Synthesis and characterization of Co(II) and Fe(II) peptide conjugates as hydrolytic cleaving agents and their preferential enantiomeric disposition for CT-DNA: Structural investigation of l-enantiomers by DFT and molecular docking studies

Article abstract of DOI:10.1039/c5ra15742k

Enantiomeric Boc l/d-valine derived peptide conjugate complexes of Co(ii) l/d-1 and Fe(ii) l/d-2 were synthesised and thoroughly characterized by spectroscopic techniques, elemental analysis, molar conductance and magnetic moment measurements. Peptide complexes l/d-1 and l/d-2 were titrated with CT-DNA by employing various biophysical methods viz., UV-vis, fluorescence and circular dichroic studies and viscosity measurements to envisage their binding mode and binding strength; the experimental results revealed strong preferential disposition of l-enantiomeric complexes via electrostatic binding mode with DNA helix. The structures of l-1 and l-2 were further optimized by applying DFT and molecular docking studies to validate their interaction by electrostatic bonding with DNA. Since l-enantiomers exhibited greater binding propensity than d-enantiomers, therefore, the cleavage efficiency of l-1 and l-2 with pBR322 DNA was evaluated by gel electrophoretic assay. l-Enantiomers of both Co(ii) and Fe(ii) complexes were found to be efficient cleaving agents and cleavage reactions were mediated by hydrolytic pathway, which were further validated by religation experiments using T4 ligase enzyme.

Full text of DOI:10.1039/c5ra15742k

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PAPER
Synthesis and characterization of Co(II) and Fe(II)
peptide conjugates as hydrolytic cleaving agents
and their preferential enantiomeric disposition for
CT-DNA: structural investigation of L-enantiomers
by DFT and molecular docking studies†
Cite this: RSC Adv., 2015, 5, 72121
*
Sabiha Parveen, Mohammad Usman, Sartaj Tabassum and Farukh Arjmand
Enantiomeric Boc L/D-valine derived peptide conjugate complexes of Co(II) L/D-1 and Fe(II) L/D-2 were
synthesised and thoroughly characterized by spectroscopic techniques, elemental analysis, molar
conductance and magnetic moment measurements. Peptide complexes L/D-1 and L/D-2 were titrated
with CT-DNA by employing various biophysical methods viz., UV-vis, fluorescence and circular dichroic
studies and viscosity measurements to envisage their binding mode and binding strength; the
experimental results revealed strong preferential disposition of L-enantiomeric complexes via
electrostatic binding mode with DNA helix. The structures of L-1 and L-2 were further optimized by
applying DFT and molecular docking studies to validate their interaction by electrostatic bonding with
DNA. Since L-enantiomers exhibited greater binding propensity than D-enantiomers, therefore, the
cleavage efficiency of L-1 and L-2 with pBR322 DNA was evaluated by gel electrophoretic assay.
L-Enantiomers of both Co(II) and Fe(II) complexes were found to be efficient cleaving agents and
cleavage reactions were mediated by hydrolytic pathway, which were further validated by religation
experiments using T4 ligase enzyme.
Received 6th August 2015
Accepted 19th August 2015
DOI: 10.1039/c5ra15742k
www.rsc.org/advances
(where Xaa is an a-amino acid) and metallopeptides [Ni(II)$
Xaa–Xaa–His] which have proven to be unique agents targeted
Introduction
to either the minor groove of B-form DNA or loop regions of
structured RNAs. Metallopeptide binding in the DNA minor
groove could be inuenced by the character of the amino acids.9
Tertiary cobalt peptide complexes viz., [Co(F-AVOH)2(H2O)2](PF6)3,
[Co(F-PLOMe)2(H2O)2](PF6)3, [Co(Z-APCONH2)2(H2O)2](PF6)3 were
found to demonstrate good photonuclease activity and have
also been used for selective recognition and cleavage of
plasmid DNA.10
The design of DNA/RNA specic agents capable of under-
going cleavage are of paramount importance due to their
potential use as drugs, regulators of gene expression and tools
for molecular biology.11–13 The cleavage of DNA by small metal
complexes is known to occur via two mechanisms, oxidative
cleavage and hydrolytic cleavage.14 The oxidative mechanism
requires the addition of co-reactant such as oxidising and
reducing agents, light or redox active metal species to initiate
cleavage, in addition to principle cleavage domain.15 Hydrolytic
cleavage mechanism cleaves the phosphodiester backbone of
DNA and nature has developed a number of hydrolytic metal-
loenzymes that efficiently catalyze the hydrolysis of P–O bonds
of the DNA phosphate backbone.16 Many divalent 3d and 4d
transition metal {Co(III), Ni(II), Cu(II), Zn(II), lanthanide}
complexes derived from a wide range of ligands of varied
Metal-based peptides are highly desirable as “molecular drug
motifs” for chemotherapeutic drug design in the pharmaceu-
tical industry1,2 owing to their unique structural and biological
features (i) the peptide scaffold with amide functionality
(
) offers coordination ability for metal ions
which could inuence its reactivity with biomacromolecules (ii)
incorporation of chiral center in the ligands make them suitable
templates to occupy specic target site and pre-orient them to
act as efficient drug entities (as chirality not only enforces
stereoselective specic drug-interaction but also promotes the
formation of active compounds with therapeutic benet due to
complementarity with biotargets viz., DNA, RNA and proteins
which are chiral in nature)3–6 (iii) better cellular uptake reduces
the high drug dosage necessary to compensate the reduced
bioavailability which oen causes adverse effects.7,8 Long and
co-workers have studied DNA binding and cleaving ability of
some basic peptide ligands [NH2–Xaa–Xaa–His–CONH2]
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India. E-mail:
farukh_arjmand@yahoo.co.in; Tel: +91 5712703893
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra15742k
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skeletal structures and geometrical environments have been moment and mass spectrometry. The complexes L/D-1 and L/D-2
utilized as catalysts for phosphodiester hydrolysis and for DNA were air stable and showed good solubility in methanol and
cleavage.17 In oxidative cleavage, reactive oxygen species ROS, DMSO. Molar conductance values of complexes in DMSO (1 ꢁ
reactive singlet oxygen (1O2), superoxide (O2ꢀ) or hydroxyl 10ꢀ3) at 25 ꢂC suggested their non-electrolytic nature (16–20 Uꢀ1
radical (OH)c species are generated and they are known to cm2 molꢀ1). The complexes L/D-1 and L/D-2 exhibited
damage the ribose sugar/nucleic bases of the DNA and this hexacoordinate geometry which was ascertained by various
hampers their use in vivo. In contrast, hydrolytic cleavage spectroscopic studies viz. UV-vis, EPR, magnetic moment
does not suffer from such drawbacks because the DNA measurements. Furthermore, L-1 and L-2 were structurally vali-
products generated by this mechanism can be enzymatically dated by DFT methods and molecular docking studies.
religated.18 Fe(EDTA)2ꢀ and other related complexes are
The IR spectra of both enantiomeric ligands L0 and L00
known to cleave DNA oxidatively in presence of H2O2 by exhibited characteristic –OH bands of R-2-amino-2-
generating hydroxyl radicals.19 However, there is a paucity of phenylethanol and secondary amide bands at 3437–3439 cmꢀ1
literature on the hydrolytic cleavage of DNA by Co(II) and Fe(II) and 1667 cmꢀ1, respectively.26,27 On complexation, amide bands
complexes. Therefore, it was thought imperative to develop shied to a lower wave number (ca. 41 cmꢀ1) which were
cobalt(II) and iron(II) complexes as hydrolase mimics for the attributed to the coordination of (N–H) of peptide to the metal
degradation of DNA.
centre28 and the coordination of ligand to the metal ion through
Among 1st row transition metal complexes, cobalt complexes hydroxyl oxygen was also ascertained by the disappearance of
are noteworthy as promising therapeutic agents.20,21 Many –OH band. Other bands observed at 758, 1043, 1160–1180,
bioactive small molecules have exhibited diverse biological 2926–2932 cmꢀ1 were assigned to –CH bending of aromatic
properties viz., antibacterial, antiprotozoal, anticancer agents ring, C–O group, –CH2, and (C–H) stretching, respectively.29 The
and furthermore, it was observed that complexation to cobalt coordination of ligand and complex formation was also
ions could improve their therapeutic potency.22 Both Co(III) and revealed by the presence of medium intensity (Co/Fe–N),
Co(II) have been shown to demonstrate therapeutic activity (Co/Fe–O) bands around ꢃ430 and ꢃ550 cmꢀ1, respectively in
while d6 Co(III) complexes were used as hypoxia-activated anti- far IR region.
1
cancer prodrugs that are capable of undergoing bioreduction
The H and 13C NMR spectra of the diamagnetic complexes
and in the reducing environment of cancer tissues could inhibit of Fe(II) L/D-2 were obtained in DMSO-d6. The 1H NMR spectra of
the cancer cells, and could also yield d7 high spin Co(II) the ligand and complexes L/D-2 revealed signals at 0.85, 1.94 and
complexes on reduction.23 Iron complexes are mainly designed 3.47 ppm attributed to –CH3, –CH2 and –CH, respectively
to model structural and functional properties of antitumor revealing the presence of valine moiety. The OH signal in the
glycopeptides antibiotics, bleomycin.24,25
ligand at 4.14 ppm was absent in the spectra of the complexes
Herein, we describe L/D-valine derived peptide conjugate L/D-2 ascertaining the deprotonation of the OH group and
complexes of Co(II) L-1, D-1 and Fe(II) L-2, D-2, the interaction coordination through the oxygen atom. The NH peptide signa-
studies of enantiomeric analogues of Co(II) and Fe(II) peptide ture which appeared at 8.2 ppm in the ligand was absent in the
complexes were carried out with CT-DNA by employing various spectra of the complexes implicating coordination of nitrogen
biophysical and spectroscopic methods. Since L-enantiomers L-1 atom to the metal via deprotonation of NH peptide. The char-
and L-2 were found to exhibit greater binding propensity than acteristic aromatic signatures were found at 7.19–7.39 ppm and
D-enantiomers D-1 and D-2, therefore, cleavage efficiency of signal at 1.38 ppm was attributed to the t-Boc group.30
L-enantiomers with pBR322 DNA was evaluated by gel electro-
The 13C NMR spectra of Fe(II) complexes were characterised
phoretic assay. The complexes cleaved DNA efficiently by by various characteristic signatures due to O–C]O, N–C]O
hydrolytic pathway, which was further supported by T4 religa- and aromatic signals at 156, 173 and 126–140 ppm, respectively.
tion assay. The metallopeptide complexes L-1 and L-2 were
The EPR spectra of Co(II) complexes L/D-1 recorded in DMSO
envisaged by applying density functional theory (DFT) calcula- at liquid nitrogen temperature (LNT) showed isotropic signals
4
tion which determined the quantum mechanical (QM) reactivity due to presence of Co(II) as d7 system and has F ground state
descriptors viz., highest occupied molecular orbital (HOMO) term (Fig. S1†). The ‘giso’ values obtained for L-1 and D-1 were
and lowest unoccupied molecular orbitals (LUMO) and further found to be 2.081 and 2.036, respectively showing deviation
investigated their effect in electrostatic mode of binding with from spin only value (g ¼ 2.0023). In an octahedral eld, it splits
DNA. Molecular docking experiments were also carried out to into three states which are connected by spin–orbit coupling,
get clear insight of binding affinity and involvement of weak therefore, the spin relaxation time was short.31 The magnetic
interactions between peptide complexes and minor groove of moment values, meff for Co(II) complexes L-1 and D-1 were found
DNA helix.
to be 4.98 and 4.86 B.M., respectively which corresponded to
presence of three unpaired electrons suggesting octahedral
geometry whereas Fe(II) as d6 complexes showed diamagne-
tism32 and ground state term was found to be 1I. With the help
of above results, the spin multiplicity of Co(II) and Fe(II)
Results and discussion
Synthesis and spectroscopic characterization
Chiral Boc L/D-valine derived peptide complexes L/D-1 and L/D-2 complexes was found to be 4 and 1, respectively.
were synthesized and thoroughly characterized (Scheme 1) by The ESI-MS spectra of complexes recorded in DMSOs
elemental analysis, IR, NMR spectroscopy, EPR, magnetic displayed molecular cation peaks at m/z 869 and 778
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Scheme 1 Synthetic scheme of complexes L/D-1 and L/D-2.
corresponding to Co(II) and Fe(II) complexes, respectively.
ESI-MS spectra of complexes exhibited some prominent peaks
at m/z 782, 446 and 720 corresponding to [C36H58CoN4O11 + H]+,
[C18H28CoN2O4 + 3H2O–H]+ and [C36H48FeN4O8–4H]+, respec-
tively (Fig. S2†).
Thermogravimetric analysis was performed to examine the
thermal decomposition behaviour of complexes unꢂder nitrogen
atmosphere in the temperature range of 20–1000 C. The TGA
prole of complexes showed 3 and 4 decomposition steps for
Co(II) and Fe(II) complexes, respectively (Fig. S3†). For both Co(II)
X-ray diffraction analysis
To get further evidence about the structure of metal complexes,
XRPD studies were carried out. The XRPD patterns indicated
amorphous nature for the complexes L/D-1 and L/D-2. The dif-
fractograms obtained for metal complexes L-1 and L-2 are given
in ESI (Fig. S4 and S5†).
DNA binding experiments
Electronic absorption studies. In the electronic spectra, the
enantiomers L-1 and D-1, the rst step revealed the degradation complexes L/D-1 and L/D-2 exhibited intense absorption bands
of six water and one methanol molecules in the temperature with maxima at ꢃ270–290 nm region which could be attributed
range 134–148 ꢂC accompanied by a net weight loss of 2.302 and to intra-ligand p / p* transitions.33 On titrating the enantio-
16.11% (calculated: 2.245 and 15.71%), respectively. However, meric analogues of complexes 1 and 2 with CT-DNA, ‘hyper-
for Fe(II) complexes L-2 and D-2, there was loss of three water chromism’ was observed with no apparent shi in the position
molecules in the range 90–110 ꢂC with a weight loss of 1.10 and of the absorption bands at their respective intra-ligand (IL)
6.9% (calculated: 1.14 and 7.3%). In the second step of bands (Fig. 1). The ‘hyperchromic’ effect is associated with
decomposition, further weight loss of 11.01 and 77.10% electrostatic binding mode typical of cationic complexes
(calculated: 10.76 and 75.30%) was observed for Co(II) involving damage of secondary structure of DNA double helix.34
complexes which was attributed to the pyrolysis of two ligand The preliminary ndings suggested that metal complexes pref-
moieties in the temperature range 255–300 ꢂC and for Fe(II) erentially bind to the polyanionic phosphate backbone of DNA
complexes weight loss of 8.06 and 50.32% (calculated: 8.23 and via electrostatic binding mode as previously proposed by Sletten
51.41%) was observed corresponding to the degradation of et al.35a whereas ‘hypochromic’ effect has been interpreted for
three water molecules and one ligand in the temperature range intercalative mode of binding indicative of stabilization of DNA
245–280 ꢂC. In the third step, total weight loss for Co(II) double helical structure.35b The binding strength of the
complexes occurred leading to the formation of metal oxides complexes was quantied by intrinsic binding constant, Kb value
and for Fe(II) complexes weight loss of 13.88 and 86.67% which was determined by using Wolfe–Shimer equation.
(calculated: 13.71 and 85.61%) was observed attributing to
The Kb values were found to be 6.11 ꢁ 104, 2.76 ꢁ 104, 2.33 ꢁ
the decomposition of both ligand molecules in the range 104 and 1.77 ꢁ 104 and followed the trend L-1 > L-2 > D-1 > D-2
545–560 ꢂC. Finally, at 780–800 ꢂC total weight loss occurred indicating that L-enantiomers L-1 and L-2 possess higher
leading to the formation of metal oxides.
propensity for DNA binding in comparison to D-enantiomers
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Fig. 1 (i) Absorption spectral traces of L/D-1 and L/D-2 complexes in Tris–HCl buffer upon addition of CT-DNA at 25 ꢂC. Inset: plots of [DNA]/3b vs.
[DNA] for the titration of CT-DNA with complexes, experimental data points; full lines, linear fitting of the data. [Complex] 6.67 ꢁ 10ꢀ6 M, [DNA]
0–33.3 ꢁ 10ꢀ6 M.
because the L-form interacts with right handed B-DNA due to 1.18 ꢁ 104, following the order L-1 > L-2 > D-1 > D-2 (Fig. 2). The
compatible molecular symmetry on the basis of two-pole binding constant K values gave a similar trend of the DNA
complementary principle.36a Since the complexes exhibit elec- binding propensity as observed in case of absorption spectral
trostatic interactions, amenable hydrogen bonding at the studies.
molecular sites could not be ruled out which is facilitated more
Ethidium bromide displacement assay. Emission quenching
in L-enantiomeric complex. The binding affinity of complex L-1 experiment has also been carried out with EB which is most
was found to be greater than complex L-2, because complex L-1 sensitive uorescence probe that can intercalate between adja-
exhibited higher H-bonding (8H-bonds) with nucleotides as cent base pairs of double stranded DNA.38 The occurrence of the
compared to L-2 (3H-bonds),36b the above rationale has also phenomenon is due to the competition of the complexes with
been supported by DFT and molecular docking studies in terms EB for the same binding sites on DNA resulting in knock out of
of H-bonding, electrostatic and van der Waals energy.
EB from DNA. The titration of DNA–EB system with complexes
Fluorescence studies. In the absence of CT-DNA, the L/D-1 and L/D-2 displayed a decrease in emission intensity of the
complexes L/D-1 and L/D-2 did not exhibit any luminescence in DNA–EB system as depicted in Fig. S6.† It can be inferred from
Tris–HCl buffer at ambient temperature with a maximum the decrease in emission intensity that there could be partial
wavelength at ꢃ380 nm when excited at 260 nm. Upon intercalation of the aromatic moiety of the peptide scaffold into
progressive addition of CT-DNA to the complexes, the the DNA helix, as complete intercalation results in the
uorescence-emission intensity was enhanced indicating that enhancement of the emission intensity. The observed results
the complexes interacted strongly with DNA. The observed implicate that the complexes cause the contraction of DNA helix
higher emission intensity was attributed to decreased vibra- via electrostatic binding mode.39,40 The quenching extents of
tional modes of relaxation due to restricted complex mobility at complexes L/D-1 and L/D-2 were determined qualitatively by
the binding site which could be due to inaccessibility of the employing Stern–Volmer equation and were found to be 0.54,
solvent DMSO molecules to reach the hydrophobic environ- 0.2, 0.31, 0.15, respectively.
ment inside the DNA helix.37 The binding constant, K values of
Circular dichroic studies. Circular dichroism (CD) spectra is
the complexes L/D-1 and L/D-2 calculated from the plots of r/cf a powerful technique to diagnose changes in DNA morphology
versus r, were found to be 5.87 ꢁ 104, 3.23 ꢁ 104, 2.14 ꢁ 104 and during drug–DNA interactions and to study various binding
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Fig. 2 (i) Emission spectra of L/D-1 and L/D-2 complexes in Tris–HCl buffer DNA in presence of DNA at 25 ꢂC. [DNA] 0–33.3 ꢁ 10ꢀ6 M. Arrows
show the intensity changes upon increasing concentration of the complexes.
modes of DNA binding viz., intercalative (negative CD), groove (Fig. S7†). All complexes exhibited a decrease in relative
binding (positive CD), and outside stacking (bisignate CD).41,42 viscosity of the DNA solution suggesting a non covalent inter-
The observed CD spectrum of calf thymus DNA exhibited a action possibly electrostatic binding mode, which produces
positive band at 275 nm (UV, lmax ¼ 260 nm) caused by base bends or kinks in the DNA chain, diminishing its effective
stacking between compounds and DNA and a negative band at
245 nm caused by helicity, which is characteristic of DNA in
right-handed B form. Intercalation enhances the intensities of
both positive and negative bands whereas groove binding and
electrostatic interaction of complexes causes less or no pertur-
bation on the base stacking and helicity bands.43 On incubation
of complexes L/D-1 and L/D-2 the CD spectra of CT-DNA displayed
changes in positive and negative (ellipticity and helicity bands)
(Fig. 3). The complexes L-1 and L-2 exhibited increase in inten-
sity of both positive and negative bands due to enantioprefer-
ential selectivity of L-form. On the other hand, D-enantiomeric
form of complexes D-1 and D-2 exhibited decrease in intensity in
both positive and negative bands as compared to unbound
CT-DNA. From these CD assays, we can virtually differentiate
between enantiopreferential binding of DNA to L- and D-form of
complexes.
Viscosity measurements. To further investigate the binding
modes of DNA, viscosity measurements on complexes L/D-1 and
L/D-2 incubated with CT-DNA were carried out and the results
are presented as (h/ho) vs. 1/R([complex]/[DNA]), where h is the
Fig. 3 CD spectra of (i) (a) CT-DNA in presence of L-1, (b) CT-DNA
alone and (c) CT-DNA in presence of D-1, (ii) (a) CT-DNA in the
presence of L-2, (b) CD spectrum of CT-DNA alone and (c) CT-DNA in
the presence of D-2 in Tris–HCl buffer at 25 ꢂC, [complex]/[DNA] ¼ 1 ꢁ
viscosity of DNA in presence of complex and ho is the viscosity of
DNA alone. The plots of relative viscosities vs. 1/R are shown in 10ꢀ5 M/1 ꢁ 10ꢀ5 M.
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length as well as the viscosity. However, a greater decrease was predict the geometry and change in free energy upon non-
observed in case of L-1 which validates its stronger binding covalent binding of a ligand to a macromolecule viz. DNA.
propensity of Co(II) complex with CT-DNA.44
The docking studies of L-1 and L-2 were performed and
Density functional theory. To obtain an insight into the combined with DFT calculations to fully explore the exibility of
geometrical and electronic structures, the complexes L-1 and L-2 the ligand and DNA to obtain more accurate binding energies,
were optimized in gas phase at the DFT/B3LYP level of theory.45 potential binding sites, mode of interaction and geometries.
The optimized structures of CoII (t25g eg2) complex L-1 and FeII The most favourable energy-minimized docked pose, Fig. 5,
(t62g e0g) complex L-2 at ground state are depicted in Fig. S8.† The revealed that complex L-1 and L-2 tted snugly into the curved
geometrical parameters viz. calculated bond lengths and bond contour of the DNA target in the minor groove.
angles are given in Table S1.† The energies of the Kohn–Sham's
The binding energy of complex L-1 was found to be larger
frontier orbitals in particular, the highest occupied molecular than complex L-2, because complex L-1 exhibited higher
orbital (HOMO) and lowest unoccupied molecular orbitals H-bonding (8H-bonds) with nucleotides as compared to L-2
(LUMO) were calculated.46 Previous literature reports revealed (3H-bonds). The electrostatic and van der Waals energy for
that higher HOMO energy of DNA and lower LUMO energy of complex L-1 were also found higher than complex L-2, shown in
the complex molecule results in much stronger interaction Table 1.
between these two molecules, because “electron-cloud” trans-
DNA cleavage activity. Since L-enantiomers possess higher
fers more easily from the HOMO of the DNA molecule to the propensity for DNA binding, therefore DNA cleavage activity was
LUMO of the complex and consequently, a stronger interaction carried out with complexes L-1 and L-2. The DNA cleavage
between DNA and the complex takes place. The results of N. studies have been investigated by gel electrophoresis using
Kurita and K. Kobayashi showed that the HOMO and HOMOꢀ1 supercoiled pBR322 DNA in 5 mM Tris–HCl/50 mM NaCl buffer
of DNA was mostly populated on p orbital of base pairs and solution at pH 7.2. DNA cleavage is controlled by relaxation of
HOMOꢀn (n ¼ 2–3) on phosphate backbone.47 From Fig. 4, it supercoiled circular, SC form (Form I) of pBR322 DNA into the
was observed that in the complexes L-1 and L-2 LUMO was nicked, NC (Form II) and linear form, LC (Form III). When a
localized in the vicinity of metal atoms and HOMO was on the circular plasmid DNA is subjected to gel electrophoresis, the
ligand moieties. Since the metal centres are devoid of p charge fastest migration is observed for supercoiled form (Form I). If
character, therefore, the possibility of intercalation between one strand of DNA is cleaved, the supercoil will relax to produce
complexes L-1 and L-2 and the DNA base pairs was ruled out. a slower-moving open circular form (Form II) and if both
Furthermore, Fukai function was calculated which revealed that strands are cleaved, a linear form (Form III) will be generated
the nucleophilic susceptible area of FMO was localized in the which migrates in between (Form I) and (Form II). Conse-
vicinity of metal. It is therefore, concluded that the dominant quently, binding of drug entities/small molecules to plasmid
force for binding was derived from electrostatic mode of inter- DNA results in a decrease in mobility of Form I and an increase
action between the electrophilic metal centre and nucleophilic in mobility of the Form II and Form III.49
DNA (Fig. S9†).48
Molecular docking. Computational docking methods have
been employed to search large conformational spaces and
Fig. 4 Frontier MOs contour plots of complexes L-1 and L-2 using the Fig. 5 Molecular docked model of complexes with DNA (a) complex
DFT method.
L-1 (b) complex L-2.
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Table 1 Electrostatic and van der Waals energies for complexes L-1
and L-2
methyl green were used to probe the potential interacting site of
complex L-1 and L-2 with supercoiled plasmid pBR322 DNA. The
patterns presented in (Fig. 6(b) and 7(d)) demonstrated that
DNA cleavage activity was inhibited in presence of DAPI in both
the complexes. However, no apparent inhibition of DNA
damage was observed with methyl green in presence of
complexes L-1 and L-2 suggesting the minor groove binding for
both the complexes, which was further validated by molecular
docking studies.
Binding energy van der Waal
Electrostatic
(kcal molꢀ1 energy (kcal molꢀ1) energy (kcal molꢀ1
)
)
Complex L-1 ꢀ892.89982
Complex L-2 ꢀ781.33356
ꢀ224.65759
ꢀ217.47395
ꢀ4315.39317
ꢀ4115.69020
DNA cleavage in presence of ROS. Reactive oxygen species
generated during the interaction between metal complexes and
dioxygen or redox reagents are the major cause of DNA
damage.51 To probe the potential mechanism of DNA cleavage
mediated by complexes L-1 and L-2, reactions were carried out in
presence of hydroxyl radical scavengers (DMSO, EtOH), singlet
oxygen quencher (NaN3) and superoxide oxygen scavenger
(SOD), under identical conditions. For both the complexes,
DMSO (Lane 2) and EtOH (Lane 3) inhibited the DNA cleavage
suggestive of involvement of diffusible hydroxyl radical as one
of the reactive species involved in the cleavage process (Fig. 6(b)
and 7(c)). These OHc free radicals participate in the oxidation of
the deoxyribose moiety, followed by hydrolytic cleavage of the
sugar phosphate backbone. On the other hand, addition of SOD
and NaN3 (Lane 4 and 5) showed no inhibitory effect on the DNA
cleavage.
The mechanism of DNA cleavage suggested for complexes L-1
and L-2 involved the participation of hydroxyl radicals in the
oxidation of the deoxyribose moiety followed by hydrolytic
cleavage of the sugar phosphate backbone (Fig. 6(d)). The
complexes L-1 and L-2 interacting with DNA base pairs could be
directly coordinated by metal cation acting as Lewis acid with
the negatively charged oxygen in the phosphodiester backbone
of DNA. These Lewis acids have the ability to activate the
phosphate group for nucleophilic attack, activate water or
Concentration dependent DNA cleavage. In a concentration
dependent experiment, cleavage of complexes L-1 and L-2 were
carried out with pBR322 DNA which exhibited conversion of
supercoiled form (Form I) into nicked form (Form II). However,
on increasing the concentration to 25 mM, a more intensied
nicked (Form II) was observed; on the other hand, complex L-2
displayed complete conversion of supercoiled form (Form I) to
nicked form (Form II) at lower concentration 15 mM. In both the
complexes linearized form (Form III) was not observed and
therefore single strand DNA scission was interpreted50 (Fig. 6
and 7(a)).
DNA cleavage in presence of activators. The nuclease effi-
ciency of metal complexes is known to depend on the activators
used for initiating the DNA cleavage. Therefore, cleavage activity
of complexes L-1 and L-2 was evaluated in presence and in the
absence of activators viz. H2O2, ascorbate(Asc), 3-mercaptopro-
pionic acid (MPA) and glutathione (GSH) (Fig. 6(c) and 7(b)).
The results revealed that cleavage activity of complexes was
signicantly enhanced in presence of these activators and fol-
lowed the order H2O2 [ MPA > Asc z GSH for complex L-1 and
H2O2 > GSH > Asc > MPA for complex L-2, respectively. The
complex L-1 in presence of H2O2 exhibited signicant DNA
cleavage activity, followed by complete degradation of DNA.
DNA cleavage in presence of recognition elements. Minor
groove binding agent, DAPI and major groove binding agent,
Fig. 6 The cleavage patterns of the gel electrophoresis showing cleavage of pBR322 supercoiled DNA (300 ng) by complex L-1 at 37 ꢂC after
30 min of incubation in buffer (5 mM Tris–HCl/50 mM NaCl, pH ¼ 7.2) (a) at different concentration; Lane 1: DNA control; Lane 2: 5 mM of L-1
+ DNA; Lane 3: 10 mM of L-1 + DNA; Lane 4: 15 mM of L-1 + DNA; Lane 5: 20 mM of L-1 + DNA; Lane 6: 25 mM of L-1 + DNA; Lane 7: 30 mM of L-1 +
DNA. (b) In presence of different ROS and groove binding agents. Lane 1: DNA control; Lane 2: L-1 + DMSO (0.4 mM) + DNA; Lane 3: L-1 + ethyl
alcohol (0.4 mM) + DNA; Lane 4: L-1 + SOD (15 U) + DNA; Lane 5: L-1 + NaN3 (0.4 mM) + DNA; Lane 6: L-1 + DNA + DAPI (8 mM); Lane 7: L-1 +
DNA + MG (2.5 mM of a 0.01 mg mlꢀ1 solution). (c) In presence of different activating agents Lane 1: DNA control; Lane 2: 25 mM of L-1 + H2O2
(0.4 mM) + DNA; Lane 3: 25 mM of L-1 + MPA (0.4 mM) + DNA; Lane 4: 25 mM of L-1 + Asc (0.4 mM) + DNA; Lane 5: 25 mM of L-1 + GSH (0.4 mM) +
DNA. (d) The proposed mechanism of hydrolytic cleavage of DNA.
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Fig. 7 The cleavage patterns of the gel electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300 ng) by complex L-2 at 37 ꢂC
after 30 min of incubation in buffer (5 mM Tris–HCl/50 mM NaCl, pH ¼ 7.2) (a) at different concentration; Lane 1: DNA control; Lane 2: 5 mM of L-2
+ DNA; Lane 3: 10 mM of L-2 + DNA; Lane 4: 15 mM of L-2 + DNA; Lane 5: 20 mM of L-2 + DNA; Lane 6: 25 mM of L-2 + DNA; Lane 7: 30 mM of L-2 +
DNA. (b) In presence of different activating agents Lane 1: DNA control; Lane 2: 15 mM of L-2 + Asc (0.4 mM) + DNA; Lane 3: 15 mM of L-2 + H2O2
(0.4 mM) + DNA; Lane 4: 15 mM of L-2 + GSH (0.4 mM) + DNA; Lane 5: 15 mM of L-2 + MPA (0.4 mM) + DNA. (c) In presence of different ROS and
groove binding agents. Lane 1: DNA control; Lane 2: L-2 + DMSO (0.4 mM) + DNA; Lane 3: L-2 + ethyl alcohol (0.4 mM) + DNA; Lane 4: L-2 +
NaN3 (0.4 mM) + DNA; Lane 5: L-2 + SOD (15 U) + DNA. (d) Lane 1: DNA control; Lane 2: L-2 + DNA + DAPI (8 mM); Lane 3: L-2 + DNA + MG (2.5
mM of a 0.01 mg mlꢀ1 solution).
yl)-1,1,3,3-tetramethyluroniumhexauorophosphate (HBTU),
tris(hydroxymethyl)aminomethane (Sigma) were used as
received. Disodium salt of calf thymus DNA (CT-DNA) was
ꢂ
purchased from Sigma Chemical Co. and was stored at 4 C.
6ꢁ loading dye (Fermental Life Science), agarose, ascorbic
Fig.
8 Gel electrophoresis pattern for the religation of pBR322
plasmid DNA linearized by complexes L-1 and L-2: Lane 1, DNA control;
Lane 2, pBR322 plasmid DNA cleaved by complex L-1; Lane 3, pBR322
plasmid DNA cleaved by complex L-2; Lane 4, ligation of linearized
pBR322 plasmid DNA by T4 DNA ligase, Lane 5, ligation of linearized
pBR322 plasmid DNA by T4 DNA ligase.
acid (Sigma) and supercoiled plasmid DNA pBR322 (Genei)
were utilized as received. All reagents were of the best
commercial grade and were used without further purication.
Methods and instrumentation
Elemental analyses (C, H and N) were performed on an
Elementar Vario EL III. Fourier-transform infrared (FTIR)
spectra were recorded on an Interspec2020 FTIR spectrometer
in KBr pellets from 400–4000 cmꢀ1. Electronic spectra were
recorded on UV-vis spectrophotometer Lambda 20 (Perkin
Elmer). Data were reported in lmax (nm). The EPR spectra of the
Co(II) complexes were acquired on a Varian E112 spectrometer
using X-band frequency (9.1 GHz) at liquid nitrogen tempera-
ture in the solid state. The 1H and 13C NMR spectra were
obtained on a BrukerDRX-400 spectrometer. ESI-MS spectra
were recorded on a Micromass Quattro II triple quadrupole
mass spectrometer. Circular dichroism (CD) spectra were
recorded on a Jasco J-815CD spectrometer operating at room
temperature at 25 ꢂC. The region of wavelength between 200
and 700 nm was scanned for each sample using a 1 cm path
quartz cell. Viscosity measurements were carried out from
observed ow time of CT-DNA containing solution (t > 100 s)
corrected for the ow time of buffer alone (t0), using Ostwald's
Viscometer at 25 ꢄ 0.01 ꢂC. Flow time was measured with a
digital stopwatch. Cleavage experiments were performed with
the help of Axygen electrophoresis supported by a Genei power
supply with a potential range of 50–500 V, visualized and
photographed by Vilber-INFINITY gel documentation system.
hydroxyl as nucleophile. A plausible explanation suggested for
the cleavage reaction could be that the nucleophilic species in
the complex–DNA system is the coordinated water linked to
cation. A proton is removed from one of the water molecules in
the complex resulting in the formation of aquo-hydroxo form.
The activated phosphorus atom is attacked by the active
nucleophilic species. Finally, one of the P–O ester bonds of the
phosphodiester in the DNA backbone is broken by the intra-
molecular charge delivery, resulting in the DNA cleavage.52
T4 DNA ligase assay. To ascertain the discernible hydrolytic
DNA cleavage pathway mediated by complexes L-1 and L-2, a
DNA religation experiment was performed in which supercoiled
pBR322 DNA was treated with a T4 DNA ligase enzyme and
subjected to gel electrophoresis.53 The complexes L-1 and L-2
supercoiled form was converted to nicked form which was
religated by T4 DNA ligase enzyme; nicked form (Form II) was
religated to a large extent in presence of T4 ligase enzyme in
comparison to control DNA alone in supercoiled form (Form I)
(Fig. 8), providing a direct evidence in favour of hydrolytic
mechanism.
Experimental section
Reagents and materials
Theoretical calculations
Boc-L-valine, Boc-D-valine, dicyclohexylcarbodiimide (DCC) The density functional theory (DFT) calculations were done with
(Fluka), R-2-amino-2-phenylethanol, 2-(1H-benzotriazol-1- the ORCA3.0.1 computational package.54,55 The geometry
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optimizations were carried out by hybrid B3LYP functional
using the Ahlrichs def2-TZVP basis set for metals and def2-SVP
basis set for C, H, O, N atoms.56–60 The optimized structures were
further recalculated using def2-TZVP basis set for all atoms. To
accelerate the calculations we utilized the resolution of identity
(RI) approximation with the decontracted auxiliary def2-TZV/J
Coulomb tting basis sets and the chain-of-spheres (RIJCOSX)
approximation to exact exchange as implemented in ORCA.61,62
The molecular docking studies have been performed by
using AutodockVina version 1.1.2.63,64 All rotatable bonds within
the ligand were allowed to rotate freely and receptor was
considered rigid. The crystal structure of the B-DNA dodecamer
d(CGCGAATTCGCG)2 (PDB ID: 1BNA) was retrieved from the
protein data bank (http://www.rcsb.org./pdb). Visualization of
minimum energy favourable docked poses has been performed
using Discovery studio 4.1 and PyMol.65,66
Syntheses
Synthesis of enantiomeric L- and D-form ligand [L0 and L00].
The ligands were synthesized according to the procedure
reported earlier in our laboratory.72 To a solution of 1.085 g of
Boc-L/D-valine (5 mmol) in 15 ml DCM was added 0.685 g
(5 mmol) of R-2-amino-2-phenylethanol at 0 ꢂC followed by the
addition of 1.031 g (5 mmol) of DCC and HBTU. The reaction
mixture was kept on stirring for 48 h and was monitored by TLC.
The residue was taken up in ethyl acetate and the dicyclohex-
ylurea (DCU) was ltered off. The ltrate was evaporated to half
the volume and kept in refrigerator overnight. White crystalline
product was isolated and washed with hexane and dried in
vacuo.
Yield: 58%. Mp 200 ꢂC. Anal. calc. for C18H27N2O5 (%): C,
64.26; H, 8.39; N, 8.33; found: (%) C, 64.37; H, 8.61; N, 8.98.
FT-IR (KBr, n, cmꢀ1): 3437 n(O–H); 2932 n(C–H); 1667 n(C]O);
1538 d(N–H) + n(C–N); 1162 n(CH2); 758 n(Ar). 1H NMR
(DMSO-d6, ppm): 8.2 (NH, 1H); 7.47–7.19 (m, Ar, 6H); 1.94 (m,
CH2, 1H); 1.38 (s, t-Boc, 9H); 0.81 (d, CH3, 6H). ESI-MS (m/z): 351
[C18H25N204 + H2O–3H]+.
In vitro DNA binding studies
DNA binding experiments include absorption spectral traces,
emission spectroscopy, viscosity and circular dichroism con-
formed to the standard methods and practices previously
adopted by our laboratory67–70 whereas DNA cleavage was per-
formed by the standard protocol as described previously.71
While measuring the absorption spectra, absorbance of inves-
tigated complexes were recorded aer successive addition of
CT-DNA. A reference cell contained DNA alone to nullify the
absorbance due to the DNA at the measured wavelength and
Tris–HCl buffer was subtracted through base line correction. All
the experiments involving interaction of the complexes with
CT-DNA were performed in double distilled buffer containing
tris(hydroxymethyl)aminomethane and adjusted to pH 7.3 with
hydrochloric acid. Solution of CT-DNA in buffer gave a ratio of
UV absorbance at 260 and 280 nm of ca. 1.9 : 1 indicating that
DNA was sufficiently free of protein. The DNA concentration per
nucleotide was determined by absorption spectroscopy with the
molar absorption coefficient 6600 Mꢀ1 cmꢀ1 at 260 nm. The
binding strength of the complexes was quantied by intrinsic
binding constant, Kb value which was determined by using
Wolfe–Shimer equation
Synthesis of complexes L-1 and D-1. To a methanolic solution
of L0/L00 (0.672 g,
2 mmol) was added a solution of
Co(NO3)2$6H2O (0.291 g, 1 mmol) in methanol. The reaction
mixture was stirred for 6 h under nitrogen atmosphere. On
completion of the reaction, the reaction mixture was reduced to
half of its volume. The pink colored product was obtained
which was washed with hexane and dried in vacuo.
ꢂ
(L-1). Yield, 62%. Mp 240 C; anal. calc. for C37H68CoN4O15
(%) C, 51.20; H, 7.90; N, 6.46. Found (%) C, 51.31; H, 7.98; N,
6.50. FT-IR data (KBr, n, cmꢀ1): 3328 n(H2O); 2928 n(C–H); 1626
n(C]O); 1538 d(N–H) + n(C–N); 1162 n(CH2); 758 n(Ar); 583 n(Co–
N), 435 n(Co–O). ESI-MS (m/z): 869 [C36H56CoN4O10 + 4H2O +
CH3OH]+. Molar conductance, LM (10ꢀ3 M, DMSO): 20.0 Uꢀ1
cm2 molꢀ1 (non-electrolyte).
ꢂ
(D-1). Yield, 65%. Mp 245 C; anal. calc. for C37H68CoN4O15
(%) C, 51.20; H, 7.90; N, 6.46. Found (%) C, 51.32; H, 7.96; N,
6.44. FT-IR data (KBr, n, cmꢀ1): 3333 n(H2O); 2930 n(C–H); 1627
n(C]O); 1539 d(N–H) + n(C–N); 1162 n(CH2); 758 n(Ar); 581 n(Co–
N), 432 n(Co–O). ESI-MS (m/z): 869 [C36H56CoN4O10 + 4H2O +
CH3OH]+. Molar conductance, LM (10ꢀ3 M, DMSO): 18 Uꢀ1 cm2
molꢀ1 (non-electrolyte).
[DNA]/3a ꢀ 3f ¼ [DNA]/3b ꢀ 3f + 1/Kb(3b ꢀ 3f)
(1)
Synthesis of complexes L-2 and D-2. These complexes were
synthesized according to procedure described above for L-1 and
D-1 with FeSO4$7H2O (0.278 g, 1 mmol).
where 3a, 3f and 3b correspond to Aobsd/[complex], the extinction
coefficient for free complex, and the extinction coefficient for
the complexes in the fully bound form, respectively. Plot of
[DNA]/(3a ꢀ 3f) vs. [DNA], where [DNA] is the concentration of
DNA in the base pairs, gives Kb as the ratio of slope to the
intercept. The cleavage experiments of supercoiled pBR322
DNA (300 ng) by 1 in Tris–HCl/NaCl (5 : 50 mM) buffer at pH
7.2 was carried out using agarose gel electrophoresis. The
samples were incubated for 45 min at 25 ꢂC and cleavage
process was monitored using agarose gel electrophoresis in a
concentration dependent manner. A loading buffer containing
25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol
was added and electrophoresis was carried out at 50 V for 1 h
in Tris–HCl buffer using 1% agarose gel containing
1.0 mg mlꢀ1 ethidium bromide.
(L-2). Yield, 65%. Mp 265 ꢂC; anal. calc. for C36H58FeN4O11
(%) C, 55.53; H, 7.51; N, 7.19; found (%) C, 55.68; H, 7.54; N,
7.29. FT-IR data (KBr, n, cmꢀ1): 3315 n(H2O); 2934 n(C–H); 1628
n(C]O); 1525 d(N–H) + n(C–N); 1165 n(CH2); 758 n(Ar); 489 n(Fe–
O); 345 n(Fe–N). 1H NMR (400 MHz, DMSO-d6, ppm): 7.22–7.30
(m, Ar, 6H), 3.5 (d, CH, 1H), 1.39 (s, t-Boc, 9H), 0.85 (d, CH3, 6H).
ESI-MS (m/z): 778 [C36H56FeN4O10 + H2O]+. Molar conductance,
LM (10ꢀ3 M, DMSO): 16.0 Uꢀ1 cm2 molꢀ1 (non-electrolyte).
(D-2). Yield, 68%. Mp 268 ꢂC; anal. calc. for C36H58FeN4O11
(%) C, 55.53; H, 7.51; N, 7.19. Found C, 55.63; H, 7.47; N, 7.23.
FT-IR data (KBr, n, cmꢀ1): 3313 n(H2O); 2934 n(C–H); 1627
n(C]O); 1526 d(N–H) + n(C–N); 1166 n(CH2); 759 n(Ar); 486 n(Fe–
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O); 343 n(Fe–N). 1H NMR (400 MHz, DMSO-d6, ppm): 7.21–7.30
(m, Ar, 6H), 3.5 (d, CH, 1H), 1.38 (s, t-Boc, 9H), 0.85 (d, CH3, 6H).
ESI-MS (m/z): 778 [C36H56FeN4O10 + H2O]+. Molar conductance,
LM (10ꢀ3 M, DMSO): 19 Uꢀ1 cm2 molꢀ1 (non-electrolyte).
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Conclusion
In this work, the synthesis and characterization of chiral valine
derived peptide conjugate complexes of Co(II) and Fe(II) L/D-1 and L/
D-2 were carried out to demonstrate the synthetic design of
hydrolytic cleavage agents that could have possible application as
chemotherapeutic drug entities. The interaction studies of enan-
tiomeric analogues of L/D-1 and L/D-2 with CT-DNA were carried out
by various spectroscopic methods viz., UV-vis, uorescence and
circular dichroic studies and viscosity measurements to envisage
their binding mode and binding strength; these experimental
results revealed strong binding of the complexes via electrostatic
binding mode with preferential disposition of L-enantiomers for
DNA. Since L-enantiomers exhibited greater binding propensity
than D-enantiomers, therefore, the cleavage efficiency of L-enan-
tiomers L-1 and L-2 with pBR322 plasmid DNA was evaluated by gel
electrophoresis assay. Both the Co(II) and Fe(II) complexes were
found to be efficient cleaving agents and cleavage reactions were
mediated by hydrolytic pathway involving direct hydrolysis of the
phosphodiester bonds by nucleophilic attack.
Molecular docking studies further revealed that both Co(II)
and Fe(II) complexes bind to DNA in the minor groove. Co(II)
complex was found to exhibit more stabilized interactions by
being in close proximity to the hydrophobic interior of the
minor groove, corroborating well with the results obtained from
various spectroscopic techniques.
17 S. S. Massoud, R. S. Perkins, F. R. Louka, W. Xu, A. Le Roux,
Q. Dutercq, R. C. Fischer, F. A. Mautner, M. Handa,
Y. Hiraoka, G. L. Kre, T. Bortolotto and H. Terenzi,
Dalton Trans., 2014, 10086–10103.
Abbreviations
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20 E. L. Chang, C. Simmers and D. A. Knight, Pharmaceuticals,
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CD
Circular dichroism
Calf thymus DNA
Electronic chemical potential
Electron volt
CT-DNA
ECP
eV
QM
Quantum mechanical
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Dalton Trans., 2007, 3983–3990.
22 M. C. Heffern, N. Yamamoto, R. J. Holbrook,
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23 T. W. Hambley, Dalton Trans., 2007, 4929–4937.
24 L. M. Mir, O. Tounekti and S. Orlowski, Gen. Pharmacol.,
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Acknowledgements
The authors are grateful to SAIF Panjab University, Chandigarh,
STIC Cochin University, Cochin and IIT Bombay, Mumbai, for
providing ESI-MS, NMR, elemental analysis facility, and EPR
measurements, respectively. The author (S. Parveen) sincerely
acknowledges nancial support from University Grants
Commission (UGC), New Delhi, for BSR Fellowship and the
Department of Chemistry, AMU through UGC assisted DRS–SAP
and DST PURSE Programme.
´
25 P. Mialane, A. Nivorojkine, G. Pratviel, L. Azema, M. Slany,
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