Development of a multifunctional biomimicking l-cysteine based oxovanadium(IV) complex: Synthesis, DFT calculations, bromo-peroxidation and nuclease actIVity

Article abstract of DOI:10.1039/c5ra19585c

An oxovanadium complex [VO(sal-l-cys)(phen)] (sal-l-cys = Schiff base derived from salicylaldehyde and l-cysteine; phen = 1,10-phenanthroline) has been synthesized and characterized by spectroscopic studies (IR, UV-vis, ESI-MS and EPR studies). The structure of the complex has been optimized by density functional theory (DFT) calculations. Time-dependent DFT (B3LYP) calculations were used to establish and understand the nature of the electronic transitions observed in UV-vis spectra of the ligand and the complex. The multifunctionality of this oxovanadium complex has been exhibited by the application of it as a catalyst in peroxidative bromination of phenol red to demonstrate vanadium dependent bromoperoxidase activity, whereas nuclease activity has been established by DNA cleavage. The interaction of DNA with this structurally characterized oxovanadium complex has been studied by various physicochemical tools like UV-vis, fluorescence, and viscosity measurement studies. The intrinsic binding constant of the complex with DNA has been determined by electronic absorption studies and calculated to be (3.51 ± 0.02) × 10⁴ M^-1. The spectroscopic studies and the viscosity measurements indicate that the complex binds CT DNA by an intercalative mode. The ability of the complex to induce DNA cleavage was studied by gel electrophoresis techniques. The complex has been found to promote cleavage of pUC19 plasmid DNA from the super coiled (SC) form I to nicked coiled (NC) relaxed form II.

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Development of multifunctional biomimicking L-
cysteine based oxovanadium(IV) complex:
synthesis, DFT calculations, bromo-peroxidation
and nuclease activity
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
Urmila saha and Kalyan K. Mukherjea*
DOI: 10.1039/x0xx00000x
An oxovanadium complex [VO(sal-L-cys)(phen)] (sal-L-cys = Schiff base derived from
salicylaldehyde and L-cysteine; phen = 1,10-phenanthroline) has been synthesized and
characterized by spectroscopic studies (IR, UV-vis, ESI-MS and EPR studies) . The structure of
the complex has been optimized by Density functional theory (DFT) calculations. Time-dependent
DFT (B3LYP) calculations were used to establish and understand the nature of the electronic
transitions observed in UV−vis spectra of the ligand and the complex. The multifunctionality of this
oxovanadium complex has been exhibited by the application of it as catalyst in peroxidative
bromination of phenol red to demonstrate vanadium dependent bromoperoxidase activity whereas
nuclease activity has been established by DNA cleavage. The interaction of DNA with this
structurally characterized oxovanadium complex has been studied by various physicochemical
tools like UV-vis, fluorescence, viscosity measurement studies. The intrinsic binding constant of
the complex with DNA has been determined determined by electronic absorption studies and
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calculated to be (3.51
0.02) × 104 M−1. The spectroscopic studies and the viscosity
measurements indicate that the complex binds CT DNA by intercalative mode. The ability of the
complex to induce DNA cleavage was studied by gel electrophoresis techniques. The complex has
been found to promote cleavage of pUC19 plasmid DNA from the super coiled (SC) form I to
nicked coiled (NC) relaxed form II.
discovery of vanadium-dependent haloperoxidase enzymes
(VHPO) in 1983 [7] from Ascophyllum nodosum marine
algae boosts the attention of researchers to explore the
Introduction
The continuous escalation of interest in the vanadium
chemistry with multidented ligands is lying on the
remarkable biological and pharmacological properties [1] of
vanadium. Vanadium an essential biometal, is involved in
various catalytic and inhibitory processes [2]. It is present in
many abiotic as well as biotic systems. It also plays several
roles such as cofactors in metalloenzymes [3] and
metalloproteins [4]. Again, many oxovanadium complexes
are known to possess potent insulin-mimetic effects [5] and
anticancer activity [6], which deserves increasing application
of vanadium complexes to biomedical sciences. The
coordination chemistry of vanadium for modeling various
vanadium containing biomimetic and biomimicking
molecules to get a better understanding on the mechanism
of interaction of vanadium with the biogenic molecules.
Vanadium haloperoxidase (VHPO) catalyses the oxidative
halogenation of organic compounds [8] (hydrocarbons and
alcohols, organic sulfide etc.) in presence of halide ions [9]
and hydrogen peroxide under physiological conditions.
Irrespective of their origin they all show a high degree of
amino acid homology with oxovanadium moiety in their
active centers. The peroxidative bromination is an important
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route for the biosynthesis of many natural brominated Synthesis of the ligand (H2sal-cys)
organic compounds [10]. Such importance of the VPHO About 1.21 g (0.01 mol) of L-Cysteine and 1.22 g (0.01 mol) of
reactions warrants active research in developing newer and salicylaldehyde were refluxed for 4 h in ethanol (50 mL). After
newer vanadium based complexes capable of mimicking cooling a pale yellow precipitate was formed, which was
VHPO activity. Moreover, development of metal based collected by filtration, washed with cold water and dried to give
synthetic nucleases laid special attention in the 2.24 g (92.18 %) of the schiff base. 1H NMR (d, ppm; 300 MHz,
chemotherapeutic research [11] since, they can be DMSO-d6): 3.13 (1H, s, -CHAHBSH), 3.17 (1H, s, -CHAHBSH),
potentially used for cancer therapy or as restriction 3.40 (1H,s,-CH(N)(CH2SH)), 5.57-5.64 (1H, m, Ar-CH=N-
nucleases. Again, many oxovanadium complexes have been (geometrical isomers)), 6.93(4H,s,CH2SH), 7.08–7.54 (4H,m)
known to be the initiators in the photo-cleavage of DNA [12]. Ar-H,
8.42–8.64(1H,
b,-COOH),
8.64(1H,b,-COOH),
The transition metal complexes of 1, 10-phenanthroline or 10.31(1H,s,Ar-OH). ESI-MS(+ve) in MeOH: m/z (relative
their modified variants have been widely employed [13] in intensity) 226.05 [m+ + 1], 248.03 [m+ +23]. UV-vis in Methanol
DNA studies due to their applicability in bioinorganic and [λmaxnm (ε M-1cm-1)]: 323(8700), 279(12400), 255(27100),
biomedicinal chemistry [14] as well as in the design of 218(36200). Elemental analysis: Calc. C 53.32 %, H 4.92 %, N
stereospecific DNA binding drugs [15]. Although, the 6.22 %; Found: C 53.40%, H 5.01 %, N 6.29 %.
synthesis of some oxovanadium complexes with various
amino acid Schiff bases has been reported earlier [16], the Synthesis of the complex [VIVO(sal-L-cys)(phen)]
detail investigation on the multifunctional activities i.e, the A methanolic solution (10 ml) of the schiff base (H2sal-cys)
VPHO mimicking as well as nuclease activity by such (0.01mol, 2.25g) was added to a 10 ml methanolic solution of
complexes are scanty [17]. In the present work, synthesis, vananadyl sulfate pentahydrate (0.01 mol, 2.53 g) with
characterization, DFT calculations, catalytic activity and continuous stirring, followed by dropwise addition of a
nuclease activity of an oxovanadium (IV) complex with the methanolic solution (5 ml) of 1,10-phenanthroline monohydrate
Schiff base derived from an amino acid (L-cysteine), and the (0.01 mol,1.98 g) and stirred for another 2 h, which on standing
1,10 phenanthroline of the formula [VIVO(sal-L-cys)(phen)] produced brown yellow solid (Scheme 1). The brown yellow
have been carried out with the aim of developing an amino solid was collected by filltration, washed with cold water and
acid based multifunctional biomimicking oxovanadium dried under vaccum. Yield: 77%. IR (KBr, cm–1): 3419.96 [
υ
(O-
(C=N)],
(V=O)]. ESI-MS(+ve) in MeOH: m/z (relative intensity)
complex.
H)], 2612.79 [υ(S-H)], 1601.93 [
968.37 [
υ as(COO)], 1645.27 [υ
υ
471.11 [m+ + 1], 493.08 [m+ + 23]. UV-vis in Methanol [λmaxnm
(ε M-1cm-1)]: 382(4760), 264(33000), 218 (49200). Elemental
analysis: Calc. C 56.17%, H 3.64%, N 8.93 %; Found: C
56.31%, H 3.61 %, N 8.89 %.
Experimental Section
Materials and Methods
Physical measurements
All reagents and chemicals were procured commercially and of
AR or GR grade (SD Fine Chemicals, India; and Aldrich) and
used without further purification. Solvents were purified by
standard procedures [18], wherever necessary. All solvents
used for chromatographic analysis were either of HPLC,
spectroscopic or GR grade and in all cases their purity was
confirmed by GC analysis before use. Calf thymus DNA (CT-
DNA) was purchased from the Sigma Chemical Company,
USA; supercoiled (SC) plasmid pUC19 DNA was obtained from
Bangalore Genei (Bangalore, India). The solutions of CT-DNA
in phosphate buffer saline (PBS) medium [0.15 M, pH 7.2] gave
a ratio of A260/A280, of ca. 1.8–1.85, indicating that the DNA was
sufficiently free from protein contamination. The DNA
concentration per nucleotide was determined by absorption
spectrophotometry using the molar absorption coefficient (ε =
6600 M−1 cm−1). Stock solutions were stored at 4 °C and used
within 4 days.
The IR spectra were taken as KBr discs at room temperature
on a Perkin Elmer RFX-I IR spectrophotometer. Elemental
analyses were carried out using a Perkin–Elmer 2400 series II
CHNS analyzer. UV-vis spectra (200–800 nm) were recorded
against appropriate reagent blank at room temperature with a
Schimadzu U-1200 spectrophotometer using 1cm quartz cell.
EPR spectra were obtained on a JEOL – JES FA200 ESR
spectrometer in DCM – toluene (1:1) glass at 77K. NMR
spectral measurements were carried out in DMSO-d6 solution
1
at ambient temperature. The H NMR spectra were taken by
Bruker 300 MHz NMR spectrometer and the mass spectral
analyses were done in methanol solvent from Waters mass
spectrometer (model: XEVO-G2QTOF#YCA351). The gel
electrophoresis study was carried out with UVP Bio Doc-It
Imaging System and nicking was analysed by UVP DOC-ItLS
software.
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Scheme 1. Synthesis of [VO(sal-L-cys)(phen)]
DFT calculations
been shown to provide a reasonable spectral feature for our
DFT study is an important tool which provides understanding complex of investigation.
on the geometry, electronic structure, and optical properties of For H atoms we used 6-31G basis set; for C, N, O and V atoms
the system in question. Ground state electronic structure basis set was employed 6-31G and for S atom we adopted 6-
calculations in methanol solution of both the ligand and the 31 + G(d,p) as basis set for the optimization of the ground
complex have been carried out using DFT [19] method state. The calculated electronic density plots for frontier
associated with the conductor-like polarizable continuum model molecular orbitals were prepared by using Gauss View 5.1
(CPCM) [20]. Becke’s hybrid function [21] with the Lee-Yang- software. All the calculations were performed with the Gaussian
Parr (LYP) correlation function [22] was used in the study. The 09W software package [23]. Gauss Sum 2.1 program [25] was
geometry of the ligand and complex was fully optimized without used to calculate the molecular orbital contributions from
any symmetry constraints. On the basis of the optimized groups or atoms.
ground state geometry, the absorption spectral properties in
methanol (CH3OH) media were calculated by time-dependent Bromination of alkenols using the complex as catalyst
density functional theory (TDDFT) [23] approach associated Halogenation was carried out by the catalytic bromination of
with the conductor-like polarizable continuum model (CPCM) phenol red to bromophenol blue using the oxovanadium
[20]. We computed the lowest 40 singlet – singlet transitions complex as a catalyst. The catalytic activity of the complex was
and results of the TD calculations were qualitatively very studied by absorption spectroscopy. Bromination reaction
similar. The TDDFT approach has been demonstrated to be activity tests were carried out at a constant temperature of
reliable for calculating spectral properties of many transition
metal complexes [24]. Due to the presence of electronic
correlation in the TDDFT (B3LYP) method it can yield more
accurate electronic excitation energies. Hence, TDDFT had
(30 0.5)
°C in presence of H2O2 and KBr in mild acidic
medium. An aliquot of 30% H2O2(final concentration 2.0 mM)
was added to a H2O-DMF solution (with a volume ratio of 4:1)
of catalyst followed by the addition of 4.0 mol/L of KBr.
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Solutions used for kinetic measurements were maintained at solution in a way so that, the ratio of [complex] / [EB-DNA] were
pH=5.8 by the addition of NaH2PO4- Na2HPO4 [26]. Reactions 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and the respective solutions were
were initiated by the addition of 0.1 mmol of phenol red. incubated for 2 h at 37◦C, thereafter, the fluorescence
Bromination of phenol red was monitored by the measurement intensities of the solutions were recorded by exciting the
of the increase in the absorbance at 592 nm for reaction at solutions at 500nm whereas slits for both the excitation and
specific time points. The spectral data show the gradual emission were 10 nm.
disappearance of the peak at 443 nm due to the loss of phenol Viscometric study
red and an increase in the absorbance of the peak at 592 nm The viscosity of sonicated DNA (average molecular weight of
due to the formation of the bromophenol blue product and ~200 base pairs obtained using a Labsonic 2000 sonicator) [28]
spectral changes were recorded at the intervals of 5 min.
was measured by a fabricated micro-viscometer maintained at
The rate of this reaction is described by the rate 28 ( 0.5) °C in a thermostatic water bath. Data were
x
y
z
equation: dc/dt = kc1 c2 c3 , from which the equation ‘‘log(dc/dt) presented as (η/ηo)1/3 vs the ratio of the concentration of metal
= logk + x logc1 + y logc2 + z logc3’’ was obtained, complex to that of the CT-DNA, where η and ηo are the
corresponding to ‘‘–log(dc/dt) = –x logc1 – b (b = logk + y logc2 viscosities of the CT-DNA solutions in the presence and
+ z logc3)’’, where k is the reaction rate constant; c1, c2, c3 are absence of the complex, respectively. The viscosity of DNA (η
the concentrations of the oxovanadium complex, KBr and = t − to) was calculated from the observed flow time of CT-DNA
phenol red,respectively; while x, y, z are the corresponding solutions (t) and buffer solution (to).
reaction orders. According to Lambert–Beer’s law, A =
which on differentiation we get dA/dt = d(dc/dt), where ‘A’ is DNA cleavage activity of the complex was monitored with the
the resultant absorbance; ‘ ’ is the molar absorption coefficient help of a Gel electrophoresis Model No. 2101, Genei,
(for bromophenol blue
= 14 500 M-1 cm-1 at 592 nm); ‘d’ is the Bangalore. The super coiled pUC19 DNA (0.5 ꢀg per reaction)
ԑdc, Gel electrophoresis study
ԑ
ԑ
ԑ
light path length of the sample cell (d = 1). The absorbance in Tris-HCl/NaCl buffer (pH 7.2) was treated with increasing
data were plotted against the reaction time, a straight line was amounts of metal complex over a range of 20–60 ꢀM alongwith
obtained and the reaction rate (dA/dt) was obtained from the H2O2 (4 ꢀM) [29]. After incubation for 2 h at 37 °C, it was mixed
slope of this line. The reaction rate constant (k) can be with a sample loading dye. The samples were run on a 0.9%
obtained from the plot of –log(dc/dt) versus –logc1. In the agarose in 1X TAE buffer for 3 h at 80 mV, then it was treated
experiment, the reaction orders of KBr and phenol red (y and z) with EB solution and the bands were visualized by UV light and
were taken as 1 according to the literature [27], c2 and c3 are photographed with UVP Bio Doc-It Imaging System. The
known as 0.4 and 10–4 mol L–1, respectively.
percentage of cleavage of super coiled (SC) pUC19 DNA
cleavage induced by the complex was determined by using
UVP BIODOC-ItLS software.
DNA binding studies and nuclease activity
UV–vis spectral study
Results and discussion
UV–vis spectra were recorded in a Schimadzu U-1200
spectrophotometer using Quartz cuvettes of 1cm path length.
The electronic absorption spectra were monitored in PBS
(0.15M, pH 7.2) buffer at room temperature 250 C by keeping
the concentration of the CT-DNA constant (10 ꢀM), while
varying amounts of complex [in 1 % methanol- PBS buffer (v/v)
solution] over a range of 1–12 ꢀM. While measuring the
absorption spectra, equal amounts of complex was added to
both the test and the reference solutions to eliminate any
absorbance of the complex itself.
IR and UV–vis spectrophotometric characterization of the
complex
The characteristic υ(V=O) vibration [9, 11] of oxovanadium (IV)
complex appears at 968.37 cm-1 as a sharp band. The
complex presents very strong bands at 1645.27 cm-1 and
1601.93 cm-1; these correspond to υ(C=N) and υas(COO)
respectively of the Schiff base ligand. The strong vibrations at
1438.25 cm−1 and at 736.03 cm−1 are due to the υ(C–N) vibration
of the phen ligand in the complex, which apprared at 1421 cm-1
and 731 cm-1 respectively in the uncoordinated phenanthroline.
The electronic absorption bands of the oxovanadium(IV)
complex in methanol solution at the 382nm, 218nm and 264
nm are assigned as intra ligand charge transfer transitions [30].
Mass spectrometric analysis
Fluorescence study
Competitive ethidium bromide (EB) binding experiment was
carried out to investigate the binding mode of the complex to
CT-DNA. The fluorescence was measured by a Perkin Elmer
LS55 spectrofluorimeter. The experiment was monitored by the
addition of 10.0 µM of CT-DNA in Tris–HCl/NaCl buffer to a 15
µM solution of ethidium bromide in the same buffer medium.
Aliquot of stock solution of the complex [1% MeOH–buffer (v/v)
solution] was added to the ethidium bromide bound CT-DNA
The molecular formulation and some structural information can
be obtained from ES-MS analysis and elemental analysis. The
molecular formula of the complex [VO(Sal-Cys)(Phen)]
obtained from elemental analysis is supported by the
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observation of peak at m/z (relative intensity): 471.11 [m+ + 1],
493.08 [m+ + 23].
O1-V-N3
O3-V-N3
O1-V-O2
79.136
163.102
152.050
N1-V-O2
N1-V-N2
O2-V-O3
78.772
161.950
103.007
Geometry optimization and electronic structure
determination by DFT calculation
Table 2 Change in bond lengths for the oxovanadium
compared to free the ligand (H2sal-cys) in the ground state
calculated at B3LYP Levels
The optimized geometry of the ligand (H2sal-cys) and its oxo
vanadium complex is shown in Fig.1a and Fig.1b respectively.
For both the ligand and the complex, only the ground state
geometries have been optimized. Main optimized geometrical
parameters of the oxovanadium complex are listed in Table 1.
The hexa-coordinated metal centre possesses a distorted
octahedral geometry in the complex. All calculated V-N
distances occur in the range 2.073-2.419 Å and V-O distances
are in the range 1.614-1.979 Å whereas, on complexation,
some C-N and C-O bond lengths are changed with respect to
that in free ligand and Table 2 describes the change in bond
lengths in the complex compared to the free ligand (H2sal-cys).
Bond Lengths (Å)
Ligand (H2sal-cys) Oxovanadium
complex
O1-C1
N1-C3
N1-C4
O2-C5
1.362
1.303
1.486
1.369
1.290
1.302
1.472
1.291
Table 1 Selected optimized geometrical parameters for the
oxovanadium complex in the ground state calculated at B3LYP
Levels.
Bond Lengths (Å)
V-N1
V-N2
V-N3
2.073
2.173
2.419
V-O1
V-O2
V-O3
1.958
1.979
1.614
Bond Angles (o)
N1-V-N3
N2-V-N3
N2-V-Ol
93.626
71.902
95.616
N2-V-O2
N2-V-O3
N1-V-O1
91.910
91.200
87.290
(a)
(b)
Fig. 1 Optimized geometry of the free ligand (H2Sal-Cys) and the Complex [VO(Sal-Cys)(Phen)] under DFT[B3LYP (6-31G)].
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In case of the free ligand (H2Sal-Cys) at the ground state, the The ligand shows four absorption bands at 323, 279, 255 and
electron density at HOMO and LUMO+2 orbitals mainly reside 218 nm in methanolic solution at room temperature. These four
on the salicylaldehyde moiety while a considerable contribution absorption bands can be assigned to the S0→S1, S0→S4,
comes from cysteine moiety along with the contribution of S0→ S7 and S0→S10 transitions (Fig. 3) from TDDFT
salicylaldehyde moiety in HOMO–2, HOMO–1, LUMO and calculations. The absorption energies along with their oscillator
LUMO+1 orbitals. The energy difference (Fig. 2) between strengths, the main configurations and their assignments
HOMO and LUMO is 4.328 eV of the ligand (H2Sal-Cys). In calculated using TDDFT method for the ground state geometry
case of the oxovanadium complex all the HOMO-3, HOMO -2 for H2L is discussed here and the related data are given in
and HOMO orbitals are mainly originating from salicylaldehyde- Table 3
.
cysteine π and π* orbital contribution while the LUMO, LUMO The complex shows three absorption bands at 382, 264 and
+1 and LUMO +3 orbitals arise from metal d orbital contribution 227 nm in methanolic solution at room temperature. The
along with the π orbital contribution of 1,10- phenanthroline. calculated absorption bands are located at 385 and 259 nm in
The energy difference between HOMO and LUMO is 3.068 eV the complex (Fig. 4), which are in good agreement with the
of the complex (Fig. 2).These compositions are useful in experimental results of 382 and 264nm (Table 4). These two
understanding the nature of transition as well as the absorption absorption bands can be assigned to the S0→S18 and S0→ S24
spectra of both the ligand and the complex (vide infra).
transitions, respectively (Table 4).
Fig.2 Frontier molecular orbital of complex as well as ligand optimized under [B3LYP (6-31G)].
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Table 3 Selected Parameters for the vertical excitation (UV-vis absorptions) of the free ligand (H2Sal-Cys); electronic excitation
energies (eV) and oscillator strengths (f), configurations of the low-lying excited states of the ligand; calculations of the S0→Sn
energy gaps are based on optimized ground-state geometries (UV-vis absorption) (CH3OH used as solvent).
Electronic
transition
Composition
Excitation
energy
Oscillator
strength
(f)
CI
λexp
(nm)
S0 → S1
S0 → S4
HOMO–2 → LUMO
HOMO–1 → LUMO
HOMO→ LUMO
3.7738 eV
(328nm)
0.0617
0.21195
0.50636
-0.42029
323
HOMO–3→ LUMO
HOMO–3→ LUMO + 1
HOMO–2→ LUMO + 1
HOMO–1→ LUMO
4.3852 eV
(282 nm)
0.0510
0.26802
0.10157
0.21231
0.25215
279
255
218
S0 → S7
S0 → S10
HOMO–3→ LUMO +1
HOMO–1→ LUMO + 1
HOMO–2→ LUMO
4.9183 eV
(252 nm)
0.1337
0.0851
0.27380
0.26486
-0.18159
5.7118 eV
(217 nm)
HOMO–1→ LUMO +2
HOMO→ LUMO +2
0.49863
-0.41682
Table 4 Simulated values of various factors of the optical transitions for the oxovanadium complex.
Electronic
transition
Composition
Excitation
energy
Oscillator
strength
(f)
CI
λexp
(nm)
S0 → S18
HOMO – 2 → LUMO
HOMO – 2 → LUMO+3
HOMO → LUMO + 3
3.2192 eV
(385 nm)
0.0891
0.23713
0.15239
-0.17791
382
264
S0 → S24
HOMO – 3→ LUMO + 1
HOMO – 2→ LUMO
4.2856 eV
(259 nm)
0.1257
0.26945
0.39122
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Fig. 3 Frontier molecular orbitals involved in the UV-vis absorption of the free ligand (H2Sal-Cys).
Fig. 4 Frontier molecular orbitals involved in the UV-vis absorption of the oxovanadium complex.
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EPR study
EPR spectroscopy is a powerful tool that provides information
about the nuclearity (Fig.5), and electronic structure of a
paramagnetic state. X-band EPR spectra of the complex
[VIVO(sal-L-cys)(phen)] was recorded in frozen (77K)
dichloromethane – toluene (1:1) glass. It gives rise to well
resolved 51V (I = 7/2) hyperfine eight lines and the spectral
parameters are listed in table 5. The spectrum has axial
symmetry with g║ < g┴. The caracteristic g║ < g┴ and a║ >>a┴
relationship corresponding to an axially compressed d1xy
configuration [31, 32] was observed. The EPR study confirms
the presence of mononuclear vanadium(IV) moiety in the
complex.
Fig.5 EPR spectrum of complex in solution (DCM / toluene) at
77K.
Table 5 EPR spectral data at 77K in solution DCM – toluene (1:1)
Compound
g║ (A║ / G)
g┴ (A┴ /G)
gav (Aav /G)
[VIVO(sal-cys)(phen)] 1.948 (71.88) 1.987 (57.25) 1.974 (61.24)
Therefore, all the above spectroscopic findings along with DFT calculations suggest that the molecular geometry of the complex is
[VIVO(sal-L-cys)(phen)] with O, N, O linkage from the schiff base, N,N coordination from o-phenanthroline and the oxo linkage.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Biomimicking catalysis by the compound
Many oxovanadium complexes are found to mimic the Mechanism of the catalytic activity
vanadium haloperoxidases catalyzes the bromination of The mechanism of catalytic activity is assumed to be similar as
organic substrates in the presence of H2O2 and bromide. The decribed in our previous work [11]. During the catalytic process,
catalytic haloperoxidase activity of vanadium complexes is the catalyst (I) was converted to a bound peroxide intermediate
often investigated by the kinetic study of the catalytic (III) in presence of peroxide in mild acidic medium. This
bromination of phenol red to bromophenol blue as a model intermediate in turn oxidizes the bromide (Br−) ion in the
system. The catalytic ability of the oxovanadium complex medium to bromonium ion (Br+) which exists in reaction
-
[VIVO(sal-L-cys)(phen)] for the oxidative bromination of phenol medium as Br3 , Br2 or HOBr [8]. Attack of a bromide ion at one
of the peroxo atoms (IV) and the uptake of a proton from a
surrounding water molecule leads to the generation of
hypobromous acid (HOBr) (V) followed by restoration of the
native state (I). The in situ generated bromonium ion reacts
with the organic substrate (phenol red) to form corresponding
brominated derivatives. The catalytic cycle has been depicted
in scheme-2.
red was monitored by UV−vis spectrophotometry (Fig. 6). The
gradual decrease in the absorbtion at 443 nm due to
disappearance of phenol red and the appearance of a new
peak at 590 nm due to formation of bromophenol indicate the
conversion of phenol red to bromophenol blue, and the reaction
was complete after
∼8 h under ambient conditions. The plots
for the kinetic studies of the above conversion are shown in
Fig. 7 and Fig. 8. The same reaction was monitored without
addition of the oxovanadium complex whereby no appreciable
change could be observed.
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Fig.8 -log(dc/dt) dependence of -logc (c is the concentration of
the oxovanadium complex; conditions used: c(phosphate
buffer) = 50 mmol L-1, pH = 5.8, c(KBr) = 0.4 mol L-1, c(phenol
red) = 10-4 mol L-1.
Fig.6 Oxidative bromination of phenol red catalyzed by the
oxovanadium complex (0.04 mmol). Spectral changes at 10
min intervals. Spectral data taken of aliquots in pH = 5.8
−
1
aqueous phosphate buffer, c(phosphate buffer) = 50 mmol L ,
−
−
−
1
1
c(KBr) = 0.4 mol L , c(phenol red) = 10 4 mol L .
Fig.7 The measurable absorbance dependence on time for the
oxovanadium complex.Conditions used: pH = 5.8, c(KBr) = 0.4
mol L-1, c(H2O2) = 2 mmol L-1, c(phenol red) = 10-4 mol L-1.
c(complex/mmol L-1) = a: 2×10-2; b: 4×10-2;c: 6×10-2; d: 8 ×10-2;
e: 1×10-1.
Scheme 2 Schematic representation of the proposed
mechanism of the catalytic bromination of the complex
[VIVO(sal-cys)(phen)] as a catalyst in presence of KBr and
H2O2 in acidic medium.
Nuclease activity of the oxovanadium complex
Spectrophotometric studies on DNA binding by the
complex
Electronic absorption spectroscopy is usually used to
determine the binding of metal complexes with DNA. A complex
bound to DNA through intercalation is characterized by the
change in absorbance (hypochromism) and red shift in
wavelength, due to the stacking interaction between the
aromatic chromophore and the DNA base pair [33, 34, 35]. To
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calculate the complex-DNA binding constant (Kb),the data are small molecule to the double-helical DNA. Ethidium
treated according to the following equations [33]:
bromide(EB) emits intense fluorescence in the presence of
DNA due to its strong intercalation between the adjacent DNA
base pairs. This enhanced fluorescence can be quenched by
the addition of a third molecule which can bind DNA by
intercalative mode by displacing EB [29, 36, 37]. The emission
spectra of EB bound to DNA in the absence and presence of
the complex are presented in Fig. 11. The addition of the
complex to DNA pretreated with EB causes a gradual
quenching in emission intensity, indicating that the complex
competes with EB in binding DNA, which leads to a quenching
in the fluorescence intensity of EB-DNA complex system. In
fact, as expected for a displacement effect, the increase in the
concentration of complex gradually quenches the fluorescence
intensity of EB-DNA complex system. This significant decrease
in fluorescence intensity lends strong support in favor of
intercalation of the complex into the DNA double helix by
displacing EB. Fluorescence quenching study in presence of
the complex was analyzed further by Stern-Volmer equation:
I0/I = 1 + KSV [Q] [29]; where I0 and I are the fluorescence
intensities in absence and presence of quencher (complex)
respectively; [Q] is the concentration of the quencher, KSV is the
Stern-Volmer quenching constant, which is obtained from the
slope of plot of I0/I vs. [Q]. A plot of I0/I vs. [complex]/[DNA]
appears linear (Fig. 12) and the Stern-Volmer quenching
constant (KSV) was found to be (5.24 0.02) × 104M-1 at 37 ºC.
This data is also in agreement with the value obtained by
electronic spectral studies [29, 36, 37].
1/ (A-A0) = 1/ (A∞-A0) + 1/ K(A∞-A0) . 1/ Cligand
Where, Ao is the absorbance of DNA at 260 nm in the absence
of complex, A∞ is the final absorbance of the ligated DNA, and
A is the recorded absorbance at different ligand concentrations.
The absorption spectrum of CT-DNA at 260 nm is monitered by
varying the concentration of the oxovanadium complex. Upon
the addition of incremental amounts of complex hypochromism
was observed (Fig.9) without any significant red or blue shift.
The double reciprocal plot of 1/(A-Ao) versus 1/Cligand is linear,
and the binding constant (K) can be estimated from the ratio of
the intercept to the slope (Fig.10). The complex shows binding
constant as (3.51 0.02) × 104.
Fig. 9 Absorption spectra of DNA upon increasing amounts of
complex, [Complex]/[DNA] = 0.2; 0.4; 0.6; 0.8; 1.0; 1.2.
Fig. 11
Fluorescence emission spectra of the EB-DNA
complex in the presence of [EB] = 15 ꢀM + [DNA] = 10.0 ꢀM;
the ratio of [VO(sal-cys)(phen)] / EB-DNA = 0, 0.2, 0.4, 0.6,
0.8, 1.0, 1.2. Excitation wave length=500 nm.
Fig. 10 Plot of 1/(A-Ao) versus [complex] ×105 (M-1)
Fluorescence emission titrations for DNA interaction
Fluorimetric measurements using ethidium bromide (EB) as a
probe are usually carried out to establish the binding mode of
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prepared so as to give total complex/base pair ratios of 0, 0.2,
0.4, 0.6, and 0.8.
Gel electrophoresis study for nuclease activity
The double-stranded plasmid pUC19 DNA exists in a compact
supercoiled (SC) form. Upon introduction of strand breaks, the
supercoiled form of DNA is disrupted into the nicked circular
(NC) form or the linear form. If one strand is cleaved, the super
coiled form will relax to produce a nicked circular form. If both
the strands are cleaved, a linear form will be produced.
Relatively fast migration is observed for super coiled form when
the plasmid DNA is subjected to electrophoresis, while the
nicked circular form migrates slowly and the linear form
migrates in between SC and NC [9, 29, 37]. Hence, DNA strand
breaks could be quantified by measuring the transformation of
the super coiled form into nicked circular and linear forms. The
ability of the vanadium complex to induce DNA cleavage was
studied by gel electrophoresis using super coiled pUC19 DNA
in Tris-HCl/NaCl buffer (pH 7.2). The results of the Gel
electrophoresis (Fig. 14) experiment and quantitative cleavage
data are presented in Table 6. In the gel electrophoresis six
bands are observed. Lane 1 contains only DNA and has 87%
supercoiled form i.e., Form I and 13 % relaxed form i.e., Form
II. On the addition of H202 (4 µM), percentage of relaxed form
increases to 28 % (Lane 2) while on addition of the compound
(20 µM to 60 µM), the percentage of relaxed form increases
through 31 to 39 to 51 in lane 3, 5, 7 respectively. Whereas, on
addition of different concentrations of compound and H202
together (4 µM H202+ 20 µM), (4 µM H202+ 40 µM) and (4 µM
H202+ 60 µM) the percentage of relaxed form of DNA increases
to 36 % (lane 4), 45 % (lane 6) and 69 % (lane 8) respectively.
This clearly indicates that the compound alone shows
moderate nuclease activity but exhibits greater nuclease
activity at higher concentration when used in combination with
H202 [9, 29, 37].
Fig. 12 Plot of I/I0 vs. [VO(sal-cys)(phen)] / DNA
Viscometric studies to confirm the DNA binding pattern
Viscometric studies play a very important role in ascertaining
DNA binding pattern in solution. More precisely, it can lend
strong support in favour of intercalative binding. The
intercalative binding by the ligand lengthens the DNA helix,
which in turn, causes an increase in the viscosity of DNA [38].
The values of relative specific viscosities of DNA in the
absence and presence of the complex are plotted against
[complex] / [DNA] and are presented in Fig.13. It is observed
that the addition of the complex to the CT-DNA solution leads
to an increase in the viscosity of the CT-DNA, thereby clearly
demonstrating the intercalative binding of the complex to CT-
DNA [38, 39].
Fig. 14 (a) Agarose gel(0.9 %) electrophoregram of supercoiled
DNA (0.5 ꢀg) incubated for 45 min at 37 °C, in PBS buffer (0.15
M, pH 7.2) at 37 °C. Lane 1: DNA control; Lane 2: DNA+
H2O2; Lane 3: DNA + complex (20 µM); Lane 4: DNA+ complex
Fig. 13 Effect of increasing amount of [VO(sal-cys)(phen)]
complex on the specific viscosity of CT DNA. Samples were
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(20 µM) + H2O2; lane 5: DNA + complex (40 µM); lane 6: DNA+
complex (40 µM) + H2O2; lane 7: DNA + complex (60 µM); lane
8: DNA+ complex (60 µM) + H2O2
1
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Table 6. Results of the cleavage of pUC19 DNA determined
by gel electrophoresis study
2
3
Sl No.
Reaction condition
Form I
(% SC)
87
72
69
Form II
(%NC)
13
28
31
Lane 1
Lane 2
Lane 3
Lane 4
control DNA
DNA + H202 (4 µM)
DNA+ complex (20 µM)
DNA+ complex (20 µM)+ 64
H202
36
Lane 5
Lane 6
DNA + complex (40 µM)
DNA + complex (40 µM) + 55
H202
61
39
45
4
5
6
Lane 3
Lane 6
DNA+ complex (60 µM)
49
51
69
DNA + complex (60 µM) + 31
H202
Conclusion
A new oxovanadium complex [VIVO(sal-cys)(phen)] has been
designed and synthesized with the aim of developing
multifunctional molecule, active both in the fields of biocatalysis
as well as in DNA knicking . The compound mimics the in vitro
bromination of organic substate giving corresponding
brominated product. The results of physicochemical studies of
DNA binding as well as DNA cleavage activity indicate that the
oxovanadium(IV) compound may be useful in DNA
manipulation studies. Hence, the newly synthesized complex
[VIVO(sal-cys)(phen)], exhibits versatile multifunctional enzyme
mimetic catalytic activities both as a bromoperoxidase as well
as a DNA nuclease.
7
8
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Acknowledgements
11 U. Saha, T.K. Si, P.K. Nandi and K.K. Mukherjea, Inorg. Chem.
Commun., 2013, 38, 43–46.
The authors are thankful to UGC, New Delhi for financial support in
the form of a Major Research Project [Sanction no.39-706/2010(SR)]
to K.K.M., where U.S. had been a project fellow..
12 (a) M. Sam, J.H. Hwang, G. Chanfreau and M.M. Abu-Omar, Inorg.
Chem., 2004, 43, 8447–8455; (b) P.K. Sasmal, S. Saha, R.
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Notes and references
Department of Chemistry, Jadavpur University, Calcutta (Kolkata), 700032, India.
∗
Corresponding author. Tel.: +91 9831129321; fax: +91 33
24146223.E-mail address: k_mukherjea@yahoo.com (K.K.
Mukherjea).
Ϯ
Electronic Supplementary Information (ESI) available: [details
of any supplementary information available should be included
here]. See DOI: 10.1039/b000000x/.
15 (a) P. Nagababu, J. Naveena, L. Latha and S. Satyanarayana,
Chemistry & Biodiversity, 2006, 3, 1219-1228.
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