Inorganic Chemistry Communications 13 (2010) 380–383

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Inorganic Chemistry Communications

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

Kaushik Ghosh *, Nidhi Tyagi, Pramod KumarDepartment of Chemistry, Indian Institute of Technology, Roorkee, Roorkee 247 667, Uttarakhand, India

a r t i c l e i n f o

Article history:Received 31 October 2009Accepted 28 December 2009Available online 11 January 2010

Keywords:Manganese(II) and manganese(III)mononuclear complexesCarboxamido nitrogenDNA bindingNuclease cleavageSuperoxide scavenging activity

1387-7003/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.inoche.2009.12.028

* Corresponding author. Tel.: +91 1332 285547; faxE-mail address: ghoshfcy@iitr.ernet.in (K. Ghosh).

a b s t r a c t

Three new ligands Pyimpy (2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)pyridine), Me-Pyimpy (1-phenyl-1-(pyridin-2-yl)-2-(1-(pyridin-2-yl)ethylidene)hydrazine) and PampH (N0-phenyl-N0-(pyridin-2-yl)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, (3�CH3OH) were synthesizedand characterized. The molecular structure of [Mn(Pamp)2](ClO4)�CH3OH, (3�CH3OH) was determinedby single crystal X-ray diffraction, which afforded distorted octahedral coordination sphere havingmeridionally 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 nitro-gen (Nam) donor, has been revealed to catalyze more effectively the dismutation of superoxide (O��2 ) ionsin xanthine–xanthine oxidase–nitro blue tetrazolium assay as compared to 1 and 2. Among the threecomplexes, complex 3 was found to be most effective in nuclease activity in the presence of H2O2.

� 2010 Elsevier B.V. All rights reserved.

Manganese is widely distributed in nature and in terms of ter-restrial abundance it is second among the 3d-block transition ele-ments [1]. In biosystem, this essential element could act as a Lewisacid like zinc and magnesium; on the other hand, it could partici-pate in redox reactions like copper and iron. Hence several metal-loenzymes in biosystem require manganese as cofactor for theircatalytic activities [2]. Among them superoxide dismutases (SOD)are important class of redox enzymes which disproportionatesuperoxide 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 shownbelow: [P-Mox and P-Mred are metalloproteins with metal in oxi-dised and reduced states, respectively]:

O��2 þ P-MOX ! O2 þ P�Mred

O��2 þ P-Mred þ 2Hþ ! H2O2 þ P-MOX;

where M ¼ Cu=Zn;Mn; Fe;Ni

Redox active copper–zinc or manganese or iron [2] or nickel [3] cen-tres were found in the active-sites of these classes of metalloen-zymes. This reactive oxygen species is involved in oxidative stressand in several diseases [4]. Coordination chemistry of manganesewas exploited for the structural and functional modelling Mn-SODmetal-site. Manganese complexes, which mimic the activity ofsuperoxide dismutase enzyme, are used for the destruction of det-

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rimental superoxide ions and in several cases as therapeutic agents[5]. It has been documented in the literature that native SOD en-zyme as well as small molecule SOD mimics interact with DNAand exhibit nuclease activity [6,7]. This prompted us to study theDNA interaction as well as nuclease activity of manganese com-plexes having SOD activity.

There has been upsurge of interest for transition metal complexand DNA interaction studies because of their potential applicationsin nucleic acid chemistry and cancer research [8]. In this regard,application of manganese chemistry is important because manga-nese is biologically relevant and lesser toxic metal [9]. We areinterested to study the interaction of manganese complexes withDNA and recently we communicated a family of mononuclearmanganese complexes which executed SOD activity as well asnuclease activity [10].

In this communication we want to report the role of deproto-nated carboxamido nitrogen (Nam) not only in SOD activity but alsoin nuclease activity and the results of our mechanistic investiga-tion on DNA cleavage. It is well known in the literature that ligandscontaining Nam donor stabilize higher oxidation states of metalions because Nam exhibits strong r-donating property [11]. Man-ganese chemistry with ligands having carboxamido nitrogen wasexploited for several applications. Mascharak and co-workersfound that Nam was responsible for the coordination and photola-bility of nitric oxide [12]. Oxo-transfer reactions were investigatedfor manganese complexes derived from a ligand having Nam byYang et al. [13]. Brown and co-workers [14] found that coppercould be substituted by manganese in prion protein in vivo and it

Fig. 1. Ball-and-stick representation of the crystal structure of [Mn(Pamp)2](-ClO4)�CH3OH (3�CH3OH), atoms are shown as sphere of arbitrary diameter. Selectedbond 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.

K. Ghosh et al. / Inorganic Chemistry Communications 13 (2010) 380–383 381

is known in the literature that copper binds to octarepeat of prionprotein through carboxamido nitrogen [15]. Workman et al. ex-plained the insight into manganese oxidation chemistry with thehelp of manganese complexes derived from ligands with Nam do-nors [16]. On the other hand, Guo and co-workers [17] studied onlySOD activity of manganese complexes having ligands containingcarboxamido nitrogen; however, DNA interaction and nucleaseactivity studies were not reported. Hecht and co-workers reported[18] oxygen mediated DNA degradation by Mn-bleomycin (Mn-BLM) and the DNA strand scission was similar to Fe-bleomycin(Fe-BLM) where iron is coordinated to deprotonated carboxamidonitrogen [19]. To the best of our knowledge, there is no other re-port available in the literature where manganese complexes hav-ing ligands containing deprotonated carboxamido nitrogendonors showing SOD activity as well as nuclease activity simulta-neously. Hence, we designed a tridentate ligand (PampH) (shownin Scheme 1) containing single carboxamido nitrogen donor alongwith two pyridine nitrogen (Npy) donors. To examine the role ofcarboxamido nitrogen we also prepared corresponding Schiff baseligands namely Pyimpy, Me-Pyimpy, both having two pyridine andone imine nitrogen (Nim) donors (Scheme 1).

Reaction of Mn(ClO4)2�6H2O with Pyimpy and Me-Pyimpy (me-tal to ligand ratio 1:2) in methanolic solution afforded complexes[Mn(Pyimpy)2](ClO4)2, 1 and [Mn(Me-Pyimpy)2](ClO4)2, 2, respec-tively. [Mn(Pamp)2](ClO4)�CH3OH, 3 was synthesized by the reac-tion of Mn(CH3COO)3�2H2O and PampH in methanol–watersolution. Details of the ligand and metal complexes syntheses aredescribed in Supporting Information. In IR, m–HC@N for 1 and 2 werefound at 1599 and 1598 cm�1, respectively. Coordination of Pamp�

to manganese in 3�CH3OH was indicated by the shift of m–C@O from1694 cm�1 in the free ligand to 1655 cm�1 for the manganese com-plex [12,20] and was further supported by disappearance of the mN–

H band (3353 cm�1). All the three metal complexes 1, 2 and3�CH3OH show bands near 1090 cm�1 together with a band at623 cm�1 for the uncoordinated perchlorate ion [10]. The molarconductivity values along with magnetic moment data were con-sistent for 1, 2 and 3 [10,21].

The molecular structure of complex, [Mn(Pamp)2](-ClO4)2�CH3OH (3�CH3OH) was determined by X-ray crystallogra-phy. Structure of 3�CH3OH is shown in Fig. 1. Matrix parametersand selected bond distances and bond angles are described inTable S3 and Table S4. Ligand Pamp� was bound to Mn(III) centremeridionally in distorted octahedral fashion through two trans car-boxamido nitrogens, four cis pyridine nitrogens. In complex3�CH3OH, Mn(III)–Nam distances were longer than the reported dis-tances, 1.9133(11) Å [12] and 1.922(4) or 1.945(3) Å [17]. In3�CH3OH, Mn(III)–Npy distances are usually shorter than reportedMn(III)–NPy distances [10,12,22]. The elongation of the transMn(III)–Nam bonds and contraction of Mn–NPy bonds may be dueto Jahn–Teller effect for a high-spin d4 electronic configurationsimilar to [MnIII(Phimp)2](ClO4) [10,23].

Examination of the MnII/MnIII redox potential, which is depen-dent on the coordination sphere of the metal centre, provided usfollowing important information (Fig. S20 and Table S1). Firstly,

N N

NNC

R

R; H - PyimpyR; Me - Me-Pyimpy

N N

N

HN

C

O

PampH

Scheme 1. Schematic drawing of tridentate ligands and abbreviations.

imine and pyridine nitrogen stabilized manganese(II) more be-cause 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 com-plexes 1 and 2, respectively. Secondly, due to the electron donatingproperty of methyl group the potential is less negative in complex2 as compared to complex 1 [24]. Thirdly, manganese(III) was morestabilized in 3 because hard carboxamido nitrogen is a strong r-donor and in general stabilizes higher oxidation state of metalsand therefore complex 3 showed MnII/MnIII couple at E1/2, 0.26 Vvs Ag/AgCl. Moreover, we found MnIII/MnIV couple for complex 3at E1/2, 1.11 V vs Ag/AgCl. A metal complex which could serve asa small molecule SOD mimic, should have redox potential in therange –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 betweenthe two solid arrows as depicted in Fig. 2(a). The E1/2 values (forMnII/MnIII redox couple) of these complexes reported here clearlyindicate (Fig. 2(a)) that the redox potential of 3 is only in the prop-er range and therefore 3 could only be a small molecule SODmimic.

Complex 3 has been shown to catalyze effectively the dismuta-tion of superoxide (O��2 ) ions in the NBT assay with an IC50 value of3.73 lM (shown in Fig. 2(b)). This result clearly indicated that thecomplex 3 exhibited an appreciable SOD activity with an IC50 valueclose to the value reported by Lin et al. [17] and can be used as afunctional SOD mimic. The inhibition study for 1 and 2 gave riseto a curve similar to the curve found by Biencenue et al. [25] andthe curve did not go beyond 65–70% inhibition. Moreover, theIC50 values calculated for 1 and 2 were higher than the values cal-culated for Mn(ClO4)2�6H2O [26]. It is clear from the electrochem-ical data that the MnII/MnIII redox couples are not in the properrange for 1 and 2 and hence some other mechanism similar to thatof SOD activity of Mn(ClO4)2�6H2O could be involved for the scav-enging of superoxide ions [26,27].

The DNA binding properties of complexes were studied by UV–visible, circular dichroism and fluorescence spectroscopy. Uponsuccessive addition of CT-DNA, the UV–visible absorption band ofmetal complexes showed modest hypochromism in case of 1 andsmall hypochromism in case of 2 and 3, indicating the moderateinteraction of complexes to DNA (Kb = 2.21 � 104, 0.15 � 104 and

0.0-0.2-0.4-0.6-0.8-1.0 0.2 0.4 0.6 0.8 1.0

O2.- + e- + 2H+ H2O2

O2 + e- O2.-

Reduction potential, V vs Ag/AgCl

Complex 3

Complex 2

Complex 1

(a)

0 2 4 6 8 100

20

40

60

80

IC50 = 3.73 µM

% I

nhib

itio

n

Concentration (µM) of 3

(b)

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 at37 �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).

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

3.32 � 104 M�1 for 1, 2 and 3 respectively) [10,28]. In fluorescencequenching studies, Stern–Volmer plots of Fo/F vs [R] are shown inFig. S22, S23, S24 and quenching data calculated are listed inTable S2. The KSV (12.8 � 104, 6.1 � 104 and 8.5 � 104 for 1, 2 and3, respectively) data are in agreement with the results form UV–visible spectroscopic data. Furthermore, the interaction of 1 and3 is stronger than that of 2 according to the different KSV valueswhich may be due to steric hindrance of –Me in 2 than that of 1and 3 [29]. The conformational changes of CT-DNA induced by 1,2 and 3 were shown by CD spectroscopy (Fig. S25) [30]. Consider-ing UV–visible, fluorescence and circular dichroism spectral datatogether, external binding with moderate intercalation wasspeculated.

We investigated the nuclease activity of these complexes 1, 2and 3, to draw a correlation between nuclease activities withSOD activity. Nuclease activity was assayed by incubating threecomplexes with supercoiled pBR322 DNA plasmid by varying theconcentration 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 pres-ence of the complexes only which excluded the possibility ofhydrolytic nuclease activity. However we found DNA cleavageactivity in presence of H2O2 and not in presence of BME (shownin Supporting Information). Nuclease activity of complexes 1 and2 was found negligible at high concentration of the complex(150 lM) as well as H2O2 (1.6 mM) without the formation of linearcircular (LC) form of DNA. However, in case of complex 3 (100 lM)we found LC as well as nicked circular (NC) form of pBR322 DNAplasmid. Mechanism of nuclease activity was examined by usingradical scavengers such as D2O, urea, DMSO, EtOH and KI (OH�

scavengers), L-histidine and NaN3 (1O2 scavenger) [31] duringDNA cleavage activity. Nuclease activity was inhibited to small ex-tent in presence of urea, DMSO, EtOH and L-histidine whereas noinhibition was found in case of D2O, NaN3 and KI. Hence, possibleparticipation of reactive oxygen species (ROS) was involved forDNA cleavage.

In conclusion, mononuclear complexes 1, 2 and 3�CH3OH weresynthesized and characterized by spectroscopic as well as electro-chemical studies. The molecular structure of 3�CH3OH was estab-

lished by X-ray crystallography. Investigation of redox propertiesafforded different redox behaviour for 3 as compared to 1 and 2and 3 was only qualified to show SOD activity, however, complexes1 and 2 were less effective in scavenging superoxide ions. Complex3 was found to be effective in nuclease activity also. Schiff basecomplexes 1 and 2 afforded lower activity for SOD as well as nucle-ase activity due to the lack of Nam donor in the ligand frames.Hence role of carboxamido nitrogen was important for theenhancement of SOD as well as nuclease activity. Detail studieson these results and related complexes and their biological appli-cations are under progress.

Acknowledgements

K.G. is thankful to DST, New Delhi, India, for SERC FAST Trackproject support. N.T. and P.K. are thankful to CSIR, India, for finan-cial assistance. We are thankful to Central Instrumental Facility, IITGuwahati for single crystal X-ray facility.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.inoche.2009.12.028.

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