have received 35
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
more and more concerns.
36
Perylene tetracarboxylic diimides (PDI) have also generated 37
great interest in the fields of sensors because of their excellent 38
photo-stability, chemical stability, and high electron-accepting 39
ability [35–44]. Most perylenediimide fluorometric sensors were 40
designed to sense photophysical changes produced upon com- 41
plexation, including photoinduced electron transfer (PET), intra- 42
molecular charge transfer (ICT) [45], and fluorescence resonance 43
energy transfer (FRET) [46]. In these sensors, the PET-based PDI 44
fluorometric sensors [47,48] have been widely utilized in the 45
design of sensors due to the inherently higher sensitivity of PET in 46
comparison with the normal fluorescence quenching motif. 47
However, PET-based PDI fluorometric sensors with dual-switch 48
sensor for proton have not been reported before.
49
Herein, based on the unique structural transformations of 50
rhodamine in acid media and the high electron-accepting ability of 51
PDI, two novel dual-switch fluorescent probes 4 and 5 were 52
designed and synthesized as shown in Fig. 1. In these probes, 53
the perylene diimide chromophore plays as an electron acceptor; 54
*
Corresponding author. School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, China.
E-mail address: yingqian@seu.edu.cn (Y. Qian).
http://dx.doi.org/10.1016/j.cclet.2016.01.039
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: H.-R. Cheng, Y. Qian, Two novel rhodamine-perylenediimide fluorescent probes: Synthesis,
photophysical properties, and cell imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.01.039

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chromatography on silica gel (CH2Cl2/MeOH, 10:1) to give 3 as a
99
pale yellow powder 4.7 g, yield: 85%. M.p. 216ꢁ218 8C (the
100
101
102
103
104
reference value 217ꢁ219 8C), yield: 75%. 1H NMR (CDCl3, ppm):
d
7.89 (m, 1H), 7.42 (m, 2H), 7.07 (m, 1H), 6.40 (dd, J = 8.9, 2.6 Hz,
4H), 6.26 (dd, 2H, J = 8.8, 2.7 Hz), 3.32 (m, 8H), 3.18 (t, 2H,
J = 6.6 Hz), 2.40 (t, 2H, J = 6.6 Hz), 1.15 (t, 12H, J = 6.9 Hz).
2.4. Synthesis of 1,7-Bis(4-tert-butylphenyloxy)perylene-3,4:9,10-
105
107
6
tetracarboxylic Acid Bisanhydride (2)
(1) A mixture of 5.0 g (13 mmol) of perylene-3,4:9,10-tetraboxylic
acid bisanhydride (1) and sulfuric acid (100 mL) was stirred at
50 8C for 12 h, and subsequently I2 (0.56 g) was added. The
reaction mixture was heated to 80 8C, and bromine 2.2 g
(15 mmol) was added dropwise over a time period of 2 h.
After bromine addition, the reaction mixture was heated for
an additional 48 h, the excess bromine was removed by
saturated aqueous solution of K2CO3, and water (100 mL) was
added carefully. The precipitate was separated by filtration,
washed with water (100 mL), and dried in a vacuum to give a
red powder 6.0 g. Without further purification it was used to
the next reaction.
(2) A mixture of compound 1,7-dibromoperylene-3,4:9,10-tetra-
carboxylic acid bisanhydride 2.0 g (3.6 mmol), 4-tert-butyl-
phenol 1.8 g (12.0 mmol), and K2CO3 2.4 g (6.8 mmol) in dry
DMF (120 mL) was heated at the refluxing temperature for 4 h
under an N2 atmosphere. The reaction mixture was poured into
water (100 mL) and neutralized with aqueous 1.2 N HCl
solutions. The formed precipitate was collected by filtration
and washed with water and methanol to give crude product 2
3.1 g, yield: 92%. As this product showed poor solubility in
common organic solvents, it was used for the next reaction
without further purification.
10190
111
112
113
114
115
116
117
118
119
120
121
Fig. 1. Structure of the compounds 4 and 5.
55
56
57
58
59
60
61
62
63
64
65
the rhodamine units serve as electron donors, and the ethylene-
diamine plays as spacer, which separates the two units. In order to
improve the solubility of PDI-core in probes, two PDI cores have
been prepared via modifying the bay region of perylene diimides
with 4-tert-butylphenol. In these cases, A PET process can occur
accompanying with the spirolactam ring-opening of rhodamine.
The probes represent off-state when the rhodamine units are in
spirocyclic, the protonation coordination of the amine fluorophore
would generate the rhodamine ring-open. At the same time, the
PET process should be blocked and the probes represented
‘‘switched on’’ state.
12223
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125
126
127
128
129
130
131
132
133
66
67
2. Experimental
2.1. Materials
68
69
70
71
72
73
74
75
76
77
78
Commercially available Rhodamine B, ethylenediamine and
perylene-3,4:9,10-tetraboxylic acid bisanhydride were used
without purification. The starting compounds 3 [49] and (1,7-
bis(4-tert-butylphenyloxy)perylene-3,4:9,10-tetracarboxylic acid
bisanhydride) 2 [50,51] were prepared according to the reported
procedures. All solvents used in spectroscopic measurements
were of analytical grade. Reactions were monitored by thin layer
chromatography using Merck TLC Silica gel 60 F254. Silica gel
column chromatography was performed over Merck Silica
gel 60. Dilute hydrochloric acid or sodium hydroxide was used
for tuning pH values.
2.5. Rhodamine-perylenediimide (4)
134
Compound 3 1.1 g (2.3 mmol), compound 2 0.42 g (0.6 mmol),
and N (C3H7)3 (0.5 mL) were dissolved in DMF (25 mL), the mixture
was heated at 120 8C for 12 h under nitrogen and the reaction was
followed by TLC (DCM: Methanol = 10:1). On completion of the
reaction the solvent was removed under reduced pressure, a red-
black solid got, washed with waters (100 mL), dry in 60 8C, a more
rigorous purification was then carried out via column chromatog-
raphy (DCM/Methanol = 20:1) to give a purple solid 4 0.79 g, yield:
135
136
137
138
139
140
141
142
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147
148
149
150
151
152
153
79
2.2. Methods
80%. m.p. > 300 8C. FT-IR (KBr, cmꢀ1): 3422 (
CH), 1696 (nasN-C=O), 1665 (nsN-C=O), and 1578(
(CDCl3, ppm):
2H), 7.43ꢁ7.41 (m, 8H), 7.14 (s, 2H), 7.05 (d, 2H, J = 6.0 Hz), 6.96 (d,
4H, J = 8.4 Hz), 6.62ꢁ6.60 (m, 4H), 6.43ꢁ6.40 (m, 4H), 6.29ꢁ6.26
(m, 4H), 3.74 (t, 4H, J = 4.5 Hz), 3.52 (t, 4H, J = 4.5 Hz), 3.28 (m,
16H), 1.65 (s, 18H), 1.13 (t, 24H, J = 6.3 Hz); 13C-NMR (CDCl3):
n
n
NH), 2950–2863
(
n
N-C=O). 1H NMR
80
81
82
83
84
85
86
87
88
89
90
91
92
93
1H NMR and 13C NMR spectra were recorded on a Bruker DMX
d
8.53(s, 2H), 8.33 (d, 4H, J = 8.1 Hz), 7.79ꢁ7.71 (m,
300 NMR spectrometer and
a Bruker ADVANCE 500 NMR
spectrometer in CDCl3 with tetramethylsilane (TMS) as internal
standard. Mass spectra were recorded on Agilent Technologies
6530 Accurate-Mass Q-TOF LC/MS. HRMS were recorded on an
Ultraflex II MALDI-TOF mass spectrometer. UV-visible absorption
spectra were determined on a Shimadu UV-3600 spectrophotom-
eter. Fluorescence spectra were measured on a HORIBA FL-4 Max
spectrometer. FT-IR spectra were recorded on a Nicolet 750 series
in the region of 4000–400 cmꢀ1 using KBr pellets. DFT calculations
of compounds were performed using the Gaussian 03 program
package. The calculations were optimized at the B3LYP/6- 31G (d)
level of theory. The molecular orbitals were visualized using
GaussView.
d
168.4 163.2, 155.7, 153.8, 152.8, 147.0, 132.8, 131.1, 128.8, 127.8,
126.5, 123.7, 122.9, 122.7, 120.1, 119.5, 119.2, 44.4, 44.3, 44.2, 39.4,
34.3, 31.6, 31.4, 29.6, and 12.5. MALDI-TOF-MS: m/z. Calculated:
[M + H]+ = 1621.7635, found: 1621.7639.
2.6. Compound 5 was prepared according the same method of
compound 4
154
155
A
oxblood red solid
5
was got 1.1 g, yield: 84%.
156
157
158
159
160
161
162
m.p. = 292ꢁ294 8C. FT-IR (KBr, cmꢀ1): 3423 (
nNH), 2969–2863
94
2.3. Synthesis of amino-functional rhodamine B (3)
(n n
CH), 1695(nasN-C=O), 1616(nsN-C=O), 1592( N-C=O). 1H NMR
(300 MHz):
d
8.45 (m, 8H, J = 8.1 Hz), 7.94ꢁ7.85 (m, 2H),
95
96
97
98
Rhodamine B 5.0 g (11.2 mmol) and ethylenediamine 9.0 mL
(134.8 mmol) were dissolved in ethanol (50 mL) in a 250 mL flask,
then the mixture was heated at 80 8C for 7 h. After ethanol was
removed under vacuum, the residue was purified by column
7.45ꢁ7.36 (m, 4H), 7.02 (dd, 2H, J = 5.1, 4.2 Hz), 6.5 (d, 4H,
J = 8.7 Hz), 6.31 (d, 4H, J = 2.1 Hz), 6.03 (dd, 4H, J = 8.7, 1.8 Hz), 4.25
(t, 4H, J = 3.2 Hz), 3.57 (t, 4H, J = 3.2 Hz), 3.12 (m, 16H), 1.04 (t, 24H,
Please cite this article in press as: H.-R. Cheng, Y. Qian, Two novel rhodamine-perylenediimide fluorescent probes: Synthesis,
photophysical properties, and cell imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.01.039

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3
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J = 6.9 Hz); 13C-NMR (CDCl3):
d 168.8, 162.5, 153.4, 148.6, 135.4,
135.4, 134.1, 132.6, 132.3, 132.1, 131.2, 130.8, 131.3, 128.0, 127.8,
126.4, 124.4, 123.7, 123.3, 122.7, 129.8, 118.3, 108.0, 97.8, 77.4,
77.0, 76.5, 73.6, 61.6, 58.8, 45.8, 41.5, 38.8, and 12.5. MALDI-TOF-
MS: m/z. Calculated: [M + H]+ = 1325.5859, found: 1325.5854.
168
169
3. Results and discussion
3.1. Synthesis of probe molecules 5 and 4
Scheme 1. Synthesis of 1,7-bis(4-tert-butylphenyloxy)perylenediimide 2.
170
171
172
173
174
175
176
177
178
179
180
181
Compounds 2, 4, and 5 were synthesized via simple steps using
standard organic reactions, as depicted in Schemes 1 and 2. Details
of the synthesis of compounds 4 and 5 have been discussed above.
Compound 2 was synthesized by refluxing 1,7-dibromoperylene-
3,4:9,10-tetracarboxylic acid bisanhydride and 4-tert-butylphenol
in DMF, as its poor solubility in common organic solvents, it was
used for the next reaction without further purification. Compounds
4 and compound 5 were prepared by refluxing compounds 3 and 2
in imidazole in the presence of NEt3, their yields were 80% and 82%,
respectively. All of the new compounds were fully characterized by
FT-IR, 1H NMR, 13C NMR and high-resolution mass spectrometry
(HRMS-MALDI-TOF).
182
183
3.2. The pH dependence of absorption and fluorescence spectra of
compounds 4 and 5
184
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194
195
196
197
198
199
200
201
202
203
204
Fig. 2 presents the absorption spectra of compounds 4 and 5 in
DMSO/H2O at different pH values. The maximum absorption peaks
of probe 4 are at 480 and 530 nm, which are corresponding to the
absorption of rhodamine and PDI units in probes, respectively. As
shown in Fig. 2-left the absorption spectra of probe 4 showed
nearly no changes when the pH value > 7.0, which is ascribed to its
spirolactam form of probe in solution; while the pH decreases to
pH value < 7.0 the absorption intensity of probe 4 at 530 nm and
480 nm increases, respectively. Meanwhile, an obvious absorption
blue shifts generates, indicating the formation of the ring-opened
amide form of rhodamine units in probe 4. The nearly same results
can be obtained in the probe 5 (Fig. 2 right). The synchronous
changes of absorption spectra at 480 nm and 530 nm maybe can
prove the dual-switch process happened in the probes.
The pH dependence of fluorescence spectra of compounds 4 and
5, obtained after excitation within the spectral region of maximal
absorption of the peripheral fluorophore (lex = 480 or 530 nm),
show two emission bands at 550 nm and 580 nm, corresponding to
the emission bands of rhodamine units and the PDI core in
compounds 4 and 5 as shown in Fig. 3. The corresponding data is
showed in Table 1. From the insets photons, we can see that the
Scheme 2. Synthesis of the probes 4 and 5.
probe 4 is non-fluorescent when the pH value > 7.0, when the 205
value of the pH falls to less than 7.0, the fluorescence intensity 206
gradually increases to about 170-fold from pH 7.8 to 5.4. The 207
quantum yield of the probe 4 is determined to be 0.46 in acidic 208
condition (pH 5.1) and < 0.01 in neutral condition with rhodamine 209
6G as a standard [52]. The similar results can be obtained for 210
compound 5, as shown in Fig. 3 right. Compared with to the 211
fluorescent spectra of the two probes, the nearly same results can 212
be obtained in probes 4 and 5, which can be attributed to the nearly 213
same molecular structure of probes. As shown in Fig. 3, the 214
synchronous enhanced fluorescence intensity of probes to 215
the various pH values at 550 nm and 580 nm further prove that 216
the dual-switch processes exactly happened in the probes.
217
ꢀ
ꢁ
IFmaxꢀIF
IFꢀIFmin
log
¼ pHꢀpKa
(1)
1.0
1.0
0.8
0.6
0.4
0.2
0.0
Compound 4
pH
Compound 5
10.2
9.3
pH
11.2
9.4
8.6
7.8
3.5
2.3
1.6
1.1
0.6
8.5
7.8
6.5
4.1
3.0
2.6
2.2
1.9
1.5
1.0
0.4
ε
ε
0.2
0.0
300
360
420
480
540
600
300
360
420
480
540
600
λ (nm)
λ (nm)
Fig. 2. The pH dependence of absorption spectra of compounds 4 and 5 (c = 10
mmol/L, in DMSO/H2O, 1:9).
Please cite this article in press as: H.-R. Cheng, Y. Qian, Two novel rhodamine-perylenediimide fluorescent probes: Synthesis,
photophysical properties, and cell imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.01.039

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2.0x106
1.0x106
10
8
Compound 4
Compound 5
16
12
8
1.6x106
1.2x106
8.0x105
8.0x105
6.0x105
4.0x105
6
4
4
2
0
0
2
4
6
8
10
12
2
4
6
pH
8
10
12
pH
4.0x105
0.0
2.0x105
0.0
560
640
720
800
500
550
600
650
700
750
λ (nm)
λ (nm)
Fig. 3. The pH dependence of fluorescence spectra of compounds 4 and 5 (c = 10
m
mol/L,
l
ex = 480 nm, in DMSO/H2O, 1:9). Inset: fluorescence intensity of probes at various
pH values.
Table 1
Fluorescent properties of compounds 4 and 5 in DMSO/H2O, 1/9.
max
1abs
e1Þ/alm2aabxs
ð
e2
Þ
l1
/
flu l2 (nm)
Dv
1/Dv2 (nm)
F1 /
ex F2
b
c
ex d
Compounds
pH
l
ð
flu
4
7.8
5.1
7.8
5.1
496/531
486/521
491/526
491/526
554/587
554/587
544/582
544/582
58/56
68/66
53/56
53/56
–
4-H+
5
0.46/0.38
–
5-H+
0.42/0.36
The fluorescence quantum yields were determined using rhodamine 6G (
The max absorption was used as the excited wavelength.
F= 0.95 in ethanol) as a standard.
a
Absorption maximum.
b
Emission maximum.
c
Stoke’s shift.
d
Fluorescence quantum yield. lex = 480 nm, (l, Dv, F, E, t)1 = Rh units in probe, (l, Dv, F, E, t)2 = PDI unit in probe.
3.3. Sensing mechanism and DFT calculations
230
21
2
89
0
The abilities of the probes to recognize proton were also
investigated. From Fig. 3, it can be seen that the linear response
range covers the acidic pH range from 5.1 to 7.0. The acidity
constants apparent pKa of probes 4 and 5 were determined by
fluorimetric titration as a function of pH, taking the part of the inset
graph in Fig. 3 located between pH 5.1 and 7.0, the pKa values of
the light harvesting probes 4 and 5 have been calculated by Eq. (1)
[53]. The calculated pKa values are 6.12 and 6.30 for probes 4 and 5,
respectively. These pKa values prove that the probes are valuable
for the study of acidic environment.
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229
In these probes, there are two important factors that lead to the
fluorescence quenching of compounds 4 and 5. The first factor is
that the PET process from the rhodamine units to the perylene-
diimide core in probes. The other is the closed spirolactam of the
rhodamine units in the probes as shown in Fig. 4. Therefore, while
the pH decreased to pH value < 7.0 the PET process in probes was
pressed and the fluorescence of PDI core in probe recovered.
Meanwhile, the closed spirolactam of the rhodamine units in the
231
232
233
234
235
236
237
238
Fig. 4. Photo-induced electron transfer (PET) process for the proposed sensors.
Please cite this article in press as: H.-R. Cheng, Y. Qian, Two novel rhodamine-perylenediimide fluorescent probes: Synthesis,
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5
environment. So, it can be concluded that the PET of probes can be 262
repressed in acid environment. 263
3.4. Interference of metal cations in the fluorescence intensity of dyes 264
Fig. 7 shows the selectivity of probes 4 and 5 to various ions, 265
respectively. Upon addition of Hg2+, Na+, K+, Ca2+, Mg2+, Co2+, Ni2+
,
266
Cu2+, Zn2+, Mg2+, Pd2+, Pb2+, Fe2+, Mn2+, and Al3+ ions to the 267
10 mol/L probe solutions, no significant fluorescence intensity 268
Fig. 5. Structure of the model compounds 6, 7, and 8.
m
changes were observed. However, the same amount of H+ led to a 269
remarkable enhancement in fluorescence intensity, and the 270
fluorescence intensity changes caused by H+ are not obviously 271
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244
245
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247
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253
254
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probes also opened, the probes show strong fluorescence with dual
emission wavelength at 550 nm and 580 nm. The fluorescence
response processes of probes proved that the design of the dual-
switch state is favorable for the probe to work as a pH indicator.
Moreover, the ring-open of rhodamine can be confirmed by 1H
NMR, a new multiplet around 9.6 ppm is assigned to ring-open of
probe 4 (Fig. S5 in supporting information).
DFT calculations were performed to understand the PET
progress of 4 at the molecular level. The HOMOs and LUMOs of
model compounds 4 and 4-H+ are showed in Fig. S10. The LUMOs
(–5.44 eV) of model compound 4 are evenly delocalized over the
rhodamine units and its HOMOs (–10.9072 eV) are mainly
delocalized over perylenediimide units. On the other hand, The
HOMOs (–8.3504 eV) of model compound 4-H+ are mainly
delocalized over rhodamine units and its LUMOs (–2.2848 eV)
are mainly delocalized over perylenediimide unit. Meanwhile, in
order to understand the PET process, three model compounds 6, 7,
and 8 (Fig. 5) are used for DFT calculations. The Optimized
geometries and calculated HOMO and LUMO density maps are
showed in Fig. 6. The calculated HOMOs of compounds 7 and 8 are
law compared with to that of compound 6. These results reflect
that the energy level of PDI units in probe 4 in acid environment
changes higher than the HOMOs of PDI units in probe 4 in neutral
influenced by the coexisting metal ions such as Hg2+, Na+, K+, Ca2+
,
272
Mg2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, Pd2+, Pb2+, Fe2+, Mn2+, and Al3+ 273
ions. Meanwhile, the fluorescence spectra of probes 4 and 5 to the 274
mixture of various metal salts were showed in Fig. 8. The results 275
indicate that probes 4 and 5 have high selectivity to protons in the 276
presence of other metal ions.
277
3.5. Reproducibility, reversibility, and response time
278
In order to study the response time, and reversibility of the 279
sensors, the reversible nature of the sensors were examined by 280
recording the ratio of fluorescence intensity at 550 nm with 281
respect to the change of pH from acidic (pH 2.0) to alkaline (pH 7.8) 282
range and vice versa up to 7 cycles. Fig. 9 shows the fluorescence 283
intensity change with time upon switching from one solution to 284
the other. The relative standard deviations from seven measure- 285
ments for blank solution of pH 7.0 was found to be 0.1% and the 286
relative standard deviations in fluorescence intensities recorded 287
from five replicates of pH 2.0 was estimated as 1.1%. The response 288
time were 5–8 s for probes of pH 2.0, meanwhile, it was found that 289
the recovering time was independent of the H+ concentration 290
change. Results indicate the reversibility between the protonated 291
Q2
Fig. 6. Optimized geometries and calculated HOMO and LUMO density maps for model compounds according to DFT calculations at B3LYP/6–31* level.
4-H+- other ions
5-H+- other ions
1.8
1.2
1.5
1.2
0.9
0.9
0.6
0.6
0.3
0.3
0.0
0.0
Hg2+
Cu
Hg2+
Cu
2+
2+
2+
2+Mn2M+ g2+
4-H+
2+Na+
Pb
2+Ag+
3+ Pd
Ni
2+Ag+
Cd
2+
Co
2+
2+
2+
Al3+
2+Na+
Zn
Mn2+Mg2+ 2+Al3+5-H+
Pb
3+ Pd
2+
Cd
Co
Ni
Zn
Cr
Cr
Metal cations
Metal cations
Fig. 7. Fluorescence responses of compounds 4 and 5 to various metal salts of metal cations, such as K+, Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, Al3+, and Co2+ (4 eq.).
Please cite this article in press as: H.-R. Cheng, Y. Qian, Two novel rhodamine-perylenediimide fluorescent probes: Synthesis,
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H.-R. Cheng, Y. Qian / Chinese Chemical Letters xxx (2016) xxx–xxx
2.0x106
Compound 4
Compound 5
1.6x106
1.2x106
8.0x105
1.2×106
pH 1.0
pH 1.2
9.0×105
6.0×105
2+ 3+
2+
2+ 2+
Hg2+, Fe3+, Zn2+, Co2+, Ni2+,
Al3+,Cr3+ and other metal ions
Hg , Fe , Zn , Co , Ni
,
3+ 3+
4.0x105
0.0
Al ,Cr and other metal ions
3.0×105
500
550
600
650
700
750
800
500
550
600
650
700
750
800
λ (nm)
λ (nm)
Fig. 8. Fluorescence responses of probes to the mixture of various metal salts, such as K+, Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, Al3+, and Co2+ (4 eq.).
×106
×106
1.5
1.2
0.9
0.6
0.3
0.0
Compound 5
Compound 4
2.0
1.5
1.0
0.5
0.0
pH 2.2
pH 2.0
pH 7.8
pH 7.8
800
0
400
1200
1600
0
300
600
Time (sec)
900
1200
1500
Time (sec)
Fig. 9. The ratio of fluorescence intensity of probes at 550 nm (c = 10
mmol/L, lex = 480 nm, in DMSO/H2O, 1:9) upon consecutive addition of HCl (1 mol/L) and NaOH (1 mol/L)
solution up to seven cycles.
292
293
and deprotonated forms of the sensors. Thus the sensors might be
applicable for real time pH monitoring.
incubated again for 30 min at 37 8C, and washed with a PBS
buffer. After treatment with pH 5.8 in the RPMI-1640 medium,
confocal fluorescence images were studied, the cells show
enhanced red fluorescence emission as shown in Fig. 11-B and
D. These results suggested that probes 4 and 5 are effective dual-
switch fluorescent pH probes intracellular imaging.
314
315
316
317
318
319
294
3.6. Long-time stability and lifetime
295
296
297
298
299
300
301
302
To investigate the short-term stability of the sensors, the
fluorescence intensity of the sensors exposed to solution of pH
2.0 and 7.8 were tested over a period of seven days. The
fluorescence emission intensities were recorded every 24 h. As
shown in Fig. 10, the relative standard error of < 1% was obtained
for the solution. The solution exhibits good stability and has a
lifetime of at least one month. Thus, the probes have remarkably
long lifetime.
×106
Compound 4 pH 2.2
1.8
Compound 5 pH 2.0
1.2
303
3.7. Confocal fluorescence images
304
305
306
307
308
309
310
311
312
313
HeLa cell was cultured in media (RPMI 1640 supplemented
with 10% PBS, 100 units/mL of penicillin and 100 units/mL of
streptomycin) at 37 8C in a humidied incubator, and culture
media were replaced with fresh media every day. The utilities of
probes 4 and 5 in living cells were studied. The HeLa cell lines
0.6
Probes pH 7.8
0.0
were incubated with receptor probes [1.0
mmol/L in DMSO/H2O
1
2
3
4
5
6
7
(1:9, v/v) buffered with HEPES, pH 7.0] in a RPMI-1640 medium
for 1 h at 37 8C and washed with a phosphate-buffered saline
(PBS) buffer (pH 7.2) to remove excess receptor 4 or 5. The cells
were then treated with pH 5.8 in the RPMI-1640 medium,
Time (day)
Fig. 10. The time courses of fluorescence intensity of probes in buffers of various pH
values (7.8, 2.2, and 2.0, respectively) ex = 480 nm, em = 550 nm.
l
l
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G Model
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H.-R. Cheng, Y. Qian / Chinese Chemical Letters xxx (2016) xxx–xxx
7
Fig. 11. Confocal fluorescence images of Hela cells incubated with 1
mmol/L probes 4 (Fig. A and B) and 5 (Fig. C and D) for 30 min (pH 5.8, Fig. B and D: fluorescence image; pH
7.2, Fig. A and C: photos in bright field).
[6] J. Hynes, T.C. O’Riordan, A.V. Zhdanov, et al., In vitro analysis of cell metabolism 367
using a long-decay pH-sensitive lanthanide probe and extracellular acidification 368
320
4. Conclusion
assay, Anal. Biochem. 390 (2009) 21–28.
369
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In summary, two novel pH fluorescent chemosensors based on
rhodamine-perylenediimide have been designed, synthesized, and
fully characterized by 1H NMR, 13C NMR, and HRMS-MALDI-TOF.
The dual-switches of the sensors were based on the structural
transformation of rhodamine units and an intramolecular photo-
induced electron transfer (PET) process that occurred with the
switching effects of rhodamine units in the probes. These probes
showed excellent fluorescent sensitivity for protons with en-
hanced emission from 550 to 580 nm. The fluorescence changes of
probes 4 and 5 were reversible within a wide range of pH values
from 2.0 to 11.0. Furthermore, the sensors exhibited short response
time, long lifetime, and high selectivity toward protons in the
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Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Zn2+, Cd2+, Ba2+, and Pb2+. The possible
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According to the experiments of confocal laser scanning micros-
copy, compounds 4 and 5 could be used to detect the acidic pH
variations in living cells with an effective dual-switches fluores-
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Acknowledgments
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343
344
345
346
347
348
We sincerely acknowledge Miss. H.L. Liu and Mr. Y. Yang for the
confocal fluorescence images. This work was financially supported
by the Fundamental Research Funds for the National Natural
Science Foundation of China (No. 61178057) and for the Central
Universities (No. CXLX12_0085). The characteristic data of
compounds were in the Supplementary information.
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Appendix A. Supplementary data
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photophysical properties, and cell imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.01.039