Journal of Molecular Liquids 213 (2016) 294–303

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Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

Characterization of Mammea A/AA in solution and in interaction with

β-cyclodextrin: UV–visible spectroscopy, cyclic voltammetry and
DFT-TDDFT/MD study
Baruch A. Ateba a,b, Daniel Lissouck a, Anatole Azébazé b, Christophe Thiery Ebelle b,c,⁎, Achille Nassi b,c,
Emmanuel Ngameni c, Guy Duportail d, Luc Mbazé b, Cyril A. Kenfack a,⁎
a
Laboratoire d'Optique et Applications, Centre de Physique Atomique Moléculaire et d'Optique Quantique, Faculté des Sciences Université de Douala, B.P. 8580 Douala, Cameroon
b
Laboratoire de Chimie bio-organique, analytique et structurale. Département de Chimie, Faculté des Sciences, Université de Douala, Cameroon
c
Laboratoire de Chimie Analytique, Faculté des Sciences, Université de Yaoundé 1, Cameroon
d
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 du CNRS Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401, Illkirch Cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: Mammea A/AA (MA) was recently introduced as a new anticancer, antioxidant and antimicrobial compound, but
Received 27 July 2015 presenting some cellular toxicity. In this work, MA in solution and interacting with β-cyclodextrin (β-CD) was
Received in revised form 26 September 2015 characterized by UV–visible absorption spectroscopy, cyclic voltammetry and theoretical density functional
Accepted 2 November 2015
theory (DFT), time dependent density functional theory (TDDFT) calculations and molecular dynamic (MD) sim-
Available online 6 December 2015
ulation. We found that the absorbance of this compound was governed by the relative wavelength position of its
Keywords:
charge transfer (CT) and locally excited (LE) states, which are respectively sensitive to polarity and H-bonding.
Mammea A/AA Comparison of the spectroscopy of MA in solution and in presence of increasing amount of β-CD was showing
UV–visible spectroscopy a systematic blue shift and hyperchromic effect, while a decrease of the voltammetry signal peak was observed,
Cyclic voltammetry which we attributed to the formation of an inclusion complex with β-CD. A binding constant of 7.2 × 104 M−1
DFT-TDDFT/MD was estimated and the stoichiometry of the complex was found to be 1:1. Theoretical calculations have shown
Inclusion complex that this complex does not affect the antioxidant potency of MA and that the structure of β-CD complex is stabi-
lized by two H-bonds between MA carbonyl groups and the β-CD wider rim hydroxyl groups, as well as attractive
Van der Waals forces. These results suggest that the binding of MA to β-CD is strong enough and hence allowing
this compound to be stored and carried by β-CD for in vivo loading.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction cyclodextrins are emerging all-purpose molecular containers for organ-

MA (Scheme 1) is a 4-phenylcoumarin extracted from the stem bark
of Mammea africana, a plant widely distributed in equatorial rain forest
regions of Cameroon and commonly used in traditional medicine.
Coumarin and some of its derivatives have been reported to have
biomedical applications [1–4]. In this respect MA has shown in vitro
antiproliferative activity on a variety of cancer cells [5], as well as anti-
oxidant [6] and antimicrobial activities [7,8].
The application of MA as a pharmaceutical drug is hampered by its
significant cytotoxicity [8]. Moreover, the solubility of coumarin deriva-
tives is often poor in water. Therefore, in order to preserve MA biological
relevant activities, it needs to be protected by a formulation with the
capacity to deliver it to physiological targets.
β-cyclodextrin (β-CD) (Scheme 2) molecule is widely used in many
applications to preserve the active compounds by the encapsulation in-
side its hydrophobic cavity to form an inclusion complex. Indeed,
⁎ Corresponding authors.
E-mail addresses: thiercebel@yahoo.fr (C.T. Ebelle), ckenf@yahoo.com (C.A. Kenfack).
ic, inorganic and organometallic compounds that may be neutral,
cationic, anionic, or even radical molecules [9–13].
CDs are cyclic oligomers composed of D-glucose units. The most
used CDs have 6, 7 and 8 glucoses and they are respectively called α-,
β- and γ-CD. These glucose units are connected to form a truncated
cone, having a hydrophobic cavity [14]. This rendered their use
extremely popular in drug manufacturing, as efficient drug delivery
vehicles, capable of enhancing the solubility, dissolution rate and mem-
brane permeability of the encapsulated drugs [15,16]. The formation of
the inclusion complex of MA with CDs and the elucidation of the struc-
ture of their complex in aqueous solution could be of some practical
interest for the possible pharmacological use of this molecule.
In the present work, UV–visible spectroscopy and cyclic voltamme-
try were used to characterize MA in solution and monitor its inclusion
in a β-CD cage in aqueous solution. The photo-absorption of MA was
first investigated in solution to locate the different electronic transitions
and to highlight the effect of polarity and specific solute solvent interac-
tion. To deeply understand the nature of the electronic transitions that
occur in MA and to gain information on the geometry of the MA-β-CD

http://dx.doi.org/10.1016/j.molliq.2015.11.006
0167-7322/© 2015 Elsevier B.V. All rights reserved.
 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303
Scheme 1. The chemical structure of Mammea A/AA.
complex, quantum mechanics calculations were undertaken using the
DFT and TDDFT approaches and the PCM method [17] to account for sol-
vent effect. The usefulness of theoretical methods has been previously
recognized as a powerful tool for conformation and electronic property
studies [18,19].
We report that the UV–visible spectra of MA are highly sensitive to
polarity and H-bonding interaction and that upon encapsulation into
the β-CD host, noticeable changes occur in the UV–visible absorption
of the MA reflecting the complexation process in aqueous solution.
The binding constant was estimated from optical density (OD) varia-
tion. Results of theoretical calculations provide useful insights regarding
the geometry and the thermodynamic and electronic transitions of MA
in solution and within the β-CD cage.
2. Materials and methods
2.1. Experimental section
MA was extracted as yellow crystal as previously indicated [5]. All
the solvent used was of spectroscopic grade. The β-CD sample was pur-
chased from Sigma-Aldrich and used as received. Distilled water was
used for preparing a 22 mM solution of β-CD at neutral pH. A
Scheme 2. Representation β-CD and its truncate aspect.
295
concentrated stock solution of MA (370 μM) was prepared in acetone
and kept in the dark. The UV–visible absorption spectra were recorded
using a fibre-optic spectrometer Avantes 2048 (Avantes, Netherlands)
with a spectral sensitivity within the 250–1100 nm range. The resolu-
tion of the spectrometer was δλ ± 0.8 nm. An optical fibre with a core
diameter of 200 μm was used to measure the transmitted light. Integra-
tion time was 50 ms and each spectrum was averaged automatically
over 100 independent consecutive measurements. The light source
was a 500 W collimated xenon lamp.
2.2. Titration experiments
Titrations were performed by adding increasing concentrations of β-
CD to a 50 μM solution of MA. The binding was monitored by the
displacement of the absorption peak and the variation of the optical
density. The band displacement was evaluated through the ratio:
ðλ−λCD Þ
L¼ ð1Þ
λW −λCD
where λW, λCD and λ are respectively the position of the longer wave-
length absorption peak of MA in pure water, in MA-β-CD complex and
at a given concentration of β-CD. The complex binding constant k was
obtained as a fit parameter by plotting OD as a function of total β-CD
concentration and fitting to the rewritten Scatchard equation:
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 
2
ð1 þ kð½Mt þ n½LtÞÞ− 1 þ kð½Mt þ n½LtÞ2 −4nk ½Lt½Mt
A ¼ εL ½Lt þ ðεM −εL Þ
2k
ð2Þ
where εL and εM are the molar absorptivities of MA and β-CD-MA
complex, respectively. [Lt] and [Mt] are the total concentration of MA
and β-CD, and n is the number of β-CD in the complex. Parameters
were recovered from non-linear fit of the above equation to the exper-
imental data set using the MATLAB software.
2.3. Cyclic voltammetry measurements
Electrochemical measurements were performed by using an Autolab
potentiostat equipped with the General Purpose Electrochemical System
(GPES) (Eco-Chemie, Netherlands) and a standard three-electrode
cell, connected to a computer. A glassy carbon electrode (GCE, 3 mm
diameter) was used as the working electrode, while platinum (Pt)
wires were used as counter electrode. A saturated calomel electrode
(SCE) was used as reference electrode. The working electrode was
polished with 0.3 μm alumina slurry on a polishing cloth, sonicated in
deionized water, rinsed with water and acetone and then dried before
296 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303
use. Solutions containing ethanol and K2HPO4/KH2PO4 (both 0.1 M) 1:1
(v/v) with pH adjusted with KOH or H3PO4 to desired values were used
as supporting electrolyte. MA was added directly to the cell (final con-
centration 10−3 M). After obtaining a cyclic voltammogram of the elec-
trode immersed in the MA-free solution, the solution was purged with
nitrogen before any further measurement. The experiments were car-
ried out at room temperature.
2.4. DFT-TDDFT and MD simulations
The geometry of MA was optimised with DFT method, using
the B3LYP functional in combination with the 6-31G basis set for
all atoms. The β-CD structure was extracted from the crystal structure
of β-CD complex obtained from Protein Data Bank (PDB ID-3CGT).
Structures of both MA and β-CD were prepared for docking in
Schrödinger Small Molecule Drug Discovery Suite 2014-1 programme
(http://www.schrodinger.com). The resulting geometry of the complex
was then optimised with DFT method.
The calculation of the electronic transitions wavelength and oscilla-
tor strength was also performed with the hybrid functional PBE0
functional with the 6-31 + G (d) basis set, using the TDDFT method.
The solvent contribution was incorporated through the PCM model. To
properly account for the hydrogen bonding in a protic solvent environ-
ment, we used, as suggested by Improta et al. [20], a cluster including
three explicit solvent molecules which are further embedded in the
dielectric continuum mimicking bulk solvent.
The binding free energy ΔG of each complex was calculated in
solution according to a procedure proposed by Lattach et al. [21]. This
binding free energy is calculated according to the thermodynamic cycle:

Ag þ Bg →Δr Gg Cg
j j j in chloroform. This variation of R is probably due to the difference in
ΔG0solv ðAÞ ΔG0solv ðBÞ ΔG0solv ðCÞ
↓−TΔSsolv ↓−TΔSsolv ↓−TΔSsolv
0
Asol þ Bsol →Δr Gsol C sol
Δr G0sol ¼ Δr Gg þ ΔGpcm ðC Þ−ΔGpcm ðAÞ−ΔGpcm ðBÞ−RT ln ð24:5Þ
solv solv solv
ð3Þ
−TΔSsolv ðC Þ þ TΔSsolv ðAÞ þ TΔSsolv ðBÞ−RT ln ðn55:5Þ
where ΔrGg⁎ and ΔGpcm solv are respectively the standard reaction free
energy in the gas phase and in solution and −TΔSsolv is the entropy con-
tribution to the binding free energy. We used a value of −6.31 kcal/mol
for solvation free energy of water molecule in water solvent and −5.1
kcal/mol for solvation energy of methanol in methanol solvent [22].
RT ln(24.4) is the energy needed to take one mole of solvent from
gas to liquid phase at a pressure of one atmosphere. RTln(n55.5) is a
term added when the solvent is considered as a reactant, n being the
number of solvent molecules involved in the solvation complex.
The binding energies were estimated including basis set superposi-
tion error (BSSE) correction. All computations were performed using
Gaussian 09 computational chemistry software package [23].
To evaluate the binding free energy of the β-CD-MA complex, the
numerical value of the different terms in Eq. (3) was retrieved from
molecular dynamic (MD) simulations in explicit solvent. This approach
was justified by the difficulty to obtain the entropy contribution with
quantum mechanic formalism, due to the large size of the system. MD
simulation was conducted as follows: The starting structure of the
complex obtained from B3LYP/6-31G optimisation was solvated in a
TIP3BOX system of 1452 water molecules, using the standard
Amber12 rules to hydrate the system [24], which was then optimised,
heated slowly, and equilibrated for 120 ps, before 10 ns of MD simula-
tion without constraint similar to Ref. [25]. Simulations were performed
in the isothermal isobaric ensemble (P = 1 atm, T = 300 K). Periodic
boundary conditions and the Particle-Mesh-Ewald algorithm were
used. [26] A 2 fs integration time step was used, and all hydrogen-
containing bonds were frozen using SHAKE [27]. The forcefield99SB
was used to represent the β-CD and MA, [28] and the TIP3P model
was used to represent water molecules [29]. MD simulations were
carried out with the AMBER12 programme. [24]. Binding free energy
of MA and β-CD complex was calculated by two methods, the General-
ised Born (GB) model [30] and the Poisson–Boltzmann equation (PBE)
[31] for the trajectory of the MD run.
3. Results
3.1. Solvatochromism
To infer the effect of solvent on the microenvironment of MA within
the β-CD cavity, we performed a solvatochromism study by comparing
the absorbance of MA in solvents of different polarities and proticities.
The solvent was characterized by two parameters, the dielectric
constant ε and Abraham's hydrogen bond acidity α. We used hexane
(ε = 1.88; α = 0.00), chloroform (ε = 4.71; α = 0.15), DMSO (ε =
46.82; α = 0.00), acetonitrile (ε = 35.69; α = 0.07), methanol (ε =
32.61; α = 0.43) and water (ε = 78.35; α = 0.82) [32] as solvents.
Fig. 1 shows the UV–visible spectra of MA in these solvents at concen-
tration 50 μM. In hexane, a weak transition appears at 340 nm as a
shoulder, accompanied by a strong peak at 317 nm. To examine the
nature of these transitions, the effect of polarity and H-bonding with a
protic solvent was investigated. In this approach, we considered the
ratio R = O.Dlong/O.Dshort of optical densities of the longer wavelength
positioned around 340 nm, and the shorter one positioned at about
317 nm, respectively. In the low polar but weakly protic chloroform,
the band positions are not modified, while the relative absorbance of
the longer wavelength band increased. This was substantiated by eval-
uating the R ratio, which was found to vary from 0.39 in hexane to 0.7
Abraham's hydrogen bond acidity parameter α which increases from
0 in hexane to 0.15 in chloroform. In the more polar acetonitrile with
a smaller value of α (0.07), R drops to 0.62. The decrease of R value is
even accentuated (R = 0.57) in the DMSO which is a more polar solvent,
but with a value of α of zero. Interestingly the value of R increases to
0.96 and 1.2 in methanol (α = 0.43) and water (α = 0.82), respectively,
with an additional red shift of the longer wavelength band in water of
about 1002 cm−1. These results indicate that the two bands that appear
in the absorption spectra of MA in solution between 290 nm and
440 nm are likely of ππ* character. The plot of R versus α (Fig. 2) in
polar solvents reveals that R varies linearly with α. Thus, the observed
spectral modifications are driven by the H-bond strength of the solvent.
We inferred from our measurements that MA readily forms H-bond
complexes with protic solvents that stability depends on the H-bond
Fig. 1. Absorption spectra of MA in different solvents.
 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303
Fig. 2. Variation of the R ratio versus Abraham's acidity parameter α. The solid line repre-
sents the linear fit to the data with a slope of 0.78 ± 0.03 and an intercept of 0.58 ± 0.01.
donor power of the solvent, thus enabling us to discriminate the
molecular environment with different H-bond acidity parameters. This
property may facilitate its inclusion in β-CD.
3.2. Spectroscopy in β-cyclodextrin
Fig. 3 depicts the absorption spectra of 50 μM MA in water in the
presence of an increasing concentration of β-CD from 0 to 130 μM. In
the addition of β-CD, the longer wavelength absorption band was
progressively blue-shifted from 352 nm to 340 nm, together with an
increase of the optical density. This suggests a modification of the H-
bond configuration and a decrease in the polarity of the region sur-
rounding the MA molecule, a behaviour which is in agreement with
the usual blue shift of the OD on complexation with β-CD, as the mole-
cule moves from a polar solvent environment to the non-polar cavity of
β-CD when it forms an inclusion complex [18]. The ratio R varies from
1.2 in pure water to 1.07 in the cyclodextrin cage. This value, reported
on the linear plot in Fig. 2, yields a value of 0.64 for α, suggesting that
the hydroxyl group of the β-CD rim is a high H-bond donor and forms
strong H-bond with the MA carbonyl group. This behaviour was also
observed in flavonoid complex with β-CD [33]. The value of α is likely
overestimated as β-CD is often described to be rather close to ethanol
in H-bonding, thus suggesting the formation of a supplementary H-
bond between MA and water molecule in solution. The variation of
the band position monitored by the value of L (Eq. (1)) appears as
uncorrelated with the ratio R, providing thus an opportunity to estimate
the binding parameters by a multi-parametric approach. First, the
Fig. 3. UV absorption spectra of MA (50 μM) in the presence of increasing concentrations
of β-CD (0–130 μM).
297
stoichiometry of the complex was investigated. In this respect, L was
plotted versus the concentration ratio of host and guest [βCD]/[MA].
We obtained a curve characteristic of a phase transition (Fig. 4). The
ratio L decreases from 1 in absence of β-CD to 0 at saturated β-CD
concentration, reaching a value of 0.5 at a concentration ratio of about
1. This indicates that the stoichiometry of the complex is 1:1. Secondly,
the binding constant is evaluated by fitting the experimental data by
Eq. (2) (Fig. 5), taking into account 1:1 stoichiometry. A binding
constant of 72,000 ± 100 M−1 was obtained, a value which is higher
as compared to that reported for other organic polycyclic compounds
[13,18,32,34], showing that the inclusion of MA in β-CD is a relatively
strong interaction.
3.3. Voltammetry measurements
To further support that MA forms inclusion complex with β-CD,
cyclic voltammetry experiments were performed. We first recorded
the cyclic voltammogram of a GCE immersed in a 1:1 (v/v) water-
acetone solution at pH 7.5, in the absence of MA, with a scan rate (v)
of 100 mV s−1 (Fig. 6). No voltammetric peak was obtained at the GCE
in the absence of MA. In contrast, well-defined cathodic and oxidation
peaks appeared respectively at −0.932 V and 0.448eV when MA was
added in solution in a concentration of 10−3 M−1. This behaviour is sim-
ilar to that reported for 1,2-benzopyrone [35]. These peaks are respec-
tively attributed to the reduction and oxidation of MA at the electrodes.
Next, a constant amount of 10−5 M of β-CD solution was added to
the MA solution. Fig. 7 shows the cyclic voltammograms obtained. It
appeared that the current from both cathodic and anodic peaks
decreases. Moreover, no shift of the peak potential was observed. This
result is similar to that obtained by Deunf [36] in the study of the
electrochemical behaviour of a cobalt salt in the presence of CDs, and
by other authors investigating the binding properties of human serum
albumin (HSA) and bovine serum albumin (BSA) by this method [37,
38]. It is likely that β-CD forms a non-electrochemical complex with
MA, which hinders the electron transfer between the electrochemical
species and the electrode.
4. Theoretical calculations
To elucidate the different transitions that occur in MA both in
solution and within the β-CD cage, quantum chemical calculations
were performed.
Fig. 4. Variation of the L (Eq. (1)) ratio with increasing concentration of β-CD (0–130 μM).
298 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303
Fig. 5. Binding titration of MA (50 μM) with β-CD. Solid line corresponds to the fit of the
experimental points with Eq. (2).
4.1. Calculations in solution
4.1.1. MA in hexane
The most stable conformation of MA in hexane as non-polar-protic
solvent, obtained from PCM-B3LYP calculation, using the 6-31G basis
set is shown in Fig. 8a. The relevant geometrical parameters are the
dihedral angles a1, a2 and a3 describing respectively the orientation of
the phenyl, the but-2-enyl and oxobutyl groups. The corresponding
values of these angles are respectively 60°, 56°, and 0°, conferring to
MA a highly distorted geometry. In hexane, the most allowed electronic
transitions occur at 335 nm (f = 0.14), 330 nm (f = 0.09), 289 nm (f =
0.36) and 278 nm (f = 0.24), corresponding to S0 → S1, S0 → S2, S0 → S5
and S0 → S7 transitions, respectively. Comparing the results of these
calculations to experimental data indicates that the first two transitions
may be involved in the longer wavelength absorption band observed in
solution at 340 nm, and the two last ones in the bright absorption band
observed in hexane at 317 nm. The molecular orbitals that participate
with these transitions are presented in Fig. 9. S0 → S1 transition arises
mainly from one electron promotion from HOMO to LUMO +1. These
two orbitals are predominantly localized on the coumarin moiety,
thus conferring to S1 a LE state character. Accordingly, the resulting
dipole moment magnitude on S1 state is 4.2 D, a value which is close
to the ground state one, 5 D. S0 → S2 transition receives contribution
from HOMO to LUMO (68%) and HOMO to LUMO + 1 (32%), while in
Fig. 6. Cyclic voltammograms of an electrode in the absence (a) and presence of 10−3 M
MA (b) using 0.1 M phosphate buffer with acetone solution at pH 7,5 (v = 100 mVs−1).
Fig. 7. Cyclic voltammograms of 10−3 M MA in the absence and presence of β-CDs at
different concentrations (20 to 160 μM).
contrast to HOMO and LUMO+1 which are localized on the coumarin
moiety, LUMO is localized on the phenyl ring. As a consequence, S2 is
a CT state. Indeed, a Mulliken population analysis performed on S2 ex-
cited state density indicates that the Mulliken charge on the coumarin
moiety increases from −0.15 a.u. in the S0 state to −0.018 a.u. in the
S2 state, while that of the phenyl ring decreases from 0.15 a.u. to
0.018 a.u., and concomitantly the dipole moment increases from 5 D
in the ground state to 11.6 D in the S2 state. S0 → S5 receives its
major contribution from HOMO−2 to LUMO (78%) and HOMO− 1 to
LUMO (22%) excitation. The one electron density on HOMO − 2 is
spread on the whole molecule, and in HOMO − 1 it is predominantly
located on the coumarin moiety. The Mulliken charge on coumarin
increases from − 0.20 a.u. to − 0.12 a.u., and at the contrary that of
but-2-enyl group decreases from −0.18 a.u. to −0.24 a.u. The resulting
permanent dipole moment magnitude changes appreciably, varying
from 5 D in the ground state to 9.3 D in the S5 excited state. These
characteristics indicate that S5 is also a CT state. S0 → S7 transition is
due to the promotion of an electron from HOMO − 1 to LUMO + 1
(63%). The two MOs are mainly localized on the coumarin moiety so
Fig. 8. Structure of MA showing bond lengths and bond angles in the ground state.
 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303
Fig. 9. Kohn–Sham frontier orbitals involved in the main electronic transitions of MA along with their energies (in eV) in water (black) and in β-CD cage (red).
that S7 state can be classified as a LE state. The charge displacement that
accompanies this transition is not important, and hence the S7 dipole
moment (4.3 D) remains very close to its value in the ground state.
According to PCM-TDDFT calculations, both the shoulder and the bright
transition respectively observed in the absorption spectra in hexane at
340 nm and at 317 nm likely each contain two ππ* transitions, one of
which (S1 respectively S7 state) being a LE state involving the coumarin
moiety, and the second (S2 respectively S5) a CT state involving the
coumarin as charge donor group and the phenyl or but-2-enyl as
acceptor groups.
4.1.2. MA in dimethyl sulfoxide (DMSO)
DMSO was chosen as a model of polar solvent. Geometry optimisa-
tion in this media shows no appreciable modifications and the dihedral
angles a1, a2 and a3 remain identical to their values in hexane. The more
significant electronic transitions were obtained at 347 nm (f = 0.31),
335 nm (f = 0.1), 296 nm (f = 0.52) and 281 nm (f = 0.16), thus pre-
senting both red shift and hyperchromicity with respect to hexane.
These transitions arise from electron excitation between identical
singlet states as in hexane. The oscillator strength increase is in line
with the hyperchromicity experimentally observed by going from
hexane to DMSO. S0 → S1 and S0 → S2 transitions in DMSO originate
from the same MOs contribution respectively as S0 → S2 and S0 → S1
transitions in hexane, indicating that when the polarity increases, the
CT state, which was positioned initially at 330 nm in hexane undergoes
an important red shift (1485 cm− 1), while the LE state remains
unchanged at 335 nm. The resulting dipole moment of the CT state
increases to 13.7 D in DMSO. In comparison with hexane, S0 → S5
transition receives an additional contribution from HOMO − 3 to
LUMO (35%) beside the HOMO − 1 to LUMO (23%) and from
HOMO−2 to LUMO (35%) excitation. The CT in this state is accordingly
accentuated that we evidenced by considering the Mulliken charge den-
sity on the coumarin moiety, which increases from 0.08 a.u. in the
ground state to 0.28 a.u. in the S5 excited state, while that of the phenyl
group moiety decreases from −0.008 a.u. to −0.26 a.u., and according-
ly, the corresponding permanent dipole moment increases from 9.3 D in
hexane to 11 D in DMSO, accompanied by a red shift (820 cm−1) for this
transition. S0 → S7 transition presents the same characteristics as in
hexane and consequently is also a LE state insensitive to polarity. Taking
together, these results suggest that in polar solvents, the CT states
299
undergo bathochromic shifts, while the LE states remain at their initial
position in neutral solvent.
4.1.3. MA in methanol
To separately appreciate the contribution of polarity and H-bonding,
MA was first considered isolated (i.e. H-Bond free) in bulk methanol.
The optimised geometry of MA does not show any appreciable modifi-
cation and conserves the same geometrical parameters. TDDFT provides
electronic transitions at 346 nm (f = 0.31), 339 nm (f = 0.1), 295 nm
(f = 0.53) and 281 nm (f = 0.27). These transitions are identical to
those obtained in DMSO, thus proving that the decrease of the polarity
on going from DMSO to methanol has no effect on MA photo-
absorption. The same electronic transitions were obtained in bulk
chloroform and acetonitrile (data not shown). This may indicate a
saturation of the polarity effect in these solvent, even in a solvent with
low polarity. In order to account for the effect of the intermolecular H-
bonding on MA spectroscopy, we then considered a solvated complex
of MA with three methanol molecules, embedded in the dielectric
continuum mimicking the bulk methanol. The conformation of MA in
interaction with three methanol molecules presented in Fig. 10A is
characterized by two OH–OC bonds, where the first one occurs between
the methanol hydroxyl group and the carbonyl of the coumarin moiety
and the second one between the hydroxyl group of the second metha-
nol molecule and the carbonyl of the oxobutyl group, while an OH–OH
bond occurs between the methanol hydroxyl and one hydroxyl group
of the coumarin group, with respective bond lengths of 1.80 Å, 1.81 Å
and 1.90 Å. The binding free energy in this complex was found to be
− 0.16 eV (− 3.9 kcal/Mol). TDDFT provides electronic transitions at
346 nm (f = 0.2), 344 nm (f = 0.21), 295 nm (f = 0.32) and 292 nm
(f = 0.32) corresponding to S0 → S1, S0 → S2, S0 → S5 and S0 → S7 tran-
sitions, respectively. When compared to the results obtained for free MA
in methanol and in DMSO, it appears that the effect of the H-bond is to
induce a red shift of the LE states (S2 and S7 states), while the CT states
(S1 and S5 states) are insensitive to H-bonding interactions. This behav-
iour reduces the gap between S2 and S1 as well as between S5 and S7, so
that the wavelength position of these transitions may coincide, resulting
in one single band with an oscillator strength about twice that of each
individual transition, respectively 0.41 and 0.64 for the long- and
short-wavelength transitions. This result may explain the important
increase of the OD of the longer wavelength band on going from
DMSO to methanol. We infer from these results that the only effect of
300 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303

Fig. 10. Geometries of MA(H2O)3 (A) and MA(methOH)3 (B) complexes obtained from B3LYP/6-31G calculations, showing the distribution of water and methanol molecules around MA
and possible hydrogen bonds in dashed lines along with their corresponding bond lengths.

the H-bonding interaction is to exclusively induce a red shift of the LE to the isolated compound presented in Fig. 8. The structure of β-CD
state. in the complex after optimisation is also slightly deformed. This
deformation was quantified by measuring the distance between the
4.1.4. MA in water neighbouring glycosidic oxygen (Og) atoms [39]. These distances are
The complex of MA with three water molecules (Fig. 10B) shows an reported in Table 1. The mean distance between the Og atoms from
identical disposition of solvent molecules around MA consecutive to H- glucose residue i to residue i + 1 is 4.42 Å and between the glucose
bonds formation as compared to methanol. The H-bond lengths are residue i and residue i + 2 is 8.02 Å, distances nearly identical to
respectively 1.83 Å, 1.88 Å and 1.94 Å. The calculated value of the bind- those observed in the X ray structure of β-CD (4.44 Å and 7.96 Å) [40],
ing energy −0.41 eV (−9.45 kcal/mol) is substantially higher as com- showing that MA minimally perturbs the structure of β-CD. The
pared to that obtained in methanol. This result is probably related to distance between the centre-of-mass of the β-CD and the MA molecule
the strong H-bond donor strength of water. Indeed the Abraham H- is approximately 4.91 Å, comparable to the height of the β-CD cavity
bond acidity parameter for water (0.82) is about two times larger than (7.9 Å), thus indicating that MA does not deeply penetrated the β-CD
for methanol (0.42). Significant electronic transitions are obtained at cavity.
353 nm (f = 0.34), 347 nm (f = 0.11) and 302 nm (f = 0.56). These Interestingly, a value of − 0.242 eV was obtained for the energy of
transitions have the same features as those described in methanol and the HOMO in water and in cyclodextrin cage. According to the Koopman
DMSO but are significantly red-shifted. The positions of the S0 → S1
and S0 → S5 transitions located respectively at 353 nm and 302 nm
match well with the maxima of the first and second absorption bands
obtained experimentally at 353 nm and 317 nm. S1 and S2 states are
respectively the CT and LE states already described, while interestingly
the third transition is an equal mixing of CT and LE characters. The gap
between S1 and S2 states is small, so that we could reasonably assume
that the corresponding wavelength positions coincide. The resulting
oscillator strength of this transition is 0.45, greater than its value in
methanol, which is in line with the hyperchromicity of the longer wave-
length absorption band observed on going from methanol to water.

4.2. Calculations in β-CD

To understand the photo-absorption of MA upon encapsulation in β-

CD, the complex of β-CD with MA was optimised at the B3LYP/6-31G
level of DFT. The most stable conformation of this complex, as shown
in Fig. 11 presents two H-bonds of 1.76 Å and 2.04 Å lengths, respective-
ly. Only slight modifications occur in the structure of MA and β-CD
consecutive to inclusion complex formation. For MA the dihedral
angle of the phenyl ring, the but-2-enyl and the oxobutyl group with
respect to coumarin moiety respectively change to 42°, 60° and 3°,
showing a variation of 18°, − 4° and 3°, respectively, as compared
with the ground state conformation. The other bond lengths and angles Fig. 11. Low energy structure calculated for the complexes between β-CD and MA obtain-
of MA in the cyclodextrin cavity were very close to those corresponding ed from B3LYP/6-31G calculation.
 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303 301

Table 1
Average distances (in Å) between neighbouring glycosidic oxygen atoms (Og) obtained
from DFT, B3LYP/6-31G calculation.

Glucose residue Distance between Ri and Distance between Ri and

number Ri+1 Ri+2

R1 4.52 8.02
R2 4.34 7.85
R3 4.45 8.97
R4 4.42 7.82
R5 4.41 8.06
R6 4.43 7.91
R7 4.39 7.98
Average 4.42 8.04

theorem [41]: IP (ionization potential) = −HOMO energy. As a conse-

quence, after the formation of the inclusion complex, IP value does not
change. This indicates that MA in the encapsulated form maintains its
antioxidant potential.
The binding free energy of the complex was calculated from 10 ns of
MD simulation. Analysis of the root mean square deviation (RMSD)
along the 10 ns MD presented in Fig. 12 shows that our system is stable
in time and achieved geometric convergence. From the MD trajectory,
the binding free energy of the complex was calculated from 2000 snap-
shot pictures of the system atomic coordinates at different times. The
most favourable binding energy was obtained from the GB model that
gives an average value of −0.23 eV (−5.44 kcal/mol), close to the ex-
perimental value of − 0.28 eV (− 6.62 kcal/mol) deduced from the
binding constant. The corresponding values of ΔH and TΔS were nega-
tive, being respectively − 17.64 and −12.2 kcal/mol, thus suggesting
that the stability of this complex was governed by H-bonding and at-
tractive Van der Waals forces. [42].
The electronic transitions that occur for MA inserted in β-CD were
also investigated. In this attempt, the cyclodextrin cage was suppressed
and the H-bond between the cavity and MA was preserved by maintain-
ing the OH groups involved in the H-bond and by mutating the carbon
atom to which they were attached to ethyl group. According to the
high value of the acidity parameter evaluated for MA in β-CD, a supple-
mentary water molecule was placed near the coumarin hydroxyl group
exposed to water solvent. This yields to a system for which MA is in in-
teraction with two ethanol molecules and one water molecule (Fig. 13).
PCM-TDDFT calculations were undertaken on this system in bulk water
(ε = 78) and chloroform (ε = 4.71). In water as solvent, electronic tran-
sitions were obtained at 354 nm (f = 0.24), 336 nm (f = 0.12), 302 nm
(f = 0.40) and 285 nm (f = 0.1). In comparison with the case of MA in
interaction with three water molecules, this result suggests that the
modification of the H-bond in our model system is accompanied by a
Fig. 13. Geometry of the model system used for the calculation of the electronic transitions
occurring for MA encapsulated in β-CD cage.
blue shift of the LE states while the CT states do not vary. The resulting
gap between the S1(CT) and the S2(LE) is 1513 cm− 1, while that of
S5(CT) and S7(LE) states is 1975 cm−1. Interestingly, electronic transi-
tions are positioned at 347 nm (f = 0.20), 335 nm (f = 0.09), 298 nm
(f = 0.45) and 287 nm (0.12) in chloroform, showing that the LE states
are fixed while the CT states undergo 573 cm−1 and 444 cm−1 blue-
shifts for the longer and shorter wavelengths, respectively. These re-
sults, which are qualitatively in agreement with the experimental
data, indicate that the environment inside the β-CD cavity is less polar
than water and confirm that the LE states are exclusively sensitive to
H-bonding, while the CT states are exclusively sensitive to polarity.
This unusual property may find some practical applications. We thus
suggest that the observed blue shift of the MA absorption spectra
when increasing β-CD concentration is the signature of MA encapsula-
tion which drives the MA molecule from polar water environment to
the less polar β-CD cage. We were not able to reproduce the
hyperchromicity of the longer wavelength band experimentally ob-
served for MA-β-CD complex, thus confirming that the contribution of
the Van der Waals interactions in the binding free energy is significant
and influences the spectroscopy of MA.

5. Discussion

In this work, the inclusion of MA in β-CD was investigated by UV–

Fig. 12. RMSD variation of the MA–β-CD complex along the MD trajectory (10 ns) with re-
spect to the average structure and the corresponding thermodynamic parameters.
visible spectroscopy, cyclic voltammetry and quantum mechanical cal-
culations. The variation of optical density, the absorption wavelength
position and the voltammogram curves were used as indicators of MA
encapsulation, whereas theoretical calculations were used to provide
information on the structural parameters and the molecular origin of
the observed spectral modifications. In this theoretical approach, the
most stable conformations of MA in solution and in β-CD cage were
obtained by using the DFT method, in combination with the B3LYP
functional and the 6-31G basis set, while the TDDFT method with the
PBE0 functional and 6-31+G(d) basis set was used for the calculation
of the electronic transitions occurring in MA. The solvent effect was
incorporated in our calculation by the PCM method. To characterize
the effect of polarity, the spectroscopy of MA was compared in hexane,
acetonitrile and DMSO. The effect of H-bonds was investigated by
considering two polar-protic solvents (methanol and water) with
302 B.A. Ateba et al. / Journal of Molecular Liquids 213 (2016) 294–303
different H-bond donor abilities that were compared to DMSO, which
further allows us to describe the mechanism of the interaction of MA
with β-CD. This approach was thus well suited to monitor the inclusion
of MA in β-CD cage.
The photophysics of MA was first investigated in solution. Compari-
son of the absorption spectra recorded in solution shows a systematic
variation in the absorbance that depends on the nature of the solvent.
In hexane a model of neutral solvent, the spectroscopy of MA was char-
acterized by the presence of two absorption bands between 280 and
450 nm. In this solvent, TDDFT calculations gave electronic transitions
in good agreement with the experimental spectra and revealed that
each of the two bands in the experimental absorption spectra contains
two electronic transitions one of which is a LE state (S1 and S7, respec-
tively) and the other a CT state (S2 and S5). The increase of the polarity
from hexane to DMSO leads to the increase of the OD of the absorption
band while the band maxima position remains unchanged. According to
TDDFT calculation, this behaviour originates from the inversion of LE
and CT states. Indeed, one important feature in DMSO was the apprecia-
ble red shift of the CT states while the LE states' wavelength positions
remain unchanged. From this mechanism, the S1 state, initially a LE
state in hexane, becomes a CT state in DMSO. As a consequence,
S0 → S1 transition becomes highly allowed due to the important varia-
tion of the dipole moment magnitude between the ground state S0 and
excited state S1 (~9 D). The mechanism of this charge transfer implies
that the coumarin moiety acts as an electron donor and the phenyl
group as an electron acceptor. The response of MA to H-bonding inter-
action was examined in methanol and water. The effect of H-bonding
interactions was quantified by evaluating the ratio of the absorbance
of the longer- and shorter-wavelength absorption bands. Our work
shows that this ratio increases linearly with the solvent acidity parame-
ter, thus indicating that MA forms H-bonding complexes with protic
solvent. This behaviour was explained by the presence of two carbonyl
groups in its structure, which are strong H-bond acceptors, and one
hydroxyl group which is H-bond donor. Indeed quantum chemical
calculations show that these groups are engaged in H-bond with meth-
anol and water molecules, which affect the electronic spectra by
inducing an exclusive red shift of the LE state, leaving the CT states
unaffected in contrast to polarity effect. This behaviour probably
reduces significantly the gap between the CT and LE states, which likely
coincides, resulting in an increase of the total oscillator strength of the
S0 → S1 transition. Interestingly, the S0 → S1 transition oscillator
strength increases in the following order: water (0.45) N methanol
(0.41) N DMSO (0.34) N hexane (0.24), in agreement with experimental
data, thus explaining the hyperchromicity of the longer wavelength
absorption band in protic solvents. The binding free energy of the
complex was found to depend on the H-bond donor ability of the protic
solvent so that MA may be used to discriminate protic media.
The encapsulation of MA in β-CD was monitored by adding a con-
stant amount of β-CD in a MA solution. Under encapsulation in β-CD
cage, the longer wavelength band of the MA absorption spectrum is
blue-shifted while the shorter wavelength band remains unchanged,
demonstrating the migration of MA from a polar water environment
to the less polar β-CD cage. As compared to water, we also observed
an increase of the absorbance indicating a relatively strong interaction
of MA with β-CD. This result was confirmed by the voltammograms
that were characterized by the decrease of both the cathodic and anodic
peaks for encapsulated MA. From the evolution of absorption spectra,
some parameters describing the binding of MA to β-CD were extracted.
The binding was determined to be in a 1:1 stoichiometry, and by plot-
ting the absorbance versus β-CD concentration we deduced the value
of the binding constant, which was found around 105 M−1, indicating
a relatively strong interaction between MA and β-CD. This result was
further supported by MD simulations, which gave a high binding free
energy for the MA–βCD complex, which was explained by the presence
of two H-bonds between MA and the hydroxyl group of the β-CD rim.
By exploiting the linearity between the band ratio and the solvent
Abraham's acidity parameter we were able to estimate the acidity pa-
rameter of the β-CD rim, which was found to be comprised between
that of water and methanol, indicating that MA may form H-bond
with a water molecule that involves the hydroxyl group attached to car-
bon at 5 position (see ref. [8] for atom numbering), which remains ex-
posed to solvent when MA binds to β-CD. According to our
calculations, the inclusion of MA in β-CD was accompanied by a mini-
mal deformation of the β-CD geometry, as the distance between
neighbouring glycosidic oxygens remains almost constant, a behaviour
which is consistent with the geometry of the complex showing that
only the phenyl group is positioned inside the β-CD cage. Furthermore,
the IP of MA in β-CD was showing no variation as compared to its value
in water, thus demonstrating that the antioxidant potency of MA is pre-
served inside the β-CD cage.
To understand the photophysics of MA in the β-CD cage, the elec-
tronic transition in a model MA–β-CD complex was undertaken in
bulk water and in chloroform. In water, only the LE states were affected
in response to H-bond modifications. In contrast to chloroform, a sol-
vent with a small dielectric constant, the CT states were exclusively af-
fected. The blue shift of the longer wavelength absorption band was
satisfactorily reproduced, thus validating our protocol. This result
shows that the spectral modification observed with increasing concen-
tration of β-CD is the consequence of MA encapsulation, with as a con-
sequence the release of the water molecules that was initially inside the
β-CD molecule, and then replaced by the guest molecules, followed by
the rearrangement of the structural parameters of MA and β-CD cage,
as supported by quantum mechanical calculations.
6. Conclusion
This paper reports a detailed study of the interaction of MA with dif-
ferent environments, by UV–visible spectroscopy, cyclic voltammetry
and theoretical calculations. The spectroscopy of MA in solution was
found to be governed by interplay between CT and LE states. The CT
states are found exclusively sensitive to polarity and LE states to
proticity. Our experiments have shown that MA can discriminate protic
solvents and form inclusion complex with β-CD, resulting in a blue shift
and hyperchromicity of the longer wavelength absorption band as well
as the decrease of the voltammogram peaks. The binding constant be-
tween β-CD and MA were determined to be 7.2 × 104 M− 1 and the
binding stoichiometry was found to be 1:1. By means of theoretical
DFT calculations, we have shown that MA forms strong H-bonds in
protic media and that the antioxidant property of MA is preserved in
the MA-β-CD. The present work is the first report of the interaction be-
tween MA and its molecular environment. We hope it will help to pro-
vide some keys for the application of MA and thus stimulate further
investigations to exploit its medicinal potency.
Acknowledgements
C. A. K. acknowledges the Abdus Salam Inter-national Centre for The-
oretical Physics (ICTP) for its support to CEPAMOQ through the OEA-AC-
71 project and the High Performance Computing Center of the University
of Strasbourg for supporting this work by providing scientific support and
access to computing resources. Part of the computing resources was
funded by the Equipex Equip@Meso project (Programme Investissements
d'Avenir).
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