Preparation of a Novel pH-responsive Fluorescent Probe Based on an Imidazo[1,2-a]indole Fluorophore and its Application in Detecting Extremely Low pH in Saccharomyces cerevisiae

Article abstract of DOI:10.1007/s10895-021-02739-8

A novel pH-responsive probe based on an imidazo[1,2-a]indole fluorophore architecture is reported. The probe was highly selective to strongly acidic pH (pK_a = 3.56) with high sensitivity and a fast response time (within 30 s). The probe did not demonstrate any fluorescence changes in the presence of interfering metal ions, and it featured excellent reversibility under strongly acidic conditions. The mechanism of detection of the probe was determined to be based on intramolecular charge transfer (ICT) at different pH. The probe was also able to be used for imaging for detecting acidic pH in Saccharomyces cerevisiae.

Full text of DOI:10.1007/s10895-021-02739-8

Journal of Fluorescence
https://doi.org/10.1007/s10895-021-02739-8
RAPID COMMUNICATION
Preparation of a Novel pH-responsive Fluorescent Probe Based on an
Imidazo[1,2-a]indole Fluorophore and its Application in Detecting
Extremely Low pH in Saccharomyces cerevisiae
Yanhao Xu1 & Ruikang Duan2 & Hao Liu1 & Chengcai Xia1 & Guiyun Duan1 & Yanqing Ge1
Received: 17 March 2021 /Accepted: 3 May 2021
#
The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
A novel pH-responsive probe based on an imidazo[1,2-a]indole fluorophore architecture is reported. The probe was highly
selective to strongly acidic pH (pKa = 3.56) with high sensitivity and a fast response time (within 30 s). The probe did not
demonstrate any fluorescence changes in the presence of interfering metal ions, and it featured excellent reversibility under
strongly acidic conditions. The mechanism of detection of the probe was determined to be based on intramolecular charge
transfer (ICT) at different pH. The probe was also able to be used for imaging for detecting acidic pH in Saccharomyces
cerevisiae.
.
.
.
.
Keywords Imidazo[1,2-a]indole pH fluorescent probe Saccharomyces cerevisiae ICT Imaging
Introduction
neurodegenerative disorders, can provoke homoestatic
pH balance in cells [6]. In addition, significant changes
in the pH in the human body can cause physiological
changes that can result in the development of cellular
metabolic disorders.
As an important parameter reflecting the acid-base
strength of a solution, pH buffering allows cells and or-
ganisms to maintain homeostasis, thereby enabling nor-
mal structure and function [1]. The pH of a normal human
body is maintained at 6.5–7.1, but the pH of different
compartments within cells varies. For example, the pH
within lysosomes and endosomes varies between 4.5 and
6.8 [2], the pH within mitochondria is approximately 8
[3], and the pH of the cytosol is maintained within 6.8–
7.4 in viable cells [4]. Changes in intracellular pH can
affect the stability of the cellular environment as well as
cellular function, such as proliferation, differentiation, ap-
optosis of cells, and ion transport; these cellular changes
can also affect tissue function, such as muscle contraction
[5]. Some diseases, such as cystic fibrosis, cancer, and
Various techniques, such as absorption spectroscopy,
electrochemistry, and nuclear magnetic resonance have
been used to measure the pH within cells [7, 8]. Offering
high sensitivity and excellent selectivity, as well as trivial
and low-cost operation, fluorescent probes have been wide-
ly employed in molecular biology, biochemistry, medicine,
and other fields [9]. To date, many small-molecule, pH-re-
sponsive fluorescent probes have been developed for mea-
suring the pH in acidic organelles (lysosomes, pH 4.5–5.0,)
and neutral organelles (mitochondria, pH 6.8–7.4) [10–16].
However, few pH fluorescent probes have been developed
to measure extremely acidic pH (pH < 4). On the one hand,
strong acidity is lethal to most organisms. Acidogenic bac-
teria and other bacterial species, such as Helicobacter
pylori, can survive the acidic environment of the stomach
of mammals and can cause infections, which can be life-
threatening [17]. On the other hand, the secretory and
endocytic pathways of certain eukaryotic cell organelles
can only be carried out under acidic conditions. Hence, it
is necessary to design a highly pH-sensitive and photostable
fluorescent probe that can effective measure very low pH.
Yanhao Xu and Ruikang Duan contributed equally to this work.
* Yanqing Ge
geyanqing2016@126.com
1
School of Pharmacy, Shandong First Medical University &
Shandong Academy of Medical Sciences, Taian, Shandong 271016,
People’s Republic of China
2
Shanghai Fengxian Central Hospital, Shanghai 201400, People’s
Republic of China

J Fluoresc
Scheme 1 Synthetic route of the probe YH-1
Indoles and other heterocyclic derivatives are often
found in natural products, such as alkaloids, auxins, es-
sential oils, and coal tar [18]. In addition, indoles are
popular moieties in drug discovery and medicinal chem-
istry [19]. While many indole-containing compounds have
demonstrated a myriad of essential biological activities,
few reports on their optical properties have been pub-
lished due to limitation in the synthetic methods of in-
doles [20, 21]. In this article, we report the development
of a new derivative of imidazo[1,2-a]indole, denoted YH-
1, which is a simple, yet novel, small-molecule fluores-
cent probe for measuring the pH in highly acidic environ-
ments using intramolecular charge transfer (ICT) as its
response mechanism. Compared to other pH-responsive
fluorescent probes [22–24], YH-1 could measure the pH
of a solution very quickly (within 30 s) with high sensi-
tivity. In addition, fluorescence imaging experiments in
Saccharomyces cerevisiae were conducted to provide a
foundation for the application of this probe in other
organisms.
7.0
6.5
120000
6.0
5.5
100000
5.0
4.7
4.4
4.1
3.8
3.5
3.2
2.9
2.6
2.3
2.0
80000
60000
40000
20000
0
Experimental Section
400
450
500
550
600
650
700
Materials
Wavelength/nm
Fig. 1 The fluorescence spectrum of the probe YH-1 (1µM) dissolved in
the B-R/DMSO (8/2, v/v) solution in the pH range of 2.0–7.0 (λex = 350
nm)
Except for special instructions, all reagents were pur-
chased from commercial sources and were used directly

J Fluoresc
Scheme 2 The mechanism of the
change in fluorescence intensity
of YH-1 after addition of H+
without further processing. To avoid any interferences
from impurities, deionized water was used to prepare all
solutions throughout the experiments. The chloride salt
was dissolved in deionized water to prepare the metal
ion solution to avoid any interferences from contamina-
tion by other metal ions. The sample solutions used in the
experiment were all prepared under natural conditions,
shaken for 15 s, and then allowed to stand for 10 min.
Then, UV-Vis and fluorescence spectroscopy experiments
were performed. Solutions of Britton-Robinson buffer (B-
R) that were used in the following experiments were pre-
pared by mixing 40 mM acetic acid, phosphoric acid, and
boric acid in deionized water. The pH of the solution was
adjusted with dilute aqueous NaOH or HCl.
An FE28-standard pH meter (Shanghai Mettler) was used
to measure pH. All cell imaging experiments were per-
formed on a microscope Ti 2 (Nikon, ECLIPSE) with an
excitation wavelength of 350 nm.
Fungus Imaging
Saccharomyces cerevisiae (abbrev. S. cerevisiae), a species
of fungus used in breadmaking (including steamed bread)
and brewing, was extracted from yeast at 30 °C in peptone
glucose (YPD) medium (containing 2 % tryptophan, 1 %
yeast extract, and 2 % glucose (w/v)) and then stirred in a
table concentrator (ZHI) at 200 rpm for 12 h. The cultured
S. cerevisiae solution was placed in a 2 mL Eppendorf tube
and centrifuged at 4500 × g for 2 min to collect the
S. cerevisiae cells. The resulting pellet was resuspended in
1 mL of Britton-Robinson buffer at different pH (3.0, 5.0,
and 7.0). The centrifuge tube was then placed in a bench top
concentrator. The YH-1 probe was dissolved in DMSO.
After 2 h, the probe solution was diluted into each tube
containing buffer solution to a final probe concentration of
5 µM, and the tubes were incubated for 30 min. Finally, a
glass microscope slide was coated with the probe solution,
Instruments
All absorption spectra were acquired on a UV-2600 spec-
trophotometer (Shimadzu), and all fluorescence spectra
were recorded on a FS5 fluorescence spectrophotometer
1
13
(Edinburgh Instruments). H NMR and C NMR spectra
were acquired on a Bruker Avance 400 (400 MHz) spec-
trometer using DMSO-d6 as the solvent and
tetramethylsilane (TMS) as the internal standard material.
Fig. 2 a The fluorescence titration pH value of 2.0 to 7.0 at 450 nm fluorescence intensity. b The linear relationship between the fluorescence intensity of
the probe YH-1 at 450 nm and the pH value (pH 3.2–4.4) (R2 =-0.9975) (λex = 350 nm, λem = 450 nm)

J Fluoresc
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
pH=5
4.4
4.2
4.0
3.8
3.6
3.4
3.2
pH=2.6
0
2
4
6
8
10
Time(min)
Fig. 5 The fluorescence intensity of the probe YH-1 changes with time in
0–10 min (λex = 350 nm, λem = 450 nm)
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
log [(I -I )/(I -I )]
x b
a
x
solution. The product was isolated by suction filtration
and dried in an oven to afford YH-1 as a yellow solid
with a yield of 82 % (0.78 g). mp: 216–218 °C. 1H
NMR (400 MHz, DMSO-d6) δ 13.27 (s, 1 H), 8.65 (d,
J = 8.2 Hz, 1 H), 8.15 (s, 1 H), 8.07 (d, J = 8.0 Hz, 1 H),
7.49 (d, J = 7.3 Hz, 2 H), 7.41 (t, J = 7.3 Hz, 2 H), 7.35
(d, J = 7.1 Hz, 1 H), 7.25 (t, J = 7.5 Hz, 1 H), 7.11 (t, J =
7.6 Hz, 1 H), 5.34 (s, 2 H), 4.06 (s, 3 H).13 C NMR (100
MHz, DMSO-d6) δ 163.66, 160.92, 144.88, 137.77,
133.56, 131.18, 128.96, 128.50, 128.30, 126.81, 123.61,
120.71, 119.60, 115.22, 114.96, 80.89, 64.78, 37.15.
HRMS ([M + H]+): Calcd for C20H17N2O4: 349.1188;
found: 349.1185.
Fig. 3 The linear regression relationship between the pH value and “(log
[(Fmax-FX)/(FX-Fmin)]”
which was observed with a microscope Ti 2 (Nikon,
ECLIPSE) at a wavelength of 350 nm.
Synthesis and Characterization of Probe
9-((benzyloxy)carbonyl)-1-methyl-1 H- imidazo[1,2-a]
indole-3-carboxylic Acid (YH-1)
From commercially available 1-fluoro-2-nitrobenzene (1),
compounds 2–7 were synthesized according to previously
published literature procedures [25]. Compound 7 (0.96 g,
2.56 mmol) and NaOH (0.12 g, 3 mmol) were added to a
solution of ethanol (20 mL) and water (10 mL), and the
resulting mixture was stirred for 4 h at 80 °C. The crude
reaction mixture was decanted in 40 mL of water, and the
pH of the solution was adjusted to 2.0 with aqueous hy-
drochloric acid, after which the product precipitated from
Results and Discussion
Synthesis of the Probe YH-1
Scheme 1 shows the general synthetic route of the probe. The
structure of the final probe was characterized by HRMS
and 1 H/13 C NMR.
Spectral Characteristics of Probe N-1 and its Optical
Response to pH
For the fluorescence experiments, all samples were dissolved
in a mixture of Britton-Robinson buffer (B-R) and DMSO
(8/2, v/v) and measured after 10 min. As shown in Fig. 1,
probe YH-1 was highly fluorescent, and the fluorescence in-
tensity at 450 nm remained unchanged at pH 4.4 and above.
Over the pH range of 2.0–4.4, the fluorescence intensity de-
creased significantly as the pH decreased. The fluorescence
intensity increased significantly from 29346.2 at pH 2.0 to
100201.8 at pH 4.4. The quantum yield at pH 4.4 was calcu-
lated to be 0.115 (Quinine sulphate dehydrate in 0.1 N H2SO4
was used as the main standard, φ = 0.546, λex = 350 nm).
Fig. 4 The reversibility of the fluorescence emission intensity of the
probe YH-1 between pH 2.6 and pH 5.0 (λex = 350 nm, λem = 450 nm)

J Fluoresc
Fig. 6 Changes in the fluorescence intensity of probe YH-1 in the
solution (8/2, B-R/DMSO, v/v) under the influence of different metal
ions and amino acids at (a) pH 2.6 and (b) pH 5.0 (probe (1µM), Zn2+
(5µM), Fe3+ (5µM), Cu2+(5µM), Mg2+ (5µM), Ca2+ (10µM), Na+
(10µM), K+ (10µM), H2O2 (5µM), GSH (5µM), Cys (5µM), Hcy
(10µM), λex = 350 nm, λem = 450 nm)
As shown in Fig. 2, the x-axis and y-axis represented the
pH and fluorescence intensity, respectively, and the data are
arranged in a “Z” arrangement (at an emission wavelength of
450 nm). In Fig. 2, over the pH range of 2.3–4.4, the fluores-
cence intensity and pH demonstrated a linear relationship
(R2 = 0.9975). The pKa of the probe was then measured in a
mixture of Britton-Robison buffer and DMSO (8:2 v/v).
Based on the Henderson-Hasselbach equation, the pKa of
the probe was calculated using the fluorescence emission in-
tensity of the probe at 450 nm fluorescence according to Eq. 1:
N ¼ 2n ðn À 1Þ þ m
ð1Þ
where F represented the probe’s emission wavelength at 450
nm. The pKa of YH-1 was determined to be 3.56 (Fig. 3),
Fig. 7 The 1H NMR spectrum of probe YH-1 in DMSO-d6 under neutral conditions and acid conditions (CF3COOH)

J Fluoresc
Fig. 8 Fluorescence images of H+
in S. cerevisiae with probe YH-1
(5 µM, 30 min). Excitation: 350
nm, Emission: 450–480 nm
which made the probe suitable for measuring the pH of strong-
ly acidic solutions. Over the pH range of 2.3–4.4, and a linear
relationship between the pH and log[(Fmax-Fx)/(Fx-Fmin)] was
obtained, with a linear regression equation of pH = −
0.8497x + 3.5605, where x was equivalent to log [(Fmax-
Fx)/(Fx-Fmin)]. This equation was used to calculate the pH
of the samples over the range of 2.3–4.4.
fluorescence images (Fig. 8), no fluorescence was observed in
the yeast cells at a pH of 3.0. As the intracellular pH increased,
the fluorescence intensity of the probe increased significantly.
These results indicated that the probe was applicable in biolog-
ical systems with low pH. Compared to other probes (Table S1),
this probe not only detected strong acidic pH but was useful for
detecting intracellular acidic pH’s in S. cerevisiae.
As shown in Fig. 4, the fluorescence emission of the probe
at 450 nm was reversible between pH 2.6 and 5.0, which
meant the probe was applicable in various acidic environ-
ments with different pH values. In addition, the pH response
time of the probe under different conditions did not exceed 30
s (Fig. 5). There was also no change in fluorescence intensity
of the probe in the presence of different metal ions and amino
acids, which indicated that the probe responded selectively to
protons (Fig. 6). These results substantiated that the probe was
capable of measuring the intracellular pH of S. cerevisiae.
Conclusions
In short, the simple and novel pH-responsive fluorescent
probe YH-1 based on an imidazo[1,2-a]indole fluorophore
was synthesized and evaluated for pH-responsiveness under
strongly acidic conditions. This was the first report of an
imidazo[1,2-a]indole derivative that was used as fluorophore
for pH detection. 1H NMR analysis of the fluorophore under
neutral and acidic conditions indicated that the response
mechanism of the probe to pH was consistent with ICT. In
addition, the probe responded quickly to protons (within 30 s),
had a high selectivity to protons over other interfering metal
ions and amino acids, was sensitive to pH, and was reversible.
More importantly, the S. cerevisiae experiments corroborated
the utility of the probe in fluorescence imagine of yeast be-
cause it demonstrated robust imaging capabilities under
strongly acidic conditions in S. cerevisiae.
Mechanism of pH Detection
1H NMR was used to compared the electronics of the probe
under neutral and acidic conditions (trifluoroacetic acid,
CF3CO2H) (Fig. 7). No significant shifts in the proton reso-
nances were observed in the spectra of two species, which
indicated that the nitrogen bridgehead was not protonated;
therefore, the indole nitrogen did not undergo protonation.
Under neutral conditions, the 1-N atom was very electron-rich
and able to be protonated by the carboxylic acid, which indi-
cated that the ring system was a push-pull system. However,
under acidic conditions, the carboxylic acid groups functioned
as Lewis bases. Compared to protons under neutral condi-
tions, the imidazo[1,2-a]indole protons absorbed under a
higher electric field in acidic conditions. Therefore, the rate
of ICT should change under acidic conditions. Scheme 2
shows the process of protonation.
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s10895-021-02739-8.
Author Contributions All authors contributed to the study conception
and design. Material preparation, data collection and analysis were per-
formed by Yanhao Xu, and Ruikang Duan. Data analysis were performed
by Hao Liu, and Chengcai Xia. The first draft of the manuscript was
written by Guiyun Duan and Yanqing Ge. All authors read and approved
the final manuscript.
To verify whether the probe can be applied in biological
systems, we tested it in S. cerevisiae under strongly acidic con-
ditions. After incubation of LB medium at different pH’s (3.0,
5.0, and 7.0) with the probe for 30 min, the medium was incu-
bated with S. cerevisiae for 30 min. Based on the confocal
Funding This work was supported by the Innovative Research Programs
of Higher Education of Shandong Province (2019KJC009), the Science
Foundation of Shandong Province for Excellent Young Scholars
(ZR2017JL015), and the Natural Science Foundation of China
(21,602,153).

J Fluoresc
Data Availability The authors declare that the data supporting the find-
ings of this study are available in the article and the supplementary
materials.
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Declarations
Competing Interests The authors declare that there are no conflicts of
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