Role of carboxamido nitrogen in mononuclear manganese complex: Superoxide scavenging activity and nuclease activity

Inorganic Chemistry Communications 13 (2010) 380–383

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Role of carboxamido nitrogen in mononuclear manganese complex: Superoxide scavenging activity and nuclease activity Kaushik Ghosh *, Nidhi Tyagi, Pramod Kumar Department of Chemistry, Indian Institute of Technology, Roorkee, Roorkee 247 667, Uttarakhand, India

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Article history: Received 31 October 2009 Accepted 28 December 2009 Available online 11 January 2010 Keywords: Manganese(II) and manganese(III) mononuclear complexes Carboxamido nitrogen DNA binding Nuclease cleavage Superoxide scavenging activity

a b s t r a c t Three new ligands Pyimpy (2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)pyridine), Me-Pyimpy (1phenyl-1-(pyridin-2-yl)-2-(1-(pyridin-2-yl)ethylidene)hydrazine) and PampH (N0 -phenyl-N0 -(pyridin-2yl)picolinohydrazide) were synthesized. Hexa-coordinated mononuclear complexes [Mn(Pyimpy)2](ClO4)2 (1), [Mn(Me-Pyimpy)2](ClO4)2 (2), and [Mn(Pamp)2](ClO4)CH3OH, (3CH3OH) were synthesized and characterized. The molecular structure of [Mn(Pamp)2](ClO4)CH3OH, (3CH3OH) was determined by single crystal X-ray diffraction, which afforded distorted octahedral coordination sphere having meridionally spanning ligands. The redox properties were exploited to examine superoxide dismutase (SOD) activity using Mn(II)/Mn(III) couple. The complex 3 having ligands containing carboxamido nitrogen (Nam) donor, has been revealed to catalyze more effectively the dismutation of superoxide (O 2 ) ions in xanthine–xanthine oxidase–nitro blue tetrazolium assay as compared to 1 and 2. Among the three complexes, complex 3 was found to be most effective in nuclease activity in the presence of H2O2. Ó 2010 Elsevier B.V. All rights reserved.

Manganese is widely distributed in nature and in terms of terrestrial abundance it is second among the 3d-block transition elements [1]. In biosystem, this essential element could act as a Lewis acid like zinc and magnesium; on the other hand, it could participate in redox reactions like copper and iron. Hence several metalloenzymes in biosystem require manganese as cofactor for their catalytic activities [2]. Among them superoxide dismutases (SOD) are important class of redox enzymes which disproportionate superoxide radical (O 2 ), a toxic by product of cellular respiration, to the non-radical products oxygen and hydrogen peroxide, through metal catalyzed oxidation–reduction mechanism shown below: [P-Mox and P-Mred are metalloproteins with metal in oxidised and reduced states, respectively]:

O 2 þ P-MOX ! O2 þ P  Mred þ O 2 þ P-Mred þ 2H ! H2 O2 þ P-MOX ;

where M ¼ Cu=Zn; Mn; Fe; Ni Redox active copper–zinc or manganese or iron [2] or nickel [3] centres were found in the active-sites of these classes of metalloenzymes. This reactive oxygen species is involved in oxidative stress and in several diseases [4]. Coordination chemistry of manganese was exploited for the structural and functional modelling Mn-SOD metal-site. Manganese complexes, which mimic the activity of superoxide dismutase enzyme, are used for the destruction of det* Corresponding author. Tel.: +91 1332 285547; fax: +91 1332 2735601. E-mail address: [email protected] (K. Ghosh). 1387-7003/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.inoche.2009.12.028

rimental superoxide ions and in several cases as therapeutic agents [5]. It has been documented in the literature that native SOD enzyme as well as small molecule SOD mimics interact with DNA and exhibit nuclease activity [6,7]. This prompted us to study the DNA interaction as well as nuclease activity of manganese complexes having SOD activity. There has been upsurge of interest for transition metal complex and DNA interaction studies because of their potential applications in nucleic acid chemistry and cancer research [8]. In this regard, application of manganese chemistry is important because manganese is biologically relevant and lesser toxic metal [9]. We are interested to study the interaction of manganese complexes with DNA and recently we communicated a family of mononuclear manganese complexes which executed SOD activity as well as nuclease activity [10]. In this communication we want to report the role of deprotonated carboxamido nitrogen (Nam) not only in SOD activity but also in nuclease activity and the results of our mechanistic investigation on DNA cleavage. It is well known in the literature that ligands containing Nam donor stabilize higher oxidation states of metal ions because Nam exhibits strong r-donating property [11]. Manganese chemistry with ligands having carboxamido nitrogen was exploited for several applications. Mascharak and co-workers found that Nam was responsible for the coordination and photolability of nitric oxide [12]. Oxo-transfer reactions were investigated for manganese complexes derived from a ligand having Nam by Yang et al. [13]. Brown and co-workers [14] found that copper could be substituted by manganese in prion protein in vivo and it

K. Ghosh et al. / Inorganic Chemistry Communications 13 (2010) 380–383

is known in the literature that copper binds to octarepeat of prion protein through carboxamido nitrogen [15]. Workman et al. explained the insight into manganese oxidation chemistry with the help of manganese complexes derived from ligands with Nam donors [16]. On the other hand, Guo and co-workers [17] studied only SOD activity of manganese complexes having ligands containing carboxamido nitrogen; however, DNA interaction and nuclease activity studies were not reported. Hecht and co-workers reported [18] oxygen mediated DNA degradation by Mn-bleomycin (MnBLM) and the DNA strand scission was similar to Fe-bleomycin (Fe-BLM) where iron is coordinated to deprotonated carboxamido nitrogen [19]. To the best of our knowledge, there is no other report available in the literature where manganese complexes having ligands containing deprotonated carboxamido nitrogen donors showing SOD activity as well as nuclease activity simultaneously. Hence, we designed a tridentate ligand (PampH) (shown in Scheme 1) containing single carboxamido nitrogen donor along with two pyridine nitrogen (Npy) donors. To examine the role of carboxamido nitrogen we also prepared corresponding Schiff base ligands namely Pyimpy, Me-Pyimpy, both having two pyridine and one imine nitrogen (Nim) donors (Scheme 1). Reaction of Mn(ClO4)26H2O with Pyimpy and Me-Pyimpy (metal to ligand ratio 1:2) in methanolic solution afforded complexes [Mn(Pyimpy)2](ClO4)2, 1 and [Mn(Me-Pyimpy)2](ClO4)2, 2, respectively. [Mn(Pamp)2](ClO4)CH3OH, 3 was synthesized by the reaction of Mn(CH3COO)32H2O and PampH in methanol–water solution. Details of the ligand and metal complexes syntheses are described in Supporting Information. In IR, m–HC@N for 1 and 2 were found at 1599 and 1598 cm1, respectively. Coordination of Pamp to manganese in 3CH3OH was indicated by the shift of m–C@O from 1694 cm1 in the free ligand to 1655 cm1 for the manganese complex [12,20] and was further supported by disappearance of the mN– 1 ). All the three metal complexes 1, 2 and H band (3353 cm 3CH3OH show bands near 1090 cm1 together with a band at 623 cm1 for the uncoordinated perchlorate ion [10]. The molar conductivity values along with magnetic moment data were consistent for 1, 2 and 3 [10,21]. The molecular structure of complex, [Mn(Pamp)2](ClO4)2CH3OH (3CH3OH) was determined by X-ray crystallography. Structure of 3CH3OH is shown in Fig. 1. Matrix parameters and selected bond distances and bond angles are described in Table S3 and Table S4. Ligand Pamp was bound to Mn(III) centre meridionally in distorted octahedral fashion through two trans carboxamido nitrogens, four cis pyridine nitrogens. In complex 3CH3OH, Mn(III)–Nam distances were longer than the reported distances, 1.9133(11) Å [12] and 1.922(4) or 1.945(3) Å [17]. In 3CH3OH, Mn(III)–Npy distances are usually shorter than reported Mn(III)–NPy distances [10,12,22]. The elongation of the trans Mn(III)–Nam bonds and contraction of Mn–NPy bonds may be due to Jahn–Teller effect for a high-spin d4 electronic configuration similar to [MnIII(Phimp)2](ClO4) [10,23]. Examination of the MnII/MnIII redox potential, which is dependent on the coordination sphere of the metal centre, provided us following important information (Fig. S20 and Table S1). Firstly,

N

N C

N

N

N

R

R; H - Pyimpy R; Me - Me-Pyimpy

C

H N

N N

O

PampH

Scheme 1. Schematic drawing of tridentate ligands and abbreviations.

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Fig. 1. Ball-and-stick representation of the crystal structure of [Mn(Pamp)2](ClO4)CH3OH (3CH3OH), atoms are shown as sphere of arbitrary diameter. Selected bond lengths are as follows, Mn1–Nam: 1.962(4), 1.954(4) and Mn1–NPy: 2.158(4), 2.117(4), 2.147(4), 2.118(4)Å.

imine and pyridine nitrogen stabilized manganese(II) more because they are soft donors to the metal centres showing MnII/MnIII couples E1/2 equals to 1.25 V and 1.08 V vs Ag/AgCl in complexes 1 and 2, respectively. Secondly, due to the electron donating property of methyl group the potential is less negative in complex 2 as compared to complex 1 [24]. Thirdly, manganese(III) was more stabilized in 3 because hard carboxamido nitrogen is a strong rdonor and in general stabilizes higher oxidation state of metals and therefore complex 3 showed MnII/MnIII couple at E1/2, 0.26 V vs Ag/AgCl. Moreover, we found MnIII/MnIV couple for complex 3 at E1/2, 1.11 V vs Ag/AgCl. A metal complex which could serve as a small molecule SOD mimic, should have redox potential in the range –0.375 V 6 E1/2 6 +0.605 V vs Ag/AgCl (0.33 V 6 E1/ 2 6 +0.65 V vs SCE) [16]; hence, E1/2 value must be in between the two solid arrows as depicted in Fig. 2(a). The E1/2 values (for MnII/MnIII redox couple) of these complexes reported here clearly indicate (Fig. 2(a)) that the redox potential of 3 is only in the proper range and therefore 3 could only be a small molecule SOD mimic. Complex 3 has been shown to catalyze effectively the dismutation of superoxide (O 2 ) ions in the NBT assay with an IC50 value of 3.73 lM (shown in Fig. 2(b)). This result clearly indicated that the complex 3 exhibited an appreciable SOD activity with an IC50 value close to the value reported by Lin et al. [17] and can be used as a functional SOD mimic. The inhibition study for 1 and 2 gave rise to a curve similar to the curve found by Biencenue et al. [25] and the curve did not go beyond 65–70% inhibition. Moreover, the IC50 values calculated for 1 and 2 were higher than the values calculated for Mn(ClO4)26H2O [26]. It is clear from the electrochemical data that the MnII/MnIII redox couples are not in the proper range for 1 and 2 and hence some other mechanism similar to that of SOD activity of Mn(ClO4)26H2O could be involved for the scavenging of superoxide ions [26,27]. The DNA binding properties of complexes were studied by UV– visible, circular dichroism and fluorescence spectroscopy. Upon successive addition of CT-DNA, the UV–visible absorption band of metal complexes showed modest hypochromism in case of 1 and small hypochromism in case of 2 and 3, indicating the moderate interaction of complexes to DNA (Kb = 2.21  104, 0.15  104 and

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(a)

80 Complex 3

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

Reduction potential, V vs Ag/AgCl O2 + eComplex 1

O2.-

O2.- + e- + 2H+

% Inhibition

Complex 2

H2O2

(b)

60 40 20 0

IC50 = 3.73 µM 0

2 4 6 8 10 Concentration (µM) of 3

Fig. 2. (a) Apparent standard redox potential for superoxide couples at pH 7. (b) SOD activity of complex 3 (in DMF) in the xanthine oxidase–nitro blue tetrazolium (NBT) assay.

Fig. 3. Gel electrophoresis separations showing the oxidative cleavage of supercoiled pBR322 DNA (200 ng) by complex 3 (100 lM) in 10% dimethylformamide incubated at 37 °C for 2 h. (a) DNA (lane 1); DNA + H2O2 (1.6 mM) (lane 2); DNA + 3 (lane 3); DNA + 3 + H2O2 (0.2 mM) (lane 4); DNA + 3 + H2O2 (0.4 mM) (lane 5); DNA + 3 + H2O2 (1.6 mM) (lane 6). (b) DNA (lane 1); DNA + H2O2 (1.6 mM) (lane 2); DNA + 3 (lane 3); DNA + 3 + H2O2 (1.6 mM) (lane 4); DNA + 3 + H2O2 (1.6 mM) + D2O (20 mM) (lane 5); DNA + 3 + H2O2 (1.6 mM) + NaN3 (20 mM) (lane 6); DNA + 3 + H2O2 (1.6 mM) + urea (20 mM) (lane 7); DNA + 3 + H2O2 (1.6 mM) + DMSO (20 mM) (lane 8); DNA + 3 + H2O2 (1.6 mM) + L-his (20 mM) (lane 9); DNA + 3 + H2O2 (1.6 mM) + EtOH (20 mM) (lane 10).

3.32  104 M1 for 1, 2 and 3 respectively) [10,28]. In fluorescence quenching studies, Stern–Volmer plots of Fo/F vs [R] are shown in Fig. S22, S23, S24 and quenching data calculated are listed in Table S2. The KSV (12.8  104, 6.1  104 and 8.5  104 for 1, 2 and 3, respectively) data are in agreement with the results form UV– visible spectroscopic data. Furthermore, the interaction of 1 and 3 is stronger than that of 2 according to the different KSV values which may be due to steric hindrance of –Me in 2 than that of 1 and 3 [29]. The conformational changes of CT-DNA induced by 1, 2 and 3 were shown by CD spectroscopy (Fig. S25) [30]. Considering UV–visible, fluorescence and circular dichroism spectral data together, external binding with moderate intercalation was speculated. We investigated the nuclease activity of these complexes 1, 2 and 3, to draw a correlation between nuclease activities with SOD activity. Nuclease activity was assayed by incubating three complexes with supercoiled pBR322 DNA plasmid by varying the concentration of the complexes, concentration of the oxidising (H2O2) and reducing agents (BME) and varying the incubation time (Fig. 3). For all complexes, no nuclease activity was found in presence of the complexes only which excluded the possibility of hydrolytic nuclease activity. However we found DNA cleavage activity in presence of H2O2 and not in presence of BME (shown in Supporting Information). Nuclease activity of complexes 1 and 2 was found negligible at high concentration of the complex (150 lM) as well as H2O2 (1.6 mM) without the formation of linear circular (LC) form of DNA. However, in case of complex 3 (100 lM) we found LC as well as nicked circular (NC) form of pBR322 DNA plasmid. Mechanism of nuclease activity was examined by using radical scavengers such as D2O, urea, DMSO, EtOH and KI (OH scavengers), L-histidine and NaN3 (1O2 scavenger) [31] during DNA cleavage activity. Nuclease activity was inhibited to small extent in presence of urea, DMSO, EtOH and L-histidine whereas no inhibition was found in case of D2O, NaN3 and KI. Hence, possible participation of reactive oxygen species (ROS) was involved for DNA cleavage. In conclusion, mononuclear complexes 1, 2 and 3CH3OH were synthesized and characterized by spectroscopic as well as electrochemical studies. The molecular structure of 3CH3OH was estab-

lished by X-ray crystallography. Investigation of redox properties afforded different redox behaviour for 3 as compared to 1 and 2 and 3 was only qualified to show SOD activity, however, complexes 1 and 2 were less effective in scavenging superoxide ions. Complex 3 was found to be effective in nuclease activity also. Schiff base complexes 1 and 2 afforded lower activity for SOD as well as nuclease activity due to the lack of Nam donor in the ligand frames. Hence role of carboxamido nitrogen was important for the enhancement of SOD as well as nuclease activity. Detail studies on these results and related complexes and their biological applications are under progress. Acknowledgements K.G. is thankful to DST, New Delhi, India, for SERC FAST Track project support. N.T. and P.K. are thankful to CSIR, India, for financial assistance. We are thankful to Central Instrumental Facility, IIT Guwahati for single crystal X-ray facility. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.inoche.2009.12.028. References [1] D.W. Christianson, Prog. Biophys. Molec. Biol. 67 (1997) 217–252. [2] J.J.R. Frausto da Silva, R.J.P. Williams, The Biological Chemistry of the Elements, Clarendon Press, Oxford, 1993. p. 4. [3] D.P. Barondeau, C.J. Kassmann, C.K. Bruns, J.A. Tainer, E.D. Getzoff, Biochemistry 43 (2004) 8038–8047. [4] D. Salvemini, C. Muscoli, D.P. Riley, S. Cuzzocrea, Pulm. Pharmacol. Therapeut. 15 (2002) 439–447. [5] D. Salvemini, Z.-Q. Wang, J.L. Zweier, A. Samouilov, H. Macarthur, T.P. Misko, M.G. Currie, S. Cuzzocrea, J.A. Sikorski, D.P. Riley, Science 286 (1999) 304–306. [6] W. Jiang, Y. Han, Q. Pan, T. Shen, C. Liu, J. Inorg. Biochem. 101 (2007) 667–677. [7] M. Devereux, D.O. Shea, A. Kellett, M. McCann, M. Walsh, D. Egan, C. Deegan, K. Kedziora, G. Rosair, H. Muller-Bunz, J. Inorg. Biochem. 101 (2007) 881–892. [8] N. Farrell, Coord. Chem. Rev. 232 (2002) 1–4. [9] D.P. Riley, Chem. Rev. 99 (1999) 2573–2588. [10] K. Ghosh, N. Tyagi, P. Kamar, U.P. Singh, N. Goel, J. Inorg. Biochem. 104 (2010) 9–18.

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