Synthesis, spectroscopy, X-ray crystallography, DFT calculations, DNA binding and molecular docking of a propargyl arms containing Schiff base

Article abstract of DOI:10.1016/j.saa.2015.06.013

A propargyl arms containing Schiff base (L) was synthesized by the condensation of 1-[2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl]ethanone with trans-1,2-diaminocyclohexane. The structure of L was characterized by IR, ¹H NMR, ¹³C NMR and U

Full text of DOI:10.1016/j.saa.2015.06.013

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, spectroscopy, X-ray crystallography, DFT calculations, DNA
binding and molecular docking of a propargyl arms containing Schiff
base
⇑
C. Balakrishnan, L. Subha, M.A. Neelakantan , S.S. Mariappan
Chemistry Research Centre, National Engineering College, K.R. Nagar, Kovilpatti 628 503, Thoothukudi District, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Propargyl arms containing Schiff base (L) was synthesized. The structure of L was characterized by spec-
tral and X-ray analysis. Molecular geometry of L was optimized by density functional method (B3LYP)
with 6-31G basis set. The DNA binding ability and molecular docking were discussed.
ꢀ
Single crystal X-ray structure of a
propargyl arms containing Schiff
base.
ꢀ
ꢀ
ꢀ
Optimized geometries, IR and NMR
spectra and NBO analysis.
Binding of the Schiff base with
CT-DNA.
Molecular docking of the Schiff base
with DNA.
a r t i c l e i n f o
a b s t r a c t
Article history:
A propargyl arms containing Schiff base (L) was synthesized by the condensation of 1-[2-hydroxy-4-(pro
p-2-yn-1-yloxy)phenyl]ethanone with trans-1,2-diaminocyclohexane. The structure of L was character-
Received 2 December 2014
Received in revised form 3 June 2015
Accepted 6 June 2015
1
13
ized by IR, H NMR, C NMR and UV–Vis spectroscopy and by single crystal X-ray diffraction analysis.
The UV–Visible spectral behavior of L in different solvents exhibits positive solvatochromism. Density
functional calculation of the L in gas phase was performed by using DFT (B3LYP) method with 6-31G basis
set. The computed vibrational frequencies and NMR signals of L were compared with the experimental
data. Tautomeric stability study inferred that the enolimine is more stable than the ketoamine form.
The charge delocalization has been analyzed using natural bond orbital (NBO) analysis. Electronic absorp-
tion and emission spectral studies were used to study the binding of L with CT-DNA. The molecular dock-
ing was done to identify the interaction of L with A-DNA and B-DNA.
Available online 12 June 2015
Keywords:
Single crystal analysis
Electronic absorption spectra
DFT calculation
DNA binding
Molecular docking
Ó 2015 Elsevier B.V. All rights reserved.
Introduction
functional group is a notable key component of the pharma-
cophoric unit in antibiotics [2], anticancer [3] and antitubercular
Acetylenic group has significant attraction in medicinal chem-
istry research and pharmaceutical industry [1]. The acetylenic
[4]. A series of propargyl derivatives exhibit significant neuropro-
tective and neurorescue activities [5,6]. Organic compounds with
propargyl functional group possesses inhibitory properties toward
flavin-linked oxidases [7]. These compounds play important role in
neuroprotection [8]. Thymidylate Synthase inhibitor, an antifolate
⇑
Corresponding author. Tel.: +91 9442505839; fax: +91 4632232749.
E-mail addresses: drmaneelakantan@gmail.com, maneels@rediffmail.com
(
M.A. Neelakantan).
http://dx.doi.org/10.1016/j.saa.2015.06.013
386-1425/Ó 2015 Elsevier B.V. All rights reserved.
1

6
72
C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
containing propargyl moiety provides a 10-fold increase in potency
when compared to its methyl analogue [9].
for 1 h. The pale yellow solid product obtained was washed with
methanol. Yellow needle like crystals suitable for X-ray diffraction
were grown in dichloromethane–methanol mixture and kept in
Schiff bases possess many interesting properties, e.g., catalytic
activity, selective recognition of metal ions, and photochromic
properties. Schiff bases are used as starting materials in the prepa-
ration of antibiotics, antiallergic, antiphlogistic and antitumor
drugs [10,11]. Schiff bases are also used as probes in investigating
the structure of DNA [12]. o-Hydroxy Schiff base compounds exhi-
bit tautomerism. The different tautomeric forms usually exist in
the solid state are enol, keto and Zwitterionic forms. The tau-
tomerism in o-hydroxy Schiff bases is due to the existence of
OHꢁ ꢁ ꢁN and NHꢁ ꢁ ꢁO type hydrogen bonds. Hydrogen bonding pre-
sent in cellular matters is important to maintain the functional
structure of biological molecules. The change in position or inten-
sity of an absorption or emission band of a compound in solution
with respect to the polarity of the medium is solvatochromism
30 4 2
refrigerator for 3 weeks. mp. 196 °C. Composition for C28H O N .
Found (Calculated) (%) C: 73.34 (73.39), H: 6.59 (6.64), N: 6.11
ꢂ1
(6.16). IR (KBr), cm : 3441, 3282, 3203, 2937, 2854, 2112, 1604,
1
1448, 1338, 1263, 1184.
H
NMR (400 MHz, CDCl
– cyclohexane ring), 3.80–3.82
(1H, d, @NACHA, J = 3.6 Hz), 6.26–6.37 (6H, m, aryl), 2.50–2.52
(2H, s, AC„CH), 4.64 and 4.65 (4H, s, –O–CH ), 2.24 (6H, s,
): d (ppm) = 161.65–
3
):
d
(ppm) = 1.45–2.01 (10H, m, CH
2
2
1
3
3
CH AC@NA).
C NMR (400 MHz, CDCl
3
170.70 (AC@N1 and AC@N2), 61.59 and 75.69 (C9 and C10),
14.21 (C25, C28), 24.15 (C22, C23), 32.45 (C21, C24), 78.13 (C1,
C18), 55.63 (C3, C16).
Crystallography
[
13]. Schiff bases with positive and negative solvatochromism are
used as probes for complex biological systems [14,15]. The binding
of small molecules and DNA has been studied extensively. The
binding interaction between small molecules and DNA may be
due to intercalation, groove binding and external electrostatic
binding. The study of interaction of small molecules with DNA is
useful in designing effective chemotherapeutic agents.
Single crystal X-ray diffraction experiment was performed on a
Bruker Kappa Apex II diffractometer using MoK
a radiation
k = 0.71073 Å at 296(2) K. A pale-yellow prism of L with dimen-
sions of 0.25 mm ꢃ 0.20 mm ꢃ 0.20 mm was used. The structure
was solved by direct method procedure using SHELXS-97 program
[20]. The refinement was carried out using Full Matrix Least Square
2
In continuation of our interest on Schiff bases [16–18], we
report herein the synthesis, spectral characterization and crystal
structure of a Schiff base derived from the condensation of propar-
gylated hydroxyacetophenone and cyclohexane diamine. This
paper describes the solvatochromism of the Schiff base in different
solvents in the UV–Visible spectra. The computational studies were
method on F , which is in correspondence with 288 parameters. All
the non-hydrogen atoms were refined anisotropically. The hydro-
gen atoms bonded to carbon were inserted at calculated positions
using a riding model. Hydrogen atoms bonded to oxygen were
located from difference map and allowed to refine with tempera-
ture factors riding on those of the carrier atoms. The geometrical
parameters were obtained using PARST [21] and SHELXL-97.
1
carried out at DFT/B3LYP/6-31G level. The IR and H NMR spectra
were computed at this level and compared with the experimental
results. The interactions of L and CT-DNA were investigated sys-
tematically. The molecular docking has been employed to get
information about the interaction of L with A-DNA and B-DNA.
Computational procedures
All the computational calculations have been performed with
Gaussian 03 W program using Density Functional Theory (DFT)
with Becke’s three-parameter exchange and Lee–Yang–Parr corre-
lation functionals (B3LYP) with a combination of 6-31G basis set
Experimental
[
22]. GaussView program has been used for the molecular visual-
Materials and instrumentation
ization of computed structures [23]. The harmonic vibrational fre-
quencies and NMR signals of the studied structure were computed
at the corresponding optimized geometry using the same theory
level. The minimum energy structures are ensured by the absence
of any imaginary frequency. In solution phase, the geometry opti-
mization of the studied structure is performed at the same level
with polarizable continuum model (PCM) [24]. Natural Bond
Orbitals (NBOs) analysis has been performed for optimized enolim-
ine and ketamine form at the DFT/B3LYP/6-31G level.
All the reagents and solvents were obtained from Sigma–
Aldrich and used as received. Doubly distilled deionized water
was used throughout the experiments. CT-DNA was purchased
from Genei, Bangalore and used without purification. Tris HCl
and ethidium bromide were obtained from HiMedia. Elemental
analysis for carbon, nitrogen and hydrogen was carried out using
a Thermo Finnigan Flash EA 1112 series CHN analyzer. FT-IR spec-
tra were recorded on a Shimadzu 8400S spectrophotometer with
KBr pellets in the range of 450–4000 cm . Electronic absorption
spectra were recorded at room temperature using a UV-2450 spec-
trophotometer. The fluorescence spectra were recorded on a Jasco
FP-8300 spectrofluorophotometer. H NMR and C NMR measure-
ments were performed in CDCl on a Bruker Avance 400 MHz spec-
trometer. The 2D-COSY and HSQC spectra were obtained by using
the standard Bruker pulse programs.
ꢂ1
DNA binding measurements
Electronic absorption spectral titrations were used to study the
binding of L with CT-DNA. The binding experiments were per-
formed in Tris–HCl/NaCl buffer (50 mM Tris HCl/NaCl buffer, pH
1
13
3
7
.2). The concentration of CT-DNA was determined from the
ꢂ1
ꢂ1
absorption intensity at 260 nm with emax value 6600 M cm
.
Stock solution of DNA was stored at 4 °C and used within seven
Synthesis of Schiff base (2,2’-{cyclohexane-1,2-diylbis[nitrilo(1E)eth-
days. Absorption titration experiments were done using fixed con-
1-yl-1-ylidine]}bis[5-(prop-2-yn-1-yloxy)phenol])
centration of L (40
lM) and varying the concentration of CT-DNA
(
10–50 M). While measuring the spectra, an equal amount of
l
The synthesis of Schiff base (L) was achieved as depicted in
DNA was added to both the compound and reference solutions to
Scheme S1. 1-[2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl]ethanone(
A) was obtained through a reaction between 3-bromopropyne
and 2,4-dihydroxyacetophenone as reported in our earlier work
eliminate the absorbance of DNA itself. From the absorption data,
b
the intrinsic binding constant K was determined using the
equation [25],
[
(
19]. A solution of A (20 mM) and trans-1,2-diaminocyclohexane
10 mM) in 10 mL of methanol was refluxed for 36 h. The resulting
½
DNAꢄ
½DNAꢄ
1
¼
þ K
f b
e e
e
a
ꢂ
e
f
e
b
ꢂ
ð
a
ꢂ
e
f
Þ
liquid was quenched by adding 5 mL of distilled water and stirred

C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
673
Fig. 1. (a) ORTEP diagram of L with thermal ellipsoids at 50% probability; (b) Optimized geometry of L in gas phase.
where
e
a
e
f
, and
e
b
are the extinction coefficients of the apparent,
was determined by comparing slope of the line in the fluorescence
intensity versus compound concentration. The apparent
free and bound L, respectively.
binding constant (Kapp
app = KEB[EB]/[L], where [L] is the concentration of the Schiff base
at which there is 50% reduction in the fluorescence intensity of
) was calculated using the equation
Fluorescence titration study
K
7
ꢂ1
The interaction of L and DNA was studied by fluorescence spec-
tral method using EB-bound CT-DNA in Tris–HCl/NaCl buffer solu-
tion (pH 7.2). The excitation wavelength was fixed at 420 nm and
the emission range was adjusted before the measurements.
EB. KEB = 1.0 ꢃ 10 M and [EB] = 25
lM.
Molecular docking analysis
Changes in the fluorescence intensities at 610 nm of EB (25
bound CT-DNA (10 M) were measured with respect to different
concentrations of L (10–50 M). The magnitude of the binding
strength of L with CT-DNA can be calculated using the linear
Stern–Volmer equation [26],
l
M)
Molecular docking analysis was carried out by using Autodock
4.2 program [27]. The PDB format of L was obtained from its CIF file
using Mercury software. The crystal data of CT-DNA were obtained
from Protein Data Bank (PDB) identifier 3V9D (A-DNA) and 1BNA
(B-DNA) [28] (http://www.rcsb.org). The water molecules and
other unsupported elements (e.g., Na, K, Hg, etc.,) were removed
from the 3V9D (A-DNA) and 1BNA (B-DNA). Gasteiger charges
were added to the compound (L) by Autodock Tools (ADT) before
subjecting to docking analysis. Ligand docking calculations were
carried out using Lamarckian genetic algorithm (LGA) [29]. The
binding area was focused on the macromolecules (DNA/L) and a
l
l
I=I
0
¼ 1 þ K
s
v
½Qꢄ
where I
0
and I represents the fluorescence intensities of EB-DNA in
the absence and presence of the quencher, respectively. Q is the
concentration of the Schiff base. Ksv is the linear Stern–Volmer
quenching constant. The relative binding tendency of L to CT-DNA

6
74
C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
Table 1
form of L were compared with the crystal data (Table 2). For L,
the experimental bond lengths of N(1)–C(8), C(6)–O(3), N(2)–
C(11), and O(4)–C(13) are comparable with that of enol form,
obtained theoretically. In the optimized structure of L, hydrogen
bond is observed in the portion of O(3)–H(3)–N(1) and O(4)–
H(4)–N(2). The D (Donor)–H, H–A (Acceptor) and <D–H–A bond
angle values are 0.972 Å, 1.057 Å, and 155.39° respectively,
whereas the values obtained from the X-ray study are 0.8207 Å,
Crystal data and structure refinement of Schiff base (L).
Structural parameters
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
a (Å)
28 30 2 4
C H N O
458.54
296(2)
0.71073
Monoclinic
30.210(7)
9.636(3)
19.828(7)
90.00
1
.7437 Å, and 148.22°, respectively. The small discrepancies
b (Å)
c (Å)
between the experimental and theoretical values are due to change
in phase. In single crystal analysis, the data corresponds to mole-
cules in solid phase, whereas the optimized geometry corresponds
to molecules in gas phase.
a
(°)
b (°)
(°)
116.979(16)
90.00
5144(3)
c
3
Unit cell volume (Å )
Space group
C2/c
F(000)
Crystal size
1952
Spectroscopic studies
FT-IR spectra
0.25 ꢃ 0.20 ꢃ 0.20 mm
1.51–28.26°
98%
1.184 Mg/m
0.079 mm
Theta range for data collection
Completeness to h = 28.26
Calculated density
Absorption coefficient
No. of formula units per unit cell, Z
Reflections collected/unique
R (int)
Final R1 values [I > 2
Final wR (F ) values [I > 2
Final R1 values (all data)
3
The FTIR spectrum of L (Fig. S2a) shows a strong peak at
ꢂ1
ꢂ1
1
604 cm corresponds to C@N stretching vibration of the Schiff
8
22,315/6276
0.0811
0.0769
0.2054
0.2087
0.2709
1.117 and ꢂ0.192
Full-matrix least-squares on F2
6276/0/309
base. The spectrum exhibits
a
broadband around 3441–
ꢂ1
3269 cm
is due to the presence of O–Hꢁ ꢁ ꢁN intramolecular
r(I)]
hydrogen bond [32]. The terminal alkyne (
m
AC„CAH) exhibits the
2
r(I)]
ꢂ1
C–H stretch as a strong and narrow band at 3203 cm . A medium
absorption band at 2112 cm is due to the presence of terminal
alkyne group (mAC„CA). The strong band at 2937 and 2854 cm
are due to the asymmetric and symmetric C–H stretching
ꢂ1
2
Final wR (F ) values (all data)
Largest diff. peak and hole (e A
Refinement method
ꢂ3
ꢂ1
)
Data/restraints/parameters
Goodness-of-fit on F2
1.031
Table 2
Experimental and computed bond lengths and bond angles of L.
grid box size of 126 ꢃ 126 ꢃ 126 was created along the x, y and z
axis, i.e., blind docking was performed. The output of the
AutoDock was further analyzed with PyMOL software package
Parameters
Experimental
X-ray data
B3LYP/6-31G
Enol
Keto
Bond lengths (Å)
O(1)–C(4)
O(1)–C(3)
O(2)–C(15)
O(2)–C(16)
O(3)–C(6)
O(4)–C(13)
N(1)–C(8)
[
30].
1.379(5)
1.420(5)
1.369(4)
1.437(5)
1.307(4)
1.319(4)
1.313(5)
1.448(5)
1.298(5)
1.464(4)
1.507(5)
1.522(6)
1.503(6)
1.516(5)
1.535(5)
1.399
1.462
1.399
1.462
1.393
1.393
1.286
1.472
1.286
1.472
1.543
1.538
1.539
1.543
1.548
1.391
1.465
1.391
1.465
1.309
1.309
1.341
1.461
1.341
1.461
1.545
1.540
1.540
1.545
1.560
Results and discussion
The L obtained as pale yellow needle crystal is a stable solid at
room temperature. It is soluble in 1,4-dioxane, chloroform, dichlor-
omethane, acetonitrile, dimethyl formamide, dimethyl sulphoxide,
N(1)–C(9)
N(2)–C(11)
N(2)–C(10)
C(10)–C(24)
C(23)–C(24)
C(22)–C(23)
C(9)–C(21)
C(9)–C(10)
2
-propanol, ethanol and methanol.
Crystal structure and optimized geometry of L
The atom numbering scheme and the thermal ellipsoid plot of L
drawn at 50% probability level using ORTEP [31] is given in Fig. 1a.
The L crystallized into a monoclinic crystal lattice system with
space group C2/c (Table 1). The presence of ortho hydroxyl group
in L favors the existence of intramolecular hydrogen bonding and
also the tautomerism accounting for the formation of either phe-
nol–imine (O–Hꢁ ꢁ ꢁN) or keto–amine (Oꢁ ꢁ ꢁH–N) tautomers. The
N(1)–C(8) and N(2)–C(11) bond length of 1.313(5) and
Bond angles (°)
C(15)–O(2)–C(16)
C(6)–O(3)–H(3)
C(13)–O(4)–H(4)
C(8)–N(1)–C(9)
N(1)–C(8)–C(28)
N(1)–C(9)–C(10)
N(2)–C(10)–C(24)
N(2)–C(10)–C(9)
C(22)–C(23)–H(23B)
C(10)–C(24)–C(23)
C(10)–C(24)–H(24A)
C(23)–C(24)–H(24A)
C(10)–C(24)–H(24B)
C(8)–C(28)–H(28A)
C(21)–C(9)–C(10)
C(11)–N(2)–C(10)
118.0(3)
109.5
109.5
125.8(3)
123.0(4)
110.3(3)
108.7(3)
107.8(3)
109.6
111.8(3)
109.2
109.2
109.2
109.5
119.68
111.53
111.53
122.35
125.63
108.64
108.18
108.64
109.17
111.88
108.85
110.92
110.46
110.46
110.79
122.35
119.31
–
–
128.71
118.58
109.21
110.56
109.21
110.87
111.62
108.94
109.87
108.72
110.31
110.69
128.70
1
.298(5) Å, respectively is more close to C@N length (1.25 Å) than
the C–N bond length (1.448(5) and 1.464(4) Å). In L, C@N group
seems to have strong electron withdrawing character. The bond
distance of O(3)–C(6) and O(4)–C(13) is consistent with the C–O
single bonding (Table 2). The C–O and C@N bond length indicates
that the phenol–imine form of L exists in the crystalline state. In
the crystal packing of L, viewed down the ‘b’ axis (Fig. S1), the
cyclohexyl ring is in the chair form. The bond angle of C(10)–
C(24)–C(23) is 111.8(3)° and torsion angle of C(22)–C(23)–C(24)–
C(10) is 56.0(5)°. The hydrogen bond interaction and symmetry
codes of L are given in Table S1.
109.7(3)
126.9(3)
Torsion angles (°)
C(11)–N(2)–C(10)–C(24)
C(11)–N(2)–C(10)–C(9)
N(1)–C(9)–C(10)–N(2)
C(22)–C(23)–C(24)–C(10)
C(4)–O(1)–C(3)–C(2)
ꢂ142.9(4)
96.5(4)
ꢂ122.70
116.97
ꢂ67.40
55.19
ꢂ126.55
111.42
ꢂ60.67
55.47
ꢂ64.9(4)
56.0(5)
The optimized geometry of L in gas phase is shown in Fig. 1b.
The bond lengths and bond angles obtained from enol and keto
ꢂ68.0(5)
ꢂ77.73
ꢂ76.35

C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
675
Table 3
Experimental and calculated vibrational frequencies and assignments of L (cm ).
calculated value is a harmonic frequency. To make a comparison
with the experimental observation, correlation graph was drawn
(Fig. S2b). It is clear from the graph that the experimental funda-
mentals are found to have good correlation with the computed
values.
ꢂ1
Assignments
Vibrational frequencies
Experimental
DFT
–
OH str.
3441
3282
3203
2937, 2854
2112
1604
1448
1263
1338
736
3792
3343
3607
3212, 3205
2370
1806
1685
1588
1444
926
C–H (aromatic) str.
AC„CAH: CAH str. (terminal)
C–H str.
AC„CA str.
C@N str.
C@C (aromatic) str.
C–O (phenolic) str.
O–H bend (in plane)
O–H bend (out of plane)
C–H(aromatic) bend (in plane)
C–H(aromatic) bend (out of plane)
AC„CAH
1D and 2D NMR spectra
1
The H NMR spectrum of L recorded in CDCl
3
is given in Fig. 2.
The 1H NMR signals for the methine hydrogens are observed at
.80 ppm (H9, H10). L exhibits a singlet at 2.24 ppm with three
3
proton count (H25, H28) corresponds to methyl protons [35]. The
multiplets observed in the range of 7.28–7.26 ppm are assigned
to the aromatic protons. The acetylenic protons AC„CH are
observed in the upfield region (2.50–2.52 ppm). The cyclohexane
ring protons (H21–H24) appears as multiplet in the region of
1184
831
630
1339
976
716
1
3
1
.45–2.01 ppm [19]. In the C NMR spectrum of L (Fig. 3), azome-
thine carbon resonances (C8 and C11) are observed at 161.65–
70.70 ppm, respectively. The signals at 61.59 and 75.69 ppm can
str. – Stretching.
1
be assigned to the –CH carbons attached to N in the cyclohexane
ring [36]. The signals at 24.15 and 32.45 ppm can be attributed
to the carbons of the cyclohexane ring [37]. The experimental
and the calculated chemical shift values are given in Table S2.
The experimental spectrum of L is comparable with the spectrum
computed for the enolimine tautomer (Fig. S3a, S3b). To make a
comparison with the experimental observation, correlation graphs
were drawn (Fig. S3c, S3d). It is clear from the graph that the
experimental values are found to have good correlation with the
computed values.
vibrations, respectively [33]. The existence of the phenolic C–O
band at 1263 cm and the non existence of the NH bands are
the other evidence for the higher percentage of phenol-imine tau-
ꢂ1
tomer of L in the solid state. The C@C stretching modes of aromatic
ꢂ1
rings of L are observed in the region of 1600–1450 cm . The bands
ꢂ1
observed at 1338 and 736 cm are assigned to the –OH in-plane
and out-of-plane bending vibrations, respectively [34].
The harmonic vibrational frequencies computed for the opti-
mized geometry of enolimine tautomer at DFT level is compared
with the experimental values (Table 3). From the computed fre-
1
1
In the H– H COSY experiment, the magnetization is transferred
through scalar mechanism. The spin–spin interaction of the adja-
cent protons can be determined from the cross-contour. The
ꢂ1
quencies, a strong peak at 1806 cm corresponds to C = N stretch-
ing vibration of L. The terminal alkyne (
stretching vibration at 3607 cm . The asymmetric and symmetric
C–H stretching vibrations appears at 3212 and 3205 cm , respec-
m
AC„CAH) shows the C–H
ꢂ1
1
1
H– H COSY spectrum (Fig. 4) displayed scalar couplings for adja-
ꢂ1
cent protons like H21 M H9, H21 M H22, H24 M H10, H24 M H23,
H19 M H20 and H26 M H27. The 1,3 interactions are observed
with propargyl group protons (H1 M H3, H16 M 18). The connec-
tion between these protons and its parent carbon is confirmed by
direct couplings in HSQC spectrum (Fig. 5).
tively. The calculated frequencies are higher than the experimental
values for the majority of the normal modes. The reason for the dis-
crepancy between the experimental and computed spectra is that
the experimental value is an inharmonic frequency, while the
Fig. 2. 1H NMR spectrum of L in CDCl
.
3

6
76
C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
Fig. 3. 13C NMR spectrum of L in CDCl
3
.
Fig. 4. 1H– H COSY NMR spectrum of L in CDCl
1
3
.
Electronic absorption spectra and solvatochromism
range are shown in Fig. 6 and the spectral data are summarized
in Table 4. The L shows three absorption bands in the UV region.
The electronic absorption spectra of L in different polar protic
methanol, ethanol and iso-propyl alcohol), polar aprotic (acetoni-
trile, dimethylformamide, dimethylsulfoxide) and non polar sol-
vents (dichloromethane, chloroform, dioxane) in the 200–500 nm
The bands in the region 220–250 and 270–290 nm (
e
max = 16,825
ꢂ1
ꢂ1
⁄
(
and 22,174 M cm ) are attributed to p–p transitions of the aro-
ꢂ1
ꢂ1
matic ring and the band at 378 nm (
e
max = 19674 M cm ) is due
⁄
to
p
–p
transition of the azomethine group. The shoulder at

C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
677
Energy and stability
In order to know the tautomeric stability of L, optimization cal-
culations were performed at B3LYP/6-31G level for the enol and
keto forms. The total energy obtained demonstrates that the eno-
limine form (ꢂ1477.78224733 a.u) is more stable than the ketoa-
mine form (ꢂ1477.7842612 a.u) in gas phase. The vibrational
frequencies, UV–Vis, NBO, HOMOs and LUMOs of
L were
3
Fig. 5. HSQC NMR spectrum of L in CDCl .
2
80 nm may be due to the delocalization of hydrogen from –OH
groups to give keto and enol form (Scheme S2). The less intense
⁄
n–p
transitions of C@N are hidden under the more intense CT
band.
The o-hydroxy Schiff bases containing azo group can exist in
keto and enol forms in both the solid and solution states [38,39].
UV–Visible spectrophotometry is a very precise method for study-
ing intramolecular proton transfer between the phenolic oxygen
and nitrogen atoms. In solution, the tautomerism depends on the
solvent polarity and the ability of the solvents to form hydrogen
bonding. In polar aprotic solvents, the compound L shows absorp-
⁄
tion band around 280 nm corresponds to
p
?p
transition of exten-
sive conjugation of benzene ring. The shoulder near 320 nm is due
to intramolecular hydrogen bonding. On increasing the polarity of
the solvent, the absorption values are shifted to higher wavelength.
This red shift suggests that L is more stable in the excited state
than in the ground state. In polar protic solvents, L shows dual
absorptions at 276 and at 289 nm, which is due to keto–enol tau-
tomerism. As the polarity of the solvent increases the compound
shows red shift suggests that L is more stable in the excited state
than in the ground state. The compound L shows a broad band in
⁄
the region 370–385 nm corresponds to
p?
p
transition of azome-
thine group. This band is commonly observed in o-hydroxyl Schiff
bases and is based on strong intramolecular hydrogen bonding
between the hydroxyl group and azomethine nitrogen [40]. The
transition energy (Emax) of L in different solvents were calculated
by using the formula [41],
E
maxðkcal molꢂ1Þ ¼ 28591:5=kmaxðnmÞ
In order to establish the sign, the value of
DE was calculated
from the Emax of the most non polar solvent and the most polar
solvent.
D
E
¼ Emax of the most non polar solvent
ꢂ Emax of the most polar solvent
When the polarity of solvent is increased, L exhibits a red shift
and positive solvatochromism. This confirms that the molecules
are stabilized by the polar solvents in the excited state [19].
Fig. 6. UV–Visible spectra of L in different solvents, (a) non-polar solvents; (b)
polar-aprotic solvents; (c) polar protic solvents.

6
78
C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
Table 4
Electronic spectral data of L in different solvents.
⁄
⁄
ꢂ1
ꢂ1
Solvents
,4-Dioxane
Chloroform
DCM
DMF
Acetonitrile
DMSO
Dielectric constant
Benzene ring
p–
p
Azomethine
p–p
E
max (kcal mol
)
D
E
max (kcal mol
)
Solvatochromism
1
2.21
4.81
8.93
36.7
37.5
47.0
18.3
24.5
32.6
247
276
271
281
270
284
276
275
276
377
380
374
376
380
382
375
376
378
75.84
75.24
76.45
75.24
75.04
75.67
76.24
76.04
75.64
+0.2
Positive
2
-Propanol
Ethanol
Methanol
Fig. 7. HOMO–LUMO plot of L by DFT/B3LYP/6-31G level of theory.
considered by using the enol form. 3D plots and energy gap values
of HOMOs and LUMOs of L are given in Fig. 7. The energy gap
between HOMO and LUMO explains the molecular chemical stabil-
ity. The energy gap values for the HOMO and LUMO and HOMOꢂ1
and LUMO+1 are 5.1901 and 5.2674 eV, respectively. In the present
investigation, the HOMO is localized over the aromatic phenyl
rings, whereas LUMO is distributed over the entire molecule except
the propargyl arms.

C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
679
C1„H1, C18„H18 bonds have the character of double and triple
bond, which is evident by B3LYP method. In benzene rings, the
C–C bonds are typical single–double arrangement, which form
the conjugate structure. All the results predicted here are consis-
tent with the information obtained from the bond distances of
the crystal structure. The total atomic charges of L were obtained
by Mulliken population analysis at DFT/B3LYP/6-31G in gas phase
in ground state for keto and enol forms. The charge distribution on
some selected atoms of keto and enol forms is represented in
Fig. S4. The higher negative charge on oxygen atoms (O3, O4) than
the nitrogen atoms (N1, N2) suggests the existence of enolimine
form in the ground state as a stable tautomer by forming the
intramolecular hydrogen bond [O–Hꢁ ꢁ ꢁN].
DNA binding measurements
The binding capability of Schiff base (L) with CT-DNA is moni-
tored by observing changes in the ligand based transitions of the
Schiff base upon varying the concentration of DNA. In general,
hypochromism and red or blue shifts are related with the intercala-
tive binding of the compound with DNA [42]. The hyperchromism
is consistent with groove binding [43]. Fig. 8a shows the
UV-absorption spectra of L in the absence and presence of DNA.
In the UV region, L exhibits strong absorption peaks at 264 and
3
44 nm. When increasing the concentration of CT-DNA the absorp-
tion intensity of L increases (hyperchromism), however there is no
change in the position of the absorptions. This suggests groove
binding between L and DNA. The DNA minor groove contains pri-
marily H-bond acceptor groups, the purine N(3) and pyrimidine
O(2) at the floor of the groove walls [44]. The L contains –OH
groups involve secondary interactions like H-bonding with DNA.
6
ꢂ1
The binding constant of L (K
b
= 1.00 ꢃ 10 M ) agrees well with
that of the reported minor groove binders [43].
Fig. 8. (a) Absorbance spectra of L in the presence of DNA; (b) Emission spectra of
EB (25 M) with DNA of L (10–50 M).
Fluorescence titration study
l
l
The interaction mode of Schiff base (L) with DNA and the rela-
tive binding ability has been studied by the ethidium bromide (EB)
displacement method. EB in Tris–HCl buffer has a low intrinsic flu-
orescence intensity. However, on addition of DNA the fluorescence
intensity of EB increases because of its intercalation with DNA. This
higher fluorescence intensity of EB-DNA can be quenched by the
addition of other DNA binding agents. The DNA binding agents
enter with the EB bound DNA leads to the decrease in emission
Natural Bond Orbital (NBO) analysis and atomic charge distributions
Selected NBO occupancies of L is listed in Table S3, which were
calculated on the B3LYP/6-31G level in order to elucidate the
hybridization and delocalization of electron density within the
molecule. NBO analysis shows that the N1@C8, N2@C11 and
Fig. 9. Docked pose of L bound to the minor groove of DNA: (a) A-DNA; (b) B-DNA.

6
80
C. Balakrishnan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 671–681
Table 5
Bonding interactions, bonding type and bonding distances of L with A-DNA.
S. No.
Name
Distance
Bonding
Bonding types
Binding Site of DNA
Binding site of ligand
Binding mode
DNA ? Ligand
1.
2.
3.
4.
5.
6.
7.
8.
A:DG11:H3 – UNK1:O4
A:DG12:H3 – UNK1:O2
B:DG5:C1 – UNK1:N2
2.45
2.67
3.30
3.24
3.30
4.98
5.48
4.99
Hydrogen
Hydrogen
Hydrogen
Hydrogen
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Conventional H-bond
Conventional H-bond
C–H bond
C–H bond
Pi–Sigma
Pi–Alkyl
Pi–Alkyl
Pi–Alkyl
A:DG11:H3
A:DG12:H3
B:DG5:C1
UNK1:O4
UNK1:O2
UNK1:N2
A:DG12:OP1
UNK1
UNK1:C18
UNK1:C28
UNK1
H-Donor ? H-Acceptor
H-Donor ? H-Acceptor
H-Donor ? H-Acceptor
H-Donor ? H-Acceptor
C-H ? Pi-Orbitals
Pi-Orbitals ? Alkyl
Pi-Orbitals ? Alkyl
Pi-Orbitals ? Alkyl
0
0
UNK1:C3 – A:DG12:OP1
UNK1:C3
A:DG11:C2
0
0
A:DG11:C2 – UNK1
B:DC4 – UNK1:C18
B:DG5 – UNK1:C28
B:DG5 – UNK1
B:DC4
B:DG5
B:DG5
DG – Guanosine, DC – Cytosine.
Table 6
Bonding interactions, bonding type and bonding distances of L with B-DNA.
S.No
Name
Distance
Bonding
Bonding types
Binding site of DNA
Binding site of ligand
Binding mode
DNA ? Ligand
0
0
1.
2.
3.
4.
5.
B:DC28:C4 – UNK1:N2
B:DA29:OP1 – UNK1
B:DA27 – UNK1
3.71
3.75
5.73
5.05
4.69
Hydrogen
C–H bond
Pi–Anion
Pi–Pi
Pi–Alkyl
Pi–Alkyl
B:DC28:C4
UNK1:N2
UNK1
UNK1
UNK1:C28
UNK1:C28
H-Donor ? H-Acceptor
Negative ? Pi-Orbitals
Pi-Orbitals ? Pi-Orbitals
Pi-Orbitals ? Alkyl
Electrostatic
Hydrophobic
Hydrophobic
Hydrophobic
B:DA29:OP1
B:DA27
A:DT13 – UNK1:C28
A:DG14 – UNK1:C28
A:DT13
A:DG14
Pi-Orbitals ? Alkyl
DC – Cytosine, DA – Adenosine, DT – Thymidine, DG – Guanosine.
intensity of EB-DNA. In this investigation, the fluorescence inten-
sity of EB-DNA complex decreases on increasing the concentrations
of L (10, 20, 30, 40 and 50 lM) (Fig. 8b). The addition of L to
EB-DNA complex causes a decrease in emission intensity indicating
that L binds to DNA. The binding strength of L with CT-DNA is
determined from the Ksv value, which is obtained from the linear
Stern–Volmer equation [26]. The apparent binding constant
is more stable than the ketoamine form in gas phase. In polar protic
solvents the compound is more stabilized in the excited state than
in the ground state. The results of NBO analysis explains the delo-
calization of electron density within the molecule. The DNA bind-
ing results suggest that the synthesized compound binds to DNA in
a groove binding mode. The molecular docking studies demon-
strate that L fits tightly into the spiral line of the DNA target in
the minor groove.
(Kapp) of L was also calculated [Kapp = KEB[EB]50%/[L]50%]. The appar-
6
ꢂ1
ent DNA binding constant of L is 6.05 ꢃ 10 M
.
Supplementary
Molecular docking
Spectra, crystal packing and computational analysis of L are
embedded in the supplementary material. Detailed crystallo-
graphic data for the structural analyses have been deposited with
the Cambridge Crystallographic Data Centre, CCDC with deposition
number 1034553. Copies of this information can be obtained from
The Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB21EZ, UK (fax: +44-1223-336033; e-mail: deposit@
ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk.
Molecular docking study was performed with AUTODOCK 4.2 to
understand the interaction mechanism between L and A-DNA and
B-DNA, and the structural features of active site of DNA.
Energetically most favorable docked structure obtained from the
rigid molecular docking of L with 3V9D (A-DNA) and 1BNA
(
B-DNA) is shown in Fig. 9a and b. It revealed the binding of L with
the minor groove of targeted DNA towards A-T rich region. The A-T
regions appear to make a suitable binding site for the Schiff base
molecule into the minor groove of DNA [45]. The docked structures
are stabilized by van der Waals interaction and hydrophobic con-
tacts with DNA. The L binds to the minor groove of the DNA with
three base pairs, specially involving the G-C residues (A-DNA)
and A-T residues (B-DNA) as exhibited by the docked structure.
The binding mode of L to the three base pairs is like A-T, A-T,
G-C (B-DNA) and A-T, G-C, G-C (A-DNA) to the outer surface, i.e.,
the groove of DNA molecule. In the docking analysis results, the
relative binding energy of docked L-DNA is found to be
Acknowledgments
Dr. M.A.N. gratefully acknowledges the financial support from
Council of Scientific and Industrial Research (CSIR), New Delhi,
India (01(2736)/13/EMR-II). C.B is thankful to CSIR, India for his fel-
lowship. Board of Research in Nuclear Sciences (BRNS), DAE-BARC,
Mumbai, India is also acknowledged for financial support (No.
35/14/03/2014). STIC, Cochin is acknowledged for performing the
SCXRD analysis.
ꢂ1
ꢂ1
ꢂ4.77 kcal mol for A-DNA and ꢂ6.52 kcal mol for B-DNA. The
bonding interaction, types of bonding and bonding distance of L
with DNA (A-DNA, B-DNA) are given in Tables 5 and 6.
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.saa.2015.06.013.
Conclusion
The present work describes the synthesis and spectral charac-
terization of a propargyl arms containing Schiff base. The single
crystal analysis shows that the compound exists in enolimine form
with monoclinic crystal lattice system and space group C2/c.
Density functional calculation on L reveals that the enolimine form
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