Anti-oxidant behavior of functionalized chalcone-a combined quantum chemical and crystallographic structural investigation

Article abstract of DOI:10.1016/j.molstruc.2017.05.100

Compound (2E)-3-(methoxyphenyl)-1-(4-methylphenyl) prop-2-en-1-one (Ch) was synthesized by the Claisen-Schmidt condensation reaction between para-methylacetophenone and para-methoxybenzaldehyde under basic condition. The structure of the molecule was eluc

Full text of DOI:10.1016/j.molstruc.2017.05.100

Accepted Manuscript
Anti-oxidant behavior of functiolised chalcone-a combined quantum chemical and
crystallographic structural investigation
Bijo Mathew, Adebayo A. Adeniyi, Monu Joy, Githa Elizabeth Mathew, Ashona Singh-
Pillay, C. Sudarsanakumar, Mahmoud E.S. Soliman, Jerad Suresh
PII:
S0022-2860(17)30711-1
10.1016/j.molstruc.2017.05.100
MOLSTR 23841
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 11 April 2017
Revised Date: 19 May 2017
Accepted Date: 21 May 2017
Please cite this article as: B. Mathew, A.A. Adeniyi, M. Joy, G.E. Mathew, A. Singh-Pillay, C.
Sudarsanakumar, M.E.S. Soliman, J. Suresh, Anti-oxidant behavior of functiolised chalcone-a combined
quantum chemical and crystallographic structural investigation, Journal of Molecular Structure (2017),
doi: 10.1016/j.molstruc.2017.05.100.
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ACCEPTED MANUSCRIPT
Anti-oxidant behavior of functiolised chalcone-a combined
quantum chemical and crystallographic structural
investigation
a*
b
c
d
Bijo Mathew , Adebayo A. Adeniyi , Monu Joy , Githa Elizabeth Mathew , Ashona
b
c
b
e
Singh-Pillay , C. Sudarsanakumar , Mahmoud E.S. Soliman Jerad Suresh
a
Division of Drug Design and Medicinal Chemistry Research Lab, Department of
Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad, Kerala, India
b
School of Health Sciences, University of KwaZulu-Natal, Westville, Durban 4001, South
Africa
c
School of Pure & Applied Physics, M.G. University-686560, Kottayam, India.
d
Department of Pharmacology, Grace College of Pharmacy, Palakkad 678004, Kerala, India
e
Department of Pharmaceutical Chemistry, Madras Medical College, Chennai-600004,
India.

ACCEPTED MANUSCRIPT
Anti-oxidant behavior of functiolised chalcone-a combined
quantum chemical and crystallographic structural
investigation
a*
b
c
d
Bijo Mathew , Adebayo A. Adeniyi , Monu Joy , Githa Elizabeth Mathew , Ashona
b
c
b
d
Singh-Pillay , C. Sudarsanakumar , Mahmoud E.S. Soliman , Jerad Suresh
a
Division of Drug Design and Medicinal Chemistry Research Lab, Department of
Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad, Kerala, India
b
School of Health Sciences, University of KwaZulu-Natal, Westville, Durban 4001, South
Africa
c
School of Pure & Applied Physics, M.G. University-686560, Kottayam, India.
d
Department of Pharmacology, Grace College of Pharmacy, Palakkad 678004, Kerala,
India.
e
Department of Pharmaceutical Chemistry, Madras Medical College, Chennai-600004, India.
Corresponding author: Dr.Bijo Mathew, Division of Drug Design and Medicinal
Chemistry Research Lab, Department of Pharmaceutical Chemistry, Ahalia School of
Pharmacy, Palakkad, Kerala, India
Highlights
•
•
•
(2E)-3-(methoxyphenyl)-1-(4-methylphenyl) prop-2-en-1-one is synthesized
Structure determination by X-ray diffraction
Antioxidant activity was evaluated and compared with standard

ACCEPTED MANUSCRIPT
•
DFT calculations were performed and the structural features towards Antioxidant
activity has been established
Graphical abstract

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Abstract
Compound (2E)-3-(methoxyphenyl)-1-(4-methylphenyl) prop-2-en-1-one (Ch) was
synthesized by the Claisen-Schmidt condensation reaction between para-
methylacetophenone and para-methoxybenzaldehyde under basic condition. The structure of
the molecule was elucidated using X-ray diffraction. Compound (Ch) demonstrated higher
antioxidant activities in the DPPH test and H O assay (IC =12.23±0.53 and 15.62±0.98)
2
2
50
than with the standard ascorbic acid (IC =17.32±0.44 and 19.07±0.35). An evaluation of the
5
0
atomic and molecular properties of ascorbic acid and Ch were computed based on their
antioxidant activities. The molecular properties give insight into possible reasons for the
enhanced antioxidant properties of Ch compared to ascorbic acid. The atomic properties
provide further insight into chemical changes of the atoms of the compounds. Such changes
include electronic shifting of the compounds electrophilic and/or nucleophilic states which
highlight chemical moieties which characterize the antioxidant activity but do not directly
relate to a variation in their antioxidant activities. The results obtained reflect oxygen atoms
having significant nucleophilic interactions of each of the compounds. This was characterized
by higher Fukui indices, isotropic and anisotropic hyperfine and orbital coupling stability
energy.
Keywords: Chalcones, Crystal structure, antioxidant activity, DFT, DPPH test, H O assay
2
2
1
. Introduction
Chalcones are a unique class of flavonoids which are characterized by an open-chain
structure constituting two aromatic ring systems separated by a three-carbon linker of an α,β-

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unsaturated carbonyl system. Introduction of various groups on both phenyl systems and the
presence of a hetero-nucleus around the enone system, can contribute to a variety of
pharmacological properties [1]. The presence of a rotatable bond coupled with a low total
polar surface area of chalcones is architectural characteristics responsible for various target-
based mechanisms of action [2]. The method of preparation of chalcones is based upon the
Claisen–Schmidt condensation reaction which involves a reaction between substituted
acetophenones and aryl aldehydes under basic conditions [3].
Many studies have proven that chalcone derivatives exhibit a wide range of cytotoxic
and antioxidant activity [4-6]. Due to this property they are considered a valuable scaffold for
chemotherapeutics against various proliferative stages of disease [7, 8]. To date, several
reports have been published on the antioxidant activity of chalcones, especially the
hydroxychalcones [9, 10]. Literature also suggests that the radical-scavenging activity of
chalcones is largely affected by the aryl groups and their respective substitution patterns. The
hydroxyl functional group is considered a key moiety which can enhance the antioxidant
activity due to its easy conversion to phenoxy-radicals through the hydrogen transfer
mechanism [11]. These factors have led to recent study suggesting that the presence of an
electron donating methoxy-group on the chalcone structure also can contribute significant
antioxidant effect [12]. In light of these facts, and as a part of our ongoing work related to the
synthesis and cytotoxicity of chalcones [13-16], herein we report the synthesis, crystal
structure, antioxidant activity and the establishment of the density functional test (DFT)
calculations. However, the crystal structure of the present chalcone derivative (Ch) was
reported elsewhere [17]. The synthesis of chalcone, Ch [(2E)-3-(methoxyphenyl)-1-(4-
methylphenyl) prop-2-en-1-one] was accomplished by the Claisen–Schmidt condensation
reaction between 4-methyl acetophenone and p-hydroxy benzaldehyde in the presence of an
aqueous basic media (scheme I).

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2
. Experimental
2
.1 Synthesis and crystallization
A mixture of 4-methyl acetophenone (0.01mol, 1 equiv.), p-hydroxy benzaldehyde
(0.01mol, 1 equiv.) and 40% aq. KOH (15ml) in ethanol (30ml) was stirred at room
temperature for about 3 h. The resulting product was kept overnight in the refrigerator. The
solid was filtered, washed with water and recrystallized from ethanol-water solution [18].
O
CH3
O
CHO
O
40 % KOH
+
CH3
CH3
O
H C
3
H C
3
Scheme 1 Synthetic protocol
.2 Structure Solution & Refinement
2
3
The dimensions of the crystal used for data collection were 0.14 x 0.24 x 0.26 mm .
The crystals belonged to a monoclinic system having space group P21/c with unit cell
dimensions, a = 11.676Å, b = 11.127Å, c = 11.672Å, α = 90˚, β = 115.5˚ and γ = 90˚. Data
was collected using the Brucker AXS Kappa Apex III CMOS detector diffractometer with a
graphite monochromatic Mo-K , radiation (λ=0. 71073Å) at 293K. The data was reduced
α
using the program SAINT-8.34A [19] and empirical absorption corrections were performed
using the SADABS [20]. The structure was solved by the intrinsic phasing method using
SHELXT-2013. The top 19 peaks formed the complete structure. The structure was refined
2
using the full-matrix least squares method based on F using SHELXL-2013 [21]. Five cycles

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of isotropic refinement were followed by anisotropic refinement of the non-hydrogen atoms
using 3025 reflections. The position of all hydrogen atoms was geometrically fixed and
treated with riding atoms; with C-H distances of 0.93 or 0.96 Å. Materials for publications
were prepared using the ORTEP 3 program [22, 23].
2
.3. Antioxidant activity
2
.3.1. DPPH Radical Scavenging Method
A α,αˈ-diphenyl-β-picryl hydrazyl (DPPH) radical was used to assess the antioxidant
activity of test compounds [24]. The test samples were reacted with the stable DPPH radical
in methanol solution. DPPH in methanol is characterized by a strong absorption band at 517
nm and the solution visibly deep violet in color. As the DPPH radical reacts with donated
hydrogen from an antioxidant compound the colour changes from deep violet to light yellow,
i.e. the absorbance decreases with an increase in antioxidant activity. A solution of 0.5 mL of
DPPH (0.2 mM) and 0.1 mL of test compound of varying concentration (10–100 ꢀL) were
mixed and made up to a final volume of 4ml with methanol. The mixture was kept at room
temperature for 30 minutes and then absorbance (OD) was measured at 517 nm against a
blank in a UV-Visible spectrophotometer (Shimadzu). A mixture of methanol (3.9 mL) and
sample (0.1 mL) served as a blank. A control solution was prepared by mixing methanol (3.5
mL) and the DPPH radical solution (0.5 mL). The percentage (%) reduction of free radical
concentration (OD) with different concentrations of test compounds was calculated and
compared with the standard and ascorbic acid (in triplicate). The % inhibition was calculated
using the following formula:
ꢉꢊꢁꢋꢁꢉꢌ
%
ꢁꢂꢃℎꢄꢅꢄꢆꢄꢇꢃꢁ = ꢈ
ꢍꢁ× 100
(1)
ꢉꢊ
Where, ꢎ is the absorbance of the control and ꢎ
ꢆ
is the absorbance in the presence of
0
the test/standard samples. The results are expressed as IC values (i.e. the concentration of
5
0
test compound required to scavenge 50 % of total free radical).

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2
.3.2. Hydrogen peroxide scavenging assay
The scavenging ability of test compounds was measured against hydrogen peroxide
according to method [25]. A solution of hydrogen peroxide (20 mM) was prepared in 50 mM
phosphate buffer (pH 7.4). A 1 ml solution of varying concentration of test compound (25-
2
50 µg/ml) was transferred into test tubes. This was followed by the addition of a 2 ml
hydrogen peroxide solution into each test tube. Each sample was vortexed and had the
absorbance measured at a wavelength of 230 nm after 10 min, against a blank which
comprised only 3 mL of the 50 mM phosphate buffer without hydrogen peroxide. A standard
sample of 1 mL ascorbic acid and 2 mL H O solution was also prepared with a measured
2
2
absorbance. The hydrogen peroxide scavenging ability of the test compounds were done in
triplicate. The % scavenging ability was calculated by the formula:
ꢉꢊꢁꢋꢁꢉꢌ
%
ꢁꢏꢐꢑꢒꢓꢃꢔꢄꢃꢔꢁꢑꢅꢄꢕꢄꢆꢖꢁ = ꢈ
ꢍꢁ× 100
(2)
ꢉꢊ
2
.4. Computational methods
The geometric optimization and computation of all the atomic and molecular
properties of ascorbic acid and Ch were carried out using Gaussian 09 (G09) [26].
Optimization was performed using the Perdew-Wang exchange functional as modified by
Adamo and Barone combined with the PW91 correlation (mPW1PW91) and basis set 6-
3
11G(d,p) [27]. The excitation properties were computed with the functional B3LYP and the
hyperfine computed using the UB3LYP.The expression for the energy (E) of a molecule
under a static electric field (F) is:
1
1
1
0
E = E − µ F − α F F − β F F F − γ F F F F −...
(3)
i
i
ij
i
j
ijk
i
j
k
ijkl
i
j
k
l
2
6
26

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0
The E represents the energy of the molecule in the absence of an electronic field, µ is
i
the dipole moment vector, α is the linear polarisability tensor, β and γ are the first and
ij
ijk
ijkl
second hyperpolarizability tensors where i, j, and k label are the x, y, and z components
respectively [28].In this project, all the terms in the energy expression under the external
electric fields were computed except the second hyperpolarizability tensor (γijkl).
The dipole was computed by:
2
2
2 1/2
µ = (µ + µ + µ )
(4)
x
y
z
The mean polarizability (isotropic polarizability) was estimated using:
<
α>= 1/3 (α + α + α )
(5)
(6)
xx
yy
zz
The anisotropy polarization was calculated using:
α = 1/2 (α + α ) – α
∆
1
xx
yy
zz
2
2
2
2
2
2
1/2
∆
α = {1/2 [(α - α ) + (α - α ) - (α – α ) + 6 (α + α + α )]}
(7)
2
xx
yy
xx
zz
yy
zz
xy
xz
yz
2
2 1/2
∆
α = [(∆α ) – (∆α ) ]
(8)
3
2
1
In addition to the mean polarizability (<α>) and polarizability anisotropies (α1,
α2, α3), the polarizability exaltation index (Γ) was also calculated using the expression:
Γ = α(mol) - Σi<α>i, (9)
where, α(mol) is the mean polarizability of a molecule and Σ <α> is the summation of each
i
i
of the atomic polarizability that made-up each molecule. Calculated values of Γ have been
used to estimate the relative aromaticity of furan homologues and the stabilities of their
atomic clusters [29]. A large negative Γ value denotes a very stable structure.

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The Quasi-Pythagorean problem was applied to calculate the single values of first
static hyperpolarizability (ß) [30] using the matrix β
, β , β , β , β , β , β
xxx xxy xyy yyy xxz xyz yyz
,
β
, β , β
xzz yzz zzz
output from G09:
2
x
2
y
2 1/2
z
ß = (ß + ß + ß ) where ß = (ß + ßxyy + ß ), ß = (ß + ßyxx + ß ) and ß = (ß +
zxx
tot
x
xxx
xzz
y
yyy
yzz
z
ßzyy + ßzzz).
(10)
The computed total ß is converted from its atomic units (a.u.) to electrostatic units (esu) i.e.
-33
1
a.u. = 8.6393 x 10 esu.
3
. Results and discussion
3
.1 Crystal structure
The geometry of Ch is slightly distorted planar in nature favoured toward one side
(Fig: 1). This is evident from the corresponding dihedral angles ∠C4-C5-C8-O2 171.02(3),
∠
C9-C10-C11-C12 2.09(5), ∠C13-C14-O1-C17 175.04(3) and ∠O2-C8-C9-C10 13.53(5).
The extensive delocalization of electron density over the molecule is catalyzed by the
electron donating effect of methyl as well as methoxy-groups attached to the two aryl groups.
2
This effect is further supported by the sp hybridized carbon atoms (C9-C10) which act as a
bridge allowing the transfer of π-electrons throughout the molecule. The bridge distance is
1
.318(4), which is within range of similar observed structures for related thiophene based
chalcones [31]. The non-covalent interactions of Ch are depicted in Fig: 2, where the ketonic
carbonyl oxygen and methoxy oxygen atoms interact with aromatic protons.

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Fig. 1: The molecular structure of ch, showing the atom-numbering scheme and displacement
ellipsoids drawn at the 50% probability level.
Fig. 2. Non-covalent interactions of ketonic carbonyl and methoxy oxygen with different
aromatic protons
Table 1. Crystal data and structure refinement details for ch.
Identification code:
Empirical formula:
Formula weight:
Temperature:
ch
C17 H16 O2
252.30
273(2) K
0.71073 Å
Monoclinic
Wavelength:
Crystal system:

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Space group:
P 21/c
Unit cell dimensions
a = 11.676(2) Å
b = 11.1270(19) Å
c = 11.672(2) Å
α= 90°.
β= 115.535(9)°.
γ = 90°.
3
Volume”:
1368.4(4) Å
4
Z
3
Density (calculated):
Absorption coefficient:
F(000)
1.225 Mg/m
-1
0.079 mm
536
3
Crystal size:
0.260 x 0.240 x 0.140 mm
3.501 to 24.997°.
Theta range for data collection:
Index ranges:
-13<=h<=13, -13<=k<=13, -13<=l<=13
25865
Reflections collected:
Independent reflections:
Completeness to theta = 24.997°
Absorption correction:
Max. and min. transmission:
Refinement method:
2395 [R(int) = 0.1747]
99.8 %
Semi-empirical from equivalents
0.981 and 0.970
2
Full-matrix least-squares on F
Data / restraints / parameters:
2395 / 0 / 175
2
Goodness-of-fit on F :
0.959
Final R indices [I>2sigma (I)]:
R1 = 0.0660, wR2 = 0.1143
R indices (all data):
Extinction coefficient:
R1 = 0.1926, wR2 = 0.1530
0.0063(14)
-3
Largest diff. peak and hole:
0.128 and -0.132 e.Å
3
.2. Antioxidant activity
The in vitro antioxidant activity of Ch was evaluated spectrophotometrically by using
DPPH and H O methods compared with the standard ascorbic acid. The results are presented
2
2
in Table 2. The values are expressed in IC , i.e. the ability of the sample compound required
5
0
to decrease the concentration of test free radical by 50%. The results show that the chalcone
under present study, exhibits potent antioxidant activity in both methods. The presence of a
methyl and methoxy groups at the para position on both phenyl systems of the chalcone may
counterbalance the free radical generated during the oxidation.
Table 2: Antioxidant activity

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Compound name
ch
IC (µg/mL) – DPPH*
IC (µg/mL)* - H O
50 2 2
5
0
12.23±0.53
17.32±0.44
15.62±0.98
19.07±0.35
Ascorbic acid
*
n = 3 values expressed in mean ±S.D
3
3
.3. DFT calculation
.3.1. The molecular properties in the three electronic states
The HOMO, LUMO, band-gap and polarizability properties of the standard ascorbic
acid and chalcone in their three electronic states of neutral, electrophilic (oxidative) and
nucleophilic (reductive) are given in Table 3. The electrophilic state of the models increases
the energy level of the HOMO, while the nucleophilic state lowers the HOMO energy level
similar to the neutral state which also lowers the LUMO energy level (Fig. 3). Both the
LUMO and HOMO of the compound Ch have lower energy in their reductive states compare
to ascorbic which indicates better stability of the compound ch after the antioxidant function.
Both the nucleophilic and electrophilic states have a lower energy band-gap compared to the
neutral state. The dipole moment of molecule ch is higher than that of the ascorbic. All the
polarizabilities of its nucleophilic states are higher than the neutral and electrophilic states
which are contrary to what was observed in the ascorbic acid. This can further suggest the
possible reasons for its better antioxidant activities. The electrophilic and nucleophilic states
of Ch have a very high positive value of calculated Γ which is a clear indication that they
have lower aromaticity and stability compared to the neutral state since a higher negative
value of Γ correspond to more aromaticity and stability [27].

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-
+
-
+
as
as
as
ch
ch
ch
Fig. 3: The energy level of the models in their respective three states
Table 3. The energy, HOMO, LUMO, band-gap, polarizability and polarizability exaltation
index of the two models in three different states.
-
+
-
+
Parameters ascorbic ascorbic
ascorbic
ch
ch
ch
Energy
LUMO (ev)
HOMO (ev)
Dipole
-684.807
-0.940
-6.440
4.038
-684.841
1.170
-0.800
-684.563
-3.010
-6.690
-807.885
-2.330
-6.110
6.641
-807.979
-0.380
-2.590
-807.661
-3.280
-5.920
Band-gap
5.500
1.970
3.680
3.780
2.210
2.640

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(ev)
<
α>
104.964
28.568
52.761
983.807
122.117
21.120
52.301
149.881
80.632
133.123
303.580
279.194
344.643
449.939
489.885
735.634
416.739
456.603
669.887
∆
∆
∆
α1
α2
α3
1144.688
5610.073 20414.843 150584.74 120131.41
5
8
Σi<α>i
137.460
-32.496
137.460
-15.343
137.460
12.421
280.870
22.710
280.870
169.069
280.870
135.869
Γ
Considering the hardness (η) of the molecules in their various states, the results further
confirm lower stability of the electrophilic and nucleophilic states of Ch compared to their
neutral state and compared to the ascorbic acid (Table 3). Lower values of η would be an
indication of lower hardness and effectively lower stability. The hardness (η) is given in
terms of ionization energy (IE) and electron affinity (EA), which are respectively
approximated as HOMO and LUMO energies (ε). The energies are computed as follows:
η=1/2 (IE - EA) ≈ 1/2(εHOMO - εLUMO
)
(11)
The molecules with a small energy gap are referred to as soft, while those with a large
energy gap are considered hard [32]. The high level of hardness of a molecule is an indicator
of more stable isomers [33, 34].This can also be an effect of the degree of electron
localization, molecular polarizability and resonance energy [35, 36]. An increase in the
HOMO-LUMO gap represents an increase in hardness which means an increase in stability
as molecules follow a rule of nature [37].
The ch has very significant hyperpolarizability (β) values in its nucleophilic state (Table
S1, Supplementary Information) in the x-direction (b ) compared to its other states. This
xxx
is an indication of more non-linear optical properties of Ch in its nucleophilic state. There is
a significantly higher extinction co-efficient of UV adsorption that appears in both the
nucleophilic and electrophilic states of the ch between the λ ranges of 600 to 700 nm as
shown in Fig: 4.

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Fig. 4: The UV spectra for the two models in their respective three states
Natural bond orbital
Natural bond orbital (NBO) analysis is an important method to study the intra- and
inter-molecular bonding interactions of a molecule and to investigate the nature of the charge
transfer [38, 39].The quantitative description of bonding and anti-bonding interactions can be
(2)
achieved using the NBO second-order perturbation interaction energy E [40-42].The
stabilization energy E(2) that describe the electron delocalisation from i(donor) to
j(acceptor) can be expressed as:
2
F
(
i; j
)
E (
2
)
= qi
(12)
ε j − εi
The F(i,j) represents the off-diagonal Fock matrix element, q is the donor orbital
i
(2)
occupancy, ε and ε are diagonal orbital energies. A high value of E indicates a donating
i
i
affinity from electron donors to electron acceptors, consequently resulting in a stronger
conjugation of the whole system. Any of the donor-acceptor conjugation in the two systems
(2)
that is found above the threshold of 10 kcal/mol of the value of E are shown in Table S2
Supplementary Information).
(

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The nucleophilic states of the two molecules have a very unique feature that describes
(2)
the most stable form of conjugate interactions. These are characterised with the highest E
values of the primary hyperconjugative interactions due to the orbital overlap n → n* of the
lone pair of electrons of the oxygen atom (O7 in ascorbic acid and O18 in Ch) with the lone
pair of their respective carbon atoms that form a direct bond. The stability values of this
unique n → n* overlap in the nucleophilic state of the systems (53.6 kcal/mol in ascorbic and
5
0.62 kcal/mol in Ch) is higher than all the other conjugate interactions such as; p → p*, n →
p* and p → n* which have been found to significantly stabilise the systems in their neutral,
electrophilic and nucleophilic states.
Nucleophilicity and electrophilicity
The electrophilicity and nucleophilicity of the two molecules were computed using
the Fukui indices. The Fukui functions were computed under a constant external potential
ν(r) as the change in the electronic density of the molecule is due to the change in the number
of its electrons (N). It is defined as:


δρ 

F
(r
)
= 
v
(13)
δN 
This function is one of the more basic and commonly used reactivity indicators to
identify the most reactive sites for electrophilic, nucleophilic or radical reactions within a
th
molecule. The condensed or atomic Fukui functions for the j atom site for the electrophilic
-
+
0
(
f ), nucleophilic (f ) or radical (f ) can be defined in using the atomic charges q(N) as
j j j
follows:
ꢀ
f j = qj
(
N
)
−qj
(
N −1
)
(14)
✛
f j = q (N+1)ꢀq (N)
j
j
(15)

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0
j
1
2
f = [q (N +1)ꢀq (Nꢀ1) ]
j
j
(16)
The sites in the chemical species with the largest values of the Fukui function (f ) have
j
been shown to have high reactivity for nucleophilic, electrophilic or radical attack, depending
on the corresponding reaction [43].
-
+
Condensed Fukui function for the electrophilic (fj ), nucleophilic (fj ) and possibly the
0
radical (fj ) are shown in Table S3 (Supplementary Information), for ascorbic acid and Ch
in their corresponding states. These terms were calculated using the ESP of the respective
compounds and their states. The results from the Fukui analysis clearly indicate the order of
the first four atoms that are significant to the nucleophilic interaction of ascorbic acid and Ch.
The order for ascorbic acid is as follows: C3 > O8 > O7 > C1 whereas the order of Ch is: C8
>
C11 > C16 > O18. The four highly nucleophilic centers of ascorbic acid include the oxygen
and carbon atoms of the hydroxyl group that are α- and β-orientated to the carbonyl unit.
However, for molecule Ch, these are the α-carbon of the carbonyl unit, the two para-carbon
atoms of the second aromatic ring that is bonded to the oxygen atom of the methoxyl group,
followed by the carbon atom of the methoxyl group. The MESP mapping of the electronic
density in Fig: 5 depict the atoms dominating the positive surface of the MESP map in the
nucleophilic states while the carbonyl region of the molecules dominates the negative surface
in their electrophilic state.
as
ch
-
-
as
ch
+
+
as
ch

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Fig. 5: The molecular electrostatic potential surfaces (MESP) of the models in their
respective three states
Hyperfine properties
It is experimentally possible to gain an insight into the electronic structure of a
molecule using electron paramagnetic resonance (EPR) spectroscopy via the nuclear electron
hyperfine couplings. Anisotropic hyperfine couplings are an important property that can give
a quantitative measurement of spin density distributions. In principle it can measure the
interaction of the nuclear spin with the unpaired spin density [44].Significant structural
features to the antioxidant properties of ascorbic acid are the hydroxyl groups and for the Ch
is the methoxy group. The isotropic and anisotropic hyperfine values of the oxygen atoms in
the two models in their electrophilic and nucleophilic states with their corresponding electron
spin density are given in Table S4 (Supplementary Information). As previously discussed,
the oxygen atoms of the hydroxyl groups in ascorbic that are α and β to the carbonyl group
(i.e. O8 and O7 respectively) and the oxygen atom of the methoxyl group in Ch (O18) are
very important. These oxygen atoms have the highest magnitude of the hyperfine values
when the molecules are in their nucleophilic state which is attributed to their antioxidant
activities. They also have the highest values of the electron spin density in the nucleophilic
states. This agrees with previous reports that the spin density can predict the relative
magnitude of the anisotropic tensor values [44].The positive values of the hyperfine

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properties is an indication that the spin polarization from an atom's own π-spin results in an
increase in α−spin at its nucleus (positive hyperfine) whereas negative values are indicative
of spin polarization from a neighbouring π-spin resulting in an increase in β-spin (negative
hyperfine)[45].In all the various states of the molecules, the anisotropic hyperfine values of
Bcc is higher in magnitude than the principal anisotropic (Baa).
The QTAIM properties
The atomic charges computed at different levels of theory and the QTAIM properties
of the selected atoms are described in Table S5, while Table S6 (Supplementary
Information) shows the QTAIM properties of the associated bonds. The oxygen atoms O7
and O8 of ascorbic acid and O18 of Ch are the main atomic centres for nucleophilic
interaction are characterized with lower atomic charges (NPA, Hirshfeld, ESP, q ), electronic
A
kinetic energy of atom (K ), approximation to virial-based total energy of atom (K_Scaled ),
A
A
and electrostatic potential from nuclei (ESP_Nuc ). The values of q(A)* in the Table S5 is
A
the atomic charges of the selected atoms with the sum of the contribution of the bonding
atoms. In the anionic state of the molecules, there is charge transfer from the neucleophilic
oxygen center of molecules to their bonding atoms which is responsible for lower q(A)*
compare to the q(A) while a reverse scenario is observed for their cationic state. The
magnitude of intra-atomic dipole moments (Mu_Intra ) of these oxygen atoms is relatively
A
higher. The atomic charges and the Laplacian of the electron density of the bond critical
points are shown in Fig: 6. The general properties for the bond critical point is an increase in
2
the electron density (rho) and Laplacian of the electron density (∇ Rho) as well as a decrease
in the ellipticity from the neutral states to the nucleophilic and electrophilic states. This is an

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indication that there was an increase in the intramolecular bonding in both the anionic and
2
cationic states due to the increase in the rho and ∇
but a decrease in the π-character of the
Rho
2
bonds dues to a decrease in their ellipticity value. In ascorbic, the rho and ∇
of the bond
Rho
critical point increase in its nucleophilic states compare to electrophilic state while a reverse
is observed in Ch. This gives further insight into possible reason for the experimentally
observed higher oxidation properties of Ch compare to ascorbic. The π-character and the
total electrostatic potential (ESP) of the neucleophilic states of both ascorbic and Ch are
higher than that of their nucleophilic state.
as
ch
-
-
as
as
ch
+
+
ch
Figure 6. The QTAIM atomic and bond properties showing the critical points of the bonds (green) and of the
existing rings (red) with the values of the atomic charge (black values) and the Laplacian derivative of the
electron density (blue values) of the selected atoms and bonds
The geometries of Ch in both the neutral and electrophilic states are associated with
new two rings formed through the H...H interactions which are reduced to one in the
nucleophilic state (Fig: 6). The two weak H...H bonds in Ch have low electron density but it
worth pointing out because it shows the possible contribution of the electrostatic interactions
(acid/base), polarization (hard/soft) effects, van der Waals (dispersion/repulsion) interactions

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and covalency (charge transfer). No significant variation is observed in the plots of the
Laplacian of the electronic density shown in Fig: 7.
0
+
as
ch
-
-
as
ch
+
+
as
ch
Fig. 7. The contour plot of the Laplacian of the electron density showing the positive plots (blue lines) and
negative plots (red lines)
Conclusion
The atomic and molecular properties of ascorbic and Ch were computed using the hybrid
DFT functional methods and the results were related to their antioxidant properties. The
atoms that play significant roles in the antioxidant activities of ascorbic acid are the hydroxyl

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groups that are α and β to the C=O group (corresponding to O8 and O7 respectively). In the
molecule Ch, the oxygen atom of the methoxyl group (O18) is very significant. In the
nucleophilic (oxidative) state, these oxygen atoms are characterized with higher hyperfine
and spin density values. Higher molecular stability is predicted for the molecules in their
nucleophilic states based on the lower HOMO energy level compared to the electrophilic
states that raised the HOMO energy level. The two states have lower band-gap, hardness and
high positive values of polarizability exaltation index which indicates lower stability
compared to their neutral states. There were three unique characteristics observed for the
oxidative state of Ch these are: 1) a very high hyperpolarizability, 2) a higher level of
adsorption extinction co-efficient, and 3) the highest orbital conjugation of n → n*.
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