Synthesis, structural characterization and catalytic activity of a multifunctional enzyme mimetic oxoperoxovanadium(v) complex

Article abstract of DOI:10.1039/c2dt12505f

The synthesis and structural characterization of a novel oxoperoxovanadium(v) complex [VO(O₂)(PAH)(phen)] containing the ligands 2-phenylacetohydroxamic acid (PAHH) and 1,10-phenanthroline (phen) has been accomplished. The oxoperoxovanadium(v) complex was found to mimic both vanadate-dependent haloperoxidase (VHPO) activity as well as nuclease activity through effective interaction with DNA. The complex is the first example of a structurally characterized stable oxoperoxovanadium(v) complex with a coordinated bi-dentate hydroximate moiety (-CONHO^-) from 2-phenylacetohydroximate (PAH). The oxoperoxovanadium(v) complex has been used as catalyst for the peroxidative bromination reaction of some unsaturated alcohols (e.g. 4-pentene-1-ol, 1-octene-3-ol and 9-decene-1-ol) in the presence of H₂O₂ and KBr. The catalytic products have been characterized by GC-MS analysis and spectrophotometric methods. The DNA binding of this complex has been established with CT DNA whereas the DNA cleavage was demonstrated with plasmid DNA. The interactions of the complex with DNA have been monitored by electronic absorption and fluorescence emission spectroscopy. Viscometric measurements suggest that the compound is a DNA intercalator. The nuclease activity of this complex was confirmed by gel electrophoresis studies.

Full text of DOI:10.1039/c2dt12505f

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PAPER
Synthesis, structural characterization and catalytic activity of a
multifunctional enzyme mimetic oxoperoxovanadium(V) complex†
Tapan K. Si,a Shiv S. Paul,a Michael G. B. Drewb and Kalyan K. Mukherjea*a
Received 29th December 2011, Accepted 17th February 2012
DOI: 10.1039/c2dt12505f
The synthesis and structural characterization of a novel oxoperoxovanadium(V) complex [VO(O2)(PAH)-
(phen)] containing the ligands 2-phenylacetohydroxamic acid (PAHH) and 1,10-phenanthroline (phen)
has been accomplished. The oxoperoxovanadium(V) complex was found to mimic both vanadate-
dependent haloperoxidase (VHPO) activity as well as nuclease activity through effective interaction with
DNA. The complex is the first example of a structurally characterized stable oxoperoxovanadium(V)
complex with a coordinated bi-dentate hydroximate moiety (–CONHO−) from 2-phenylacetohydroximate
(PAH). The oxoperoxovanadium(V) complex has been used as catalyst for the peroxidative bromination
reaction of some unsaturated alcohols (e.g. 4-pentene-1-ol, 1-octene-3-ol and 9-decene-1-ol) in the
presence of H2O2 and KBr. The catalytic products have been characterized by GC-MS analysis and
spectrophotometric methods. The DNA binding of this complex has been established with CT DNA
whereas the DNA cleavage was demonstrated with plasmid DNA. The interactions of the complex with
DNA have been monitored by electronic absorption and fluorescence emission spectroscopy. Viscometric
measurements suggest that the compound is a DNA intercalator. The nuclease activity of this complex
was confirmed by gel electrophoresis studies.
Introduction
There has been a continuous upsurge of interest in peroxo com-
pounds of vanadium(V) since the discovery of vanadium-depen-
dent haloperoxidase (VHPO) enzymes with a five coordinated
VO4N moiety (Fig. 1) as an active site,1 as well as the antiproli-
ferative and cytotoxic effects of peroxovanadium(V) compounds2
via interaction with DNA. The VHPO enzyme catalyzes the oxi-
dation of halides in the presence of hydrogen peroxide under
physiological conditions.3–6 This is an important route for the
biosynthesis of many useful natural halogenated organic com-
pounds.7 In addition, the oxovanadium(V) complexes function as
good insulin enhancing agents8–11 and are also used as catalysts
for the oxidation of various organic compounds (hydrocarbons
and alcohols,12–19 organic sulfide20–22 etc.). Transition metal
complexes are of current interest because of their potential appli-
cations as probes of DNA structure, for DNA-dependent electron
transfer and site specific cleavage of nucleic acids23 with the aim
of developing novel therapeutic and diagnostic agents. With
Fig. 1 Proposed structure of the active site in peroxo state of the
natural vanadium dependent bromoperoxidase enzyme.
aDepartment of Chemistry, Jadavpur University, Kolkata 700032, India.
E-mail: k_mukherjea@yahoo.com; Fax: +(91)+(33)24146223;
Tel: +(91)9831129321
bDepartment of Chemistry, The University of Reading, PO Box 224,
White Knights, Reading RG6 6AD, UK
regard to DNA cleavage activity, more attention has been
focused on the middle to late transition metals than the earlier
members of the series.24 Among the latter, the vanadium com-
pounds belong to a new class of nonplatinum metal antitumor
agents which exert antiproliferative and cytotoxic effects via
interaction with DNA.25–27 Therefore, the reactivity of
†Electronic supplementary information (ESI) available: The figures of
GC-MS (Fig. S1–S3), gas chromatography (Fig. S4–S6), 1H NMR
(Fig. S7), are available in the supplementary materials. CCDC 628820.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c2dt12505f
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oxoperoxovanadium(V) complexes is receiving renewed attention
in the fields of both bromination of organic compounds via
bromide oxidation28 in the presence of H2O2 and the develop-
ment of DNA cleavage reagents.29
The natural vanadium-bromoperoxidase (VBPOs) enzyme,
particularly that isolated from the brown algae Ascophyllum
nodosum, has generated interest due to its high turnover of Br−
oxidation30 at pH = 6.5 in the presence of H2O2. The propensity
of vanadium(V) to coordinate peroxides is known31 but very few
peroxovanadium(V) complexes have been tested for their enzyme
mimetic properties.32 Herein, we report the synthesis and struc-
tural characterization of a novel oxoperoxovanadium(V) complex
[VO(O2)(PAH)(phen)] with 2-phenylacetohydroxamic acid
(PAHH) and phenanthroline, including its multifunctional
enzyme mimetic properties. The hydroxamic acid derivatives are
good analytical reagents33 and have important biological func-
tions due to their siderophoric role.34 There is no earlier report
of structurally characterized oxoperoxovanadium(V) complexes
with hydroxamate based ligands.35 The oxoperoxovanadium(V)
complex catalyzes the peroxidative bromination of olefinic alco-
hols (e.g. 4-pentene-1-ol, 1-octene-3-ol and 9-decene-1-ol) in
the presence of H2O2 and KBr in a mild acidic medium, just like
the natural VBPO enzymes36 and has good catalytic efficiency at
low pH range. The conversion of homoallylic alcohols and
olefinic alcohols into their β-brominated cyclic ethers and other
oxidized derivatives has been reported recently, where oxovana-
dium(V) Schiff base complexes have been used as catalyst pre-
cursors.37,38 Apart from VHPO activity, the nuclease activity of
the present compound has also been established. The DNA
binding of this complex has been studied with CT DNA whereas
the DNA cleavage is demonstrated with plasmid DNA. The be-
havior of the complex interacting with DNA is explored by elec-
tronic absorption and fluorescence emission spectroscopy as well
as gel electrophoresis and viscometric measurements.
Scheme 1 The synthetic pathway of the oxoperoxovanadium(V)
complex [VO(O)2(PAH)(Phen)].
(O22−) appears at 935 cm−1. The ν(CvO) vibration occurs at
1634 cm−1 in free 2-phenylacetohydroxamic acid (PAHH),
whereas after its coordination with vanadium(V) the ν(CvO) of
coordinated PAH− appears at 1605 cm−1. The shifting of the
ν(CvO) absorption band43 to a lower energy region indicates a
decrease in the CvO bond order due to drainage of electron
density from the carbonyl oxygen to the vanadium(V) center.
Similarly, the ν(C–N) vibration of uncoordinated PAHH
(1546 cm−1) also shifts to lower wave number (1517 cm−1) after
coordination with the vanadium(V) ion. The strong vibration
bands at 1426 cm−1 and at 727 cm−1 are due to the C–N
vibration of the phen ligand in the complex. The electronic spec-
trum of the orange-red vanadium compound exhibits a new (not
exhibited by the ligands) higher wavelength band44 at λmax
=
455 nm; (ε = 400 M−1 cm−1), which is due to the LMCT tran-
2−
sition of O2 → vanadium(V). In the compound, the two
shoulders at 343 and 324 nm and the strong absorption band at
291 nm (ε = 11 960 M−1 cm−1) may be assigned as the intra
ligand π → π* transitions of the aromatic ring and the CvO
function of the coordinated ligand.
Solid state X-ray crystallographic structure
The molecular structure of [VO(O2)(PAH)(phen)] is illustrated
in Fig. 2 and selected bond lengths and bond angles are listed in
Tables 1 and 2 respectively. The structure of the complex is
mononuclear, with a distorted pentagonal-bipyramidal arrange-
ment around the vanadium atom. The vanadium atom is coordi-
nated by two bidentate ligands (O,O and N,N donors): one is the
uni-negative 2-phenylacetohydroxamate ligand providing the O,
O donor and the other is the neutral 1,10-phenanthroline (phen)
ligand providing the N,N donor. The ligands form two five-
membered chelate rings orthogonal to each other. The average
standard deviation of atoms from the pentagonal plane is
0.034 Å. The vanadium atom is displaced from the plane
towards the apical O1 oxygen atom by 0.223(9) Å. This is less
than the average value, 0.236 Å, found for other mono oxoper-
oxovanadium(V) complexes with polydentate amino-polycarbox-
ylates.45 In addition, two water molecules, one on a two fold axis,
were located and refined with 50% occupancy but their hydrogen
atoms could not be located. The oxoperoxovanadium(V)
complex containing a O,O donor hydroxamate (–CONHO−)
moiety from the bidentate ligand 2-phenylacetohydroxamic acid
Results and discussion
Synthesis of the complex [VO(O2)(phen)(PAH)]
The reaction of an aqueous solution of NH4VO3 in an acidic
medium may produce a variety of polyoxovanadates39 but in the
presence of both acid (pH 2) and H2O2 under cold conditions
(0–5 °C), it is converted to the red-brown peroxovanadate(V)
species40 [VO(O2)(H2O)3]+. The coordinated water molecules of
the [VO(O2)(H2O)3]+ species are replaced by two strong biden-
tate ligands ortho-phenanthroline (N,N donor) and 2-phenylace-
tohydroximate (O,O donor) after the addition of a methanol
solution of the PAHH ligand and 1,10-phenanthroline (phen)
ligand (1 : 1 ratio), resulting in the orange–red oxoperoxovana-
dium(V) complex [VO(O2)(phen)(PAH)] as a solid residue. The
synthesis of the complex is shown in Scheme 1. The compound
is soluble in highly polar solvents like DMF, DMSO and THF
and behaves like a non-electrolyte. It is diamagnetic at 298 K.
(PAHH) is a new example of this class of compounds.35,46
A
IR and UV–Vis spectroscopic characterization of the complex
structural representation of the ligand, 2-phenylacetohydroxamic
acid (PAHH) is shown in Fig. 3. The length of V1–O1 double
bond is 1.604 Å which is consistent with typical oxovanadium(V)
complexes.47,48 The trans effect can be observed in the
As recorded in the Experimental section, the ν(VvO)
vibration41 in the complex appears at 947 cm−1, while the
characteristic absorption band ν(O–O) for the peroxo group42
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Fig. 3 (PAHH) N-(1-phenyl acetyl)hydroxamic acid
Table 3 Relevant hydrogen bonds in the present compound
d(D–H)/ d(H⋯A)/ d(D⋯A)/
D–H⋯A/Å
Å
Å
Å
∠(DHA)/º
N(33)–H(33)⋯O(2)i
C(12)–H(12)⋯O(1)
0.86
0.93
1.98
2.53
2.40
2.58
2.789(5)
2.979(6)
3.283(7)
3.271(8)
157
110
159
132
Fig. 2 The ORTEP structure of the complex, [VO(O2)(phen)(PAH)]
with ellipsoids at 30% probability.
C(24)–H(24)⋯O(34)ii 0.93
C(37)–H(37)⋯O(3)ii
0.93
Symmetry codes (i) x, −y, −1/2 + z, (ii) 1/2 − x, 1/2 + y, 1/2 − z, (iii)
π(pi) interaction: C(40)–H(4))⋯Cg(1)iii where Cg(1) is the centroid of
the ring C(36)–C(37). (iii) 1 − x, y, 1/2 − z.
Table 1 Selected bond lengths (Å) of the complex
V(1)–O(1)
V(1)–O(3)
V(1)–O(2)
V(1)–O(34)
V(1)–O(31)
V(1)–N(11)
V(1)–N(22)
O(2)–O(3)
1.604(3)
1.864(4)
1.891(4)
1.963(3)
2.062(3)
2.146(4)
2.306(4)
1.417(5)
N(11)–C(12)
N(11)–C(16)
C(17)–N(22)
C(21)–N(22)
O(31)–C(32)
C(32)–N(33)
N(33)–O(34)
1.325(5)
1.360(5)
1.332(6)
1.335(6)
1.264(5)
1.297(6)
1.373(5)
Table 2 Selected bond angles (°) of the complex
O(1)–V(1)–O(3)
O(3)–V(1)–O(2)
O(3)–V(1)–O(34)
O(3)–V(1)–N(11)
O(34)–V(1)–O(31)
O(34)–V(1)–N(11)
O(31)–V(1)–N(11)
O(3)–V(1)–N(22)
O(3)–V(1)–O(31)
103.50(19)
44.32(16)
76.84(16)
123.51(16)
77.73(13)
150.50(14)
77.27(12)
91.19(15)
152.77(15)
O(1)–V(1)–O(2)
O(1)–V(1)–O(34)
O(2)–V(1)–O(34)
O(1)–V(1)–N(11)
O(2)–V(1)–O(31)
O(2)–V(1)–N(11)
O(1)–V(1)–N(22)
O(2)–V(1)–N(22)
O(31)–V(1)–N(22)
101.87(18)
104.34(18)
119.60(15)
91.97(16)
153.28(14)
79.54(14)
163.38(17)
83.13(14)
77.61(12)
Fig. 4 Packing of the complex showing intermolecular π–π stacking
interaction of aromatic rings over a centre of symmetry.
via intermolecular hydrogen bonds thus forming a macrocyclic
grid structure (Fig. 6).
Peroxidative bromination of alkenols
elongation of the apical V1–N22 bond [2.306(4) Å], which is
trans to the V1–O1 bond when compared with the equatorial
V1–N11 distance [2.146(4) Å], and the elongation of the V1–
O31 bond distance [2.062(3) Å], which is trans to oxygen atoms
of the η2-peroxo ligand compared to V1–O34 bond distance
[1.963(3) Å]. The intermolecular aggregation in the complex is
determined (Table 3) by a combination of N–H⋯O, C–H⋯O
hydrogen bonds and π–π stacking interactions (Fig. 4). Intermo-
lecular N–H⋯O hydrogen bonds link the molecules related by
glide symmetry into an infinite one-dimensional chain propagat-
ing along the [001] direction (Fig. 5). Dimensions for N(33)–
H⋯O(2) (x, −y, z − 0.5) are N⋯O 2.791(7) Å, H⋯O 1.98 Å
and N–H⋯O 156°. The solvent H2O molecules form a chain
The [VO(O2)]+ core of the peroxovanadium(V) complex [VO-
(O2)(phen)(PAH)] is to some extent equivalent to the respective
mono-peroxovanadium(V) intermediate species of the VHPO
enzymes which plays the key role in the enzymatic
processes49–53 in the presence of H2O2. Here, the catalytic reac-
tion mixture, as indicated in the Experimental section, contains
the peroxovanadium(V) complex (the catalyst), H2O2 (the term-
inal oxidant), KBr and the olefinic alcohol, 4-pentene-1-ol (the
substrate to be brominated) in DMF and acetonitrile at
pH 2. Here, the substrate-to-catalyst ratio is 200 : 1. Firstly, the
peroxovanadium(V) complex oxidizes the bromide (Br−) ion by
two electrons to Br+ ions54 in the presence of H+ ions, which
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Fig. 6 Themacrocyclic grid structure through intermolecular H-bonds
with the solvent H2O molecules in the packing of the complex. The
hydrogen atoms on the water molecules could not be located. The two
water molecules O1W and O2W have 50% occupancy. The O1W–O2W
distance of two oxygen atoms of two solvent water molecules is 2.59(2)
Å and the O3–O2W distance is 2.78(2) Å. For clarity of the figure, H
atoms are not included.
Fig. 5 The formation of an infinite one-dimensional chain propagating
along the [001] direction in the complex through intermolecular
N–H⋯O hydrogen bonds.
then forms the species HOBr or Br2 (Scheme 2a). Now the Br+
ion or Br2 molecule reacts with the π-electrons of the olefinic
double bond and produces the normal brominated products
(Scheme 2b) via the formation of a bromocyclic intermediate. In
case of the substrate, 4-pentene-1-ol, the cleavage of the inter-
mediate bromocyclic species via nucleophilic attack by the
hydroxyl oxygen of 4-pentene-1-ol results in the formation of
brominated cyclic ethers55 namely 2-bromomethyl-tetrahydro-
furan and 3-bromo-pentahydropyran (Scheme 3). This type of
ring closure reaction of alkenols via bromination is known as the
brominative cyclization38 of organic compounds. The yields of
the catalytic reactions are quite good (Table 4). After reacting for
5 h the yield percentage of total brominated products is 39%. 2-
Bromomethyl-tetrahydrofuran is 67% of the total brominated
products while 3-bromo-pentahydropyran is 33%. From the
GC-MS analysis, it was observed that along with the brominated
products [m/z 165, 167 in the mass spectrum (Fig. S1†)] a small
amount of epoxides (5%) was also obtained during the catalytic
bromination reaction of 4-pentene-1-ol (Table 4). In case of the
substrate 1-octene-3-ol (Scheme 4), the acyclic brominated alco-
hols [m/z 207, 209 in the mass spectrum (Fig. S2†)], 1-bromo-
octane 3-ol (35%) and 2-bromo-octane 3-ol (7%) were the major
products with a small amount of acyclic epoxides (5%). After
5 h of reaction time the cyclic brominated products were not
obtained due to the instability of the cyclic ether of the large ring
structure. For the substrate 9-decene-1-ol (Scheme 5), the acyclic
brominated alcohols [m/z 238, 240 in the mass spectrum
Fig. S3†)], 9-bromo-decane 1-ol (8%) and 10-bromo-decane 1-
ol (15%) were obtained with a small amount of epoxidic alco-
hols (10%). The details of the catalytic results are shown in
Table 4. The gas chromatography images (Fig. S4–S6†) for the
above catalytic reaction mixtures are included in the ESI.†
shift for the two H atoms attached with one ethereal carbon
linked to chiral carbon of 3-bromo pentahydropyran is observed
at δ 3.87 ppm (dd, 2H, J1 = 4.34 Hz, J2 = 4.27 Hz). The chemi-
cal shifts for two H atoms of another ethereal carbon far away
from the chiral atom is observed at δ 3.72 ppm (t, 4H, J = 5.985
Hz). The chemical shift for the single H atom attached to the
chiral carbon of 2-bromomethyl tetrahydrofuran is observed at δ
4.23 ppm (m, 1H) while that of 3-bromo pentahydropyran is
observed at δ 3.48 ppm (m, 1H). The integration of the NMR
signals indicates that the yield of 3-bromo pentahydropyran is
less than that of 2-bromomethyl tetrahydrofuran which is also
observed by GC-MS (Table 4).
DNA binding studies
The establishment of the nuclease activity and the DNA double
strand breaks by the oxoperoxovanadium(V) complex demands a
thorough study on the DNA binding pattern of the said complex.
The oxygen rich vanadium(V) center with coordinated hydroxi-
mate ligand of the complex enhances the binding interaction
with the base pairs of the DNA strand via hydrogen bonding and
the planar moiety of coordinated phenanthroline ligand helps the
compound to intercalate between the DNA base pairs by facili-
tating the cleavage of DNA strands (Fig. 8). The interaction of
DNA with this complex has been studied by various physico-
chemical tools, the results of which are presented herein.
Electronic absorption spectroscopy for DNA binding with the
complex
Electronic absorption spectrophotometry is usually used to deter-
mine the binding of metal complexes with DNA. A complex
bound to DNA through intercalation is characterized by a change
in absorbance (hypochromism) and a red shift in the wave-
length56 due to the stacking interaction between the aromatic
chromophores and the DNA base pairs. The electronic spectra of
the complex in the presence and absence of DNA was monitored
at 268 and 292 nm upon the addition of incremental amounts of
1H NMR spectroscopy of brominated cyclic ether
The isomeric brominated cyclic ethers are finally identified by
1H NMR spectrometry (Fig. 7). The chemical shift for the bro-
momethyl group of 2-bromomethyl tetrahydrofuran is observed
at δ 3.64 ppm (dd, 2H, J1 = J2 = 10.05 Hz) while the chemical
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Scheme 2 (a) Schematic representation of the reaction mechanism51a of the catalytic bromination of olefinic alcohol using the [VO(O2)(PAH)-
(phen)] complex as a catalyst in the presence of H2O2 and KBr in an acidic medium. (b) Schematic representation of the reaction mechanism of the
catalytic bromination of 4-pentene-1-ol using the [VO(O2)(PAH)(phen)] complex as catalyst in the presence of H2O2 and Br− ions in an acidic
medium after 5 h.
enhancement was around 1.5 times the initial luminescence, I0
(Fig. 12). This magnitude of change in luminescence due to the
movement of the complex from bulk aqueous solvent to the
more hydrophobic environment of DNA, is typical for intercalat-
ing complexes. The increase in luminescence allowed a second
estimate of the binding parameters for the interaction of complex
Scheme 3 The catalytic bromination of 4-pentene-1-ol using the [VO-
with CT DNA. The luminescence titration data were fitted using
eqn (3) (vide the Experimental section). This process gave an
estimate of Kb = 7.8 × 104 M−1, which matches very well the
evaluation from absorption titration analysis. This is also pro-
duced a site size of 2 bp (base pair), which is consistent with the
intercalative binding mode and is comparable with other metal
complexes.57–59
(O2)(PAH)(phen)] complex as catalyst in the presence of H2O2 and KBr
in an acidic medium at room temperature after 5 h.
DNA. A considerable drop in the absorptivity and a blue shift
was observed (Fig. 9). A shift in the λmax value of 2 nm was
observed on addition of DNA. The binding constant of the
complex with DNA was calculated to be (5.3 0.02) × 104 M−1
from the plot of [DNA]/(εa − εf) vs. [DNA] (Fig. 10). The
binding constant of this complex is comparable to other classical
intercalators and metal complex-intercalators.
Viscometric studies for assigning the DNA binding pattern
Viscometric studies play a very important role in ascertaining the
DNA binding pattern in solution. More preciously it lends strong
support in favour of intercalative binding. The intercalative
binding by the ligand lengthens the DNA helix which in turn
increases the viscosity of DNA.60 Partial or nonclassical interca-
lation of ligands may bend or kink the DNA helix, thereby
Fluorescence emission titrations for DNA interaction
The complex exhibits fluorescence when excited at 268 nm with
an emission maxima at 363 nm. The emission intensity for the
complex is enhanced on progressive addition of CT DNA
(Fig. 11), and a plot of I/I0 vs. [DNA] revealed that this
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Table 4 Details of the catalytic bromination of olefinic alcohol using the [VO(O2)(PAH)(phen)] complex as catalyst in the presence of H2O2 and
KBr in an acidic medium at room temperature are shown below. The reaction period was 5 h
Products
Entry
I
Substrate
Epoxides/diols
Brominated
Yield of epoxidic products (%)
5
Yield of bromo products (%)
39
A(27) + B(12)
II
3
42
C(7) + D(35)
III
10
23
E(8) + F(15)
Scheme 4 The catalytic bromination of 1-octene-3-ol using the [VO-
(O2)(PAH)(phen)] complex as catalyst in the presence of H2O2 and KBr
in an acidic medium at room temperature after 5 h.
Scheme 5 The catalytic bromination of 9-decene-1-ol using the [VO-
(O2)(PAH)(phen)] complex as catalyst in the presence of H2O2 and KBr
in an acidic medium at room temperature after 5 h.
Fig. 8 Probable intercalative interaction of the [VO(O2)(PAH)(phen)]
complex with the DNA base pairs.
decreasing its effective length and subsequently the viscosity.61
The values of relative specific viscosities of DNA in the absence
and presence of complexes are plotted against [complex]/
[DNA]. The results of the viscometric studies 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
binding60,61 of CT DNA by the present complex.
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
Fig. 7 1H NMR spectrum of the brominated products of 4-pentene-
1-ol.
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Fig. 9 Absorption spectra of the [VO(O2)(PAH)(phen)] complex
(20 μM) in the presence of increasing amounts of CT DNA, [DNA]/
[complex] = (a − l): 0.0; 0.2; 0.4; 0.6; 0.8; 1.0; 1.2; 1.4; 1.6; 1.8; 1.9;
2.0.
Fig. 11 Emission titration of the [VO(O2)(PAH)(phen)] complex with
CT DNA in Tris buffer, [complex] = 0.7 μM, in the presence of increas-
ing amounts of CT DNA, [DNA] = 700 μM, over the range of
0–240 μM, excitation wavelength = 268 nm.
Fig. 10 Plot of [DNA]/(εa − εf) × 108 M/M−1 cm−1 vs. [DNA] ×
105 M.
Fig. 12 Plot of I/I0 vs. [DNA].
coiled form will relax to produce a nicked circular form. If both
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. The nicked circular
form migrates slowly and the linear form migrates between SC
and NC.62 Hence, DNA strand breaks were quantified by
measuring the transformation of the super coiled form into
nicked circular and linear forms.
complex produces a very small amount of form II (NC) 15% and
23%, respectively. Higher concentrations (100 μM, 200 μM) of
the complex are capable of inducing a substantial amount of
DNA cleavage up to 53% DNA cleavage (Table 5). The metal
solution and the ligand alone are not capable of inducing any
cleavage.
The ability of the vanadium complex to induce DNA cleavage
was studied by gel electrophoresis using super coiled pUC19
DNA in 50 mM Tris–HCl/50 mM NaCl buffer (pH 7.2). Fig. 14
shows the results of the gel electrophoresis experiment carried
out with super coiled DNA and the cleavage induced on DNA
by various concentrations of the complex. Control experiments
suggest that untreated DNA (lane 1) does not produce form II
(NC), whereas treatment of DNA with 10 μM and 50 μM of the
Conclusions
As the reactivity of oxoperoxovanadium(V) complexes has been
receiving renewed attention recently both in the field of VHPO
model studies as well as in the development of DNA cleavage
reagents, we have synthesized the oxoperoxovanadium(V)
complex with a hydroximate based ligand (2-phenylacetohydrox-
imate) with the aim of developing compounds that can act both
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Table 5 Extent of DNA SC pUC19 cleavage by the complex
Lane
no.
Form I
(% SC)
Form II
(% NC)
Reaction condition
1
2
DNA CONTROL
DNA + 10 μM [VO(O)2(PAH)
(Phen)] complex
90
85
10
15
3
4
5
DNA + 50 μM [VO(O)2(PAH)
(Phen)] complex
DNA + 100 μM [VO(O)2(PAH) 64
(Phen)] complex
DNA + 200 μM [VO(O)2(PAH) 47
(Phen)] complex
77
23
36
53
Experimental
Materials
Extrapure ammonium metavanadate and hydroxylamine hydro-
chloride were obtained from the Sisco Research Laboratory
(India). Methyl benzoate was obtained from S.D. Fine Chem.
Ltd (India). Potassium hydroxide pellets and methanol (G.R.)
were products of E. Merck (India) and were used directly.
Ethanol (95%) was obtained from Bengal Chemical and Pharma-
ceutical Works (Calcutta), and was lime distilled before use. All
other chemicals were obtained from E. Merck (India). Aceto-
nitrile, dichloromethane and acetone were further purified using
a literature method63 for the physico-chemical studies. Iolar II
grade dioxygen, dihydrogen, zero air, and dinitrogen gas used
for chromatographic analysis were obtained from Indian Oxygen
Co. (Calcutta). All the solvents used for chromatographic analy-
sis were either of HPLC, spectroscopic, or GR grade and in all
cases their purity was confirmed by GC analysis before use. The
analytical grade solvents used for physico-chemical studies were
further purified by the literature method before use, wherever
necessary. CT DNA was purchased from the Sigma Chemical
Company, USA; supercoiled plasmid pUC19 DNA was obtained
from Bangalore Genei (Bangalore, India).
Fig. 13 Effect of increasing the amount of the [VO(O2)(PAH)(phen)]
complex on the specific viscosity of CT DNA. Samples were prepared to
give total complex/base pair ratios of 0.35, 0.70, 1.15, 1.40 and 1.75.
Fig. 14 Agarose gel (9%) electrophoregram of supercoiled pUC19
DNA (0.5 μg) incubated for 45 min at 37 °C, in a buffer containing
50 mM Tris–HCl and 50 mM NaCl at 37 °C pH 7.2 with increasing
complex concentrations. Lane 1: DNA control; lanes 2–5: complex 10,
50, 100, 200 μM.
Synthesis of the ligand, 2-phenylacetohydroxamic acid (PAHH)
The ligand was prepared by following a literature method.64 The
reaction of methyl phenylacetate ester with NH2OH·HCl in
methanolic KOH solution resulted in the formation of 2-phenyl-
acetohydroxamic acid and was followed by the neutralization of
the reaction mixture. The white crystalline solid precipitate was
filtered off, washed with water and dried in vacuo. Yield 6.7 g
(45%). Anal. calcd for C8H9NO: C, 63.57; H, 5.96; N, 9.27;
Found: C, 63.49; H, 6.05; N, 9.15; IR (KBr disc, cm−1). 3190
(b), 3009(m), 2909(w), 1948(w), 1806(w), 1634(s), 1546(m),
1493(w), 1458(w), 1420(w), 1366(m), 1296(w), 1193(w), 1075
(m), 1052(s), 979(s), 927(w), 774(m), 716(s), 697(m), 624(s),
610(m), 541(m), 469(m).
as VHPO-mimicking and DNA-cleaving agents. To our knowl-
edge, this is the first example of a complex with hydroximate
ligands. The compound mimics the in vivo brominative cyclisa-
tion of homoallylic alcohols giving the β-bromocyclic ether, a
prototype of terpinols (VHPO activity). The results of physico-
chemical studies of DNA binding efficiency as well as DNA
cleavage activity indicate that the oxoperoxovanadium(V) com-
pound is also capable of DNA cleavage. This observation
suggests that the compound may be a good representative of the
new class of nonplatinum, metal antitumor agents and may also
be useful in DNA manipulation studies. The [VO(O)2(PAH)
(Phen)] complex exhibits versatile multifunctional enzyme
mimetic catalytic activity both as a bromoperoxidase as well as a
nuclease.
Synthesis of the complex [VO(O2)(PAH)(phen)]
NH4VO3, 0.284 g (2.5 mmol) was dissolved in 5 ml H2O and
then 30% 0.5 ml (6.5 mmol) H2O2, was added. The reaction
mixture was placed in an ice bath with continuous stirring while
5812 | Dalton Trans., 2012, 41, 5805–5815
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Table 6 Crystal data and structure refinement parameters of the
complex
20 ml ethanolic solution of 0.371 g (2.5 mmol) PAHH and
0.495 g (2.5 mmol) 1,10-phenanthroline (phen) was added to
the reaction mixture. This was followed by acidification with 4
M HNO3. A reddish precipitate was obtained after 30 min. The
precipitate was filtered off and washed with water and ethanol.
The yield was 0.67 g (60%). Good quality orange-red diffract-
able grade crystals were obtained by slow diffusion of isopropa-
nol into a DMF solution of the solid. Anal. calcd for
C20H17.5N3O5.75V: C, 54.20; H, 3.98; N, 9.48; V, 11.50; Found:
C, 53.37; H, 4.22; N, 9.07; V, 10.97; IR (KBr disc, cm−1). 3441
(s), 3146(w), 3085(w), 2930(w), 2821(w), 1651(w), 1605(s),
1517(s), 1456(w), 1426(s), 1366(w), 1328(w), 1225(w), 1141
(w), 1103(w), 1054(m), 1002(m), 947(s)[VvO], 935(s)[O–O],
853(m), 776(w), 727(s), 696(w), 647(w), 538(s), 471(w).
UV-Vis; λmax nm (ε M−1 cm−1); 455(400), 343(sh), 324(sh),
291(11 960).
Empirical formula
Mr
C20H17.5N3O5.75
442.81
V
T/K
λ/Å
150(2)
0.71073
Crystal system
Space group
Monoclinic
C2/c
Unit cell dimensions
a/Å
b/Å
c/Å
18.367(2)
17.309(2)
13.316(3)
90.00
α/°
β/°
108.17(16)
90.00
4022.00(12)
2, 1.457
1820
γ/°
V/Å3, Z
Dc/g cm−3
F(000)
Crystal size/mm
θ Range for data collection/°
Reflections collected
0.05 × 0.05 × 0.30
2.5–28.5
9238
Independent reflections (Rint
Completeness to θ = θmax/%
Refinement method
)
3657(0.043)
78.3
X-Ray crystallographic details
Full-matrix-least-squares on F2
3657/0/268
1.081
Well-formed orange-red narrow needle shaped crystals of the
compound were obtained from slow diffusion of isopropanol
into a DMF solution of the compound. The x-Ray intensity data
for the complex was measured at 150 K using the Oxford
X-Calibur CCD System65 with MoKα radiation. The structures†
were solved using direct methods with the program SHELXS
and refined using the program SHELXL.66 The non-hydrogen
atoms were refined with anisotropic thermal parameters. The
hydrogen atoms bonded to carbon were included in geometric
positions and given thermal parameters equivalent to 1.2 times
those of the atom to which they were attached. Empirical absorp-
tion corrections were carried out using the ABSPACK
program.67 The crystal data, structure solution and refinement
parameters for the complex are summarized in Table 6.
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R1 = 0.0740
wR2 = 0.1728
R1 = 0.1072
wR2 = 0.1905
0.788–0.282
R indices (all data)
Largest diff. peak, hole/e Å−3
Schimadzu U-1200 spectrophotometer. The electronic spectra of
the complex were monitored both in the presence and absence of
DNA. The binding constant for the interaction of complex with
DNA was obtained from absorption titration data at 268 nm as
well as at 292 nm. A fixed concentration of complex was titrated
with increasing amounts of DNA over a range of 20–200 μM.
While measuring the absorption spectra, equal amounts of DNA
were added to both complex and the reference solution to elimin-
ate the absorbance of DNA itself. The intrinsic binding constant
was determined by using eqn (1).68
Bromination of alkenols using the complex as catalyst
The complex in 2 ml DMF solution (0.010 g, 0.022 mmol) was
mixed with 10 mmol of alkenol (e.g. 4-pentene-1-ol, 9-decene-
1-ol, 1-octene-3-ol) that was separately dissolved in 8 ml
CH3CN solution. After that, 2.39 g (20 mmol) KBr and 2 ml
(25 mmol) 30% H2O2 was added to each of the alkenol-complex
solutions which was acidified with dilute nitric acid. The reaction
mixture was stirred continuously at room temperature for 5 h fol-
lowed by quantitative extraction of the organic part with ether
and the products were characterized by GC-MS (SHIMAD-
ZU-QP-5050A) and 1H NMR (BRUKER-300 MHz)
spectrometry.
½DNAꢀ=ðεa ꢁ εf Þ ¼ ½DNAꢀ=ðεb ꢁ εf Þ þ 1=½Kbðεb ꢁ εf Þꢀ ð1Þ
Here, [DNA] is the concentration of DNA in base pairs, the
apparent absorption coefficients εa, εf and εb correspond to
Aobsd/[complex], the extinction coefficient for the free complex,
and the extinction coefficient for the complex in the fully bound
form respectively. Plots of [DNA]/(εa − εf) vs. [DNA] gave a
slope of 1/(εb − εf) with a Y-intercept of 1/[Kb(εb − εf)]. The
intrinsic binding constant Kb was obtained from the ratio of the
slope to the intercept.68
Viscometric studies. The viscosity of sonicated69 DNA
(average molecular weight of ∼200 base pairs using a Labsonic
2000 sonicator) was measured by a fabricated micro-viscometer
maintained at 28 ( 0.5) °C in a thermostatic water bath. Data
were presented as (η/ηo)1/3 vs. the ratio of the concentration of
metal complex to that of the CT DNA, where η and ηo are the
viscosities of the CT DNA solutions in the presence and absence
of the complex, respectively. The viscosity value (η = t − to) was
calculated from the observed flow time of CT DNA solutions (t)
and corrected for the buffer solution (to).
DNA binding measurements
UV–Vis spectral study. The solutions of CT DNA in Tris–
HCl/NaCl (50 mM Tris–HCl and 50 mM NaCl, pH 7.2) buffer
medium gave a ratio of A260/A280, of ca. 1.8–1.9, 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) at 260 nm. Stock solutions were stored at 4 °C
and used within 4 days. UV–Vis spectra were recorded in a
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Dalton Trans., 2012, 41, 5805–5815 | 5813

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experiments, a 5% DMF solution of [VO(O2)(PAH)(phen)] was
used and the concentration was so adjusted that the final concen-
tration of the complex was 0.70 μM. This was titrated with
increasing concentrations of DNA over the range 0–240 μM
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Cb ¼ C½ðI ꢁ I0Þ=ðImax ꢁ I0Þꢀ
ð2Þ
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where, C is the total vanadium-complex concentration, I and I0
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concentration of the free complex, Cf, is equal to C − Cb.
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nꢁ1
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Gel electrophoresis study. The DNA cleavage activity of the
complex was monitored using agarose gel electrophoresis. The
super coiled pUC19 DNA (0.5 μg per reaction) in Tris–HCl
buffer (50 mM) with 50 mM of NaCl (pH 7.2) was treated with
the indicated amount of metal complex followed by dilution
with the Tris–HCl buffer to a total volume of 15 μl and then
incubated for 2 h at 37 °C. After incubation it was mixed with a
loading buffer containing 25% bromophenol blue, 30% glycerol
(3 μl) and was loaded on a 0.9% agarose gel containing 1.0 μg
ml−1 ethidium bromide. Electrophoresis was carried out at 60 V
for 3 h in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM
EDTA, pH 7.2). The bands were visualized by UV light and
photographed. The extent of super coiled (SC) pUC19 DNA
cleavage induced by the complex was determined by measuring
the intensity of the bands with the UV pro Platinum 2.0 Gel
Documentation System.
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Acknowledgements
The authors are thankful to Late Prof. R. Bhattacharyya for
encouragement, and constructive suggestions. T. K. S. is thankful
to CSIR, India for Research Associateship (Award No.-9/96
(0624)2K10-EMRI dated 10.03.2010). Funding by DST, India
for the Agilent 6890N Gas Chromatograph and EPSRC and the
University of Reading for the X-Calibur system are thankfully
acknowledged.
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