Sensitive detection of strong acidic condition by a novel rhodamine-based fluorescent pH chemosensor

Article abstract of DOI:10.1002/bio.3043

A novel rhodamine-based fluorescent pH probe responding to extremely low pH values has been synthesized and characterized. This probe showed an excellent photophysical response to pH on the basis that the colorless spirocyclic structure under basic condit

Full text of DOI:10.1002/bio.3043

Research article
Received: 12 July 2015,
Revised: 24 August 2015,
Accepted: 2 September 2015
Published online in Wiley Online Library: 15 October 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.3043
Sensitive detection of strong acidic condition
by a novel rhodamine-based fluorescent pH
chemosensor
Jia-Lian Tan,a Ting-Ting Yang,a Yu Liu,a Xue Zhang,a Shu-Jin Cheng,a
Hua Zuoa* and Huawei Heb*
ABSTRACT: A novel rhodamine-based fluorescent pH probe responding to extremely low pH values has been synthesized
and characterized. This probe showed an excellent photophysical response to pH on the basis that the colorless spirocyclic
structure under basic conditions opened to a colored and highly fluorescent form under extreme acidity. The quantitative
relationship between fluorescence intensity and pH value (1.75–2.62) was consistent with the equilibrium equation
pH = pKa + log[(Imax – I)/(I – Imin)]. This sensitive pH probe was also characterized with good reversibility and no interac-
tion with interfering metal ions, and was successfully applied to image Escherichia coli under strong acidity. Copyright ©
2015 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: extreme acidity; rhodamine-derived probe; pH; E. coli
body from ingested microorganisms (20). The dilemma of the
Introduction
detection of precise pH values in the cellular compartments
Intracellular pH plays an essential and sophisticated role in many
above prompts the development of novel fluorescent probes
cellular events, such as cellular metabolism (1,2), proliferation (3),
that can be applied to accurately monitor the acidic pH values
signal transduction (4), chemotaxis (5), apoptosis (6) and autoph-
in microorganisms.
agy (7). Monitoring pH changes inside living cells, therefore, con-
tributes to exploring cellular functions and understanding
physiological and pathological processes in organisms.
Rhodamine derivatives have been widely used as ‘turn-on’ fluo-
rescence probes (23–28) due to their unparalleled photophysical
properties, such as high quantum yields, relatively long absorption
Among the developed methods for measuring the intracellular
and emission wavelengths in the visible region, large extinction
pH like microelectrodes, nuclear magnetic resonance (NMR),
coefficients and great photostability. The spirocyclic structure in
absorbance spectroscopy and fluorescence spectroscopy, fluores-
rhodamine derivatives plays a major role in the response to the
cence techniques have attracted the most attention due to their
change of solvent pH value. It remains the spirocyclic form that is
high selectivity and sensitivity. We have noticed that some fluores-
cent pH probes were developed and even commercially available,
non-fluorescent and colorless at high pH (pH ≥7.0), while the
ring-opened form exhibits strong fluorescence and a pink color
such as green fluorescent protein and micromolecular pH sensors
at very low pH (29). Given that rhodamine derivatives are used
(8) derived from benzoxanthenes, cyanine (9), fluorescein (10) and
to measure acidic pH values, we designed and synthesized a
others (11,12). However, these fluorescent pH sensors are only
applicable to aqueous solutions in the pH range 4–9 (13–18) and
it remains challenging to detect the very low intracellular pH.
Challenges to develop fluorescent sensors that respond to
extreme acidity are ascribed to the weak fluorescence and
novel fluorescent pH-sensitive probe L constructed from rhoda-
mine hydrazine, carbonylethylene and 1-phenylpiperazine, this
chemical instability of fluorescein or coumarin under strongly
* Correspondence to: H. Zuo, College of Pharmaceutical Sciences, Southwest
acidic conditions, the loss of selectivity due to the coordination
with interfering metal ions of those reported pH-sensitive
fluorescent probes containing carboxyl or hydroxyl functional
groups and the lack of linear relationship between fluorescence
intensity and pH due to the incorporation of too many receptors
(19). In spite of the fact that most living species could hardly sur-
vive in extremely acidic environments, certain microorganisms,
such as Helicobacter pylori and ‘acidophiles’ favor such harsh
conditions (20–22). The stomach, the strongly acidic organ of
mammals, even contains gastric juices with acidity pH 1.5–3.0,
thereby activating digestive enzymes and also protecting the
University, Chongqing 400715, China.
E-mail: zuohua@swu.edu.cn
* H. He, State Key Laboratory of Silkworm Genome Biology, Southwest University,
Chongqing 400715, China.
a
College of Pharmaceutical Sciences, Southwest University, Chongqing
400715, China
b
State Key Laboratory of Silkworm Genome Biology, Southwest University,
Chongqing 400715, China
Abbreviations: ICT, intramolecular charge transfer; MS, mass spectra; NMR,
nuclear magnetic resonance; TLC, thin-layer chromatography.
Luminescence 2016; 31: 865–870
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J.-L. Tan et al.
probe exhibited much higher fluorescent intensity and quantum
yield than that of certain probes working under strong acidic
conditions (6,30–33). Compared with the sensor reported by
our group (34), the probe is less prone to being interfered by
coexisting metal ions due to the removal of the fluorine atom
from the phenylpiperazinyl moiety.
Synthesis of probe L
The synthetic route of probe L is outlined in Scheme 1.
Chloroacetyl chloride (9.50 mmol, 2.0 equiv) was added dropwise
to the solution of rhodamine hydrazide 2 (4.75 mmol, 1.0 equiv)
(35) in 70 mL dichloromethane and the reaction mixture was
then stirred for 10 h at room temperature, and excess
triethylamine (47.50 mmol, 10.0 equiv) was added slowly into
the solution. After completion of the reaction as indicated by
TLC, 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 (v/v) to
Experimental
Materials
Deionized water was used throughout the experiments. All re-
agents were purchased from commercial suppliers and used with-
out further purification. All samples were prepared at room
temperature. Solvents were dried and purified by known conven-
tional methods. The solutions of metal ions were prepared from
nitrate salts, which were dissolved in deionized water. Britton-
Robinson (B-R) buffer was a mixture of 40mM acetic acid, boric
acid and phosphoric acid. Dilute hydrochloric acid or sodium hy-
droxide was used for tuning pH values.
give the intermediate 3. The solution of compound
3
(0.43 mmol, 1.0 equiv) and 1-phenylpiperazine (0.99 mmol, 0.7
equiv) was then heated in refluxing acetonitrile (18mL) and
generated the desired product in the presence of potassium
carbonate (2.3mmol, 2.0 equiv). The recrystallization from aceto-
nitrile (20 mL) afforded probe L as white solid in 82.1% yield.
m.p. 214.0–216.0 °C.
Instruments
Melting point (uncorrected) was determined on a micromelting
point apparatus (Shanghai Shenguang Instrument Co., Ltd., China).
1H and 13C NMR spectra (at 400 MHz and 100 MHz, respectively)
were recorded in CDCl3 with tetramethylsilane as internal reference
on a Bruker Advance 500 FT spectrometer. Chemical shifts were
reported in parts per million. Mass spectra (MS) were measured
by the electrospray ionization (ESI) method on an Agilent 6510
Q-TOF mass spectrometer. CDCl3 delivered from Adamas Co., Ltd.
(Shanghai, China) was used. Aluminium oxide (100–200 mesh)
was used for flash column chromatography. All reactions were
monitored by thin-layer chromatography (TLC) using 0.25 mm silica
gel plates with an ultraviolet (UV) light indicator (Shanghai Jiapeng
Technology Co., Ltd., China). UV–vis spectra were recorded on a
UV-2550 spectrometry (Hitachi, Japan). Fluorescence was measured
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).
Figure 1. The fluorescence spectrum of L (25 μM) in solution (buffer:EtOH, v/v = 1:1)
at different pH, λex = 561 nm.
Scheme 1. The synthetic route for probe L. Reagents and conditions: (a) ethanol, reflux, 24 h; (b) Et3N, CH2Cl2, r.t., 24 h; (c) K2CO3, CH3CN, reflux, 3 h.
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rhodamine-based fluorescent pH chemosensor for strong acidic condition
Bacteria culture and imaging
subsequently opened to form a conjugated xanthene ring
(Scheme 2), which was similar to the reported ring-opening pro-
cess (36–39) .
E. coli cells were cultured at 37 °C in Luria-Bertani culture (tryptone
10 g/L, yeast extract 5 g/L, NaCl 10 g/L) in a table concentrator
(AO HUA, ZD-85, China) shaking for 5 h at 180 rpm. The culture
was then centrifuged (Heal Force TGL-16 M, China) at 10 000rpm
for 5 min and the obtained sediment was resuspended with B-R
buffer at pH1.75, 2.30 or 4.83, respectively. The probe dissolved
in ethanol was then added into each buffer, at a final probe con-
centration of 25μM after resuspension. The E. coli cells were incu-
bated with the probe for 1 h, washed with deionized water and
smeared onto slides. The obvious color change in E. coli cells was
captured at 555 nm wavelength under laser confocal microscopy
(Carl Zeiss lsm-700, Germany).
The fluorescence intensity also showed a linear response to pH
values between 1.75 and 2.62 with the function Y = À3008.96 pH
+ 8442.80 and R2 = 0.9909, as shown in Fig. 2. This function further
allowed us to calculate the pH value of any sample with pH ranged
from 1.75 to 2.62. Therefore, probe L could be used as a highly sen-
sitively colorimetric and fluorescent pH indicator.
Results and discussion
Synthesis of probe L
Scheme 1 illustrates the general synthetic route for probe L. Firstly,
rhodamine B (1) was reacted with hydrazine hydrate and cyclized
to form the five-member ring in rhodamine hydrazide 2, which
then nucleophilically attacked the electron-deficient carbonyl of
chloroacetyl chloride. The obtained amide 3 underwent N-
alkylation with 1-phenylpiperazine to afford the probe L, which
1
was characterized by H NMR, 13C NMR (Fig. S3, Fig. S4, Fig. S5)
and HRMS. 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 7.97 (d,
J= 6.4Hz, 1H), 7.54–7.46 (m, 2H), 7.23 (d, J = 8.0Hz, 2H), 7.13 (d,
J= 6.5Hz, 1H), 6.88–6.83 (m, 3H), 6.65 (d, J = 8.7Hz, 2H), 6.33–6.28
(m, 4H), 3.30–3.24 (m, 8H), 3.04 (s, 2H), 2.92 (s, 4H), 2.51–2.44 (m,
4H), 1.08 (t, J= 7.0Hz, 12H). 13C NMR (101 MHz, CDCl3) δ 167.5,
165.3, 153.6, 151.5, 151.3, 149.0, 133.2, 129.5, 129.4, 129.0, 128.4,
124.1, 123.5, 120.0, 116.3, 108.2, 104.1, 97.4, 66.1, 60.4, 53.2, 49.5,
44.3, 31.0, 12.6. ESI-HRMS m/z: calcd for [M + H]+ C40H47N6O3+:
659.3710, found: 659.3713.
Figure 2. Linear regression based Intensity versus pH value. Equation: Y = À3008.96
pH + 8442.80, R2 = 0.9909 (The data are the same with those in Fig. 1).
Spectroscopic properties and optical responses to pH
It was observed that the fluorescence intensity increased dramati-
cally when the pH value of the buffer was decreasing from 3.06 to
1.75, in marked contrast to the slight increase of fluorescence in-
tensity in the buffer ranging from basic to weakly acidic condition
(Fig. 1). Simultaneously, the obvious pink color appeared under
highly acidic condition, which was consistent with the proposed
ring-opening mechanism of the probe. Specifically, the alkyl nitro-
gen atom of the piperazine was protonated upon the addition of
trifluoroacetic acid and with the further protonation of the amide
nitrogen atom in the spirolactam ring, the spirolactam ring
Figure 3. Absorption spectra of L (25 μM) in solution (buffer:EtOH, v/v = 1:1) with dif-
ferent pH.
Scheme 2. Proposed ring-opening mechanism of probe L.
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J.-L. Tan et al.
As illustrated in Fig. 3, the UV–vis absorption intensity increased
upon the acidification of the neutral solution of probe L, especially
under extremely acidic conditions (from pH 3.01 to 1.75), in agree-
ment with the trend in fluorescence intensity. When the pH value
varied from 3.01 to 1.75, both the absorbance at 560nm and the
fluorescence intensity at 575 nm were remarkably increased. How-
ever, the more significant increasing tendency in the fluorescence
spectra than in the absorption spectra permitted us to efficiently
evaluate pH values through fluorescence spectra.
To investigate the reversibility of the fluorescence intensity ver-
sus pH, we changed the pH value of the buffer for the probe L from
2.25 to 7.15 by slowly adding NaOH solution and reversely acidify-
ing the solution from 7.15 to 2.25 through HCl solution. The corre-
sponding fluorescence intensity was detected and depicted in
Fig. 4. The fluorescence intensity decreased dramatically from
about 1600 to less than 250 with the neutralization of the
strongly acidic buffer, accompanied by the disappearance of
the noticeable pink color, which resulted from the conversion
from the spirolactam form existing predominantly in neutral
buffered media to the ring-opened amide structure upon the
acidification of the solution. Despite the reversible changes of
pH value between 2.25 and 7.15, the figures for fluorescence
intensity remained nearly the same level at the same pH.
The probe L responded to extreme acidity quickly (less than
1 min) as illustrated by the time course of fluorescence intensity
(Fig. S1). Since various metal ions, including Zn2+, Ni2+, Na+, Mn2+,
Mg2+, Li+, K+, Hg2+, Fe3+, Cu2+, Ba2+, Cs+, Al3+, Ag+, Co2+, or Ca2+
could not coordinate with the probe, they proved not to affect
the pH-sensing properties of L (Fig. S2), which guaranteed the
successful application of the probe L in complex ionized solutions.
pH bioimaging in live E. coli
The images of E. coli cells were captured by scanning confocal mi-
croscopy to investigate the ability of the probe to monitor pH
value inside living cells. To simulate the extremely acidic
Figure 4. The fluorescence intensity at 575 nm of probe L (25 μM) between pH 2.25
and 7.15 in B-R buffer (excitation wavelength: 561 nm).
Figure 5. Imaging acidity in E. coli cells with probe L. (a–c) pH = 1.75; (d–f ) pH = 2.30; (g–i) pH = 4.83. (a, d, g) are the fluorescence images; (b, e, h) are bright field images; (c, f, i)
are merged images (scale bar: 5 μm).
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rhodamine-based fluorescent pH chemosensor for strong acidic condition
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Conclusions
In summary, we have developed a novel fluorescent pH probe L
based on rhodamine B with excellent selectivity and sensitivity to
extremely acidic conditions. Apart from the strong selectivity due
to no interference with the coexisted metal ions, good reversibility
and short response time (<1 min) are also valuable characters of
the probe. The fluorescence 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.
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We would like to thank the Technology and Program Project of the
Science and Technology Department of Sichuan Province
(2015FZ0070) and the Fundamental Research Funds for the
Central Universities (SWU112111, XDJK2013A019) for financial
support.
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