2,6-Dinitroaniline and β-cyclodextrin inclusion complex properties studied by different analytical methods

Article abstract of DOI:10.1016/j.carbpol.2014.07.062

The formation of supramolecular host-guest inclusion complex of 2,6-Dinitroaniline (2,6-DNA) with nano-hydrophobic cavity of β-cyclodextrin (β-CD) in solution phase were studied by UV-visible spectrophotometer and electrochemical method (cyclic voltammetr

Full text of DOI:10.1016/j.carbpol.2014.07.062

Carbohydrate Polymers 113 (2014) 577–587
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
2,6-Dinitroaniline and ␤-cyclodextrin inclusion complex properties
studied by different analytical methods
Krishnan Srinivasana, Krishnamoorthy Sivakumarb, Thambusamy Stalina,∗
a Department of Industrial Chemistry, School of Chemical Sciences, Alagappa University, Karaikudi 630 003, Tamilnadu, India
b Department of Chemistry, Sri Chandrasekharendra Saraswathi Viswa Mahavidyalaya University, Enathur, Kanchipuram 631 561, Tamilnadu, 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 supramolecular host-guest inclusion complex of 2,6-Dinitroaniline (2,6-DNA) with
nano-hydrophobic cavity of ␤-cyclodextrin (␤-CD) in solution phase were studied by UV-visible spec-
trophotometer and electrochemical method (cyclic voltammetry, CV). The prototropic behaviors of
2,6-DNA with and without ␤-CD and the ground state acidity constant (pKa) of host-guest inclusion com-
plex (2,6-DNA-␤-CD) was studied by Spectrophotometrically. The binding constant of inclusion complex
at 303 K was calculated using Benesi–Hildebrand plot and thermodynamic parameter (ꢀG) were also
calculated. The solid inclusion complex formation between ␤-CD and 2,6-DNA was confirmed by 1H
NMR, FT-IR, XRD and SEM analysis. The ␤-CD:2,6-DNA inclusion complex obtained by molecular docking
studies is in good correlation with the results obtained through experimental methods.
© 2014 Elsevier Ltd. All rights reserved.
Received 9 December 2013
Received in revised form 27 June 2014
Accepted 20 July 2014
Available online 12 August 2014
Keywords:
␤-Cyclodextrin
2,6-Dinitroaniline
Inclusion complex
Patch dock server
1. Introduction
several advantages in other areas, such as the food, cosmetics
industries and agro chemistry (Zhang, Lerner, Rustrum, & Hofman,
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 diameters 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 hydropho-
bic and the outer side is hydrophilic. The hydrophobic 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 (Szejtli, 1998; Shuai, Porbeni, Wei, Shin, &
Tonelli, 2001; Harada, Nishiyama, Kawaguchi, Okada, & Kamachi,
1997; Do Nascimento, Da Silva, De Torresi, Santos, & Temperini,
2002). 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,
etc.) between the host (CDs) and guest molecules (Rekharsky &
Inoue, 1998; Schneider, Hacket, & Rudiger, 1998). CDs also have
1999; Loukas, Jayasekera, & Gregoriadis, 1995; Loukas, Vraka, &
Gregoriadis, 1996), 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).
2,6-Dinitroaniline (2,6-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 2,6-DNA was mainly used as inter-
mediates in the synthesis of dyestuff, pharmaceuticals, pesticides,
and herbicides (Kataoka, 1996), and they are released into the envi-
ronment directly as industrial waste or indirectly as degradation
products of herbicides and pesticides. After enter into the envi-
ronment, this compound can experience complex environmental
transformations at trace level, and it is very harmful to the envi-
ronment potentially. Acute or chronic exposure to 2,6-DNA can
produce symptoms of headache, dizziness and nausea. With the
growing use of these compound in different industries, the 2,6-
DNA have been included in the list of priority pollutants in many
countries. In view of the environmental importance of separation
of the 2,6-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
∗
Corresponding author. Tel.: +91 9944266475; fax: +91 4565 225202.
E-mail address: drstalin76@gmail.com (T. Stalin).
http://dx.doi.org/10.1016/j.carbpol.2014.07.062
0144-8617/© 2014 Elsevier Ltd. All rights reserved.

578
K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
Scheme 1. 3D view of chemical structures of ␣-CD, ␤-CD and ␥-CD.
selectively include the 2,6-DNA by hydrophobic interaction. 2,6-
DNA is a good candidate as a model compound to characterize
inclusion formation by 1H NMR and FT-IR spectroscopy.
In this present study, we report the inclusion complex formation
in solution phase by UV-visible spectroscopy and cyclic voltammet-
ric technique. The solid complex were prepared and characterized
by 1H NMR, 2D 1H NMR (ROESY), FT-IR, XRD, SEM techniques and
molecular docking technique (using PatchDock server) in virtual
state. We found that the virtual state analysis results correlates
well with the liquid and solid-state analysis results.
from crystallographic databases. The guest molecule (2,6-DNA)
was docked in to the host molecule (␤-CD) cavity using Patch-
Dock server by submitting the 3D coordinate data of 2,6-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 complementarily. PatchDock algorithm
separates the Connolly dot surface representation (Connolly, 1983)
of the molecules into concave, convex and flat patches. These
divided complementary patches are matched in order to gener-
ate candidate transformations and evaluated by geometric fit and
atomic desolvation energy scoring (Zhang, Vasmatzis, Cornette, &
DeLisi, 1997) function. RMSD (root mean square deviation) cluster-
ing is applied to the docked solutions to select the non-redundant
results and to discard redundant docking structures.
2. Experimental
2.1. Instruments
The UV–vis spectra (absorption spectral measurements) were
carried out with Shimadzu UV-2401PC double-beam spectropho-
tometer, the pH values in the range 2.0–12.0 were measured on
Elico pH meter LI-120; Electrochemical studies were carried out
using Auto lab electrochemical analyzer, it used to apply potential
on the working electrode equipped with a three-electrode glassy
carbon electrode (diameter: 1 mm) is served as a working elec-
trode 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 elec-
trolyte was pH ∼ 7 (0.1 M KH2PO4 + 0.1 M NaOH). 10 mg of sample
were dissolved in 0.7 ml of D2O solvent and then filtered through a
Pasteur pipette, equipped with a glass wool plug that discharges
into an NMR tube. Two-dimensional rotating-frame Overhauser
effect spectroscopy (ROESY) experiments were performed using
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 1s and mixing time 300 ms under the spin lock
conditions. Powder X-ray diffraction spectra were taken by XPert
PRO PANalytical diffractometer (2Theta: 0.001; minimum step size
Omega: 0.001). The surface morphology was taken by Hitachi S
3000 H SEM.
2.3. Reagents
␤-Cyclodextrin {(␤-CD), was obtained from Sd fine chemical
company} and used without further purification. 2,6-Dinitroaniline
(2,6-DNA) purchased from Alfa Aesar 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 mod-
ified 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 mea-
surements. The concentrations of the solutions were of the order
(10−4 to 10−5 mol/dm3). The stock solution of 2,6-DNA and ␤-CD
preparation for spectral and electrochemical studies was prepared
by adopting the procedure detailed in our previous report (Stalin,
Srinivasan, Vaheethabanu, & Manisankar, 2011a; Stalin, Srinivasan,
Kayalvizhi, & Sivakumar, 2011b).
2.4. Preparation of solid inclusion complex of 2,6-DNA with ˇ-CD
Accurately weighed 1 g of ␤-CD was placed into 50 ml conical
flask and 30 ml triply distilled water added and then oscillated
this solution enough. After that, 0.1774 g 2,6-DNA was put in to
a 50 ml beaker and 20 ml ethanol added and put over electro-
magnetic stirrer to stir until it was dissolved. Then slowly poured
2,6-DNA solution into ␤-CD solution. The above mixed solution was
continuously stirred for 48 h at room temperature and the follow-
ing equilibriums can take place G (solution) + CD (solution) ↔ G-CD
2.2. Molecular docking study
The most probable structure of the 2,6-DNA:␤-CD inclusion
complex was determined also by molecular docking studies using
the Patch Dock server (Duhovny, Inbar, Nussinov, & Wolfson,
2005). The 3D structural data of ␤-CD and 2,6-DNA was obtained

K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
579
Table 1
Table 2
Prototropic maxima (absorption spectra) of 2,6-DNA in without and with ␤-CD
medium and pKa value of 2,6-DNA (without ␤-CD).
Absorption maxima (nm) and log ε of 2,6-DNA at different concentrations of ␤-CD
in pH ∼ 7 solution.
S. No.
pH ∼ 7
Without ␤-cyclodextrin
With ␤-cyclodextrin
Species
Concentration of ␤-CD (M)
ꢁmax (nm)
Log ε
ꢁmax (nm)
H0/pH/H
ꢁmax (nm)
H0/pH/H
1
2
3
4
5
6
7
0
432
254
225
433
225
225
433
255
226
433
256
226
434
256
226
434
256
228
435
257
227
17.8
−7.25
3.94
4.01
4.20
3.95
4.03
4.23
3.95
4.06
4.24
3.95
4.07
4.24
3.95
4.08
4.25
3.96
4.09
4.25
3.96
4.11
4.26
432
227
213
432
325
254
225
–
−4.89
–
–
Monocation
Neutral
0.002
0.004
0.006
0.008
0.010
0.012
−4.65–15.97
0.83–15.97
435
257
227
–
Monoanion
pKa
–
–
−4.89
(solution) G-CD (solid) the first equilibrium is Kbind, and the sec-
ond one is Ksolid
.
The reaction mixture was put and kept in to refrigerator for 48 h.
At this time, we observed that yellow precipitate was formed. The
precipitate was filtered by G4 crucible and washed with triply dis-
tilled water. After dried in oven at 50 ◦C for 12 h, yellow powder
was obtained. This is solid inclusion complex of 2,6-DNA with ␤-
CD and it further analyzed by 1H NMR, 2D 1H NMR (ROESY), FT-IR,
XRD and SEM analysis.
Binding constant (M−1
ꢀG (kJ/mol)
)
3. Results and discussion
of electrons on N atom is in conjugation with the ␲-bond system of
the benzene ring. In alkali medium, there is no change in absorp-
tion maxima even in −4.65. So there is no formation of monoanion;
due to deprotanation is not possible, because the nitro groups are
present in the ortho position and it makes steric hindrance to amine
group, so that the formation of monoanion was restricted. The
prototropic equilibrium is shown in Scheme 2. The pKa value for
the equilibria is determined spectrophotometrically and is given in
Table 1.
3.1. Effect of pH
The absorption spectra of 2,6-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 the
spectra observed in non-aqueous solvents and thus can be assigned
to the neutral species. In the pH range −4.65 (H0) to pH ∼ 12 the
absorption maxima were observed at 432 nm. With a decrease on
pH from 7.0, there is no change in the absorption maxima up to in
−4.65 solution. The absorption intensity is decreases at 432 nm in
H0 −4.85 this is due to the formation of monocation. The absorp-
tion intensity is decreases due to the protanation with lone pair
of electrons present on the amine group and also removal of con-
jugation by the presence of electron withdrawing group ( NO2)
in two adjacent side (ortho position). In the case of 2,6-DNA (neu-
tral), absorption maxima observed at 432 nm because of lone pair
3.2. Effect of ˇ-cyclodextrin
Table 2 and Fig. 2 show the absorption maxima of 2,6-DNA
(2 × 10−4 mol/dm3) in pH ∼ 7 (neutral) solution containing differ-
ent concentrations of ␤-CD. At pH ∼ 7, 2,6-DNA exists as a neutral
form only. In pH ∼ 7, no clear isosbestic point was observed in the
Fig. 2. Absorption spectra of 2,6-DNA (conc. 2 × 10−4 M) in pH ∼ 7 solutions at dif-
ferent ␤-CD ((a)–(g) conc. 0–12 × 10−3 M). Inset (a) magnified view of ␤-CD effect
in pH ∼ 7 solution at 432 nm.
Fig. 1. Absorption spectra of different prototropic species of 2,6-DNA at 303 K; con-
centration 2 × 10−4 M (MC) monocation and (N) neutral.

580
K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
Scheme 2. Prototropic equilibria of 2,6-DNA in aqueous medium.
absorption spectra even in the presence of higher ␤-CD concentra-
tion.
In pH ∼ 7 solution the absorption maxima is increased and 1:1
inclusion complex is formed, whereas the absorption spectra of
2,6-DNA in pH ∼ 7 there was slight red shift in the absorption max-
ima was observed and also the absorption intensity was increased
when increasing the ␤-CD concentration. At pH ∼ 7, 2,6-DNA exist
as neutral form only with 1:1 complex form, In pH ∼ 7 the 2,6-DNA
located inside the ␤-CD cavity and the NH2 group located at above
the ␤-CD rim, which will cause the increase of the absorbance. This
behavior has been attributed to the enhanced dissolution of the
2,6-DNA molecule through the hydrophobic interaction between
2,6-DNA and ␤-CD. These results indicate that 2,6-DNA molecule is
entrapped into ␤-CD cavity to form an inclusion complex. The bind-
ing constant for the formation of 2,6-DNA:␤-CD complex has been
determined by analyzing the changes in the intensity of absorbance
with the ␤-CD concentration. Fig. S1 plotting 1/(A − A0) versus
1/[␤-CD] for a 1:1 host:guest inclusion complex. In 1:1 inclusion
complex gives straight line at pH ∼ 7. The binding constant (K) can
be obtained by using the modified Benesi–Hildebrand equation
(Benesi & Hildebrand, 1949) for the 1:1 complex Eq. (1) between
2,6-DNA and ␤-CD as shown below.
Scheme 3. The proposed structure of inclusion complex of (a) 2,6-DNA with ␤-CD.
The oxygen atoms are shown as red, nitrogen as blue and carbon as gray color,
hydrogen atoms are white.
in Scheme 3. Such 1:1 inclusion complex structure gains further
stabilization energy by releasing of water from hydrophobic cav-
ity. The inclusion complex has further stabilized by the interaction
of lone pair electrons of secondary hydroxyl group of ␤-CD with
␲ electrons (benzenoid) of 2,6-DNA. The pH dependent changes in
the absorption of the 2,6-DNA molecule in ␤-CD solution have also
been recorded (Table 1). Even in higher alkali (H 15.65) medium
there is no formation of monoanion in ␤-CD medium which was
same behavior as without ␤-CD medium, due to the 2,6-DNA was
included into the nano hydrophobic cavity of ␤-CD and the NH2
group present above the ␤-CD rim. To substantiate the above dis-
cussion, the effect of ␤-CD on the prototropic equilibrium, the
2,6-DNA is present as neutral in pH range −4.65 (H0) to H 15.65. The
absorption spectra of 2,6-DNA in ground state shows the pH range
{−4.65 (H0) to 15.65(H )} as neutral form, neutral to monocation
(without ␤-CD) are hyperchromic effect is observed and neutral pH
red slight shifted in ␤-CD medium this was similar to its in aqueous
medium (Scheme 2), its indicating 2,6-DNA molecule entrapped in
␤-CD cavity.
1
1
ꢀε
1
ꢀ
ꢁ
=
)
+
(1)
A − A
(
0
K 2, 6 − DNA 0 ꢀε ␤ − CD
[
]
0
where A − A0 is the difference between the absorbance of 2,6-
DNA in the presence and absence of ␤-CD, ꢀε is the difference
between the molar absorption coefficient of 2,6-DNA and the inclu-
sion complex [2,6-DNA]0 and [␤-CD]0 are the initial concentration
of 2,6-DNA and ␤-CD, respectively. A good correlation was obtained
for pH ∼ 7 (Fig. S1, R2 = 0.9576), it’s also confirm that the formation
of 1:1 inclusion complex. From the intercept and slope value of
this plot K was calculated [pH ∼ 7 = 17.8 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 the following equation:
ꢀG = −RT ln K
(2)
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 negative which suggests that the inclusion process
proceeded spontaneously at 303 K. This indicates that the forma-
tion of inclusion complex is an exergonic process.
3.4. Semi empirical quantum mechanical calculations
˚
The internal diameter of the ␤-CD is approximately 6.5 A and its
˚
height is 7.8 A (Scheme 4). Considering the shape and dimensions
of ␤-CD, the ground state of 2,6-DNA molecules were optimized
using AM1 method (Scheme 4). In 2,6-DNA; the vertical distances
˚
between H14–H17 and H15–H17 is 5.8 A. The horizontal distance
3.3. Possible inclusion complex of 2,6-DNA in ˇ-CD cavity
˚
˚
between O9–O12 is 7.1 A, O10–O12 and O9–O13 is 6.3 A. The vertical
distance and the horizontal distance measured from the terminal
atoms of 2,6-DNA are less than the height and vertical diameter of
␤-CD. Since, the height of 2,6-DNA are lower than upper-lower rim
In 1:1 inclusion complex, the aromatic moiety is 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

K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
581
Scheme 4. Schematic representation of semi empirical bond distance of (a) ␤-CD, (b) 2,6-DNA. (For interpretation of the references to color in this scheme legend, the reader
is referred to the web version of this article.)
Table 3
of ␤-CD, the insertion of 2,6-DNA in the ␤-CD cavity is possible as
CV for 2,6-DNA:␤-CD in pH ∼ 7 buffer solution, scan rate 100 mV/s, 2,6-DNA (conc.
shown in Scheme 3.
2×10−4 M) and (a–g) 0–12×10−3 M ␤-CD concentrations.
pH ∼ 7
Concentration of
␤-CD (M)
3.5. Molecular docking studies of inclusion process
Epa (V)
Ipa (␮A)
Epc (V)
Ipc (␮A)
0
0.197
7.262
−0.630
−0.816
−0.630
−0.818
−0.633
−0.823
−0.633
−0.826
−0.635
−0.826
−0.635
−0.826
−0.638
−0.828
−16.92
−16.04
−17.50
−16.59
−18.78
−17.16
−19.13
−18.21
−19.33
−18.42
−20.86
−19.35
−21.09
−19.99
The 3D structure of 2,6-DNA was obtained from crystallo-
graphic databases are shown in Scheme 5(a). The guest molecules
of 2,6-DNA was docked into the cavity of ␤-CD using PatchDock
server. The PatchDock server program gave several possible docked
models for the most probable structure based on the energetic
parameters; geometric shape complementarity score, approximate
interface area size and atomic contact energy of the 2,6-DNA:␤-
CD inclusion complexes. The docked 2,6-DNA:␤-CD 1:1 model
(Scheme 5b) with the highest geometric shape complementar-
0.002
0.004
0.006
0.008
0.010
0.012
0.200
0.205
0.234
0.234
0.234
0.258
6.888
6.637
6.201
5.675
5.438
4.934
ity score 2346 for 2,6-DNA:␤-CD, the approximate interface area
2
˚
size of 243.3 A for 2,6-DNA:␤-CD and atomic contact energy of
Binding constant (M−1
ꢀG (kJ/mol)
)
66
−10.5
−850 kJ/mol for 2,6-DNA:␤-CD was calculated. This is highly proba-
ble and energetically favorable model and it was in good correlation
with results obtained through experimental methods.
3.6. Electrochemical studies
The redox behaviors of both 2,6-DNA were clearly represented
in Scheme 6. The cathodic peak current (ipc) of 2,6-DNA (Table 3
and Fig. 3) was increased with increasing the ␤-CD concentration
in pH ∼ 7. The cathodic peak potential (Epc), shifted toward positive
direction in pH ∼ 7. This result shows that the inclusion complex
between 2,6-DNA and ␤-CD was formed when 2,6-DNA was added
into ␤-CD aqueous solution. In addition, the cathodic peak current
increases with increasing ␤-CD concentration; this is due to the
nitro groups are present inside of the ␤-CD cavity and the catalytic
behavior of ␤-CD to the included guest molecule.
The cyclic voltammograms (CV) show the electrochemical
behavior of 2,6-DNA in pH ∼ 7 with ␤-CD (Table 3 and Fig. 3). For-
mation of the inclusion complex of 2,6-DNA with ␤-CD were also
confirmed by electrochemical method. In pH ∼ 7 the cyclic voltam-
mograms shows an anodic peak during the forward scan (toward
positive potential) at 0.197 V and two reduction peaks during the
reverse scan (toward negative potential) at −0.630 V and −0.816 V.
These peaks are ascribed as oxidation of 2,6-DNA (NH2; 0.197 V)
and the reduction of anilinum anion (−0.630 V) into NH2 with
another one reduction peak of NO2 (−816 V) into hydroxylamine.
The binding constant (K) and stoichiometric ratio of the inclu-
sion complex of 2,6-DNA can be determined according to the

582
K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
Scheme 5. Ball and stick representation of (a) 2,6-DNA (b) ␤-CD (c)2,6-DNA:␤-CD inclusion complex; the oxygen atoms are shown as red ball, nitrogen as blue and carbon
as gray 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 this article.)
Table 4
1H NMR chemical shifts of ␤-CD in free and complexed state determined in D2O at
303 K.
Protons
␤-CD
2,6-DNA:␤-CD
ı (ppm)
ꢀı
ı (ppm)
H1
H2
H3
H4
H5
H6
4.966
3.554
3.877
3.535
3.748
3.797
4.964
3.553
3.860
3.476
3.736
3.796
−0.002
−0.002
−0.017
−0.002
−0.012
−0.001
Table 5
1H NMR chemical shifts of 2,6-DNA in free and complexed state determined in D2O
at 300 K.
Substances
Ha ı (ppm)
Hb ı (ppm)
Hc ı (ppm)
2,6-DNA
2,6-DNA:␤-CD
ꢀı
8.335
8.285
−0.050
–
–
–
7.080
6.996
−0.084
Fig. 3. Cyclic voltammograms for 2,6-DNA:␤-CD in pH ∼ 7 buffer solution, scan rate
100 mV/s, 2,6-DNA (conc. 2 × 10−4 M) and ␤-CD ((a)–(g) conc. 0–12 × 10−3 M).
typical 1H NMR spectra of (a) ␤-CD, (b) the solid inclusion complex
of 2,6-DNA with ␤-CD (chemical shift changes with ␤-CD), (c) 2,6-
DNA and (d) the solid inclusion complex (chemical shift changes
with 2,6-DNA). 10 mg of sample were dissolved in 0.7 ml of D2O
solvent and then filtered through a Pasteur pipette, equipped with
a glass wool plug that discharges into an NMR tube. The values
of chemical shifts, ı for different protons in ␤-CD with solid inclu-
sion complex and 2,6-DNA with solid inclusion complex were listed
in Tables 4 and 5. It can be seen from the 1H NMR that in solid
inclusion complex, a great upfield shift was occurred for H3 and H5
protons of ␤-CD experience magnetic perturbation due to the guest
molecule while those at the H2 and H4 positions have no significant
change in the chemical shift. Which (H3 and H5) locate in the nano
hydrophobic cavity of ␤-CD (Scheme 7b). The changes of chemi-
cal shift (ꢀı) of H3 and H5 suggested that the 2,6-DNA monomer
entered into the nano hydrophobic cavity of ␤-CD. The phenyl ring
of 2,6-DNA made the signals of ␤-CD protons (H3 and H5) upfield
Benesi–Hildebrand (14) relation assuming the formation of a 1:1
host–guest complex.
1
1
ꢀI
1
ꢀ
ꢁ
=
+
(3)
IHG − IG
K 2, 6-DNA 0 ꢀI ␤-CD
[
]
0
where IG is the reduction peak current of guest molecule of 2,6-
DNA, and IHG is the reduction peak current of inclusion complex
of 2,6-DNA:␤-CD. IHG − IG is the difference between the reduction
peak current of inclusion complex and 2,6-DNA. ꢀI is the differ-
ence between the molar peak current coefficient of the inclusion
complex and 2,6-DNA. The [2,6-DNA]0 and [␤-CD]0 are the initial
concentration of 2,6-DNA and ␤-CD, respectively. Plot of [1/IHG − IG]
verses 1/[␤-CD] gives a straight line in pH ∼ 7 solution as shown in
Fig. S2. Good linear correlation was obtained (R2 = 0. 9945, pH ∼ 7),
it’s also confirm that the formation of a 1:1 inclusion complex for
pH ∼ 7 solution. From the intercept and slope values of this plot
K was evaluated, the binding constant values for 2,6-DNA:␤-CD
is 66 M−1 in pH ∼ 7. This value indicate that 2,6-DNA molecule is
encapsulated in the ␤-CD cavity to form an inclusion complex.
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 as shown as in Scheme 7b, were only slightly affected by
the guest molecule. Similarly, the chemical shifts of Ha, Hb and Hc
of 2,6-DNA (Scheme 7c) locate in the nano hydrophobic cavity of
␤-CD was also moved in upfield significantly because the inter-
action between hydrophobic interactions of aromatic ring of the
2,6-DNA and hydrogen atoms in the ␤-CD cavity should be present
in the complex. Since the C H bonds of positions 3 and 5 of the
3.7. 1H NMR studies of 2,6-DNA with ˇ-CD
The formation of solid inclusion complex can be analyzed from
1H NMR spectra (Chen, Diao, & Zhang, 2006). Fig. 4 showed the

K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
583
Fig. 4. 1H NMR spectra of (a) ␤-CD, (b) the solid complex (chemical shift with respect to ␤-CD); (c) 2,6-DNA, (d) the solid complex (chemical shift with respect to 2,6-DNA).
3.8. 2D 1H NMR (ROESY) studies of 2,6-DNA with ˇ-CD
glucopyranosyl unit point toward the ␤-CD cavity, shielding effects
due to the aromatic ring current should influence the chemical
shifts of the host protons. The upfield-shifted signals of H3 and H5
suggested that the majority of those protons should not be exactly
in the plane of the ring current. The H2 and H4 protons do not
directly interact with 2,6-DNA because they are exposed to bulk
environments. The significant distinguish for these 1H NMR spec-
tra strongly confirmed that the solid inclusion complex formation
of 2,6-DNA with ␤-CD.
The 2D 1H NMR (ROESY) is a powerful technique for investi-
gation of inter and intra molecular interactions. The chemical shift
changes was shown by Ha, and Hc protons of aromatic (hydrophobic
part) moiety (Sohajda et al., 2009) of 2,6-DNA may play a major role
in the inclusion process. To verify this hypothesis, 2D 1H NMR spec-
tra were recorded. The presence of NOE cross-peaks between pro-
tons of two different species in 2D 1H NMR spectra is an indication

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K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
Fig. 5. The 2D 1H NMR (ROESY) spectra of inclusion complex of (a) 2,6-DNA:␤-CD, (b) partial counter plot of 2,6-DNA:␤-CD in DMSO-d6 at 300 K.
Scheme 6. Reaction mechanism of 2,6-DNA in pH ∼ 7 buffer solution at glassy carbon electrode.

K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
585
Fig. 6. FT-IR Spectra of (a) 2,6-DNA, (b) ␤-CD and (c) solid inclusion complex of
2,6-DNA:␤-CD complex in KBr.
that they are in spatial contact through space within the cavity of ␤-
CD. Fig. 5 shows the 2D spectra of (a) 2,6-DNA:␤-CD and (b) counter
part of 2,6-DNA:␤-CD systems, two groups of intermolecular NOE
cross-peaks were observed.
Fig. 7. Powder X Ray diffraction spectra of (a) 2,6-DNA, (b) ␤-CD and (c) solid inclu-
sion complex of 2,6-DNA:␤-CD.
In 2,6-DNA:␤-CD complex two groups of inter molecular NOE
cross peaks were observed. The first NOE cross peak belongs
to the interaction between the H3 (3.860 ppm) proton of ␤-CD
with the meta positioned protons (8.285 ppm) of 2,6-DNA and the
another NOE cross peak was assigned the interaction between the
H5 protons (3.736 ppm) of ␤-CD with the para positioned proton
(6.996 ppm) of 2,6-DNA indicates their proximity. In all cases the
altered. Fig. 6 is the FT-IR spectra of (a) ␤-CD, (b) 2,6-DNA, (c) solid
inclusion complex of 2,6-DNA.
From the Fig. 6 the two characteristic stretching of -NO2 peaks
were observed at 1562 cm−1, 1359 cm−1. These stretching bands
were shifted to 1564 cm−1, 1365 cm−1 and also the intensity of
these peaks were reduced this is due to the nitro group of 2,6-DNA
was present (included) in the nanocavity of ␤-CD. The characteris-
tics stretching of C N ( NH) appeared at 1265 cm−1and its shifted
to 1269 cm−1, this is due to the phenyl ring of 2,6-DNA was present
into the nanocavity of ␤-CD. The stretching band of C N ( NO2)
group appeared at 889 cm−1 and it shifted to 892 cm−1 and also its
intensity was reduced, because of the nitro group was entrapped
interaction of 2,6-DNA with only internal protons of ␤-CD (H3
&
H5) were observed. In addition the H6ab protons of ␤-CD were not
affected by the inclusion process. So that we can confirmed that
the 2,6-DNA is included in to the ␤-CD cavity via wider rim. From
the above fact the 2,6-DNA are interact with ␤-CD through space
contact, not by bonding.
into the nanocavity of ␤-CD. The stretching vibration of
C C
appeared at 1635 cm−1 and it shifted to 1637 cm−1, and its inten-
sity also reduced, due to the hydrophobic part of benzene ring of
2,6-DNA was included into the nanocavity of ␤-CD. As can be seen
above discussions it can be concluded that of 2,6-DNA molecule
was included into ␤-CD cavity.
3.9. FT-IR spectral studies
The solid inclusion complex formation has been confirmed by
FT-IR spectroscopy, because the bands resulting from the included
part of the guest molecule are generally shifted or their intensities
Fig. 8. Scanning electron microscope photographs (Pt. coated) of (a) 2,6-DNA (b) ␤-CD and (c) solid inclusion complex of 2,6-DNA:␤-CD.

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K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
Scheme 7. (a) The stereo-configuration of ␤-CD, (b) truncated-cone shape of ␤-CD and (c) 2,6-DNA.
3.10. Powder X-ray diffraction pattern
3.11. Scanning electron microscope morphological observations
The lack of crystallinity is an added evidence for the formation
of inclusion complex (Williams, Mahaguna, & Sriwongjanya, 1998).
Fig. 7 shows the XRD patterns for guest (2,6-DNA), host (␤-CD)
and their complex systems were prepared by co-precipitation tech-
niques at molar ratio of 1:1. The powder X-ray diffraction pattern of
2,6-DNA (Fig. 7a) revealed that several sharp high intensity peaks
at different diffraction angles (2Â) of 12.64, 19.07, 25.57, and 38.84
suggesting that the 2,6-DNA existed as crystalline nature. The ␤-CD
(Fig. 7b) showed a crystalline diffractogram, while a diffuse halo-
pattern was recorded for 2,6-DNA-␤-CD (Fig. 7c) demonstrating its
amorphous nature. The diffraction patterns of the investigated in
the 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 preparation in
solution phase of the pure crystalline components. Few of diffrac-
tion peaks of 2,6-DNA was matches with those of ␤-CD was evident.
A typical diffuse pattern indicating the entirely amorphous nature
of 2,6-DNA in complexed state.
Scanning electron microscope (SEM) are well suited for visual-
ize the surface texture of the deposited film. The SEM analysis is
ideal for quantitatively measuring the surface roughness and for
visualizing the surface texture of the substance. First we observed
surface morphological structure of (a) 2,6-DNA,(b) ␤-CD (Fig. 8) by
SEM, and then we also observed the surface morphological struc-
ture 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 nano hydrophobic cavity of ␤-CD.
4. Conclusions
The following conclusion can be arrived at from the above stud-
ies: (i) The ground state acidity constant (pKa) value for prototropic
reaction of 2,6-DNA was calculated in absence of ␤-CD by spec-
trophotometricaly. (ii) 2,6-DNA forms 1:1 inclusion complex with
␤-CD in pH ∼ 7. (iv) The electrochemical studies also confirmed the

K. Srinivasan et al. / Carbohydrate Polymers 113 (2014) 577–587
587
formation of inclusion complex in pH ∼ 7 (v) 1H NMR, 2D 1H NMR
(ROESY), FT-IR, XRD and SEM results suggest 2,6-DNA formed a
solid inclusion complex with nano hydrophobic cavity of ␤-CD. (vi)
The energetically favorable complex obtained by molecular dock-
ing studies is in good correlation with the inclusion model predicted
through experimental investigations.
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One of the authors K. Srinivasan acknowledges to UGC-BSR
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