Synthesis and photophysical investigation of 2-hydroxyquinoline-3-carbaldehyde: AIEE phenomenon, fluoride optical sensing and BSA interaction study

Article abstract of DOI:10.1016/j.jphotochem.2018.03.039

Nowadays compounds showing aggregation-induced emission enhancement (AIEE) are extensively studied due to their huge range of applications in material science. AIEE and the anion recognition ability of 2-hydroxyquinoline-3-carbaldehyde (1) were explored. Unique AIEE phenomenon is observed in CH₃CN with water as the cosolvent and the highest emission was noted in the solvent volume ratio 2:8 (CH₃CN: H₂O). The compound is a selective time dependent turn on fluoride ion sensor in acetonitrile medium with a distinct color change from colorless to yellow, fabricating it as a visible sensor. Ion sensing ability was monitored through UV–vis, steady state emission, life time studies and ¹H NMR spectroscopy. The limit of detection of fluoride ion is 4.09 × 10^?6 M. Moreover the compound exhibited notable quenching of fluorescence intensity with bovine serum albumin. Thus small molecule like the quinoline motif can be used in extensive applications in the scientific research.

Full text of DOI:10.1016/j.jphotochem.2018.03.039

Accepted Manuscript
Title: Synthesis and photophysical investigation of
2-hydroxyquinoline-3-carbaldehyde: AIEE phenomenon,
fluoride optical sensing and BSA interaction study
Authors: Nilanjan Chakraborty, Sutanwi Bhuiya, Arijit
Chakraborty, Deep Mandal, Suman Das
PII:
S1010-6030(18)30167-9
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.03.039
JPC 11208
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
6-2-2018
26-3-2018
27-3-2018
Please cite this article as: Nilanjan Chakraborty, Sutanwi Bhuiya, Arijit Chakraborty,
Deep Mandal, Suman Das, Synthesis and photophysical investigation of 2-
hydroxyquinoline-3-carbaldehyde: AIEE phenomenon, fluoride optical sensing
and BSA interaction study, Journal of Photochemistry and Photobiology A:
Chemistry https://doi.org/10.1016/j.jphotochem.2018.03.039
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Synthesis and photophysical investigation of
-hydroxyquinoline-3-carbaldehyde: AIEE phenomenon,
fluoride optical sensing and BSA interaction study
2
a,c
c
b*
c
Suman Dasc*
Nilanjan Chakraborty , Sutanwi Bhuiya , Arijit Chakraborty , Deep Mandal
,
aDepartment of Chemistry, Maulana Azad College 8, Rafi Ahmed Kidwai Road, Kolkata 700
0
13, India.
bDepartment of Chemistry, Acharya B N Seal College, Cooch Behar, West Bengal 730 161,
India.
cDepartment of Chemistry, Jadavpur University, Kolkata 700 032, India.
*
To whom all correspondence should be addressed.
Suman Das
Physical Chemistry Section
Department of Chemistry
Jadavpur University
Raja S. C. Mullick Road, Jadavpur
Kolkata 700 032
India
Tel.: +91 94 3437 3164, +91033 2457 2349
Fax: +91 33 2414 6266
E-mail:sumandas10@yahoo.com
Graphical abstract
1
| P a g e

Highlights

Aggregation induced enhancement in emission intensity of the flurophore 2 ­
hydroxyquinoline­3­carbaldehyde was reported.


Solvent effect on UV-vis and emission spectrum of the fluorophore were studied.
The compound 2­hydroxyquinoline­3­carbaldehyde showed selective naked-eye,
－
UV-vis and fluorescence spectral responses towards F ion.

Interaction of the compound with protein bovine serum albumin (BSA) in buffer tris­
HCl was reported.
Abstract
Nowadays compounds showing aggregation-induced emission enhancement (AIEE) are
extensively studied due to their huge range of applications in material science. AIEE and the
anion recognition ability of 2-hydroxyquinoline-3-carbaldehyde (1) were explored. Unique
AIEE phenomenon is observed in CH CN with water as the cosolvent and the highest
3
2
| P a g e

emission was noted in the solvent volume ratio 2:8 (CH
3
CN : H O). The compound is a
2
selective time dependent turn on fluoride ion sensor in acetonitrile medium with a distinct
color change from colorless to yellow, fabricating it as a visible sensor. Ion sensing ability
1
was monitored through UV-vis, steady state emission, life time studies and H-NMR
-6
spectroscopy. The limit of detection of fluoride ion is 4.09 × 10 M. Moreover the compound
exhibited notable quenching of fluorescence intensity with bovine serum albumin. Thus small
molecule like the quinoline motif can be used in extensive applications in the scientific
research.
Keywords:
2-hydroxyquinoline-3-carbaldehyde;
aggregation-induced
emission
enhancement; naked eye sensing; fluorescence sensor; fluoride ion, bovine serum albumin
interaction.
1
. Introduction
In the very recent years there have been a drift towards designing of molecular systems
(fluorophores) with emission properties in solid state/ aggregate in solution. Compounds
showcasing aggregation-induced emission (AIE) or aggregation-induced emission
enhancement (AIEE) [1-4] have found their applications in organic light emitting devices
(OLEDs) [5, 6], bioimaging applications [7, 8], nonlinear optical materials [9, 10], logic
system modulation [11, 12] and many others. Generally, the fluorophores which are
non-emissive or weakly emissive in nature in solution (solvents with higher solubility) emit
highly upon addition of solvents like water (solvents with lower solubility) due to molecular
aggregation forming hydrosol.
Among anions, fluoride ion tops the list due to its wide range of applications in
biological and medicinal fields. It has been important in dental care, osteoporosis treatment
etc [13], whereas acute exposure results in hypocalcemia, change in thyroid hormone status,
3
| P a g e

fluorosis, nervous system problems, weak bones and ligaments etc [14]. Fluoride being the
most electronegative anion the host-guest interaction mainly occurs through hydrogen
bonding [15-18], so while designing detectors this factor was kept in mind. Most of the
fluoride ion detectors response colorimetrically [15-17, 19]. Many of them shows quenching
[
15, 17, 20] while some of them show enhancement in fluorescence intensity [16, 19, 21, 22].
In many cases they are also responsive to anions like acetate or dihydrogen phosphate [22]
along with fluoride. So, designing selective and sensitive fluoride ion sensor is a challenge.
In the field of drug discovery and pharmaceutical sciences binding of various drugs to
plasma protein like serum albumin is of special interest. The drug-albumin binding
interaction decreases bioavailability and increases the in vivo half life of the drug [23]. The
importance of serum albumin is due to its abundance as a multifunctional protein in blood
plasma. It also act as carrier of many exogenous and endogenous compounds like fatty acids,
amino acids, steroids, etc to blood and transport of drugs and ligands to cells [24-26]. In the
family of serum albumins BSA (bovine serum albumin) being the most extensively studied
one due to its repeating pattern of disulphide linkages and of about 76% of structural
homology to HSA (human serum albumin) [27]. BSA is composed of 583 amino acid
residues in a single polypeptide chain and is sensitive towards intrinsic fluorescence due to
Tryptophan (Trp) residues Trp 134 and Trp 212 [28]. Therefore its binding affinity can be
studied through fluorescence technique.
Quinoline and its derivatives have been a good fluorophore, schiff bases and other
functionalities
-hydroxyquinoline-3-carbaldehyde [29] from which various metal complexes and
fluorescent sensors have been developed [30, 31]. To the best of our knowledge still now
-hydroxyquinoline-3-carbaldehyde itself have neither been targeted as fluorescent sensor nor
its AIEE properties been investigated. We have synthesized
have
been
derived
involving
formyl
group
of
2
2
4
| P a g e

2
-hydroxyquinoline-3-carbaldehyde from reported methods [32] and have proven it to be an
efficient and selective turn on chemosensor for fluoride ions along with AIEE phenomenon in
hydrosol form. We have also explored the studies on the interaction of this compound with
BSA (bovine serum albumin) which revealed quenching of fluorescence intensity of BSA.
2
. Experimental section
2
.1. Materials
All solvents of analytical grade were purchased from Merck, India and reagents including
tetrabutyl ammonium fluoride were obtained from Spectrochem, India and were used without
further purification. Distilled water was used for AIEE studies. Bovine serum albumin (BSA)
was obtained from Sigma Aldrich Corporation. By using known extinction value of 43824
•1
•1
M cm at 280 nm concentration of BSA solution was determined. Tris base was obtained
from Sigma Aldrich and to the 50 mM solution of the tris base appropriate amount of HCl
was added drop wise to make tris-HCl buffer of pH=7.4. The buffer solution was filtered
through milipore filter paper of 0.45 μm pore size. Distilled and deionized water was used
throughout the experiments.
2
.2. Methods
2
.2.1. Physical measurements
1
13
H NMR and C spectra were acquired using a Bruker 300 MHz (Bruker AVANCE 300)
spectrometer and were recorded at an ambient temperature. ESI_MS was recorded on
Q-tof-micro quadrupole mass spectrometer. IR spectra were obtained in KBr discs on a
-1
Spectrum Two FT-IR spectrometer in the 4000 - 400 cm region. The melting points were
determined on a LabX, India Digital Melting Point apparatus and are uncorrected. All
weighing was done in Mettler Toledo Analytical Balance Band (Model: MS 2045).
2
.2.2. UV-vis and spectrofluorimetric studies
5
| P a g e

All UV-vis spectra of receptor were recorded in HPLC grade acetonitrile on a
Perkin-Elmer UV/VIS spectrometer (Model: Lambda 25) in matched quartz cells of 1 cm
path length.
Steady state fluorescence spectra were generated on a Perkin Elmer Fluorescence
spectrometer (Model: LS 55). Measurements were made in fluorescence free quartz cells of
path length 1 cm. All the measurements were done keeping an excitation and emission band
pass of 10 nm and 5 nm. The excitation wavelength was fixed at 378 nm for anion
recognition and other physical studies of compound 1. For studies on the interaction with
BSA excitation was made at 290 nm.
2
.2.3. Fluorescence quantum yield measurement
The fluorescence quantum yield of the compound 1 was measured with reference to
quinine sulphate in 0.1 N H
2
SO
4
using the following equation [33, 34]:
2
 s
 r
I s
I r
Ar  s



    (1)
2
As  r
Where ϕ
is the absorption maxima of the reference, A
integrated peak area at the maximum intensity of the reference, I
at the maximum intensity of the sample, η is the refractive index of the reference medium, η
is the is the refractive index of the sample medium.
.2.4. Determination of binding constant
The spectrophotometric titration data were used to construct Benesi-Hildebrand [35] plot
to determine binding constant from the following equation
s
is the quantum yield of the sample, ϕ
r
is the quantum yield of the reference used, A
r
s
is the absorption maxima of the sample, I
r
is the
s
is the integrated peak area
r
s
2

A
m ax
1

1 
    (2 )

A
K [C ]
ΔA= |A -A
x 0
| and ΔAmax= |A
∞
-A
0
|. A
0
, A
x
and A are the absorbances of compound 1 in
∞
-
absence of F , at an intermediate anion concentration and at a concentration of complete
6
| P a g e

-
saturation with F respectively. K represents the binding constant of the complexation
-
-
between compounds 1 and F , [C] is the concentration of the variant (here in F ). Plot was
made with ΔAmax/ΔA as a function of 1/[C]. The binding constants can be obtained from the
slope.
The binding constant for BSA protein binding studiers was obtained from emission
titration profile utilizing Benesi-Hildebrand equation:

I m ax
1

1 
    (3)
I
K [C ]
ΔI = |I -I
x 0
| and ΔImax= |I
∞
-I
0
|. I
0
, I
x
and I are the emission intensities of BSA in absence of
∞
compound 1, at an intermediate anion concentration and at a concentration of complete
saturation with compound 1 respectively. K represents the binding constant of the complex
formation between compounds 1 and BSA and [C] is the concentration of the variant (here in
compound 1).
2
.2.5. Determination of binding stoichiometry
Job’s method [36] of continuous variation was employed to estimate the stoichiometry of
－
complexation of F with compound 1 using absorbance spectroscopy. The difference in
absorbance (ΔA) between the absorbance of free ligand and absorbance after anionic
-
interaction was plotted against the increasing mole fraction of F . Sum of the concentrations
of ligand and anion was maintained at 50 M with varying their mole fractions from 0 to 1.
The corresponding absorbances were taken at max 378 nm for studies with receptor 1. The
-
-
break point in the resulting plot corresponds to the mole fraction of F in the F :1 complex.
Therefore, the stoichiometry is obtained in terms of anion:ligand [anion:(1-anion)] where anion
is the mole fraction of anions calculated as the ratio of anions to the total molar concentration
of receptors and anions
2
.2.6. Limit of detection
7
| P a g e

The detection limit wascalculated using calibration curve obtained from the UV-vis titration
profile with the help of the equation:
3

L.O .D 
    (4 )
S
Where σ is the standard deviation of the blank solution (relative absorbance ratio at 302 nm
-5
to 378 nm of 5 × 10 M solution of 1 in acetonitrile only), and S is the slope of the relative
-
absorbance versus [F ] calibration curve in the lowest concentration range.
2
.2.7. Fluorescence lifetime studies
Time correlated single photon counting (TCSPC) measurements were carried out in
various water fractions for AIEE properties, in acetonitrile for anionic interaction and in
tris-HCl buffer for BSA interaction. For both the studies the photo excitation was made at 336
nm using a picosecond diode laser (IBH Nanoled 07) in an IBH fluorocube apparatus. The
fluorescence decay data were collected on a Hamamatsu MCP photomultiplier (R3809) and
were analyzed by using IBH DAS6 software using the following equation [37]:

t 
F

t


  i e
    (5)



 
Where, α
time of the excited species.
.2.8. Synthesis
i
is the i-th pre-exponential factor and τ is the decay time. The decay time is the life
2
Synthesis of 2-chloroquinoline-3-carbaldehyde:
To a solution of acetanilide (5 mmoles) in dry DMF (15 mmoles) at 0-5 ºC POCl (60
3
mmoles) was added drop wise with stirring and the mixture was further stirred at 80-90 ºC for
6 hrs. The mixture was poured into crushed ice with stirring. After 5 minutes the resulting
1
solid was filtered, washed and dried. The compound was purified by recrystallization from
ethyl acetate and the melting point was found to be 148 ºC.
Synthesis of 2-hydroxyquinoline-3-carbaldehyde (1):
8
| P a g e

A suspension of 2-chloroquinoline-3-carbaldehyde (1 mmole) in 70% aqueous acetic acid (10
mL) was heated under reflux for 4 hours. The completion of the reaction was checked by
TLC. Upon cooling the reaction mixture, a solid product precipitated out which was filtered,
1
washed with water, dried and purified by recrystallisation from DMF. mp: 303-305ºC. H
NMR (300MHz, DMSO-d
6
):  (ppm) 12.22 (s, 1H), 10.23 (s, 1H,), 8.49 (s, 1H) 7.90 (d, J =
7
.68, 1H), 7.64 (d, J = 7.47, 1H), 7.34 (d, J = 8.25, 1H), 7.25 (d, J = 7.56, 1H) (Fig. S1), FT
̵
13
IR (KBr) ν cm ): 3139 (N-H), 1681 (CHO), 1612 (CO) (Fig. S2), C NMR (300 MHz,
DMSO-d
6
,  (ppm)) : 190.25, 161.91, 142.91, 141.63, 134.16, 131.39, 126.08, 123.13,
+
1
18.62, 115.89 (Fig. S5), HRMS (m/z): [M+H] : Calculated: 174.06, Found: 174.13 (Fig.
S6).
3
. Results and discussion
3
.1 Synthesis
We have synthesized 2-hydroxyquinoline-3-carbaldehyde from acetananilide through the
formation of 2-chloroquinoline-3-carbaldehyde by using Vilsmeier-Haack formylation
method and subsequent hydrolysis of the chloro derivative (scheme 1) using methods
reported in literature [32]. The compound 1 is a lactam system which can be easily converted
to the corresponding iminol system. From the literature it is unclear which specific form be
predominant in a particular solution condition. In fact there are several reports [30, 31, 38,
3
9] for the existence of both the forms. Probably, the change of tautomeric forms depends on
the interaction with analyte.
9
| P a g e
Scheme 1. Synthesis of 2-hydroxy quinoline-3-carbaldehyde (1)

3
3
.2. AIEE characteristics of compound 1
.2.1. UV-vis and fluorescence studies on AIEE characteristics of compound 1
10 | P a g e

UV-vis and emission spectra of the fluorophore 1 were taken in different proportions of
CH CN/water (Fig. 1) to study the effect on its photo physical properties upon aggregation.
Compound 1 was well dissolved in a good solvent CH CN where it gave a characteristic
3
3
spectrum having two absorption bands at 378 and 302 nm and it displayed a moderately weak
emission spectrum with emission maxima at 436 nm with a very low quantum yield of
ϕ=0.0042. With the increase in fraction of poor co solvent water emission maxima showed a
positive stoke shift from 437.5 nm to 473 nm with increase in emission intensity. The positive
stoke shift continued to 493 nm up to 70% of water fraction with drastic increase in emission
intensity in a continuous manner. Then there was a sudden shift to 502 nm with maximum
intensity (8.5 fold of intensity in pure CH
However, upon further increase in water fraction the intensity decreases (7.2 fold of intensity
in pure CH CN) with no further shift up to 100% of water fraction. AIEE feature was also
photographed under UV lamp at 366 nm (Fig. 1C). In absorption spectra there were small
3
CN) at 80% of water fraction with a ϕ=0.098.
3
11 | P a g e

changes upon on increment of water fraction with a slight blue shift up to370 nm from 378
nm in pure water. In our quinoline based compound 1 the free carbonyl group may serve as
the rotatable unit which weaken the molecule relaxation channels for deactivation of the
exitons. So in this case the enhancement of emission is probably not due to cause of restricted
intramolecular rotation (RIR) but of a more generalized concept of Restricted intramolecular
motion (RIM) [40]. With increase in water fraction the faint emission gradually got increased
with a gradual red shift as discussed before. It may be due to the lowering of the band gap
with increase in solvent polarity which is the expected characteristics of AIEE behavior. At
water fraction above 80% the decrease in intensity is probably due to insolubility of the
compound.
3
.2.3. Time course of Fluorescence response to AIEE properties: Lifetime studies
To further confirm the AIE nature time resolved fluorescence spectra was taken where
representative decay profiles of intensity (log scale) versus time (ns) are presented in the
Fig. 1. AIEE spectral properties depicted by various fractions of hydrosols of compound 1. A:
Changes in absorption spectra, B: Changes in emission spectra, C: Naked eye AIEE
characteristics under UV light (366 nm) (increasing water fractions from left to right)
12 | P a g e

Fig.2. and the corresponding data are summarized in Table 1. Compound 1 in free form was
found to exhibit bi-exponential decay profile with average lifetime 0.86 ns in pure
acetonitrile. The bi-exponential decay was also found to be the characteristic of
fluorohore1inhydrosol form. The probable explanation of bi-exponential decay profiles of
fluorohore1 both the conditions indicate two different forms [30] i.e., the keto and enol form
of the fluorohore 2-hydroxy quinoline-3-carbaldehyde in the excited state. The average life
time (τ) of the hydrosol were found to be 1.91 ns and 1.54 ns for 50% and 80% water
fraction. The life times of the hydrosols are much more than pure acetonitrile solution of
compound 1 which is 0.86 ns. 80% water containing hydrosol flourished more than its 50%
counterpart but stability of 50% water containing hydrosol in excited state is the maximum as
depicted from the fluorescence life time studies. Pure water solution of the fluorophore 1
exhibited a life time value of 1.46 ns which is less than 80% hydrosol which indicates at
reduction in stability of the hydrosol in the excited state with increasing water fraction. From
the average lifetime of the hydrosols and their quantum yield the radiative rate constant (K
and the non radiative rate constants (Knr) were calculated from the following equations:
r
)

1

 K r  K n r     (6 )

K r

    (7 )

The data related to decay profile have been depicted in the table where α
1
and α represent the
2
13 | P a g e
Fig. 2. Decay profile of fluorophore 1 in acetonitrile and other hydrosol fractions.

relative amplitude of the decay components in the excited state and other terms have their
usual meaning as mentioned in the text. From the life time study it is evident that at 0 %
water fraction the non radiative decay constant was much higher compared to radiative decay
constant but with increase in water fraction the non radiative decay constant decreased from
1
.155 ns to 0.615 ns. This fact supports that through the RIM mechanism is operative in
aggregate solution the non-radiative decay process is getting hampered enhancing the
fluorescence emission.
3
.2. Effect of viscosity and solvent polarity on emission spectra
Effect of viscosity on emission spectra was observed by increasing viscosity of the solvent by
increasing fraction of glycerol in ethanol. With increasing viscosity in solvent, the emission
intensity also got enhanced up to 75% and drops down at pure glycerol medium (Fig. 3).
Enhancement due to increase in viscosity is probably due to restricted intramoleclar motion
(RIM) in a more viscous solvent and reducing the nonradiative emissive pathways. In pure
glycerol the intensity drops due to agglomeration of particles in the medium of maximum
viscosity.
Table 1. Decay parameters of the time resolved study of various hydrosol fractions of the fluorophore 1.
%
of water
in the
Average life
time (τ) the Quantum
fluorophore
1 (ns)
Radiative
rate
Non
radiative rate
constants
τ
(ns)
1
τ
(ns)
2
(α
1
)
(α
2
)
χ2
CH
medium
3
CN
yield (ϕ)
constant
-1
-1
(K
r
) (ns ) (Knr) in ns .
0
0.17 78.47 3.38
21.53 1.34
22.3 0.99
0.86
0. 0042
0. 0786
0. 0980
0. 0950
0. 0049
0. 0408
0. 0637
0. 0646
1.155
0.479
0.586
0.615
5
8
0
0
1.02
0.08
77.7 5.00
8.24 1.68
1.91
1.54
1.46
91.76 1.22
77.42 1.00
100
0.04 22.58 1.87
Fig. 3. Emission spectra of compound 1 in various fractions of glycerol in ethanol.
14 | P a g e

While studying effect of solvent on the absorption and emission spectra of the quinoline
-5
moiety 1 the spectra were recorded in various solvent with solute concentration of 5×10 M
(Fig. 4). In absorption studies there is no remarkable change on the two absorption bands.
The higher energy band II remained more or less unchanged whereas slight changes were
visible on the lower energy band I, but no clear-cut trend of solvent dependency of the
compound 1 on the solvent polarity was observed. But noticeable changes can be found in the
emission spectrum with different solvents. The wavelength at emission maximum for
chloroform, toluene, dimethyl formamide, dimethyl sulfoxide centered between 461.5 nm to
4
63 nm. Emission maxima for hexane and acetonitrile were at 426.5 and 436 nm respectively.
-5
Fig. 4. Solvent effect on absorption and emission spectrum of compound 1 (5 × 10 M).
Only for polar protic ethanol emission maximum was observed at a longer wavelength of
82.5 nm. The fluorophores are generally weak fluorescent in hydrophilic (polar) solvents
and high fluorescent in hydrophobic (non-polar) solvents but in some cases the reverse trend
strong fluorescence in protic solvents and weak fluorescence in aprotic solvents) is observed
4
(
like acridine and 7-methoxy-4-methylcoumarin [41, 42]. In case of our compound 1 the
reverse trend was noticed where maximum emission intensity was observed in polar protic
ethanol followed by aprotic dimethyl formamide, acetonitrile, toluene dimethyl sulfoxide,
chloroform and hexane. The solvatochromism data are depicted in Table 2.
15 | P a g e

3
3
.3. Anion recognition
.3.1. Naked eye sensing
16 | P a g e

Naked eye experiment of anion interaction was carried out in acetonitrile. To a solution of
-4
-4
-
-
-
-
-
PO -
compound 1 (9 × 10 M) in acetonitrile 15 × 10 M anions (F , Cl , Br , I , AcO , H
2
4
,
-, NO
-, AsO
-, N
-
) were added. It was observed that for F ion the colorless receptor
̵
HPO
4
3
2
3
solution changed to yellow color indicating a host-guest type of interaction between charge
-
neutral receptor 1 and charged F ion. No such obvious change of color was observed with
-
-
-
-
PO -
2-, NO -
, AsO -
, N -
) which may be due to feeble
other anions (Cl , Br , I , AcO , H
2
4
, HPO
4
3
2
3
17 | P a g e

Table 2. Parameters related to UV-vis and emission spectra of compound 1 in various solvents.
Dielectric
constant (ε) Band I
at 20 ℃
20.7
λ
max at
λ
max at
λ
maxat
Intensity at
emission
maxima
102.79
Solvent
Acetonitrile
Band II in
nm.
emission
maxima
436
(nm)
378
302
Dimethlformamide
Chloroform
Dimethylsulfoxide
Ethanol
36.7
4.81
46.7
24.5
2.38
1.89
382
378
385
377
384
382
303
302
305
301
303
307
462.5
461.5
463
161.64
57.92
85.04
254.12
113.39
1.97
482.5
462.5
426.5
Toluene
Hexane
or no interaction with the receptor (Fig. 5). This colorimetric sensing indicated some sort of
-
host-guest interaction between the neutral receptor and charged anion F . Similar experiment
was also performed under ultraviolet radiations of 366 nm where weak turn on emission was
-
visible with F in comparison to free receptor 1. No turn on effect was evident in case of
-
-
-
-
PO -
2-, NO -
,
interaction of compound 1 with other anions viz. Cl , Br , I , AcO , H
2
4
, HPO
4
3
AsO
2
-, N -.
3
-4
-4
Fig. 5. Colorimetric changes of compound 1 (9 × 10 M) in acetonitrile in presence of 15 × 10 M
anions A: naked eye B: under UV light at 366 nm.
18 | P a g e

1
3
.3.2. H NMR studies
1H NMR spectra of the receptors 1 was recorded in DMSO-d6 both in the presence and absence
1
of fluoride ion to know the nature of interaction between them. H NMR spectrum of the
receptor 1 showed -OH proton signal at δ 12.2 ppm (Fig. 6). To investigate anion binding
1
-
properties of the receptor 1, the H NMR spectral change caused by the addition of F as
-
tetrabutylammonium salts were studied. The addition of F was increased gradually from 0 to 1
-
equivalent. On adding 1 equivalent of F the proton signals corresponding to -OH proton at δ
1
2.2 disappeared.
1
-
Fig. 6. Partial H-NMR studies before and after addition of 1 equivalent of F to receptor 1.
The disappearance of the proton signal is possibly due to H-bonding interactions between
-
slightly acidic -OH protons and F ions. As a result of this interactions the electron density in
the ring increases thus shielding also increases which further caused an upward shift of field
for the ring protons.
3
.3.3 Absorbance studies on anionic interaction
1
To corroborate with colorimetric and H-NMR studies anionic interaction was investigated
through absorption studies. The receptor itself showed two absorption maxima at 378 nm and
3
02 nm in acetonitrile medium.
19 | P a g e

-3
UV-vis studies were done with 10 × 10 M standard solution of anions in acetonitrile. To
-5
5
× 10 M solution of receptor 1 in acetonitrile in the cuvette 10 equivalent of each of the
anions were added and changes in the absorption spectra were noticed (Fig. 7).
The free receptor exhibited absorption band at 378 and 302 nm which corresponds to two
isomeric forms of the quinoline moiety. Upon excess addition of anions, the absorption band
at 378 nm got drastically diminished and absorption band at 302 nm increased in intensity
-
with a blue shift to 292 nm for F ion. There was insignificant change in the absorption band
of the sensor negligible for other anions (Fig. 7) suggesting that the interaction is occurring
-
only for F ion.
-5
Fig. 7. Changes in absorption spectra of 1 (5×10 M) after addition of 10 equivalent of each anion.
-
-5
UV-vis titration was performed with F ion in which 5 × 10 M solution of 1 in acetonitrile
-
-6
was titrated by gradual addition of F in acetonitrile upto 52 × 10 M (Fig. 8). The absorption
band at 378 nm got gradually diminished and enhancement of the absorption band at 302 nm
happened. Ultimately the absorption maximum at 302 outlives. Towards saturation a positive
blue shift of the band at 302 nm to 292 nm occurred accordingly the faint colored solution of
the receptor 1 changed to deep yellow color. The course of titration was associated with two
20 | P a g e

isosbsetic points at 425 nm and 329 nm indicating equilibrium between receptor 1 and 1.F
complex formed in due course of titration. The obtained fact indicates at strong hydrogen
-
bonding interaction and further deprotonation upon excess addition of F ion. All of these
observation points at acidic nature of OH present in the receptor and highly basic nature of F-
ion.
Fig. 8. Spectral titration of 1 (5.0 × 10-5 M) in CH
CN (2 mL) (curve 1) with the addition of
3
-
-5
incremental amount of F (52 × 10 M) (curve 12). Inset: Variation of maximum absorbance with
fluoride ion concentration in μM at 302 nm and 378 nm.
The extent of interaction was understood from the determination of binding constant
-
form Benesi-hildebrand plot (Fig. 9) keeping in mind 1:1 complexation of 1 and F (which
1
was indicative from H NMR studies and Jobs plot which is discussed a little later). The value
3
-1
of binding constant obtained is 2.641 × 10 M . The value of binding constant indicates at the
acidity of OH protons which are participating in anionic interaction.
21 | P a g e

-
Fig. 9. Benesi-Hildebrand plot for binding of F ion with 1 (absorbance taken at 378 nm).
-
The detection limit of the receptor 1 towards F was determined from the calibration curve
(Fig.10) as mentioned in the experimental section. The limit of detection (L.O.D) was
-6
determined to be as low as 4.09 × 10 M.
-
Fig. 10. Linear fit of ratiometric absorbance with increasing F concentration.
22 | P a g e

Job’s method of continuous variation was employed to determine the stoichiometry of
-
complex formation between anion F and compounds 1 using absorption profile. In our study,
the inflection point was found to be at 
-=0.49. From this inflection point stoichiometry of
F
-
the F :1 complex as calculated are ~1:1 (Fig. 11).
-
Fig. 11. Job’s plot for binding of F ion with 1 (absorbance taken at 378 nm).
3
.3.4. Emission Studies on anionic interaction
The receptor 1 showed two emission maxima at 436 nm and 461 nm when excited at 378
nm in acetonitrile medium (Fig. 12). The emission slit was kept at 5 nm and excitation slit
was kept at 10 nm. The two emission bands correspond to two isomers of the quinoline
-5
-
-
-
-
-
PO -
, HPO
2-,
moiety. On one-time addition of the 1 × 10 anions viz. F , Cl , Br , I , AcO , H
2
4
4
-, AsO
-, N
-
to 1 × 10 of the solution of sensor 1 was done to monitor the selectivity of
-5
NO
3
2
3
the sensor already noticed from absorption studies. The emission band of the free receptor at
-
4
36 nm and 461 nm showed fluorescence enhancement upon addition of F , whereas similar
addition of other anions resulted in some minor changes in intensity of the existing emission
spectra of the receptor.
23 | P a g e

-5
-5
Fig. 12. Emission studies of 1 × 10 M solution receptor 1 with 1 × 10 M of anions.
The sensor 1 showed a time dependent enhancement of fluorescence intensity upon
-
-
addition of F ions. To 10 µM solution of the receptor in acetonitrile 10 µM of F ions was
added for kinetic study (Fig. 13). In the kinetic study the valley was obtained after 50 scans at
an interval of 1 minute. At initial level the emission maxima both at 437.5 nm and 461 nm
-5
Fig. 13. Steady state increase in emission intensity of 1 × 10 M solution receptor 1 with
-5
-
1
× 10 M of F with increase in time.
24 | P a g e

showed enhancement of emission intensity. As the time progress the band at 461 nm saturates
while the band at 437.5 nm continued to show enhancement until stabilization. At the
beginning two emission bands showed enhancement due to hydrogen bond formation of
F-with both the isomeric forms of ligand 1. With progress of time a stable hydrogen bonded
form outlives till the stability in enhancement with time was reached.
After attainment of stability in spectra with time fluorescence titration with incremental
-
addition of F ion was performed. The emission band at 437.5 nm started to diminish in
intensity with a positive Stoke shift to 496 nm where the intensity enhances until saturation
-5
-
upto addition of 155×10 M of F (Fig. 14). This phenomenon probably corresponds to
-
deprotonation with increase in F ion concentration.
-5
-
Fig. 14. Fluorescence titration of ligand 1 (1 × 10 M) after attainment of stability with time with F
-5
-5
from 5 × 10 M (curve 1) to 155 × 10 M (curve 32) fluoride ion concentration. Inset: Variation of
maximum intensity with fluoride ion concentration in μM at 437.5 nm and 496.5 nm.
3
.3.5. Fluorescence Lifetime studies on anionic interaction
In our life time studies representative decay profiles of intensity (log scale) versus time (ns)
are presented in the Fig. 15 and the lifetime data are presented in Table 3. α and α represent
1
2
25 | P a g e

Fig. 15. Decay profile of fluorophore 1 in acetonitrile before and after interaction with F-.
the relative amplitude of the decay components in the excited state. Lifetime signifies the
average amount of time fluorophore herein compound 1 resides in the excited state during
excitation. Moreover for multi-exponential decay, lifetime is a statistical average. As stated
earlier compound 1 in free form was found to exhibit bi-exponential decay profile with a
-
lifetime of 0.86 ns. In our experimental condition the fluorophore 1 upon binding with F also
exhibited a bi-exponential decay and the lifetime value was increased, which provide two
lifetime values like free fluorophore. Most probable explanation of this observation is that
along with the presence of two different forms of the ligand [24] in the free form and
presence of bound fluorophore along with trace amount of free fluorophore after anionic
interaction. Lifetime value was increased from 0.86 ns to 1.03 ns. Increase in lifetime value
-
indicated the stronger binding of fluorophore 1 with F in the experimental condition.
-
Table 3. Life time decay parameters of fluorophore1 upon interaction with F ion.
Average life time (τ)
Compounds
τ
1
(ns) (α
0.17 78.47 3.38
0.15 68.8 2.98
1
)
τ
2
(ns)
(α
2
)
of the fluorophore 1
ns)
χ2
(
1
21.53
31.20
0.86
1.34
1.46
1
+[F-]
1.03
26 | P a g e

3
.3.6. Selectivity of fluoride sensing: Competitive experiment
Competitive experiments were carried out after attainment of stability with time. To the
-5
-
-5
-5
mixture of receptor 1 (1 × 10 M) and F (5 × 10 M) various anions (5 × 10 M) were added
-
to find their effect on the F interaction with ligand 1. In the competitive there were no or
slight changes in the fluorescent intensity at 437.5 nm (Fig. 16). Thus selectivity and
uniqueness of sensor 1 for fluoride ion sensing was once again established.
-5
Fig. 16. Competitive experiment with various anions (5 × 10 M) to the mixture of receptor 1
-5
-
-5
(
1 × 10 M) and F (5 × 10 M).
3
.4. Interaction of compound 1 with BSA
The compound 1 was found to interact with protein bovine serum albumin (BSA) in
-5
tris-HCl (pH=7.4) medium. The quinoline 1 (5.0 × 10 M) exhibited two absorption maxima
at 302 nm and 369 nm (Fig. 17). Absorption intensities at the maxima got reduced upon
-7
addition of BSA solution (Upto 3.81 × 10 M). Two clear isosbestic points were noted at 320
nm and 347 nm indicating an equilibrium between free and BSA bound compound 1.
-7
In fluorescence titration experiment 3.81 × 10 M BSA was titrated with increasing
-5
concentration of compound 1 upto 10 × 10 M (Fig. 18.). BSA exhibited emission maxima at
27 | P a g e

-5
-7
Fig. 17. UV-vis titration spectra of 1 (5.0 × 10 M) (curve 1) with 3.81 × 10 M BSA
(curve 6).
3
46.5 nm in tris-HCl (pH=7.4) medium which upon interaction with compound 1 got
quenched. The association constant was evaluated from the fluorescence titration of BSA with
compound 1 (mentioned later). From the fluorescence titration we have also estimated the
Stern Volmer quenching constant using the Stern-Volmer equation since there was reduction of
BSA fluorescence on interaction with the said compound.
-7
-5
Fig. 18. Emission titration spectra of BSA with 1. (Curve 1) 3.81 × 10 M BSA. (Curve 11) 10 × 10
M of compound 1.
28 | P a g e

The Stern-Volmer equation is:
I
0

1  K S V [ X ]     (8)
I
where, I and I are the fluorescence intensities of the free fluorophore and complex
o
between fluorophore and quencher, [X] is the concentration of the quencher ion and KSV is the
Stern–Volmer quenching constant. KSV is indicative of the accessibility of the bulky quencher
to the fluorophore 1. The slope of the I
/I versus [X] obtained for the quenching is more or less a linear one throughout indicating
the quenching to be static in nature. From the slope obtained in the plot the KSV value obtained
0
/I versus [X] plot yields the value of KSV. The plot of
I
0
5
-1
obtained is 4.01×10 M for quenching of emission intensity of free BSA upon binding with
compound 1 (Fig.19). The quenching efficiencies were calculated to be 82.22%. Binding
constant of protein-1 complex formation was obtained from emission profile with the help of
Benesi-Hildebrand plot (Fig. S3) where ΔImax/ΔI was plotted as a function of 1/[C] as
5
-1
mentioned in the experimental section. The binding constants came out to be 4.21×10 M
from the slope which indicates at strong interaction between compound 1 and protein BSA.
Fig. 19. Stern-Volmer plot for quenching of fluorescence intensity of BSA at 346.5 nm.
29 | P a g e