Preparation and characterizations of solid/aqueous phases inclusion complex of 2,4-dinitroaniline with β-cyclodextrin

Article abstract of DOI:10.1016/j.carbpol.2014.01.091

The formation of host-guest inclusion complex of 2,4-dinitroaniline (2,4-DNA) with nano-hydrophobic cavity of β-cyclodextrin (β-CD) in solution phase were studied by UV-visible spectrophotometer and electrochemical method (Cyclic Voltammetry, CV). The pro

Full text of DOI:10.1016/j.carbpol.2014.01.091

Carbohydrate Polymers 107 (2014) 72–84
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Preparation and characterizations of solid/aqueous phases inclusion
complex of 2,4-dinitroaniline with ␤-cyclodextrin
Thambusamy Stalina,∗, Krishnan Srinivasana,
Krishnamoorthy Sivakumarb, S. Radhakrishnanc
a Department of Industrial Chemistry, School of Chemical Sciences, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
b Department of Chemistry, Sri Chandrasekharendra Saraswathi Viswa Mahavidyalaya University, Enathur, Kanchipuram 631 561, Tamil Nadu, India
c Central Electrochemical Research Institute, Karaikudi 630 006, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The formation of host–guest inclusion complex of 2,4-dinitroaniline (2,4-DNA) with nano-hydrophobic
cavity of ␤-cyclodextrin (␤-CD) in solution phase were studied by UV–visible spectrophotometer and
electrochemical method (Cyclic Voltammetry, CV). The prototropic behaviors of 2,4-DNA with and with-
out ␤-CD was studied by spectrophotometrically. The binding constant of the inclusion complex at 303 K
was calculated using Benesi–Hildebrand plot and thermodynamic parameter (ꢀG) were also calculated.
The inclusion complex formation between ␤-CD and 2,4-DNA was confirmed by 1H NMR, 2D ROESY NMR,
FT-IR, XRD and SEM analysis. The 2,4-DNA:␤-CD inclusion complex was obtained by molecular docking
studies and it was good correlation with the results obtained through experimental methods.
© 2014 Elsevier Ltd. All rights reserved.
Received 31 October 2013
Received in revised form 24 January 2014
Accepted 28 January 2014
Available online 5 February 2014
Keywords:
␤-Cyclodextrin
2,4-Dinitroaniline
Inclusion complex
2D ROESY NMR
Patch dock server
1. Introduction
etc.) between the host (CDs) and guest molecules (Rekharsky &
Inoue, 1998; Schneider, Hacket, & Rudiger, 1998). The CDs also
Cyclodextrins (CDs) are cyclic oligosaccharides consisting of 6,
7, and 8 units of 1,4-linked glucose units, and are named alpha (␣),
beta (␤) and gamma (␥)-Cyclodextrins, respectively (Scheme 1).
These macromolecules, which can be spatially represented as a
torus with wide and narrow openings corresponding to secondary
and primary hydroxyl groups respectively, can encapsulate a large
variety of compounds due to the hydrophobic character of their
internal cavity (Szejtli, 1998). Although the depth of the cavities
for the three CDs is the same (∼0.78 nm), their cavity diame-
ters are ∼0.57, 0.78 and 0.95 nm, respectively. Due to the unique
chemical structure of CD molecules, the inner side of the cavity
is hydrophobic and the outer side is hydrophilic. The hydropho-
bic nature of the CD cavities facilitates the ability of CDs to act
as host for both nonpolar and polar guests, which include small
molecules as well as polymers (Do Nascimento, Da Silva, De Torresi,
Santos, & Temperini, 2002; Harada, Nishiyama, Kawaguchi, Okada,
& Kamachi, 1997; Shuai, Porbeni, Wei, Shin, & Tonelli, 2001; Szejtli,
1998). Once the inclusion compound is formed, the stability of
the guest molecules increases due to the binding forces (van der
Waals attractions, hydrogen bonding, hydrophobic interactions,
have several advantages in other areas, such as the food, cosmetics
industries and agro chemistry (Loukas, Jayasekera, & Gregoriadis,
1995; Loukas, Vraka, & Gregoriadis, 1996; Zhang, Lerner, Rustrum,
& Hofman, 1999), especially owing to their capacity to protect the
guest molecules against oxidation, light-induced reaction and loss
by evaporation. Additionally, they usually enhance the aqueous
solubility of poorly soluble or even insoluble compounds such as
organic and drug molecules (Choi, Nyongryu, Ryoo, & Pillee, 2001).
2,4-Dinitroaniline (2,4-DNA), as dinitro-substituted derivatives
of aromatic amines, have become more and more significant in
environmental studies due to their highly toxic nature and their
suspected carcinogenic properties (Dimou, Sakkas, & Albanis, 2004;
Xiang, Tong, & Lin, 2007). This 2,4-DNA was mainly used as inter-
mediates in the synthesis of dyestuff (Xifeng et al., 2006; Xu, Liu,
Wu, & Feng, 2004), pharmaceuticals (Armson et al., 1999), pesti-
cides (Ayala, El-Din, & Smith, 2010; Benbow, Bernberg, Korda, &
Mead, 1998), and herbicides (Kataoka, 1996; Kearney, Isensee, &
Koktson, 1977; Wang & Arnold, 2003), and they are released into
the environment directly as industrial waste or indirectly as degra-
dation products of herbicides and pesticides. After entering into
the environment, this compound can experience complex envi-
ronmental transformations at trace level, and it is very harmful
to the environment potentially. Acute or chronic exposure to 2,4-
DNA can produce symptoms of headache, dizziness and nausea.
∗
Corresponding author. Tel.: +91 9944266475; fax: +91 4565 225202.
E-mail addresses: tstalinphd@rediffmail.com, drstalin76@gmail.com (T. Stalin).
0144-8617/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2014.01.091

T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
73
Scheme 1. 3D view of chemical structures of ␣-CD, ␤-CD and ␥-CD.
With the growing use of these compounds in different industries,
the 2,4-DNA have been included in the list of priority pollutants in
many countries. In view of the environmental importance of sep-
aration of the 2,4-DNA present in the wastewaters, surface waters
and other environments at trace level or ultra trace level. The ␤-
CD can be used to separate this compound from aqueous medium
by preparing their inclusion complex, because the ␤-CD can selec-
tively include the 2,4-DNA by hydrophobic interaction. 2,4-DNA is
a good candidate as a model compound to characterize inclusion
formation by 1H NMR and FT-IR spectroscopy.
voltammetric technique. The solid complex was prepared and char-
acterized by 1H NMR, 2D ROESY NMR, FT-IR, XRD, SEM techniques
and molecular docking technique (using PatchDock server) in a vir-
tual state. We found that the virtual state analysis results correlates
well with the liquid and solid-state analysis results.
2. Experimental
2.1. Instruments
In this present study, we report the inclusion complex for-
mation in solution phase by UV–visible spectroscopy and cyclic
The UV–Vis spectra (absorption spectral measurements)
were carried out with Shimadzu UV-2401PC double-beam
Scheme 2. Various prototropic equilibria of 2,4-DNA in aqueous and ␤-CD medium.

74
T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
Table 1
Various prototropic maxima (absorption spectra) and pKa values of 2,4-DNA in without and with ␤-CD medium.
Without ␤-cyclodextrin
ꢁmax (nm)
With ␤-cyclodextrin
species
pH
ꢁmax (nm)
pH
–
Monocation
400 (Sh)
348
−4.4
–
233
206
Neutral
348
263
227
−4.0 to 14.02
347
261
224
212
519
350
262
224
−4.0 to 14.02
Monoanion
516
349
262
225
14.37
14.37
pKa
−4.4
Sh – Shoulder.
spectrophotometer, the pH values in the range 2.0–12.0 were
measured on Elico pH meter LI-120; Electrochemical studies were
carried out using an Auto lab electrochemical analyzer, it used
to apply potential on the working electrode equipped with a
three-electrode glassy carbon electrode (diameter: 1 mm) serves
as a working electrode system. Reference electrode was saturated
calomel electrode (SCE) and platinum wire as counter electrode. All
experiments were carried out at 30 1 ◦C. The working electrode
was polished to a mirror with 0.05 ␮m alumina aqueous slurry,
and rinsed with triply distilled water before each experiment. The
supporting electrolyte was pH ∼ 7 (0.1 M KH2PO4 + 0.1 M NaOH)
and H 14.37 (2 M NaOH). 1H NMR (D2O, 500 MHz) spectra were
taken by BRUKER-NMR instrument operating at 300 K using a
5 mm probe. The sample solutions for 1H NMR were prepared by
dissolving the 2,4-DNA and its complex in D2O solvent to obtain
the final concentration of 20 mM. Two-dimensional rotating-frame
Owerhauser effect spectroscopy (ROESY) experiments were per-
formed by BRUKER-NMR 400 MHz, instrument operating at 300 K
and the standard Bruker program was used, DMSO-d6 was used as
a solvent, relaxation delay of 1 s and mixing time 300 ms under the
spin lock conditions. Powder X-ray diffraction spectra were taken
by XPert PRO PANalytical diffractometer. FT-IR was recorded using
Nicolet 380 Thermo Electron Corporation Spectrophotometer
2.3. Reagents
␤-Cyclodextrin {(␤-CD), was obtained from the Sd fine chemical
company} and used without further purification. 2,4-Dinitroaniline
(2,4-DNA) purchased from the SRL chemical reagents company.
Triply distilled water was used to prepare all solutions. Solutions
in the pH range 2.0–12.0 were prepared by adding the appropriate
amount of NaOH and H3PO4. Yagil basicity scale (H–) (Yagil, 1967)
for solutions above pH ∼ 12 (using a NaOH–H2O mixture) and A
modified Hammett’s acidity scale (H0) (Jorgenson & Hartter, 1963)
for the solutions below pH ∼ 2 (using a H2SO4–H2O mixture) was
employed. The solutions were prepared just before taking measure-
ments. The concentrations of the solutions were of the order (10−4
to 10−5 mol/L). The stock solution of 2,4-DNA and ␤-CD preparation
for spectral and electrochemical studies were prepared by adopt-
ing the procedure detailed in our previous report (Stalin, Srinivasan,
Kayalvizhi, & Sivakumar, 2011; Stalin, Srinivasan, Vaheethabanu, &
Manisankar, 2011).
2.4. Preparation of solid inclusion complex of 2,4-DNA with ˇ-CD
Accurately weighed 1 g of the ␤-CD was placed into 100 ml coni-
cal flask and 30 ml triply distilled water added and then oscillated to
completely dissolve the ␤-CD. After that, 0.1612 g of 2,4-DNA was
put into a 10 ml beaker and 5 ml ethanol was added and put over
electromagnetic stirrer to stir until it was dissolved. Then slowly
poured 2,4-DNA solution into ␤-CD solution. The above mixed solu-
tion was continuously stirred for 48 h at room temperature. The
reaction mixture was kept in to refrigerator for 48 h. At this time, a
yellow precipitate was formed. The precipitate was filtered by G4
crucible and washed with triply distilled water. After drying in oven
at 50 ◦C for 12 h, yellow powder was obtained. This is an inclusion
complex of 2,4-DNA with ␤-CD and it further analyzed by 1H NMR,
2D ROESY NMR, FT-IR, XRD and SEM analysis.
using KBr pellets between 4000 and 400 cm−1
.
2.2. Molecular docking study
The most probable structure of the 2,4-DNA:␤-CD inclusion
complex was determined also by molecular docking studies using
the PatchDock server (Duhovny, Inbar, Nussinov, & Wolfson, 2005).
The 3D structural data on ␤-CD and 2,4-DNA was obtained from
crystallographic databases. The guest molecule (2,4-DNA) was
docked into the host molecule (␤-CD) cavity using PatchDock
server by submitting the 3D coordinate data of 2,4-DNA and
␤-CD molecules. Docking was performed with complex type con-
figuration settings. PatchDock server follows a geometry-based
molecular docking algorithm to find the docking transformations
with good molecular shape complementarity. PatchDock algo-
rithm separates the Connolly dot surface representation (Connolly,
1983a; Zhang, Vasmatzis, Cornette, & DeLisi, 1997) of the molecules
into concave, convex and flat patches. These divided comple-
mentary patches are matched in order to generate candidate
transformations and evaluated by geometric fit and atomic des-
olvation energy scoring (Connolly, 1983b) functions. RMSD (root
mean square deviation) clustering is applied to the docked solu-
tions to select the non-redundant results and to discard redundant
docking structures.
3. Results and discussion
3.1. Effect of pH
The absorption spectra of 2,4-DNA were recorded at different pH
solution and the relevant data are compiled in Table 1 and Fig. 1.
In the pH range 2.0–12 absorption maxima nearly resemble with
the spectra observed in non-aqueous solvents (e.g. in 2-propanol at
335.5 nm, acetonitrile at 335 nm and DMF at 345 nm) and thus can
be assigned to the neutral species, and also the absorption maxima
was observed in aqueous medium at 348 nm, its due to the exists of
the neutral form of 2,4-DNA. With a decrease in pH from 2.0, there

T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
75
on N atom is in conjugation with the ␲-bond system of the ben-
zene ring. In an alkali medium (at H 14.37), a new absorption peak
was observed at 517 nm; this is due to the formation of monoanion,
due to deprotanation of the aniline group. Here the more conjuga-
tion is established and the negative charge attracted by the nitro
group in the para position through the benzenoid system. The pro-
totropic equilibrium was shown in Scheme 2. The pKa value for the
equilibrium was determined spectrophotometrically (Table 1).
3.2. Effect of ˇ-cyclodextrin
Table 2 and Fig. 2 show the absorption maxima of 2,4-DNA
(2 × 10−4 mol/L) in pH ∼ 7 (neutral) and H 14.37 (monoanion)
solutions containing different concentrations of ␤-CD. At pH ∼ 7,
2,4-DNA exists as a neutral form only, hence we also recorded spec-
tra at H 14.37 as an anionic form. The absorption spectra of these
two forms (neutral, monoanion) are drastically different. In pH ∼ 7
and H 14.37, no clear isosbestic point was observed in the absorp-
tion spectra even in the presence of higher ␤-CD concentration. The
equilibrium between neutral and monoanion forms of 2,4-DNA in
␤-CD medium.
Fig. 1. Absorption spectra of different prototropic species of 2,4-DNA at 303 K; con-
centration 2 × 10−4 M (a) monocation (b) neutral and (c) monoanion.
+
−H
␤-CD + 2, 4-DNA ꢀ ␤-CD : 2, 4-DNA
ꢀ
␤-CD : 2, 4-DNA(−)
pH 7
H 14.37
(1)
is no change in the absorption maxima in higher acidic medium (up
to H0 −3.87). The absorption intensity was decreased at H0 −4.4,
also a shoulder peak was observed at 400 nm, this is due to the for-
mation of monocation. The absorption intensity was decreases (H0
−4.4) due to the protanation with a lone pair of electrons present on
the amino group. In the case of 2,4-DNA (neutral), absorption max-
ima was observed at 348 nm because of the lone pair of electrons
In pH ∼ 7 solutions the absorption intensities are increased due
to the formation of an inclusion complex. In the absorption spec-
tra of 2,4-DNA (pH ∼ 7), there was no considerable spectral shift
in the absorption maxima, because there is no hydrogen bond for-
mation between ␤-CD and 2,4-DNA, At pH ∼ 7, 2,4-DNA exist as
Table 2
Absorption maxima (nm) and log ε of 2,4-DNA at different concentrations of ␤-CD in H 14.37 and pH ∼ 7 solutions.
S.No
Concentration of ␤-CD (mM)
H 14.37
pH ∼ 7
ꢁmax (nm)
log ε
ꢁmax (nm)
log ε
1
Without ␤-CD
516
349
262
225
3.12
4.23
4.06
4.36
348
263
227
4.22
4.03
4.05
2
3
4
5
6
7
2
517
349
262
222
3.17
4.21
4.06
4.40
348
262
213
4.24
4.05
4.11
4
517
349
262
226
3.18
4.23
4.07
4.39
347
262
225
213
4.25
4.05
4.11
4.12
6
517
350
262
225
3.19
4.21
4.07
4.39
347
262
224
211
4.26
4.06
4.10
4.11
8
517
350
262
226
3.21
4.23
4.09
4.40
347
262
224
212
4.26
4.06
4.10
4.11
10
12
517
350
263
223
3.23
4.24
4.06
4.32
347
262
224
212
4.26
4.06
4.10
4.11
519
350
262
224
3.32
4.19
4.05
4.31
347
261
224
212
4.26
4.06
4.11
4.12
Binding constant (M−1
ꢀG (kJ mol−1
)
81
−11
380
−15
)

76
T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
the neutral form only and also 2,4-DNA located inside the ␤-CD
cavity and the NH2 group located at above the ␤-CD rim, which
will cause the increase of the absorbance. In H 14.37; 2,4-DNA
exist as an anionic form with formation of an inclusion complex. In
H 14.37 the 2,4-DNA located inside the ␤-CD cavity and the NH(−)
group located at above the ␤-CD rim, which will cause the increase
in the absorbance intensities. In these two pH solutions (neutral
and monoanion) the UV–Vis absorption behavior of the inclusion
complex was same as in aqueous medium. This behavior has been
attributed to the enhanced dissolution of the 2,4-DNA molecule
through the hydrophobic interaction between 2,4-DNA and ␤-CD.
These results indicate that 2,4-DNA molecule was entrapped into
␤-CD cavity. In both cases, the binding constant for the formation
of 2,4-DNA:␤-CD inclusion complex has been determined by ana-
lyzing the changes in the intensity of absorbance with the ␤-CD
concentration. In this Fig. S1 plotting 1/(A − A0) versus 1/[␤-CD] for
the host: guest inclusion complex and it gives straight line at pH ∼ 7
and H 14.37, The binding constant (K) can be obtained by using the
modified Benesi–Hildebrand equation (Benesi & Hildebrand, 1949)
for the 1:1 complex Eq. (2) between 2,4-DNA and ␤-CD as shown
below.
1
1
ꢀε
1
=
+
(2)
A − A0
K[2, 4-DNA]0ꢀε[␤-CD]0
where A − A0 is the difference between the absorbance of 2,4-DNA
in the presence and absence of ␤-CD, ꢀε is the difference between
the molar absorption coefficient of 2,4-DNA and the inclusion
complex [2,4-DNA]0 and [␤-CD]0 are the initial concentration of
2,4-DNA and ␤-CD respectively. A good correlations were obtained
(Fig. S1; r2 = 0.9864 and 0.978 for pH ∼ 7 & H 14.37, respectively), it
confirms that the formation of the inclusion complex as 1:1 in the
molar ratio. From the intercept and slope values of this plot K were
calculated [pH ∼ 7 = 380 M−1, H 14.37 = 81 M−1] at 303 K (Table 2).
The determination of the thermodynamic parameter for inclusion
process change in the free energy (ꢀG) can be calculated from the
binding constant ‘K’ by the following Eq. (3).
ꢀG = −RT ln K
(3)
The thermodynamic parameter ꢀG, for the binding of the guest
molecule to ␤-CD cavity is given in Table 2. As can be seen from
Table 2, ꢀG is a negative value, which suggests that the inclusion
process proceeded spontaneously at 303 K. This indicates that the
formation of inclusion complex is an exergonic process.
Fig. 2. Absorption spectra of 2,4-DNA (Conc. 2 × 10−4 M) in (a) pH ∼ 7 and (b)
H 14.37 (c) maginfication of ␤-CD effect solutions at different ␤-CD (a – g Conc.
0–12 × 10−3 M).
Scheme 3. The proposed structure of inclusion complex of 2,4-DNA with ␤-CD for 1:1 inclusion complex. The oxygen atoms are shown as red ball, nitrogen as blue and
carbon as gray color balls; hydrogen atoms are white balls. (For interpretation of the references to color in this scheme legend, the reader is referred to the web version of
the article.)

T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
77
Scheme 4. Rasmol 3D model of (a) ␤-CD (b) 2,4-DNA (c) 2,4-DNA:␤-CD inclusion complex and bond distance of (d) ␤-CD, (e) 2,4-DNA and (f) 2,4-DNA:␤-CD. The oxygen
atoms are shown as red ball, nitrogen as blue balls. (For interpretation of the references to color in this scheme legend, the reader is referred to the web version of the article.)
3.3. Possible inclusion complex of 2,4-DNA in ˇ-CD cavity
only in aqueous medium and neutral to monoanion are red shifted
in ␤-CD medium, it was similar to in aqueous medium (Scheme 2),
its indicating 2,4-DNA molecule entrapped in ␤-CD cavity.
In 1:1 inclusion complex, the aromatic moiety was embedded
in ␤-CD cavity and the NH2 group present above the ␤-CD rim. The
formation of the 1:1 host: guest complex is clearly demonstrated
in Scheme 3. Such 1:1 inclusion complex structure gains further
stabilization energy by releasing of water from hydrophobic cav-
ity. In 1:1 complex, the binding constant for pH ∼ 7 is considerably
greater than that of in H 14.37. The binding constant is very sensi-
tive to changes of pH values, which further supported the selective
inclusion associated with the species form of 2,4-DNA. Of the two
species, we should note that the ␤-CD can readily accept the neu-
tral species than the anionic species, because the anionic species is
more hydrophilic than the neutral species. The inclusion complex
has further stabilized by the interaction of lone pair electrons of
secondary hydroxyl group of ␤-CD with ␲ electrons (benzenoid) of
2,4-DNA. The pH dependent changes in the absorption of the 2,4-
DNA molecule in ␤-CD solution have also been recorded (Table 1).
The formation of monoanion in ␤-CD medium with absorption
maxima was same behavior as without ␤-CD medium, due to the
2,4-DNA was included into the nano hydrophobic cavity of ␤-CD
and the NH(−) group present above the ␤-CD rim. Hence there is
no more difficult to deprotonate in ␤-CD medium. To substantiate
the above discussion, the effect of ␤-CD on the prototropic equilib-
rium, the 2,4-DNA is present as neutral in pH range −4.0 to 14.02,
monoanion at H 14.37 (Scheme 2). The absorption spectra of 2,4-
DNA in ground state shows the above pH (−4.0 to 14.0) as, neutral to
monocation (without ␤-CD) are hyperchromic effect was observed
3.4. Molecular docking study of inclusion process
The 3D structure of ␤-CD and 2,4-DNA obtained from crys-
tallographic databases are shown in Scheme 4a and b. The guest
molecule, 2,4-DNA was docked into the cavity of ␤-CD using
PatchDock server. The PatchDock server program gave several pos-
sible docked models for the most probable structure based on
the energetic parameters; geometric shape complementarity score,
approximate interface area size and atomic contact energy (Armson
et al., 1999) of the 2,4-DNA:␤-CD inclusion complex (Table 3). The
docked 2,4-DNA:␤-CD 1:1 model (Scheme 4c) with the highest
geometric shape complementarity score 2344, approximate inter-
2
˚
face area size of the complex 245 A and atomic contact energy
−819 kJ mol−1 were the highly probable and energetically favorable
model and it is in good correlation with results obtained through
experimental methods.
3.4.1. Semi empirical quantum mechanical calculations
˚
The internal diameter of the ␤-CD is approximately 7.8 A and its
˚
height is 7.8 A (Scheme 4d). Considering the shape and dimensions
of ␤-CD, 2,4-DNA can be completely embedded in the ␤-CD cavity.
The ground state of 2,4-DNA (Scheme 4e) molecule was optimized
using the AM1 method. The vertical distances between H17–O12
,
˚
H18–O13 and O3–O13 is 7.0 A. The horizontal distance between

78
T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
Table 3
Scores of the top 10 docked models of 2,4-DNA:␤-CD inclusion complex computed
using PatchDock server.
Model
Geometric
shape comple-
mentarity
score
Approximate
interface area
size of the
Atomic contact
energy
(kcal/mol)
complex (Å2)
1
2
3
4
5
6
7
8
9
2344
2330
2266
2234
2062
1978
1760
1708
1684
1560
245.00
246.30
237.00
244.50
233.60
201.90
238.90
241.30
237.40
176.90
−195.65
−196.48
−178.38
−184.13
−91.20
−144.53
−183.85
−182.20
−178.94
−158.40
10
˚ ˚ ˚
H16–O1 is 5.6 A, H15–H14 is 4.3 A and H16–O3 is 4.9 A The vertical
distance and the horizontal distance measured from the terminal
atoms of 2,4-DNA is less than the height and vertical diameter of
␤-CD. Since, the height of 2,4-DNA is lower than upper-lower rim
of ␤-CD, the insertion of 2,4-DNA in the ␤-CD cavity is possible as
shown in Scheme 4f.
3.5. Electrochemical studies
The cyclic voltammograms shows an electrochemical behavior
of 2,4-DNA in pH ∼ 7 and H 14.37 with ␤-CD (Fig. 3). Formation
of the inclusion complex of 2,4-DNA with ␤-CD also confirmed
by electrochemical method. In pH ∼ 7 the cyclic voltammograms
shows an anodic peak during the forward scan (toward positive
potential) at −0.104 V and two reduction peaks during the reverse
scan (toward negative potential) at −0.363 V and −0.775 V. These
peaks are ascribed as oxidation of 2,4-DNA (NH2; −0.104 V) and the
reduction of anilinium ions (−0.363 V) into NH2 and another one
reduction peak of NO2 (−0.775) into hydroxylamine.
In H 14.37, from the cyclic voltammograms two-reduction peak
was observed during the reverse scan (toward negative potential)
at −0.465 V and −0.712 V. These peaks were ascribed as reduc-
tion of NH(−) into NH2 and NO2 (−0.712 V) into hydroxylamine.
The oxidation and reduction mechanism of 2,4-DNA was clearly
explained in Scheme 5.
The cathodic peak current ipc (Fig. 3 and Table 4) was increased
with increasing the ␤-CD concentration in both pH solutions
(pH ∼ 7 and H 14.37). The cathodic peak potential, Epc, shifted
toward a positive direction in pH ∼ 7 and negative direction in
H 14.37 when ␤-CD concentration was increased in both cases.
The result showed that the inclusion complex between 2,4-DNA
and ␤-CD was formed when 2,4-DNA was added into ␤-CD aque-
ous solution. In addition, the cathodic peak current was increased
with increasing ␤-CD concentration; this is due to the nitro groups
Fig. 3. CV for 2,4-DNA:␤-CD in (a) pH ∼ 7 and (b) H 14.37 buffer solution, scan rate
100 mV s−1, 2,4-DNA (Conc. 2 × 10−4 M) and ␤-CD (a – g Conc. 0–12 × 10−3 M).
are encapsulated in the ␤-CD cavity and the catalytic behavior of
␤-CD to the included guest molecule (2,4-DNA).
The binding constant (K) and the stoichiometric ratio of the
inclusion complex of 2,4-DNA can be determined according to the
Benesi–Hildebrand (Benesi & Hildebrand, 1949) relation assuming
the formation of a 1:1 host–guest complex.
1
1
ꢀI
1
=
+
(4)
IHG − IG
K[2, 4-DNA]0ꢀI[␤-CD]0
Table 4
CV for 2,4-DNA:␤-CD in H 14.37 and pH ∼ 7 buffer solution, scan rate 100 mVs−1, 2,4-DNA (Conc. 2×10−4 M) and (a-g) 0 –12×10−3 M ␤-CD concentrations.
H 14.37
Epc (V)
pH ∼ 7
Concentration of
␤-CD (mM)
Ipc (␮A)
Epa (V)
Ipa (␮A)
Epc (V)
Ipc (␮A)
Without ␤-CD
2
4
6
8
10
12
−0.465−0.712
−0.470−0.719
−0.478−0.719
−0.480−0.719
−0.482−0.719
−0.492−0.719
−0.495−0.720
−0.524−10.070
−0.608−11.140
−0.746−11.920
−0.777−11.990
−0.816−12.470
−0.820−13.890
−0.988−15.380
−0.104
−0.104
−0.102
−0.097
−0.094
−0.094
−0.094
6.242
6.186
5.780
4.443
4.268
4.268
4.095
−0.363−0.775
−0.363−0.775
−0.358−0.775
−0.356−0.775
−0.35 −0.761
−0.314−0.761
−0.314−0.761
−0.808−14.260
−0.808−14.370
−1.734−14.590
−1.843−14.760
−1.863−16.800
−1.912−17.500
−1.972−18.010
Binding constant (M−1
)
11.8
−6.2
12
−6.3
ꢀG (kJ mol−1
)

T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
79
Scheme 5. Reaction mechanism of 2,4-DNA in pH ∼ 7 and H 14.37 buffer solutions at glassy carbon electrode.

80
T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
Fig. 4. 1H NMR spectra of (a) ␤-CD, (b) solid complex (chemical shift of ␤-CD); (c) 2,4-DNA, (d) inclusion complex (chemical shift of 2,4-DNA).
where IG is the reduction peak current of the guest molecule of 2,4-
DNA, and IHG is the reduction peak current of inclusion complex
of 2,4-DNA:␤-CD. IHG − IG is the difference between the reduction
peak current of the inclusion complex and 2,4-DNA. ꢀI is the dif-
ference between the molar peak current coefficient of the inclusion
complex and 2,4-DNA. The [2,4-DNA]0 and [␤-CD]0 are the initial
concentration of 2,4-DNA and ␤-CD, respectively.
Plotting of [1/IHG − IG] verses 1/[␤-CD] gives a straight line for
both pH solutions as shown in Fig. S2. Good linear correlations
were obtained (r = 0.9501, 0.9228 for H 14.37 and pH ∼ 7 respec-
tively), its confirm that the formation of a 1:1 inclusion complex
for both pH (H 14.37 and pH ∼ 7) solutions. This plot of K was eval-
uated from the intercept and slope value, the binding constant
values for 2,4-DNA: ␤-CD is 11.8 M−1and 12 M−1 in H 14.37 and

T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
81
Table 5
2,4-DNA (Scheme 4c) are located in the nano hydrophobic cavity
of ␤-CD was also moved in upfield significantly, because when 2,4-
DNA monomer entered into the nano hydrophobic cavity of ␤-CD,
the change of the micro-environment of 2,4-DNA protons leaded
the phenyl ring protons moved upfield shift. The significant distin-
guish for these 1H NMR spectra strongly confirmed that the solid
inclusion complex formation.
1H NMR chemical shifts of ␤-CD and 2,4-DNA in free and complexed state deter-
mined In D2O at 300 K.
Name of the
substance
Protons
Free ı (ppm)
Complex ı
ꢀı
(ppm)
H1
H2
H3
H4
H5
H6
4.966
3.554
3.877
3.535
3.748
3.797
4.965
3.555
3.855
3.528
3.734
3.796
−0.001
−0.001
−0.022
−0.007
−0.014
−0.001
␤-CD
3.6.1. ROESY NMR (2D)
The 2D ROESY NMR is a powerful technique for investigation of
inter and intra molecular interactions. The chemical shift changes
were shown by Ha, Hc and Hd protons of aromatic (hydrophobic
part) moiety (Sohajda et al., 2009) of 2,4-DNA may play a major role
in the inclusion process. To verify this hypothesis, 2D ROESY NMR
spectra were recorded. The presence of NOE cross-peaks between
protons of two different species in 2D ROESY spectrum is an indica-
tion that they are in spatial contact through space within the cavity
of ␤-CD.
2,4-DNA
Ha
Hc
Hd
8.982
8.105
6.982
8.908
8.086
6.905
−0.074
−0.019
−0.077
pH ∼ 7 respectively. These values indicate that 2,4-DNA molecule
is encapsulated in the ␤-CD cavity to form an inclusion complex.
3.6. 1H NMR spectrum
Fig. 5 shows the 2D spectrum of 2,4-DNA:␤-CD system, two
groups of intermolecular NOE cross-peaks were observed. In the
complex of 2,4-DNA:␤-CD the first peak was assigned to the inter-
action between the H3 protons of the ␤-CD with ortho positioned
protons of the 2,4-DNA. The other peak was assigned to the inter-
action between the H5 protons of the ␤-CD with meta positioned
protons of the 2,4-DNA. In all cases the interaction of 2,4-DNA with
internal protons of the ␤-CD was observed. In addition the H6ab pro-
tons of the ␤-CD were not affected by the inclusion process. From
this result we can confirm that 2,4-DNA was included into the ␤-CD
cavity via wider rim. From the above fact the 2,4-DNA interact with
␤-CD through space contact, not by bonding.
The formation of the solid inclusion complex can be analyzed
from 1H NMR spectra (Chen, Diao, & Zhang, 2006). Fig. 4 showed
the typical 1H NMR spectra of (a) ␤-CD, (b) solid inclusion com-
plex of 2,4-DNA with ␤-CD (chemical shift changes with ␤-CD), (c)
2,4-DNA and (d) solid inclusion complex (chemical shift changes
with 2,4-DNA). The values of chemical shifts, ␦ for different pro-
tons in a ␤-CD with solid inclusion complex and 2,4-DNA with
solid inclusion complex were listed in Table 5. It can be seen from
the 1H NMR that in solid inclusion complex, a great upfield shift
was occurring for H3 and H5 protons, which locate in the nano
hydrophobic cavity of ␤-CD (Scheme 6b). The changes of chemi-
cal shift (ꢀı) of H3 and H5 suggested that the 2,4-DNA monomer
encapsulated into the nano hydrophobic cavity of ␤-CD. The phenyl
ring of 2,4-DNA made the signals of ␤-CD protons (H3 and H5)
upfield shift. On the contrary, the chemical shifts of H1, H2, H4,
and H6ab, which are on the outer surface of ␤-CD and the narrow
opening of ␤-CD (Scheme 6b), which were only slightly affected by
the guest molecule. Similarly, the chemical shifts of Ha, Hb and Hc of
3.7. FT-IR spectral studies
The solid inclusion complex formation has been character-
ized by FT-IR spectroscopy, because the bands resulting from
the included part of the guest molecule are generally shifted
or their intensities altered. Fig. 6 is the FT-IR spectra of (a) ␤-
CD, (b) 2,4-DNA, (c) solid inclusion complex of 2,4-DNA. From
Scheme 6. (a) The stereo-configuration, (b) truncated-cone of ␤-CD and (c) 2,4-DNA.

82
T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
Fig. 5. 2D ROESY spectrum of inclusion complex of (a) 2,4-DNA:␤-CD and (b) Partial counter plot of 2,4-DNA:␤-CD in DMSO at 300 K.
the Fig. 6 the two characteristic stretching of -NO2 peaks were
observed at 1585 cm−1, 1332 cm−1. These stretching bands were
shifted to 1589 cm−1, 1332 cm−1 and also the intensity of these
peaks were reduced this is due to the nitro group of 2,4-DNA
was present (included) in the nanocavity of ␤-CD. The charac-
teristics stretching of C N ( NH) appeared at 1257 cm−1and its
shifted to 1263 cm−1, this is due to the phenyl ring of 2,4-DNA
was present in the nanocavity of the ␤-CD. The stretching band
of C N ( NO2) group appeared at 833 cm−1 and it shifted to
835 cm−1 and also its intensity was reduced, because of the nitro
group was entrapped into the nanocavity of ␤-CD. The stretch-
3.8. Powder X-ray diffraction pattern
The lack of crystallinity is an added evidence for the forma-
tion of inclusion complex (Williams, Mahaguna, & Sriwongjanya,
1998). Fig. 7 show the XRD patterns for guest (2,4-DNA), host (␤-
CD) and their solid inclusion complex systems were prepared by
co-precipitation techniques at a molar ratio of 1:1. The powder
X-ray diffraction pattern of 2,4-DNA (Fig. 7a) revealed that sev-
eral sharp high intensity peaks at different diffraction angles (2Â)
of 13.7, 14.2, 18.5, 19.6, 20.6, 23.3, 28.0, 28.9 and 31.3 suggesting
that the 2,4-DNA existed as crystalline nature. The ␤-CD (Fig. 7b)
showed a crystalline diffractogram, while a diffuse halo-pattern
was recorded for 2,4-DNA-␤-CD (Fig. 7c) demonstrating its amor-
phous nature. The diffraction patterns of the investigated in the
solid inclusion complex correspond to the correct position of those
of the pure components. The lower intensities of the diffraction
peaks indicate that the particle sizes were reduced during complex
ing vibration of
C
C
appeared at 1627 cm−1 and it shifted to
1629 cm−1, and its intensity also reduced, due to the hydropho-
bic part of the benzene ring to 2,4-DNA was included into the
nanocavity of the ␤-CD. As can be seen above discussions it can
be concluded that of 2,4-DNA molecule was included in ␤-CD cav-
ity.
Fig. 6. FT-IR spectra of (a) 2,4-DNA, (b) ␤-CD and (c) inclusion complex of 2,4-
DNA:␤-CD complex in KBr pellet.
Fig. 7. Powder X-ray diffraction pattern of (a) 2,4-DNA, (b) ␤-CD and (c) inclusion
complex of 2,4-DNA:␤-CD.

T. Stalin et al. / Carbohydrate Polymers 107 (2014) 72–84
83
Fig. 8. Scanning electron microscope photographs (Pt. coated) of (a) 2,4-DNA (b) inclusion complex of 2,4-DNA:␤-CD and (c) ␤-CD.
preparation in solution phase of the pure crystalline components.
Few of diffraction peaks of 2,4-DNA was matched with those off
␤-CD was evident. A typical diffuse pattern indicating the entirely
amorphous nature of 2,4-DNA in complexed state.
of ␤-CD, (vi) The energetically favorable complex was obtained by
molecular docking studies and it was good correlation with the
inclusion model predicted through experimental investigations.
Acknowledgements
3.9. Scanning electron microscope morphological observations
One of the authors K. Srinivasan acknowledges to UGC-BSR for
providing Research Fellowship for Science Meritorious (AU/UGC-
BSR/IC/1069/2011) student and we are thankful to SAIF IIT Madras
for utilizing the 1H NMR spectra facility.
Scanning electron microscope (SEM) is well suited for visualiz-
ing the surface texture of the deposited film. The SEM analysis is
ideal for measuring the surface roughness and for visualizing the
surface texture of the substance. First we observed surface morpho-
logical structure of (a) 2,4-DNA,(b) ␤-CD (Fig. 8) by SEM, and then
we also observed the surface morphological structure of (c) solid
inclusion complex (Fig. 8c). These pictures clearly elucidated the
difference of each other. Modification of crystals can be assumed
as a proof of the formation of a solid inclusion complex with the
nano hydrophobic cavity of ␤-CD.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.carbpol.2014.
01.091.
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