Imidazolyl–benzocoumarins as ratiometric fluorescence probes for biologically extreme acidity

Article abstract of DOI:10.1016/j.saa.2020.119088

A rational approach to develop a fluorescent probe for sensing biologically “extreme” acidity (pH <3) is disclosed. The probe, a push–full type 3-(imidazolyl)benzocoumarin dye, has the lowest pK_a = 1.3 among ratiometric probes known so far, which is ascribed due to a unique sensing mechanism. The probe has high quantum yields, high chemical stability and good aqueous solubility. The probe was successfully applied to ratiometric fluorescence imaging of intrabacterial acidity from pH 4.0–1.0, offering a practical means for studying biological systems under the extreme pH conditions.

Full text of DOI:10.1016/j.saa.2020.119088

SAA-119088; No of Pages 9
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Imidazolyl–benzocoumarins as ratiometric fluorescence probes for
biologically extreme acidity
Hyerim Kim a,1, Sourav Sarkar a,1, Madhurima Nandy , Kyo Han Ahn ⁎
b
a,
a
Department of Chemistry, Pohang University of Science and Technology (POSTECH), 37673, Republic of Korea
Department of Life Sciences, Pohang University of Science and Technology (POSTECH), 37673, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2020
Received in revised form 9 October 2020
Accepted 11 October 2020
Available online xxxx
A rational approach to develop a fluorescent probe for sensing biologically “extreme” acidity (pH <3) is disclosed.
The probe, a push–full type 3-(imidazolyl)benzocoumarin dye, has the lowest pK = 1.3 among ratiometric
a
probes known so far, which is ascribed due to a unique sensing mechanism. The probe has high quantum yields,
high chemical stability and good aqueous solubility. The probe was successfully applied to ratiometric fluores-
cence imaging of intrabacterial acidity from pH 4.0–1.0, offering a practical means for studying biological systems
under the extreme pH conditions.
Keywords:
pH probe
©
2020 Elsevier B.V. All rights reserved.
extreme acidity
Ratiometric probe
Benzocoumarin
Coumarin
1
. Introduction
to monitor two signals at a time, can provide reliable data. Herein, we
disclose a rational approach to develop a ratiometric fluorescent probe
for detecting the extreme acidity.
Cellular processes such as metabolism, growth, apoptosis, endocyto-
sis, ion transport, etc., are uncompromisingly controlled by a narrow
fluctuation in the intracellular pH [1]. Imbalance in the intracellular
pH is known to cause physiological malfunction such as cardiopulmo-
nary disorders, cancer, Alzheimer's disease, etc. [2] Therefore, it is neces-
sary to monitor proton activity in biological systems for understanding
associated processes. To monitor biological pH changes, several fluores-
cence microscopy–based assay methods have been developed, which
are highly advantageous for their noninvasiveness, high sensitivity,
high selectivity, and fast response time [3]. The fluorescent pH probes
known so far mostly work in a pH range from near neutral [4] to mod-
erately acidic, a compatible pH range for most biological systems [5].
Besides, there are many microorganisms such as acidophiles and
helicobacter pylori that have evolved to live in a strongly acidic environ-
ment [6]. Particularly, the stomach in our body secretes gastric acid and
maintains the extreme acidity of pH ~ 2, which is necessary for protein
digestion [7]. A few fluorescent probes for detecting the “extreme” bio-
logical acidity are known so far, which have been used to investigate
stomach–related disorders and function of acidophiles in body [8].
Most of the pH probes, however, provide the single emission
intensity–based assay, which suffers from data fluctuations owing to
the inherently environment–sensitive and concentration–dependent
fluorescence intensity. Conversely, ratiometric probes, which allow us
Recently, linear–shape, push–full type benzocoumarins have
evolved as a promising dye platform for the development of ratiometric
fluorescent probes for biological analytes. Benzocoumarins containing
an amine or an alcohol as the electron–donating group conjugated
with electron acceptor groups such as carboxy, benzothiazole, or pyri-
dine served as excellent fluorophores in the development of fluorescent
probes [9]. From our experience, benzocoumarins containing intramo-
lecular H–bonding between the fluorophore core and 3–heterocylic
substituent have high quantum yields in solution and provide bright
cellular images [9a]. Keeping in mind these advantages, we have inves-
tigated the imidazole–substituted benzocoumarins where similar intra-
molecular H–bonding is possible (Fig. 1). We reasoned that protonation
at the imidazole would make the amine–donor at the opposite side less
basic, allowing us to detect the extreme acidity. Indeed, the 3–
(imidazol–5–yl)benzocoumarins are found to detect an extreme pH
range, pH 0.5–4.0, with well-resolved ratiometric signal changes as well.
2
. Experimental section
2.1. Materials and methods
The chemical reagents were purchased from Aldrich or TCI. Com-
mercially available reagents were used without further purification.
Anhydrous solvents were used for reactions. All reactions were per-
formed under argon atmosphere unless otherwise stated. Thin–layer
⁎
Corresponding author.
E-mail address: ahn@postech.ac.kr (K.H. Ahn).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.saa.2020.119088
386-1425/© 2020 Elsevier B.V. All rights reserved.
1
Please cite this article as: H. Kim, S. Sarkar, M. Nandy, et al., Imidazolyl–benzocoumarins as ratiometric fluorescence probes for biologically
extreme acidity, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2020.119088

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Fig. 1. Design strategy to develop a pH probe for an extreme acidity.
chromatography was performed on pre–coated silica gel 60F–254
3H), 7.51 (s, 1H), 7.25 (d, J = 8 Hz, 1H), 6.92 (d, J = 1.0 Hz, 1H), 4.56 (s,
1H), 3.54 (s, 2H) 3.48 (s, 2H), 3.04 (s, 3H), and 1.72 (s, 2H). C NMR
1
13
13
glass plates. H and C NMR spectra were measured with Bruker
AVANCE III FT–NMR spectrometer. Coupling constants (J value) are
reported in Hertz. The chemical shifts (δ) are shown in ppm, multi-
plicities are indicated by s (singlet), d (doublet), t (triplet), dd (dou-
blet of doublets) and m (multiplet). UV/Vis absorption spectra were
obtained using a HP 8453 UV/Vis spectrophotometer. Fluorescence
emission spectra were recorded on a Photon Technical International
Fluorescence System using a 1.0 cm standard quartz cell. High–
resolution mass spectra were recorded on a JEOL JMS–700 spectrom-
eter at the Korea Basic Science Center, Kyungpook National Univer-
sity and the values are reported in units of mass to charge (m/z).
6
(125 MHz, DMSO–d , 298 K): δ 159.4, 149.9, 148.4, 136.1, 136.0, 134.8,
129.3, 127.6, 123.1, 115.9, 115.5, 108.5, 103.4, 58.2, 48.7, 38.0, and 29.8.
+
19 3 3
HRMS (EI positive): m/z, calculated for C20H N O [M] 349.14; found
349.14.
2.2.3. 8–(2–(Hydroxymethyl)pyrrolidin–1–yl)–3–(1H–imidazol–5–yl)–
2H–benzo[g]chromen–2–one (ImBC3)
This compound was prepared in a similar way as above, starting
from
3–hydroxy–6–(2–(hydroxymethyl)pyrrolidin–1–yl)–2–
naphthaldehyde (100 mg, 0.37 mmol), as an orange solid (40 mg,
1
3
0%). H NMR (500 MHz, DMSO–d
1H), 8.14 (s, 1H), 7.85–7.79 (m, 2H), 7.74 (s, 1H), 7.49 (s, 1H), 7.15
dd, J = 5.1 1.0 Hz, 1H), 6.84 (d, J = 1.0 Hz, 1H), 4.88–4.85 (m, 1H),
3.94–3.89(m, 1H), 3.57–3.49 (m, 2H), 3.27–3.19 (m, 2H), and
2.08–1.91 (m, 4H). 13C NMR (75 MHz, DMSO–d
, 298 K): δ 159.4,
6
, 298 K): δ 12.29 (s, 1H), 8.52 (s,
2.2. Synthesis and characterization
(
2
.2.1. 7–(Dimethylamino)–3–(1H–imidazol–5–yl)–2H–chromen–2–one
(
ImC)
To a solution of 4–(dimethylamino)–2–hydroxybenzaldehyde
20 mg, 0.12 mmol) and methyl 2–(1H–imidazol–5–yl)acetate hydro-
6
149.9, 146.6, 136.1, 136.1, 134.9, 129.4, 127.8, 123.2, 116.4, 115.3,
(
108.3, 103.2, 61.0, 60.0, 48.1, 27.8, and 22.5. HRMS (EI positive): m/z,
+
chloride salt (25 mg, 0.14 mmol) in EtOH (5 mL) were added two
drops of piperidine. The resulting mixture was refluxed for 48 h and
then subjected to extraction with dichloromethane. The organic layer
calculated for C21H N O
19 3 3
[M] 361.14; found 361.14.
2.3. UV–Vis absorption and fluorescence spectroscopy
was washed with brine, dried over anhydrous Na
2 4
SO , and concen-
trated. The residue was purified by silica gel column chromatography
UV–Vis absorption spectra were obtained using a HP 8453 UV–Vis
spectrophotometer. Fluorescence emission spectra were recorded on a
Photon Technology International fluorimeter with a 1.0 cm standard
quartz cell. The excitation and emission wavelength band paths were
both set at 4 nm. All of the solvents used were of analytical grade. A
stock solution of each of the ImC and ImBC dyes was prepared in
DMSO at a concentration of 1.0 mM. A required amount of the mixed so-
lution was transferred to a cuvette (1.0 mL) for spectroscopic
measurement.
1
(
eluent: MeOH/CH
2 2
Cl = 2/98) to afford ImC as a yellow solid. H
NMR (300 MHz, MeOD, 298 K): δ 9.03 (s, 1H), 8.37 (s, 1H), 8.29 (s,
1
3
2
H), 8.11 (s, 1H), 7.6 (d, J = 8.7 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), and
.04 (s, 6H). HRMS (EI positive): m/z, calculated for C14H N O [M]
13 3 2
55.10; found 255.10.
+
8
–(Allyl(methyl)amino)–3–(1H–imidazol–5–yl)–2H–benzo[g]
chromen–2–one (ImBC1).
A solution of 6–(allyl(methyl)amino)–3–hydroxy–2–naphthaldehyde
56 mg, 0.23 mmol) and 4–imidazole acetic acid (56 mg, 0.35 mmol) in
DMF (5 mL) was treated with Et N (96 μL, 0.69 mmol). After being stirred
(
3
2.4. Quantum yield calculation
for 10 min, the reaction mixture was treated with 1–(3–dimethy
laminopropyl)–3–ethylcarbodiimide (EDC; 66 mg, 0.35 mmol) and 4–
dimethylaminopyridine (DMAP; 0.5 mg, 0.04 mmol). The resulting mix-
ture, after being stirred for 8 h at 60 °C, was subjected to extraction with
dichloromethane. The organic layer was washed with brine, dried over
Quantum yield was calculated using the following equation using
rhodamine 6G as reference dye (Φ = 0.91 in EtOH). Where Φ is the
F
quantum yield, I is the measured integrated emission intensity, η is
the refractive index of the media and A is the absorbance. Subscript ‘r’
stands for reference.
anhydrous Na
by silica gel column chromatography (eluent: MeOH/CH
2
SO
4
, and concentrated. The crude product was purified
Cl = 3/97) to
2
2
ꢀ
ꢁ ꢀ ꢁꢀ
ꢁ
2
1
η
ηr
I
Ir
Ar
A
afford ImBC1 as an orange solid (33 mg, 43%). H NMR (500 MHz,
DMSO, 298 K): δ 12.29 (s, 1H), 8.54 (s, 1H), 8.16 (s, 1H), 7.83–7.8 (m,
Φ ¼ Φr
2H), 7.75 (s, 1H), 7.53 (s, 1H), 7.22 (dd, J = 9.5 & 2.5 Hz, 1H), 6.96 (d,
J = 2 Hz, 1H), 5.9–5.85 (m, 1H), 5.16–5.12 (m, 2H), 4.12 (d, J = 4.5 Hz,
2.5. Theoretical calculation
H), and 3.06 (s, 3H). 1 C NMR (125 MHz, DMSO–d
3
, 298 K): δ 148.6,
2
1
1
6
36.2, 135.0, 133.9, 133.7, 129.5, 127.8, 124.8, 123.4, 116.7, 116.3, 116.2,
15.7, 108.8, 103.9, 54.0, 38.1. HRMS (EI positive): m/z, calculated for
Theoretical calculation was performed with Gaussian 09. The geo-
metric and electronic structures of molecules were investigated with
the density functional theory (DFT) using B3LYP functional. All calcula-
tions were performed under 6–31G (d) basis set in vacuum.
+
C
20
H
17
N
3
O
2
[M] 331.13; found 331.13.
2
.2.2. 8–((3–Hydroxypropyl)methylamino)–3–(1H–imidazol–5–yl)–2H–
benzo[g]chromen–2–one (ImBC2)
2.6. Preparation of pH solutions
This compound was prepared in a similar way as above, starting from
3
–hydroxy–6–((3–hydroxypropyl)methylamino)–2–naphthaldehyde
Universal buffer was used for preparing pH solutions. 40 mM univer-
sal buffer was titrated with 6.0 M HCl to prepare pH 1–4 solutions. The
pH was measured using an Orion three star benchtop electrode.
1
(100 mg, 0.39 mmol), as an orange solid (25 mg, 20%). H NMR (500 MHz,
DMSO–d , 298 K): δ 12.28 (s, 1H), 8.52 (s, 1H), 8.13 (s, 1H), 7.83–7.74 (m,
6
2

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Previously reported
This work
Ph
N
N
N
N
O
Ph
Ph
N
O
N
H
N
H
N
H
Ph
O
O
O
O
O
O
Imidazol-2-yl
Imidazol-2-yl
Imidazol-1-yl
Imidazol-5-yl
Fig. 2. Known imidazolyl–coumarins and new imidazolyl–benzocoumarins.
2
.7. E. coli culture and imaging
imidazole–5–yl group at C–3, along with the corresponding coumarin
analogue (ImC) for comparison. We have evaluated their photophysical
properties and pH sensing behavior. Further, we demonstrate that one
of the benzocoumarin dyes is useful in probing a biological system in
the extreme acidity range.
E. coli were cultured at 37 °C in Luria–Bertani medium (yeast extract
g/L, Tryptone 10 g/L, and NaCl 10 g/L) for 16 h. Then the E. coli were
5
collected by centrifuging the culture for 5 min at 2000 rpm. The sedi-
ment is washed with sterile water, and then resuspended in a solution
of ImBC3 (20 μM) in pH 7.4 PBS. It was then incubated for 1 h at
3.2. Synthesis
37 °C, followed by centrifugation, washing and resuspension in different
pH buffers containing both nigericin and monensin (each at 10 μM). It
was then incubated for 30 min prior to imaging. Imaging samples
were irradiated with 405 nm confocal laser and the emissions were col-
lected in two different windows: green, 460–540 nm; red, 600–675 nm.
The synthetic routes to ImC and ImBC are outlined in Scheme 1. ImC
was prepared through Vilsmeier–Haack formylation on 3–
(dimethylamino)phenol, followed by Knoevenagel condensation with
4–imidazoleacetic acid methyl ester. On the other hand, ImBC1–
ImBC3 dyes were prepared by directed formylation on the respective
aminophenols, followed by coupling with 4–imidazoleacetic acid. The
advanced intermediate 1 was prepared through the established syn-
thetic route [13].
3. Results and discussion
3.1. Design of the imidazolyl-benzocoumarin dyes
3.3. Photophysical properties
Imidazole–substituted coumarin dyes are widely used in laser tech-
nology, probe development and bioimaging applications [10]. The con-
ventional imidazole–substituted coumarin dyes are imidazol-2-yl
derivatives, which are known to form planner structures due to the in-
tramolecular H–bonding between the imidazole-NH and carbonyl oxy-
gen (Fig. 2) [11]. Recently, a coumarin derivative with an imidazole–
We expected that the imidazolyl–benzocoumarins (ImBC1–ImBC3)
would have high quantum yields and provide bright cellular images:
The imidazole-NH is assumed to interact with the lactone carbonyl
through intramolecular hydrogen bonding, which would inhibit the
free–rotation of the imidazole group and thus increase the fluorescence
quantum efficiency.
1–yl substituent was also reported [12]. Still, coumarin dyes with an
imidazole–5–yl substituent remains unexplored. Coumarin dyes absorb
and emit at rather shorter wavelengths. To make them emit in the lon-
ger wavelengths, we have been investigating benzene-fused coumarin
analogues, called benzocoumarins [13]. In this work, we have synthe-
sized three benzocoumarin dyes (ImBC1–ImBC3) substituted with an
The reference dye ImC has the absorption and emission maxima
(λabs and λem) at 380 nm and 475 nm, respectively, in ethanol (Fig. 3).
Whereas ImBC1 has the absorption and emission maxima at 425 nm
and 555 nm, respectively, which are substantially red–shifted from
those of ImC. Both ImC and ImBC1 showed large Stokes shifts (95 nm
N
a
b
N
H
O
Me N
OH
Me N
OH
Me N
O
ImC
O
2
2
2
O
c
d
e
R1
R1
R1
N
R2
OH
N
R2
OMOM
N
N
R
OMOM
HO
OH
2
f
N
H
O
g
R1
R1
N
O
O
N
R2
OH
R2
1
=
Me, R = Allyl
= Me, R = CH
= Pr
ImBC1: R
ImBC2: R
1
1
1
2
2
CH OH
olinol
2
2
ImBC3: R , R
2
Scheme 1. Syntheses of ImC and ImBC1–ImBC3. Reagents and conditions: a) POCl
c) Na , HNR , H O; 150 °C, 8 h. d) NaH, DMF, ClCH OCH ; 0 °C, 1 h. e) t–BuLi, Et
DMF; 60 °C, 8 h.
3
, DMF; 0–70 °C, 1 h. b) 4–Imidazoleacetic acid methyl ester, MeOH, piperidine; reflux, overnight.
S O
2 2 5
R
1 2
2
2
3
2 3
O, DMF; −15 °C, 2 h. f) HCl, i–PrOH; 60 °C, 3 h. g) 4–Imidazoleacetic acid, EDC, DMAP, Et N,
3

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
red–shift when the medium was changed from nonpolar DCM to polar
DMSO (Fig. 4d). The ICT nature of ImBC1 accounts for its solvatochromic
behavior.
EtOH
ImC
1
30 nm
ImBC1
1
0
0
0
.2
.8
.4
.0
9
5 nm
3.4. Response to acidic pH
a
Imidazole has pK of 6–7 depending on the medium [15]. The pro-
tonation results in symmetrical resonance structures; hence, the emis-
sion properties of imidazole–substituted dyes are readily influenced
by pH changes even in the neutral pH range. Therefore, we investigated
the effect of pH on the photophysical properties of ImC and ImBC1 dyes.
At both pH 4.0 and 7.0, the absorption spectra of ImC displayed two
overlapped peaks at 380 and 425 nm, plausibly from the neutral and
mono–protonated (at the imidazole) forms, respectively (Fig. 5a).
Upon excitation at 380 nm, the fluorescence peak of ImC at 495 nm de-
creased as the acidity increased (Fig. 5b). This result suggests that, in the
case of ImC, protonation at the amine donor results in fluorescence
quenching, plausibly owing to the diminished ICT [16]. ImBC1 at
pH 4.0 and pH 7.0 displayed two absorption peaks well separated at
Absorption
00 400
Wavelength (nm)
Emission
3
500
600
700
Fig. 3. Normalized absorption and emission spectra of ImC and ImBC1, each at 10 μM in
ethanol. The emission spectra were obtained under excitation at the maximum
absorption wavelength of each dye.
4
50 nm and 430 nm, respectively (Fig. 5c). At pH 1.0, a new absorption
and 130 nm, respectively, in ethanol), reflecting their intramolecular
charge–transfer (ICT) nature. The larger Stokes shift in the case of
ImBC1 suggests that it experiences a larger geometric relaxation during
de-excitation in comparison with ImC.
Absorption spectra of ImC in dichloromethane (DCM) and acetoni-
trile exhibited two peaks at 335 nm and 400 nm, the former peak disap-
peared when the medium was changed to ethanol and dimethyl
sulfoxide (Fig. 4a). Interestingly, ImC showed negligible emission spec-
tral shifts upon changing solvent, similarly to the case of other
imidazolyl–coumarins (such as coumarin–7) (Fig. 4b) [14]. On the
other hand, ImBC1 produced very similar absorption spectra in the
four organic solvents assessed, which suggested existence of a relatively
nonpolar ground state (Fig. 4c): Its emission spectra underwent 35 nm
peak at 345 nm appeared with no other appreciable peaks. This new
peak is ascribed due to the di-protonated form of ImBC1, both at the im-
idazole and the amine donor sites. Then, among the two absorption
peaks observed both at pH 7.0 and pH 4.0, the mono-protonated species
is expected to appear at the longer wavelength owing to the enhanced
ICT. In the case of ImC, the absorption maxima of the neutral and
mono-protonated species seem to be close. Gratifyingly, in the case of
ImBC1, the di-protonated species also emitted significant fluorescence
at 485 nm, in addition to that of the mono-protonated species at
610 nm (Fig. 5d). Therefore, ImBC1 could enable ratiometric monitoring
of acidity changes from pH 4.0 to pH 1.0 or lower.
Theoretical calculation data suggests that at pH 4, the transition from
HOMO to LUMO is responsible for the molecular excitation in ImBC1.
(b) 400
(
a) 0.09 ImC
DCM
ImC
DCM
MeCN
EtOH
DMSO
MeCN
EtOH
DMSO
300
0
0
0
.06
.03
.00
2
1
00
00
0
4
70 nm
300
350
400
450
500
550
400
500
600
700
Wavelength (nm)
Wavelength (nm)
(
c) 0.3
(d) 400
ImBC1
DCM
ImBC1
DCM
MeCN
EtOH
DMSO
MeCN
EtOH
DMSO
300
0
0
0
.2
.1
.0
2
1
00
00
0
3
00
350
400
450
500
550
4
00
500
600
700
Wavelength (nm)
Wavelength (nm)
Fig. 4. Absorption and emission spectra of ImC (a, b) and ImBC1 (c, d), each at 10 μM in the organic solvents. The emission spectra were obtained under excitation at the maximum
absorption wavelength of each dye.
4

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
(
a)
(b) 600
ImC
pH 1.0
pH 4.0
pH 7.0
ImC
495 nm
pH 1.0
pH 4.0
pH 7.0
0
0
0
0
.03
.02
.01
.00
4
2
00
00
0
ex: 380 nm
700
3
00
400
500
600
500
600
Wavelength (nm)
Wavelength (nm)
(
c) 0.09 ImBC1
(d) 450
ImBC1
pH 1.0
pH 4.0
pH 7.0
pH 1.0
pH 4.0
pH 7.0
6
10 nm
3
1
00
50
0
0
0
0
.06
.03
.00
485 nm
ex: 375 nm
500
3
00
400
500
600
600
700
Wavelength (nm)
Wavelength (nm)
Fig. 5. Absorption and emission spectra of ImC and ImBC1 in universal buffer at pH 1.0, 4.0 and 7.0. Fluorescence spectra were obtained under excitation at 380 nm for ImC (5.0 μM) and at
75 nm for ImBC1 (5.0 μM).
3
Distinct orbital distribution between HOMO and LUMO supports that
ImBC1 belongs to ICT fluorophores. At pH 1.0, the transition takes
place from HOMO–1 to LUMO, which suggests, not an ICT, but a local-
ized excited (LE) state is responsible for fluorescence (Fig. 6).
3.5. Rational modifications toward ImBC3
Limited water–solubility of organic fluorophores is a critical issue for
their application to biological systems. Therefore, we assessed solubility
E (eV)
-
-
-
4
6
8
LUMO+1
93%
LUMO
HOMO
-
-
10
12
85%
HOMO-1
H+
+
+
ImBCH (pH~4)
ImBCH (pH~1)
2
(
ICT ꢀluorophore)
(LE ꢀluorophore)
Fig. 6. Orbital energy distribution of an ImBC prototype (8-(N,N-dimethyl)-analogue), obtained by TD–DFT calculation using B3LYP functional and 6–31G(d) basis set in vacuum.
5

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Scheme 2. From ImC to ImBC3, leading to the extreme pH probe with improved solubility and ratiometric signaling capability.
bands in ethanol from the others, plausibly due to the stronger donor
character of the geometrically–strained prolinol moiety (Fig. S3, SI)
Table 1
a
Photophysical properties of ImBC1–ImBC3. Absorption and emission wavelength max-
ima in EtOH at 25 °C. Molar absorptivity in the unit, L·mol ·cm
tum yields in different solvents ( CH
rhodamine 6G as a reference dye (Φ
[17]. Being a dipolar donor–acceptor (D–A) type dye, the fluorescence
b
−1
−1 c–f
.
Fluorescence quan-
c
d
e
f
intensity of ImBC dyes was minimal in aqueous media. However, all of
them exhibited moderate–to–strong fluorescence in organic solvents.
The quantum yields of ImBC dyes were highest in dichloromethane
among the solvents evaluated (Table 1).
2 2 3
Cl , CH CN, EtOH, and DMSO), determined using
F
= 0.91 in EtOH).
b
c
d
e
f
λ
abs (nm)a
λ
em (nm)a
ε
ɸ
F
ɸ
F
ɸ
F
ɸ
F
ImBC1
ImBC2
ImBC3
425
432
438
552
559
561
22,299
18,142
12,516
0.52
0.44
0.37
0.45
0.30
0.33
0.33
0.21
0.22
0.43
0.32
0.37
3.6. Sensing extreme acidity with ImBC3
Next, we investigated optical response of ImBC2 and ImBC3 toward
of ImBC1, which was found to be even lower than 2.0 μM level in PBS 7.4
Fig. S1, SI). The planer structure of ImBC1 seems to facilitate intermo-
a
acidic pH and calculated their pK values. Similarly to ImBC1, the strong
(
absorption peak of ImBC3 at 450 nm gradually decreased, while a new
peak at 340 nm appeared with increasing acidity (Fig. 7a). The fluores-
cence emission spectra were recorded by exciting at 375 nm, the
isosbestic point: the emission peak at 620 nm gradually decreased
while that at 480 nm increased when the acidity increased from
pH 4.0 to pH 0.5 (Fig. 7b). ImBC2 behaved very similarly (Fig. S4, SI).
lecular π– π stacking interaction, leading to the poor solubility.
To increase the solubility, we replaced the donor amine group with
hydroxyl–containing ones: N–(hydroxypropyl)methylamine and
prolinol. The resulting analogues, ImBC2 and ImBC3 respectively,
were synthesized similarly. As we expected, both the compounds had
much better solubility than ImBC1 (Fig. S1, SI). Particularly, ImBC3
showed a linearity in the absorbance–concentration plot above 10 μM
level, plausibly due to the steric hindrance caused by the prolinol ring
that inhibits the π–π stacking (Scheme 2).
The pK
spectral changes: ImBC2 has pK
lower pK = 1.3 (Fig. 8). From the pH-dependent ratio value graph,
we could estimate the extreme acidity from pH 4.0 to 0.5. A plausible
reason for the pK difference is likely due to the different electron–
a
values were calculated from the pH–dependent emission
a
= 2.4, whereas ImBC3 has much
a
ImBC2 and ImBC3 displayed very similar absorption and emission
spectra to those of ImBC1 (Fig. S2, SI). ImBC3 showed slightly red–
shifted absorption (λabs = 438 nm) and emission (λem = 561 nm)
a
donating ability of the amine donors: Previously, we delineated that
the electron–donating ability of the secondary–amine donor in dipolar
(
a) 0.24 ImBC3
pH 0.5
(b) 150
ImBC3
pH 4.0
pH 0.5
pH 0.5
pH 4.0
pH 0.5
1
00
50
0
pH 4.0
0
0
0
.16
.08
.00
pH 4.0
3
00
350
400
450
500
450 500 550 600 650 700
Wavelength (nm)
Wavelength (nm)
Figure 7. Changes of (a) absorption and (b) emission spectra of ImBC3 (10 μM) in universal buffers, upon changing the pH from 4.0 to 0.5. All the measurements were conducted at 25 °C
by irradiating at the isosbestic point (λex = 375 nm).
6

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
1
We further monitored H NMR spectral changes of the probe upon
addition of DCl in DMSO-d (Fig. 9), which also supported the pH-
6
dependent optical changes observed above. The spectral changes calcu-
lated for the probe, and its mono- and di-protonated species (using
ChemDraw program) indicated that the imidazole proton at C-2
ImBC2
ImBC3
0
0
0
0
.9
.6
.3
.0
(
Im_H-2) would undergo a notable chemical shift upon protonation at
the imidazole nitrogen (δ 7.64 → 8.92 ppm). Indeed, upon addition of
0 μL DCl to the probe solution (3 mg in 0.5 mL DMSO-d ), Im_H-2,
1
6
which was buried in the other aromatic protons in the probe's spec-
trum, appeared at δ 9.3 ppm. This notable downfield shift, that is, an en-
hancement in the induced magnetic field in the imidazole aromatic ring
current, seems to be caused by an increase in the ICT from the prolinol
nitrogen to the imidazole moiety, upon the imidazole protonation.
When additional 300 μL DCl was added, which was expected to further
protonate the prolinol nitrogen, Im_H-2 underwent an upfield shift
1
2
3
4
pH
(
presumably, the peak at δ 8.69 ppm), that is, a decrease in the induced
Fig. 8. Plots of normalized intensity ratio values (I475/I620) for ImBC2 and ImBC3 at
different pH values.
magnetic field. This up-field shift may be explained by a decrease in the
ICT upon the protonation at the electron-donor site of such a dipolar
dye. Upon the di-protonation, the pyrrolidine ring methylene protons
nearby the nitrogen underwent down-field shifts with peak splitting.
These down-field shifts also support the second protonation. Addition-
ally, the peak splitting can be explained by the second protonation,
which generates diastereomeric salts and thus further splitting take
place. In addition to the spectral change, upon protonation we also ob-
served absorption and emission color changes of the probe, which also
correlate well with the stepwise protonation processes.
dyes can be influenced by the 1,3–allylic strain between the donor
group and the aryl moiety (ortho–hydrogens) connected [17]. In this re-
gard, the five–membered cyclic amine donor will experience less 1,3–
allylic strain and thus can donate more electron density to the aryl
core, in comparison with the corresponding acyclic amine donor. Ac-
cordingly, the electron density on the prolinol nitrogen is less than
that on the acyclic amine nitrogen. Thus, ImBC3 has the lower pK
a
than ImBC2, as observed. Compared with the known ratiometric probes
that sense biologically extreme acidity (pH <3), ImBC3 has the lowest
pK value. A fundamental difference between the known ratiometric
a
probes and ImBC3 lies at the center of protonation: The known probes,
except one FRET probe, are dipolar dyes and sense the protonation at
the nitrogen of an electron-acceptor site, which takes place even at
Note that ImBC3 is stable at pH 1.0 when monitored up to 30 min
(Fig. S6, SI). It also showed good selectivity toward proton in presence
of various biologically relevant analytes including metal cations
(Fig. S7, SI). Surely, the pH detection with ImBC3 is reversible, as mon-
itored for four cycle between pH 4 and pH 1 (Fig. S8, SI).
the rather weakly acidic pH (pK
.6) (Table S1, SI). In contrast, ImBC3 senses protonation at the donor
site, which takes place at the extreme acidity (pK = 1.3).
When moved from pH 7.0 to pH 4.0, both absorption and emission
spectra showed red shifts of ~15 nm (Fig. S5, SI), plausibly owing to an
increase in the ICT caused by protonation at the imidazole nitrogen.
a
= 2.3–3.2) except one case (pK
a
=
3.7. Intrabacterial pH imaging
1
a
Given that ImBC dyes respond to the extreme acidity with
ratiometric optical signal changes, then we evaluated an applicability
of ImBC3 in sensing a biological system at the extreme acidity. To this
end, we chose E. coli, a bacterium species known to survive well in a
Vis. Flu.
Im_H-2
Im_H-2
Fig. 9. 1H NMR spectral changes of ImBC3 in DMSO-d
upon addition of DCl. Images of the solutions, taken under visible and 365 nm light irradiation, respectively.
7
6

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Green ch.
Red ch.
(
a)
(b)
Green ch.
pH 1
pH 4
(460 nm – 540 nm) (600 nm – 675 nm)
/ Red ch.
Bright ꢀield
High
1
0
0
.0
.8
.6
pH 1
100
100
100
100
pH 2
pH 3
pH 4
5
00
550
600
650
700
Wavelength (nm)
(
c)
0
0
0
0
.9
.6
.3
.0
pH 1
pH 2
pH 3
pH 4
Low
Fig. 10. (a) Fluorescence spectra measured directly for the E. coli incubated with ImBC3 and then suspended in pH 1 and in pH 4 buffers, respectively. (b) Ratiometric fluorescence images
of E. coli incubated with ImBC3 and then suspended in different pH solutions (universal buffer) containing both nigericin and monensin (each at 10 μM), obtained by CLSM. The images
were obtained by exciting at 405 nm and collecting of emissions through green (460–540 nm) and red (600–675 nm) channels. The pixel–to–pixel ratiometric images were constructed
using Matlab. Scale bar: 100 μm. (c) Emission intensity ratios (Igreen/Ired) plotted against respective pH value. The intensity was calculated using software, imageJ. E. coli were incubated
with ImBC3 (20 μM) for 1 h and then centrifuged at 2000 rpm, washed with water, and finally resuspended in different pH solutions.
broad range of pH perturbation and thus has been used in related stud-
ies [18]. E. coli were cultured for 16 h prior to incubating with ImBC3
means for studying biological systems associated with the extreme
acidity.
(
20 μM) for 1 h, which were then washed and resuspended in different
pH buffer solutions containing both nigericin and monensin (each at
0 μM) for rapid proton equilibration [19]. Fluorescence imaging by
CRediT authorship contribution statement
1
confocal laser–scanning microscopy (CLSM) was conducted by exciting
at 405 nm and collecting emissions through green (460–540 nm) and
red (600–675 nm) channels (Fig. 10a). When E. coli were suspended
in pH 4.0 buffer, emissions majorly came from the red channel, whereas,
upon lowering the medium pH, the emissions from the green channel
became stronger (Fig. 10b). The green over red channel ratiometric
Hyerim Kim: Conceptualization, Investigation. Sourav Sarkar:
Writing - original draft, Conceptualization, Investigation, Validation.
Madhurima Nandy: Investigation. Kyo Han Ahn: Conceptualization,
Supervision, Resources, Writing - review & editing.
Declaration of competing interest
(Igreen/Ired) images indicate a gradual change in the intrabacterial pH
when moved from pH 4.0 to pH 1.0 (Fig. 10c). The results demonstrate
that ImBC3 has potential for studying pH changes of biological species
in the extreme pH region.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
4. Conclusion
This work is financially supported by Global Research Laboratory
Program (NRF2014K1A1A2064569) funded by Ministry of Science, ICT
and Future Planning; Basic Science Research Program funded by the
Ministry of Education (NRF2019R1A2C2085438); Technology Develop-
ment Program (S2632282) funded by the Ministry of SMEs and Startups
(MSS), Korea.
Fluorescent probes for studying biological systems at extreme
acidity are of interest but relatively unexplored. We have developed
such a probe that responds to an extreme pH down to pH 0.5, by in-
troducing an imidazole–5–yl group to a linear–shape benzocou
marin dye. In comparison with a coumarin–based analogue that re-
sponds to pH changes with turn–on type fluorescence enhancement,
the benzocoumarin probe provides ratiometric signal changes. The
pH probe, which is chemically stable even at pH 1.0 and highly solu-
ble in buffer, was applied to ratiometric imaging of E. coli under pH
changes from pH 4.0 to pH 1.0. The pH probe thus offers a reliable
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.119088.
8

H. Kim, S. Sarkar, M. Nandy et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
(
(
c) W.J. Zhang, L. Fan, Z.B. Li, T. Oua, H.J. Zhaia, J. Yang, C. Dong, S.M. Shuang,
Thiazole–based ratiometric fluorescence pH probe with large Stokes shift for in-
tracellular imaging, Sensors Actuators B Chem. 233 (2016) 566–573;
d) S.L. Shen, X.F. Zhang, S.Y. Bai, J.Y. Miao, B.X. Zhao, A novel ratiometric pH probe
for extreme acidity based on FRET and PET, RSC Adv. 5 (2015) 13341–13346.
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