One pot synthesis of cu(ii) 2,2′ bipyridyl complexes of 5-hydroxy-hydurilic acid

Journal of Inorganic Biochemistry 105 (2011) 256–267

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o

One pot synthesis of Cu(II) 2,2′-bipyridyl complexes of 5-hydroxy-hydurilic acid
and alloxanic acid: Synthesis, crystal structure, chemical nuclease activity
and cytotoxicity
Namrata Dixit a, R.K. Koiri b, B.K. Maurya b, S.K. Trigun b, Claudia Höbartner c, Lallan Mishra a,⁎
a
Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India
b
Department of Zoology, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India
c
Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: A barbiturate derivative [1,5-dihydro-5-[5-pyrimidine-2,4(1H,3H)-dionyl]-2H-chromeno[2,3-d] pyrimidine-2,4
Received 7 July 2010 (3H)-dione] (LH4) was allowed to react with 2,2′-bipyridyl-dinitrato-Copper(II)-dihydrate which provides two
Received in revised form 4 November 2010 complexes, characterized as [Cu(bpy)(L1)]·3H2O (1) and [Cu(bpy)(L2)]·H2O (2), where bpy = 2,2′-bipyridine,
Accepted 5 November 2010
L1 = 5-hydroxy-hydurilic acid and L2 = alloxanic acid. In a separate reaction of LH4 with Cu(NO3)2·H2O another
Available online xxxx
type of complex [Cu(LH3)2·(H2O)2]·4H2O (3) is formed. The complexes were characterized by single crystal
Keywords:
X-ray crystallography, physicochemical and electrochemical studies. The interaction of complexes 1 and 3 with
One pot synthesis DNA was monitored using absorption and emission titrations as well as circular dichroism spectroscopy. The
Transformation of barbiturate derivative complexes were found to cleave supercoiled plasmid DNA to nicked circular and linear DNA. Complexes 1 and 3
Chemical nuclease property were also tested against T-cell lymphoma (Dalton lymphoma DL) and showed significant cytotoxic activity with
Cytotoxicity studies IC50 values of ~9.0 nM and 0.6 nM.
© 2010 Elsevier Inc. All rights reserved.

1. Introduction nucleosidic bond and subsequent strand breakage. Among purine
nucleobases, guanine is most susceptible to oxidation. However, most
Chemical nucleases show potential applications in the fields of of the cleavage reagents that exhibited outstanding DNA cleavage
biotechnology and therapeutic reagents [1–5]. They are efficient tools activity require the addition of either external oxidant (dihydrogen
for the cleavage of DNA. A large number of transition metal complexes peroxide, molecular oxygen) or external reductant (ascorbic acid, 3-
have been explored with good DNA cleavage activities through either mercaptopropionic acid). However, in few cases, the photo-induction of
hydrolytic or oxidative pathways [6–13]. The transition metal DNA cleavage was also reported. Thus, in vivo applications of these
complexes are known to bind DNA via both covalent and non- reagents are limited [15–18]. Therefore, the development of chemical
covalent interactions. In covalent binding, the labile ligand of the nucleases that work without any external stimuli is a challenge for
complexes is replaced by a DNA nucleobase, e.g. coordination via chemists, and only few examples are known to us [19–22].
guanine N7. On the other hand, the non-covalent interactions with In this context, it was noted that barbiturates besides their
DNA include intercalative, electrostatic and groove (surface) binding biological significance, can also be exploited as building blocks in
of cationic metal complexes along the major or minor grooves of DNA the construction of supramolecular structures owing to their both
helices. The transition metal complexes can also induce cleavage of DNA H-bond donor and acceptor capabilities. Therefore, such molecules lie
under physiological conditions. This property is of interest, especially in at the forefront of modern chemical research [23–30].
the areas of genomic research, footprinting and development of Thus, in view of excellent precedence of barbiturate chemistry, it
therapeutic agents [14]. Hydrolytic cleavage of DNA involves scission was considered worthwhile to synthesize a barbiturate derivative
of phosphodiester bonds to generate fragments which can subsequently [1,5-dihydro-5-[5-pyrimidine-2,4(1H,3H)-dionyl]-2H-chromeno[2,3-d]
be re-ligated. The compounds which enable hydrolytic cleavage mimic pyrimidine-2,4(3H)-dione)] LH4 which was initially complexed with
restriction enzymes. The oxidative DNA cleavage involves either Zn(bpy)(NO3)2 2H2O. The X-ray diffraction study of the resulting
oxidation of the deoxyribose moiety by abstraction of sugar hydrogen complex provided a supramolecular structure of type [Zn(bpy)2·
atoms or oxidation of nucleobases, followed by cleavage of the 2H2O]·(LH3)2·7H2O [31]. Enthused by this interesting observation, a
reaction of barbiturate ligand (LH4) was carried out with a Cu(II)
ion coordinatively protected with 2,2′-bipyridine and bearing two
⁎ Corresponding author. Tel.: +91 542 6702449; fax: +91 542 2368174. substitutable NO−groups. Ligand LH4 on reaction with 2,2′-bipyridyl-
3
E-mail address: lmishrabhu@yahoo.co.in (L. Mishra). dinitrato-copper(II)-dihydrate, yielded two new metal complexes in a

0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.11.006

N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 257

one-pot-reaction. Complex 1 possesses 5-hydroxy-[5,5′]-bipyrimidinyl- 2.4. Synthesis of complex 1
2,4,6,2′,4′,6′-hexaone (5-hydroxy-hydurilic acid) whereas the other
complex, 2 contains 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic A methanolic solution (10 mL) of Cu(bpy)(NO3)2·2H2O(0.361 g,
acid (alloxanic acid) as co-ligand. Since none of the complexes bear the 1 mmol) was added drop wise to a solution of LH4 (0.374 g, 1 mmol) in
framework of LH4, the original ligand must have been transformed 5 mLdimethylformamide(DMF).Thereactionmixturewasthenrefluxed
during the reaction. In order to explore the effect of 2,2′-bipyridyl groups for 2 h. The resulting solution was kept at room temperature for slow
on the transformation of LH4, a reaction between LH4 and copper nitrate evaporation. After 5–6 days, the dark green coloured crystals were
salt was also carried out. This reaction provided complex 3 of type [Cu obtained. These crystals were washed with methanol and dried in air.
(LH3)2(H2O)2], which retained the original ligand framework. Yield: 54%, M.P. 220 °C, elemental analysis calculated for C18H11CuN6O10
In view of the aforementioned reports and owing to the biological (%): C, 40.44; H, 2.05; and N, 15.73. Found (%): C, 41.24; H, 2.17; and
significance of Cu(II) ions [32], the nuclease property of the novel N, 16.21. UV–vis absorptions: λmax (DMSO, 10−4 M), nm (ε/104 M−1
complexes was studied. In addition, the present article embodies the cm−1) 266 (4.0), 312 (2.922) and 620 (0.023). IR (KBr): νmax, cm−1
spectroscopic and single crystal characterization of the newly synthesized 3436 (OH, H2O), 3201 (NH), 3086 (CH, Ph), 1695 (CO), and 1603 (2,2′-bpy).
complexes. The DNA binding and cleavage properties of copper(II)
complexes 1 and 3 have been studied (complex 2 is insoluble in common 2.5. Synthesis of complex 2
organic solvents and was not investigated further). The cytotoxic effects of
complexes 1 and 3 against Dalton's lymphoma cell lines are also reported. After isolation of complex 1, the filtrate thus obtained provided a
bluish brown solid product after two weeks. It was then redissolved in
2. Experimental section MeOH and left for slow evaporation at room temperature. After 24 h,
block shaped blue colour crystals were formed which were found
2.1. Materials insoluble in all common organic solvents. The crystals were then
washed with diethyl ether and dried in air. Yield: 25%, M.P. N250 °C,
Barbituric acid, 2,2′-bipyridine and salicylaldehyde were purchased elemental analysis calculated for C14H10CuN4O6 (%): C, 42.74; H, 2.54;
from Sigma Aldrich Chem. Co and copper(II) nitrate dihydrate was and N, 14.24. Found (%): C, 42.85; H, 3.08; and N, 14.84. IR (KBr): νmax,
purchased from S.D. Fine Chemicals, India and used as received. Solvents cm−1 3303 (NH), 1731 and 1656 (CO), 3037 (CH, 2,2′-bpy), 2929
were purchased from E. Merk and were freshly distilled prior to their (CH), 1266, 1024 (C–O–C), and 3378 (OH, water).
use. The barbiturate ligand (LH4) was synthesized using slight
modification of the reported procedure [33]. Calf thymus (CT) DNA 2.6. Synthesis of complex 3
and supercoiled (SC) plasmid DNA pBR322 (as a solution in Tris buffer
and cesium chloride purified), with a length of 4361 base pairs were A solution of Cu(NO3)2·2H2O (0.241 g, 1 mmol) in MeOH (10 mL)
purchased from Bangalore Genei, India. pUC19 plasmid DNA with a was added drop wise to a solution of LH4 (0.374 g, 1 mmol) in DMF
length of 2686 base pairs was purchased from Fermentas. Restriction (5 mL). The reaction mixture after stirring for 5–6 h at room
enzymes were purchased from New England Biolabs and DNA temperature was left for slow evaporation. Fluorescent block shaped
oligonucleotide primers were purchased from Sigma Aldrich Chem. Co. green colour crystals were grown in solution after 4–5 days. The crystals
were washed with MeOH followed by diethyl ether and then dried
2.2. Physical measurements in air. Yield: 72%, M.P. N250 °C, elemental analysis calculated for
C30H38CuN8O22 (%): C, 38.87; H, 4.10; and N, 12.09. Found (%): C, 39.20;
IR (KBr disc, 400–4000 cm−1) spectra were recorded on a Varian FTIR H, 4.76; and N, 12.98. UV–vis absorptions: λmax (DMSO, 10−4 M), nm
3100 spectrometer; elemental analysis was done on Carbo-Erba 1108 (ε/104 M−1 cm−1) 329 (4.059), 379 (0.088) and 408 (0.056). IR (KBr):
elemental analyzer, UV-visible (UV-vis) spectra were recorded on a νmax, cm−1 3225 (NH), 1705 and 1658 (CO), 3020 (CH, Ph), 2937 (CH),
Shimadzu UV-1601 spectrometer while TGA plots were taken on a DU- 1266, 1039 (C–O–C), and 3409 (OH, water).
PONT9900thermalanalyzingsystem(heatingrate10 °C/min)upto400 °C.
Cyclic voltammetric measurements were performed on a CHI 620c 2.7. X-ray structural studies
Electrochemical Analyzer using glassy carbon as working electrode, a
platinum wire auxiliary electrode, and Ag/Ag+ reference electrode in a Single crystal X-ray diffraction data for the complexes were collected
standard three-electrode configuration. Tetrabutylammonium perchlo- in the temperature range of 100(2) K to 293(2) K on an Enraf Nonius
rate(TBAP)wasusedasthesupportingelectrolyte,andtheconcentrationof MACH 3 diffractometer using graphite monochromatized Mo Kα
solutionsofthecomplexesinDMSOwasmaintainedas10−3 M.ESRspectra radiation (λ = 0.71073 ) from block shaped crystals in the ω–2θ scan
were recorded at 273 K and 77 K on a Varian E-line Century Series ESR mode for complexes 1, 2 and 3. Intensities of these reflections were
spectrometer equipped with a dual cavity and operating at X-band of measured periodically to monitor crystal decay. The crystal structures
100 kHz modulation frequency. Tetracyanoethylenewas used asthe field were solved by direct methods and refined by full matrix least squares
marker (g = 2.00277). The CD measurements of DNA with and without (SHELX-97) [34]. Due to high degree of hydration, thermal motion and
complexeswerecarriedoutwithaJascoJ500spectropolarimetercalibrated disorder, hydrogen atoms of water of crystallization could not be
withammonium(+)-10-camphorsulfonate. located. Drawings were carried out using MERCURY [35] and special
computations were carried out with PLATON [36]. The crystal
2.3. Equipments used for DNA cleavage studies refinement data are collected in Table 1 while selected bond distances
and bond angles are reported in Table 2.
PCR amplification was performed on an Eppendorf Mastercycler ep
gradient S. Polyacrylamide gel electrophoresis was carried out with 2.8. Interaction of complexes 1 and 3 with DNA
20× 30 cm self-cast denaturing polyacrylamide gels (5–20% acrylamide,
7 M urea, 1× TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 2.8.1. Absorption titration
8.3) on CBS Scientific DNA sequencing systems using PowerPac HV The binding of complexes 1 and 3 with DNA was measured in a Na-
power supply from Biorad. Gels were dried on a Whatman 3MM filter phosphate buffer solution (pH 7.2). The absorption ratio at 260 nm
paper using a gel dryer model 583 from Biorad at 80 °C for 30 min. and 280 nm of calf thymus DNA (CT DNA) solutions was found as
Phosphorimaging was performed with a Storm 820 Phosphorimager 1.9:1, demonstrating that DNA is sufficiently free of protein. The
from GE Healthcare. concentration of DNA was then determined by UV-visible absorbance

258 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267

Table 1
Crystal data for 1, 2 and 3.

Compound 1 2 3

Chemical formula C18H11N6O10Cu C14H10N4O6Cu C30H38N8O22Cu
Formula weight 534.87 393.80 926.22
Temperature 150(2) K 273(2) K 150(2) K
Wavelength 0.71073 A 0.71073 A 0.71073 A
Crystal system Triclinic Monoclinic Triclinic
Space group P-1 P 1 21/n 1 (n = 14) P-1
a(Å) 9.201(2) 13.5470(11) 7.7024(2)
b(Å) 9.201(2) 7.2157(6) 8.9975(4)
c(Å) 14.331(4) 14.8634(12) 13.9532(6)
α(°) 72.31(2) 90.00 99.533(4)
β(°) 72.31(2) 90.187(3) 97.280(3)
γ(°) 73.159(2) 90.00 104.157(3)
Volume(Å3) 1074.9(4) 1452.9(2) 910.42(6)
Z 2 18 2
Absorption coefficient 1.086 mm−1 6.497 mm−1 0.705 mm−1
F(000) 540 918 479
Theta range for data collection 2.98 to 25.00° 2.03 to 23.74° 2.91 to 25.00°
Reflections collected/unique 10974/3727 [R(int) = 0.0859] 11283/1578 [R(int) = 0.0576] 8621/3192 [R(int) = 0.0207]
Completeness to theta 98.4% 71.2% 99.7%
Goodness-of-fit on F2 1.021 0.92 1.021
Final R indices [I N 2σ(I)] R1 = 0.0638, wR2 = 0.1613 R1 = 0.0472, wR2 = 0.1213 R1 = 0.0262, wR2 = 0.0653
R indices (all data) R1 = 0.1058, wR2 = 0.1818 R1 = 0.0795, wR2 = 0.1409 R1 = 0.0329, wR2 = 0.0686
Largest diff. peak and hole 2.003 and −0.959 e. Å3 0.347 and −0.470 e. Å3 0.296 and −0.356 e. Å3

using the molar absorptivity (6600 M−1 cm−1) at 260 nm [37]. The [Complex], the extinction coefficient for the free copper(II) complex
absorption titration of 1 and 3 (100 μM) in Na-phosphate buffer (pH initially, after sequential addition of DNA and extinction coefficient for
7.2) with 10% DMSO against CT DNA were performed by monitoring the copper(II) complex in the fully bound form, respectively [38]. Kb is
the changes in absorption spectra. The titration experiments were the ratio of slope to the intercept.
performed by maintaining the concentration of metal complexes
constant at 100 μM while the concentration of CT DNA was varied 2.8.2. Competitive binding studies
within 25–225 μM. An equal quantity of CT DNA was also added to the Relative binding of the copper complexes to CT DNA was studied by
reference solution to eliminate the absorption by DNA. From the fluorescence spectroscopy using ethidium bromide (EB) bound to CT
absorption data, the intrinsic binding constant Kb was calculated from DNA in a Na phosphate buffer solution (pH 7.2). In a typical experiment,
a plot of [DNA] / (εa − εf) vs. [DNA] using the equation: 20 μL of CT-DNA solution (A260 = 2.0) was added to 2.0 mL of EB buffer
solution (pH 7.2) and the fluorescence intensity was measured upon
½DNAŠ = ðεa −εf Þ = ½DNAŠ = ðεb −εf Þ + ½Kb ðεb −εf ÞŠ
−1 excitation at 510 nm; maximum emission was observed at 600 nm. The
complex concentration was increased by addition of aliquots from a
0.1 mM stock solution until the fluorescence intensity did not decrease
where [DNA] represents the concentration of DNA in base pairs. The
any further. Stern–Volmer quenching constants were calculated using
apparent absorption coefficients εa, εf and εb correspond to Aobsd/
the following equation [39],

Iο = I = 1 + Ksv r;
Table 2
Selected bond lengths (Å) and angles (°).
where Iο and I are the fluorescence intensities in absence and
Complex 1
presence of complexes, respectively, Ksv is a linear Stern–Volmer
Cu1–O2 1.873(9) O2–Cu1–O1 97.27(18) quenching constant and r is the ratio of the total concentration of
Cu1–O1 1.877(19) O2–Cu1–N1 89.43(19) complex to that of DNA. The value of Ksv is given by the ratio of slope
Cu1–N1 1.985(24) O1–Cu1–N1 169.87(20)
Cu1–N2 1.987(7) O2–Cu1–N2 168.93(22)
to intercept in a plot of Iο/I vs. [Complex]/[DNA].
O1–C14 1.418(15) O1–Cu1–N2 92.3(2)
O2–C18 1.288(17) N1–Cu1–N2 81.86(21) 2.8.3. DNA cleavage study
The nuclease activity of the copper(II) complexes was studied
Complex 2
using supercoiled pBR322 and pUC19 plasmid DNA. Electrophoresis in
Cu1–O1 1.881(5) N2–Cu1–O4 95.66(23) native agarose gel was used to quantify the unwinding of plasmid
Cu1–N2 1.971(6) O1–Cu1–N1 94.58(21)
DNA induced by copper(II) complexes. The cleavage reactions on
Cu1–O4 1.973(6) N2–Cu1–N1 81.58(23)
Cu1–N1 1.998(6) O4–Cu1–N1 165.84(23)
pBR322 were carried out for 24 h at 37 °C in a total volume of 25 μL
Cu1–O6i 2.303(5) O1–Cu1–O6i 95.08(19) containing 0.5 μg pBR322 DNA and different concentrations of
O6–Cu1ii 2.303(5) N2–Cu1–O6i 93.07(21) complexes (ranging from 10 to 500 μM) in 5 mM Tris–HCl buffer
O1–Cu1–N2 171.70(21) O4–Cu1–O6i 87.72(19) (pH 7.2), 50 mM NaCl and 10% DMSO. The samples were analyzed by
O1–Cu1–O4 86.27(21) N1–Cu1–O6i 106.25(22)
electrophoresis for 3 h at 50 V on 1% agarose gel in 1× TAE buffer
Complex 3 (40 mM Tris acetate and 1 mM EDTA) pH 8.3. The gel was stained with
a 0.5 μg/ml ethidium bromide and visualized by UV light and then
Cu1–O1 1.963(3) O1–Cu1–O1i 179.99(5)
Cu1–O1i 1.963(3) O1–Cu1–O8i 90.55(6) photographed for analysis. The extent of cleavage was determined
Cu1–O8i 1.976(2) O1i–Cu1–O8i 89.45(6) from the intensities of the bands using the AlphaImager 2200
Cu1–O8 1.976(2) O8i–Cu1–O8 179.99(6) software [40]. However, cleavage study on pUC19 was carried in a
Cu1–O7i 2.415(7) O1–Cu1–O7i 95.43(5) total reaction volume of 10 μL, containing 100 ng (1 μL) of pUC19
Cu1–O7 2.415(7) O8i–Cu1–O7i 89.11(6)
DNA, and different concentrations of complexes 1 and 3 in 5 mM Tris–


HCl buffer (pH 7.2) containing 25% DMSO for 24 h at 37 °C. The triphosphate) mix (2 mM), 1 μL Taq buffer (10 mM Tris–HCl, 50 mM
samples were analyzed by electrophoresis for 1.3 h at 75 V on 1% KCl, and 1.5 mM MgCl2) pH 8.3 and 0.5 μL Taq DNA polymerase (5 U/μL)
agarose gel in 1× TAE buffer. The gel was stained with 1:20000 stain G, in a final volume of 10 μL. The primer extension reactions were run
visualized by UV light, and photographed for analysis. under PCR conditions with temperature cycling (30 cycles of denatur-
ation at 94 °C (30 s), annealing at 50 °C (30 s), and extension at 72 °C
2.8.4. Determination of site of DNA cleavage (30 s), followed by a final extension at 72 °C for 5 min). After the
For this study pUC19 plasmid DNA was used. The linearization of completion of PCR reaction, 3 μL of stop solution was added to both the
pUC19 with complexes was studied first and then primer extension tubes and heated at 90 °C for 2 min. The tubes were then cooled in an ice
reactions were carried out to locate the probable site of cleavage. The bath for 2 min and the samples were loaded on a 10% denaturing
details of expected extended product and primers and restriction polyacrylamide gel. The gel was run for 1.5 h at 35 W. The gel was
enzyme combinations used are given in Table 3. soaked on a filter paper and then dried in a gel dryer (80 °C for 30 min)
and exposed overnight to a phosphor screen. The screen was scanned to
2.8.4.1. Restriction digestion of pUC19 visualize the DNA bands.
2.8.4.1.1. Eco-RI restriction digestion. In a reaction tube, 100 ng
(1 μL) of pUC19 DNA, 1 μL of Eco buffer (50 mM Tris–HCl pH 7.5, 2.8.5. In vitro cytotoxicity assay
10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100 and 0.1 mg/mL BSA), The DL (Dalton's lymphoma: a transplantable T cell lymphoma) cells
0.5 μL of EcoRI restriction enzyme (10 U/μL) and 7.5 μL of deionized were collected from the mouse ascite. The viable DL cells, determined by
water were mixed together. Then the tube was incubated at 37 °C for trypan blue exclusion test, were seeded onto 96 well plates in 100 μL of
1 h. This reaction mixture was used for a control lane for visualizing the RPMI-1640 culture medium supplemented with 10% fetal bovine
linear pUC19 DNA. serum, penicillin G(100 U/mL), and streptomycin(100 μg/mL). The cells
2.8.4.1.2. PvuII restriction digestion. 50 ng of pUC19 (1 μL), 1 μL of were then allowed to grow in a CO2 incubator with 5% CO2 at 37 °C. After
buffer G (10 mM Tris–HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.1 mg/ 24 h incubation, different concentrations (10−15 to 10−8 M) of the
ml bovine serum albumine), 0.5 μL of PvuII restriction enzyme (10 copper (II) complexes, made by serial dilutions in the culture medium,
units/μL) and 7.5 μL of deionized water were incubated for 1 h at were added and the plates were incubated for another 24 h. Cell viability
37 °C. This PvuII digested DNA was used as template in control primer was determined by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
extension reactions. diphenyltetrazolium bromide) assay, which is based on the ability of
the viable cells to reduce a soluble yellow tetrazolium salt to blue
2.8.4.2. pUC19 linearization with complex 1. 50 ng of pUC19 was formazan crystals [41]. Briefly, after 24 h of the treatment, the MTT dye
incubated with 100 μM of complex 1 at 37 °C for 24 h in 5 mM Tris– (10 μL/100 μL of medium), prepared in phosphate buffered saline (PBS),
HCl buffer (pH 7.2), 25% DMSO. This linearized DNA was used as was added to all the wells. The plates were then incubated for 4 h at
template in primer extension reactions. 37 °C, the medium was discarded and 100 μL of DMSO was added to
each well. Optical density was measured at 570 nm. As described in our
2.8.4.3. 5′-32P-Labeling of primers with T4 polynucleotide kinase (PNK). previous report [42] the percentage of viable cells was determined by
In a reaction tube, 25 pmol (0.25 μL) of the primer, 1 μL of PNK buffer taking the cell counts in the untreated sets as 100%. The semi logarithmic
(70 mM Tris–HCl, 10 mM MgCl2 and 5 mM dithiothreitol) pH 7.6, dose–response plots, constructed using the Graphpad Prism5 software
0.5 μL of [γ-32P] ATP (10 mCi/mL) of specific activity 3000 Ci/mmol, [43], were used to determine the IC50 values as the complex
0.5 μL of PNK enzyme (10 U/μL) and 7.75 μL of deionized water were concentrations that inhibited DL cell growth by 50%.
added together. After incubation for 1 h at 37 °C, stop solution (10 μL)
containing 95% formamide, 1 mg/ml bromophenol blue and 1 mg/ml 3. Results and discussion
xylene cyanol was added. The enzyme was deactivated by incubation
at 90 °C for 2 min and the reaction mixture was loaded on 12% 3.1. Synthesis and characterization
denaturing polyacrylamide gel. The gel was run at 35 W for 1 h. The
labeled DNA was extracted by crush-and-soak using TEN buffer In our earlier study it has been reported that LH4 reacts with Zn(bpy)
(10 mM Tris–HCl pH 8.0, 1 mM EDTA, and 300 mM NaCl) and then (NO3)2 2H2O, and provides a supramolecular structure consisting of two
precipitated using three volumes of cold absolute ethanol. LH− anion and one [Zn(bpy)2·2H2O]2+ cation together with seven co-
3
crystallized water molecules [31]. Enthused by this study, a reaction of
2.8.4.4. Primer extension studies. In a typical primer extension LH4 was carried with another metal precursor 2,2′-bipyridyl-dinitrato-
experiment, two reactions were performed in parallel using Taq DNA copper(II)-dihydrate in anticipation that the Cu(II) ion, due to its
polymerase and linearized pUC19 DNAs as templates. For the control distorted configuration and its redox active nature, may interact with
reaction, pUC19 was linearized with PvuII restriction enzyme, whereas the ligand LH4 in a different way compared to Zn(II) ion. The reaction
the reaction product from incubation of pUC19 with complex 1 was used between Cu(bpy)(NO3)2·2H2O and LH4 in DMF containing MeOH
for locating the cleavage site. Primer extensions were run with both resulted initially in a dark green solution, from which two products
templates using 1 μL 32P-labeled primer, 2 μL dNTP (deoxynucleotide crystallized, a dark green complex 1 and a blue coloured complex 2.

Table 3
Primers of pUC19 and restriction enzymes used for primer extension studies.

Primer 5′–3′ Sequence Comment Restriction enzyme used Length of extended
to prepare template DNA product (bp)

P1 GTAAAACGACGGCCAGT M13/pUC fwd 379–395 PvuII 249
P2 AACAGCTATGACCATC M13/pUC rev 476–461 PvuII 170
P3 GGAGACGGTCACAGC pUC19 fwd 50–64 PvuII 256
P4 TCGGAACAGGAGAGC pUC19 rev 1000–986 PvuII 372
P5 GGTACCTGTCCGCC pUC19 fwd 1016–1029 BSaI 750
P6 AAGCATCTTACGGATG pUC19 rev 2162–2147 BSaI 396
P7 CAATAACCCTGATAAATGC pUC19 rev 2531–2513 ScaI 354
P8 CACATTTCCCCGAAAAGT pUC19 fwd 2592–2610 PvuII 400


These complexes were characterized initially by their IR spectra.
Complex 1 showed sharp peaks at 1695 and 1602 cm−1, whereas
complex 2 showed a distinct peak at 1731 cm−1 in addition to other
major peaks at 1665 and 1611 cm−1. Thus, the IR spectra suggested
that both complexes 1 and 2 contain ligands of different constitution.
This was conﬁrmed by solving their X-ray crystal structure, which
demonstrated that the original framework of LH4 was no longer
present. The ligand LH4 was transformed into two different com-
pounds which in situ coordinated with Cu(II)-2,2′-bipyridine to give
two new complexes (Scheme 1).
In addition, LH4 was allowed to react with copper nitrate in the
absence of the bipyridyl ligand. This resulted in formation of green
coloured complex 3, which was characterized by the presence of two
molecules of the original barbiturate derivative LH4 acting as Cu(II)
ligands.

3.2. Structural description of complexes

Complex 1 consists of a tetra coordinated Cu(II) ion having a
N2O2 coordination core (Fig. 1a), involving 2 nitrogen atoms from 2,2′-
bipyridine and 2 oxygen atoms from 5-hydroxy-hydurilic acid. It
crystallizes into a triclinic P-1 space group and Cu–N and Cu–O distances
are lying in the reported range (Table 2). It has a Kitaigorodskii Packing
Index (KPI) of 70.2% which shows compact packing with few solvent
Fig. 1. (a) Molecular structure of 1 (30% probability ellipsoid), hydrogen atoms are
accessible voids [44]. Several H-donor and acceptor functional groups
omitted for clarity and (b) a perspective of water clusters in crystal lattice of 1.
present on the skeleton of the ligand form ten hydrogen bonds (S-1).
The formation of six non-conventional hydrogen bonds involves C–H as
H-donor and oxygen as H-acceptor whereas four conventional H-bonds
are formed using N–H as donors and O as acceptors. The co-crystallized Like complex 1, complex 3 also crystallizes in a triclinic P-1 space
water molecules are arranged in a C3 chain water cluster in packing group. Its Cu(II) ion is surrounded by 4 oxygen atoms, two originate
diagram forming a water hexamer (Fig. 1b). from two monodentate ligands as LH−, whereas two other oxygen
3
Complex 2 is monoclinic with P-1 space group. It consists of a penta- atoms are from two coordinated water molecules (Fig. 3a). Complex 3
coordinated Cu(II) ion with N2O3 coordination core from 2,2′-bipyridyl exhibits square planar geometry and also contains four co-crystallized
(2N) and alloxanic acid (3O) (Fig. 2a). The Cu–O and Cu–N bond water molecules (Fig. 3b) which stabilize the structure by formation
distances (Table 2) are found in range as reported for other penta- of hydrogen bonds (S-2).
coordinated Cu(II) complexes [45]. The assembly of monomeric unit
leads to a helical 1D polymeric framework (Fig. 2b). The study of weak 3.3. UV–vis spectroscopy and electrochemical studies
interactions using PLATON indicates the presence of seven hydrogen
bonds in crystal packing of complex 2. Five non-conventional hydrogen The complexes were characterized by UV–vis spectroscopy and
bonds involve C–H donor groups, and two conventional hydrogen bonds their electrochemical properties were determined. The paramagnetic
involve O–H as donors (Fig. 2c). copper(II) complexes 1 and 3 in solution (10−4 M in DMSO) exhibited

O
O

HN NH O
HN NH
H
O O N N O
Cu(bpy)(NO3)2.2H2O O
O O O Cu O
N
DMF, MeOH O O N Cu
N O
HN N
HN N O
O N O
H
O
1 2
Cu(NO3)2.2H2O

O O
NH
DMF, MeOH HN
NH H2O HN O
O

O Cu O
O O

H2O HN
NH O
O NH
N
H O O
3

Scheme 1. Synthetic strategy for complexes 1-3.


Fig. 2. (a) Molecular structure of a single unit of 2 (30% probability ellipsoid), hydrogen atoms are omitted for clarity, (b) zigzag polymeric structure of 2 and (c) conventional
hydrogen bonds in crystal lattice of 2.

a broad d–d band in the range of 590–690 nm with a molar extinction complex 2 in powder displayed well resolved four lines at liquid N2
coefficient of 145–235 M−1 cm−1. However, intense absorption bands temperature. The axial g and A tensor values with g∥ N g⊥ suggest that
are observed at 260–270 nm, which are attributed to π–π* transitions. dx2–y2 is a ground state while g0 values are calculated using the
Absorption bands observed in the region of 300–400 nm are assigned relationship g0 = (g∥ + 2g⊥) [46]. The values of calculated ESR
to n–π* transition overlapping with ligand to metal charge transfer parameters are shown in Table S-6. Although the ratio g∥/|A∥| is
(LMCT) transition between the heterocyclic base and the metal ion. normally taken as an indication of the stereochemistry of the copper
Since, the DNA binding and cleavage studies were carried out in (II) complexes, yet it is suggested that this ratio may be an empirical
aqueous medium, UV–vis spectra of complexes 1 and 3 were also indication of the tetrahedral distortion of a square planar geometry
recorded in DMSO/water (v/v, 1:10) mixture. It showed (S-3) that [47]. The values of hyperfine splitting lower than 135 cm−1 are
complexes retain their structures in DMSO as well as in DMSO/water observed for square planar structures and those higher than 150 cm−1
mixture. for tetrahedrally distorted complexes. The data shown in Table S-6 are
The complexes display a quasi-reversible cyclic voltammetric found in consistence with earlier reports as well as structure observed
response in the range of 0.2 to 0.5 V (vs. silver reference electrode) in from their X-ray diffraction studies.
DMSO (10−4 M). The redox peak is assigned to Cu(II)/Cu(III) couple in
view of reported redox potential data (S-4). 3.5. Thermo-gravimetric studies

3.4. Electron spin resonance Thermo gravimetric analysis (TGA) (S-7) of the complexes showed
that the loss of crystallized water molecules starts at ~90 °C in each
The ESR spectra of complexes 1 and 3 in DMSO at 66 K displayed complex. The weight loss continues up to 165 °C in complex 1 and the
the typical four-line pattern as expected from 63Cu nucleus (S-5). magnitude of the weight loss (%) corresponds to three water
Three parallel hyperfine lines were well resolved in both complexes molecules (observed 11.1, calculated 10.4). In complex 2 weight
while the fourth line overlapped with g⊥ signal. The spectrum of loss of 4.2% corresponds to removal of one water molecule (calcd.


Fig. 3. (a) Molecular structure of 3 (30% probability ellipsoid), hydrogen atoms are omitted for clarity and (b) a perspective of water clusters in crystal lattice of 3.

3.8%) and it continues up to 150 °C. However, in complex 3 six water
molecules (two coordinated and four co-crystallized) are lost
between 90 and 130 °C (observed weight loss 12.3%, calculated
weight loss 11.6%). The TGA data thus showed that the water
molecules are bound weakly in the lattice of complex 3 as compared
to complexes 1 and 2 [48].

3.6. DNA binding studies

In general, intercalation of a complex into DNA results in a
hypochromic red shift of its absorption band. This may occur due to
strong stacking interactions between the aromatic chromophore of
the complex and the base pairs of the DNA. On increasing the
concentration of CT DNA, the hypochromicity increased in the ligand-
centred (LC) band of complex 1. In contrast, a hyperchromic effect
was observed in LC band of complex 3 (Fig. 4). The copper(II)
complexes can bind to the double-stranded DNA in different modes
on the basis of their structure, charge and type of ligands. As DNA
double helix possesses many hydrogen bonding ligands accessible
both in the minor and major grooves, it is likely that the N–H group of
barbiturate ligand might be forming hydrogen bonds with DNA.
Hence, it may contribute to the hyperchromic shift in its absorption
spectrum. In order to compare the binding strength of the complexes
with CT DNA, the intrinsic binding constants Kb were obtained from
the ratio of slope to the intercept from the plots of [DNA] / (εa − εf) vs.
[DNA]. The calculated Kb values of 1.9 × 106 M−1 and 1.7 × 105 M−1 for
complexes 1 and 3 respectively show that DNA binds complex 1
stronger than complex 3.

3.7. Competitive binding with ethidium bromide

The ability of a complex to affect the fluorescence intensity of EB-
DNA adduct is a reliable tool for the measurement of its affinity
towards DNA. Intense fluorescent light is emitted from EB in presence
of DNA owing to its strong intercalation between adjacent DNA base
pairs. A complex binds with DNA by the displacement of EB bound to
DNA. Consequently, the intensity of emission is reduced as emission
from free EB is readily quenched by surrounding water molecule [49].
Fig. 4. UV–vis absorption spectra of (a) [complex 1] = 25 μM in the absence and in
The emission quenching from DNA bound ethidium bromide is due to presence of increasing amounts of DNA = 0–225 μM and (b) [complex 3] = 25 μM in the
displacement of ethidium bromide from the DNA helix. The emission absence and in presence of increasing amounts of DNA = 0–225 μM. Arrow shows the
spectra of EB-DNA system in the presence and absence of copper absorbance changes upon increasing DNA concentrations.


Fig. 5. Emission spectra of EB bound to DNA in the absence (—) and in the presence of, [complex 1] 0–4 μM, [EB] 10 μM, [DNA] 10 μM. Arrow shows changes in the emission intensity
upon addition of increasing concentration of the complex.

complexes 1 and 3 are shown in Figs. 5 and 6. The addition of shows two conservative CD bands in the UV region, a positive band at
complexes to DNA pretreated with EB shows appreciable reductions 278 nm due to base stacking and a negative band at 246 nm due to
in emission intensity. On the addition of 4 μM of complex 1 to 10 μM of poly nucleotide helicity [50]. The changes in CD pattern of DNA
CT DNA pretreated with EB, ~ 80% displacement of ethidium bromide observed after interaction with these complexes is considered to
was observed. This suggests that complex 1 is a good intercalator. assign the corresponding changes in structure of DNA [51]. Simple
However, complex 3 brings about only ~ 40% displacement of groove binding and electrostatic interaction of small molecules show
ethidium bromide at the same concentrations of both CT DNA and less or no perturbation on the base-stacking and helicity bands, while
the complex. The quenching plots of Iο/I vs. [Complex]/[DNA] (insets intercalation enhances the intensities of both bands and stabilizes the
in Figs. 5 and 6) are in good agreement with the linear Stern–Volmer right-handed B conformation of CT DNA, as observed for classical
equation. Stern–Volmer quenching constants (Ksv) were calculated to intercalator methylene blue [52].
be 3.8 and 1.2 for complex 1 and complex 3 respectively. CD spectral variations of calf thymus DNA (50.0 μM, in 0.1 mM Na-
phosphate buffer (pH = 7.4), were recorded in the presence of
3.8. CD spectral studies increasing amounts of complexes 1 and 3 until [complex]/[DNA]
molar ratios approached approximately 0.4. By addition of complex 1,
Circular dichroism measurements were conducted in order to a blue shift of the positive CD band of DNA was observed (Fig. 7a).
determine the extent of changes which occur in DNA conformation These ﬁndings indicate that a subtle change of the DNA double helix
upon binding of complexes 1 or 3. The B form conformation of DNA occurs owing to the interaction of the metal complex with DNA [53].

Fig. 6. Emission spectra of EB bound to DNA in the absence (—) and in the presence of, [complex 3] 0–4 μM, [EB] 10 μM, [DNA] 10 μM. Arrow shows changes in the emission intensity
upon addition of increasing concentration of the complex.


Fig. 8. (a) Gel electrophoresis diagram showing the cleavage of SC pBR322 DNA (0.5 μg) by
complex 1 on 12 h of incubation in 50 mM Tris–HCL buffer (pH 7.2): lane 1, DNA control;
lane 2, DNA + 10 μM; lane 3, DNA + 25 μM; lane 4, DNA + 50 μM; lane 5, DNA + 100 μM;
and lane 6, DNA + 500 μM, (b) cleavage of supercoiled pBR322 DNA showing the decrease
in SC DNA and the formation of NC DNA with increasing concentration of complex 1.

activity in the physiological pH range. Though, future experiments
will be needed to characterize the cleavage mechanism in detail.
However, a preliminary experiment showed that neither Cu(bpy)
(NO3)2·2H2O nor LH4 separately caused DNA cleavage (S-8).

3.10. Determination of site of DNA cleavage

The gel electrophoretic separation of plasmid pUC19 DNA induced
Fig. 7. Circular dichroism spectra of CT-DNA (50 μM) in the absence (—) and presence of by complexes 1 and 3 and EcoRI is shown in Figs. 10 and 11.
complex 1 (a) and complex 3 (b) in 0.1 mM Na-phosphate buffer. Complexes 1 and 3 linearize pUC19 at concentrations of 25 μM and
10 μM respectively on incubation for 24 h in a medium of Tris–HCl/
NaCl pH 7.2 containing 25% DMSO. The intensity of linear form
Therefore, it can be inferred that complex 1 tightly binds to DNA. increases with the increase in the concentration of complexes.
However, binding of DNA with complex 3 induces a decrease in the Primer extension reactions were performed to assay the site of
intensity of both positive and negative bands with a red shift in the DNA cleavage by complex 1. In these experiments, 5′-32P-labeled
position of the band [54]. primers annealed to template DNA are extended from their 3′-end
with Taq DNA polymerase until the 5′-end of the template DNA is
3.9. DNA cleavage study reached. This “end” in the template DNA can either be generated by
a restriction enzyme (linearization of the plasmid DNA) or by Cu-
The DNA-cleaving ability of the copper(II) complexes has been complex catalyzed cleavage of the DNA. A set of 8 primers in
studied by the relaxation of supercoiled pBR322 DNA to the nicked combination with three restriction enzymes was used to probe the full
circular DNA. When circular plasmid DNA is subjected to electropho- length of the 2686 base-pair long pUC19 plasmid (Fig. 12). Primer
resis, relatively fast migration is observed for intact supercoiled form extension of 32P-labeled primer 8 from the DNA template that had
(S form). However, if scission of DNA occurs at one strand (nicking), been treated with complex 1 yielded an extension product that was
the supercoiled DNA will relax to generate a slower-moving open/ much shorter than the product generated from a control reaction with
nicked circular (NC form). If both strands are cleaved, a linear form (L) PvuII-digested DNA (S-9). No stop was observed on DNA treated with
will be generated [55]. The gel electrophoretic mobility assay (Figs. 8 complex 1 in other primer extension reactions using the primers of
and 9) showed that both copper(II) complexes convert supercoiled Table 3. A likely explanation for this observation is that complex 1
(SC) plasmid pBR322 DNA into nicked circular (NC) DNA after cleaves pUC19 speciﬁcally within the ca 400 bp region between the
incubation at 37 °C for 24 h in a medium of Tris–HCl/NaCl pH 7.2. primer 8 binding site and the ﬁrst PvuII cleavage site (position 306,
Complex 1 converts more than 90% of SC form into NC form at a see Fig. 12).
concentration of 100 μM, whereas, for a similar level of conversion,
only 50 μM of complex 3 is required. Thus, both complexes show 3.11. Evaluation of cytotoxicity in vitro
nuclease activity without addition of any external oxidizing or
reducing agent unlike most of the other Cu(II)-based nucleases. In presence of copper(II) complexes, IC50 values were determined
These results suggest that the copper(II) complexes show nuclease against Dalton's lymphoma (DL) cell lines. The MTT assay measures


Fig. 11. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (0.1 μg) by
complex 3 on 24 h of incubation in 5 mM Tris–HCL buffer (pH 7.2): lane 1, marker; lane
2, DNA control; lane 3, EcoRI treated DNA; lane 4, DMSO control; lane 5, DNA + 10 μM;
lane 6, DNA + 25 μM; lane 7, DNA + 50 μM.

Fig. 9. Gel electrophoresis diagram showing the cleavage of SC pBR322 DNA (0.5 μg) by and the IC50 values are determined using the GraphPad Prism5
complex 3 on 12 h of incubation in 50 mM Tris–HCL buffer (pH 7.2): lane 1, DNA software.
control; lane 2, DNA + 10 μM; lane 3, DNA + 25 μM; lane 4, DNA + 50 μM; and lane 5,
The results thus obtained suggested that after 24 h of incubation,
DNA + 100 μM, (b) cleavage of supercoiled pBR322 DNA showing the decrease in SC
DNA and the formation of NC DNA with increasing concentration of complex 3. the copper(II) complexes are cytotoxic against DL cells with an IC50
values ~9.0 nm and 0.6 nm for complexes 1 and 3 respectively. Copper
(II) complexes decreased viability of DL cells in a concentration-
mitochondrial dehydrogenase activity as an indication of cell viability. dependent manner (with increasing concentration from 10−15 M to
It has been carried out with the copper complexes using murine 10−8 M). A ~40% decrease in cell viability is observed in the presence
Dalton's lymphoma cells which are T cell lymphoma of spontaneous of Cu(II) complexes as compared to control. The values of IC50
origin in the thymus. Dalton's lymphoma cells have often been indicate that complex 3 is a stronger cytotoxic agent than complex 1
successfully used to identify the anticancer potential of newly when tested against DL cell (S-10). These values are found to be
synthesized compounds both in vitro and in vivo [56]. Hence, the significantly higher than the IC50 value of cisplatin against DL cell
effect of Cu(II) complexes on the viability of DL cell lines has been lines [57].
measured after 24 h of treatment as a function of concentration. The
experiments have been performed in triplicates for all the complexes
4. Conclusion

Three new copper(II) complexes of different geometry were
prepared and characterized. Complexes 1 and 2 bearing 2,2′-bipyridyl
as terminal ligand were isolated in a one pot synthesis as a result
of transformation of the original barbiturate ligand LH4 in the
presence of Cu(bpy)(NO3)2·H2O. The complexes contain a significant
number of co-crystallized water molecules in their crystal lattice
which stabilize the corresponding supramolecular structures through
H-bonds.
Complexes 1 and 3 bind with the calf thymus DNA strongly though
the binding constant for complex 1 is little higher than that of
complex 3. These complexes also transform supercoiled DNA to
nicked and linear forms under physiological conditions and possess
considerable chemical nuclease activity. In contrast to DNA binding
results, DNA cleavage studies indicated that complex 3 is a better
nuclease in comparison to complex 1. The better binding affinity of
complex 1 with DNA could be due to the presence of 2,2′-bipyridine
ligand which reportedly intercalates well with DNA. However, the
better nuclease property of complex 3 could be attributed to the
presence of ligand LH4 bearing various H donor and acceptor
functionalities in its structure. The findings also suggest that the
DNA cleavage property of the described complexes is region-specific.
Such molecules may offer new prospects for controlled manipulation
Fig. 10. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (0.1 μg) by
complex 1 on 24 h of incubation in 5 mM Tris–HCL buffer (pH 7.2): lane 1, marker; lane
of the genome and therefore, can be of great interest in biotechnology
2, DNA control; lane 3, EcoRI treated DNA; lane 4, DMSO control; lane 5, DNA + 10 μM; and therapeutics. Both complexes 1 and 3 are also active against
lane 6, DNA + 25 μM; lane 7, DNA + 50 μM. Dalton's lymphoma cell lines at nano-molar concentrations.


Fig. 12. Schematic representation of pUC19 plasmid target with location of cleavage sites of restriction enzymes and lengths of primer extension products using appropriate primers.
Arrows represent the direction of primer extension.

Abbreviations [6] R. Tawa, D. Gao, M. Takami, Y. Imakura, K.-H. Lee, H. Sakurai, Bioorg. Med. Chem. 6
(1998) 1003–1008.
bpy 2,2′-bipyridine [7] M. Pitié, C.J. Burrows, B. Meunier, Nucleic Acids Res. 28 (2000) 4856–4864.
CD circular dichroism [8] M.E. Branum, A.K. Tipton, S. Zhu, L.J. Que, J. Am. Chem. Soc. 123 (2001) 1898–1904.
CT DNA calf thymus DNA [9] E. Boseggia, M. Gatos, L. Lucatello, F. Mancin, J. Am. Chem. Soc. 126 (2004)
4543–4549.
DL Dalton's lymphoma: a transplantable T cell lymphoma
[10] U.S. Singh, R.T. Scannell, H. An, B.J. Carter, S.M. Hecht, J. Am. Chem. Soc. 117 (1995)
DMF dimethyl formamide 12691–12699.
EB ethidium bromide [11] S. Oikawa, S. Kawanishi, Biochemistry 35 (1996) 4584–4590.
ESR electron spin resonance [12] C.A. Detmer III, F.V. Pamatong, J.R. Bocarsly, Inorg. Chem. 35 (1996) 6292–6298.
[13] B.C. Bales, T. Kodama, Y.N. Weledji, M. Pitié, B. Meunier, M.M. Greenberg, Nucleic
L1 5-hydroxy-hydurilic acid Acids Res. 33 (2005) 5371–5379.
L2 alloxanic acid [14] D.S. Sigman, A. Mazumder, D.M. Perrin, Chem. Rev. 93 (1993) 2295–2316.
LH4 1,5-dihydro-5-[5-pyrimidine-2,4(1H,3H)-dionyl]-2H-chro- [15] U.S. Singh, R.T. Scannell, H. An, B.J. Carter, S.M. Hecht, J. Am. Chem. Soc. 117 (1995)
12691–12699.
meno[2,3-d] pyrimidine-2,4(3H)-dione [16] B.C. Bales, T. Kodama, Y.N. Weledji, M. Pitié, B. Meunier, M.M. Greenberg, Nucleic
MeOH methanol Acids Res. 33 (2005) 5371–5379.
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium [17] P.J. Benites, R.C. Holmberg, D.S. Rawat, B.J. Kraft, L.J. Klein, D.G. Peters, H.H. Thorp,
J.M. Zaleski, J. Am. Chem. Soc. 125 (2003) 6434–6446.
bromide [18] M. Tanaka, K. Ohkubo, S. Fukuzumi, J. Am. Chem. Soc. 128 (2006) 12372–12373.
NC nicked circular [19] P.U. Maheswari, S. Roy, H.D. Dulk, S. Barends, G.V. Wezel, B. Kozlevčar, P. Gamez,
PCR polymerase chain reaction J. Reedijk, J. Am. Chem. Soc. 128 (2006) 710–711.
[20] K. Li, L. Zhou, J. Zhang, S. Chen, Z. Zhang, J. Zhang, H. Lin, X. Yu, Eur. J. Med. Chem.
PNK T4 polynucleotide kinase
44 (2009) 1768–1772.
SC super coiled [21] J. Wang, Q. Xia, X. Zheng, H. Chen, H. Chao, Z. Mao, L. Ji, Dalton Trans. 39 (2010)
TBAP tetra butyl ammonium perchlorate 2128–2136.
[22] D. Li, J. Tian, W. Gu, X. Liu, S. Yan, J. Inorg. Biochem. 104 (2010) 171–179.
TGA thermo gravimetric analysis
[23] A. Burger, Medicinal Chemistry, 3 rd Ed.Wiley-Interscience, New York, 19708
1367.
[24] X. Cheng, K. Tanaka, F. Yoneda, Chem. Pharm. Bull. 38 (1990) 307–311.
[25] P. Poitier, Chem. Soc. Rev. 21 (1992) 113–1198 and references therein.
Acknowledgements [26] K.S. Gulliya, US Patent 5 (1997) 674 870. (e) M.Z. Teixeira, Med. Hypotheses 60(2)
(2003) 276-283.
[27] D.N. Reinhoudt, M. Crego-Calama, Science 295 (2002) 2403–2407.
Financial support received from DAE-BRNS (grant no. 2005/37/33/ [28] L.J. Prins, C. Thalacker, F. Würthner, P. Timmerman, D.N. Reinhoudt, Proc. Natl
BRNS/1807 to L. M.), Mumbai, India and partial ﬁnancial support from Acad. Sci. 98 (2001) 10042–10045.
Max Planck Society, Germany for the short term stay of N. D., SRF to R. [29] A. Zafar, S.J. Geib, Y. Hamuro, A.D. Hamilton, New J. Chem. (1998) 137–141.
[30] M.A. Mateos-Timoneda, M. Crego-Calama, D.N. Reinhoudt, Chem. Soc. Rev. 33
K. K. and JRF to B.K. M. by CSIR, New Delhi, India and the help received (2004) 363–372.
from IITB, Powai, Mumbai, India for recording the single crystal X-ray [31] N. Dixit, K. Goto, L. Mishra, H.W. Roesky, Polyhedron 29 (4) (2010) 1299–1304.
diffraction data of the complexes are gratefully acknowledged. [32] B. Champin, P. Mobian, J. Sauvage, Chem. Soc. Rev. 36 (2007) 358–366.
[33] J.D. Figueoa-Villar, C.E. Cruz, L.N. Santos, Synth. Comm. 22 (1992) 1159–1164.
[34] G.M. Sheldrick, SHELEX-97: Programme for Structure Solution and Reﬁnement of
Appendix A. Supplementary data Crystal Structure, Gottingen, Germany, 1997.
[35] MERCURY, New software for searching the Cambridge structural database and
visualizing crystal structures, I.J. Bruno, J.C. Cole, P.R. Edgington, M.K. Kessler, C.F.
CCDC reference numbers 650285, 708173 and 665796 contains the Macrae, P. McCabe, J. Pearson, R. Taylor, Acta Cryst B58 (2002) 389–397.
supplementary crystallographic data for 1, 2 and 3 respectively. These [36] PLATON, A.L. Spek, Acta Crystallogr. A46 (1990) 194–201.
[37] J. Marmur, J. Mol. Biol. 3 (1961) 208–218.
data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/ [38] A. Wolfe, G.H. Shimer, T. Meehan, Biochemistry 26 (1987) 6392–6396.
retrieving.html (or from the Cambridge Crystallographic Data Centre, [39] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed.Plenum Press,
12, Union Road, Cambridge CB2 1EZ, UK; fax: +44-1223/336-033; New York, 1999.
[40] AlphaImager 2200 software, Alpha Innotech Corporation 2401 Merced St. San
e-mail: deposit@ccdc.cam.ac.uk). The table of weak interactions; Leandro, CA 94577.
packing diagram of 1 and 3, UV–vis spectra of 1 and 3 in DMSO/H2O, [41] T. Mosman, J. Immunol, Methods 65 (1983) 55–63.
ESR data, TGA diagrams of complexes, polyacrylamide gel image [42] R. Prajapati, S.K. Dubey, R. Gaur, R.K. Koiri, B.K. Maurya, S.K. Trigun, L. Mishra,
Polyhedron 29 (2010) 1055–1061.
showing probable DNA cleavage site for complex 1 and MTT assay
[43] GRAPHPAD PRISM5, GraphPad Software, Inc., 2236 Avenida de la Playa La Jolta, CA
graph are available. Supplementary data to this article can be found 92037, USA.
online at doi:10.1016/j.jinorgbio.2010.11.006. [44] A.I. Kitaigorodskii, Molecular Crystals and Molecules, Academic Press, New York,
1973.
[45] C. Gkioni, A.K. Boudalis, Y. Sanakis, C.P. Raptopoulou, Polyhedron 26 (2007)
References 2536–2542.
[46] H. Yokoi, A.W. Addison, Inorg. Chem. 16 (1977) 1341–1349.
[1] A. Sreedhara, J.D. Freed, J.A. Cowan, J. Am. Chem. Soc. 122 (2000) 8814–8824. [47] U. Sagakuchi, A.W. Addison, J. Chem. Soc. Dalton Trans. (1979) 600–608.
[2] J.A. Cowan, Curr. Opin. Chem. Biol. 5 (2001) 634–642. [48] G. Xu, Y. Ding, Y. Huang, G. Liu, W. Sun, Micro. Meso. Mater. 113 (2008) 511–522.
[3] J.A. Cowan, Y. Jin, J. Am. Chem. Soc. 127 (2005) 8408–8415. [49] J.-B. LePecq, C. Paoletti, J. Mol. Biol. 27 (1967) 87–106.
[4] F. Manicin, P. Scrimin, P. Tecilla, U. Tonellato, Chem. Commun. (2005) 2540–2548. [50] V.I. Ivanov, L.E. Minchenkova, A.K. Schyolkina, A.I. Poletayer, Biopolymers 12
[5] D.S. Sigman, Biochemistry 29 (1990) 9097–9105. (1973) 89–110.


[51] P. Lincoln, E. Tuite, B. Norden, J. Am. Chem. Soc. 119 (1997) 1454–1455. [55] B. Selvakumar, V. Rajendiran, P.U. Maheswari, H.S. Evans, M. Palaniandavar,
[52] B. Norden, F. Tjerneld, Biopolymers 21 (1982) 1713–1734. J. Inorg. Biochem. 100 (2006) 316–330.
[53] A. Silvestri, G. Barone, G. Ruisi, D. Anselmo, S. Riela, V.T. Liveri, J. Inorg. Biochem. [56] R.K. Koiri, S.K. Trigun, L. Mishra, K. Pandey, D. Dixit, S.K. Dubey, Invest. New Drugs
101 (2007) 841–848. 27 (2009) 503–516.
[54] Q. Jiang, Z. Wu, Y. Zhang, A.C.G. Hotze, M.J. Hannon, Z. Guo, Dalton Trans. (2008) [57] S. Sarna, R.K. Bhola, Curr. Sci. 93 (2007) 1300–1304.
3054–3060.

One pot synthesis of cu(ii) 2,2′ bipyridyl complexes of 5-hydroxy-hydurilic acid

Recomendados

Recomendados

Mais conteúdo relacionado

Mais procurados

Mais procurados (17)

Destaque

Destaque (7)

Semelhante a One pot synthesis of cu(ii) 2,2′ bipyridyl complexes of 5-hydroxy-hydurilic acid

Semelhante a One pot synthesis of cu(ii) 2,2′ bipyridyl complexes of 5-hydroxy-hydurilic acid (20)

Mais de rkkoiri

Mais de rkkoiri (11)

Último

Último (20)

One pot synthesis of cu(ii) 2,2′ bipyridyl complexes of 5-hydroxy-hydurilic acid