DNA binding, cytotoxicity and DNA cleavage promoted by gold(III) complexes

Article abstract of DOI:10.1016/j.inoche.2012.12.013

Square planar Au(III) complexes of 2,2′-dipyridylamine (A ¹), di(2-pyridyl)ketone (A²), 2-(4-chlorophenyl)-1H- imidazo[4,5-f] [1,10] phenanthroline (A³) and 2-(4-bromophenyl)-1H- imidazo[4,5-f][1,10] phenanthroline (A⁴) of type [Au(A ⁿ)Cl₂].Cl were synthesized and characterized using conductivity measurement, C,H,N elemental analysis, FT-IR, LC-MS, ¹H and ¹³C NMR spectroscopy. The compounds manifested significant cytotoxic properties in vitro for brine shrimp lethality bioassay. The metal complexes were screened for series of DNA binding activity using UV-vis. absorption titration, hydrodynamic measurement and thermal DNA denaturation study. The nucleolytic activity was performed on plasmid pUC19 DNA. The Michaelis-Menten kinetic studies were performed to evaluate rate of enhancement in metal complexes mediated DNA cleavage over the noncatalyzed DNA cleavage.

Full text of DOI:10.1016/j.inoche.2012.12.013

Inorganic Chemistry Communications 29 (2013) 190–193
Contents lists available at SciVerse ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
DNA binding, cytotoxicity and DNA cleavage promoted by gold(III) complexes
⁎
Mohan N. Patel , Bhupesh S. Bhatt, Promise A. Dosi
Department of Chemistry, Sardar Patel University Vallabh Vidyanagar–388 120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Square planar Au(III) complexes of 2,2′-dipyridylamine (A1), di(2-pyridyl)ketone (A2), 2-(4-chlorophenyl)-
1H-imidazo[4,5-f] [1,10] phenanthroline (A3) and 2-(4-bromophenyl)-1H-imidazo[4,5-f][1,10] phenanthroline
(A4) of type [Au(An)Cl2].Cl were synthesized and characterized using conductivity measurement, C,H,N elemental
analysis, FT-IR, LC–MS, 1H and 13C NMR spectroscopy. The compounds manifested significant cytotoxic properties
in vitro for brine shrimp lethality bioassay. The metal complexes were screened for series of DNA binding activity
using UV–vis. absorption titration, hydrodynamic measurement and thermal DNA denaturation study.
The nucleolytic activity was performed on plasmid pUC19 DNA. The Michaelis–Menten kinetic studies
were performed to evaluate rate of enhancement in metal complexes mediated DNA cleavage over the
noncatalyzed DNA cleavage.
Article history:
Received 22 October 2012
Accepted 12 December 2012
Available online 5 January 2013
Keywords:
Au(III) complexes
Covalent binding
Reaction kinetics
© 2013 Elsevier B.V. All rights reserved.
Gold(III) compounds are emerging as a new class of metal complexes
with outstanding cytotoxic properties and are presently being evaluated
as potential antitumor agents. Interest in gold coordination chemistry
has been growing in recent years with the successful use of auranofin in
oral administration, for the treatment of rheumatoid arthritis [1,2].
Auranofin has also shown evidence of anticancer activity in in vivo studies
of P388 leukemia in mice [3]. Unfortunately, in spite of the abundant
literature existing on gold drugs; only a few reports concern the biological
actions of gold(III) complexes [4]. The anticancer activity of Pt(II)
complexes containing nitrogen ligands is well documented, but its use
is limited by the development of drug resistance and associated toxic
side effects. The metal centers other than platinum (i.e. gold) might
produce specific and/or improved anticancer effects in vitro and in vivo
and may be developed as clinically useful drugs [5]. Some literature
suggests that gold(III) complexes can bind to DNA [6–9].
In recent years, the interest for gold(III) complexes can be judged from
the increased number of new gold(III) complexes synthesized using
N-ligands [10,11]. Therefore, to explore nuclease and cytotoxic profile of
gold complexes as an alternative of cisplatin and in continuation of our
earlier work [12], we synthesized Au(III) complexes of N-donor ligands
and characterized them using different analytical and spectroscopic
techniques. These compounds manifested significant cytotoxic properties
in vitro for brine shrimp lethality bioassay. The metal complexes were
screened for series of DNA binding activity to consider DNA interaction
of metal complexes as the possible reason for their cytotoxicity. The
nucleolytic activity was performed on plasmid pUC19 DNA. The reaction
kinetic for DNA cleavage was determined by performing time dependent
gel electrophoresis and also by performing Michaelis–Menten studies.
The complexes [Au(An)Cl2]Cl were isolated from the ethanolic
solution containing chloro auric acid as the starting material. Washing
with ether gave the product in sufficiently pure form to record NMR
spectra. All the complexes were obtained in good yield and were charac-
terized by conductivity measurement, elemental analysis, UV–vis., FT-IR,
LC–MS, 1H and 13C-NMR spectroscopic techniques. The molar conductiv-
ity data reveal that the complexes are 1:1 electrolytes, in accordance with
the proposed formulations. The reaction sequences are outlined in
Scheme 1. The experimental details are provided in the Supplementary
material (Supplementary S.1).
Au(III) with its d8 electronic configuration forms a variety of square–
planar complexes. The low energy LMCT transitions are quite important
for the study of Au(III) complexes. The MLCT transitions do not play
any role owing to the lack of reducing properties of Au(III). The time
dependent spectrophotometric analyses of ligand to metal charge
transfer transitions clearly show that these compounds are essentially
stable for at least 24 h in DMSO (Supplementary S.2). The spectra of
the samples dissolved in the reference buffer undergo some changes
at room temperature, implying that the metal complexes hydrolyzed
and converted into their dihydroxy form (Supplementary S.3).
Such hydrolysis phenomenon for Au(III) complexes has been also
reported by Messori et al. [13]. The hydrolyzed metal chromophore
[Au(A)(OH)2]+ fragment was sufficiently stable for at least 24 h in
10 mM phosphate buffer (pH 7.2) (physiological conditions). The LMCT
region of metal complexes with their extinction coefficient values are
shown in the experimental part.
Considering that a bioassay is the first step necessary for the drug
discovery process, the complexes were screened for in vitro toxicity
using the microwell assay. This method allows the use of smaller quanti-
ties of pure compounds and permits a larger number of samples with
dilutions within a shorter time. Brine shrimp lethality bioassay is a recent
development in the assay of bioactive compounds [14,15]. The LC50
⁎
Corresponding author. Tel.: +91 2692 226856 218.
E-mail address: jeenen@gmail.com (M.N. Patel).
1387-7003/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.inoche.2012.12.013

M.N. Patel et al. / Inorganic Chemistry Communications 29 (2013) 190–193
191
Scheme 1. Reaction sequence for synthesis of complexes.
(median lethal concentration) values obtained after the analysis of com-
plexes 1, 2, 3 and 4 are 14.4, 11.8, 6.8 and 8.9 μM respectively. The
reported LC50 values of some known drugs i.e. podophyllotoxin, cyclo-
phosphamide and etoposide in the literature are 6.7 μM (2.76 μg/mL)
[16], 364 μM (95 μg/mL) and 10.2 μM (6 μg/mL) [17], respectively.
From the data, it is evident that all the complexes are more cytotoxic
than cyclophosphamide, comparable to etoposide, and lower than
podophyllotoxin. The compound 3 displays potent cytotoxic activity
against Artemia Cysts in this assay.
It is generally accepted that DNA is the primary target in the cisplatin
mechanism [18]. Similarly, interactions between small molecules and
DNA rank among the primary action of mechanisms for cytotoxic activity.
UV–vis. spectra of compound 1 exhibits its characteristic absorption in
buffer solution as broad bands at 253 nm, 311 nm and 377 nm, which
can be ascribed to the charge transfer electron transition. With increasing
concentration of DNA added, significant hyperchromicity and red-shift
was observed (Supplementary S.4). At the ratio of [DNA]/[M] up to 5
times, 12% hyperchromicity with a 4 nm red-shift was achieved. This sug-
gests that complexes may covalently bind to DNA in the major or minor
groove. Hyperchromism results from breakage of secondary structure of
DNA due to the fact that phosphate group can provide the suitable an-
chors for coordination with complexes. Presence of red shift indicates co-
ordination of complex with DNA through N7 position of guanine. Such
type of binding was observed in many cisplatins, chloro-ruthenium and
aqua-ruthenium complexes [19,20]. The binding constant (Kb) values
for complexes 1–4 are 1.99×105, 2.43×105, 4.36×105 and 3.51×105
respectively, as calculated from Eq. (1) (Supplementary S.1).
To understand the nature of the interaction between the complexes
and DNA, viscosity measurements were done. Due to its sensitivity to
the change of length of DNA, viscosity determination may be the most
effective means to study the binding mode of complexes to DNA, espe-
cially in the absence of X-ray crystallographic or NMR structural data
[21]. Viscosity experiments results indicate that all the complexes
decrease the relative viscosity of HS-DNA, consistent with partial
intercalation or covalent nature of DNA binding (Supplementary S.5).
The results obtained from viscosity studies and thermal DNA denatur-
ation studies as well as UV–vis. spectral titration validate the covalent
binding nature of DNA interaction for all the complexes.
All the Au(III) complexes of different ligands were used to test the
plasmid DNA cleavage. All complexes showed significant DNA cleavage,
where the supercoiled DNA (form I) was cleaved to the relaxed open
circular DNA (form II) over a period of 6 h as illustrated in Fig. 1. During
this incubation period, complexes showed pronounced capability to
further cleave form II to linear DNA (form III). The cleavage ability of
the complexes decreases in the order 3>4>2>1.
As a result of the very high efficiency of the complexes in cleaving the
strands of plasmid DNA, all complexes were subjected for further detailed
kinetics studies in order to explore their activities in the catalytic cleavage
of plasmid DNA. The pUC19 DNA (50 μM) was incubated with 200 μM
complex 1 in TE buffer (pH 8) at 37 °C for 50–350 min. The increase in
the amount of OC and L forms of DNA was observed to be associated
with the increase of reaction time (Fig. 2). The amount of linear DNA
was 22% when the reaction time was 350 min. The decrease in the
amount of SC form and the formation of OC form of DNA with time
Thermal behavior of DNA in the presence of metal complexes can give
information about the interaction strength of the complexes with DNA.
The double-stranded DNA tends to gradually dissociate to single strands
on increasing solution temperature and generates a hyperchromic effect
on the absorption spectra of DNA bases (λmax =260 nm). In order to
identify this transition process, the melting temperature Tm, which is
defined as the temperature where half of the total base pairs gets
non-bonded, is a valuable parameter. Strong interaction of compounds
to DNA generally results in a considerable increase in the melting temper-
ature (Tm). The DNA melting studies with the present complexes 1–4
show a moderate positive shift in the melting temperature (ΔTm) of 5.5,
6.1, 6.9, and 6.6 °C respectively suggesting primarily intercalation or
covalent binding nature.
Fig. 1. Agarose gel (1%) of pUC19 (20 μM) at 37 °C in TE buffer (pH 8) incubated
with 150 μM complexes 1–4 for 6 h. Lane 1, DNA control; lane 2, DNA+metal
salt; lane 3, DNA+complex 1; lane 4, DNA+complex 2; lane 5, DNA+complex 3;
lane 6, DNA+complex 4.

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M.N. Patel et al. / Inorganic Chemistry Communications 29 (2013) 190–193
Fig. 4. Michaelis–Menten Reaction kinetics for the cleavage of plasmid DNA (incuba-
tion time: 100 min): lanes 1–7, pseudo Michaelis–Menten kinetic, at constant DNA
concentration ([DNA]=50 μM) and varying complex concentration ([Complex]=
50–350 μM); lanes 8–14, true Michaelic–Menten kinetic, at constant complex concen-
tration ([Complex]=150) and varying DNA concentration ([DNA]=20–80 μM).
Fig. 2. Agarose gel (1%) of pUC19 (20 μM) at 37 °C in TE buffer (pH 8) in presence of
150 μM complex 1 with increasing incubation time. Lanes 1–7: 50, 100, 150, 200,
250, 300 and 350 min, respectively.
Gel electrophoresis of plasmid DNA in presence of complexes shows
that the complexes are effective DNA cleavage agent. The Michaelis–
Menten kinetic study on plasmid DNA shows rate enhancement ratio
of the complexes in the order of 107 in cleaving DNA over noncatalyzed
DNA. These studies represent the attractive possibility of activating the
cleavage chemistry of covalently bound complexes.
shows the exponential nature of the curves (Fig. 3). The plot of ln(%SC
DNA) versus time is linear, which confirms that the process is pseudo
first-order (Supplementary S.6). The rate constant k1 (7.5×10−5 s−1),
the slope of the linear plot, was obtained using a complex concentration
of 200 μM.
Plasmid DNA cleavage was monitored under pseudo Michaelis–
Menten kinetic conditions using a constant DNA concentration (50 μM)
and varying complex concentration (50–350 μM) (Fig. 4). The kinetic
parameters for the pseudo-Michaelis–Menten conditions were derived
from the plots of kobs versus [Complex] and fit to Eq. (3) (Supplementary
S.1). Under these experimental conditions, values of KM were obtained in
the range of 172–238 μM for all the complexes (Supplementary S.7),
where these values represent pseudo Michaelis–Menten conditions. The
calculated value of complexes fall in the range reported for the cleavage
of DNA by other metal complexes (1.0×102–3.0×102 μM) [22,23].
The rate enhancements obtained for complexes 1–4 are 2.7×107,
3.0×107, 3.2×107, 3.2×107 respectively, over the non-catalyzed DNA
(k=3.6×10−8 h−1 at 37 °C) [23] clearly reveals the efficiency of the
complexes to cleave the double-stranded DNA.
Acknowledgments
Authors thank the Head of the Department of Chemistry Sardar Patel
University, India for making it convenient to work in laboratory and the
CSIR, India for providing financial support under “CSIR-Senior Research
Fellowship” scheme.
Appendix A. Supplementary material
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.inoche.2012.12.013.
References
Plasmid DNA cleavage was also monitored under true Michaelis–
Menten kinetic conditions using a constant complex concentration
(150 μM) and varying DNA concentration (20–80 μM) (Fig. 4). The
cleavage rate constants kobs were estimated using Eq. (2) (Supplementary
S.1) and then plot of kobs versus [DNA] was drawn. Under the experimen-
tal conditions, values of KM were obtained in the range of 73–87 μM for all
the complexes (Supplementary S.7), which is comparable to the value
reported by Kumbhar et al. [22]. The rate enhancements obtained for
complexes 1–4 are 2.2×107, 2.3×107, 2.7×107 and 2.3×107 respective-
ly, over the non-catalyzed DNA.
In summary, four new metal compounds of Au(III) have been synthe-
sized and characterized. All the compounds are monoionic in nature and
formed by coordination through N of different neutral bidentate ligands.
The UV–vis spectra of complexes in the DMSO and buffer shows that the
complexes convert into their dihydroxy form i.e. [Au(A)(OH)2]+ in the
physiological condition. Also the dihydroxy chromophore is stable for
24 h under the physiological conditions. The complexes show potent
cytotoxic properties against Artemia Cysts and DNA could be the possi-
ble target of the Au(III) complexes. The dihydroxy species of Au(III)
complexes exhibit covalent binding mode of interaction with DNA.
[1] I. Ott, On the medicinal chemistry of gold complexes as anticancer drugs, Coord.
Chem. Rev. 253 (2009) 1670–1681.
[2] K.C. Dash, H. Schmidbauer, Metal Ions in Biological Systems, Marcel Dekker, New
York, 1982.
[3] V. Gandin, A.P. Fernandes, M.P. Rigobello, B. Dani, F. Sorrentino, F. Tisato, M. Bornstedt,
A. Bindoli, A. Sturaro, R. Rella, C. Marzano, Cancer cell death induced by phosphine
gold(I) compounds targeting thioredoxin reductase, Biochem. Pharmacol. 79 (2010)
90–101.
[4] P.J. Sadler, R.E. Sue, The chemistry of gold drugs, Met. Based Drugs 1 (1994)
107–144.
[5] E.A. Pacheco, E.R.T. Tiekink, M.W. Whitehouse, Gold chemistry—applications and
future directions in the life sciences, John Wiley & Sons, 2009.
[6] M. Navarro, C. Hernandez, I. Colmenares, P. Hernandez, M. Fernandez, A. Sierraalta, E.
Marchan, Synthesis and characterization of [Au(dppz)2]Cl3. DNA interaction studies
and biological activity against Leishmania (L) mexicana, J. Inorg. Biochem. 101 (2007)
111–116.
[7] K. Palanichamy, A.C. Ontko, Synthesis, characterization, and aqueous chemistry of
cytotoxic Au(III) polypyridyl complexes, Inorg. Chim. Acta 359 (2006) 44–52.
[8] C. Gabbiani, A. Guerri, M.A. Cinellu, L. Messori, Dinuclear gold(III) complexes as
potential anticancer agents: structure, reactivity and biological profile of a series
of gold(III) oxo-bridged derivatives, Open Crystallogr.J. 3 (2010) 29–40.
[9] G. Marcon, S. Carotti, M. Coronnello, Gold(III) complexes with bipyridyl ligands:
solution chemistry, cytotoxicity, and DNA binding properties, J. Med. Chem. 45
(2002) 1672–1677.
[10] L. Ronconi, C. Marzano, P. Zanello, M. Corsini, G. Miolo, C. Macca, A. Trevisan, D.
Fregona, Gold(III) dithiocarbamate derivatives for the treatment of cancer: solu-
tion chemistry, DNA binding and hemolytic properties, J. Med. Chem. 49 (2006)
1648–1657.
[11] L. Messori, G. Marcon, P. Orioli, Gold(III) compounds as new family of anticancer
drugs, Bioinorg. Chem. Appl. 1 (2003) 177–187.
[12] M.N. Patel, B.S. Bhatt, P.A. Dosi, Topoisomerase inhibition, nucleolytic and electrolytic
contribution on DNA binding activity exerted by biological active analogue of coordina-
tion compounds, Appl. Biochem. Biotechnol. 166 (2012) 1949–1968.
[13] L. Messori, P. Orioli, C. Tempi, G. Marcon, Interactions of selected gold(III) com-
plexes with Calf Thymus DNA, Biochem. Biophys. Res. Commun. 281 (2001)
352–360.
[14] B.N. Meyer, N.R. Ferrigni, J.E. Putnam, J.B. Jacobsen, D.E. Nicholsand, J.L. Mclaughlin,
Brine shrimp; a convenient general bioassay for active plant constituents, Planta
Med. 45 (1982) 31–34.
[15] B. Jaki, J. Orjala, H.R. Bürji, O. Sticher, Biological screening of cyanobacteria for
antimicrobial and molluscicidal activity, brine shrimp lethality and cytotoxicity,
Pharm. Biol. 37 (1999) 138–143.
[16] Y. Lu, Y.P. Zhao, C.X. Fu, Biological activities of extracts from a naturally wild kiwifruit,
Actinidia macrosperma, Afr. J. Agric. Res. 6 (2011) 2231–2234.
[17] J.M. Mbaria Nguta, D.W. Gakuya, P.K. Gathumbi, J.D. Kabasa, S.G. Kiama, Cytotoxicity
of antimalarial plant extracts from Kenyan biodiversity to the brine shrimp, Artemia
salina L. (Artemiidae), Drugs Ther. Studies 2 (2012) 64–67.
Fig. 3. Decrease in the SC form and formation of the OC and L forms of pUC19 DNA in
presence of the complex 1 (150 μM) with incubation time.

M.N. Patel et al. / Inorganic Chemistry Communications 29 (2013) 190–193
193
[18] C.X. Zhang, S.J. Lippard, New metal complexes as potential therapeutics, Curr.
Opin. Chem. Biol. 7 (2003) 481–489.
[19] M. Chauhan, F. Arjmand, Stannoxane capping derived from chiral tridentate NNO
donor ligand for nickel and copper macrocycles: comparative binding studies of
stannoxane moiety and its modulated copper complex with CTDNA, J. Organomet.
Chem. 692 (2007) 5156–5164.
[20] N. Grover, T.W. Welch, T.A. Fairley, M. Cory, H.H. Thorp, Covalent binding of
aquaruthenium complexes to DNA, Inorg. Chem. 33 (1994) 3544–3548.
[21] S. Satyanaryana, J.C. Daborusak, J.B. Chaires, Tris(phenanthroline)ruthenium(II)
enantiomer interactions with DNA: mode and specificity of binding, Biochemistry
32 (1993) 2573–2584.
[22] A. Barve, A. Kumbhar, M. Bhat, B. Joshi, R. Butcher, U. Sonawane, R. Joshi, Mixed-ligand
copper(II) maltolate complexes: synthesis, characterization, DNA binding and
cleavage, and cytotoxicity, Inorg. Chem. 48 (2009) 9120–9132.
[23] W. Xu, F.R. Louka, P.E. Doulain, C.A. Landry, F.A. Mautner, S.S. Massoud, Hydrolytic
cleavage of DNA promoted by cobalt(III)?tetraamine complexes: synthesis
and characterization of carbonatobis[2-(2-pyridylethyl)]-(2-pyridylmethyl)
aminecobalt(III) perchlorate, Polyhedron 28 (2009) 1221–1228.