Nuclear blebbing of biologically active organoselenium compound towards human cervical cancer cell (HeLa): In vitro DNA/HSA binding, cleavage and cell imaging studies

Article abstract of DOI:10.1016/j.ejmech.2014.12.014

New pharmacophore organoselenium compound (1) was designed, synthesized and characterized by various spectroscopic methods (IR, ESI-MS, ¹H, ¹³C and ⁷⁷Se NMR) and further confirmed by X-ray crystallography. Compound 1 consi

Full text of DOI:10.1016/j.ejmech.2014.12.014

Accepted Manuscript
Nuclear blebbing of biologically active organoselenium compound towards human
cervical cancer cell (HeLa): In vitro DNA/HSA binding, cleavage and cell imaging
studies
Masood Ahmad Rizvi , Mehvash Zaki , Mohd. Afzal , Manoj Mane , Manjeet Kumar ,
Bhahwal Ali Shah , Saurabh Srivastav , Saripella Srikrishna , Ghulam Mustafa
Peerzada , Sartaj Tabassum
PII:
S0223-5234(14)01123-4
10.1016/j.ejmech.2014.12.014
EJMECH 7574
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 July 2014
Revised Date: 6 December 2014
Accepted Date: 9 December 2014
Please cite this article as: M.A. Rizvi, M. Zaki, M. Afzal, M. Mane, M. Kumar, B.A. Shah, S. Srivastav,
S. Srikrishna, G.M. Peerzada, S. Tabassum, Nuclear blebbing of biologically active organoselenium
compound towards human cervical cancer cell (HeLa): In vitro DNA/HSA binding, cleavage and cell
imaging studies, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2014.12.014.
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Graphical abstract

ACCEPTED MANUSCRIPT
Nuclear blebbing of biologically active organoselenium compound towards
human cervical cancer cell (HeLa): In vitro DNA/HSA binding, cleavage and
cell imaging studies
†
‡
‡
§
∥
Masood Ahmad Rizvi, Mehvash Zaki, Mohd. Afzal, Manoj Mane, Manjeet Kumar, Bhahwal
†
∥
⊥
⊥
Ali Shah, Saurabh Srivastav, Saripella Srikrishna, Ghulam Mustafa Peerzada and Sartaj
‡
Tabassum *
†
Department of Chemistry, University of Kashmir, Hazratbal, Srinagar–190006, J&K India.
Department of Chemistry, Aligarh Muslim University, Aligarh–202002, India
Centre for Material Characterization, CSIR National Chemical Laboratory (NCL) Pune India.
Natural Product Microbes, CSIR–Indian Institute of Integrative Medicine (IIIM), Jammu Tawi–
‡
*
§
∥
1
80001, J&K, India.
⊥Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005,
India
*
Corresponding author: Tel.: +91 9358255791
E–mail address: tsartaj62@yahoo.com (S. Tabassum)
ABSTRACT
New pharmacophore organoselenium compound (1) was designed, synthesized and characterized
1
13
77
by various spectroscopic methods (IR, ESI–MS, H, C and Se NMR) and further confirmed
by X–ray crystallography. Compound 1 consists of two 3,5–bis(trifluoromethyl)phenyl units
which are connected to the selenium atom via the organometallic C–Se bond. In vitro DNA
binding studies of 1 was investigated by absorption and emission titration methods which
revealed that 1 recognizes the minor groove of DNA in accordance with molecular docking
studies with the DNA duplex. Gel electrophoretic assay demonstrates the ability of 1 to cleave
pBR322 DNA through hydrolytic process which was further validated by T4 religation assay. To
understand the drug–protein interaction of which ultimate molecular target was DNA, the
affinity of 1 towards HSA was also investigated by the spectroscopic and molecular modeling
techniques which showed hydrophobic interaction in the subdomain IIA of HSA. Furthermore,
the intracellular localization of 1 was evidenced by cell imaging studies using HeLa cells.
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Keywords: Organoselenium compound; X–ray crystallography; DFT studies; DNA binding;
pBR322 hydrolytic cleavage; GPx activity.
Abbreviations
UV–vis
CT DNA
Tris
UV–visible
Calf thymus DNA
Tris(hydroxymethyl)aminomethane
Ethidium bromide
EB
1
. Introduction
Medicinal inorganic chemistry is an interdisciplinary thrust area of chemical research; is
currently much more known for its many applications in catalysis and also has enormous
potential to act as therapeutic and diagnostic agents. There is a growing interest in the field of
small molecules capable of binding to deoxyribonucleic acid (DNA) has received immense
influence in the development of new therapeutic modalities for cancer chemotherapy owing to
the fact that many present treatment regimes (Platinum based drugs) in chemotherapy have failed
or fall short either in terms of efficiency or toxicity problems [1–3]. Therefore, other non–
platinum complexes have been evaluated in cancer chemotherapy, and in particular
organometallic compounds have been extensively investigated for their medicinal properties [4–
6
]. Koepf and Koepf–Maier started to explore the anticancer activity of titanocene dichloride, in
the 1970s with several clinical trials conducted (trials on actual patients with various forms of
cancer that have not responded to existing treatments, including surgery, chemotherapy, and
radiotherapy), although finally the compound was not approved for use [7]. Nevertheless, the
5 5 5 2 2
early and highly promising research on Ti(η –C H ) Cl inspired other researchers to study the
anticancer properties of organometallic compounds, who brought rational design and many new
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ideas for the development of cancer chemotherapeutic drugs. Among the non–platinum
complexes for metal based chemotherapy, organometallic selenium (Se)–containing molecules
are emerging as new chemotherapeutic agents due to their ability to modulate multiple
physiological effects on a wide range of cancer cell types as well as reverse the activity of drug
resistance mechanisms to potentiate chemotherapy/radiotherapy efficacy [8]. The trace element
selenium (Se), at very high doses is potential toxic and fatal for the human body, but the low
doses of Se supplementation (nutritional intake of 50–350 ꢀg per day) is seems to be beneficial
not only for cancer prevention, but also influence other functions in organism by reducing
inflammations, heart diseases and regulating the blood pressure. Moreover, selenium is essential
for cell metabolism as a constituent of several seleno–proteins, including glutathione peroxidase,
thioredoxin reductase etc which are primarily involved in antioxidant functions and maintenance
of redox state [9]. Since the discovery of the first GPx (Glutathione Peroxidase) mimics
organoselenium
compound,
2–phenyl–1,2–benzoisoselenazol–3–(2H)–one
(ebselen),
considerable efforts have been made for the design and development of several organoselenium
compounds as GPx mimics for the treatment of oxidative stress initiated by excess reactive
oxygen species (ROS), and thereby preventing the cellular damage [10].
DNA is the essential carrier of genetic information which is concerned with most
cancers resulting from DNA damage, and thus it is considered as a main target for anticancer
drugs development. Interaction between small molecules and DNA is of great significance in the
elucidation of the mechanisms of anticancer drugs and the screening of DNA targeted drugs [11].
In view of the aforementioned fact, the present work embodies the spectroscopic and
single
crystal
X–ray
diffraction
of
the
newly
synthesized
bis(3,5–
bis(trifluoromethyl)phenyl)selane. It would be pertinent to mention here that synthesis of
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compound 1 was based on our understanding of a series of diselenides synthesized by our group,
wherein we found that the molecule having bistrifluoromethyl phenyl moiety displayed most
potent anticancer activity [12]. With this rationale, in vitro DNA binding profile of
organoselenium compound 1 with CT–DNA and cleavage studies of 1 with pBR322 plasmid
DNA by agarose gel electrophoresis was evaluated. The role of organoselenium compound 1 was
not only merely antioxidative (by detoxification of reactive oxygen species as part of glutathione
peroxidases), but rather acting as a DNA binding and cleaving agent. Furthermore, affinity of 1
towards human serum albumin (HSA) was also investigated since the drug–albumin interaction
plays an important role in drug distribution and pharmacokinetics which influences the solubility
of the prospective drug, extend its in vivo half–life, slow down or prevent its passive
extravasations to the target tissues [13]. These studies provide an important rationale for the
design of new lead anticancer drugs and their specific delivery at the active site of action, besides
providing the pharmacological profile in vitro.
2
. Results and Discussion
We report the synthesis of new organoselenium bis(3,5–bis(triflouromethyl)phenyl)selane
compound 1 and the detailed procedure was described in experimental section (Scheme I) [14].
The product was isolated as a yellowish red solid, and suitable crystals for X–ray diffraction
analysis was grown by slow evaporation of its solution in CHCl /MeOH (8:2 v/v), over a period
3
of one week at room temperature. The resulting compound 1 was stable towards air and moisture
and readily soluble in DMSO and MeOH. The single–crystal X–ray analysis reveals that the
compound 1 crystallizes in a triclinic crystal system with P–1 space group. The unit cell
parameters were found to be a = 8.7402(5) Å, b = 8.9544(5) Å, c = 12.4403(6) Å, α =
3
1
07.674(4)°, β = 102.497(4)°, γ = 99.079(5)° with unit cell volume, V = 879.16(9) Å . A
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perspective view of the ORTEP diagram and molecular structure of compound 1 was shown in
Figure 1. From the crystal structure it has been observed that two 3,5–bis(trifluoromethyl)phenyl
moieties are attached to the Selenium atom. The C–Se–C bond angle is 100.9(2)°, as expected
3
for the divalent sp Se atom with lone pair–lone pair repulsions. Selected crystallographic data
for the compound were summarized in Table S1. The crystal packing of compound 1 displayed
the common feature of layers of weakly interacting stacked phenyl groups of one molecule with
the other. Besides, 3D supramolecular architecture of compound 1 was generated which shows
two types of secondary interactions, hydrogen and weak halogen bonding between the identical
moieties of the same molecules (Figure 2). The distance between the hydrogen atoms of one
molecule with the fluorine atom of another molecule is 2.658(2) Å (H–F), and fall in the range of
moderate hydrogen bonding distance. Interestingly, the halogen (F–F) distance in compound 1
was 2.713(3) Å, which was quite favorable for halogen interactions thereby helping the
molecules to interact and stay closer. Density functional theory (DFT) is a popular computational
method of medicinal chemists widely used today for screening of biologically relevant molecular
systems in a time and cost effective manner [15, 16]. The DFT studies were performed using
Turbomole 6.0 program with B3LYP functional and three different basis sets {6-31G(d,P),
LanL2MB, TZVP} for a close agreement of the optimized structure with the experimental crystal
structure. To have an idea about ionization potential, electron affinity and any potential optical
properties of the newly reported molecule the HOMO LUMO energies were also
computationally worked out. The most appropriate match of optimized structure with the crystal
structure was achieved at the B3LYP/TZVP level of theory in accordance with findings of
Taubert et al [17]. In order to improve our calculations for HOMO and LUMO energies at a
reasonably lower computational cost, we performed single point energy calculation of
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B3LYP/TZVP optimized structure of compound 1 with larger TZVPP basis set [18]. Comparison
of selected geometrical parameters of the X–ray crystallographic structure and DFT optimized
structure of the bis(3,5–bis(triflouromethyl)phenyl)selane was depicted in Table 1. A high band
gap of 4.6 eV, predicted that the compound was stable with respect to electron transfer. The
electron density plots and molecular surfaces of HOMO and LUMO (Figure 3), indicated that the
electron density in case of HOMO is concentrated on selenium metal and in case of LUMO the
electron density is predominantly on the phenyl rings. Thus electron rich selenium atom of
compound 1 has a nucleophillic character which also supports the proposed pathway of its DNA
cleavage (see the Supporting Information Figures S1 and S2 and Scheme II).
In order to assess the possible pharmacological properties of compound 1, a toxicity
profile evaluation was performed by employing the OSIRIS software (http://www.organic–
chemistry.org/prog/peo/). As depicted in Table 2, compound 1 does not show any toxicity risk
that is non–mutagenic, non–tumorigenic, non–irritant and no reproductive effect. The log P
(4.85) value was under the acceptable criteria (log P<5) indicating that the compound was quite
hydrophilic in nature. Compound 1 also shows moderate to good drug likeness (–27.9) and drug
score (0.17) which revealed that it could be considered as a drug candidate for oral absorption
(
Figure S3, Supporting Information).
.1. DNA binding studies
2
DNA binding is very important in the development of new metal based chemotherapeutic agents.
The electronic absorption spectrum of compound 1 in DMSO–buffer mixture exhibit an intense
transition at 270 nm, attributed to the π–π* transition of the ligand. Upon increasing the
–5
concentration of CT DNA (0–4.66) × 10 M, a concomitant increase in absorption intensities
was observed with hyperchromism of 31.1–75.2 % with red shift of 4 nm in the intraligand band
6

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(
Figure 4). The observed hyperchromic effect suggests that compound binds to DNA by external
contact, and red shift was due to the participation of aromatic chromophores [19]. Since, DNA
possesses several hydrogen bonding sites which are accessible both in major and minor grooves
[
20]. So it is likely that –CF group present in the compound favor hydrogen bonding with the
3
base pairs of DNA helix in the grooves. To compare quantitatively the binding affinity of
compound, the intrinsic binding constant K was calculated by monitoring the changes in
b
absorbance in the intraligand band at corresponding λmax with increasing concentration of DNA.
4
–1
The K
b
value of compound 1 was found to be 4.2 × 10 M . The magnitude of DNA binding
was greater for compound 1 than previously synthesized Cu(II)–Sn(IV)
2
heterobimetallic
4
–1
complex by our group (3.0 × 10 M ).
Small molecules that bind and cleave at specific DNA sequences are widely used as fluorescent
molecular probes for assessing the local structure of biomacromolecules such as nucleic acids
and proteins [21]. Therefore, the binding ability of the small molecules with DNA was also
investigated by fluorescence spectroscopic titration. Compound 1 emits intense luminescence
when excited at 270 nm in 0.01 Tris–HCl/50mM NaCl buffer at room temperature with an
emission maximum appearing at 361 nm. On addition of increasing amount of CT DNA,
emission intensity of 1 decreases by 3.14 times than those in the absence of DNA and saturates at
a ratio of [DNA]/[1] is 1.5, respectively (Figure 5). The quenching of emission intensity of 1
suggested the occurrence of molecule–molecule excitation interactions in the presence of DNA
or changes in compound environment, such as hydrophobic interactions with the DNA binding
sites versus hydrophilic interactions with the solvent molecules [22]. The binding constant (K)
5
–1
value for 1 was determined from Scatchard equation and found to be 2.07 × 10 M . To further
investigated the interaction of compound 1 with the DNA three−dimensional (3D) fluorescence
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spectroscopy was performed on compound 1 in the absence and presence of CT−DNA (Figure
S4, Supporting Information). The 3D fluorescence spectrum and contour ones of compound 1
consisted of two prominent peaks, peak A (λ = 270 nm) and peak B (λ = 361 nm) showing
ex
em
the fluorescence behavior. However, upon addition of DNA, the fluorescence intensity of both
the peaks excitation peak A and emission peak B decreases significantly indicating either partial
intercalation or electron/energy transfer with double–stranded DNA which was in good
agreement with that derived by absorption spectral and fluorescence measurements.
Competitive binding of drugs to DNA with EB could provide rich information with
regard to the DNA binding affinity [23]. The EB fluorescence intensity will be enhanced in the
presence of DNA because of its intercalation into the helix, and it is quenched by the addition of
another molecule either by replacing the EB [24]. The extent of reduction of the emission
intensity gives a measure of the binding propensity of the compounds to CT–DNA. The emission
spectra of EB bound to DNA in the absence and presence of compound 1 was shown in Figure 6.
Upon increasing concentration of the 1, the emission band at 591 nm was quenched up to
4
4.71%, of the initial fluorescence intensity accompanied by red shift of 22 nm. The observed
decrease in the fluorescence intensity with a strong red shift clearly indicates that the EB
molecule was displaced by the 1 from their DNA binding sites [25]. Furthermore; the quenching
extents was evaluated quantitatively by employing Stern–Volmer equation. The K value for
sv
compound 1 was found to be 1.2 revealing that some of the EB was displaced from their DNA
binding sites.
2
.2. Fluorescence quenching studies with HSA
Human serum albumin is the most abundant plasma protein (∼60% of total plasma protein)
providing a depot and carrier for many endogenous and exogenous compounds with a crucial
8

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effect on the drug distribution and pharmacokinetics [26]. The emission quenching experiment of
HSA was carried out to study the interaction of compound 1 with HSA (Figure 7) [27]. The
fluorescence spectra of HSA in the absence and presence of compound 1 as a quencher in Tris–
HCl buffer (pH 7.4) was monitored with an excitation wavelength of 293 nm.
–
6
–5
Upon the addition of increasing concentration of compound 1 (3.33×10 to 26.6×10 M) to
fixed amount of HSA, significant decrease in the intrinsic fluorescence intensity of HSA was
observed at 362 nm with a slight blue shift in the wavelength clearly indicating that the
secondary structure of protein could be altered [28]. The observed blue shift was mainly
attributed to the reduction in the polarity of the microenvironment around the Trp residues after
the addition of 1 and the binding of 1 to the hydrophobic sites of HSA near the subdomain IIA
[29].
In order to speculate the possible fluorescence quenching mechanism of HSA in presence of
compound 1, Stern–Volmer equation was applied:
Fo
=
1+ Kqτo[Q] =1+ K [Q]
(7)
sv
F
o
where F and F are the fluorescence intensities in the absence and presence of quencher, while
q
K , is the biomolecular quenching rate constant of the biomolecules and so is the average life
–
8
time of the molecule without quencher ( o
τ =10 s), K is the Stern–Volmer quenching constant,
sv
and [Q] is the concentration of the quencher, respectively. The Stern–Volmer plots of F /F versus
o
[Q] for the quenching of HSA fluorescence by compound
1 was depicted in Figure S5 (See
3
–1
Supporting Information) and the calculated KSV and K value was found to be 7.63 × 10 M and
q
12
–1 –1
7
.63 × 10 M s , respectively. The observed K
q
value was larger than the limiting diffusion
1
0
–1 –1
constant Kdif of the biomolecules (Kdif = 2.0×10 M s ), [30] indicating that the fluorescence
quenching was caused due to the specific interaction of compound
1 with HSA, consistent with
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the static quenching mechanism [31]. For static quenching, the Scatchard equation was employed
to calculate the binding constant and number of binding sites [32]:
Fo − F
log[
] = log K + nlog[Q]
(8)
F
where F and F are the fluorescence intensities of HSA in the absence and presence of quencher,
o
K and n are the binding constant and the number of binding sites, respectively. Thus, a plot of
o
log[(F –F)/F] versus log[Q] was used to determine K (binding constant) from the intercept on Y–
axis and n (binding sites) from the slope (Figure S6, Supporting Information). From the
corresponding Scatchard plot, the K values were found to be 0.416 for compound
1 while the n
was calculated to be 1.46, respectively.
In order to further gain insight in the conformational changes of HSA, 3D fluorescence
spectral studies was carried out in the presence and absence of compound . The 3D fluorescence
spectra and contour ones of HSA and compound –HSA system were shown in Figure 8. Peak A
was the Rayleigh scattering peak ( em). Peak B (295, 332 nm, em) dominatingly
displays spectral behavior of the Trp residue, and the fluorescence intensity of this residue was
associated with its micro–environment s polarity. The results revealed that fluorescence intensity
of both peaks peak A and peak B (295, 332 nm, λex λem) decreases significantly indicating the
quenching of fluorescence induced by Trp residue of HSA. However, the fluorescence intensity
of peak B was strongly quenched revealing that binds to HSA near the tryptophan residues [33].
These results revealed that the interaction of with HSA induced micro–environmental changes
in the structure of HSA, corroborated well with our spectroscopic results.
.3. DNA cleavage activity
1
1
λ
ex
=
λ
ex
λ , λ
′
,
1
1
2
1
0

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The ability of compounds to cleave supercoiled (SC) pBR322 DNA was assayed by gel
electrophoresis in the absence and/or presence of external agents. If one strand is cleaved, the SC
DNA produces a slower–moving nicked circular (NC) form. If both strands are cleaved, the
linear (L) form is generated which migrates in between the SC and NC forms [34]. With
increasing concentration of
conversion to NC form with simultaneous increase in the intensity of the latter form. It was
clearly observed that at 25 M concentration, was found to promote the cleavage of DNA from
1, the amount of SC form decreases gradually and there was
ꢀ
1
supercoiled form (SC) to nicked circular form (NC) (Figure S7, Supporting Information)
revealing the single strand DNA cleavage. Quantification of the both the forms originating from
SC and NC plasmids by using Vilber–INFINITY gel documentation system were depicted in
Figure S7c and S7d (See Supporting Information).
To predict the cleavage mechanism of pBR322 plasmid DNA by compound
1, reactions
were carried out in the presence of hydroxyl radical scavengers (DMSO and EtOH), singlet
•
–
oxygen scavenger (NaN ) and superoxide dismutase (SOD) as superoxide ion radical (O )
3
2
scavenger (Figure 9). The significant inhibition of cleavage in the presence of DMSO and EtOH
was observed indicating that cleaves DNA might be through hydrolytic pathway. The addition
1
of NaN (lane 6) decreases the strand scission implying that the singlet oxygen or a singlet
3
oxygen–like entity may be also involved in the cleavage reaction. However, SOD did not show
•–
any effect on DNA cleavage which clearly rule out the participation of superoxide radical (O ).
2
The results revealed that the presence of electron rich organoselenium compound
1 provides an
inbuilt nucleophile Se, which facilitates the nucleophilic attack towards the phosphorus, followed
by the formation of five–coordinate phosphate intermediate and subsequent rearrangement of the
phosphate allows the DNA to be cleaved readily (Scheme SI, Supporting Information). The
1
1

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DNA groove binding propensity of the compound was also studied using compound 1, DNA
minor groove binder DAPI and major groove binger methyl green. The cleavage pattern
demonstrated that the inhibition in the DNA cleavage activity was observed in the presence of
DAPI revealing the minor groove binding.
To further confirm the hydrolytic pathway mediated by compound 1, DNA religation
experiment was performed in which supercoiled pBR322 DNA was treated with T4 ligase
enzyme and subjected to gel electrophoresis. It is well known that in DNA hydrolytic cleavage,
3
′
–OH and 5
these fragments can be enzymatically ligated [35]. In some cases, the hydrolytic products either
did not end at the required 5 –phosphate and 3 –OH (ribose) termini or the compound sometimes
bound to the termini of cleaved DNA. These reasons would result in the religation being
incomplete or even failing completely [36]. Compound yielded nicked DNA which was
religated by using T4 DNA ligase enzyme. Under our experimental conditions, the nicked form
Form II) was religated to a large extent in the presence of T4 ligase enzyme which was
3
′–OPO (5′–OH and 3′–phosphate) fragments must be exclusively afford and that
′
′
1
(
quantitatively ascertained by comparison of compound treated DNA and control DNA alone in
supercoiled form providing direct evidence in favor of hydrolytic mechanism (Figure 10).
Moreover, addition of T4 ligase showed significant retardation in mobility of religated DNA
relative to that of control supercoiled DNA as a relatively sharp band during electrophoresis.
Change in the electrophoretic mobility of plasmid DNA might be due to the strong binding of
DNA T4 ligase at the ligation site.
2
.4. Glutathione Peroxidase Activity
Glutathione peroxidase (GPx) belongs to the family of selenoproteins and plays an important
role in the defense mechanisms oxidative damage by catalyzing the reduction of a variety of
1
2

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hydroperoxides, using glutathione as the reducing substrate. Therefore, much effort has been
made to mimic the function of GPx activity using small organoselenium molecules as a scaffold
in which the catalytic selenium is introduced for therapeutic applications. The catalytic site of
GPx enzyme includes a selenocysteine residue in which the selenium undergoes a redox cycle
involving the selenol as the active form that catalyzes the reduction of harmful peroxides in the
presence of cofactor glutathione (GSH) and thereby protects the lipid membranes as well as
biologically important molecules against oxidative stress [37, 38]. The enzyme–catalyzed
reduction of peroxide by GSH was studied according to the method reported by Tomoda et al by
using organoselenium compound
1 as a GPx mimic [39]. In this assay, the catalytic GPx model
reaction was initiated by the addition of an excess amount of H O to a solution of GSH
2
2
containing a selenium catalyst and was monitored by UV spectroscopy at 305 nm. The initial
reduction rates of H O (v ) were obtained by monitoring the increase in UV absorption at 305
2
2
0
nm due to the formation of corresponding disulfide (GSSG). However, the reduction rate of
–
1
H O without any compound was very slow (1.21 mM min ) but a considerable enhancement in
2
2
the Gpx activity was observed on increasing the concentration of organoselenium compound
1
–
1
(
14.2 mM min ) revealing that
1
act as excellent GPx mimic (Figure 11). Therefore, the
synthetic organoselenium compound involves the rapid cleavage of the aliphatic carbon–
selenium bond when employed as catalysts for the reduction of hydroperoxides. The 3,5–
bis(trifluoromethyl)benzene was used as cofactors and the catalytic mechanism involves the
formation of different intermediates which was responsible for the reduction of hydrogen
peroxide with GSH (Scheme II).
2
.5. Docking studies with DNA and HSA
1
3

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To ascertained the groove binding mode of compound
results, compound was docked on DNA duplex of sequence d(CGCGAATTCGCG)2
dodecamer (PDB ID: 1BNA). The resulting model showed that compound recognizes DNA in
the minor groove situated within G–C rich region from chain A (G2A and C3A) through
aromatic benzene ring and stabilized by van der Waal s interactions and hydrophobic contacts
with DNA functional groups. Moreover, fluorine atoms of the compound may be engaging in
1 obtained from gel electrophoretic
1
1
′
1
hydrogen–bonding interactions with DNA nucleobases DG14 (3.55, 2.71 Å), DG16 (2.51, 3.00
Å) and DG10 (2.46, 3.55 Å) available in the minor grooves in GC rich sequences (Figure 12).
The resulting relative binding energy of docked metal compounds
Kcal/mol, indicated that compound strongly binds to the DNA, which is correlated well with
the experimental DNA binding results.
Similarly, the quenching of Trp214 residues in presence of compound
1 DNA was found to be –3.77
1
1
was also
validated by molecular docking technique. HSA comprises three homologous domains (denoted
I, II, and III): I (residues 1–195) II (196–383) and III (384–585); each domain has two
subdomains (A and B) that assemble to form heart shaped molecule. The principal region of drug
binding sites of HSA are located in hydrophobic cavities in subdomain IIA and IIIA,
corresponding to site I and site II, respectively and tryptophan residue (Trp–214) of HSA in
subdomain IIA [40, 41]. The model revealed that the compound
1 was inserted in the
hydrophobic cavity of site I, and was close proximity to the tryptophan residue of HSA (Trp214),
suggesting the existence of hydrophobic interaction between them. Such finding provides a good
structural basis to explain the very efficient fluorescence quenching of HSA emission in the
presence of compound
1. At the same time, there were considerable number of hydrogen bonds
and electrostatic interactions between Arg–160 and Arg–257 and Ala–291 and fluorine atoms of
1
4

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compound
1
. On the other hand, the amino acid residues with aromatic ring could match with
that of the benzene ring of compound
the binding environment of the compound
.6. Docking studies with Glutathione peroxidase
To gain insights into the biological effects of organoselenium compound serve as Glutathione
peroxidase mimics, compound was successively docked with the target enzyme to understand
the interaction of compound with the target active–site. The active sites of GSH peroxidase are
1
and stabilized by
π–π staking interaction in space to firm
1
(Figure 13 and Figure S8, Supporting Information).
2
1
1
found in flat depressions on the molecular surface and are readily accessible from solvent
channels in the crystals. The catalytic triad of GPXs is composed of the selenocysteine, a
glutamine and tryptophan amino acid residues [42]. Exposure of the catalytically active
selenocysteine residues at the molecular surface is consistent with the easy access of the
substrates and thus the high reaction rates of GSH peroxidase [43]. The resulting docked model
clearly indicated that some aromatic amino acid residues Ala138, Cys152, Ser157, Arg153,
Met140, Thr137, Asn114, Asn154, Asp155 and Trp148 are found in the close proximity with
that of compound
amino acid residues Pro143, Ile146, Val151 of GPx and was involved in the van der Waal
interactions with compound . Similarly, the selenium atom was involved in van der Waal
interactions with Leu139 amino acid residues. Furthermore, hydrogen bonding of Tryp148,
Asn154 and Asp155 residues at the active site with the fluorine atom of the compound
1 showing the electrostatic interactions in the active site (Figure 14). The
1
1
enhances the reactivity of Se towards hydroperoxides [44]. The total interaction energy was
found to be –10.39 kcal/mol, indicating that the non–covalent interactions are important criteria
for the catalytic reduction of hydrogen peroxide and organic peroxides. The results suggested
that high GPx activity of compound
1 was due to the presence of hydrogen bonding, electrostatic
1
5

ACCEPTED MANUSCRIPT
and van der Waal interactions within the active site of GPx catalytic triad. Therefore, compound
1
was acting as a GPx mimic, which e
mitochondria against oxidative damage.
.7. Cell imaging studies
The anticancer activity of organoselenium compound
ffectively scavenged hydroperoxides and protected the
2
1
was investigated against HeLa cell line
by using the MTT assay and confocal microscopy for cell viability and cell imaging studies
respectively for documenting intracellular changes due to apoptosis. The HeLa cells were
incubated with an increasing concentration of
concentration–dependent decline in the number of viable HeLa cells. It was found that at 30
concentration cell viability was reduced to around 68 percent. While, at 40 M concentration cell
1 separately for 24 h. The results revealed
µM
µ
viability was reduced to less than 50 percent. Increasing concentration further resulted in
progressive decrement in cell viability as shown in Figure S9 (See Supporting Information).
To gain further insight in the mechanism of cell death, confocal microscopy was performed as a
preliminary investigation of its applicability as an anticancer therapeutics. The control and
treated cells were stained with PI (Propidium Iodide), a nucleus specific dye that helps in
understanding nuclear morphology. Cell imaging of Propidium Iodide stained cell that were
already treated with compound
of nuclei, which was typical of late apoptosis (inset in Figure 15 c). However, no such blebbing
was observed in control (inset in Figure 15 f). Therefore, the potential compound to initiate
1 revealed nuclear condensation with blebbing and fragmentation
1
nuclear blebbing in HeLa cancer cell line suggests its efficacy for utilizing it in anti–cancer
chemotherapeutics.
3
. Experimental
1
6

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3
.1. Reagents and Materials
All reagents were commercially available and used as supplied without further purification. 6X
loading dye (Fermental Life Science) and Supercoiled plasmid DNA pBR322 (Genei) were
utilized as received.
HSA (fatty acid free, 99%) was purchased from Sigma and used without further purification.
–3
Human serum albumin of 1×10 M was prepared by dissolving protein in Tris–HCl bu
ffer
solution at pH 7.3. The protein concentration was determined spectrophotometrically using an
–
1
–1
extinction coe
used to maintain the ionic strength of bu
Working standard solution was obtained by appropriate dilution of the stock solution.
.2. Methods and instrumentation
The Crystal structure was solved using Super Nova Dual source X-ray Diffractometer system
Agilent Technologies) equipped with a CCD area detector and operated at 250 W power (50 kV,
fficient of 35219 M cm at 280 nm. NaCl (analytical grade, 1 M) solution was
ff
er at 0.1 M, pH was adjusted to 7.3 by using HCl.
3
(
0
.8 mA) to generate Mo K
α
radiation (
λ = 0.71073 Å) and Cu Kα radiation (λ = 1.54178 Å) at
1
13
2
93(2) K. H and C NMR spectra were recorded on Bruker–Avance DPX FT–NMR 400 MHz
spectrometer with TMS as an internal standard. The IR spectra were recorded as KBr pellets with
–
1
Shimadzu IR–408 Perkin–Elmer 1800 (FTIR) and its values are given in cm . Chemical shifts
are expressed in parts per million ( ppm). HRESIMS were recorded on G6540–UHD LC/MS
δ
Q–TOF Agilent Technologies. Silica gel coated aluminum plates were used for TLC. All the
reagents used were of analytical grade. Electronic spectrum was recorded on UV–1700
PharmaSpec UV–vis spectrophotometer (Shimadzu) in DMSO cuvettes of 1 cm path length.
Data were reported in λmax/nm. Fluorescence measurements were determined on a RF–5301 PC
spectrofluorophotometer (Schimadzu). DNA cleavage experiments were performed with the help
1
7

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of Axygen made electrophoresis supported by a Genie power supply with a potential range of
5
0–500 volts, visualized and photographed by a Vilber–INFINITY gel documentation system.
.3. Synthesis of Bis(3,5–bis(triflouromethyl)phenyl)selane ( ). Selenium powder (1.98 g, 25.0
3
1
mmol) was stirred for 15 min in dry THF, in a two necked 50 ml round bottomed flask, followed
o
by addition of distilled SO Cl (2.00 g, 28.0 mmol) at 0 C. The mixture was kept on stirring for
2
2
another 1 h to obtain a clear brownish red solution of SeCl In another two necked 100 ml round
2
.
bottomed flask freshly activated Mg turnings were vigorously stirred in THF for 5 min, followed
by addition of 1–bromo–3,5–bis(trifluoromethyl)benzene (7.28 g, 25 mmol) and kept on stirring
2
for 6 h. After formation of Grignard reagent, SeCl (0.5 eqiv.) solution was added drop wise via
o
cannulation at –78 C and allowed to warm to room temperature. The progress of the reaction
was monitored by thin layer chromatography. After completion, the mixture was poured into
water and extracted with ethyl acetate (3 × 50 ml). The combined organic layers were dried over
sodium sulfate and solvents were removed under vaccum through rotary evaporator. The crude
extract was purified by column chromatography using hexane:ethyl acetate (5:1) to afford pure
1
bis(3,5–bis(triflouromethyl)phenyl)selane as a yellowish red solid in 85% yield (10.73g). H
1
3
NMR (400 MHz, CDCl
3
):
δ
7.78 (s, 4H), 7.92 (s, 2H). C NMR (100 MHz, CDCl
3
):
δ 121.6,
7
7
1
22.3, 123.7, 131.5, 132.1, 132.3, 132.6, 132.8. Se NMR:
δ 485.185. HRESI–MS found for
+
+
C H F Se [M+H] : 506.1634 (calcd. C H F Se [M+H] : 506.1671) {See Supporting
16
6
12
16
6 12
Information Figures S10–S13}.
3
3
.4. Computational Details
.4.1. DFT studies
The DFT calculations were performed using TURBOMOLE 6.0 computational chemistry
program [45]. The geometry optimization of the Bis(3,5–bis(triflouromethyl)phenyl)selane was
carried out with B3LYP functional [46] which has a proven track record of predicting the
1
8

ACCEPTED MANUSCRIPT
structures and the energetics accurately [47]. Three basis sets 6–31G(d,p) [48], LanL2MB [49]
and TZVP [50] were examined for the perfect matching of the optimized structure of compound
1
to its crystal structure [51]. The single point energy calculation of the B3LYP/ TZVP
optimized structure was attempted using TZVPP basis set [52].
.4.2. Molecular docking studies
3
The molecular docking studies were performed using Argus lab software, [53–55] an interactive
molecular graphics program for calculating and displaying feasible docking modes of pairs of
protein, enzymes and DNA molecule. Minimization was performed by geometry
convergence function of ArgusLab software according to Hartree–Fock calculation
method. Semi–emperical PM3 method was used for the geometrical optimization of ligand and
charm force field was used for the preparation of receptor after that we have performed docking
in which ligand is used as a flexible mode. The crystal structure of the B–DNA dodecamer
2
d(CGCGAATTCGCG) (PDB ID: 1BNA), human serum albumin (PDB ID: 1h9z) and
glutathione peroxidase (PDB ID: 1GP1) were downloaded from the protein data bank.
Visualization of the docked file has been done using Discovery studio molecular graphics
programs.
3
.5. DNA Binding and cleavage Experiments
DNA binding experiments include absorption spectral traces and emission spectroscopy
conformed to the standard methods and practices previously reported in literature, [56–58]
whereas DNA cleavage experiment has been performed by the standard protocol [59]. While
measuring the absorption spectra an equal amount of DNA was added to both the compound
solution and the reference solution to eliminate the absorbance of the CT DNA itself, and Tris
buffer was subtracted through base line correction.
1
9

ACCEPTED MANUSCRIPT
3
.6. Fluorescence quenching studies with HSA
Fluorescence quenching measurements were carried out at excitation and emission wavelengths
–
5
–5
set at 293 and 200–800 nm, respectively. The interaction of compound
1
(0.67×10 to 4.6×10
M) with the HSA content of fixed concentration (6.67 × 10 M) was studied. The intensity at
62 nm (Trp–214) was used to calculate the binding constant (K) and 3D fluorescence spectra of
HSA (1.5 mM) in the absence and presence of compound (11.5 mM) were recorded in Tris–
HCl bu er (pH 7.3) at room temperature by using Hitachi F–2700 fluorescence
spectrophotometer.
.7. Glutathione Peroxidase Assay
The GPx activity of the organoselenium compound
–
6
3
1
ff
3
1
was determined through a
spectrophotometric method at 305 nm. The test mixture contained GSH (2 mM), EDTA (20 mM),
glutathione reductase (2.5 U/mL), and NADPH (2 mM) in 0.05 M potassium phosphate buffer,
pH 7.0. The GPx samples (30
reaction was started through the addition of H
calculated from the oxidation rate of NADPH. The initial reduction rate was determined at least
ꢀl–0.3 ml) were added to the test mixture at 25 °C, and the
2 2
O
(2.5 mM). The initial reduction rates were
–
1
–1
three times and calculated from the first 5–10% of the reaction using 6220 M cm as the
extinction coefficient for NADPH.
3
.9. In–vivo study of Bis(3,5–bis(triflouromethyl)phenyl)selane (1)
The efficacy of compound
in HeLa cell line.
1 as an anti–cancerous chemotherapeutics in live cell was undertaken
3
.10. Maintenance of cell lines
2
0

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HeLa cell line was maintained in DMEM (Dulbeco’s Modified Eagle Medium) medium
supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics and antimycotic
(
Himedia) as per manufacturer’s protocol. Cells were incubated in CO incubator at 5% CO and
2 2
o
3
7 C temperature.
3
.11. Cell Proliferation and viability assay
To measure cell viability and growth MTT assay was done. In this assay, cells seeded in 96–well
3
plates (5x10 cells/ well) in duplicate were cultured in complete DMEM medium. Cells were
treated with different concentrations of compound
1 (10, 20, 30, 40, 50 µM) separately for 24
hours. The cells, after treatment were incubated with MTT for 2 hours. The yellow tetrazolium
salt (MTT) is reduced in metabolically active cells to form insoluble purple formazan crystals,
which are solubilised by the addition of DMSO (Sigma). The colour was then quantified by
spectrophotometric measurement at 570 nm wavelength on a microtiter plate reader (Bio–Rad
model 680 microplate reader).
3
.12. Confocal imaging of compound 1 treated HeLa cells
We proceeded for utilization of “compound
cell line treated with “compound ”. In this study HeLa cells were incubated separately with 30
M of compound for 60 min in dark. Cells were washed for 5min with 1X PBS (Phosphate
1” as an anti–cancerous chemotherapeutics in HeLa
1
ꢀ
1
buffered saline, pH 7.4) twice. Cells were stained with 1µg/ml concentration of Propidium
Iodide (PI) for 5 min in dark. Cells were washed for 5min with 1X PBS (Phosphate buffered
saline, pH 7.4) twice. Cells collections after each step was done by centrifugation at 2000 rpm
for 2min. Cells were mounted with DABCO on slide. Control for compound
1 was also taken.
Slides were observed under 63X oil objective lens of Zeiss, LSM 510 Meta Confocal
microscope.
2
1

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4
. Conclusion
The new organoselenium compound
1
was synthesized and thoroughly characterized by
spectroscopic (IR, H, C, Se NMR, HRESI–MS), analytical techniques and further validated
by X–ray crystallography. The DFT study of was also performed by using Turbomole
computational chemistry program (B3LYP/TZVP method) and the HOMO–LUMO gap of 4.6
eV indicated the stability of molecule. In vitro DNA binding profile of compound was carried
out by using various biophysical techniques in accordance with molecular docking studies which
revealed that binds to DNA through external contact and hydrogen bonding, in addition to
selective recognition towards the minor groove of DNA. Compound cleaves supercoiled
1
13
77
1
1
1
1
plasmid pBR322 DNA via hydrolytic cleavage mechanism induced by the nucleophilic Se atom
which was further supported by DNA relegation assay by employing T4 DNA ligase.
Furthermore, affinity of
understand the carrier role of serum albumin for
antitumor activity of was investigated by using MTT assay and confocal cell imaging studies
which showed nuclear condensation and blebbing inside the HeLa cells typical of late apoptosis
or necrosis. Therefore, compound act as a promising candidate for GPx mimic and its potential
1
towards HSA was also investigated by fluorescence spectroscopy to
1
in blood under physiological conditions. The
1
1
to initiate nuclear blebbing in HeLa cancer cell line warrants further in vivo investigation.
Acknowledgements
Masood Rizvi thankfully acknowledges Dr. Rahul Banerjee, National Chemical Laboratory,
Pune, India for the X–ray crystallographic studies and Discussion. We also thankfully
acknowledge Prof. M.A. Khuroo, coordinator DST, University of Kashmir for financial
assistance to this work. We gratefully acknowledge the financial support received from the DST–
PURSE, DST–FIST programme and DRS-I (SAP) from UGC, New Delhi, India. Author
2
2

ACCEPTED MANUSCRIPT
Mehvash Zaki is also highly grateful to CSIR–SRF (SRF scheme–2013: 9/112(503) 2K13–
EMR–I), New Delhi for providing financial assistance.
Notes and References
*
Department of Chemistry, Aligarh Muslim University, Aligarh, UP–202202, India. Tel.: +91
9
358255791: E-mail address: tsartaj62@yahoo.com
†
Electronic supplementary information (ESI) available: Tables, and a CIF file and
crystallographic data. Crystallographic data are also available from the CCDC as file CCDC
023336. Crystallographic data for the structural analyses have been deposited with the
1
Cambridge Crystallographic Data Centre, CCDC Copies of this information may be obtained
free of charge from: The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: (int.
code)
+44
1223–336–033;
e–mail:
deposit@ccdc.cam.ac.uk
or
www:
http://www.ccdc.cam.ac.uk]. Details of DFT studies, in vitro screening, DNA and HSA binding,
concentration dependent DNA cleavage and mechanism for the hydrolytic cleavage.
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Figure and Scheme Captions
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Scheme I. Synthesis of bis(3,5–bis(trifluoromethyl)phenyl)selane.
Scheme II. Proposed mechanism for the Glutathione Peroxidase activity of compound 1.
Fig. 1. ORTEP diagram of compound
Fig. 2. Molecular structure of compound
Fig. 2. Diagramatic representation of 3D (a) view in wire frame model and (b) CPK model
in compound
Fig. 3. Electrostatic potential map and HOMO–LUMO Energy separation of compound
Fig. 4. Absorption spectra of compound in 5mM Tris HCl/ 50 mM NaCl buffer upon the
addition of calf thymus DNA; Inset: Plots of [DNA]/
titration of CT DNA with compound 1 ▲, experimental data points; full lines, linear
1.
1
showing Ball and stick model.
1
.
1.
1
2
a f
ε –ε (m cm) vs [DNA] for the
–6
–5
fitting of the data. [compound
1] = 6.67×10 M, [DNA] = (0–4.66) × 10 M. Arrow
shows change in intensity with increasing concentration of DNA.
Fig. 5. Emission spectra of compound
1 in Tris–HCl buffer (pH 7.3) in the presence and absence
of CT DNA at room temperature. Arrow shows change in intensity with increasing
concentration of DNA.
Fig. 6. Emission quenching spectra of CT DNA bound ethidium bromide in the presence of (a)
compound
1 in buffer 5 mM Tris–HCl/50 mM NaCl, pH = 7.3 at 25 ºC. Arrow shows
change in intensity with increasing concentration of ethidium bromide.
Fig. 7. The fluorescence quenching spectra of HSA by different concentrations of compound
1
with the excitation wavelength at 293 nm in 5 mM Tris–HCl/50 mM NaCl buffer, pH 7.3,
–
6
at room temperature: [HSA], 6.67 × 10 M; the concentration of compound
1 was
2
8