Synthesis, structure and DNA interaction studies of bisphosphoramides: Theoretical and experimental insights

Article abstract of DOI:10.1016/j.ica.2017.02.017

New bisphosphoramides (having phenyl (EDAPOPh₂) and ethoxy (EDADEP) substituents attached to phosphoryl groups bridged with ethylenediamine spacer) are synthesized and structurally characterized by spectroscopic techniques as well as elemental analysis. The molecular structure of EDAPOPh₂was determined by single crystal X-ray diffraction technique. The interaction of these bisphosphoramides with calf thymus DNA (ct-DNA) is investigated using UV–Visible absorption and fluorescence spectral data as well as the DFT calculations. These studies reveal that EDAPOPh₂and EDADEP interact with DNA in a partial intercalation mode. The intrinsic binding constants K_bof two different bisphosphoramides with ct-DNA were determined by fluorescence spectroscopy as 2.08 × 10⁴and 3.86 × 10⁴M^?1respectively. The results indicated that the two compounds bind to ct-DNA with different binding affinities, i.e. EDAPOPh₂> EDADEP. The binding mechanism of these bisphosphoramides to ct-DNA is also discussed.

Full text of DOI:10.1016/j.ica.2017.02.017

Accepted Manuscript
Research paper
Synthesis, Structure and DNA Interaction Studies of Bisphosphoramides: The-
oretical and Experimental Insights
E. Veerashekhar Goud, Akella Sivaramakrishna, Kari Vijayakrishna, C.V.S.
Brahmmananda Rao, Vijay M. Khedkar, Prakash C. Jha
PII:
S0020-1693(17)30226-8
http://dx.doi.org/10.1016/j.ica.2017.02.017
ICA 17443
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
4 August 2016
4 February 2017
15 February 2017
Please cite this article as: E. Veerashekhar Goud, A. Sivaramakrishna, K. Vijayakrishna, C.V.S. Brahmmananda
Rao, V.M. Khedkar, P.C. Jha, Synthesis, Structure and DNA Interaction Studies of Bisphosphoramides: Theoretical
and Experimental Insights, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.02.017
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Synthesis, Structure and DNA Interaction Studies of
Bisphosphoramides: Theoretical and Experimental Insights
1
1
1
E. Veerashekhar Goud, Akella Sivaramakrishna*, Kari Vijayakrishna,
2
3,4
5
C.V.S. Brahmmananda Rao, Vijay M. Khedkar, Prakash C. Jha
1
Department of Chemistry, School of Advanced Sciences, VIT University, Vellore-632014, Tamil
Nadu, India
2
Chemistry Group, Indira Gandhi Centre for Atomic Research, Kalpakkam-603102, Tamil Nadu,
India
3
School of Health Sciences, University of Kwa Zulu Natal, Westville Campus, Durban 4000, South
Africa
4
Combi-Chem Resource Centre, CSIR-National Chemical Laboratory, Pune, 411008 Maharashtra,
India
5
School of Chemical Sciences, Central University of Gujarat, Sector-30, Gandhinagar, Gujarat,
India
E-mail: asrkrishna@vit.ac.in
=
================================================================
Abstract
New bisphosphoramides (having phenyl (EDAPOPh
2
) and ethoxy (EDADEP) substituents attached
to phosphoryl groups bridged with ethylenediamine spacer) are synthesized and structurally
characterized by spectroscopic techniques as well as elemental analysis. The molecular structure of
EDAPOPh was determined by single crystal X-ray diffraction technique. The interaction of these
2
bisphosphoramides with calf thymus DNA (ct-DNA) is investigated using UV-Visible absorption
and fluorescence spectral data as well as the DFT calculations. These studies reveal that
EDAPOPh
constants K
spectroscopy as 2.08 x 10 and 3.86 x 10 M respectively. The results indicated that the two
compounds bind to ct-DNA with different binding affinities, i.e. EDAPOPh > EDADEP. The
binding mechanism of these bisphosphoramides to ct-DNA is also discussed.
2
and EDADEP interact with DNA in a partial intercalation mode. The intrinsic binding
b
of two different bisphosphoramides with ct-DNA were determined by fluorescence
4
4
-1
2
Key words: Bisphosphoramides, crystal structure, molecular docking, DNA interaction.

1
. Introduction
Deoxyribonucleic acid (DNA) is an important genetic material for the transfer of genetic
information over generations with a double stranded helical structure in living organisms. It plays a
vital role in the transcription, replication and regulation of genes which are important steps for
living organisms. When small organic molecules bind to DNA that leads to a significant change in
the DNA sequence and further damages the growth of cancer cells [1-2]. Among the molecules
reported, the importance of organophosphorus reagents was elucidated in terms of anti-bacterial,
anti-viral, anti-oxidant activity [3-4], anti-cancer [5], anti-inflammatory agents [6], pesticides,
herbicides, insecticides [7-8] and anti HIV agents [9]. There are three types of possible interactions
i.e. non-covalent connections such as intercalative binding between DNA base pairs and ligand,
groove binding and static electronic effects [10]. Ligand planarity is the key factor in intercalative
binding [11]. The intercalative is the most important binding which is similar to the anti-tumor
activity of drug molecules. Various researchers have been working on the intercalative binding
which involves antibiotics, transition metal complexes and N-donor containing molecules [12].
Since the investigations on interactions of smaller organic molecules and DNA is of fundamental
importance [1], there is an increasing demand for synthesis of new molecules in molecular biology
as well as in medical field to act as sequence selective DNA binding agents and DNA targeting
anti-tumor drugs. As shown in the literature, various heterocyclic compounds containing
phosphorus derivatives with more electron donating groups exhibited significant antitumor activity
[
13]. The successful history of cisplatin and platinum derivatives as anticancer drugs in clinical
chemistry is due to their covalent binding with DNA even though it has some draw backs such as
serious cellular side effects, toxicity and acquired drug resistance [14]. In order to minimize these
drawbacks of platinum based drugs, the research is intensified based on replacing them with other
molecules or complexes having covalent binding [15]. In this connection, we have studied earlier
the DNA interactions with various poly(ionic liquids), hydazide and semicarbazide derivatives as
well as some selected quinine derivatives [16]. The research in the past is mainly focused on the
involvement of various transition metal complexes, heterocyclic compounds, and dye molecules in
the interaction of DNA, but the information on binding of phosphoramides, phosphine oxides and
phosphates with DNA is very meager [17]. As it has been shown earlier that phosphoryl groups are
very good electron donors [18], it would be interesting to investigate the influence of
aminophosphine oxides with multi donor sites on DNA interaction. The present work deals with the

synthesis and the structural characterization of two different bisphoshoramides and their
involvement in DNA interactions.
2
. Experimental part
2
.1. Materials and methods
All synthetic procedures were carried out under inert atmosphere using Schlenk line techniques.
The commercially available solvents were distilled from Na/benzophenone before the usage. The
reagents including magnesium turnings, HCl, diethyl phosphite, isopropyl alcohol, ethyl acetate and
cyclohexyl bromide (Merck, India) were used as received. Ethylene diamine, Ethidium bromide,
NaOH, TRIS-HCl buffer and sodium salt of calf thymus DNA (ct-DNA) were procured from
Sigma-Aldrich.
Diphenylphosphine oxide was prepared from diethylphosphite using Mg turnings and
bromobenzene as per the modified literature procedure [19].
The spectrophotometric titrations were performed by maintaining the concentration of
bisphosphoramide (10.0 µM) throughout by varying the concentration of ct-DNA from 10.0 to 100
µM at room temperature.
2
.3. Instrumentation
1
13
31
1
H, C, and P{ H} NMR spectra were recorded on a Bruker DMX-400 spectrometer and all 1H
chemical shifts were reported relative to the residual proton resonance in deuterated solvents (all at
2
98 K, CDCl ). Infrared spectra were recorded on a Shimadzu Affinity 1 FT-IR Spectrometer. UV-
3
Visible absorption spectrum was recorded on UNICAM UV4-100 type double-beam
spectrophotometer (ATI UNICAM, Cambridge, UK) controlled by software VISION 3.4 and
instrument equipped with 1.0 cm quartz cuvettes. Blank buffer was placed in the reference cell and
sample was placed in the sample cell. The fluorescence quenching studies were carried out using
the HITACHI F-700 fluorescence spectrophotometer. Infrared spectra were recorded on a
Shimadzu Affinity 1 FT-IR Spectrometer. Heraeus CHNS rapid micro analyzer was used for
elemental analysis. GC analyses were recorded on a Perkin Elmer Clarus 680 (GC) and Clarus 600
(
EI, mass) equipped with an FID and a 30 m x 0.32 mm CP-Wax 52 CB column (0.25 µm film
thickness). The carrier gas was helium at 5.0 psi. The oven was programmed to hold at 32°C for 4
min and then to ramp to 200°C at 10 deg/min and hold 5 min.

2
.3.1. Single crystal X-ray diffraction analysis
X-Ray diffraction intensity data was collected at room temperature (293K) on a Brukeraxs SMART
APEXII single crystal X-ray diffractometer equipped with graphite monochromatic MoKα
3
(
λ=0.71073 Å) radiation and CCD detector. A crystal of dimensions 0.30 X 0.25 X 0.20 mm was
mounted on a glass fiber using cyanoacrylate adhesive. The unit cell parameters were determined
from 36 frames measured (0.5° phi-scan) from three different crystallographic zones using the
method of difference vectors. The intensity data were collected with an average four-fold
redundancy per reflection and optimum resolution (0.80 Å). The intensity data collection, frames
integration, Lorentz and polarization corrections and decay correction were carried out
using SAINT-NT (version 7.06a) software. An empirical absorption correction (multi-scan) was
performed using the SADABS program. The crystal structure was solved by direct methods
using SHELXS-97 and refined by full-matrix least-squares using SHELXL-97. Molecular geometry
was calculated using PARST. All non-hydrogen atoms were refined using anisotropic thermal
parameters. The hydrogen atoms were included in the structure factor calculation at idealized
positions by using a riding model, but not refined. Images were created with the ORTEP-PLATON
program. The CCDC deposition number is 1486557.
2
.3.2. Molecular Docking studies
The experimental results clearly showed the interaction of DNA with two different phosphoramides
EDADEP and EDAPOPh ) at varied concentrations. These observations further prompted us to
(
2
investigate the mechanistic steps involved through the molecular docking, which is generally used
to predict the orientation of ligands to their protein active sites in order to predict their efficiency
and activity in the drug designing. An interactive molecular graphics program Glide (Grid-Based
Ligand Docking With Energetic) incorporated in the Schrödinger molecular modeling package
(
Schrödinger, Inc., USA, version- 2015) installed on a Windows workstation with an Intel (R)
xenon 2.8 GHz processor and 32 GB physical memory was used to predict the mode of binding and
the associated interactions of the bisphosphoramide derivatives with DNA. The crystal structure of
DNA duplex receptor (B-DNA) used for docking was retrieved from the Protein Data Bank
(
www.rcsb.org) (PDB code 425D) [20]. The B-DNA crystal structure was preprocessed for docking
calculation by deleting the crystallographically observed water molecules since no water molecule
was found to be conserved and adding hydrogen atoms corresponding to pH 7.0. Following the
assignment of appropriate charge and protonation state, the structure was energy minimized (to

relieve the steric clashes introduced due to addition of hydrogen atoms) using the Optimized
Potentials for Liquid Simulations (OPLS-2005) force field until an root mean square deviation
(
RMSD) value of 0.30 Å was reached. The 3D-strucutres of bisphosphoramide derivatives were
sketched using the build panel in Maestro and their geometries were optimized using LigPrep
module. The partial charges were ascribed using the OPLS-2005 force-field and the structures were
then subjected to energy minimization until their RMSD reached 0.01 Å. The shape and properties
of the binding site of B-DNA were defined for docking using the receptor grid generation panel.
This grid was defined by a 14x14x14 Å box that was centered on the geometric centroid of the B-
DNA structure and was sufficiently large to include a significant part of the B-DNA. The
bisphosphoramide derivatives were docked flexibly into the B-DNA structures using with extra
precision (i.e. with GlideXP) scoring function to rank the docking poses and to estimate their
binding affinities.
3
. Synthesis of Bisphosphoramides
3
.1. General procedure
Scheme 1. Synthesis of bisphosphoramides
2
.1.1. Synthesis of bis(diphenyloxophosphino)ethylenediamine (EDAPOPh )
3
To a 2-neck 500 mL RB flask, 6.72 g (33.0 mmol) of diphenylphosphine oxide in 150 mL of THF
and 6.42 mL (66.0 mmol) of carbon tetrachloride were transferred under N atm. To this, 1.1 mL
16.6 mmol) of ethylenediamine and 9.26 mL (66.0 mmol) of triethylamine (dissolving in 100 ml of
2
(
THF) were added drop wise over a period of 30-60 minutes at ice cold temperature. There after the
temperature of the vessel was allowed to attain the room temperature and contents of the reaction
vessel were stirred for 12 h. The reaction mixture was filtered to remove hydrochloride salt of
triethylamine and solvent traces were removed under high vacuum. The desired compound was
obtained as crystalline solid in quantitative yields. Further the recrystallization of product was
carried out using a solvent mixture of dichloromethane and n-hexane (1:1). The yield of the product

-
1
1
was 90% and melting point is 154–156 °C. FT-IR (KBr, cm ): 1180, H NMR (400 MHz, CDCl
00 K, relative to TMS): δ 3.15-3.18 (q, 4H, –CH –N), 7.86-7.91 (d, 8H, o-CH), 7.50-7.52 (t, 4H,
p–CH), 7.43-7.47 (t, 8H, m–CH). P NMR (162 MHz ppm relative to H PO in CDCl ): δ 30.85.
C NMR (100 MHz): δ 42.13, 128.33-128.45, 131.48-131.56, 131.65-131.23, 132.80, 134.06.
Mass: Positive-ion EI (m/z): 460.16. Anal. Calcd. for C26 : C, 67.82; H, 5.69; N, 6.08.
3
,
3
2
3
1
3
4
3
1
3
H
26
N
2
O P
2 2
Found-: C, 68.12; H, 5.75; N, 6.11. The characterization data of this product closely agrees with the
literature report as they have followed a different procedure [21].
3
.1.2. Synthesis of bis(diethylphosphino)ethylenediamine (EDADEP)
N,N-Bis(diethylphosphoryl)ethylenediamine was synthesized according to the literature report by
modifying the synthetic procedure [22]. The title compound was synthesized by taking the 7.25 g
(
52.5 mmol) of diethylphosphite, 8.08 g (52.4 mmol) of carbon tetrachloride, 2.001 g (26.2 mmol)
of ethylenediamine and 5.30 g (52.4 mmol) of triethylamine in THF. The colorless crystalline
1
compound was obtained in 98% yield with a melting point of 80-82 °C. H NMR (400 MHz,
chloroform-d, 300 K, relative to TMS): δ 1.29–1.32 (t, 12H, CH
3
), 2.98–3.01 (q, 4H, –CH
–O–), 3.53 (s, 2H, –NH). P NMR (162 MHz, relative to H PO ): δ 9.25.
CNMR (100 MHz, D O): δ 15.3 (CH ), 41.8 (–CH2–NH), 63.7 (–CH –O–P). 135DEPT (100
MHz, D2O): δ 15.3 (CH ), 41.7 (-CH -NH), 63.7 (-CH -O-P). Mass: Positive-ion EI (m/z):
: C, 36.15; H, 7.89; N, 8.43. Found: C, 36.18; H, 7.92; N,
2
–N),
3
1
4
.01-4.08 (p, 8H, –CH
2
3
3
1
3
2
3
2
3
2
2
3
8
32.21.Anal.Calcd. for C10
.47.
H
26
N
2
O P
6 2
4
. General procedure used for DNA interaction studies
The 10 mM TRIS-HCl buffer was prepared using double distilled deionized water, and the pH was
adjusted to 7.4 using 0.1 M NaOH solution. ct-DNA (1% w/w) in buffer was prepared and stored at
°
5
2
C. The concentration of ct-DNA was estimated by using its known molar extinction coefficient at
−
1
−1
60 nm (ε260 = 6600 M cm ) and the absorbance was recorded as (λmax = 0.42) followed by
concentration of 63.8 µM. Then the final concentration of ct-DNA stock solution was obtained as
2.7 mM. The ct- DNA used in this study was sufficiently free from protein which is determined
from the absorbance ratio (A260/A280) = 1.8.45 [21]. EDADEP ligand was prepared in phosphate
buffer and EDAPOPh was prepared in 10% DMSO in phosphate buffer.
1
2

Chart 1.Bisphosphoramides employed in DNA interaction studies
The EB-DNA complex was titrated against various concentrations (0-100 µM) of phosphoramide
ligands by keeping the concentration of ct-DNA (10 µM) constant. The prepared solutions were
mixed thoroughly and incubated for 5 min, following by measuring the fluorescence intensities at
room temperature. EDADEP has an emission maximum at 604 nm when excited at 530 nm and for
EDAPOPh
2
was excited at 500 nm followed by emission at 606 nm.
. Results and Discussion
.1. Synthesis and structural characterization of bisphosphoramides
4
4
The bisphosphoramides (EDAPOPh
2
and EDADEP) were prepared by the reaction of two moles of
either diphenylphosphine oxide or diethylphosphite with ethylenediamine in the presence of
3
1
triethylamine. The purity of the products was assessed by P-NMR spectral data based on the
formation of a single peak due to the presence of two equivalent phosphorus atoms. These
1
13
31
compounds were characterized by UV/Vis, FT-IR, H, C, P NMR and mass spectral data and
further the composition was confirmed by elemental analysis.
All the absorption spectra were recorded using a matrix containing phosphate buffer and 10%
DMSO as a blank to nullify the influence of the matrix on absorption spectra. The EDAPOPh and
2
EDADEP ligands showed an intense absorption bands at 205 nm and 209 nm respectively due to
intra-ligand π–π* transition of the coordinated groups (NH, P=O) [23].
The compounds EDAPOPh
2
and EDADEP showed strong infrared absorptions at 1180 and 1172
-1
31
cm for their phosphoryl groups respectively. P-NMR spectra exhibited a single peak for each
compound at δ 30.85 (EDAPOPh2) and δ 9.25 (EDADEP). The mass spectra showed the formation
of fragmented species (EtO)
2
P(H)=O of m/z 138.27 and 202.19 for Ph P(H)=O.
2

Single crystals of EDAPOPh
n-hexane (1:1). The molecular structure of EDAPOPh
analysis. The perspective view of EDAPOPh is shown in Fig.1.
2
were obtained from the solvent mixture of dichloromethane and
2
was determined by single crystal X-ray
2
Fig.1. Molecular structure of EDAPOPh : Hydrogen atoms were removed for clarity. Some
2
selected bond lengths (Å): P=O, 1.486; P-N, 1.626; P-C, 1.797 and bond angles (°): O-P-C, 109.8;
O-P-N, 112.8; O-P-C, 114.9; C-P-N, 111.1 and C-P-C, 104.8.
Fig. 2. A drawing of the molecular structure of EDAPOPh with hetero atoms and intermolecular
2
hydrogen bonds. Hydrogen atoms are omitted for clarity; Brown color ellipsoids are carbons, pink
color indicates nitrogen atom, royal blue indicates phosphorous atom and red indicates oxygen
atom.
As shown in the molecular structure of EDAPOPh
tetrahedral environment and four phenyl groups exist in the same plane. The P=O bond length was
.486 Å and in good agreement with the literature reports. Interestingly, the monomers are
2
(Fig.1), the phosphorous centers are present in
1
connected to each other via two P=O-----H-N bonds through prominent hydrogen bonds in the solid
state structure and the bond lengths were observed as H(2)-N(2)------O(1)-P(1) 2.094, H(1)-N(1)----

-
-O(2)-P(2) 1.959 Å. The strong hydrogen bonds are formed due to the presence of highly
electronegative oxygen atom (Fig. 2). The packing arrangement of molecules in crystal lattice is
given in Fig. 3.
Fig. 3. Crystal packing of EDAPOPh molecules in the lattice
2
4
.2. DNA interaction studies
UV-Visible absorption spectroscopy is an important tool for DNA binding studies. The interaction
of EDADEP and EDAPOPh with ct-DNA was investigated in vitro by UV spectroscopy and
2
indirectly by the evaluation of the EB-displacing ability of these multi-dentate molecules examined
by fluorescence emission spectroscopy. The absorption studies of EDADEP in the presence and in
absence of DNA are shown in Fig. 4 and Fig. 5. EDADEP and EDAPOPh ligands showed an
2
intense absorption bands at 205 and 209 nm respectively due to intra-ligand π–π* transition of the
coordinated groups [24,25]. These bands were considered to characterize the DNA interaction
studies in phosphate buffer.
A decrease in the absorption was observed by the addition of ct-DNA with varying concentration
(
0-100 µM) of ligands. The hypochromic shifts (or hypsochromic shift) in the wavelength of the
bands suggest that there was a strong intercalative binding of bisphosphoramide ligands with DNA
base pairs, indicating the direct formation of a new complex-DNA [24]. The DNA binding affinities
with EDAPOPh
accordance with earlier report [25] In order to compare the binding affinities quantitatively, the
intrinsic binding constant K was calculated by using the following equation [26]:
2
or EDADEP show strong hypochromism and a prominent red-shift, which is in
b
[
DNA]/(ε
a
-ε
f
) = [DNA]/(ε
b
-ε
f
)+ 1/K
b
(ε
b
-ε )………Eq. 1
f

Where ε
a
refers to apparent extinction coefficient calculated by using Aobs/[complex], ε
b
refers to
extinction coefficient of the complex in the bound form, ε
f
refers to the extinction coefficient of the
free form of complex and [DNA] refers to the concentration of DNA in base-pairs.
By substituting the obtained values in the above equation gives a plot of [DNA]/(ε
a
-ε
is obtained from
is calculated from the graphs for EDADEP and
are 2.08 x 10 and 3.86 x 10 M respectively (Fig. 4 and 5).
f
) vs. [DNA]
with a straight line with a slope of 1/(ε
b
-ε
f
) and a y-intercept of 1/K
b
(ε
b
-ε
f
) and K
b
the ratio of the slope to the y-intercept. K
b
4
4
-1
EDAPOPh
2
-3
Fig. 4. UV spectrum of EDADEP (1x10 M) in the absence (R0) and presence of (R1-R9) ct-DNA
0-100 µM) in 10 mM phosphate buffer (pH = 7.2). Inset: the plot of [DNA]/(ε -ε ) vs. [DNA] for
(
a
f
EDADEP. The arrow depicts the decrease in the absorption with increasing the ct-DNA
concentration.

-3
Fig. 5. UV-Vis spectrum of EDAPOPh
DNA (0-100 µM) in 10 mM phosphate buffer (pH = 7.2). Inset: the plot of [DNA]/(ε
for EDAPOPh . The decrease in the absorption with increasing the ct-DNA concentration was
shown with arrow mark.
2
(1x10 M) in the absence (R0) and presence of (R1-R9) ct-
a
-ε )vs. [DNA]
f
2
The FTIR analysis shows the binding of EDADEP to ct-DNA, which is evident by shifting the
-1
characteristic bands at 1209, 1174 and 1092 cm are due to the increase in the intensity of the
phosphate stretching vibrations as a result of its interaction with EDADEP ligand. The sharp DNA
-1
band at 978 cm is a characteristic of C-C and C-O vibration stretch of deoxyribose sugar and gets
-1
shifted to 964 cm due to the interaction of EDADEP with deoxyribose of ct-DNA (Fig. 6).
A
B

Fig. 6: The infrared spectral data of ct-DNA (A) and a combination of ct-DNA-EDADEP (B)
4
.3. Fluorescence quenching studies
The fluorescence titration is an important task in binding studies to investigate the mode of
EDADEP and EDAPOPh binding to the ct-DNA. For this purpose competitive binding experiment
2
was carried out using very well-known ethidium bromide (EB) (20 µM) as an intercalative probe. It
is a known that the most sensitive fluoresce is due to its planar structure that binds to DNA in
intercalative mode followed by weak emission of fluorescence in aqueous medium. In spite of this
the fluorescence ability will increase in presence of DNA due to intercalative mode bind between
DNA base pairs and EB led to formation of EB-DNA complex [27]. The enhancement of EB-DNA
complex fluorescence can be quenched by adding other ligand molecule. If the added ligand
intercalates into DNA, there was a clear diminution in the fluorescence spectrum because of
decrease in the binding sites of DNA available for EB [28].
A regular decrease in the fluorescence intensity without change in the fluorescence emission
wavelength of EB-DNA complex by increasing the concentration of EDADEP and EDAPOPh is
2
observed as shown in Fig.7 and Fig.8. Small organic molecules can interact with DNA by
intercalative when the concentration of organic molecules to DNA (cM/cDNA) is less than 100 and
the fluorescence intensity of EB-DNA complex decreases by 50% [24] here, the quenching extent
has been reached up to 35%. This clearly indicates that phosphoramides are competing with the
ethidium bromide in ct-DNA binding.
-3
There is a change in the absorbance of fluorescence spectrum of EDADEP (1x10 M) and
-3
EDAPOPh
phosphoramides (from top to bottom, 0-100µM). According to the classical Stern-Volmer equation
29]:
2
(1x10 M) in 10 mM phosphate buffer at pH 7.0, in the absence and presence of
[
F
0
/F = 1 + K [Q]…………….Eq. 2
q
Here, both F
respectively. K
quencher. The K
the Fig. 7 and 8 insets. The K
0
and F denote the fluorescence intensities in the absence and presence of the complex,
is a linear Stern-Volmer quenching constant, [Q] is the concentration of a
value can be obtained from a linear plot drawn against F /F vs. [Q] as shown in
values for EDADEP and EDAPOPh are 1.12 x 10 , 7.7 x 10 M
q
q
0
4
4
-1
q
2
respectively. These results suggest that the mode of binding is intercalative with ct-DNA by
releasing EB from EB-DNA complex, which closely agrees with the absorption results.

-3
Fig.7. Fluorescence spectrum of EDADEP (1x10 M) in the absence and presence of ct-DNA (0-
00 µM) in 10 mM phosphate buffer (at pH 7.2). The inset is Stern-Volmer quenching plot of
1
DNA-EB system of EDADEP. A steady decrease in the emission intensity upon increasing the
EDADEP concentration is observed.
-3
Fig.8. Fluorescence spectrum of EDAPOPh
2
(1x10 M) in the absence and presence of EDAPOPh
2
(
0-100 µM) in 10 mM phosphate buffer (pH=7.2). The inset is Stern-Volmer quenching plot of
DNA-EB system of EDAPOPh
2
. With the addition of EDAPOPh , it is revealed that the emission
2
intensity decreases gradually.

2
.4. Molecular Docking studies
The molecular docking is a very well-known computational tool to predict the interaction energy
between two molecules and ligand orientation with overall minimum energy. This computational
method mainly requires algorithm like molecular dynamics, Monte stimulation, genetic algorithms,
point complementary methods, fragment based searching methodologies, distance geometry
mythologies and systematic searching [30]. The small molecules (considering them as ligands)
generally fit into the protein cavities and this is predicted by the search algorithms. These protein
cavities can be activated automatically when they contact with external molecules so, they are
known as active sites. The docking results are analyzed by a statistical function which is converting
the interacting energies into numerical values (docking scores) which are based on non-covalent
interactions. The 3-D view of the bound complex can be visualized using various tools like Pymol,
Rasmol etc. which is helpful in the identification of the best fit within the complex. Hence docking
studies have great importance in the designing and discovery of new drugs [31].
Molecular docking study was performed to simulate the modes of interactions between the
bisphosphoramide derivatives and B-DNA which can corroborate the experimental results.
Structures of the bisphosphoramide derivatives as well as B-DNA were kept flexible to explore the
conformational space in order to predict the best fit orientation and the best docked complex has
been analyzed. The lowest energy docked complexes revealed that the bisphosphoramide
derivatives could snuggly fit into the curved contour of B-DNA engaging in intimate electrostatic
and van der Waals interactions with the walls of the groove. Table-1 shows the optimal energy
ranked results of EDADEP and EDAPOPh interactions with B-DNA.
2
Table 1. The binding affinity data for the bisphosphoramide derivatives (EDADEP and
EDAPOPh
2
)
-1
S. No. Compound Binding Constant [M ] Docking score Binding
energy(kcal/mol)
4
4
1
2
.
.
EDADEP
EDAPOPh
2.08 x 10
3.86 x 10
-7.378
-9.333
-37.374
-44.752
2

From the ensuing docked structures it is revealed that the binding energy of EDAPOPh
than that of EDADEP which indicates that EDAPOPh binds strongly to B-DNA compared to
EDADEP. Analysis of the docked complex of EDAPOPh (Fig.9) showed that it could slide into the
2
is lower
2
2
minor grove of B-DNA forming significant van-der Waals interactions with G22 (-4.214 kcal/mol),
C21 (-6.617 kcal/mol), C20 (-8.444 kcal/mol), A19(-4.496 kcal/mol), C8(-2.323 kcal/mol), A7 (-
5
.870 kcal/mol), T6 (-6.261 kcal/mol), G5 (-3.632 kcal/mol), G4 (-1.170 kcal/mol) while favorable
electrostatic interactions observed with G22 (-2.477 kcal/mol), C21 (-5.716 kcal/mol), C20 (-3.247
kcal/mol), A19 (-1.221 kcal/mol) residues further stabilized the complex. The per-residue
interaction analysis revealed that van-der Waals interactions (-36.397 kcal/mol) surpassed
electrostatic component (-8.354 kcal/mol) in the overall binding affinity (-44.752 kcal/mol) of
EDAPOPh
2
towards B-DNA. In addition, there are two hydrogen bonding interactions observed
and oxygen atoms of C20 and C21 with a bonding
between amide hydrogens of EDAPOPh
2
distance of 2.221 Å and 1.726 Å respectively. The hydrogen bonding interactions are crucial in that
they act as "anchors" guiding the 3D orientation of a ligand in its binding site, thereby facilitating
the steric and electrostatic interactions. Further the interaction of amide hydrogen with DNA
through hydrogen bonding is also feasible. On the other hand, docking of EDADEP (Fig. 10)
revealed that the compound binds to B-DNA at the same site as EDAPOPh with no significant
2
difference in their binding mode but with a weaker strength. Inspection of the non-bonded
interaction energies revealed that even for EDADEP, the contribution of van-der Waals interactions
(
-31.425 kcal/mol) dominated over the electrostatic component (-5.948 kcal/mol) in the overall
binding energy (-37.374 kcal/mol). The per residue interaction analysis showed that it is stabilized
within the active site through extensive Vander Waals contacts with G22 (-1.73 kcal/mol), C21 (-
6
.052 kcal/mol), C20 (-5.567 kcal/mol), A19 (-3.122 kcal/mol), C8 (-1.185 kcal/mol), A7 (-5.531
kcal/mol), T6 (-4.853 kcal/mol), G5 (-2.476 kcal/mol) and G4 (-0.780 kcal/mol) and favorable
electrostatic interactions with G22 (-2.112 kcal/mol), C21 (-3.603 kcal/mol), C20 (-2.852 kcal/mol)
and A19 (-0.835 kcal/mol). Even EDADEP was found to be anchored to the minor groove of B-
DNA through two hydrogen bonding interactions observed between amide hydrogens of EDADEP
and oxygen atoms of C20 and C21 with a bonding distance of 1.983Å and 1.879 Å respectively. An
additional hydrogen bond was observed between the amine H of G5 and the O atom of EDADEP
with a bond length of 1.832 Å. Inspection of the per-residue interaction energy break up clearly
justify that significantly higher values observed for EDAPOPh contributed to its stronger binding
2
affinity to B-DNA over EDADEP which is in harmony with the experimentally observed data.
Furthermore, analysis of the binding modes of these bisphosphoramide derivatives also revealed

that the B-form was well retained by the DNA with no significant change in the base pairs even
after complexation with bisphosphoramides.
Fig. 9. Binding mode of EDAPOPh into the groove of B-DNA
2
Fig. 10. Binding mode of EDADEP into the groove of B-DNA.
7
. Conclusions
In this paper, the interaction of bisphosphoramides with B-DNA was studied by experimental and
molecular modeling techniques. Combining the results from UV/Visible absorption spectral and
molecular modeling, it is concluded that bisphosphoramides bind to B-DNA through an
intercalation mode. The results of molecular modeling studies indicate that the primary driving

force for mechanical interlocking was the steric complementarities between the bisphosphoramides
and B-DNA as evidenced from the relatively higher contribution of van-der Waals interaction over
electrostatic components in the overall binding scores while hydrogen bonding forces may also play
an essential role in the binding. The binding pattern predicted by molecular docking complemented
with a detailed per residue interaction analysis provides a strong a basis for the rational design of
bisphosphoramides with higher binding affinity to B-DNA.
Acknowledgements
Dr ASRK is highly thankful to DAE-BRNS (INDIA) (No. 2012/37C/6/BRNS/623) for the financial
assistance. Mr. E.V. Goud is grateful to DAE-BRNS for the senior research fellowship. We also
thank DST-VIT-FIST for NMR and SIF-VIT University for other instrumental facilities.
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Graphical Abstract
New bisphosphoramides (having phenyl (EDAPOPh ) and ethoxy (EDADEP) substituents attached
2
to phosphoryl groups bridged with ethylenediamine spacer) are synthesized and structurally
characterized by IR and NMR spectroscopic techniques as well as elemental analysis. The
molecular structure of EDAPOPh was determined by single crystal X-ray diffraction technique.
2
The interaction of these bisphosphoramides with calf thymus DNA (ct-DNA) is investigated using
UV-Visible absorption and fluorescence spectral data as well as the DFT calculations. These
studies reveal that EDAPOPh
The intrinsic binding constants K
determined by fluorescence spectroscopy as 2.08 x 10 and 3.86 x 10 M respectively. The results
indicated that the two compounds bind to ct-DNA with different binding affinities, i.e. EDAPOPh
EDADEP. The binding mechanism of these bisphosphoramides to ct-DNA is also discussed.
2
and EDADEP interact with DNA in a partial intercalation mode.
b
of two different bisphosphoramides with ct-DNA were
4
4
-1
2
>

Graphical Pictogram
ct-DNA
Bisphosphoramide

Highlights
ꢀ
Two new bisphosphoramides with phenyl and ethoxy substituents attached to phosphoryl
groups bridged with ethylenediamine spacer are synthesized.
ꢀ
These molecules are structurally characterized by IR and NMR spectroscopic techniques as
well as elemental analysis including the molecular structure of phenyl substituted
bisphosphoramide.
ꢀ
ꢀ
The interaction of these bisphosphoramides with ct-DNA is investigated using UV-Visible
absorption and fluorescence spectral data.
Both experimental and DFT calculations reveal that both the phosphoramides interact with
DNA in a partial intercalation mode, but the binding affinity is more for the phenyl
substituent than the ethoxy analogue.