Synthesis, Characterization, and DNA Binding Studies of a Chromium(III) Complex Containing a Tridentate Ligand

Article abstract of DOI:10.1002/ejic.200300170

[Cr(bzimpy)₂]Cl, where bzimpy is 2,6-bis(benzimidazol-2-yl)-pyridine, has been synthesized and characterized by ESI-MS, UV/Visible, and fluorescence spectra. Absorption titration and thermal denaturation experiments indicate that the complex binds to DNA with moderate strength, while viscosity measurements show that it may undergo surface binding. The fluorescence intensity of the complex increases with increasing DNA concentration, in contrast to [Cr(phen)₃]³⁺ and [Cr(bpy)₃]³⁺. The complex cleaves pBR322 DNA in the presence of H₂O₂. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003.

Full text of DOI:10.1002/ejic.200300170

FULL PAPER
Synthesis, Characterization, and DNA Binding Studies of a Chromium(III
Complex Containing a Tridentate Ligand
)
Vaidyanathan Ganesan Vaidyanathan[a] and Balachandran Unni Nair*[a]
Keywords: Chromium / Tridentate ligands / DNA cleavage / Radicals
[Cr(bzimpy)2]Cl, where bzimpy is 2,6-bis(benzimidazol-2-yl)-
pyridine, has been synthesized and characterized by ESI-
MS, UV/Visible, and fluorescence spectra. Absorption titra-
tion and thermal denaturation experiments indicate that the
complex binds to DNA with moderate strength, while viscos-
ity measurements show that it may undergo surface binding.
The fluorescence intensity of the complex increases with in-
creasing DNA concentration, in contrast to [Cr(phen)3]3+ and
[Cr(bpy)3]3+. The complex cleaves pBR322 DNA in the pres-
ence of H2O2.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2003)
The interaction of metal complexes with biomolecules inhibits enzymatic activity in the biosynthesis of nucleotide
has been extensively studied in recent decades.[1Ϫ13] Several pools, which in turn is responsible for mutations in
metal complexes, which bind to DNA through different cells.[37Ϫ38] We have demonstrated that a chromium()
modes, have been used as probes for DNA structure in solu- complex of a Schiff base having a donorϪacceptor group
tion, as agents for mediation of strand scission of duplex exhibits nuclease activity, while [Cr(salen)(H2O)2]ϩ can
DNA and as chemotherapeutic agents.[14Ϫ16] [M(LL)3]nϩ cleave DNA only in the presence of peroxide.[39Ϫ40] Fur-
metal complexes, where LL is 1,10-phenanthroline (phen) thermore, Sudgen et al. showed that CrIII aromatic biden-
or a modified phenanthroline ligand, are particularly at- tate amines that can enter cells are mutagenic agents in the
tractive species to recognize and cleave DNA.[17Ϫ22] Some Ames test, as such complexes can generate a CrIII/CrII ac-
transition metal complexes that bind covalently to DNA tive redox center.[41] These observations indicate that modi-
function as anti-tumor agents.
fication of the ligand environment would alter the binding
There has been renewed interest in biological aspects of affinities and DNA cleaving properties of the metal com-
chromium, and its extensive use in the tanning and electro- plex. Our present study focuses on the interaction of DNA
plating industries has aroused much environmental concern with chromium() complex 1 which has a pyridine-type tri-
over effluents bearing CrVI and CrIII. Epidemiological and dentate ligand, bzimpy.
animal studies have firmly established hexavalent chromium
compounds as potent carcinogens.[23Ϫ25] Chromate can
form several DNA lesions, including DNA, DNAϪprotein,
and DNAϪamino acid crosslinks, strand breaks and alkali
labile sites, which are responsible for its mutagenic and car-
cinogenic effects.[26,27] The carcinogenic activity of CrVI is
due to its reduction by intracellular reductants to generate
CrV and CrIV intermediates and finally CrIII. The chro-
mium(/) intermediates are proposed to cause DNA dam-
age that leads ultimately to tumor development.[28Ϫ32]
Chromium() is believed to be non-toxic and is an essential
trace element for glucose and lipid metabolism.[33] While
chromium() picolinate complex is a therapeutic in the
treatment of adult-onset diabetes,[34Ϫ35] Wetterhahn and co-
workers have demonstrated that Cr(pic)3 causes chromoso-
mal damage in Chinese hamster ovary cells (CHO).[36]
Chromium() not only affects DNA replication but also
Results and Discussion
[a]
Chemical Laboratory, Central Leather Research Institute,
Adyar, Chennai 600 020, India
Compound 1 was synthesized in good yield by reaction
Fax: (internat.) ϩ91-44-24911589
E-mail: bcunchem@rediffmail.com
of the ligand bzimpy with [Cr(dmso)6]3ϩ generated in situ.
Eur. J. Inorg. Chem. 2003, 3633Ϫ3638
DOI: 10.1002/ejic.200300170
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3633

V. G. Vaidyanathan, B. U. Nair
FULL PAPER
The ESI mass spectrum shows the base peak at m/z ϭ 672
due to [Cr(bzimpy)]ϩ. The ESI spectrum thus confirms the
authenticity of the complex. It is noteworthy that the ligand
behaves like a monoanion (through the loss of a proton
from a ϪNH group). Similar behaviour has been observed
in MnII complex of this ligand.[42] However, with
[Ru(bzimpy)2]2ϩ the ligand coordinates as a neutral triden-
tate ligand without such a loss of a proton.[43] Despite our
best efforts we could not grow diffraction quality crystals.
However, the MnII complex of the same ligand has been
crystallographically characterized and found to have a reg-
ular octahedral geometry.[42] The CrIII complex reported
here is expected to have a similar geometry. The cyclic vol-
tammogram of the complex shows a reduction of CrIII to
CrII at a cathodic peak potential, Epc, of Ϫ1.05 V versus
SCE. Reoxidation of the CrII species occurred at Ϫ0.81 V
upon reversal of the scan (Figure 1). The separation of an-
odic and cathodic peak potentials, ∆Ep ϭ 240 mV for
[Cr(bzimpy)2]ϩ, indicates a quasi-reversible redox process.
Such behavior is typical of many CrIII/CrII couples because
of the JahnϪTeller distortion expected for the CrII ion.
Figure 2. Electronic spectra of [Cr(bzimpy)2]ϩ in Hepes buffer
upon addition of calf thymus DNA, [Cr] ϭ 10 µ (—), [DNA] ϭ
20Ϫ150 µ (- - -)
[DNA]/(εA Ϫ εF) ϭ [DNA]/(εB Ϫ εF) ϩ 1/Kb (εA Ϫ εF)
(1)
A plot of [DNA]/(εA Ϫ εF) versus [DNA] revealed
moderate intrinsic binding constant Kb of
a
1.013Ϯ0.03 ϫ 104 Ϫ1, which is similar to those observed
for many other chromium and ruthenium complexes
(1Ϫ4 ϫ 104 Ϫ1),[40,45] and is more in keeping with groove
binding than intercalative binding. Further, since the com-
plex is octahedral, and upon coordination the planarity of
the ligand is lost, intercalative binding to DNA is highly un-
likely.
Figure 1. Cyclic voltammogram of [Cr(bzimpy)2]ϩ
Thermal Denaturation Studies
Absorption Titration
DNA melting is observed when double-stranded DNA
molecules are heated and separate into two single strands;
it occurs due to disruption in intermolecular forces such as
π stacking and hydrogen bonding interactions between
DNA base pairs. Here, a DNA melting experiment revealed
that Tm of calf thymus DNA was 65Ϯ0.2 °C and 68Ϯ0.2
°C in the absence and presence of the complex, respectively.
However, for the ligand alone, there is no change in DNA
melting temperature. The ∆Tm of 3 °C indicates that the
binding cannot be intercalative. Such a small ∆Tm is gener-
ally associated with surface binding of the metal complex.
Generally one would expect a much larger increase in Tm
for classical intercalators.[46Ϫ49] A small ∆Tm also indicates
weak binding of the complex to DNA. This is also reflected
in the binding constant.
Absorption titration can monitor the interaction of a me-
tal complex and DNA. In general, hypochromism and red-
shift are associated with the intercalative binding of the
complex to the helix, due to strong stacking interactions
between the aromatic chromophore of the complex and the
base pairs of DNA. The chromium complex 1 shows an
absorbance at 317 nm due to the intraligand (πϪπ*) tran-
sition of the ligand bzimpy. Binding of chromium() with
DNA is expected to change markedly its electronic spec-
trum. Upon addition of CT DNA, the chromium() com-
plex absorbance increases, and is accompanied by a small
shift of 3 nm in λmax, from 317 to 320 nm (Figure 2), that
is consistent with groove binding, leading to small pertur-
bations. Such a small increase in λmax and hyperchromicity
occurs with some porphyrin and copper complexes on their
interaction with DNA.[44] This hyperchromism can be attri-
buted to external contact (surface binding) with the duplex.
The absorption data were analyzed to evaluate the intrinsic
Viscosity Measurement
Optical photophysical probes generally provide support
binding constant Kb, which can be determined from Equa- for a binding model. Hydrodynamic measurements that are
tion (1), where εA corresponds to Aobsd/[Cr] and εF, and εB sensitive to length change are, in the absence of crystallo-
are the molar absorption coefficients for the free chromium graphic data, regarded as the most critical tests of a binding
complex and the chromium complex in the fully bound model in solution. Intercalating agents are expected to de-
form, respectively.
stack the base pairs to accommodate the ligands, causing
3634
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Inorg. Chem. 2003, 3633Ϫ3638

DNA Binding Studies of a Chromium(III) Complex Containing a Tridentate Ligand
FULL PAPER
elongation of the double helix and an increase in the vis- potential corresponding to the zero-zero spectroscopic en-
cosity of the DNA. However, some complexes, which bind ergy of the excited state.
through non-intercalative modes, decrease the viscosity in-
itially and then increase it at higher concentrations. With
chromium complex 1, the viscosity of DNA showed a com-
plicated dependence on complex concentration. The change
in viscosity with the addition of the complex to DNA is
shown in Figure 3. The viscometric data rules out classical
intercalation. Similar behaviour has been observed on the
addition of [Cu(bcp)2]ϩ to ST DNA.[50] The viscometric re-
sults could be explained in terms of a bridged structure to
promote the extension of duplexes. Here the binding may
involve inner-sphere complex formation via phosphate oxy-
gen, indicating that the binding of CrIII complex with DNA
could be surface binding or by forming bridged adducts.
Adducts formed by bridging duplexes are stabilized by
hydrophobic interaction or coulombic interaction, as with
[Cu(bcp)2]ϩ and [Cu(dpsmp)2(H2O)].[51Ϫ53]
Figure 4. Emission spectra of [Cr(bzimpy)2]ϩ in aqueous solution
in a) absence and b) presence of DNA. [Cr] ϭ 10 m, [DNA] ϭ
200Ϫ800 m. Inset: Relative emission intensity of [Cr(bzimpy)2]ϩ
versus [DNA]/[Cr]
E0(*Cr3ϩ/Cr2ϩ) ϭ E0(Cr3ϩ/Cr2ϩ) Ϫ E0Ϫ0(Cr3ϩ/*Cr3ϩ
)
(2)
The reported excited state potentials of [Cr(bpy)3]3ϩ and
[Cr(phen)3]3ϩ are 1.44 and 1.42 V, which are much greater
than the 0.92 V in the present case. Hence, compared to
*[Cr(bpy)3]3ϩ and *[Cr(phen)3]3ϩ, the excited state of
[Cr(bzimpy)2]ϩ is not a good oxidizing agent to oxidize the
guanine base; as the excited state potential is low compared
to that of guanine base.
Figure 3. Effect of increasing amounts of [Cr(bzimpy)2]ϩ (Ϫ·Ϫ) on
the relative viscosity of DNA
؉
DNA Cleavage by Cr(bzimpy)2
The ability of [Cr(bzimpy)2]ϩ to serve as a metallonucle-
ase has been examined. The reaction of [Cr(bzimpy)2]ϩ with
pBR322 DNA was monitored by observing the conversion
of supercoiled plasmid DNA (fastest migrating species) into
the circular, nicked form (slowest migrating species). All re-
actions were evaluated by comparing the amount of circular
plasmid to the amount present in plasmid controls. Neither
relaxation nor nicking is observed in control reactions of
DNA alone or with DNA and either [Cr(bzimpy)2]ϩ or hy-
drogen peroxide (Figure 5. lanes 1Ϫ3). However, with hy-
Fluorescence Spectroscopic Study
The emission spectra of [Cr(bzimpy)2]ϩ in the absence
and the presence of CT DNA is shown in Figure 4. The
luminescence spectrum of complex 1 shows an emission
band at 717 nm when excited at 320 nm. The band at
717 nm is attributable to the 2EgǞ4A2g transition of
[Cr(bzimpy)2]ϩ. Addition of DNA causes a gradual in-
crease in the fluorescence intensity and the emission maxi-
mum also shifts by 2 nm to a longer wavelength. This is
because, in the presence of DNA, the metal complex is
bound in a relatively non-polar environment compared to
water. Such an increase in fluorescence intensity has been
reported for certain intercalators upon their binding to
DNA. The increase observed here, however, is less than that
observed for classical intercalators. In contrast,
[Cr(bpy)3]2ϩ and [Cr(phen)3]2ϩ show a decrease in lumi-
nescence intensity in the presence of DNA, indicating that
the excited state of these complexes are strong oxidizing ag-
ents that could oxidize guanine base.[54Ϫ55] The excited state
potential for complex 1 was calculated as 0.92 V from
Figure 5. Nicking of pBR322 DNA by [Cr(bzimpy)2]ϩ over time.
Lane 1, control DNA alone; lane 2, DNA ϩ complex 1; lane 3,
DNA ϩ H2O2; lanes 4 and 5, DNA ϩ complex 1 ϩ H2O2 incu-
bated for 30 and 60 min, respectively. Lane 6, DNA ϩ complex 1
ϩ H2O2 ϩ EtOH (1 ); lane 7, DNA ϩ complex 1 ϩ -mannitol
Equation (2), where E0Ϫ0(Cr3ϩ/*Cr3ϩ) is the one-electron (500 m)
Eur. J. Inorg. Chem. 2003, 3633Ϫ3638
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3635

V. G. Vaidyanathan, B. U. Nair
FULL PAPER
drogen peroxide, [Cr(bzimpy)2]ϩ relaxes supercoiled Experimental Section
pBR322 DNA in a time-dependent manner (lanes 4,5). At
Materials: o-Phenylenediamine, 2,6-pyridinedicarboxylic acid, and
calf thymus DNA were obtained from Sigma chemicals, USA. Tris,
Hepes, agarose, and pBR322 DNA were obtained from Bangalore
Genei, Bangalore. All solvents were obtained from Ranbaxy, India.
Milli Q water was used for preparing buffers.
longer times, the supercoiled form is completely converted
into the open circular form (form II) in the presence of CrIII
complex. The time dependence of the reaction suggests that
[Cr(bzimpy)2]ϩ acts as a catalyst. In the presence of CrIII
complex, H2O2 produces radical species, which cause the
DNA cleavage. The addition of radical traps (1  EtOH or
-mannitol) to reaction mixtures containing complex and
H2O2 inhibited the cleavage of DNA (lanes 6 and 7 of Fig-
ure 6). Gel electrophoresis of similar DNA-cleaving experi-
ments with CT DNA showed that complex 1 cleaved the
CT DNA in a non-specific manner, in the presence of
H2O2. The presence of hydroxyl radicals has been shown
to lead to hydrolysis of p-nitrophenyl phosphate ester.[56]
Treatment of p-nitrophenyl phosphate with complex 1 and
H2O2 generates p-nitrophenol, which has an absorption at
404 nm (Figure 6). This confirms the formation of hydroxyl
radicals from hydrogen peroxide in the presence of com-
plex 1.
Synthesis of [Cr(bzimpy)2]Cl (1): 2,6-Bis(benzimidazo-2-yl)pyridine
was synthesized according to the published procedure.[57] An excess
of DMSO was added to chromium() chloride salt (1 mmol,
0.267 g) and boiled under reflux for 1 h. The ligand bzimpy
(2 mmol, 0.622 g) was then added to the resulting mixture, and re-
fluxing was continued for a further hour. The volume of solvent
was then minimised by rotary evaporation and a reddish orange
precipitate was obtained on addition of diethyl ether. The precipi-
tate was filtered off and dried in vacuo. The compound was then
recrystallised from acetonitrile (yield 0.66 g, 75%); found C 64.33,
H 3.34, N 19.80, Cr 7.32% calcd. for C38H24ClCrN10 (M.wt. 707.5):
C 64.43, H 3.41, N 19.78, Cr 7.34%. IR (KBr pellet): 3401 (NϪH),
3057 (CϪH), and 1615 (CϭN) cmϪ1
.
Physical Measurements: UV/Visible spectra of the complex and
DNA binding studies were recorded on a PerkinϪElmer Lambda
35 spectrophotometer at 25 °C. Elemental analysis was performed
using a Heraeus-CHN-Rapid Analyzer at RSIC, IIT, Madras. The
emission spectra were recorded on a Hitachi 650Ϫ40 spectrofluori-
meter. The electrospray ionization (ESI) mass spectrum of the com-
plex was recorded with a HewlettϪPackard 1100 mass spec-
trometer equipped with an electron spray source. The infrared spec-
trum of the complex was recorded on a Perkin-Elmer FT-IR spec-
trometer. Cyclic voltammetry was performed on an EG and G PAR
173 potentiostat/Galvanostat analyzer. Tetrabutylammonium per-
chlorate (TBAP) was used as supporting electrolyte. The sample in
dried DMSO was purged with nitrogen prior to measurement. A
standard three-electrode system consisted of a glassy carbon
working electrode, platinum auxiliary electrode and a saturated
calomel reference electrode (SCE).
Figure 6. Kinetics of phosphate ester hydrolysis of p-nitro-
phenylphosphate by complex 1 in the presence of hydrogen
peroxide
DNA Binding Experiments: All experiments involving the interac-
tion of the complex with DNA were carried out in Hepes buffer
(10 m, pH 7.5). A solution of calf thymus DNA in the buffer gave
a ratio of UV absorbance at 260 and 280 nm of about 1.8Ϫ1.9:1,
indicating that the DNA was sufficiently free from protein.[58] The
DNA concentration per nucleotide was determined by absorption
spectroscopy using the molar absorption coefficient (6600
Conclusions

Ϫ1cmϪ1) at 260 nm.[59]
The electronic spectra of the CrIII complex were monitored in both
the presence and absence of DNA. The binding constant for the
interaction of CrIII complex 1 with DNA was obtained from ab-
sorption titration data. A fixed concentration of complex 1 (10 µ)
was titrated with increasing amounts of DNA over the range
20Ϫ150 µ.
Chromium complexes having a tridentate ligand can bind
DNA with moderate strength. Spectroscopic and viscosity
measurements show that the CrIII complex interacts with
the DNA surface. A fluorescence study shows that the emis-
sion intensity of the complex increases on addition of DNA
as the environment around the complex is more hydro-
phobic in the presence of DNA. The emission of both
[Cr(phen)3]3ϩ and [Cr(bpy)3]3ϩ is quenched on binding to
DNA. The emission of [Cr(bzimpy)]2ϩ, however, is not
quenched by DNA as it has a lower excited state potential
than that for guanine base. Gel electrophoresis shows that
[Cr(bzimpy)2]ϩ nicks DNA in the presence of H2O2. The
amount of nicked DNA formed increases with increasing
incubation time of the CrIII complex and H2O2 with DNA.
The nicking of DNA is inhibited by radical scavengers such
as ethanol and -mannitol.
During measurement of the absorption spectra, an equal amount
of DNA solution was added in both complex solution and refer-
ence solution to eliminate the absorbance of DNA itself.
Thermal denaturation studies were conducted in a PerkinϪElmer
Lambda 35 spectrophotometer supplied with thermostatted cell
holder. The temperature was controlled by a Peltier system (Ϯ0.1
°C). The absorbance at 260 nm was monitored for DNA (100 µ)
in the absence and presence of the complex (10 µ).
Viscosity measurement was carried out on an Ostwald viscometer,
immersed in a thermostatted water bath maintained at 28Ϯ0.1 °C.
3636
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Inorg. Chem. 2003, 3633Ϫ3638

DNA Binding Studies of a Chromium(III) Complex Containing a Tridentate Ligand
FULL PAPER
[17]
D. S. Sigman, Acc. Chem. Res. 1986, 19, 180Ϫ186.
A. M. Pyle, J. K. Barton, Prog. Inorg. Chem. 1990, 38,
413Ϫ475.
The DNA concentration was kept constant (150 µ) and that of
the metal complex was varied from 5 to 75 µ. The flow time was
measured with a stop-watch operated manually, and each sample
was measured five times and an average flow time calculated. Data
are presented as (η/ηo)1/3 versus [Cr]/[DNA], where η is the vis-
cosity of DNA in the presence of complex, and ηo is the viscosity
of DNA alone. Viscosity values were calculated from the observed
flow time of DNA-containing solutions corrected for the flow time
of buffer alone (to), η ϭ t Ϫ to.[60]
[18]
[19]
[20]
[21]
[22]
S. Sigman, A. Mazumdar, D. M. Perrin, Chem. Rev. 1993, 93,
2295Ϫ2316.
K. Naing, M. Takashani, M. Taniguchi, A. Yamagishi, Inorg.
Chem. 1995, 34, 350Ϫ356.
F. L. Liu, K. A. Meadows, D. R. Millin, J. Am. Chem. Soc.
1993, 115, 6699Ϫ6704.
Y. Xiong, X.-F. He, X.-H. Zou, J.-Z. Wu, X.-M. Chen, L.-N.
Ji, R.-H. Li, J.-Y. Zhou, K.-B. Yu, J. Chem. Soc., Dalton Trans.
1999, 19Ϫ24.
IARC, 1990, 49, 49Ϫ256.
U. Glaser, D. Hochrainer, H. Kloppel, H. Oldiges, Toxicology
1986, 42, 219Ϫ232.
A. Leonard, R. R. Lauwreys, Mutat. Res. 1980, 76, 227Ϫ239.
S. De Flora, M. Bagnasco, D. Serra, P. Zanacchi, Mutat. Res.
1990, 238, 99Ϫ172.
J.-L. Yang, Y.-C. Hsieh, C.-W. Wu, T.-C. Lee, Carcinogenesis
1992, 12, 2053Ϫ2057.
S. Kawanishi, S. Inoue, S. Sano, J. Biol. Chem. 1986, 261,
5952Ϫ5958.
X. Shi, N. S. Dalal, Environ. Health. Perspect. 1994, 102,
231Ϫ236.
A. Kortenkamp, G. Oetken, D. Beyersmann, Mutat. Res. 1990,
232, 155Ϫ161.
A. Mikalsen, J. Alexander, H. Wallin, M. I. Sundberg, R. A.
Andersen, Carcinogenesis 1991, 12, 825Ϫ831.
M. Sugiyama, K. Tsuzuki, R. Ogura, J. Biol. Chem. 1991,
266, 3383Ϫ3386.
R. A. Anderson, Biol. Trace Element Res. 1992, 32, 19Ϫ24.
R. A. Anderson, N. Cheng, N. A. Bryden, M. M. Polansky, N.
Cheng, J. Chi, J. Feng, Diabetes 1997, 46, 1786Ϫ1791.
A. Ravina, L. Slezack, Harefuah 1993, 125, 142Ϫ145.
D. M. Stearns, J. P. Wise, S. R. Patierno, K. E. Wetterhahn,
FASEB J. 1995, 9, 1643Ϫ1648.
In steady state fluorescence experiments, the complex concen-
tration (10 µ) was fixed while that of DNA was varied (200Ϫ800
µ). The chromium() complex in the presence of DNA was ex-
cited at 320 nm and the fluorescence spectra were recorded between
650 and 850 nm.
[23]
[24]
[25]
[26]
Phosphate ester hydrolysis was monitored on a PerkinϪElmer
Lambda 35 spectrophotometer at 25 °C by measuring the pro-
duction of p-nitrophenol at 404 nm. The background reaction was
carried out with the peroxide and ester alone. A reaction mixture
containing CrIII complex 1 (0.025 m), peroxide (0.1 m, freshly
prepared) and p-nitrophenylphosphate (1 m) in Hepes (10 m,
pH 7.0) was used to determine the rate of p-nitrophenol formation
at 404 nm.
[27]
[28]
[29]
[30]
[31]
[32]
For gel electrophoresis experiments, supercoiled pBR322 DNA
(800 ng) was treated with CrIII complex (100 µ) in Tris-EDTA
buffer (10 m, pH 8.0) and the solution was incubated for 16 h at
room temperature. H2O2 (500 µ) was then added and a timed
assay was carried out. The reactions were quenched at various
times by adding loading buffer. The samples were analyzed by elec-
trophoresis for 4 h at 50 V on a 0.8% agarose gel in TrisϪboric
acidϪEDTA buffer. The gel was stained with 0.5 µg/mL ethidium
bromide and photographed under UV light.
[33]
[34]
[35]
[36]
[37]
S. E. Kohlami, M. Gattke, E. M. McIntosh, B. A. Kunz, J.
Mol. Biol. 1991, 220, 933Ϫ946.
Acknowledgments
V. G. V thanks the CSIR for a research fellowship.
[38]
[39]
J. Singh, E. T. Snow, Biochemistry 1998, 37, 9371Ϫ9378.
R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, B. U.
Nair, Biochem. Biophys. Res. Commun. 2000, 271, 731Ϫ734.
R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, B. U.
Nair, Biochim. Biophys. Acta 2000, 1475, 157Ϫ162.
K. D. Sugden, R. D. Geer, S. J. Rogers, Biochemistry 1992,
31, 11626Ϫ11631.
R. Rajan, R. Rajaram, B. U. Nair, T. Ramasami, S. K. Mandal,
Dalton Trans. 1996, 2019Ϫ2020.
V. G. Vaidyanathan, B. U. Nair, J. Inorg. Biochem. 2002, 91,
405Ϫ412.
R. F. Pasternack, E. J. Gibbs, J. J. Villafranca, Biochemistry
1983, 22, 2406Ϫ2414.
M. Erikkson, M. Leijon, G. Hiort, B. Norden, A. Graslund, J.
Am. Chem. Soc. 1992, 114, 4933Ϫ4934.
M. Cory, D. D. McKee, J. Kagan, D. W. Henry, J. A. Miller, J.
Am. Chem. Soc. 1985, 107, 2528Ϫ2536.
M. J. Waring, J. Mol. Biol. 1965, 13, 269Ϫ282.
J. M. Kelly, A. B. Tossi, D. J. McConell, C. OhUigin, Nucleic
Acids Res. 1985, 13, 6017Ϫ6034.
G. A. Neyhart, N. Grover, S. R. Smith, W. A. Kalsbeck, T.
A. Fairley, M. Cory, H. H. Thorp, J. Am. Chem. Soc. 1993,
115, 4423Ϫ4428.
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[1]
S. M. Hecht, Acc. Chem. Res. 1986, 19, 383Ϫ391.
[2]
T. D. Tullius, B. A. Dombroski, Science 1985, 235, 679Ϫ681.
[3]
A. M. Burkoff, T. D. Tullius, Nature 1988, 331, 455Ϫ457.
[4]
A. M. Burkoff, T. D. Tullius, Cell 1987, 48, 935Ϫ943.
[5]
W. K. Pogozelski, T. K. Tullius, Chem. Rev. 1998, 98,
1089Ϫ1108.
[6]
C. J. Burrows, J. G. Muller, Chem. Rev. 1998, 98, 1109Ϫ1152.
[7]
A. E. Friedman, J. K. Barton, J. Am. Chem. Soc. 1990, 112,
4960Ϫ4962.
[8]
R. M. Hartshorn, J. K. Barton, J. Am. Chem. Soc. 1992, 114,
5919Ϫ5925.
C. J. Murphy, J. K. Barton, Methods Enzymol. 1993, 226,
576Ϫ594.
[9]
[10]
Y. Jenkins, A. E. Friedman, N. J. Turro, J. K. Barton, Biochem-
[47]
[48]
istry 1992, 31, 10809Ϫ10816.
C. Turro, S. H. Bossmann, Y. Jenkins, J. K. Barton, N. J. Turro,
[11]
J. Am. Chem. Soc. 1995, 117, 9026Ϫ9032.
N. Gupta, N. Grover, G. A. Neyhart, W. Liang, P. Singh, H.
[49]
[12]
H. Thorpe, Angew. Chem. Int. Ed. Engl. 1992, 31, 1048Ϫ1050.
X.-H. Zou, B.-H. Ye, H. Li, Q.-L. Zhang, H. Chao, J.-G. Liu,
[13]
[50]
[51]
[52]
[53]
F. Liu, K. A. Meadows, D. R. McMillin, J. Am. Chem. Soc.
1993, 115, 6699Ϫ6704.
P. G. Schultz, J. S. Taylor, P. B. Dervan, J. Am. Chem. Soc.
1982, 104, 6861Ϫ6863.
S. Mahadevan, M. Palaniandavar, Inorg. Chem. 1998, 37,
693Ϫ700.
G. Yang, F. Z. Wu, L. Wang, L. NianFi, X. Tina, J. Inorg.
Biochem. 1997, 66, 141Ϫ144.
L.-N. Ji, J. Biol. Inorg. Chem. 2001, 6, 143Ϫ150.
[14]
C. J. Burrows, S. E. Rokita, Acc. Chem. Res. 1994, 27,
295Ϫ301.
[15]
T. D. Tullius, Metal-DNA chemistry, ACS Symposium Series
402, American Chemical Society, Washington DC, 1989, pp.
1Ϫ23.
[16]
A. R. Banerjee, J. A. Jaeger, D. H. Turner, Biochemistry 1993,
32, 153Ϫ163.
Eur. J. Inorg. Chem. 2003, 3633Ϫ3638
www.eurjic.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3637

V. G. Vaidyanathan, B. U. Nair
FULL PAPER
[54]
[57]
M. A. Jamieson, N. Serpone, M. Z. Hoffman, Coord. Chem.
A. W. Addison, P. J. Burke, J. Heterocycl. Chem. 1981, 18,
803Ϫ805.
J. Marmur, J. Mol. Biol. 1961, 3, 208Ϫ218.
M. F. Reichmann, S. A. Rice, C. A. Thomas, P. Doty, J. Am.
Chem. Soc. 1954, 76, 3047Ϫ3053.
G. Cohen, H. Eisenberg, Biopolymers 1969, 8, 45Ϫ55.
Received March 29, 2003
Rev. 1981, 39, 121Ϫ179.
R. T. Watson, N. Desai, J. Wildsmith, J. F. Wheeler, N. A. P.
[55]
[58]
[59]
Kane-Maguire, Inorg. Chem. 1999, 38, 2683Ϫ2687.
A. Parand, A. C. Royer, T. L. Cantrell, M. Weitzel, N. Memon,
[56]
[60]
J. B. Vincent, M. W. Crowder, Inorg. Chim. Acta 1998, 268,
211Ϫ219.
3638
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
Eur. J. Inorg. Chem. 2003, 3633Ϫ3638