Binding of 6-methyl-3-phenyliminomethyl-4H-chromen-4-one with bovine serum albumin in free and β-cyclodextrin-complexed forms: Modulation of the binding by β-cyclodextrin

Article abstract of DOI:10.1016/j.molliq.2016.07.056

We report in this paper that β-cyclodextrin modulates the binding of a Schiff's base derivative, 6-methyl-3-phenyliminomethyl-4H-chromen-4-one, with the protein bovine serum albumin. The stoichiometry, the association constant, and the mode of association

Full text of DOI:10.1016/j.molliq.2016.07.056

Journal of Molecular Liquids 222 (2016) 398–406
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Binding of 6-methyl-3-phenyliminomethyl-4H-chromen-4-one with
bovine serum albumin in free and β-cyclodextrin-complexed forms:
Modulation of the binding by β-cyclodextrin
Sowrirajan Chandrasekaran a, Natesan Sudha a, Dhanaraj Premnath b, Israel V.M.V. Enoch a,
⁎
a
Nanotoxicology Research Lab, Department of Science and Humanities, Karunya University, Coimbatore 641 114, Tamil Nadu, India
b
Department of Biosciences & Technology, Karunya University, Coimbatore 641 114, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report in this paper that β-cyclodextrin modulates the binding of a Schiff's base derivative, 6-methyl-3-
phenyliminomethyl-4H-chromen-4-one, with the protein bovine serum albumin. The stoichiometry, the associ-
ation constant, and the mode of association of the derivative with β-cyclodextrin are investigated by ultraviolet-
visible absorption, steady-state and time-resolved fluorescence, and proton nuclear magnetic resonance and two
dimensional correlation spectroscopic techniques. The structure of the host–guest complex is proposed. The
binding of the chromen-4-one with bovine serum albumin and the influence of the added β-cyclodextrin on
the binding are reported. β-cyclodextrin is found to decrease the Stern–Volmer quenching constant and the bind-
ing strength of the compound with the protein. The donor-to-acceptor distance is altered by the addition of β-cy-
clodextrin as studied by Förster resonance energy transfer. The binding sites of the chromen-4-one with bovine
serum albumin are reported by molecular modeling.
Received 17 January 2016
Received in revised form 13 July 2016
Accepted 15 July 2016
Available online 19 July 2016
Keywords:
Schiff's base
β-cyclodextrin
bovine serum albumin
fluorescence spectroscopy
ROESY
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
We consider such an investigation substantial due to the following
reasons: (i) gathering information on whether cyclodextrin complexa-
Albumins form drug–protein complexes and transport the drugs to
their destination. Since serum albumins are present in much higher
amounts in the blood than the other proteins, most of the studies focus-
ing on protein–drug binding is centered on serum albumins. The fate of
therapeutic drug molecules in the blood stream needs to be studied in
detail in order to understand their pharmacological behavior [1]. The
bio-distribution, and the elimination of drugs occurring in the body de-
pends on the strength of the protein–drug binding [2,3]. Bovine serum
albumin (BSA) is a model protein used for studying the serum protein
binding of drugs [4]. It is mostly identical to human serum albumin in
its sequence of amino acids. About 52–62% of the total plasma protein
fraction are represented by BSA [5]. There are three domains in BSA
viz., I, II, and III domains and these in turn contain sub-domains [6,7].
Trp-134 and Trp-212 are the tryptophans present in BSA, with Trp-
134 the sub-domain IB [7–9].
tion increases or decreases the strength of binding of drugs with carrier
proteins can help understanding the release rate of drugs, (ii) the idea of
blocking a part of the small molecule can lead to better knowing the
pharmacophore activity, and (iii) the mechanism of action of specific
pro-drugs can be exactly known by blocking their cleavage by cyclodex-
trins and studying their pharmacokinetics. Our lab has been active in
this area of research since the work hinges on the rationale of novel
sustained release of drugs from carrier molecules and the tuning of
drug binding to macromolecular targets [19–21] .
Coumarins form a structural part of many drugs. After oral adminis-
tration coumarin is absorbed and metabolized, only 2 to 6% of it reaches
the systemic circulation [22]. Due to the importance of coumarin in its
in-vivo action, it has now been accepted as a pro-drug. About 35% of cou-
marins and 47% of 7-hydroxycoumarins bind to plasma proteins [23].
Pertaining to the pharmacological significance of aminocoumarin deriv-
atives, we synthesized a new coumarin Schiff's base, 6-methyl-3-
phenyliminomethyl-4H-chromen-4-one (MPIMC). In host-guest com-
plexes, the part of guest molecules which remain outside the cyclodex-
trin cavity can bind to bio-macromolecules. Encapsulation by CD can
tune the binding of small molecules with DNA [24–26]. However, stud-
ies on the binding of organic molecules in cyclodextrin–encapsulated
form with protein are scarce [27]. In this paper, we extend insight into
the Schiff's base's encapsulation by β-cyclodextrin (β-CD) and the bind-
ing of the compound in free and β-CD–encapsulated forms with BSA.
β-Cyclodextrin is a cyclic doughnut shaped compound having seven
D-glucopyranose units [10–12]. This molecule has a hollow center
which can accommodate a variety of drug molecules based on their
size and hydrophobicity. The complexes formed by β-cyclodextrin can
enhance the solubility and stability of drugs in water [13–16] and in-
crease their bio-availability [17,18].
⁎
Corresponding author.
E-mail address: drisraelenoch@gmail.com (E. Israel V.M.V.).
http://dx.doi.org/10.1016/j.molliq.2016.07.056
0167-7322/© 2016 Elsevier B.V. All rights reserved.

C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
399
Fig. 1. The reaction scheme for the synthesis of MPIMC.
The binding strengths of MPIMC and MPIMC/β-CD with BSA are calcu-
lated using fluorescence spectroscopic studies. The resonance energy
transfer (FRET) efficiencies between the BSA and MPIMC in water and
in aqueous β-CD solution are explained.
room temperature. Pale yellow crystals were separated out. The com-
pletion of the reaction was monitored by TLC and purified by column
chromatography using hexane:ethyl acetate mixture (80:20) as eluting
solvent. The 1H and 13C-NMR spectra of MPIMC are shown in Figs. SI 1
and 2 respectively (in the supporting information). 1H-NMR (500 MHz,
CDCl3): Chemical shift, δ 2.342 (s, 6–CH3), δ 11.897 (d, 2–CH), δ 13.755
(d, 11–CH), δ 7.111–8.998 (Aromatic protons). 13C-NMR (500 MHz,
CDCl3): Chemical shift, δ 20.32 (6–CH3), δ 163.75 (2–C), δ 125.18 (3–
C), δ 154.46 (11–C), δ 181.63 (4 C = O), δ 116.2–152.68 (Aromatic
carbons).
2. Experimental
2.1. Chemicals
Sigma–Aldrich product of 6-methyl-3-formylchromone (AR) was
purchased from Bangalore. Aniline (AR) was obtained from Merck,
India and used as such without further purification. Solutions of various
pH were made with phosphate buffer mixing phosphoric acid and sodi-
um hydroxide (Qualigens). A modified Hammett's acidity scale [28] is
used for the measurement of H0 and pH value below the pH 2. Crystal-
line bovine serum albumin, HEPES buffer and β-cyclodextrin were pur-
chased from Hi Media. All the solvents (products of Merck) were of
spectral grade and used as received.
2.3. Preparation of MPIMC/β-CD solid complex
In a 50 ml beaker, MPIMC (0.25 g, 0.95 mmol) was dissolved in 5 ml
of methanol. 5 ml of aqueous β-CD (1.0 g, 0.95 mmol) was prepared in
another 50 ml beaker separately. A solution of MPIMC was added slowly
to the solution of β-CD at room temperature in an Ultra-sonicator and
maintained for 30 min to get homogenous solution medium. Then the
mixture was warmed to 50 °C for 10 min and kept at room temperature
for two days. The solid obtained was re-crystallized and analyzed.
2.2. Synthesis and characterization of MPIMC
6-Methyl-3-phenyliminomethyl-4H-chromen-4-one was synthe-
sized as per the reported procedure [29] and the reaction scheme is
given in Fig. 1. An equimolar (2.66 mmol) amount of 3-formyl-6-
methylchromone (0.5 g) and aniline (0.25 g) were stirred with 50 ml
of methanol. The mixture was heated to 60 °C for 0.5 h and cooled to
2.4. Preparation of test solutions
Test solutions were prepared by appropriate dilution of a stock solu-
tion of MPIMC. The stock solution of MPIMC was made with methanol
due to its poor solubility of in water. The test solutions were having
Fig. 2. (a). Absorption spectra of MPIMC in varying concentrations of β-CD (b). Fluorescence spectra of MPIMC in varying concentrations of β-CD (c). Benesi – Hildebrand plot of MPIMC/β-
CD complex.

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C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
Table 1
Absorption and fluorescence spectral data of MPIMC in various concentrations of β-CD.
Conc. of β-CD,
Absorption
maximum, nm
Absorbance, a.u.
Fluorescence
maximum, nm
mol dm−3
0
403
398
398
398
398
398
397
397
0.0130
0.0172
0.0196
0.0207
0.0229
0.0240
0.0241
0.0250
473.0
472.0
472.0
472.0
472.0
472.0
471.5
471.5
1.0 × 10−3
2.0 × 10−3
4.0 × 10−3
6.0 × 10−3
8.0 × 10−3
1.0 × 10−2
1.2 × 10−2
the concentration of methanol as 1% (v/v). HEPES buffer (0.1 M) is
used to prepare stock solutions of the BSA of concentration
3.0 × 10−5 mol dm−3. All reagents and solvents used were of spec-
tral grade, which were used without further purification. Twice-
distilled water was used throughout the experiments. All experi-
Fig. 3. Time–resolved fluorescence spectra of MPIMC.
3. Results and discussion
ments were carried out at ambient temperature of 25
2 °C. The
test solutions were homogeneous after the addition of all additives.
The absorption and the fluorescence spectra were recorded against
appropriate blank solutions.
The absorption spectrum of MPIMC in water and in various amounts
of the added aqueous β-CD solution is shown in Fig. 2 (a). Table 1 lists
out the absorption spectral data. The absorption at 262 nm shows a
small red shift with an increase in the concentration of β-CD is ascribed
to the host-guest association of MPIMC in β-CD and due to the surfactant
action of β-CD. Moreover, the absorbance continuously increases on each
addition of β-CD up to a concentration of 1.2 × 10−2 mol dm−3 of β-CD.
The fluorescence spectra of MPIMC with various amounts of β-CD are
shown in Fig. 2 (b). An enhancement of fluorescence is observed at the
addition of β-CD to MPIMC in water. The fluorescence band at 473 nm
is shifted by 1.5 nm towards the blue (Table 2). Small blue shifts are gen-
erally indicative of non-bonded interactions and the dislodging of the
guest molecule from the polar environment to the non-polar cavity of
the cyclodextrin. If there had been any bond formations, there would
have been large shifts of bands or formation of new bands. The stoichiom-
etry and the association constant of the complex of MPIMC with β-CD
was found out from the Benesi–Hildebrand equation [32] given as
2.5. Instrumentation
Jasco V-630, double beam UV–Visible spectrophotometer was used
for absorption measurements using 1 cm path length cells. A Perkin-
Elmer LS55 spectrofluorimeter equipped with a 120 W Xenon lamp
for excitation served the measurement of fluorescence. Both the excita-
tion and the emission bandwidths were set up at 2.5 nm. Time–resolved
fluorescence measurements were done on a time–correlated single
photon counting (HORIBA) spectrofluorimeter. Ultra-sonicator (PCI
9 L 250H, India) was used for sonication. pH solutions were adjusted
using an Elico LI 120 pH meter (India). 1H, 13C and two dimensional ro-
tating frame Overhauser effect (ROESY) NMR spectroscopic technique
were recorded on a Bruker AV III instrument operating at 500 MHz.
CDCl3 was used as the solvent for 1H and 13C-NMR spectra and
DMSO–d6 for MPIMC/β-CD complex in recording 2D ROESY NMR spec-
tra. The internal standard was Tetramethylsilane (TMS) and the chemi-
cal shift values were obtained downfield from TMS in parts per million
(ppm). The 2D ROESY experiments were performed using the prepared
solid complex of MPIMC–β-CD. The mixing time for ROSEY spectra was
200 ms under the spin lock condition.
Molecular docking was done using a Schrodinger Maestro (Version
8.5) QSite QM/MM Program. The structure of BSA [PDB: 4F5S] was
used from the Protein Data Bank database in which single sequence is
used for the molecular docking studies. The structure of MPIMC was op-
timized to three dimensional orientations for the docking with the BSA.
MPIMC was docked with the single sequence BSA by standard precision
and extra precision methods. The best poses of the molecular docking
for the binding of drug–BSA was considered from the Glide score (G-
score) value [30]. G-score approximates a systematic search of active
binding sites of the guest with the host binding site using a series of hi-
erarchical filters [31].
1
I−I0
1
1
1
¼
þ
ð1Þ
I0−I0 I0−I0 K β−CD
2
½
ꢀ
where I is the intensity at each concentration of β-CD, I0 is the fluores-
cence intensity of MPIMC in water and I′ is the fluorescence intensity at
the highest concentration of β-CD. K is the association constant and its
value is calculated as 4.14 × 104 mol−2 dm6. The linearity observed for
the plot 1/(I-I0) vs. 1/[β-CD]2 implies that the stoichiometry of the inclu-
sion complex is 1:2 complexation is given in Fig. 2 (c).
The fluorescence decays of MPIMC–β-CD were recorded in aqueous
medium at the same excitation and observation wavelengths. Fig. 3
shows the time–resolved fluorescence spectra of MPIMC in pure water
and at low and high concentrations of β-CD. The fluorescence decay
profile in water is bi-exponential with the relative amplitudes of 90.16
and 9.84 with the corresponding fluorescence lifetimes of 1.49 ns and
8.57 ns respectively. The decay profile becomes tri-exponential due to
the new species formed i.e., β-CD complexed–MPIMC. This analysis
Table 2
Time–resolved fluorescence spectral data of MPIMC in water and β-CD.
Conc. of β-CD, mol dm−3
Energy states
Lifetime (s)
Relative Amplitude (%)
χ2
Standard deviation (s)
0
T1
T2
T1
T2
T3
T1
T2
T3
1.4857 × 10−9
8.5712 × 10−9
7.2393 × 10−10
1.7424 × 10−9
9.2534 × 10−9
4.4244 × 10−10
1.5247 × 10−9
7.8639 × 10−9
90.16
9.84
1.1247
4.7467 × 10−12
1.0018 × 10−10
1.1204 × 10−10
1.6942 × 10−11
8.9920 × 10−11
2.7085 × 10−11
1.2145 × 10−11
9.7986 × 10−11
1.0 × 10−3
18.79
66.03
15.17
23.65
66.25
10.10
1.1765
1.1242
1.2 × 10−2

C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
401
Fig. 4. (a) Absorption spectra of MPIMC at various pH in water (b) Fluorescence spectra of MPIMC at various pH in water (c) I/I0 versus H0/pH plot of MPIMC in water.
supports the view that two emitting individuals are present in solution
possessing different fluorophore populations viz., the free and the β-
CD–complexed form of MPIMC. The formation of a new species with a
different lifetime is an evidence for the formation of the host–guest
complex. The lifetime of this species increases in high concentration of
β-CD whereas the status corresponding to the free MPIMC remains
unmodulated between low and high concentrations of β-CD. These
changes are related to the host–guest association between MPIMC and
β-CD.
In order to add further evidence to the result that the complex
formed between MPIMC and β-CD is host-guest complex and not any
non-inclusional association, the absorption and the fluorescence spectra
Fig. 5. (a) Absorption spectra of MPIMC at various pH in β-CD (b). Fluorescence spectra of MPIMC at various pH in β-CD (c). I/I0 versus H0/pH plot of MPIMC in β-CD.

402
C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
Fig. 6. 2D ROESY spectra of MPIMC-β-CD complex.
of MPIMC are recorded with various amounts of D-(+)-glucose. The
fluorescence intensity is not significantly altered due to the addition of
D-(+)-glucose. This suggests that the hydrophobic cavity of β-CD in-
deed plays a role in the changes observed in the spectra.
In order to find out low acidic strength affects the spectra of MPIMC
with and without β-CD and to extend insights into the mode of binding
of MPIMC with β-CD; we measured the absorption and fluorescence
spectra at various H0/pH in water and in β-CD. With the decrease of
pH, the absorption band at 262 nm showed a decrease of absorbance
in Fig. 4 (a) which indicates that equilibrium exists between the neutral
and the cationic forms of MPIMC. The ground state pKa value is calculat-
ed using the expression [33] (Eq. (4)) derived as follows:
existence of equilibrium between the free and the protonated forms of
the MPIMC molecule. The ground state pKa calculated is 0.98. Quite con-
trary to the effect of acidity on MPIMC in water, at the addition of β-CD,
the fluorescence gets quenched on increase of acid strength. This may
be due to some phenomenon other than collisional quenching. This oc-
curs due to the inaccessibility of the guest molecule being encapsulated
strongly by two β-CD molecules. The change in the relative intensity of
fluorescence with pH is shown in Fig. 5 (c).
In order to confirm 1:2 stoichiometry of the MPIMC/β-CD complex
and to optimize the structure of the inclusion complex, 2D ROESY spec-
trum was recorded (Fig. 6). Cross correlation peaks are observed be-
tween the signals of methyl protons in the positions of 6 of MPIMC
and H4 protons of β-CD (marked with a triangle). This reveals that the
methyl group of MPIMC is surrounded by one β-CD molecule. The aro-
matic protons of imine-linked phenyl ring interact with the H4 proton
of β-CD which is due to the engulfing of phenyl ring to the hydrophobic
cavity of another β-CD. This occurs due to the free availability of the
phenyl ring. This cross peak is marked with a circle in Fig. 6. Here we
could not observe the correlation peak between the protons of another
β-CD molecule and the aromatic protons of the chromone moiety in
MPIMC. This may be to the steric hindrance offered by chromone ring
Aðλ1Þε2ðλ2Þ−Aðλ2Þε2ðλ1Þ
C1
¼
ð2Þ
ð3Þ
ε2ðλ1Þε2ðλ2Þ−ε1ðλ2Þε2ðλ1Þ
C2 ¼ CT−C1
where CT is the total concentration of the compound in both forms and
ε1(λ1), ε2(λ2), ε2(λ1), ε2(λ2) are the molar extinction coefficients of the
protonated and neutral forms at wavelengths λ1 (262 nm) and λ2
(225 nm) respectively.
pKa ¼ pH þ logC1=C2
ð4Þ
The calculated ground state pKa is 1.12. Decrease of pH shows a
quenching of fluorescence as seen in Fig. 4 (b). This may be due to pro-
ton–induced fluorescence quenching. However, a new band in the pro-
tonated form of MPIMC could not be found and hence the excited state
pKa cannot be calculated. Fig. 4 (c).
The effect of acidity on the absorption and fluorescence of MPIMC in
the presence of β-CD are shown in Fig. 5 (a) and (b) respectively. There
is an isosbestic point at 270 nm in the absorption spectra indicating the
Fig. 7. Structural representation of 1:2 stoichimetry complex of MPIMC-β-CD.

C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
403
Fig. 8. (a) Absorption spectra of BSA with various concentrations of MPIMC (b). Quenching of tryptophan fluorescence of BSA due to binding with MPIMC (c). The Stern–Volmer quenching
plot of BSA by MPIMC (d). The plot of log (1/[Dt] – (F0-F)[Pt]/F0) versus log (F0-F)/F for the binding of MPIMC with BSA.
in covering the aromatic group by β-CD. The cross peak observed for the
methyl protons in position 6 is due to the partial coverage of β-CD.
Based on the above arguments, the 1:2 inclusion complex of MPIMC
withβ-CD could have the structure as given in Fig. 7.
The binding titration of MPIMC with bovine serum albumin was car-
ried out with a constant concentration of BSA kept at 3.0 × 10−5 mol/
dm3 and adding aliquots of MPIMC [Fig. 8 (a)]. At the maximal concentra-
tion of MPIMC there were 0.3 absorbance units (more than 0.1) both at
400 nm (absorption maximum) and at 473 nm (fluorescence maximum).
Therefore the inner filter effect was corrected before further experiments.
The inner filter effect was nullified with a correction of fluorescence in-
tensity by including a correction factor from the equation [34] which con-
siders re-absorption of the emitted photons.
where Fcor is the corrected fluorescence intensity and Fobs is the observed
intensity of fluorescence, Aex is the absorbance at excitation wavelength
and Aem is that at the emission wavelength.
The absorption band at 265 nm is distinctly seen in water, but it dis-
appears at the addition of BSA with the band shifting to the blue contin-
uously. The shoulder at the shorter wavelength on complete binding of
MPIMC with BSA is centered at 255 nm which is a large shift from
265 nm. The absorbance continuously increased with the addition of
BSA. The longer wavelength band observed at 403 nm is
bathochromically shifted only by 2 nm. These results are due to the
highly hydrophobic environment offered by the BSA macromolecule
for MPIMC.
The quenching of tryptophan fluorescence of BSA by MPIMC is
shown in Fig. 8 (b). The fluorescence quenching is linear which is attrib-
uted to the binding of MPIMC with BSA. There is no other process except
Fcor ¼ Fobs ꢁ eðAexþAemÞ=2
ð5Þ
Scheme 1. Energy transfer between BSA and MPIMC.

404
C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
Stern–Volmer plot for the quenching of fluorescence by MPIMC is
shown in Fig. 8 (c). The calculated KSV is 1.18 × 104 mol−1 dm3.
The apparent association constant for the binding of MPIMC with
BSA the stoichiometry of the binding is calculated from the plot [Fig. 8
(d)] of log{1/[Dt]-(F0-F)[Pt]/F0} vs. log(F0-F) following Eq. (7).
ꢀ
ꢁ
ꢀ
ꢁ
F0−F
F
1
log10
¼ n log10KA−n log10
ð7Þ
½Dtꢀ−ðF0−FÞ½Ptꢀ=F0
In this equation, F0 and F are the fluorescence intensities without
and with the quencher respectively, [Dt] is the total quencher concen-
tration and [Pt] is the total protein concentration [35]. The association
constant (K) is calculated to be 1.37 × 104 mol−1 dm3 and the stoichi-
ometry is ≈1.
Forster's non-radiative energy transfer theory aids the estimation of
the distance between the buried BSA and the bound MPIMC.
There can be an energy transfer between the tryptophan residues of
BSA and the bound MPIMC with the BSA acting as donor and the MPIMC
as acceptor [Scheme 1]. The overlap of the fluorescence spectrum of BSA
and the absorption spectrum of MPIMC is shown in Fig. 9. The efficiency
of energy transfer (E), the donor – acceptor binding distance and the
critical distance (R0) are calculated [36] using the following Eqs. (8) to
(10),
Fig. 9. The spectral overlap of the absorption spectrum of MPIMC (A) and the fluorescence
emission spectrum of BSA (F).
binding of these two. The fluorescence spectrum shows a blue shift of
8 nm due to the non-polar environment offered by the BSA molecule.
Fluorescence quenching is well known to be classified into dynamic
and static types. The quenching mechanism between MPIMC and BSA
was analyzed using the titration data, applying the Stern–Volmer equa-
tion (Eq. (6))
E ¼ 1−F=F0 ¼ R60 þ r60
ð8Þ
F0
F
where R0 is the Förster distance at 50% energy transfer and r0 is the dis-
tance between the donor and the acceptor. R0 is calculated as follows,
¼ 1 þ Kq τ0½Qꢀ ¼ 1 þ KSV½Qꢀ
ð6Þ
ꢂ
ꢃ
where F0 and F are the fluorescence intensities of BSA without and with
the quencher respectively. Kq is the bimolecular quenching rate
constant and τ0 is the lifetime of the BSA in water. [Q] is the quencher
concentration and KSV is the Stern–Volmer quenching constant. The
R60 ¼ 8:8 ꢁ 10−25 κ2n‐4ΦD JðλÞ in Å6
ð9Þ
where n is the refractive index of the medium, ɸD is the relative interac-
tion with acceptor, κ2 is the relative orientation of the donor and
Fig. 10. (a). Absorption spectra of BSA with various concentrations of MPIMC-β-CD complex (b). Quenching of tryptophan fluorescence of BSA due to binding with MPIMC-β-CD complex
(c). The Stern–Volmer quenching plot of BSA by MPIMC-β-CD complex (d). The plot of log (1/[Dt] – (F0-F)[Pt]/F0) versus log (F0-F)/F for the binding of MPIMC-β-CD complex with BSA.

C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
405
1.18 × 103 mol−1 dm3. The association constant value is smaller than
that for the binding of free MPIMC with BSA (in water). Hence cyclodex-
trin complexation decreases the strength of binding of the MPIMC mol-
ecule with BSA, due to the reason that MPIMC is covered up by β-CD
from the approach of BSA molecule for binding. This observation leads
to the idea that cyclodextrin encapsulation can help the transport of
drugs in blood stream by binding with the BSA and the bound drug is
more readily released than the free drug stronger bound with BSA.
Fig. 11 shows the overlap of the fluorescence spectrum of BSA with
the absorption spectrum of MPIMC-β-CD complex. The calculated values
of n, Φ, J, E, R0, and r0 are 1.33, 0.15, 2.01 × 10−21 M−1 cm−1 nm4, 0.010,
2.875 nm and 6.115 nm, respectively with the method discussed earlier
in the case of MPIMC–BSA binding. These values are different from the
values obtained for MPIMC binding with BSA in water. Hence cyclodex-
trin clearly modulates the binding.
Further insight into the mode of MPIMC–BSA binding was offered by
molecular modeling. BSA consists the major domains I, II, and III with
the residues 1–183 in domain I, 184–376 in domain II, and 377–583 in
domain III. The hydrophobic cavity in sub-domain IIA can allow the
drug molecule to get accommodated, which plays a vital role in the
transportation of drugs in BSA. The best energy ranked results (Fig.
12) reveal that MPIMC is located within the sub-domain III hydrophobic
cavity in close proximity to the residues, such as Leu-505, Ala-527, Leu-
543, and Thr-545 suggesting that there occurs a hydrophobic contribu-
tion to the binding. Moreover, hydrogen bonding is revealed at these
sites. Hence, this finding explains the effective quenching of fluores-
cence of BSA by MPIMC. Furthermore, there are many of hydrophobic
interactions; many several apolar residues close to the ligand aid in sta-
bilizing the molecule via phobic interactions. The hydrogen bonds in-
crease the stability of the MPIMC–BSA bound system. Therefore, the
interaction between the MPIMC and BSA is dominated both by hydro-
phobic binding and hydrogen bonds. Since β-CD blocks some of the pos-
sible hydrogen bond formation between MPIMC and BSA, the binding
strength of MPIMC–β-CD with BSA is relatively low compared to that
of MPIMC in water.
Fig. 11. The spectral overlap of the absorption spectrum of MPIMC-β-CD (A) and the
fluorescence emission spectrum of BSA (F).
acceptor, and J(λ) is the overlap integral of the absorption spectrum of
the acceptor and the fluorescence spectrum of the donor. J(λ), the over-
lap integral is given by
Z
∞
FDðλÞεAðλ0Þλ4dλ
0
Z
J ¼
ð10Þ
∞
FDðλÞdλ
0
In the above equation FD(λ) refers to the corrected fluorescence in-
tensity of the donor between the wavelengths λ and (λ + Δλ), and εA
(λ) is the molar extinction coefficient of the acceptor at the wavelength
λ. Random orientations of the donor and the acceptor are assumed. The
results obtained are n = 1.33, J = 7.39 × 10−21 M−1 cm−1 nm4, E =
0.029, R0 = 2.433 nm, and r0 = 4.354 nm. The donor–to–acceptor dis-
tance (4.354 nm) confirms that static quenching occurs with the energy
transfer from BSA to MPIMC.
According to the literature, there is almost no influence of β-CD on
BSA [37]. The absorption spectrum of MPIMC–β-CD complex with vari-
ous amounts of BSA is shown in Fig. 10 (a). The blue shift of absorbance
is less compared to the shift in water. This is due to the protonated en-
vironment offered by β-CD to MPIMC from BSA. However, the absor-
bance increases, which indicate that binding with BSA occurs. Fig. 10
(b) shows the quenching of tryptophan fluorescence of BSA due to bind-
ing with MPIMC. There is an isosbestic point due to the equilibrium be-
tween MPIMC–β-CD and its complex with BSA. The quenching
magnitude in the presence of β-CD is lesser compared to that in
water. The Stern–Volmer quenching plot for the above mentioned inter-
action is shown in Fig. 10 (c). The calculated Stern–Volmer constant is
9.94 × 103 mol−1 dm3. This is lesser when compared the case of binding
of MPIMC with BSA in water. The plot of log (1/[Dt] – (F0-F)[Pt]/F0) vs.
log (F0-F)/F for the binding of MPIMC-β-CD with BSA following Eq. (5)
is shown in Fig. 10 (d). The association constant is determined as
4. Conclusions
MPIMC forms 1:2 inclusion complex with β-CD and the association
constant is determined as 4.14 × 104 mol−2 dm6. This is evidenced by
fluorescence spectroscopy and 2D ROESY correlation spectroscopy.
The decrease of pKa for the equilibrium of MPIMC in the presence of
β-CD than in water, revealing the restriction to protonation offered by
the β-CD. Study of the binding of MPIMC with BSA is carried out in the
absence and the presence of β-CD. The calculated KSV for the quenching
of fluorescence of BSA by MPIMC is 1.18 × 104 mol−1 dm3. The associa-
tion constant is calculated as 1.37 × 104 mol−1 dm3 and the stoichiom-
etry is 1. In the presence of β-CD the association constant is determined
as 1.18 × 103 mol−1 dm3. Förster resonance energy transfer between
BSA and MPIMC is studied in the absence and the presence of β-CD.
The donor–to–acceptor distance confirms that static quenching occurs
with the energy transfer from BSA to MPIMC. This distance is smaller
in the case of free MPIMC than the β-CD-complexed form of MPIMC
binding with BSA. Hence the encapsulation by cyclodextrin decreases
the strength of binding of the MPIMC molecule with BSA. Molecular
modeling reveals that the binding of MPIMC through the hydrophobic
and the hydrogen bonding sites of BSA. The hydrogen bonds increase
the stability of the MPIMC–BSA bound system. Since β-CD blocks
some of the possible hydrogen bonding sites between MPIMC and
BSA, the binding strength of MPIMC–β-CD complex with BSA is relative-
ly low with that of free MPIMC.
The Supplementary Material contains the 1H and 13C NMR spec-
trum for the synthesized MPIMC. Supplementary data associated with
this article can be found in the online version, at http://dx.doi.org/10.
1016/j.molliq.2016.07.056.
Fig. 12. Molecular docking poses of MPIMC with BSA.

406
C. Sowrirajan et al. / Journal of Molecular Liquids 222 (2016) 398–406
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This work is financially supported by the Department of Science and
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