A reusable zigzag copper(II) coordination polymer with bio-essential constituents as a facile DNA scission agent

Article abstract of DOI:10.1016/j.ica.2005.04.003

A copper(II) coordination polymer has been synthesized in the form of an infinite zigzag chain where each metal is bonded to one glycine, two adenine, one nitrate anion and a water molecule giving a distorted octahedral coordination geometry with N₃O₃ donor set. Each chain is hydrogen-bonded through C-H?O and N-H?O interactions on both sides leading to an overall polymeric structure. This compound not only shows facile chemical nuclease activity for pBR322 supercoiled DNA but also can be reused keeping its scission activity unchanged.

Full text of DOI:10.1016/j.ica.2005.04.003

Inorganica Chimica Acta 358 (2005) 3236–3240
www.elsevier.com/locate/ica
A reusable zigzag copper(II) coordination polymer with
bio-essential constituents as a facile DNA scission agent
*
Sanjib Das, C. Madhavaiah, Sandeep Verma , Parimal K. Bharadwaj
*
Department of Chemistry, Indian Institute of Technology Kanpur, 208016, India
Received 18 February 2005; accepted 1 April 2005
Available online 23 May 2005
Abstract
A copper(II) coordination polymer has been synthesized in the form of an infinite zigzag chain where each metal is bonded to one
glycine, two adenine, one nitrate anion and a water molecule giving a distorted octahedral coordination geometry with N3O3 donor
set. Each chain is hydrogen-bonded through C–HÁ Á ÁO and N–HÁ Á ÁO interactions on both sides leading to an overall polymeric
structure. This compound not only shows facile chemical nuclease activity for pBR322 supercoiled DNA but also can be reused
keeping its scission activity unchanged.
ꢀ 2005 Elsevier B.V. All rights reserved.
Keywords: Copper(II) complex; Coordination polymer; Plasmid DNA cleavage; Reusability; Intermolecular interactions
1. Introduction
used as a footprinting reagent for DNA, RNA, and
important cellular processes [4]. But unfortunately very
little attention is paid on coordination polynuclear com-
plexes [5]. Modular synthesis of coordination polynu-
clear complexes is a subject of considerable current
interest [6–10] for potential application in opto-electron-
ics, supramolecular storage, catalysis, magnetic materi-
als and so on. These structures can be rapidly,
accurately and efficiently built from relatively simple
sub-units where metal ions, multi-dentate organic li-
gands and coordinate bonding are the parameters for
directing the self-assembly process. Of these, polynu-
clear copper complexes are attracting attention because
of their interesting magnetic properties and their rele-
vance to the active centers of a number of metallo-pro-
teins [11]. Adenine is an important nucleobase forming a
large family of biological compounds. Recently, it has
been found that the potential of adenine moieties ad-
sorbed on inorganic surfaces to act as coding and recog-
nition elements at the air–water interface [12]. Besides,
copper complexes of amino acids are of continuing
interest since they are simple model systems for the
Designing of metal complexes for cleaving DNA is
currently an area of considerable interest from chemical
as well as biological standpoints and offers potential
applications in the field of medicine in the post-genomic
era. Transition metal complexes as an artificial nucleases
are a matter of an extensive research due to their diverse
structural features and reactivities [1]. Exploration of
copper(II) complexes as chemical nucleases have seen
an upsurge in recent years due to biologically accessible
redox potential and high affinity toward nucleobases [2].
There are several reports in the literature on copper-in-
duced DNA cleavage by using various exogenously
added co-reactants such as hydrogen peroxide, peroxy-
acids and thiols [3]. The best studied examples are
mononuclear copper(II) complexes containing 1,10-phe-
nanthroline and its derivatives since they are extensively
*
Corresponding author. Tel.: +91 512 259 7304; fax: +91 512 259
7436.
E-mail address: pkb@iitk.ac.in (P.K. Bharadwaj).
0020-1693/$ - see front matter ꢀ 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2005.04.003

S. Das et al. / Inorganica Chimica Acta 358 (2005) 3236–3240
3237
study of metal protein interactions [13]. Deriving inspi-
ration from these, we have synthesized a new mixed li-
gand copper(II) insoluble coordination polymer
containing both adenine and glycine to explore the cat-
alytic potential of nucleic acid cleavage, a fundamental
biochemical reaction.
anisotropically while the hydrogens were treated as rid-
ing atoms using SHELXL default parameters (see Table 1).
2.4. pBR322 cleavage assay
Plasmid cleavage reactions were performed in sodium
cacodylate buffer (10 lM, pH 7.5, 32 ꢂC), containing
pBR322 (8 ng/lL, New England Biolabs), crystalline
copper(II) polymer (100 lg) and activating agents mag-
nesium monoperoxyphthalate (MMPP, 100 lM). For
each cleavage reaction, 16 lL of pBR322 supercoiled
DNA, 2 lL buffer and 100 lg of crystalline polymer
was used and they were initiated by adding 2 lL of mag-
nesium monoperoxyphthalate in an eppendorf tube. For
scavenger experiments, concentrations used were
100 mM. All cleavage reactions were quenched with
5 lL of loading buffer containing 100 mM of EDTA,
50% glycerol in Tris–HCl, pH 8.0 and the samples were
loaded onto 0.7% agarose gel (Biozym) containing ethi-
dium bromide (1 lg/1 ml). Electrophoresis was done for
1 h at constant current (80 mA) in 0.5· TBE buffer. Gels
were imaged with a PC-interfaced Bio-Rad Gel Docu-
mentation System 2000.
2. Materials and experimental
2.1. Chemicals and reagents
Supersoiled plasmid DNA (pBR322) was purchased
from New England Biolabs. Ethidium bromide, catalase
and superoxide dismutage (SOD) were purchased from
Sigma Aldrich Ltd. Magnesium monoperoxyphthalate
hexahydrate (MMPP) was procured from Lancaster
and sodium cacodylate buffer (SRL, Mumbai) were used
as supplied. Ethylenediaminetetraacetic acid (EDTA)
and D-manitol were purchased from S. D. Fine Chemi-
cals, Mumbai. All buffer solutions were prepared using
Milliporeꢁ water.
2.2. Synthesis of {Cu(gly)(adenine)(NO3)(H2O)}n (1)
2.5. Assay for catalyst recycles
A mixture of Cu(NO3)2 Æ3H2O (0.24 g; 1 mmol), gly-
cine (0.07; 1 mmol) and adenine ( 0.14 g; 1 mmol) were
dissolved in 10 ml of hot water to obtain a dark blue
solution. This solution was cooled to room temperature
and filtered. The filtrate was allowed to stand at room
temperature whereupon blue crystals of 1 appeared after
7 days in the form of rectangular parallelopiped in 50%
yield. Anal. Calc. for C7H11N7O6Cu1: C, 23.83; H, 3.14;
N, 27.79. Found: C, 23.75; H, 3.19; N, 27.84%. IR (KBr
phase): 3304m, 3120m, 2363m, 1674vs, 1611s, 1574s,
1523m, 1465vs, 1384vs cmÀ1 (vs, very strong; s, strong;
m, medium).
In a typical recycle experiment, reaction mixture was
centrifuged (6400 rpm) for 3 min after completion of the
Table 1
Crystal data and details of structure refinements for 1
Compound
Empirical formula
Formula weight
1
C7H11Cu1N7O6
352.77
monoclinic, P21/c
Crystal system, space group
Unit cell dimensions
˚
a (A)
8.561(5)
10.060(5)
13.561(5)
90.000(5)
99.628(5)
90.000(5)
1151.5(10)
4
˚
b (A)
˚
c (A)
a (ꢂ)
b (ꢂ)
c (ꢂ)
2.3. X-ray data collection and crystal structure
determination
À3
˚
V (A
)
Single crystal X-ray data on 1 was collected at 100 K
temperature on a Bruker SMART APEX CCD diffrac-
tometer using graphite monochromated Mo Ka radia-
Z
F(000)
Dcalc (g cmÀ3
716
2.035
)
Crystal size (mm3)
T (K)
0.12 · 0.11 · 0.08
100
1.945
˚
tion (k = 0.71073 A). The linear absorption coefficients,
scattering factors for the atoms, and the anomalous dis-
persion corrections were taken from International
Tables for X-ray Crystallography. The data integration
and reduction were processed with SAINT [14] software.
An empirical absorption correction was applied to the
collected reflections with SADABS [15] using XPREP [16].
The structure was solved by the direct method using
SHELXTL [17] and was refined on F2 by full-matrix
least-squares technique using the SHELXL-97 [18] pro-
gram package. All non-hydrogen atoms were refined
l(Mo Ka) (mmÀ1
)
2h Range (ꢂ)
Reflections measured
60
2838
Unique reflections used [I > 2r(I)]
Parameters (N)
2471
234
R1
wR2
0.0324
0.0796
0.0388
0.0841
1.035
R1(all data)
wR2(all data)
S
À3
˚
Maximum/minimum residual (e A
)
0.702/À0.321

3238
S. Das et al. / Inorganica Chimica Acta 358 (2005) 3236–3240
cleavage of the cleavage reaction to leave the catalyst in
the pellet. After the removal of the supernatant, pellet
was repeatedly washed with cacodylate buffer
(4 · 100 lL) and then reused for subsequent cleavage
reactions, under identical conditions. Gel electrophore-
sis taking of wash fraction was performed to ensure
complete removal of modified plasmid from the catalyst
pellet.
The complete decomposition of 1 is achieved above
300 ꢂC.
The Cu(II)–N distances are normal compared to
other copper complexes of N donor ligands [Table 2].
Adenine molecule acts as a bridging ligand and tethered
in a zigzag way by copper ions coordinated to N(1) and
N(9) positions propagating the polymeric chain along
the crystallographic b-axis, to reveal a structure resem-
bling the periodicity of single stranded nucleic acids
[21]. Each polymeric chain is hydrogen-bonded on both
sides with other neighboring chains leading to a poly-
meric structure in the crystallographic bc-plane (Fig. 2).
Nitrate anion and carbonyl oxygen atom of glycine
units are involved in an intricate array of C–HÁ Á ÁO
and N–HÁ Á ÁO hydrogen bonding interaction that lie in
3. Results and discussion
The structure of 1 is a zigzag coordination polymer of
copper(II) where each metal ion is equatorially coordi-
nated to one glycine and two adenine molecules, while
the axial sites are occupied by a nitrate anion and a
water molecule showing an overall N3O3 coordination
(Fig. 1(a) and (b)). The compound once formed is insol-
uble in most of the solvents including water. The
amount of impregnated copper was determined to be
11 mg copper (g polymer)À1 by atomic absorption spec-
troscopy. The compound displays an EPR signal [19]
(X-band, Bruker 300E) with gav = 2.15 that sharpens
somewhat as the temperature is lowered to 120 K with-
out revealing any detail indicative of its stereochemistry.
In the solid state, it exhibits a broad absorption band
[19] centered at 670 nm and effective magnetic moment
value is found to be 1.93 lB at 300 K which is normal
for Copper(II) complexes [20]. Thermal gravimetric
analysis [19] in air on a 13.95 mg sample of 1 shows that
it is stable upto ꢀ190 ꢂC and loses the lone coordinated
water at ꢀ200 ꢂC. Coordinated glycine is lost only
above ꢀ240 ꢂC showing the robustness of the structure.
˚
the range between 2.069(7) and 2.449(4) A [Table 3].
Time-course experiments reveal rapid conversion of
supercoiled pBR322 DNA form I to nicked form II
within 2 min by 1 upon addition of magnesium monop-
eroxyphthalate (MMPP) (Fig. 3, lanes 2–5).
Several examples [3] on copper-based artificial nucle-
ase activity through oxidative or hydrolytic pathways
are described in the literature. In view of this fact, we
further probed for cleavage mechanism by performing
reactions in the presence of commonly employed reac-
tive species (radical) scavengers to ascertain the proba-
ble mechanistic pathway of DNA modification. To
reveal the nature of reactive species involved in the
DNA scission, the cleavage reactions are performed in
the presence of known the hydroxyl radical scavengers.
Dimethyl sulphoxide (DMSO), D-mannitol and tert-
butylalcohol do not inhibit cleavage reactions any more
(Fig. 4, lanes 4–6). We further performed the cleavage
Fig. 1. (a) The zigzag coordination polymer of copper(II) extending along the crystallographic b-axis. (b) An ORTEP diagram of complex 1 with
50% probability thermal ellipsoids showing the asymmetric unit and atom numbering schemes for the metal and hetero-atoms.

S. Das et al. / Inorganica Chimica Acta 358 (2005) 3236–3240
3239
Table 2
Table 3
Geometrical parameters for various interactions found in the structure
of 1
˚
Bond lengths (A) and angles (ꢂ) of compound 1
Cu(1)–N(1)
Cu(1)–N(9)
Cu(1)–N(11)
Cu(1)–O(2)
Cu(1)–O(3)
Cu(1)–O(W)
2.025(4)
2.020(3)
1.995(2)
1.986(3)
2.609(3)
2.348(5)
a
b
c
˚
(A)
˚
(A)
Interaction
D
d
h (ꢂ)
C–HÁ Á ÁO
O5Á Á ÁH5 [2 À x, 1 À y, 2 À z]
2.976(9) 2.460(7) 116.61(2)
3.248(7) 2.449(4) 140.18(3)
O5Á Á ÁH1 [x, 1 + y, z]
N–HÁ Á ÁO
N(1)–Cu(1)–N(9)
N(1)–Cu(1)–O(3)
N(1)–Cu(1)–O(W)
N(9)–Cu(1)–O(3)
N(9)–Cu(1)–O(W)
N(11)–Cu(1)–O(2)
N(11)–Cu(1)–O(3)
N(11)–Cu(1)–O(W)
O(2)–Cu(1)–O(3)
O(2)–Cu(1)–O(W)
O(3)–Cu(1)–O(W)
91.71(8)
94.30(5)
95.54(7)
96.72(3)
95.17(7)
83.56(8)
79.58(5)
89.37(8)
86.81(5)
86.95(6)
164.32(6)
O1Á Á ÁH3 [1 À x, Ày, 1 À z]
O1Á Á ÁH2 [1 À x, Ày, 1 À z]
O4Á Á ÁH7 [1 À x, 1 À y, 2 À z]
2.857(5) 2.110(4) 170.43(3)
2.767(9) 2.069(7) 154.61(3)
2.997(6) 2.238(5) 148.44(3)
O–HÁ Á ÁO
O4Á Á ÁHW1 [1 À x, 0.5 + y, 1.5 À z] 2.845(8) 2.196(6) 140.31(3)
D is the distance between C or N or O and the acceptor (O atom).
a
b
c
d is the distance between H and the acceptor (O atom).
h is the angle at H in C–HÁ Á ÁX or N–HÁ Á ÁX or O–HÁ Á ÁX (X = O
atom).
Fig. 3. pBR322 DNA cleavage experiment by 1 at different time
intervals. Lane 1, DNA alone; lanes 2–5, DNA + coordination
polymer + MMPP (1, 2, 3 and 5 min, respectively).
Fig. 4. pBR322 cleavage experiments in presence of free radical
scavengers and singlet oxygen quencher assisted by 1 in a 3 min reaction.
Lane 1, DNA alone; lane 2, pBR322 + 1 + MMPP + EDTA; lane 3,
pBR322 + 1 + MMPP + NaN3; lane 4, pBR322 + 1 + MMPP +
DMSO; lane 5, pBR322 + 1 + MMPP + D-mannitol; lane 6,
pBR322 + 1 + MMPP + tert-BuOH; lane 7, pBR322 + 1 + MMPP.
Fig. 2. Packing diagram of 1 in the bc crystallographic plane.
reactions in presence of enzymatic scavengers such as
catalase and superoxide dismutase (SOD) to reveal the
possible role of peroxide species and superoxide radical
anion in plasmid scission reactions.
Fig. 5. pBR322 cleavage experiments in presence of enzymatic
scavengers by 1 in a 3 min reaction. Lane 1, DNA alone; lane 2,
cleavage reaction; lanes 3 and 4, cleavage reaction in presence of
catalase and SOD, respectively.
Fig. 5 clearly demonstrates the lack of direct involve-
ment of such diffused reactive species, because complete
conversion of form I to form II is observed. But, NaN3
displayed complete cleavage inhibition induced by co-
oxidant. NaN3 is well known as a potent quencher of
singlet oxygen and these results indicate towards in situ
generation and possible involvement of such reactive
species for the plasmid modification (Fig. 4, lane 3)
[22]. In presence of EDTA, the cleavage reaction is also
completely inhibited demonstrating a crucial role of
coordinated copper for plasmid modification (Fig. 4,
lane 2).
Due to insoluble and robust nature of this coordina-
tion polymer provides an opportunity for its recycling
for multiple scission reactions. In a typical recycle exper-
iment, the reaction mixture was centrifuged (6400 rpm,
3 min) after completion of the cleavage reaction to leave
the polymer in a pellet form, and the supernatant was

3240
S. Das et al. / Inorganica Chimica Acta 358 (2005) 3236–3240
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We have shown that a zigzag coordination polymer
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