X-ray structurally characterized quinoline based fluorescent probes for pH sensing: Application in intracellular pH imaging; DFT calculations and fluorescent labelling

Article abstract of DOI:10.1016/j.jphotochem.2020.113074

Quinoline based fluorescent probes QNOH-MO and QNOH-OME were synthesized, and their photo-physical properties were carefully investigated. The probes were well characterized by ¹H-NMR, ¹³C-NMR, ESI-MS, and single-crystal X-ray structure analysis. QNOH-MO and QNOH-OME can identify small changes in pH by colorimetric and fluorescence methods. In low pH range, these probes display a red fluorescence in both live and fixed cells. However, with increasing pH, the observed red fluorescence decreased with a simultaneous increase in green fluorescence. The pH-dependent switching from red to green fluorescence is well supported by theoretical calculations and ¹H NMR titration experiments. Furthermore, the ability of these probes to image pH changes in live cells was also scrutinized. Moreover, QNOH-OME was successfully utilized for fluorescent labelling of cholesterol molecule which is an important component of biological membranes and is used in the liposomal formulation for drug delivery applications. Additionally, QNOH-MO was quaternized to induce an intrinsic cationic property to the molecule. The cationic probe QNOH-MO-CA exhibited improved water solubility and pH sensing efficiency.

Full text of DOI:10.1016/j.jphotochem.2020.113074

Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
X-ray structurally characterized quinoline based fluorescent probes for pH
sensing: Application in intracellular pH imaging; DFT calculations and
fluorescent labelling
,
Subhajit Guriaa, Avijit Ghosha b, Tanushree Mishraa, Manas kumar Dasa, Arghya Adhikaryb,
,
Susanta Adhikaria *
a Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700 009, West Bengal, India
b Centre for Research in Nanoscience & Nanotechnology, (CRNN), University of Calcutta, Technology Campus, Sector-III, Block-JD 2, Salt Lake, Kolkata 700098, West
Bengal, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Quinoline based fluorescent probes QNOH-MO and QNOH-OME were synthesized, and their photo-physical
properties were carefully investigated. The probes were well characterized by 1H-NMR, 13C-NMR, ESI-MS, and
single-crystal X-ray structure analysis. QNOH-MO and QNOH-OME can identify small changes in pH by colori-
metric and fluorescence methods. In low pH range, these probes display a red fluorescence in both live and fixed
cells. However, with increasing pH, the observed red fluorescence decreased with a simultaneous increase in
green fluorescence. The pH-dependent switching from red to green fluorescence is well supported by theoretical
calculations and 1H NMR titration experiments. Furthermore, the ability of these probes to image pH changes in
live cells was also scrutinized. Moreover, QNOH-OME was successfully utilized for fluorescent labelling of
cholesterol molecule which is an important component of biological membranes and is used in the liposomal
formulation for drug delivery applications. Additionally, QNOH-MO was quaternized to induce an intrinsic
cationic property to the molecule. The cationic probe QNOH-MO-CA exhibited improved water solubility and pH
sensing efficiency.
Quinoline
Fluorescent probe
DFT calculation
Intracellular pH imaging
Fluorescent labeling
1. Introduction
lysosomes, mitochondria, and endosomal vesicles, maintain pH that is
different from the cytoplasmic pH (~7.0) and this pH gradient have
Acid-base (pH) balance plays a pivotal role in a myriad of cellular
events, like cell growth [1] and apoptosis [2], endocytosis [3,4], signal
transduction [5,6], ion transport and homeostasis [7,8]. Cells have
established highly controlled mechanisms to regulate the pH of the
intracellular medium and maintain normal cellular functions [9]. The
tumor microenvironment is generally considered for its hypoxic nature
which upsets the metabolism of the cancer cells and consequently affects
the pH of the microenvironment. Cancer cell also has a higher intra-
cellular (pH ≥ 7.4) and lower extracellular pH (~6.7–7.1) compared to a
normal cell. This reverse pH gradient promotes cell proliferation, cancer
cell invasion, and prevents apoptosis [10,11]. It also affect the uptake
and efficacy of pH-sensitive chemotherapeutic drugs favouring the
preferential accumulation of drugs that are weak acids [12–14].
Abnormal pH values can also result in cardiopulmonary and neurologic
problems [8,9]. Within a cell, subcellular compartments such as
important functional consequences for these organelles [15,16]. Thus
monitoring the pH in live cells is a basic need for a better understanding
of physiological and pathological processes [17].
A reliable, well-established tool to measure the pH of the solution is
to use an electrochemical pH sensor [18]. However; there are many
disadvantages with these types of sensors including the need for
frequent calibration, susceptibility to electrical interference, and
corrosion in the presence of alkaline solution. They are also limited by
their use in a biological environment.
To overcome these obstacles, several optical pH sensors have been
developed using commercially available fluorophores that exhibit
spectroscopic changes triggered by the reversible transformation in the
structure of the indicator induced by pH variation. This allows acidity of
the solution to be monitored using a spectrophotometer, or to visualize
the pH gradient of the cellular microenvironment in real-time using a
* Corresponding author.
E-mail address: adhikarisusanta@yahoo.com (S. Adhikari).
https://doi.org/10.1016/j.jphotochem.2020.113074
Received 10 October 2020; Received in revised form 13 November 2020; Accepted 22 November 2020
Available online 3 December 2020
1010-6030/© 2020 Elsevier B.V. All rights reserved.

S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
fluorescence microscope [19–24]. However, only limited probes are
available that can image the event of pH changes in a ratiometric
approach within live cells [25–28]. Sometimes the pH sensitive probes
are also synthesized using tedious synthetic protocols [29,30]. Hence,
there is adequate requirement to develop cost friendly and easy to
synthesize probes that can effectively measure pH changes in real sys-
tem. Herein, we have designed three pH-responsive quinoline de-
rivatives (Fig. 1a) by employing the ICT process to bring about a
ratiometric colour change for distinguishing the acidic and alkaline
environment. The ability of the Intramolecular Charge Transfer (ICT)
probes to produce a significant stokes shift makes them suitable for
ratiometric sensing of biologically important analytes [31–34] and
intracellular pH [35–40]. Protonation of the quinoline nitrogen is an
excellent strategy to promote an enhanced ICT process which would
induce significant red-shifts in absorption spectra. Initially we have
synthesized two probes QNOH-MO and QNOH-OME. Further introduc-
tion of cationic property in QNOH-MO produced QNOH-MO-CA which
showed excellent water solubility and effectiveness. All these probes
showed red fluorescence in low pH range and green in high pH range.
The photophysical properties and sensitivity towards pH changes were
carefully examined. To understand the origin of unique red colour
observed during cells imaging experiments, we also carried out theo-
retical calculations and 1H NMR titration. All these probes showed
almost similar response and effectiveness towards pH changes. Hence,
they could be used as excellent probes for measurement of pH.
biological membranes and liposomal formulation.
2. Experimental
2.1. Materials and Methods
Chemicals and solvents were purchased from commercial suppliers
and are used as received. 1H and 13C NMR spectra were recorded on a
Bruker Avance III (300 MHz, 400 MHz, and 500 MHz) spectrometer.
Chemical shifts were reported in parts per million (ppm). Multiplicity
was indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), dd (doublet of doublet), bs (broad singlet). Coupling
constants were reported in Hertz (Hz). High resolution mass spectra
were obtained on a XeVO-G2S QTof Waters spectrometer. For thin layer
chromatography (TLC), Merck pre-coated TLC plates (Merck 60 F254)
were used, and compounds were visualized with a UV light at 254 nm.
Further visualization was achieved by staining with iodine. Flash
chromatography separations were performed on SRL 230–400 mesh
silica gel. Milli-Q Milipore 18.2 MΩ cm-1 water was used throughout all
experiments and in preparation of buffer solutions. A Hitachi UH-5300
spectrophotometer and a Hitachi-F7100 spectrofluorometer were used
for UVꢀ Vis and fluorescence measurements. A Labman digital pH meter
(LMPH-10) was used to measure the pH of the solutions.
2.2. Cell line and cell culture
All the Probes were characterized using 1H-NMR, 13C-NMR, Elec-
trospray Ionization Mass Spectrometry (ESI-MS) (Figs. S1-S18, ESI), and
X-ray single-crystal structure analysis (for QNOH-MO and QNOH-OME).
Moreover, QNOH-OME was also successfully utilized for fluorescent
labelling of cholesterol molecule which is an important component of
Human cervical cancer HeLa was procured from NCCS, Pune and was
well maintained in heat inactivated FBS (fetal bovine serum, 10 %)
containing Dulbecco’s Modified Eagle’s Medium (DMEM) added with
100 mg/mL concentration of antibiotics viz. penicillin, streptomycin,
Fig. 1. (a) Synthetic route to pH sensitive quinoline derivatives QNOH-MO, QNOH-MO-CA and QNOH-OME; Single-crystal X-ray structure of (b) QNOH-MO (CCDC
2024825) and (c) QNOH-OME (CCDC 2024824).
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S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
gentamycin and amphotericin B (fungizone). Cells were incubated in a
chamber humidified with 5% CO2 to achieve 80 % of confluency, earlier
to the experiment. For re-plating, cells were trypsinised; viable numbers
of cells were counted (Trypan blue exclusion test) and allowed to grow
in fresh medium for overnight.
3. Result and discussion
3.1. Synthesis of the quinoline derivatives
Fig. 1a describes the synthesis of QNOH-MO, QNOH-MO-CA, and
QNOH-OME using commercial precursor 4-(diethylamino)salicylalde-
hyde and 2-methyl-8-quinolinol. Substituting the chlorine atom of 4-(2-
chloroethyl)morpholine hydrochloride with the oxygen atom of 4-
(diethylamino)salicylaldehyde using K2CO3 as a base in (1:1) acetoni-
trile/DMF medium gave the intermediate compound 1 as an off-white
solid in 64 % yield. This on further reaction with 2-methyl-8-quinolinol
under aldol condensation gave QNOH-MO as red solid in 24 % yield. The
compound was further recrystallized using a chloroform/diethylether
solvent system. The crystal structure of QNOH-MO is shown in Fig. 1b.
Quaternization of QNOH-MO by iodomethane in dry DCM at room
temperature gave compound QNOH-MO-CA in 41 % yield. On the other
hand, a substitution reaction between 4-(diethylamino)salicylaldehyde
and iodomethane gave the intermediate compound 2 in 80 % yield. This
under aldol reaction condition with 2-methyl-8-quinolinol gave QNOH-
OME in 49 % yield, and the corresponding crystal structure is shown in
Fig. 1c.
2.2.1. In vitro live cell imaging of pH
In vitro imaging of pH in live cells was executed according to pre-
vious methods [41]. Human cervical cancer HeLa cells were cultured in
Dulbecco’s modified eagle medium (DMEM) supplemented with 10 %
fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 ◦C and
5% CO2. For in vitro imaging studies, the cells were seeded in 12-well
tissue culture plates with a seeding density of 105 cells per well. After
reaching 60 %–70 % confluence, the previous DMEM medium was
replaced with serum free DMEM medium. Then QNOH-MO-CA/
QNOH-MO/ QNOH-OME (5 μM) was incubated for another 1 h to
facilitate cellular uptake. After an hour of incubation, the cells were
rinsed three times with potassium rich PBS at different pH values of 4.5,
5.5, 6.5, 7.5 or 8.5. The cells were incubated further with nigericin
(1 μg/mL) for 5 min in respective potassium rich PBS buffer. Images of
live cells were then taken by using an EVOS® FL Cell Imaging System,
Life Technologies, USA.
3.2. Crystal structure of QNOH-MO and QNOH-OME
2.2.2. In vitro cell imaging of pH in fixed cells
In vitro imaging of pH in fixed cells was carried out according to
literature methods [42]. Human cervical cancer cells (HeLa) were
cultured in Dulbecco’s modified eagle medium (DMEM) supplemented
with 10 % fetal bovine serum (FBS) and 1% penicillin/streptomycin at
37 ◦C and 5% CO2. For in vitro imaging studies, the cells were seeded in
12-well tissue culture plates with a seeding density of 105 cells per well.
After reaching 60 %–70 % confluence, the previous DMEM medium was
The crystal structure of QNOH-MO (CCDC 2024825) shows the trans
arrangement between the nitrogen (N3) atom of quinoline and the ox-
ygen (O1) atom of the N,N-diethylamino benzene moiety (Fig. 1b). This
is in sharp contrast to the QNOH-OME crystal structure (CCDC 2024824)
where the nitrogen atom of quinoline (N1) and the oxygen (O2) atom are
on the same side of the double bond (Fig. 1c). QNOH-MO also has a
lattice water molecule which is hydrogen bonded to the N3 (distance
~2.040 Å) and O1 (distance 1.920 Å) atom of quinoline moiety and to
the N2 (distance ~2.040 Å) atom of the morpholine unit of adjacent
molecule. Such hydrogen bonding interaction leads to the formation of
dimeric arrangement of the QNOH-MO molecules involving two lattice
water molecules (Fig. 2a). This arrangement allows the molecule to
attain a nearly planar structure between the quinoline and N,N-dieth-
ylamino benzene unit along the double bond with a planar angle of 5.07◦
(Fig. 2c), whereas for QNOH-OME the angle is 22.2◦ (Fig. 2d). The
packing diagram of QNOH-OME revealed that the hydrogen atom (H6)
opposite to quinoline nitrogen is bonded to the oxygen atom (O1) of the
adjacent molecule to create a chain-like structure (Fig. 2b).
replaced with serum free DMEM medium. Then Dye (5 μM) was incu-
bated for another 1 h to facilitate the dye uptake by cells. After that, the
cells were rinsed three times with PBS and fixed in 4% para-
formaldehyde for 30 min. The buffer solutions at a series of different pH
levels were added in different wells to adjust the pH of fixed cells and to
make sure that the intracellular pH was consistent with the surrounding
buffer solution. Images of live cells were then taken by using an EVOS®
FL Cell Imaging System, Life Technologies, USA.
2.2.3. Cytotoxicity assay
In vitro cytotoxicity was measured by using the colorimetric methyl
thiazolyltetrazolium (MTT) assay against HeLa cells according to our
previous report [43]. The Cells were seeded into 24-well tissue culture
3.3. Effect of pH on the QNOH-MO and its derivatives
plate in presence of 500 μL Dulbecco’s modified eagle medium (DMEM)
supplemented with 10 % fetal bovine serum (FBS) and 1% pen-
icillin/streptomycin at 37 ◦C temperature and 5 % CO2 atmosphere for
overnight and then incubated for 12ꢀ 24 hours in presence of
QNOH-MO-CA / QNOH-MO/ QNOH-OME at different concentrations
The optical response of QNOH-MO with varying pH conditions (pH
2–10) was studied by performing a standard pH titration experiment.
The absorption spectrum of QNOH-MO is shown in Fig. 3a. In highly
acidic conditions (pH~2) an intense absorption band was appeared in
the region of ~375 nm and a slight hump at ~505 nm is observed. As the
pH of the solution was up-regulated, the colour of the solution switches
from an almost colourless to red, with an intense band at ~505 nm
(Fig. 3a). On further increasing the pH, the red colour declines with the
band at ~505 nm losing its intensity and stabilizing as a blue-shifted
broad-band at ~425 nm (Fig. 3a: Inset). Simultaneously the bare eye
colour of the solution changed from red to yellow. Furthermore, the
reversible nature of the proton addition was studied by measuring the
UV–vis spectrum of QNOH-MO by alternating treatment with acid and
base (Fig. S19, ESI).
(10–100
plemented DMEM medium was added. Subsequently, 50
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(5 mg/mL) was added to each well and incubated for 4 h. Next, violet
μ
M). Then cells were washed with PBS buffer and 500
μ
L sup-
L of 3-(4,
MTT
μ
formazan was dissolved in 500 μL of sodium dodecyl sulfate solution in
water/DMF mixture. The absorbance of solution was measured at
570 nm using microplate reader.
2.3. Synthesis
Detail synthetic procedures and characterization files of the com-
pounds and intermediates could be found in supporting information
section.
Similarly, the fluorescence emission spectrum of QNOH-MO was also
recorded (Fig. 3b). In acidic pH region, the ligand is weakly emissive
with an emission maximum at ~615 nm (λex = 490 nm) due to the facile
charge transfer that can occur between the protonated quinoline and the
donor NEt2 group [Fig. 3(b), Inset]. As the pH of the solution is gradually
increased, the band at ~615 nm first increases and then decreased with a
blue shift to ~575 nm (λex = 490 nm).
A
band at ~550 nm
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S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
Fig. 2. (a) Hydrogen bonded dimeric arrangement of QNOH-MO; (b) packing diagram of QNOH-OME; Inter-planar angle between the quinoline moiety and the
benzene ring in (c) QHOH-MO: 5.07◦; (d) QNOH-OME: 22.18◦ and (e) packing diagram of QNOH-MO.
(λex = 380 nm) appears which further intensifies and moves to ~530 nm
on increasing the pH to ~10 (Fig. 3b).
derivatives upon pH variation are shown in the Figs. S20-S21, ESI. To
our surprise, we found that the UV–vis signature of the derivatives
closely follow the response produced by QNOH-MO in the pH titration
experiment. The corresponding pKa value was found out to be 3.4 and
6.8 for QNOH-MO-CA, and 4.4 and 7.2 for QNOH-OME. Furthermore,
the core unit of the donor (NEt2) and the acceptor (quinoline-N) remain
unchanged in all these derivatives. This leads us to conclude that the
charge transfer process in the ground state is independent of the sub-
stitution present in the electron-donating and electron-withdrawing
group.
As two distinct colour change (i.e. from colourless to red and finally
to yellow) was observed during the pH titration experiment, the working
pH range was divided into two separate portions; one between pH
2.0–4.5 and the another ranging from pH 3.5–8.0 to determine the pKa
values for the probe (Fig. 3c,d). The ratio between the absorbance bands
at ~505 and ~370 nm was used to calculate the pKa in the pH range
2.0–4.5 whereas bands at ~505 and ~430 nm were taken for pH range
of 3.5ꢀ 8.0. The corresponding pKa values were found to be 3.5 and 5.8
for QNOH-MO.
Additionally, a similar derivative of QNOH-MO was synthesized to
systematically study the effect of the substitution present on the donor
and on the acceptor moiety that is participating in the ICT process
(Fig. 1a). Substituting the morpholine unit with a methoxy group pro-
duced QNOH-OME. Furthermore, quaternization ofthe nitrogen atom of
the morpholine group in QNOH-MO by iodomethane produced QNOH-
MO-CA which showed improved water solubility and in turn, is more
efficient in pH sensing. UV–vis and fluorescence response of the
3.4. UV–vis and fluorescence study of the quinoline derivatives
Steady-state absorption and fluorescence spectra of the quinoline
derivatives were carefully examined in the presence and absence of acid
in solvents of varying polarities (Figs. S22 and S23, ESI). The absorption
spectrums for all the quinoline derivatives show a strong absorption
band at ~388ꢀ 407 nm. However, in the case of a polar protic solvent
such as ethanol, an additional red-shifted band at 525 nm was observed
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S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
Fig. 3. (a) UVꢀ Vis absorption spectra of QNOH-MO (20
μ
M) with varying pH of the buffer solution (pH ~2-10) at 25 ◦C; (b) Fluorescence emission spectra of QNOH-
MO (20
μ
M) in buffer solution (pH ~2-10) (λex = 380 nm) at 25 ◦C; (Inset: Fluorescence emission in the pH region ~2-5 (λex = 490 nm); (c) Ratio of absorbance value
(A505 nm/A370 nm) of QNOH-MO in the pH range of 2.2 to 4.5; (d) Ratio of absorbance value (A430 nm/A505 nm) of QNOH-MO in the pH range of 3.5 to 8.0.
for all the derivatives along with the above mention bands. The addition
of acid to these derivatives resulted in the formation of a new band at
527 nm with a concomitant decrease of the band at ~388ꢀ 407 nm, in all
solvents. Thus it can be assumed that the red-shifted band is due to the
intramolecular charge transfer (ICT) state of the molecules induced by
the protonation of quinoline nitrogen. The fluorescence spectra of these
quinoline compounds in different solvents show emission maximum in
the range 500ꢀ 520 nm. However, in the presence of an acid, a bath-
ochromic shift of around ~75ꢀ 90 nm is observed in the steady-state
fluorescence spectra. Hence, the systematic bathochromic shift in
emission wavelength with a variation of solvent polarity suggests an ICT
character of the emissive excited state of the derivatives.
similarly to a poplar protic medium (ethanol) as indicated by the
absence of fluorescence lifetime as shown in Table S2, ESI. The decay
profile of QNOH-MO in presence of acid in different solvents is shown in
Fig. S24b, ESI. The average lifetime and the decay profile in presence
and in absence of acid for QNOH-MO-CA and QNOH-OME were sum-
marised in Table S3-S4 and in Figs. S25 to S26, ESI.
The polarity dependent bathochromic shift in the emission maxima
could be attributed to the higher dipole moment of the excited state in
the quinoline derivatives. To verify this, we constructed the Lippert plot,
using the Lippert equation which describes the Stokes shift (νa–νf) in
terms of the changes in the dipole moment, occurring due to
photoexcitation.
The relative quantum yield of QNOH-MO was calculated in a polar
protic solvent viz. ethanol in the presence (Φ = 0.01, Φref = 0.5 for
Rhodamine-B, λex = 500 nm), and absence of acid (Φ = 0.02, Φref = 0.38
for Coumarin-153, λex = 421 nm). However, in chloroform the quantum
yield is found to be 0.1 (10 times increase) even in presence of acid
(Table S1, ESI). This indicates that a facile non-radiative decay process is
operative in polar protic solvents. The TCSPC data indicates that QNOH-
MO (@375 nm excitation) undergoes a non-radiative decay of the
excited state in the polar protic solvents (e.g. Ethanol) due to the
favourable charge transfer process initiated by the protonation of the
quinoline moiety. But in polar aprotic solvents (acetonitrile: ACN;
dimethylformamide: DMF) an average lifetime (τavg) of ~0.46 ns was
observed (Table S2 ESI). The corresponding decay profile in different
solvents for QNOH-MO is shown in Fig. S24a, ESI. When the solution of
QNOH-MO in acetonitrile was acidified with 1eq of acid, the molecule
undergoes a nonradiative decay (@450 nm excitation), and behaves
(
)
(ṽA ꢀ ṽF) =
ꢀ
2 + Constant
2
ε
ꢀ 1
η2 ꢀ 1
(μE
ꢀ
μG)
hc
2ε
+ 1
2
η2 + 1
a3
The terms in the equation have their usual meaning. The quinoline
derivative QNOH-MO shows a linear response with increasing values of
orientation polarizability (Δf) (Fig. 4). The Onsager cavity radius
necessary for the calculation of the excited state dipole moments were
found to be 6.20, and 5.65 Å for QNOH-MO, QNOH-OME using the
theoretical volume calculation done on the ground state optimized ge-
ometry which is directly obtained from the density functional theory
(DFT) calculations using B3LYP hybrid functional and 6ꢀ 31 G(d) basis
set in CPCM solvent model. The calculated values for the ground and
excited-state dipole moments were found out to be 10.93 and 23.64 D
for QNOH-MO, and 9.46 and 20.60 D for QNOH-OME. The corre-
sponding Lippert ꢀ Mataga plot for QNOH-OME is shown in Fig. S27,
ESI.
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S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
followed by a restricted rotation around the single bond in presence of
PEG-300, facilitates the charge transfer process from the donor NEt2
group to the solvated nitrogen of the quinoline moiety. On further
increasing the concentration of PEG-300, the absorbance band at
~525 nm gradually decreased with a concomitant increase in the band
at ~425 nm (Fig. 5a). Similarly, the fluorescence spectrum recorded
(λex = 490 nm) also revealed a fluorescence maxima at ~615 nm which
corresponds to the red color observed in 20 % (V/V) of PEG-300 me-
dium. Fluorescence intensity of this band increased upon increasing the
ratios of PEG-300 followed by a hypsochromic shift to ~525 nm
(Fig. 5c). Hence, the red emission could be attributed to the optimum
viscosity of the polar protic medium.
3.6. NMR titration and probable sensing mechanism
To recognize the specific site of protonation which regulated the
1
remarkable change in the photophysical property of QNOH-MO, a H
NMR titration experiment was executed using CDCl3 as the solvent and
Acetic acid-d4 (CD3COOD) as a proton source.
Fig. 4. Lippertꢀ Mataga plot for QNOH-MO.
Upon addition of 1 equivalent of acid, a downfield shift from δ 2.72
to 2.83 was observed in (f) proton, indicating initial protonation to the
morpholine unit. On further addition of acid protonation of the quino-
line moiety occurs as indicated by the appearance of the signal (m) at δ
8.32. This results in the initiation of an intramolecular charge transfer
(ICT) process as indicated by the upfield shift from δ 3.46 to 3.32 for the
(c) protons of the NEt2 group. No further changes were observed in the
(f) protons indicating complete protonation of the morpholine nitrogen
centre. The (g) and (h) protons were upfield shifted from δ 6.2–6.09 and
from δ6.39ꢀ 6.30 indicating an increase in electron density on the
benzene ring attached to the NEt2 group. Similarly, the (k) and the (l)
protons were also upfield shifted from δ7.62 to 7.52 (Fig. 6). All the
other changes in NMR δ values are given in the Table S5, ESI.
3.5. Effect of viscosity on the photo-physical property of QNOH-MO
The effect of viscosity on the absorption and the emission spectra of
QNOH-MO were investigated in variable concentration of PEG-300:
water medium (Fig. 5). In the absence of PEG-300, the UV–vis spec-
trum (Fig. 5a) of the ligand showed a peak centered at ~425 nm.
However after reaching the concentration of PEG to approximately 20 %
(V/V), a remarkable red-shifted absorption band appeared at ~525 nm
with immediate change in the color of the solution from yellow to red.
This specific band was also observed upon the addition of acid (proton
source) to the solution of QNOH-MO in various solvents (Fig. S22, ESI).
Thus it could be concluded that the solvation of the quinoline moiety
Fig. 5. Effect on absorbance (a) and fluorescence spectra of QNOH-MO (b: λex = 390 nm) (c: λex = 490 nm) with variation of viscosity of the medium using Peg-300/
water (V/V) mixture; (d) Naked eye colour of QNOH-MO at varying concentration of PEG-300.
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S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
Fig. 6. 1H NMR titration of QNOH-MO in CDCl3; Shift of aromatic protons upon addition of increasing amount of CD3COOD (0, 0.5, 1, 2 equivalent).
Considering all the above observations a schematic representation of the
proposed mechanism is shown in Fig. 7.
theoretical calculations were carried out using Density Functional
Theory (DFT) as implemented in Gaussian 09 software [44]. Geometry
optimization of QNOH-MO was carried out using Becke’s
three-parameter hybrid functional with LYP correlation functional
(B3LYP), along with a 6ꢀ 31 G(d) basis set in ethanol medium using
conductor-like polarizable continuum model (C-PCM). Further, to shed
light on the origin of the absorption bands of the ligand in the presence
3.7. Theoretical study
To understand the charge transfer process associated with the
remarkable colour change of QNOH-MO in presence of an acid,
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S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
Fig. 7. (a) The proposed mechanism for colorimetric response of QNOH-MO at different pH in aqueous medium; (b) Change in fluorescence emission of QNOH-MO at
different pH under UV light excitation.
and in absence of acid, TDDFT calculations were performed on the
ground state optimized geometry using the above-mentioned hybrid
functional and basis set. The ground state optimized structures are
shown in Figs. S28 and S29. Furthermore, a comparison table describing
the similarities found in solid-state and theoretical structures have been
incorporated in Table S6, ESI. A planar arrangement was observed from
the dihedral angle value of ~179◦ between the quinoline moiety and the
N,N-diethylamino benzyl group (C9-C17-C10-C8), indicating the pres-
ence of a facile geometry for the charge transfer process.
The corresponding dihedral angle between the N,N-diethylamino
Fig. 8. HOMO-LUMO energy gap of QNOH-MO in presence and in absence of proton.
8

S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
group, and the benzene group (C13-C12-N4-C34) was found to be -5.9◦.
The Mulliken charges for the quinoline nitrogen (N2) and N,N-dieth-
ylamino group nitrogen (N4) were found to be -0.678 and -0.481. In
presence of an acid, the C9-C17-C10-C8 dihedral angle remains un-
changed but the C13-C12-N4-C34 dihedral angle was modified to -4.8◦
indicating the attainment of planarity of the electron-donating N,N-
diethylamino group Table S6, ESI.
penicillin/streptomycin at 37 ◦C and 5% CO2. Then the cells were seeded
in 12-well tissue culture plates with a seeding density of 105 cells per
well. After reaching 60 %–70 % confluence, the DMEM medium was
replaced by serum free DMEM medium. Then 5 μM of each QNOH-MO-
CA was incubated for 1 h. After incubation, the cells were rinsed three
times with potassium rich PBS at different pH values of 4.5, 5.5, 6.5, 7.5
or 8.5. The cells were incubated further with nigericin (5 μg/mL) for
The equivalent molecular orbital pictures are shown in Fig. 8. It can
be seen that the HOMO of QNOH-MO was present on the N,N-dieth-
ylamino group, and the corresponding LUMO resides on the quinoline
fragment. Thus a charge transfer process can take place from HOMO to
LUMO. Vertical excitation values corresponding to the first three excited
states are summarized in Table S7, ESI. The percentage that a given
transition contributes to the excited state is given by 2x2, where x rep-
resents the normalized coefficient of the transition. The first excited
state of the ligand QNOH-MO comprised of two transitions, having
major contribution (88 %) from (HOMO→LUMO) transition. However
the oscillator strength of the transition was found to be 0.4. The corre-
sponding theoretical wavelength was found to be ~516 nm which was in
close agreement with the experimentally observed value of ~525 nm.
Similarly the second excited state was also found to have been consti-
tuted by two transitions, with HOMO→LUMO+1 as the dominant one
(87 %), with oscillator strength of 0.84. The corresponding theoretical
and the experimental wavelength were found to be 428 and 415 nm as
shown in Fig. S30, ESI. In presence of acid, the HOMO→LUMO transition
of the first excited state increases in the oscillator strength to 0.9, indi-
cating feasibility of the transition and supporting the experimental
observation of the increase in the intensity of the red shifted absorbance
band (Fig. S30, ESI).
5 min in respective potassium rich PBS buffer. Finally, the images of live
cells were then taken using a fluorescence microscope. From Fig. 9, it
could be seen that at pH 4.5, fluorescence intensity in red channel was
much higher compare to green channel. However, with increase in pH
the emission from red channel decreases. On the other hand, the emis-
sion from green channel gradually rose at pH 5.5 and continued to in-
crease up to pH 7.5. Hence, this cationic probe owing to its high
solubility in aqueous medium could be used as an efficient marker for pH
changes in live cells. It is important to mention that analogous experi-
ments were carried out using QNOH-MO and QNOH-OME. To our
delight, we found almost similar observations in both these cases. Re-
sults are summarised in Figs. S31-S32, ESI.
In the same way, the pH sensitivity of the probes at a pH lower than
4.5 was carried out by fixed cell imaging experiments. In this procedure,
5 μM of QNOH-MO-CA was incubated for 1 h. After that, the cells were
rinsed three times with PBS and fixed with 4% paraformaldehyde for
30 min. The buffer solutions of different pH levels (pH 3–7.4) were
added in different wells to adjust the pH of fixed cells keeping the
intracellular pH consistent with the surrounding buffer medium. Images
of fixed cells were then taken by a fluorescence microscope (Fig. 10).
From the experiment, we found that the probe showed emission at
red channel up to pH 4.6. The emission from the green channel was
negligible in this pH range. Conversely, from pH 5, the fluorescence
from green channel increases and reaches maximum at pH 7.5. In this
pH ranges, no significant emission from red channel was observed. All
these cumulative results suggested that these probes could be used to
measure intracellular pH changes in both live and fixed cells. The other
two probes QNOH-MO and QNOH-OME also showed the similar ob-
servations (Figs. S33-S34, ESI).
4. Application
4.1. Intracellular pH imaging study
Live-cell imaging experiments were carried out to scrutinize the ef-
ficiency of these quinoline probes to image changes in the intracellular
pH. The HeLa cells were cultured in Dulbecco’s modified eagle medium
(DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1%
Nevertheless, as we discussed earlier that water solubility of the
probe QNOH-MO-CA is much higher than other two probes and
Fig. 9. Imaging of QNOH-MO-CA at different pH in live cells.
9

S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
Fig. 10. Imaging of QNOH-MO-CA at different pH in fixed cells.
therefore QNOH-MO-CA is much easier to use in biological experiments.
However, probes QNOH-MO and QNOH-OME are also equally effective
towards measurement of pH in live or fixed cells. Furthermore, we
carefully examined the localization of the probe QNOH-MO-CA in live
cells with Hoechst stain (Fig. 11).
4.2. Fluorescent labelling application of CHQN-OME
We further utilized the fluorescent property of QNOH-OME to label
biologically significant molecules like cholesterol. Several fluorescent
lipids and cholesterol derivatives were synthesised that are used in
developing fluorescent liposomes to image the delivery of therapeutic
agents to cancer cells, to track the dynamics of cationic lipids in the
cellular environment, and to study lipid microdomains [45–48]. How-
ever, all these methods require tedious multistep synthesis and purifi-
cation protocols for obtaining the fluorescent lipid/cholesterol
derivatives. Hence, there is still a requirement for a cost-friendly,
nontoxic fluorophore to label cholesterol. We have tagged
QNOH-OME with a cholesterol molecule by a simple substitution reac-
tion (Fig. 12). The photophysical property of the fluorescent cholesterol
derivative (CHQN-OME) is studied in varying pH conditions
(Figs. S39-S40 ESI). The live-cell imaging application of CHQN-OME
(Fig. 12) highlights the excellent fluorescence property and good cell
permeability of the ligand. Thus QNOH-OME could serve as an excellent
From the experiment, it was observed that the probe mainly localizes
in the lysosome of cells at biological pH of 7.4. It could be assumed that
the presence of a morpholine unit in the structure of QNOH-MO-CA,
allows it to preferentially concentrate in the lysosome. However, it is
interesting to note that at a lower pH range (3–4.5), QNOH-MO-CA
specifically restricts to the nucleus of cells (Fig. S35, ESI). Hence, it
could be used as a nucleus staining for fixed cells at a low pH range.
Nevertheless, the toxicity of each probes were carefully scrutinized
in HeLa cells by MTT assay. From the results, it was observed that these
probes are nontoxic in live cells (Figs. S36-S38, ESI). Therefore, all these
quinoline derivatives could be used as an excellent probe for detection of
pH changes in live cells.
Fig. 11. Localization of QNOH-MO-CA in live cells.
10

S. Guria et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113074
Fig. 12. (upper panel) Synthetic route to CHQN-OME; (lower panel) Fluorescence cell images of Hela cells using CHQN-OME (a) blue channel image of cells after
incubating with Hoechst and CHQN-OME (20 M); (b) green channel image of the cells; (c) Overlay images of (a) and (b) (scale bar 200 m).
μ
μ
marker for labelling molecules like cholesterol. Moreover, we also hope
that CHQN-OME could be used in future to develop fluorescent lipo-
somes for image-guided therapy.
Data curation. Arghya Adhikary: Visualization, Formal analysis, Re-
sources. Susanta Adhikari: Supervision, Project administration, Fund-
ing acquisition, Writing - review & editing.
5. Conclusions
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgements
Overall we have developed quinoline based fluorescent probes
QNOH-MO and QNOH-OME and their photo-physical property was
examined carefully. The probes were characterized by 1H NMR, 13C
NMR, HRMS and single crystal X-ray structure. QNOH-MO and QNOH-
OME are able to identify small changes in pH by colorimetric and
fluorescence methods. In low pH range, these probes displayed red
fluorescence in both live and fixed cells. However, with increasing pH,
the observed red fluorescence decreases with a simultaneous increase in
green fluorescence. The appearance of unique red fluorescence from
cellular environment may attribute from combined effect of acidic pH
and viscosity or hydrophobicity. This phenomenon was resolved by
theoretical calculations and 1H NMR titration experiments. Addition-
ally, quaternization of the morpholine unit in QNOH-MO induced an
intrinsic cationic property to the molecule. The cationic probe QNOH-
MO-CA showed improved water solubility as well as pH sensing effi-
ciency. Furthermore, QNOH-OME has been utilized as an excellent
marker for fluorescent labelling of cholesterol molecules which is an
important component of all biological membranes as well as in lipo-
somal formulation for drug delivery applications. Therefore, all these
cumulative result suggested that the molecules could be used as cost
effective and easily available probes for measurement of pH changes.
S.G. acknowledges CSIR for financial support. S.A. is thankful to
SERB-DST, New Delhi, India (project no. EMR/2017/002140), for
financial assistance, and T.M. acknowledges the same for the JRF
fellowship. A.G. and A.A. are grateful to DST Nanomission and M.K.D. to
DSKPDF (UGC) for financial support. The authors are thankful to Ms.
Somali Mukherjee for helping them with the crystal data.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2020.
113074.
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