Dicopper(II) complexes showing DNA hydrolase activity and monomeric adduct formation with bis(4-nitrophenyl)phosphate

Article abstract of DOI:10.1016/j.ica.2011.04.046

Ferromagnetic dicopper(II) complexes [Cu₂(μ-O ₂CCH₃)(μ-OH)(L)₂(μ-L¹)] (PF₆)₂, where L = 1,10-phenanthroline (phen), L ¹ = H₂O in 1 and L = dipyrido[3,2-d:2′,3′-f] quinoxaline (dpq), L¹ = CH₃CN in 2, are prepared and structurally characterized. Crystals of 1 and 2 belong to the monoclinic space group of P2₁/n and P2₁/m, respectively. The copper(II) centers display distorted square-pyramidal geometry having a phenanthroline base and two oxygen atoms of the bridging hydroxo and acetate group in the basal plane. The fifth coordination site has weak axially bound bridging solvent molecule H₂O in 1 and CH₃CN in 2. The Cu·· ·Cu distances are 3.034 and 3.046 in 1 and 2, respectively. The complexes show efficient hydrolytic cleavage of supercoiled pUC19 DNA as evidenced from the mechanistic studies that include T4 DNA ligase experiments. The binuclear complexes form monomeric copper(II) adducts [Cu(L)₂(BNPP)](PF ₆) (L = phen, 3; dpq, 4) with bis(4-nitrophenyl)phosphate (BNPP) as a model phosphodiester. The crystal structures of 3 and 4 reveal distorted trigonal bipyramidal geometry in which BNPP binds through the oxygen atom of the phosphate. The kinetic data of the DNA cleavage reactions of the binuclear complexes under pseudo- and true-Michaelis-Menten conditions indicate remarkable enhancement in the DNA hydrolysis rate in comparison to the control data.

Full text of DOI:10.1016/j.ica.2011.04.046

Inorganica Chimica Acta 375 (2011) 173–180
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Inorganica Chimica Acta
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Dicopper(II) complexes showing DNA hydrolase activity and monomeric adduct
formation with bis(4-nitrophenyl)phosphate
⇑
Mithun Roy, Shanta Dhar, Basudev Maity, Akhil R. Chakravarty
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Ferromagnetic dicopper(II) complexes [Cu2(l-O2CCH3)(l-OH)(L)2(l
-L1)](PF6)2, where L = 1,10-phenan-
Article history:
throline (phen), L1 = H2O in 1 and L = dipyrido[3,2-d:20,30-f]quinoxaline (dpq), L1 = CH3CN in 2, are pre-
pared and structurally characterized. Crystals of 1 and 2 belong to the monoclinic space group of P21/n
and P21/m, respectively. The copper(II) centers display distorted square-pyramidal geometry having a
phenanthroline base and two oxygen atoms of the bridging hydroxo and acetate group in the basal plane.
The fifth coordination site has weak axially bound bridging solvent molecule H2O in 1 and CH3CN in 2.
The CuꢀꢀꢀCu distances are 3.034 and 3.046 Å in 1 and 2, respectively. The complexes show efficient hydro-
lytic cleavage of supercoiled pUC19 DNA as evidenced from the mechanistic studies that include T4 DNA
ligase experiments. The binuclear complexes form monomeric copper(II) adducts [Cu(L)2(BNPP)](PF6)
(L = phen, 3; dpq, 4) with bis(4-nitrophenyl)phosphate (BNPP) as a model phosphodiester. The crystal
structures of 3 and 4 reveal distorted trigonal bipyramidal geometry in which BNPP binds through the
oxygen atom of the phosphate. The kinetic data of the DNA cleavage reactions of the binuclear complexes
under pseudo- and true-Michaelis–Menten conditions indicate remarkable enhancement in the DNA
hydrolysis rate in comparison to the control data.
Received 28 January 2011
Received in revised form 25 April 2011
Accepted 30 April 2011
Available online 7 May 2011
Keywords:
Copper
Hydrolytic DNA cleavage
Phenanthroline bases
Crystal structures
Bis(4-nitrophenyl)phosphate adduct
Reaction kinetics
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
The synthetic metallonucleases catalyzing phosphodiester bond
hydrolysis are useful for biochemical and biomedical applications,
Transition metal complexes that are able to cleave DNA under
physiological conditions are of importance for various applications
in nucleic acids chemistry [1–13]. The double helical structure of
DNA could be damaged by two major pathways: (i) hydrolytic
cleavage of the phosphodiester linkage or (ii) oxidative DNA cleav-
age due to sugar and/or nucleobase oxidation. The half-life for
hydrolysis of the phosphodiester bond in DNA at neutral pH and
25 °C is estimated to be ꢁ16 million years. The hydrolytic stability
of DNA is primarily due to the negatively charged phosphate back-
bone that inhibits attack by the nucleophiles. DNA hydrolysis could
be readily achieved enzymatically. Natural nucleases like purple
acid phosphatase, P1 nuclease, S1 nuclease, endonuclease IV, etc.
containing transition metal ions are known to enhance the rate
of hydrolysis of DNA to a significant order for cellular function
[14–16]. The nucleases which are activated by metal ions are
known as metallonucleases. The natural restriction enzymes have
been used for cellular applications like recombinant DNA technol-
ogy. But their large size and limited sequence selectivity prevent
their applications for general purpose. It is thus of great interest
and importance to develop artificial metallonucleases that show
efficient DNA hydrolase activity.
as DNA conformational probes and chemotherapeutic agents.
Earlier reports have shown that lanthanide ions are suitable for
hydrolysis of DNA [17–22]. Ruthenium-based complexes are
known as efficient synthetic hydrolases [23]. Mononuclear Zn(II)-
binding peptide, tethered to a rhodium complex promotes plasmid
DNA cleavage with a rate constant of 2.5 ꢂ 10ꢃ5 sꢃ1 at 37 °C (pH
6.0) [24–26]. Although Fe(III) is present in the active site of some
phosphatases, its model complexes are virtually unknown as arti-
ficial nucleases. Binuclear diiron(III) complexes of benzimidazolyl-
methyl derivatives of 1,3-diamino-2-hydroxypropane (HPTB) and
1,4,7-triazaheptane (DTPB) are known to show significant nuclease
activity with a rate constant of ꢁ10ꢃ3 sꢃ1 [27,28]. The cobalt(III)
complex of a polyamine ligand cleaves plasmid DNA with a first
order rate constant of 5 ꢂ 10ꢃ5 sꢃ1 [29]. Copper(II) complex of tri-
azacyclononane cleaves DNA at 50 °C (pH 7.8) with a rate constant
of ꢁ1.5 ꢂ 10ꢃ5 sꢃ1 [30,31]. Copper(II) complexes of natural amino-
glycosides such as neamine efficiently cleave DNA hydrolytically
with a rate constant of 5.2 ꢂ 10ꢃ4 sꢃ1 [32,33]. We have recently
reported [Cu(dpq)2(H2O)](ClO4)2 as a potent model DNA hydrolase
showing rate enhancement of 1.55 ꢂ 108 fold (dpq, dipyridoqui-
noxaline) [34].The dppz analog cleaves DNA at a comparatively
lower rate than the dpq complex [35].
The present work stems from our continued interest to explore
the hydrolytic DNA cleavage potential of copper(II) complexes. We
⇑
Corresponding author. Tel.: +91 80 22932533; fax: +91 80 23600683.
E-mail address: arc@ipc.iisc.ernet.in (A.R. Chakravarty).
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.04.046

174
M. Roy et al. / Inorganica Chimica Acta 375 (2011) 173–180
Scheme 1. Dicopper(II) complexes [Cu2(
l
-OAc)(
l
-OH)(L)2(L1)](PF6)2, where L and L1 are phen, H2O in 1 and dpq, CH3CN in 2, respectively, and copper(II) complexes
[Cu(L)2(BNPP)](PF6), where L is phen in 3, dpq in 4 and BNPP is bis(4-nitrophenyl)phosphate.
have chosen hydroxo and acetato-bridged dicopper(II) complexes
considering the presence of labile site(s) in these complexes to sig-
nificantly promote the rate of hydrolytic DNA cleavage. Herein, we
present the synthesis, magnetic properties, crystal structure and
hydrolytic DNA cleavage activity of two hydroxo and acetato-
Experimental susceptibility data were corrected for diamagnetic
contributions [37]. The molar magnetic susceptibilities were fitted
by Bleaney–Bowers expression:
N , where
v
Cu = [Ng2b2/kT][3 + exp(ꢃ2J/kT)]ꢃ1
+
v
is the molar magnetic susceptibility per copper and J is
a
the magnetic exchange parameter, by means of a least-squares
program [38].
bridged dicopper(II) complexes [Cu2(
L1)](PF6)2 (1 and 2), where L = 1,10-phenanthroline (phen),
L1 = H2O in and L = dipyrido[3,2-d:20,30-f]quinoxaline (dpq),
l-O2CCH3)(l-OH)(L)2(l-
1
L1 = CH3CN in 2 (Scheme 1). Significant result of this study is the
remarkable enhancement in the DNA hydrolysis rate in compari-
son to the control data. We have studied the mechanistic aspects
of the DNA cleavage reactions using bis(4-nitrophenyl)phosphate
(BNPP) as a model phosphate ester. Structural study on the reac-
tion product show formation of novel monomeric BNPP adduct
[Cu(L)2(BNPP)](PF6) (L = phen in 3; dpq in 4) having copper(II)
bound to the phosphate moiety of BNPP (Scheme 1).
2.3. Synthesis
2.3.1. Preparation of [Cu2(phen)2(
and [Cu2(dpq)2( -O2CCH3)( -OH)(l
l
-O2CCH3)(
-CH3CN)](PF6)2 (2)
l-OH)(l-H2O)](PF6)2 (1)
l
l
Complexes 1 and 2 were prepared by a general procedure in
which a 0.2 g (0.5 mmol) quantity of dimeric copper(II) ace-
tate.hydrate in 10 ml of aqueous CH3CN (1:9 v/v) was reacted with
the appropriate heterocyclic base (0.9 mmol; phen, 180 mg; dpq,
210 mg) in 15 ml acetonitrile. The solution was stirred for 30 min
at 25 °C. The product was obtained as a blue solid in ꢁ85% yield
on addition of a solution of NaPF6 (1.0 mmol) in CH3CN. The solid
was isolated, washed with aqueous methanol and dried in vacuum
2. Experimental
2.1. Materials
over P4O10
.
Anal. Calc. for C26H22N4O4P2F12Cu2 (1): C, 35.83; H, 2.54; N, 6.43.
Found: C, 36.02; H, 2.61; N, 6.21%. FT-IR (KBr phase, cmꢃ1): 3355br,
2950w, 2000w, 1830w, 1795w, 1620w, 1580 m, 1425s, 1295s,
1225w, 1150m, 980w, 835vs (PF6ꢃ), 775m, 670s, 550m, 460w
(br, broad; vs very strong; s, strong; m, medium; w, weak). ESI-
MS in aqueous CH3OH: m/z 290.6 [(M-2(PF6)]2+. UV–Vis in Tris–
All reagents and chemicals were purchased from commercial
sources and used without further purification. Calf thymus (CT)
DNA, supercoiled (SC) pUC19 DNA (cesium chloride purified), T4
ligase and 5ꢂ ligation buffer were purchased from Bangalore Genie
(India). Agarose (molecular biology grade), distamycin and ethi-
dium bromide (EB) were from Sigma (USA). Tris(hydroxy-
methyl)aminomethane–HCl (Tris–HCl) buffer was prepared using
deionized and sonicated triple distilled water. Dipyrido[3,2-
d:20,30-f]quinoxaline (dpq) was prepared following a literature
method [36].
HCl buffer [kmax, nm (e
, Mꢃ1 cmꢃ1)]: 272 (63 280), 293sh (21
290), 708 (80). KM in CH3CN (S m2 Mꢃ1): 255. leff at 298 K: 2.06
l
B/Cu (2J = 51 cmꢃ1).
Anal. Calc. for C32H23N9O3P2F12Cu2ꢀH2O (2ꢀH2O): C, 37.87; H,
2.48; N, 12.40. Found: C, 37.62; H, 2.61; N, 12.28%. FT-IR (KBr
phase, cmꢃ1): 3360br, 3100w, 2360w, 1630w, 1795w, 1560s,
1485m, 1410s, 1210w, 1130m, 1030w, 980w, 830vs (PF6ꢃ),
730m, 555m, 430w. ESI-MS in H2O: m/z 354.1 [(M-2(PF6)]2+. UV–
2.2. Physical measurements
The elemental analysis was done using a Thermo Finnigan Flash
EA 1112 CHNSO analyzer. The infrared and electronic spectra
were recorded on Bruker Equinox 55 and Hitachi U-3000 spectro-
photometers, respectively. Molar conductivity measurements
were done using a Control Dynamics (India) conductivity meter.
Variable temperature magnetic susceptibility data in the tem-
perature range 20–300 K were obtained for polycrystalline
samples using a George Associates Inc. (Berkeley, USA) Lewis-
coil-force magnetometer. Hg[Co(NCS)4] was used as a standard.
Vis in Tris–HCl buffer [kmax, nm (
296sh (30 680), 337 (910), 640 (90). KM in CH3CN (S m2 Mꢃ1):
265. leff at 298 K: 1.98
B/Cu (2J = 48 cmꢃ1).
e
, Mꢃ1 cmꢃ1)]: 256 (87 590),
l
2.3.2. Preparation [Cu(L)2(BNPP)](PF6) (L = phen, 3; dpq, 4)
The precursor complexes were dissolved in water and 1.5
equivalent of bis(4-nitrophenyl)phosphate (BNPP) was added.
The reaction mixture was stirred for 4 h at room temperature.

M. Roy et al. / Inorganica Chimica Acta 375 (2011) 173–180
175
Crystalline complexes were obtained in moderate yield (ꢁ30%) by
slow evaporation of the solvent.
were corrected for Lorentz-polarization effects. Empirical absorp-
tion corrections were made using the SADABS program [39].
Anal. Calc. for C36H24N6O8P2F6Cu (3): C, 47.61; H, 2.66; N, 9.25.
Found: C, 47.43; H, 2.78; N, 9.02%. FT-IR (KBr phase, cmꢃ1): 3380br,
3078w, 1582m, 1520w, 1430w, 1345m, 1220s, 1160m, 1090m,
835vs (PF6ꢃ), 640 m, 736s, 624s, 440w. ESI-MS in aqueous DMF:
2.4.2. Structure solution and refinement
The structures were solved by heavy-atom method and refined
by full matrix least-squares using SHELX system of programs [40]. All
non-hydrogen atoms were refined anisotropically. However, the
m/z 761.93 [(M-(PF6)]+. UV–Vis in Tris HCl buffer [kmax, nm (
e,
Mꢃ1 cmꢃ1)]: 272 (115 640), 294 (70 480), 544sh (310), 691br. KM
in CH3CN (S m2 Mꢃ1): 140. leff (298 K): 1.88 lB
ꢃ
F1, F1A, F4 and F4A atoms of the PF6 anion in 3 were refined iso-
.
tropically due to positional disorder. The hydrogen atoms attached
to the carbon atoms were placed at their fixed positions, assigned
isotropic thermal parameters and refined using a riding model for
structure factor calculation only. The perspective views of the com-
plexes were obtained using ORTEP program [41]. Selected crystallo-
graphic data for the complexes are given in Table 1.
Anal. Calc. for C40H24N10O8P2F6Cuꢀ2H2O (4ꢀH2O): C, 45.83; H,
2.69; N, 13.36. Found: C, 45.62; H, 2.59; N, 13.18%. FT-IR (KBr
phase, cmꢃ1): 3360br, 2985w, 2361w, 1585m, 1400w, 1341w,
1225m, 1090m, 895m, 840vs (PF6ꢃ), 730m, 555m, 435w. UV–Vis
in Tris HCl buffer [kmax, nm (e
, Mꢃ1 cmꢃ1)]: 297 (78 800), 342
(19 920), 435 (600), 659br. ESI-MS in aqueous DMF: m/z 866.07
[(M-(PF6)]+. KM in CH3CN (S m2 Mꢃ1): 155. leff (298 K): 1.91 lB
.
2.5. DNA cleavage experiments
The hydrolytic DNA cleavage activity of the complexes 1 and 2
was studied by 0.8% agarose gel electrophoresis in a dark room.
Supercoiled pUC19 DNA in Tris–HCl/NaCl buffer (pH 7.2, 50 mM)
was treated with varied concentration of the complexes (10–100
2.3.3. Solubility and stability
The complexes showed good solubility in water and DMF. The
complexes showed stability in the solution phase and this was evi-
denced from the time dependent UV–Vis spectral measurements.
The complexes showed essentially single molecular ion peak in
the mass spectra in aqueous DMF suggesting the stability of the
complexes in solution.
lM) in double-distilled water in absence of any external additives.
The hydrolytic DNA cleavage reaction was carried out under pseu-
do Michaelis–Menten condition. Subsequently, the reaction was
performed under the true Michaelis–Menten condition with vary-
ing the concentration of DNA (30–150 lM) using the complex con-
2.4. X-ray crystallography
centration where maximum rate of the reaction was observed [42].
The samples were incubated for different duration (0–120 min) at
37 °C to determine respective observed rate constants. The concen-
tration of the complexes in water corresponded to the quantity in
2 lL stock solution used prior to dilution to 20 lL final volume
using Tris–HCl buffer. After incubation, loading buffer containing
2.4.1. Data collection and processing
The green colored block-shaped single crystals of 1 and 2ꢀH2O
were obtained by diffusion technique in which diethyl ether was
layered on the top of the CH3CN solution of the complexes. Single
crystals of 3 and 4ꢀH2O were obtained from slow evaporation of the
mother liquor in a period of ꢁ2 days. Crystals of respective sizes
0.39 ꢂ 0.31 ꢂ 0.27, 0.34 ꢂ 0.26 ꢂ 0.23, 0.37 ꢂ 0.31 ꢂ 0.24 and
0.38 ꢂ 0.33 ꢂ 0.28 mm3 for 1–4 were mounted on glass fibers
using epoxy cement and the intensity data were collected using a
Bruker SMART APEX CCD diffractometer having a fine focus
0.25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol
(2
l
L) was added, and the solution was finally loaded on 0.8% aga-
rose gel containing 1.0
l
g mLꢃ1 ethidium bromide (EB). Electro-
phoresis was carried out in a dark room for 2 h at 45 V in TAE
(Tris–acetate–EDTA) buffer. The bands were visualized by UV light
and photographed. The extent of cleavage of SC DNA was deter-
mined by measuring the intensities of the bands using a UVITECH
Gel Documentation System. Due corrections were made for the low
1.75 kW sealed tube Mo Ka X-ray source with increasing x (width
of 0.3° frameꢃ1) at a scan speed of 15 s per frame. Intensity data
Table 1
Selected crystallographic data for the complexes [Cu2(l-OAc)(l
-OH)(L)2(L1)](PF6)2 (L = phen, L1 = H2O, 1; L = dpq, L1 = CH3CN, 2) and [Cu(L)2(BNPP)](PF6) (L = phen, 3; dpq, 4).
1
2ꢀH2O
3
4ꢀ2H2O
Molecular formula
Formula weight (g Mꢃ1
Crystal system
Space group
a (Å)
C
26H19Cu2F12N4O4P2
C32H25Cu2F12N8O4P2
1002.62
monoclinic
P21/m
8.494(4)
24.676(11)
9.443(4)
90.00
108.459(8)
90.00
C36H24CuF6N6O8P2
908.09
monoclinic
P21/c
12.9613(18)
16.756(2)
17.493(3)
90.00
107.789(3)
90.00
C40H28CuF6N10O10P2
1048.20
triclinic
)
868.47
monoclinic
P21/n
8.5089(12)
18.430(3)
20.530(3)
90.00
98.312(3)
90.00
ꢀ
P1
7.495(4)
11.159(5)
25.048(12)
93.261(9)
91.323(9)
101.124(9)
2050.9(17)
2
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
V (Å3)
3185.7(8)
4
1877.3(14)
4
3617.4(9)
4
Z
T (K)
293(2)
1.811
293(2)
1.774
293(2)
1.667
293(2)
1.697
qcalc (g cmꢃ3
)
k (Å) (Mo K
a
)
0.71073
1.547
0.71073
1.328
0.71073
0.787
0.71073
0.713
l
(cmꢃ1
)
Data/restraints/parameters
F(0 0 0)
5331 / 0 / 451
1724
3395/0/282
1002
6175/0/530
1836
6829/0/622
1062
Goodness-of-fit (GOF)
1.151
0.1156 [0.1970]
0.1823 [0.2249]
1.211
0.0932 [0.1973]
0.1256 [0.2128]
1.066
0.0968 [0.1932]
0.1652 [0.2255]
1.024
0.0974 [0.2102]
0.1493 [0.2406]
R (Fo)a, I > 2
r
(I) [wR (Fo)b]
R (all data) [wR (all data)]
P
P
a
R = ||Fo| ꢃ Fc||/ Fo.
P
P
P
wR = { [w(F2o ꢃ Fc2)2]/ [w(Fo)2]}
;
w = [ 2(Fo)2 + (AP)2 + BP]ꢃ1
,
where P = (F2o þ 2Fc2)/3, A = 0.0875, B = 4.8244 for 1; A = 0.0931; B = 2.2471 for 2ꢀH2O; A = 0.0966,
b
½
B = 5.6159 for 3 and A = 0.1348, B = 0.9479 for 4ꢀH2O.

176
M. Roy et al. / Inorganica Chimica Acta 375 (2011) 173–180
level of nicked circular (NC) form present in the original super-
coiled (SC) DNA sample and for the low affinity of EB binding to
SC compared to NC and linear forms of DNA [43]. The error range
observed in determining %NC form from the gel electrophoresis
experiments was 3–5%.
MeCN)dicopper(II) core in which each copper(II) center displays
distorted square pyramidal geometry with a structural distortion
parameter (
tural distortion parameter is defined as
coordinate structure giving the degree of distortion ranging be-
tween trigonal bipyramidal (TBP with = 1.0 for 100% TBP) and
square pyramidal (SPY with = 0.0 for 100% SPY) structure [44].
s
) value of 0.13 and 0.19 for 1 and 0.035 for 2. Struc-
s
= (b ꢃ )/60 in a five
a
For the T4 religation experiments, the NC DNA obtained from
the hydrolytic cleavage reaction, was recovered from the agarose
gel using a gel extraction kit (obtained from Bangalore Genie, In-
dia) and this was followed by addition of 5ꢂ ligation buffer and
s
s
The perspective views of the complexes are shown in Figs. 1 and
2. The N,N-donor heterocyclic base, viz. 1,10-phenanthroline
(phen) in complex 1 and dipyrido[3,2-d:20,30-f]quinoxaline (dpq)
in complex 2, binds at the basal plane. The phenanthroline base
T4 DNA ligase (1 lL, 4 units). The solution was incubated for
10 h at 16 °C prior to gel electrophoresis. In the inhibition reac-
tions, the additive like distamycin-A or DMSO was added initially
to the SC DNA and incubation was performed for 20 min at 37 °C
prior to the addition of the complex.
is involved in
ing to another molecule. The distances between two copper cen-
ters in the complexes and are 3.034 Å and 3.049 Å,
p–p stacking interaction with another base belong-
1
2
respectively, with respective Cu–OH–Cu angle of 103.70(3)° and
104.78(6)°. The axial coordination site of the copper centers is
occupied by a solvent water molecule in 1 and CH3CN in 2. The ax-
ial ligands are showing
[45–50]. Aqua ligand showing such a binding mode is known for
3. Results and discussion
l–
g2 binding mode to the metal centers
3.1. Synthesis and general properties
other dicopper(II) complexes [45–48]. The MeCN ligand showing
a
sten complexes [49,50]. The distances of the donor atom of the
weakly bound solvent molecule from the copper(II) centers are
2.443(6) Å and 2.413(7) Å in 1 and 2.590(8) Å in 2. The structural
features of complex 1 are similar to those reported for the 2,2’-
Hydroxo and acetato-bridged dicopper(II) complexes, [Cu2
l–
g2 binding mode is reported for dimolybdenum and ditung-
(
l
-OAc)(
l-OH)(L)2(l
-L1)](PF6)2, where L = 1,10-phenanthroline
(phen), L1 = H2O in
1
and L = [3,2-d:20,30-f]quinoxaline (dpq),
L1 = MeCN in 2, are synthesized and their hydrolytic DNA cleavage
activity studied (Scheme 1). The complexes are characterized from
analytical, spectral and magnetic data (Table 2). To study the bind-
ing aspects of the binuclear complexes to DNA, we have isolated
products from the reaction of the complexes 1 and 2 with a model
phosphodiester, viz. bis(4-nitrophenyl)phosphate (BNPP). The
mononuclear copper(II) complexes [Cu(L)2(BNPP)](PF6) (L = phen,
3; dpq, 4) are structurally characterized by X-ray crystallography.
The IR spectra of the complexes display characteristic PF6 stretch-
ing band near 835 cmꢃ1. The complexes are 1:2 electrolytes. The
ESI MS spectra of the complexes display essentially the molecular
ion peak suggesting stability of the binuclear structure in a solu-
tion phase. The electronic spectra of the complexes are recorded
in Tris–HCl buffer medium (pH 7.2). Complexes 1 and 2 display vis-
ible band at 708 nm and 640 nm, respectively. The bands are
assignable to the d–d transition. The intense electronic bands ob-
served in the UV region are due to
transitions.
p–
p⁄ or n–p⁄ electronic
3.2. X-ray crystallography
Fig. 1. An ORTEP view of the cationic complex in [Cu2(l-O2CCH3)(l-OH)(phen)2(l-
H2O)](PF6)2 (1) with 50% probability thermal ellipsoids and the atom numbering
scheme for the metal and hetero atoms. Hydrogen atoms are not shown for clarity.
Binuclear copper(II) complexes 1 and 2 are crystallized in P21/n
and P21/m space group of the monoclinic crystal system, respec-
tively. The complexes have
a (l-hydroxo)(l-acetato)(l-H2O/
Table 2
Selected physicochemical data for the complexes [Cu2(l-OAc)(l
-OH)(L)2(L1)](PF6)2
(L = phen, L1 = H2O, 1; L = dpq, L1 = CH3CN, 2) and [Cu(L)2(BNPP)](PF6) (L = phen, 3;
dpq, 4).
Complex
1
2
IRa (cmꢃ1) [
l
(PF6ꢃ)]
835
290.6
830
354.1
ESI-MSb (m/z)
[(Mꢃ2(PF6)]2+
708 (80)
[(Mꢃ2(PF6)]2+
640 (90)
Visible bandc: kmax (nm)
(e
(Mꢃ1 cmꢃ1))
KM (S m2 Mꢃ1
)
255
2.06
51
265
1.98
48
d
e
leff
(
lB
)
2J (cmꢃ1
)
f
a
b
c
d
e
f
KBr phase.
In aqueous MeOH.
In Tris–HCl buffer. The bands are assigned to the copper(II) d–d transition.
Molar conductivity in CH3CN.
Magnetic moment at 298 K using solid powdered samples of the complexes.
Magnetic exchange parameter.
Fig. 2. An ORTEP view of the cationic complex in [Cu2(l-O2CCH3)(l-OH)(dpq)2(l-
CH3CN)](PF6)2ꢀH2O (2ꢀH2O) with 50% probability thermal ellipsoids and the atom
numbering scheme for the metal and hetero atoms. Hydrogen atoms are not shown
for clarity.

M. Roy et al. / Inorganica Chimica Acta 375 (2011) 173–180
177
bipyridine and 1,10-phenanthroline complexes having (
xo)( -acetato)dicopper(II) core [46,47].
l
-hydro-
gle between the dpq (containing N5–N8 atoms) and the phen ring
of BNPP (C35–C40 atoms) is 2.78°, whereas the other dpq ring
gives a dihedral angle of 5.93° with another phenyl ring of the
BNPP moiety. Two dpq rings of 4 have a dihedral angle of 44.07°.
Complex 4ꢀH2O shows significant hydrogen-bonding interactions
involving the solvent water molecule, one phosphate oxygen atom
of BNPP and another solvent water molecule giving distances of
2.742 Å and 2.855 Å. Crystal structures of 3 and 4 suggest conver-
sion of the binuclear core of 1 and 2 to the stable mononuclear
phosphate adduct having 1:2 ratio of copper and the phenanthro-
line base.
l
Complexes 3 and 4 crystallize in the monoclinic space group
ꢀ
P21/c and triclinic space group P1, respectively. The perspective
views of the molecules are shown in Figs. 3 and 4. The complexes
are monomeric having each copper(II) center bound to two phe-
nanthroline bases and bis(4-nitrophenyl)phosphate binds to cop-
per via phosphate oxygen atom. The copper centers have an
essentially trigonal bipyramidal (TBP) structure with distortion
parameter (
s
) values of 0.87 and 0.57 for 3 and 4, respectively
value that
(s
= 1.0 for 100% TBP) [44]. It is apparent from the s
complex 3 has an essentially TBP geometry. The axial Cu–N dis-
tances (1.996(6) Å and 1.979(6) Å) of complex 3 are shorter than
the equatorial Cu-N and Cu–O distances (2.043(6) Å, 2.058(5) Å
and 2.098(6) Å). The N2–Cu–N4 angle of 178.7(3)° is essentially
linear, while the other angles involving the equatorial atoms range
within 108.4(2)° to 126.5(2)°. The axial N1–Cu1–N5 angle in 4 is
172.5(2)°. The angles among the equatorial atoms are 96.4(2)°,
124.9(2)° and 138.5(2)°. The angles observed between the axial
and equatorial atoms are in the range of 81.7(2)° to 97.9(2)°. The
Cu–O distances involving BNPP for the complexes 3 and 4 are
2.043(6) Å and 2.143(5) Å, respectively. The phenanthroline rings
3.3. Magneto-structural properties
The variable temperature magnetic susceptibility data of the
complexes 1 and 2 show the presence of ferromagnetically coupled
dicopper(II) units giving 2J values of 51 and 48 cmꢃ1, respectively,
with a g value of 2.0023 (Fig. 5). The magnetic behavior compares
well with the 2J values reported for other hydroxo/alkoxo and
carboxylato-bridged dicopper(II) complexes [51]. The magneto-
structural correlation on complexes having (
l-hydroxo/alkox-
o)( -acetato)dicopper(II) cores has revealed that the 2J value is
l
in 3 are involved in
nanthroline ring of a different molecule. Each of the dpq ring in 4
is involved in stacking interaction with one of BNPP nitroben-
p–p stacking interactions with another phe-
dependent on the monoatomic hydroxo/alkoxo bridge angle.
Besides, the presence of a 3-atom carboxylato bridge makes the
superexchange pathways non-complementary thus reducing
the magnitude of 2J [52–54]. A Cu–OH–Cu angle of <110° in the
‘‘essentially tribridged’’ dicopper(II) cores makes the complexes
ferromagnetic. The significantly high value of J for the complexes
1 and 2 could be due to the presence of low Cu–OH–Cu angle of
ꢁ104°. Christou and coworkers have reported a 2J value of
38.6 cmꢃ1 for [Cu2(OH)(O2CMe)(H2O)(bpy)2](ClO4)2 [45]. Based
on the linear correlation: ꢃ2J = 10.77/ ꢃ 1198 cmꢃ1, where / is
the Cu–OH–Cu angle, we expect 2J value of 78 cmꢃ1 for a / angle
p–p
zene ring as well as one ring of another molecule. The dihedral an-
Fig. 3. An ORTEP view of the cationic complex in [Cu(phen)2(BNPP)](PF6) (3) with
50% probability thermal ellipsoids and the atom labeling scheme for the metal and
hetero atoms. Hydrogen atoms are not shown for clarity.
Fig. 5. Plots showing molar magnetic susceptibility per copper (
effective magnetic moment per copper ( eff/Cu) versus temperature (T) for the
complexes 1 (a) and 2 (b). Theoretical fits of the magnetic data are shown by
the solid line in the M/Cu plots.
vM/Cu) and the
Fig. 4. An ORTEP view of the cationic complex in [Cu(dpq)2(BNPP)](PF6)ꢀH2O (4ꢀH2O)
with 50% probability thermal ellipsoids and the atom numbering scheme for the
metal and hetero atoms. Hydrogen atoms are omitted for clarity.
l
v

178
M. Roy et al. / Inorganica Chimica Acta 375 (2011) 173–180
of 104° [51]. The observed lower 2J value from the predicted one
could be due to the presence of an axially bound bridging solvent
molecule that could enhance the counter-complementary effect
of the acetate bridge.
3.4. DNA cleavage activity
The ability of the complexes 1 and 2 to cleave supercoiled plas-
mid DNA under hydrolytic reaction condition has been assessed by
1% agarose gel electrophoresis. The DNA cleavage reaction has
been carried out in both dose and time dependent manner. When
Fig. 6. (a) Agarose gel (0.8%) electrophoresis diagram showing the cleavage of SC
pUC19 DNA (60, 120
l
M) by complex 1 in dark in Tris buffer (pH 7.2): lane-1, DNA
M) control; lane-2, DNA (120 M) + 1 (40 M), 5 min; lane-3, DNA
M) + 1 (40 M), 30 min; lane-4, DNA (120 M) + 1 (80 M), 5 min; lane-5,
M) + 1 (80 M), 30 min; lane-6, DNA (60 M) control; lane-7, DNA
M) + 1 (80 M), 60 min; lane-9,
M) + NaN3
L) + 1 (80 M),
M) + 1 (80 M)
as control without addition of T4 DNA ligase; lane-13, conversion of NC (obtained
from DNA (120 M) + 1 (80 M)) to SC DNA on treatment with T4 DNA ligase (4
units). (b) Agarose gel (0.8%) electrophoresis diagram showing the cleavage of SC
pUC19 DNA by complex 2 in dark in Tris buffer (pH 7.2): lane-1, DNA (120 M)
control; lane-2, DNA (120 M) + 2 (20 M), 5 min; lane-3, DNA (120 M) + 2
(20 M), 15 min; lane-4, DNA (120 M) + 2 (60 M), 5 min; lane-5, DNA
(120 M) + 2 (60 M), 15 min; lane-6, DNA (60 M) control; lane-7, DNA
(60 M) + 2 (60 M), 5 min; lane-8, DNA (60 M) + 2 (60 M), 15 min; lane-9,
DNA (120 M) + 2 (60 M), 15 min (under argon); lane-10, DNA (120 M) + NaN3
(500 M) + 2 (60 M), 15 min; lane-11, DNA (120 M) + DMSO (4 L) + 2 (60 M),
15 min; lane-12, NC form obtained from the treatment DNA (120 M) + 2 (60 M)
as control without addition of T4 DNA ligase; lane-13, conversion of NC (obtained
(120
(120
l
l
l
l
pUC19 DNA (4 lg, 120 lM) is incubated with 80 lM of complex 1
l
l
l
for 30 min, a significant cleavage of supercoiled (SC) DNA is ob-
served in comparison to the control reaction (Fig. 6(a)). The dpq
complex 2 is more active in cleaving DNA hydrolytically than the
phen complex 1 (Fig. 6(b)). Control experiments with copper(II)
acetate or the phenanthroline base do not show any DNA cleavage
activity under similar experimental conditions suggesting the
involvement of the complexes in the DNA cleavage reaction. The
complexes cleave DNA in dark under argon medium thus excluding
formation of any reactive oxygen species (ROS) [25]. Addition of
additives like NaN3 as singlet oxygen scavenger or DMSO as hydro-
xyl radical scavenger does not inhibit the DNA cleavage activity
indicating non-oxidative nature of the DNA cleavage. Hydrolytic
nature of the DNA cleavage is confirmed from the T4 ligase exper-
iments [55]. T4 ligase is an enzyme that specifically religates the
fragmented DNA into the supercoiled form. Hydrolytic cleavage
of DNA involves hydrolysis of the phosphodiester bond forming
DNA (120
(60 M) + 1 (80
DNA (120
(500 M) + 1 (80
30 min; lane-12, NC form obtained from the treatment DNA (120
l
l
l
l
l
M), 10 min; lane-8, DNA (60
l
l
l
M) + 1 (80
l
M), 30 min (in argon); lane-10, DNA (120
M) + DMSO (4
l
l
l
M), 30 min; lane-11, DNA (120
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
from DNA (120
units).
lM) + 2 (80 lM)) to SC DNA on treatment with T4 DNA ligase (4
Fig. 7. (a) (i) Plot showing saturation kinetics for the cleavage of pUC19 DNA (120
(pH 7.2) under pseudo Michaelis–Menten condition and (ii) saturation kinetics of the cleavage of pUC19 DNA using 80
pUC19 DNA (30–150 M) at 37 °C in Tris–HCl buffer (pH 7.2) under true Michaelis–Menten condition. (b) (i) Plot showing saturation kinetics for the cleavage of pUC19 DNA
(120 M) with different complex concentrations (10–100 M) of 2 at 37 °C in Tris–HCl buffer (pH 7.2) under pseudo Michaelis–Menten condition and (ii) saturation kinetics
M complex 2 with different concentrations of the plasmid pUC19 DNA (30–150 M) at 37 °C in Tris–HCl buffer (pH 7.2) under true
l
M) with different complex concentrations (10–100
lM) of 1 at 37 °C in Tris–HCl buffer
lM complex 1 with different concentrations of the
l
l
l
of the cleavage of pUC19 DNA using 60
l
l
Michaelis–Menten condition.

M. Roy et al. / Inorganica Chimica Acta 375 (2011) 173–180
179
fragments that could be subsequently religated. In contrast, the
oxidative cleavage of DNA leads to degradation of the sugar and/
or base making the DNA to be permanently damaged and the frag-
ments cannot be religated by T4 ligase. In our T4 ligase experiment,
the NC form obtained from the cleavage of SC DNA with the com-
plexes has been reacted with T4 ligase enzyme for overnight and
we have observed complete conversion of the NC DNA to its origi-
nal SC form (Fig. 6).
The observed DNA hydrolysis rate is in good agreement with that
of other known metal-based synthetic nucleases (Table 3) [34,56–
61]. The higher DNA hydrolase activity of the dpq complex 2 than
its phen analog seems to be due to higher DNA binding strength of
the dpq ligand with an extended quinoxaline moiety. The observed
rate for 2 is less than the mononuclear dpq complex [Cu(dpq)2(-
H2O)]2+ [34]. This could be due to larger steric bulk of 2 inhibiting
its binding to DNA phosphate linkage than its monomeric analog
[Cu(dpq)2(H2O)]2+ or due to greater lability of the axial aqua ligand
in the bis-dpq complex than the tri-bridged core in complex 2.
3.5. Kinetic investigation
3.6. Reaction with bis(4-nitrophenyl)phosphate (BNPP)
The kinetic aspects of the hydrolytic DNA cleavage are studied
to obtain the rate of the hydrolytic cleavage. The reactions were
done under pseudo-Michaelis–Menten conditions by using various
The Lewis acidity of the metal ions in metallonucleases plays a
fundamental role in their catalytic action. The commonly accepted
mechanism of DNA hydrolysis involves a nucleophilic attack of
water oxygen to phosphorus to give a five-coordinate phosphate
intermediate and subsequent rearrangement of the phosphate
leading to the DNA cleavage [62]. We have studied the mechanistic
aspects of the hydrolytic DNA cleavage using bis(4-nitro-
phenyl)phosphate (BNPP) as a model phosphodiester. The reaction
of BNPP with complexes 1 and 2 leads to the formation of stable
monomeric copper(II) BNPP adduct. The complexes isolated are
structurally characterized by X-ray crystallography. Since the
ESI-MS spectral data suggest solution stability of the binuclear
complexes, isolation of monomeric complexes 3 and 4 suggest deg-
radation of the binuclear core on binding to the BNPP molecule and
the intermediate species could be a mononuclear bis-phen/dpq
complex of copper(II).
concentrations of 1 (10–100
lM) and constant DNA concentration
(120 M), which results in the formation of nicked circular (NC)
l
DNA from supercoiled (SC) pUC19 DNA [34,35]. The decrease of
SC DNA fits well into a single-exponential decay curve and follows
pseudo-first-order kinetics as shown in Fig. 7(a, i). The reaction
rate constant obtained from the plot is 1.88( 0.03) hꢃ1 with a
complex concentration of 80
conditions, in which the complex concentration is kept constant
at 80 M and the DNA concentration is varied from 30–150 M,
we were able to obtain the highest rate constant of
1.90( 0.02) hꢃ1 using 120
M SC DNA (Fig. 7(a, ii)). The Michae-
lM. Under true Michaelis–Menten
l
l
l
lis–Menten constant (Km) is obtained by means of Lineweaver–
Burk method under true Michaelis–Menten conditions [55]. The
Km value and maximum velocity (Vmax) of the hydrolytic DNA
cleavage reaction were calculated to be 1.08 ꢂ 10ꢃ4
M and
3.03 hꢃ1. Similarly, kinetic aspects of 2 were evaluated under both
4. Conclusions
pseudo and true Michaelis–Menten conditions giving respective
rate values of 3.94( 0.03) hꢃ1 using 60
l
M
complex and
4.07( 0.02) hꢃ1 at DNA concentration of 120
l
M (Fig. 7(b)). The
The hydroxo and acetato-bridged binuclear copper(II) com-
plexes having planar phenanthroline bases are efficient cleavers
of plasmid DNA following hydrolytic pathway without involving
any external additives or reactive oxygen species. The rate of
hydrolysis of DNA has been evaluated under true Michaelis–Men-
ten conditions. The complexes cleave DNA with a rate constant of
Km and Vmax of the hydrolytic DNA cleavage by 2 under true
Michaelis–Menten condition from Lineweaver–Burk plot are
8.54 ꢂ 10ꢃ5 M and 5.95 hꢃ1, respectively. Complexes 1 and 2 show
5.2 ꢂ 107 and 1.13 ꢂ 108 fold enhancement in the rate of hydroly-
sis of DNA compared to that of control hydrolysis data.
1.90 0.02 hꢃ1 using 120
lM SC pUC 19 DNA at a complex concen-
tration of 80 lM for 1 with the Michaelis–Menten constant (Km) of
Table 3
1.08 ꢂ 10ꢃ4 M. Similarly, the rate constant for complex 2 is
A comparison of the rate values for the hydrolytic cleavage of DNA by selected metal
complexes.
4.07 0.02 hꢃ1 at a complex concentration of 60
lM with Km value
of 8.54 ꢂ 10ꢃ5 M. We have observed ꢁ108-fold rate enhancement
in the hydrolytic DNA cleavage reaction compared to that of the
control data. Possible intermediate in the hydrolysis of DNA has
been explored using bis(4-nitrophenyl)phosphate (BNPP) as a
model phosphodiester. The X-ray structures of the products iso-
lated from the reactions of BNPP with the binuclear complexes
show formation of monomeric copper(II) complexes of the phe-
nanthroline bases having BNPP bound to copper(II) in trigonal
bipyramidal geometry.
Complex
Complex
Concentration constant enhancementa
M)
(hꢃ1
Rate
Rate
References
(l
)
(Pr2
)
2000
0.90
2.5 ꢂ 107
[56]
3+
ionophore
Cu (9aneN3)
Eu3+
1000
500
ꢁ0.25
0.25
2.10
0.79
0.18
0.09
1.27
0.11
0.76
3.57
5.58
ꢁ1.1 ꢂ 106
7.0 ꢂ 106
5.83 ꢂ 107
2.00 ꢂ 107
5.00 ꢂ 106
2.5 ꢂ 107
3.52 ꢂ 107
3.06 ꢂ 106
–
[30]
[57]
[57]
[58]
[29]
[23]
[59]
[60]
[61]
[33]
[34]
(Eu3+) ionophore 500
Co3+-cyclen
Co3+-tamen
Rh-P-Znb
5000
1000
–
Ni2-complex
55
Acknowledgements
[Ru(bpy)2BPG]2+c 20
Cu-histidine
Cu-neamine
[Cu(dpq)2(H2O)]
(ClO4)2
1000
100
55
We thank the Department of Science and Technology (DST),
Government of India, for financial support (SR/S5/MBD-02/2007)
and the CCD diffractometer facility. A.R.C. thanks DST for J.C. Bose
national fellowship.
9.99 ꢂ 107
1.55x108
[Cu(dppz)2Cl]Cld
1
55
80
60
2.87
1.90
4.066
7.97 ꢂ 107
5.2 ꢂ 107
1.13 ꢂ 108
[35]
this work
this work
2
Appendix A. Supplementary material
a
Rate enhancement compared to the unhydrolyzed ds-DNA rate of
3.6 ꢂ 10ꢃ8 hꢃ1
.
b
CCDC 768666, 768667, 768668 and 768669 contain the supple-
mentary crystallographic data for complexes 1, 2, 3 and 4, respec-
tively. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
Rh–P, Rh(phi)2bpy-peptide. In situ reaction using rhodium complex (5 lM) and
ZnCl2 (2.5–20 lM).
c
BPG = bipyridine-glycoluril.
dppz = Dipyrido-[3,2-a:20,30-c]phenazine.
d

180
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