Synthesis, spectral characterization and DNA interactions of 5-(4-substituted phenyl)-1,3,4-thiadiazol-2-amine scaffolds

Article abstract of DOI:10.1016/j.molstruc.2019.126999

Five 5-(4-Substituted phenyl)-1, 3, 4-thiadiazole-2-amines have been prepared and structure of the compounds confirmed by spectroscopy studies. On the basis of spectroscopic studies, confirmed the formation of compounds. The DNA binding affinity of the prepared compounds was undertaken by absorption titration method and increasing the amount of CT-DNA observed hyperchromism or hypsochromism. The binding affinity compounds (except 5) are in the order of 4[13.341 × 10⁴ M^?1]>3 [8.505 × 10⁴ M^-1]>2 [3.567 × 10⁴ M^-1]>1[3.525 × 10⁴ M^?1]. The ethidiumbromide quenching results indicates CT-DNA was quenched by thiadiazoles via a static quenching mechanism. The DNA cleavage studies of the compounds were carried out in the presence and absence of H₂O₂ using gel electrophoresis experiment.

Full text of DOI:10.1016/j.molstruc.2019.126999

Journal of Molecular Structure 1199 (2020) 126999
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, spectral characterization and DNA interactions of 5-(4-
substituted phenyl)-1,3,4-thiadiazol-2-amine scaffolds
,
, *
N. Shivakumara a b, P. Murali Krishna a
a Department of Chemistry, M S Ramaiah Institute of Technology, Bengaluru, 560054, India
b Visvesvaraya Technological University, Belagavi, 590018, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Five 5-(4-Substituted phenyl)-1, 3, 4-thiadiazole-2-amines have been prepared and structure of the
compounds confirmed by spectroscopy studies. On the basis of spectroscopic studies, confirmed the
formation of compounds. The DNA binding affinity of the prepared compounds was undertaken by
absorption titration method and increasing the amount of CT-DNA observed hyperchromism or hyp-
sochromism. The binding affinity compounds (except 5) are in the order of 4[13.341 ꢀ 104 Mꢁ1]>3
[8.505 ꢀ 104 M-1]>2 [3.567 ꢀ 104 M-1]>1[3.525 ꢀ 104 Mꢁ1]. The ethidiumbromide quenching results in-
dicates CT-DNA was quenched by thiadiazoles via a static quenching mechanism. The DNA cleavage
studies of the compounds were carried out in the presence and absence of H2O2 using gel electrophoresis
experiment.
Received 17 October 2018
Received in revised form
27 August 2019
Accepted 27 August 2019
Available online 2 September 2019
Keywords:
Thiadiazoles
DNA binding
Fluorescence studies
Nuclease activity
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
anhydrase inhibitors [26], anti-depressant activity [15,27], anti-
parkinsonism [28], anticancer [29e31], anti-diabetic activity
Over the years, a chemist has shown interest in the synthesis of
various five-membered heterocyclic compounds and their de-
rivatives due to potential applications in pharmacology, biology etc.
The biological activity may be due to their properties such as
conformational, lipophilicity, metabolic stability, and oral
bioavailability. Among five-membered heterocyclic compounds,
thiadiazoles, oxadiazoles, and triazoles are an important one, which
exhibit a wide spectrum of biological activity [1] and also DNA
binding activity [2].
Among five-membered heterocyclic compounds, Thiadiazoles
and its derivatives, which consist of two nitrogen and one-sulphur
atoms, play an important role in biological applications. These
thiadiazoles exist several isomeric forms including 1, 2, 3; 1, 2, 4; 1,
2, 5 and 1, 3, 4-isomeric forms (Fig. 1) [3,4].
Based on literature survey, the isomer 1,3,4-thiadiazole ring
skeleton have been recognized as potential molecule found diverse
applications in therapeutic activities such as anti-inflammatory
[5e7], antiviral [8,9], analgesic [7,10], anti-microbial [11e13], anti-
convulsant [14e20], anti-oxidant properties [21], anti-hypertensive
[22e24], anti-neoplastic and anti-tubercular agents [25], carbonic
[32,33]. In addition, various naturally occurring and synthetic
compounds, which contain thiadiazole moiety, show interesting
pharmacological properties. The biological and pharmacological
activities of thiadiazoles may be due to the presence of N2C2S
moiety [34] and its high aromaticity, in vivo stability, and lack of
toxicity and its “hydrogen binding domain” and “two electron
donor system” ability makes the usage of 1,3,4-thiadiazole as one of
the potential moiety in many commercially available drugs such as
Desaglybuzole (anti-diabetic), Acetazolamide or Diamox (for glau-
coma), Methazolamide (Carbonic anhydrase inhibitor) etc. (Fig. 2)
In recent years, the interaction between drug and DNA is one of
the essential factors in order to design chemotherapeutic agents.
Nowadays, the efficiency and type of binding between the small
drug molecule and calf thymus DNA can be studied by using the
various techniques such as UVeVisible absorption, Fluorescence,
FT-IR, Circular dichromic, Viscosity measurements, Melting studies
etc. [35e40].
In the light of their importance in Agriculture, biology, phar-
macology [41], and in the continuation of our research on sulphur
and nitrogen-containing molecules [42e49], herein reporting
synthesis, characterization and biological studies of 5-(4-
substituted phenyl)-1,3,4-thiadiazole moieties.
* Corresponding author.
E-mail address: muralikp@msrit.edu (P. Murali Krishna).
https://doi.org/10.1016/j.molstruc.2019.126999
0022-2860/© 2019 Elsevier B.V. All rights reserved.

2
N. Shivakumara, P. Murali Krishna / Journal of Molecular Structure 1199 (2020) 126999
described in the literature reported by same group [42].
2.2.1. 5-[(E)-2-phenylethenyl]-1,3,4-thidiazol-2-amine (1)
IR (cmꢁ1): 3091e3296 (NH2), 1504 (C]N), 1450 (C]C), 748
(CeS); 1H NMR: 7.62e7.64 (d, NH2 J ¼ 8 Hz), 7.29e7.4 (m, 5H, Aro-
matic), 703-7.09 (d, 2H, alkenes’ protons, J ¼ 16 Hz). 13C NMR: 31,
120, 127, 129, 134, 136, 157 and 168 ppm; LCMS (m/z) Calculated for
C10H9N3S 203.257, Found 204.
Fig. 1. Isomeric forms of Thiadiazole moeity.
2.2.2. 5-Phenyl-1,3,4-thiadiazol-2-amine (2)
IR (cmꢁ1): 3060e3272(NH2), 1509(C]N), 1632(C¼Cstr),
687(CeS); 1H NMR: 7.408 (m, 5H aromatic), 7.722 (s, 2H); 13C NMR:
126,129,130,131,157,169 ppm; LCMS (m/z) Calculated for C8H7N3S:
177.221, Found 178.
2. Experimental section
2.1. Materials and methods
All the starting materials, calf thymus DNA purchased from
Sigma Aldrich and pUC18 from Bangalore Genie. The uncorrected
melting points were measured by the open capillary method, FT-IR
spectral data were recorded using Bruker alpha with KBR pellet
method. NMR spectra were recorded using Agilent with ATB probe
in a DMSO‑d6 solvent (400 MHz for 1H and 100 MHz for 13C), LC-MS
was obtained by Agilent 1200 series LC-MicromasszQ spectrom-
eter. EtBr quenching studies performed on F-2300 spectrofluo-
rimeter (Hitachi, Japan) equipped with 1.0 cm quartz cell at 298k.
The excitation and emission slit widths were kept at 5 nm, and the
excitation wavelength fixed to 500 nm in the range of 520e700 nm
for ethidium bromide and by excitation at 350 nm in the range of
390e600 nm for thiadiazoles. The EV-Visible spectroscopy recor-
ded on Elico SL159 in the range of 190e500 nm using 1 cm quartz
cell at room temperature.
2.2.3. 5-(4-Methylphenyl)-1,3.4-thiadiazol-2-amine (3)
IR (cmꢁ1): 3060e3270(NH2), 1634(C]N), 1470(C¼Cstr),
816(CeS); 1H NMR: 2.467 (s, 3H), 7.2e7.318 (d, 4Har.), 7.589 (s, 2H);
13C NMR: 21, 126, 128, 130, 139, 157 &169; LCMS (m/z) Calculated for
C9H9N3S: 191.247, Found 192.
2.2.4. 5-(4-Methoxylphenyl)-1,3.4-thiadiazol-2-amine (4)
IR (cmꢁ1): 3091e3292 (NH2), 1601(C]N), 1505(C]C),
826(CeS); 1H NMR 3.801 (s, 3H), 7.025-7.006 (d, 2H Aromatic,
J ¼ 7.6 Hz), 7.26 (s, NH2), 7.685-7.667 (d, 2H Aromatic, J ¼ 7.2 Hz);
13C NMR: 31, 55, 114, 124, 128, 160 & 206 ppm; LCMS (m/Z) Calcu-
lated for C9H9N3OS: 207.247, Found 207.95.
2.2.5. 5-(4-Ethylphenyl)-1,3,4-thidiazol-2-amine (5)
IR (cmꢁ1): 3085e3268(NH2), 1633 (C]N), 1507 (C]C), 829
(CeS); 1H NMR: 1.173e1.210 (t, 3H, J ¼ 7.6 Hz), 2.609e2.665 (q, 2H,
J ¼ 7.6 Hz), 7.282e7.32 (m, 4H), 7.64e7.66 (s, 2H); 13C NMR: 15, 27,
2.2. Synthesis of the thiadiazoles
The substituted 1,3,4-thiadiazoles (Scheme 1) will be synthe-
sized by cyclization of thiosemicarbazones using the procedure
126, 128, &145 ppm); LCMS (m/z) Calculated for C10H11N3S:
205.273, Found 206.05.
Fig. 2. Thiadiazole moeity containing drugs.
Scheme 1. Sythesis of 2-amino-5-substituted thiadiazoles.

N. Shivakumara, P. Murali Krishna / Journal of Molecular Structure 1199 (2020) 126999
3
2.3. DNA binding studies
2.4. DNA cleavage studies
2.3.1. Absorption titrations
The Nuclease activity of the prepared thiadiazoles was per-
formed on gel electrophoresis method using pUC18 DNA in TAE
buffer (pH 8.0). The samples were incubated for 60 min at 37 ꢂC and
The absorption titrations were performed to investigate the
DNA binding efficiency of thiadiazole compounds. The CT-DNA was
dissolved in tris-buffer and stored at 4 ꢂC for complete dissolution.
The ratio of absorbance values at 260 and 280 nm in buffer [5 mM
tris-buffer/50 mM NaCl, pH 7.2] gave between 1.8 and 1.9 indicates
nucleic acid is sufficiently free of protein. Measuring the absor-
bance at 260 nm and using the molar extinction coefficient value of
6600 dm3 molꢁ1cmꢁ1 calculated the concentration of DNA per
nucleotide. The DNA-thiadiazole interactions were performed with
a fixed concentration of the compounds (1.67e6.07 mM) and
added 2
0.25% xylene cyanol and 60% glycerol to the reaction mixture, then
introduced in 1% gel containing 1 g/mL of EtBr dye. The electro-
mL of loading buffer containing 0.25% bromophenol blue,
m
phoresis was carried out at 100 V in tris-acetic acid- EDTA (TAE)
buffer till the bromophenol blue reached to 3/4th of the gel. The
bands were visualized by using UV trans-illuminator and photo-
graphed. For comparison purposes, the cleavage reaction for com-
pounds was carried out in the absence and presence of hydrogen
peroxide. The ability of DNA cleavage was determined based on the
capacity of thiadiazole moieties in the conversion of open circular
(OC) or nicked circular (NC) nucleic acid from its super coiled (SC)
structure.
increasing in the concentration of DNA (0e350 mL of 400 mM). The
binding efficiency (Kb) was evaluated from the plotting the graph
between [DNA] and [DNA]/(Ɛa - Ɛf) using the following equation
½DNAꢃ
2a ꢁ 2f
½DNAꢃ
2b ꢁ 2f
1
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
¼
þ
(1)
Kb 2b ꢁ 2f
In the above equation, [DNA] is the concentration of DNA in base
3. Results and discussion
pairs, the absorption extinction coefficients Ɛa, Ɛf and Ɛb are for the
apparent, free compounds and the totally bound form, respectively.
On plotting a graph between [DNA]/(εaeεf) and [DNA] gave a slope
and the intercept equal to 1/(εaeεf) and (1/Kb)(1/(εbeεf)), respec-
tively. Hence Kb was obtained from the ratio of the slope to inter-
cept [43]. The percentage of hyperchromicity or hyperchromicity
for the DNA/[Ligand] was obtained using (Afree-Abound)/Afree ꢀ100.
3.1. Synthesis and characterization
2, 5-Disubstituted thiadiazoles (1e5) were prepared through
the cyclization of thiosemicarbazones (Scheme 1). The prepared
compounds are stable, readily soluble in methanol, chloroform,
DCM, DMSO, and DMF etc. The melting point, yield of the prepared
compounds are shown in Table 1. The structure of the thiadiazoles
was deduced from the spectroscopy techniques and data are
compiled in the synthesis part.
2.3.2. DNA quenching studies
The infrared spectral data of these compounds (Figs. S1eS5)
were observed as follows: NeH stretching vibration observed in the
range of 3296e3060 cmꢁ1, CeH aromatic stretching vibration be-
tween 2960 and 2958 cmꢁ1, C]N pertaining to the thiadiazole ring
observed 1601-1634 cmꢁ1, and stretching of CeSeC in the range of
748e829 cmꢁ1. The infrared data of the compounds indicates the
formation of thiadiazole moieties.
The proton and carbon-13 NMR spectra of the compounds were
recorded using Agilent with ATB probe NMR spectrometer
(400 MHz for 1H and 100 MHz for 13C) in DMSO‑d6. In 1H NMR
spectra (Fig. 3 and S6-S9), the aromatic protons come to resonance
at 6.9e7.41 ppm and the thiadiazole amine protons appeared at the
range of 7.4e7.77 ppm, the ethyl group protons observed at 0.98
and 2.443 for 3H (triplet) and 2H (quartet) respectively and the
methyl (CH3) protons appeared at 3.76. In 13C NMR spectra (Fig. 4
and S10-S13) peaks between 157 and 169 ppm indicate the pres-
ence of cyclic compound containing C]N group. The NMR spectral
data of the prepared compounds matching with literature values
[51]. The LC-MS data were obtained by Agilent 1200 series LC-Micro
mass Q spectrometer. In mass spectra (Figs. S14eS18), the molec-
ular ion peaks of the thiadiazole compounds are matching with the
calculated mass.
An ethidiumbromide-DNA complex quenching technique is a
superior technique to compare the DNA binding mode with the
small molecules. The widths of the excitation and emission slit
were set to 5 nm. The response time (0.04s), excitation voltage
(700 V) and scan rate (1500 nm/min) kept constant for each data
set. The fluorescence background correction made using appro-
priate blank solution in a buffer. The possible inner filter effect
arising from UVeVis absorption of CT-DNA was eliminated, the
fluorescence data were corrected before analysis of the binding and
quenching data [50]. A quartz cell of One-centimeter diameter was
used throughout the experiments. The fluorescence spectra of the
compounds were recorded by using the excitation wavelengths
241 nm (for 1), 295 nm (for 2), 302 nm (for 3 and 4), the emission
wavelength was as follows: 597.5 nm for 1, 602.5 nm for 2 and 4,
602 nm for 3. In the EtBr (EB) displacement method, to a mixture of
10
mL of the EB in tris-buffer solution (50
mM) and 10 mL of DNA
solution (10
mL at saturated binding level) [51], the compound was
added to EB-DNA mixture and was mixed for 5 min before mea-
surements. The spectrum of EB bound to DNA was obtained at an
excitation wavelength of 540 nm and an emission wavelength of
592 nm.
Table 1
Analytical data of the thiadiazoles, 1e5.
Compound
Mol. Formula
Melting point (0C)
Molecular weight
Calculated
Found
5-[(E)-2-phenylethenyl]-1,3,4-thidiazol-2-amine (1)
5-Phenyl-1,3,4-thiadiazol-2-amine (2)
5-(4-Methylphenyl)-1,3.4-thiadiazol-2-amine (3)
5-(4-Methoxylphenyl)-1,3.4-thiadiazol-2-amine (4)
5-(4-Ethylphenyl)-1,3,4-thiadiazol-2-amine (5)
C10H9N3S
C8H7N3S
C9H9N3S
C9H9N3OS
210e215
208e212
180e185
175e180
190e195
203.257
177.221
191.247
207.247
205.273
204.0
178.0
192.0
207.95
206.05
C10H11N3S

4
N. Shivakumara, P. Murali Krishna / Journal of Molecular Structure 1199 (2020) 126999
Fig. 3. 1H NMR of compound 1.
3.2. DNA binding studies
Generally, when a drug molecules bind to DNA with intercalation, it
lowers the intensity of absorption along with red shift but either
hyperchromism or hypochromism without red or blue shift
observed when it binds through non-intercalative or electrostatic
mode [54,55].
3.2.1. DNA binding studies by UVeVisible spectroscopy
The absorption titration is a widely used technique to investi-
gate the binding ability between thiadiazoles and CT-DNA [52,53].
Fig. 4. 13C NMR of compound 1.

N. Shivakumara, P. Murali Krishna / Journal of Molecular Structure 1199 (2020) 126999
5
Fig. 5. The electronic absorption spectra of compound 1 in the absence and presence of increasing amount of CT-DNA (with increase in the DNA concentration decreasing the
absorbance) Inset: Plot of [DNA]/(εa-εf) Vs [DNA].
In present studies (Fig. 5 and S19-21), absorption spectra were
recorded in 1.0 cm quartz cell, containing a fixed concentration
(1.67e6.07 mM) of the compound, except compound 5, with calf
D
G ¼ ꢁRT ln Kb
2
where R ¼ Universal gas constant (8.314 JKꢁ1molꢁ1) and T ¼ Tem-
thymus DNA (0e350
blank solution containing the same concentration of DNA was used
as a reference. A * intra-ligand transition observed between
mL of a 400 mM stock CT-DNA solution). A
perature in K (298 K).
Negative Free energy values indicate the thiadiazoles and DNA
interacted spontaneously during DNA adducts formation. Further
to investigate the mode of binding carried out fluorescence studies
(see Fig. 3).
p
/p
240 and 304 nm used to calculate the intrinsic binding constant
(Kb) for thiadiazoles. On addition of CT-DNA observed hyper-
chromism/hypochromism no blue/red shift indicates (except
compound 2 which shows 9 nm red shift) thiadiazoles are strongly
interacting with CT DNA non-intercalative mode i.e. via groove
binding mode [56] but in case of compound 2, maybe interacts with
mixed (intercalative and/or groove) binding mode and the absence
of an isosbestic point in spectra indicate the mixed binding mode.
The aromatic rings of the thiadiazole will facilitate the interac-
tion of non-covalent pep interaction results higher intrinsic bind-
ing constants values (Table 2) for the compounds [46]
3.525 ꢀ 104 Mꢁ1 (1), 3.567 ꢀ 104 M-1 (2), 8.505 ꢀ 104 M-1 (3) and
13.341 ꢀ 104 M-1 (4) (except 5)] (The detailed calculation of the
compound 1 provided in supplementary information, Table S1).
The order of the binding constants:
3.2.2. DNA binding studies by fluorescence method
The EB quenching assay results are shown in Fig. 6 and
Figs. S22eS24. On increase in concentration of the compounds
(1e4), decreased the fluorescence intensity of DNA bound EB at
598 nm indicates the quenching of EB molecules due to binding of
thiadiazole moiety to DNA [57].
If thiadiazole are the quenchers, the quenching of fluorescence
intensity of DNA bound EB can be described by the linear Stern-
Volmer equation [58].
F0
F
¼ 1 þ kqt0½Qꢃ ¼ 1 þ Ksv½Qꢃ
4 [13.341 ꢀ 104 Mꢁ1] > 3 [8.505 ꢀ 104 M-1] > 2 [3.567 ꢀ 104 M-
1] > 1 [3.525 ꢀ 104 Mꢁ1].
where F0 and F are fluorescence intensities in the absence and
presence of quencher, respectively; KSV is a linear Stern-Volmer
quenching constant; [Q] is the concentration of quencher and to
is the average fluorescence lifetime of the biomolecule in absence
of the quencher. A plot between F0/F and [Q] gave a slope, which is
Based on the binding constant (Kb), the Gibbs free energies (DG)
of thiadiazoles and DNA complexes were calculated using classical
vant Hoff's equation:

6
N. Shivakumara, P. Murali Krishna / Journal of Molecular Structure 1199 (2020) 126999
Table 2
Electronic absorption spectral data with addition of CT-DNA to compounds, 1e4.
Compound
l
max (nm)
Dl (nm)
H (%)b
Binding constant (Kb) Mꢁ1
Gibbs Free Energies (DG) (J/mol)
Free
Bound
1
2
3
4
241
295
302
302
241
304
302
302
0
9
0
0
4.68
1.42
2.07
ꢁ0.51
3.525 ꢀ 104
3.567 ꢀ 104
8.505 ꢀ 104
13.341 ꢀ 104
ꢁ1.991 ꢀ 108
ꢁ2.015 ꢀ 108
ꢁ4.804 ꢀ 108
ꢁ7.535 ꢀ 108
H (%)b ¼ [Afree e Abound/Afree]ꢀ100, ‘-’ Hypochromism and ‘þ’ hyperchromism.
Fig. 6. (a) Fluorescence titration of CT-DNA and EB (intercalator) complex with compound 1 (0e10
absence and presence of CT-DNA and Plot of log (F0 ꢁ F)/F as a function of log [Q].
mL). (b) Stern-Volmer plot for fluorescence quenching of compound 1 by EB in
equal to KSV and the higher values (1.156 ꢀ 104-3.661 ꢀ 104 M-1
)
fluorescence lifetime of the biomolecule (10ꢁ8s) indicates the
quenching process is static rather than dynamic [59].
The binding efficiency of thiadiazoles with CT-DNA by plotting a
graph between log (F0eF/F) and log[Q] which gave an intercept and
slope can calculate intrinsic binding constant (Kb) and number of
binding sites (n) using the following equation [60].
indicates the stronger quenching efficiency between thiadiazole
and DNA (Table 3). A linear SterneVolmer plot indicates quenching
process is occurring either static or dynamic mechanism [55].
Further, the bimolecular quenching rate constant (Kq) values
found to be greater than (in the order of 1012
M
ꢁ1sꢁ1) the

N. Shivakumara, P. Murali Krishna / Journal of Molecular Structure 1199 (2020) 126999
7
Table 3
Fluorimetric spectral data with addition of CT-DNA to compounds, 1e4.
Compound Stern-Volmer quenching constant KSV Quenching rate constant Kq
x
R2 (a) Binding constant Kb
(Mꢁ1) x 105
R2 (b) Number of Binding Gibbs Free Energies
sites (n) G) (J/mol)
x 104 (Mꢁ1
)
1012 (Mꢁ1 sꢁ1
)
(D
1
2
3
4
1.156 0.071
2.399 0.215
2.519 0.316
3.661 0.268
1.156 0.181
2.399 0.129
2.519 0.099
3.661 0.067
0.996 0.126
0.993 3.147
0.984 8.350
0.994 4.393
0.995 1.01
ꢁ0.711 ꢀ 108
ꢁ17.775 ꢀ 108
ꢁ47.164 ꢀ 108
ꢁ24.813 ꢀ 108
0.998 1.32
0.999 1.44
0.999 1.31
a
R is the correlation coefficient for the KSV values.
R is the correlation coefficient for the Kb values.
b
Fig. 7. Gel electrophoresis of compounds 1e4, Lane 1: Control DNA, Lane 2: Control DNA þ H2O2. Lane 3: 100
Lane 5: 100 M of 2 þ DNA þ buffer þ H2O2, Lane 7: 100
M of 2 þ DNA þ buffer, Lane 6: 100
4 þ DNA þ buffer, Lane 10: 100 M of 4 þ DNA þ buffer þ H2O2.
m
M of 1 þ DNA þ buffer, Lane 4: 100
m
M of 1 þ DNA þ buffer þ H2O2,
m
m
m
M of 3 þ DNA þ buffer, Lane 8: 100
m
M of 3 þ DNA þ buffer þ H2O2, Lane 9: 100
mM of
m
4. Conclusion
ꢂ
ꢃ
ðF0 ꢁ FÞ
log
¼ log Kb þ n log½Qꢃ
F
In this paper, reported the synthesis and characterization of new
five thiadiazole moieties. The DNA binding studies reveal that the
molecules are avid binders to CT-DNA. The DNA cleavage studies
indicate that the process of DNA cleavage may be closely related to
the oxidative type of cleavage.
The Kb and free energy values obtained at 298 K are in very close
agreement from the one obtained using absorption spectroscopy.
The negative Free energy values indicate the thiadiazole and DNA
interacted spontaneously during DNA adducts formation. The
values of n are very close to 1, which implies the existence of a
single binding site between thiadiazole and CT-DNA (The detailed
calculation of the compound 1 provided in supplementary infor-
mation, Table S2).
The prepared compounds might be important biologically, and
their further applications should be investigated.
Acknowledgments
Based on absorption and fluorescence methods CT-DNA and
thiadiazole moieties interacted via groove binding mode.
The authors thank the Department of Chemistry and Physics for
providing lab facilities. NS is thankful to the department of OBC
Government of Karnataka for a doctoral Scholarship award.
3.3. DNA cleavage studies
Appendix A. Supplementary data
The DNA Cleavage ability of the thiadiazole compounds was
studied using Gel electrophoresis technique using pUC18 DNA in
triseacetic acid-EDTA (TAE) buffer (pH 8.0). The samples were
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.126999.
incubated for 1 h at 37 ꢂC. After incubation, 2
mL of loading buffer
(0.25% bromophenol blue, 0.25% xylene cyanol and 60% glycerol)
was added to the reaction mixture and loaded onto a 1% agarose gel
Conflicts of interest
containing 1.0 mg/mL of ethidium bromide. The electrophoresis was
The authors declare that there is no conflict of interests
carried out at 100 V in Tris-acetic acid- EDTA (TAE) buffer till the
bromophenol blue reached 3/4th of the gel. Bands were visualized
by using UV transilluminator and photographed. For comparison
purposes, the cleavage reaction for compounds was carried out in
the absence and presence of H2O2 and is shown in Fig. 7. The ability
of DNA cleavage was determined based on the capacity of thia-
diazole moieties in the conversion of open circular (OC) or nicked
circular (NC) nucleic acid from its supercoiled (SC) structure.
From, Fig. 7 it is observed that the thiadiazoles do not show any
cleavage activity in the absence of H2O2 but in the presence of H2O2,
the activity enhanced moderately. The results indicate that the
thiadiazole molecules were able to convert supercoiled DNA into
open circular DNA and cleavage process may be closely related to
the oxidative type of cleavage.
regarding the publication of this paper.
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