A novel "off-on" colorimetric and fluorescent rhodamine-based pH chemosensor for extreme acidity

Article abstract of DOI:10.1016/j.saa.2014.12.110

A novel "off-on" colorimetric and fluorescent rhodamine analogue was synthesized and characterized, and used to monitor extreme acidity (below pH 3.5) via the photophysical response to pH. The colorless spirocyclic structure at high pH (pH ≥ 7.0) opened t

Full text of DOI:10.1016/j.saa.2014.12.110

Accepted Manuscript
A novel “off-on” colorimetric and fluorescent rhodamine-based pH chemosen-
sor for extreme acidity
Jia-Lian Tan, Mu-Xue Zhang, Fang Zhang, Ting-Ting Yang, Yu Liu, Zhu-Bo
Li, Hua Zuo
PII:
S1386-1425(14)01898-8
http://dx.doi.org/10.1016/j.saa.2014.12.110
SAA 13160
DOI:
Reference:
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
7 September 2014
14 December 2014
28 December 2014
Please cite this article as: J-L. Tan, M-X. Zhang, F. Zhang, T-T. Yang, Y. Liu, Z-B. Li, H. Zuo, A novel “off-on”
colorimetric and fluorescent rhodamine-based pH chemosensor for extreme acidity, Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2014.12.110
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A novel “off-on” colorimetric and fluorescent rhodamine-based
pH chemosensor for extreme acidity
a
Jia-Lian Tan , Mu-Xue Zhang , Fang Zhang , Ting-Ting Yang , Yu Liu , Zhu-Bo Li * and Hua
b
a
a
a
a,
a,
Zuo *
a
College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
b
College of Resources and Environment, Southwest University, Chongqing 400715, China
Corresponding author
Prof. Zhu-Bo Li and Hua Zuo
College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
Tel: +86-23-6825-1225; Fax: +86-23-6625-1225; E-mail: zb63@163.com; zuohua@swu.edu.cn.
1

Abstract
A novel “off-on” colorimetric and fluorescent rhodamine analogue was synthesized and
characterized, and used to monitor extreme acidity (below pH 3.5) via the photophysical response
to pH. The colorless spirocyclic structure at high pH (pH ≥7.0) opened to the colored and highly
fluorescent form at very low pH (pH<3.0). This sensitive pH probe was characterized with short
response time, good reversibility and no interaction with interfering metal ions, and
the quantitative relationship between the fluorescence intensity and pH value was consistent with
the equilibrium equation pH = pKa-log[(Imax-I)/(I-Imin)]. The fluorescent response to strong
acidity was further verified by fluorescent imaging of bacteria, E.coli, which contributed to the
development of more useful colorimetric and fluorescent sensors based on the rhodamine platform
for measuring intracellular pH in extremely acidic conditions.
Keywords
Rhodamine analogue; Fluorescence chemosensor; Strong acidity; E. coli.; Imaging
2

1.
Introduction
Intracellular pH plays a crucial role in numerous cell physiological processes, including cellular
[
growth , proliferation , apoptosis , phagocytosis , cellular metabolism
1]
[2]
[3]
[4]
[5,6]
and signal
[7]
transduction . Given these intimate connections between cellular events and internal pH of living
cells, the accurate measurement of pH inside living cells provides an access to the critical
information for understanding the physiological and pathological processes in organisms.
[8,9]
Small fluorescent organic molecules, nanoparticles , and fluorescent proteins, e.g. GFP, have
[
10]
,
been developed to measure intracellular pH since 1980s, among which benzoxanthene dyes
[11,12]
fluorescein and fluorescein derivatives
[13]
cyanine-based pH indicators
and rhodamine
derivatives are well known molecular fluorescent sensors. Nevertheless, it is still challenging to
monitor the intracellular pH in extremely acidic conditions since the majority of the existing
[14-16]
[17-19]
probes can only indicate neutral pH or subacid pH, ranging from 6 to 8
and 4 to 6
,
respectively. In spite of the fact that the extreme acidity (pH<4) is fatal for most living species,
certain microorganisms, such as helicobacter pylori and “acidophiles” function well under such
[20]
harsh condition . For another example, enteric pathogens are acidic organelles able to reach
small intestine by passing through the highly acidic mammalian stomach, resulting in
[
20, 21]
life-threatening infections
. Stomach, the strongly acidic organ of mammals even contains
gastric juices with acidity pH 1.5–3.0. This very acidic environment activates digestive enzymes
and also serves to protect the body from ingested microorganisms by acting as a barrier for
[20]
bacteria . Abnormal pH ranges interfere with the function of stomach, which results directly in
stomach disorders.
Certain molecular fluorescent probes working for strong acidic condition have been developed
in recent years [22–24], however, some of them suffer from several drawbacks, such as a narrow
working range and lower quantum yield. Developing new fluorescent sensors that can be used to
accurately detect extremely acidic pH in microorganisms is, therefore, increasingly important and
pressing.
Herein, we designed and synthesized a novel rhodamine-based “off-on” colorimetric and
fluorescent probe, which is excellently sensitive and selective to strongly acidic conditions and
can be applied to image the extremely acidic bacteria.
2. Experimental
3

2.1. Chemicals and apparatus
All of the reagents were obtained from commercial sources. Solvents were dried and purified
with known conventional methods. The solutions of metal ions were prepared from nitrate salts
which were dissolved in deionized water. Britton-Robinson (B-R) buffer was mixed by 40 mM
acetic acid, boric acid, and phosphoric acid. Dilute hydrochloric acid or sodium hydroxide was
used for tuning pH values. Unless otherwise noted, other reagents were obtained from commercial
suppliers and used without further purification.
Melting point (uncorrected) was determined on a micro melting point apparatus (Shanghai
1 13
Shenguang Instrument Co., Ltd., China). H and C NMR (nuclear magnetic resonance) spectra
(
at 400 MHz and 100 MHz, respectively) were recorded in CDCl with tetramethylsilane as
3
internal reference on a Bruker Advance 500 FT spectrometer. Chemical shifts were reported in
parts per million. Mass spectra (MS) were measured by the ESI method on an Agilent 6510
3
Q-TOF mass spectrometer. CDCl was used as delivered from Adamas Co., Ltd. (Shanghai,
China). Aluminium oxide (100–200 mesh) was used for flash column chromatography. All
reactions were monitored by TLC (thin-layer chromatography) using 0.25 mm silica gel plates
with UV indicator (Shanghai Jiapeng Technology Co., Ltd., China). UV-vis spectra were recorded
on a UV-2550 spectrometry (Hitachi, Japan). Fluorescent measurements were recorded on a
F-4500 FL spectrophotometer (Hitachi, Japan). The pH measurements were performed on a
PHS-3C digital pH-meter (Mettler Toledo, Switzerland). Images of E. coli cells were captured
with a laser confocal microscope (Carl Zeiss LSM-700, Germany).
2.2. Synthesis of probe L
The synthetic route of probe L is shown in Scheme 1. To the solution of rhodamine hydrazide 2
[
25]
(
4.75 mmol, 1.0 eq) in 70 mL dichloromethane, was added chloroacetyl chloride (9.50 mmol,
2.0 eq) dropwise. The reaction mixture was stirred at room temperature for 10 h and then excess
triethylamine (47.50 mmol, 10.0 eq) was added slowly into the solution. When the reaction was
completed the solvent was evaporated and the obtained solid was washed by 30 mL deionized
water, dried and purified by column chromatography (elute: ethyl acetate: petroleum=1: 6) to give
the intermediate 3, after which, the solution of compound 3 (0.43 mmol, 1.0 eq),
1-(2-fluorophenyl)piperazine (0.43 mmol, 1.0 eq) and potassium carbonate (0.86 mmol, 2.0 eq) in
acetonitrile (8 mL) was heated to reflux. After completion of the reaction as indicated by TLC,
4

acetonitrile was removed under vacuum, and the crude product was purified by recrystallization in
ethyl acetate. The target probe L was obtained as white solid in 85.7% yield. mp 230.0–231.8°C.
1
H NMR (CDCl
H), 7.06–6.98 (m, 2H), 6.97–6.90 (m, 1H), 6.84 (t, J = 8.3 Hz, 1H), 6.69–6.63 (m, 2H), 6.31 (dd,
J = 6.0, 2.5 Hz, 4H), 3.36–3.19 (m, 8H), 3.04 (s, 2H), 2.83 (s, 4H), 2.49 (t, J = 4.3 Hz, 4H), 1.08 (t,
3
) δ: 8.38 (s, 1H), 7.97 (dd, J = 6.2, 2.0 Hz, 1H), 7.55–7.45 (m, 2H), 7.15–7.11 (m,
1
1
J = 7.0 Hz, 12H) ppm; C NMR (CDCl
3
3
) δ: 12.6, 44.3, 50.7 (d, J = 3.1 Hz ), 53.1, 60.4, 66.1,
9
1
1
2
7.4, 104.1, 108.2, 116.1 (d, J = 20.9 Hz), 118.9 (d, J = 2.9 Hz), 122.5 (d, J = 7.9 Hz), 123.5,
24.1, 124.3 (d, J = 3.5 Hz), 128.4, 129.4, 129.5, 133.1, 140.0 (d, J = 8.6 Hz), 149.0, 151.5, 153.6,
+
: 677.3537 [M + H] ).
65.3, 167.5 ppm. ESI-HR-MS m/z: 677.3618 (Calcd for C40
H
45
N
6
O
3
.3. Bacteria culture and imaging
E. coli was incubated at 37°C in Luria-Bertani (LB) culture (Trptone 10 g/L, yeast extract 5 g/L,
NaCl 10 g/L) in a table concentrator (AO HUA, ZD-85, China) at 180 rpm for 5 h, and the culture
was centrifuged (Heal Force TGL-16M, China) in 10 mL Eppendorf tubes at 10000 rpm for 5 min.
The sediment was then resuspended with B-R buffer at pH 1.70, 2.95, 6.35, respectively. The pH
probe dissolved in ethanol was added into each tube, with a final concentration at 25 uM after
resuspension. E. coli cells with the probe were then incubated in the table concentrator as
mentioned above for 30 min, washed by deionized water and smeared on slides. The color
changes of E. coli cells were observed at the wavelength of 555 nm under laser confocal
microscopy (Carl Zeiss LSM-700, Germany).
3. Results and discussion
3.1. Synthesis of probe L
The synthetic route of probe L is given in Scheme 1. Rhodamine B was reacted with hydrazine
hydrate and cyclized to form the rhodamine hydrazide 2 with a five-member ring, which then
nucleophilically attacked the electron-deficient carbonyl of chloroacetyl chloride. The obtained
amide 3 underwent N-alkylation with 1-(2-fluorophenyl)piperazine to afford the probe L, which
1 13
was characterized by H NMR, C NMR and HRMS spectra (Fig S8, Fig S9, Fig S10).
5

Scheme 1. The synthetic route of probe L. Reagents and conditions: (a) ethanol, reflux, 24 h; (b)
Et N, CH Cl , r.t., 24 h; (c) K CO , CH CN, reflux, 3 h.
3
2
2
2
3
3
3.2. Spectroscopic properties and optical responses to pH
After slight increases of fluorescence intensity at pH values ranging from 11.00 to 4.93, the
fluorescence intensity for probe L began to dramatically rise under strongly acidic condition (pH <
.0), peaking at 4519 in the buffer at pH 1.70 (Figure 1), and a noticeable pink color appeared
Figure 2), which is, to a large extent, consistent with the ring-opening process from the
spirolactam ring in the probe L, to the structure of N-(3’,6’-bis(diethylamino)-3-oxospiro
isoindoline-1,9’-xanthen]-2-yl)-2-(4-(2-fluorophenyl)-1-yl)acetamide (Scheme 2). The
3
(
[
fluorescence intensity of L with varied concentrations of 25 uM and 10 uM at various pH values
remained the same tendency as shown in Figure 1 and Figure S6. Specifically, the xanthene ring in
3
probe L is non-conjugated as a result of the sp hybridization of the carbon at 9-position with a
1
chemical shift at 66.1 ppm in the C NMR when the buffer was basic, neutral and weakly acidic,
3
while the carbonyl was protonated at the presence of hydrogen ions, resulting in the ring opening
[
24]
.
of the spirolactam and the conjugation of xanthene ring when the pH value fell to less than 3.0
However, an unexpected red-shift (562–575 nm) in the emission maximum observed under very
acidic conditions. The quantum yield decreased from 0.97 (pH = 1.70) to 0.73 (pH = 2.96), using
rhodamine B as a standard sample.
1.70
1.91
5000
4000
3000
2000
1000
0
2
2
2
.18
.34
.37
2.68
.79
2.84
2
L
pH1.7
2.95
3.24
3.52
3.65
3.81
4.10
4.93
5.67
6.35
7.35
8.50
9.21
9.86
1.00
1
480
500
520
540
560
580
600
620
640
660
Wavele6ngth (nm)

Figure 1. The fluorescence spectrum of L (25 µM) in solution (buffer-EtOH, v/v = 1:1) with
different pH, λex = 562 nm.
pH=1.70 pH =1.91 pH=2.23 pH=2.95 pH =3.52 pH=4.10 pH =4.93 pH =5.67 pH =6.30 pH =7.35 pH =8.50 pH=9.21 pH =9.86 pH =12.56
L
Figure 2. The color change of probe L (25 uM, buffer-EtOH, v : v = 1:1 ) at different pH.
A sharp decrease was observed for the fluorescence intensity at 575 nm when pH value
increased from 1.70 to 4.10 according to the fluorescent pH titration (pH 1.70–11.0, λex = 562 nm,
Figure 3) and this relationship can be quantified and fit well to the Hendersone–Hasselbach-type
[
26]
:
mass action equation as follow
pKa = pH-log[(Imax-I)/(I-Imin)]
where I is the observed fluorescence intensity at a fixed wavelength, Imax and Imin are 4519 and
95.7, respectively. According to the equation, the pKa value of the probe L was 3.4, suitable for
3
[
27]
the study on extremely acidic organelles . The fluorescence intensity log[(Imax-I)/(I-Imin)] also
showed a linear response to pH values between 1.70 and 4.10 in accordance with the function, Y =
2
Y = 1.4034X-3.4189, R = 0.9911, as shown in Fig S1. This function further allows us to calculate
the pH value of any sample with pH ranged from 1.70 to 4.10 based on their fluorescent intensity.
5000
4000
3000
2000
1000
0
0
2
4
6
8
10
12
pH
Figure 3. The fluorescence intensity at 575 nm versus pH at different pH, λex=562 nm. (The data
are the same as the Fig 1).
7

In exploring the reversibility of fluorescence versus pH, we firstly neutralized the acidic
solution of the probe at pH 2.18 and then acidified it to pH 2.18, and we found that the
fluorescence rose to the initial intensity and the solution again appeared pink, which indicated
+
that the coordination between L and L+H is excellently reversible. The effects of the
environmental factors (air, light, humidity) and various metal ions on the pH-sensing properties
of L also are proved to be negligible, since these metal cations did not coordinate to the probe L,
as we expect, to form the conjugated amide structures (Fig S3, Fig S4).
In agreement with the tendency in fluorescence intensity, the absorption intensity increased
+
significantly upon the addition of H to the neutral solution of probe L, especially under extremely
acidic condition (Figure 4, Figure S7). It was thus inferred that the spirolactam form existed
predominantly in neutral buffered media, whereas the ring-opened amide structure was formed
upon acidification of the solution. However, the more significantly rising tendency in the
fluorescence spectra than in the absorption spectra allowed us to efficiently evaluate pH values
through fluorescence spectra.
1
.70
.86
1.91
.18
2.34
.37
2.68
.79
2.84
1
0
.18
2
0.16
2
0.14
2
0.12
3.03
3.24
3.52
3.65
3.81
3.96
4.88
5.69
6.55
8.50
0
.10
0.08
0.06
0.04
0.02
450
500
550
600
650
Wavelength (nm)
Figure 4. Absorption spectra of L (25 µM) in solution (buffer-EtOH, v/v = 1:1) with different pH.
3.4 Mechanism for ring-opening
Generally, the transformation from spirolactam into ring-opening form explains the mechanism
[28-30]
for the fluorescence change of rhodamine derivatives
and this also works for probe L
1
according to the H NMR data (Figure 5). The stabilized spirolactam structure at the high pH
value (pH ≥ 7.0) quickly opened to a conjugated form (ring-opening form, Scheme 2) and the
electron cloud density of xanthene was changed after 1.0 equivalent of trifluoroacetic acid (TFA)
8

was added, with the appearance of a new active proton and down-field shifts for xanthene protons
H1, H2, H3), methylene near the amide (H13), the active proton (H12) and methylenes in the
(
piperazine (H15, H17), while no obvious shift was observed when the TFA was increased to 2
equivalents. The protons of xanthene, the methylenes near the amide and the methyl, methylenes
in the piperazine and both active protons further shifted to down-field upon the addition of 5.0
equivalents of TFA, because the N4, N14 and N16 atoms were all protonated.
Scheme 2. The proposed spirolactam ring-opening mechanism of probe L upon the addition of
+
H .
1
Figure 5. H NMR titration of probe L with trifluoroacetic acid (TFA) in solution of CDCl
3
; (a): L;
(
b): L+TFA (1.0 eq); (c): L+TFA (2.0 eq); (d): L+TFA (5.0 eq).
.5 pH bioimaging in E. coli
The imaging of E. coli by the probe L was studied, to investigate its ability to monitoring the
intracellular pH value. To simulate the extremely acidic environment in E. coli, buffer with pH at
.70, 2.37 and 3.85, respectively, was used to incubate bacteria. The fluorescence microscopy
3
1
subsequently captured images after the probe L was added into above system. Red fluorescence
9

was clearly observed in E. coli under acidic conditions, and the strongest fluorescence intensity
was found at pH 1.70. While with the increase of pH to 2.37 and 3.85, the intensity significantly
decreased, as determined by scanning confocal microscopy (Figure 6(a)), in accordance with the
tendency shown in the fluorescence spectrum. In addition, the quantitative analysis through
ImageJ illustrated that the fluorescence intensity gradually increased with the drop of pH (Figure
6(b)). This method allows for direct, real-time estimation of extremely low pH in biological
systems.
2
1
1
1
1
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
.8
.6
.4
.2
.0
(
b)
2
3
4
pH
Figure 6. (a). Imaging acidity in E. coli cells with probe L. a–c. pH = 1.70; d–f. pH = 2.37; g–i.
1
0

pH = 3.85; a, d, g are the fluorescence images; b, e, h are the bright field; c, f, i are merged images
Scale bar: 5 µm). (b). The quantitative analysis of fluorescence intensity in E. coli cells.
(
4. Conclusion
In summary, we described the synthesis, characterization, the optical properties and cellular
application of a new rhodamine-based probe. The probe displayed fluorescence enhancement
response to extremely low pH value and its selectivity to strong acidic environment was
remarkably high owing to no interference with the coexisted metal ions. The spirocycle in the
probe opened when protonated, with accompany by the pink color, fluorescent and ultraviolet
absorption enhancement. The fluorescent imaging of bacteria E. coli by the probe contributed to
the development of more useful colorimetric and fluorescent sensors based on the rhodamine
platform for measuring intracellular pH under extremely acidic conditions.
Acknowledgements
We would like to thank the Fundamental Research Funds for the Central Universities, P. R. China
(
XDJK2012B012 and 2362014xk07) for financial support.
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[
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(
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[
29]. M. Tian, X. Peng, J. Fan, J. Wang, S. Sun, A fluorescent sensor for pH based on rhodamine
fluorophore, Dyes Pigments. 95 (2012) 112–115.
30]. Y. Yang, S. Shim, J. Tae, Rhodamine-sugar based turn-on fluorescent probe for the detection
of cysteine and homocysteine in water, Chem. Commun. 46 (2010) 7766–7768.
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Highlights
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We designed a novel “off-on” colorimetric and fluorescent pH chemosensor.
The probe displayed strong fluorescence and absorption enhancement response to
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strongly acidic pH.
The optical property response to pH is reversible, without interference from common
metal ions and environmental factors.
The probe can be used as the indictors for the extreme acidity in E.coli.
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