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Synthesis, single crystal structure and DNA cleavage studies on first 4N-ethyl substituted three coordinate copper(I) complex of thiosemicarbazone
Inorganica Chimica Acta 362 (2009) 4185–4190
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Synthesis, single crystal structure and DNA cleavage studies on first 4N-ethyl substituted three coordinate copper(I) complex of thiosemicarbazone Panchangam Murali Krishna, Katreddi Hussain Reddy * Department of Chemistry, Sri Krishnadevaray University, Anantapur 515 003, AP, India
a r t i c l e
i n f o
Article history: Received 20 December 2008 Received in revised form 7 June 2009 Accepted 16 June 2009 Available online 21 June 2009 Keywords: Copper(I) complex Cuminaldehyde-4-ethyl-3thiosemicarbazone X-ray structure Three coordinate ‘Y-shaped’ complex Nuclease activity
a b s t r a c t A copper(I) complex [Cu(CETH)2Cl] (Ia), where CETH = cuminaldehyde-4-ethyl-3-thiosemicarbazone (I), is prepared and structurally characterised. The complex crystallizes in orthorhombic space group pna2(1) with the unit cell parameters; a = 9.8598(14) Å, b = 15.411(2) Å, c = 0.817(3) Å, V = 2055.9(4) Å3 and Z = 4. The neutral complex has the copper(I) centre bonded to two thioketonic sulphur donor in g1-S bonding mode and one chloride giving ‘Y’ shape geometry. The complex is diamagnetic and exhibits a copper to ligand charge transfer bands at 351 and 398 nm in dimethylformamide. The complex shows quasireversible cyclic voltammetric response at 0.41 V (DEp = 300 mV) at 50 mVS 1 in DMF for the Cu(II)/ Cu(I) oxidation couple. Complex Ia shows marginal nuclease activity with pUC18 DNA in the presence of reducing agent (Dithiotretal) and hydrogen peroxide. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Of the sulphur donor ligands thiosemicarbazones and their derivatives are very interesting molecules ranging from bonding and structural features presented by their transition metal complexes in coordination chemistry. Thiosemicarbazones are biologically active pharmacophores and their activity enhanced on complexation with metal ions [1–10]. They have been used for the analysis of metals, device application in telecommunications, optical communicating, optical storage and optical information process [11,12]. Therefore, the transition metal complexes with these ligands have raised interest amongst many researchers, and they continue to be subjected of many studies, especially in anticancer chemotherapeutic agents [13] and as superoxide dismutase-like radical scavengers [14]. Thiosemicarbazones usually act as chelating ligands for transition metal ions by bonding through the sulphur and azomethine nitrogen atoms, although in some cases they behave as mono dentate ligand where bind through sulphur only. Thiosemicarbazones present in thione – thiol equilibrium that confers the presence of two configurations i.e. E and Z. If the neutral thiosemicarbazone binds to the metal via sulphur in E form and in thiol form binds via enolic sulphur and azomethine nitrogen in Z form. Copper is bio-essential metal for living organisms [15] with two oxidation states +1 and +2. Coordination compounds of copper * Corresponding author. Tel.: +91 8554 224347x255742; fax: +91 8554 229043. E-mail address: [email protected] (K.H. Reddy). 0020-1693/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2009.06.022
have been extensively used in metal ion mediated DNA cleavage through the generation of hydrogen ion abstracting activated oxygen species [16]. The most widely exploited oxidative cleavage agent is the bis (phenonthroline) copper(I) cation [17] and effectively cleaves in presence of oxidative or dioxygen plus reducing agent. Copper(II) complexes of thiosemicarbazones have been widely investigated. However, reports on copper(I) complexes are very limited [18–23]. Copper(I) complexes displays wide diversity in structural chemistry. The coordination number of copper is in the range from two to six. Copper(I) usually forms four-coordinate tetrahedral complexes [24], but three coordination numbers are known but less common [23,25–28]. Copper(I) complexes are diamagnetic, since Cu(I) has d10 electronic configuration and no d–d transitions are possible. Copper(I) complexes plays very important role in coordination chemistry and in catalytic reactions [29]. As a part of our research programme concerning the chelating behaviour of neutral or deprotonated thiosemicarbazones [30–34], herein we reported synthesis, structural characterisation and nuclease activity of three coordinate ‘Y’ shaped [Cu(CETH)2Cl] complex. 2. Experimental 2.1. Materials and techniques 4-Ethyl-3-thiosemicarbazide and cuminaldehyde (p-isopropyl benzaldehyde) were of reagent grade purchased from Sigma–Aldrich. All other chemicals were of AR grade and used as supplied. The solvents were distilled before use. The plasmid pUC18 DNA
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was isolated from E. coli DH5a strains in Lubria Broth (LB) medium supplemented by ampicillin cells from 5 ml culture by Qiagen column following the manufacturer’s protocol [35]. Elemental analysis was carried out on a Perkin–Elmer 2400 CHNS elemental analyser. Magnetic susceptibility measurements were carried out on a magnetic susceptibility balance (Sherwood scientific, Cambridge, England), high purity CuSO45H2O was used as a standard. Molar conductance (10 3 M) in DMF at 30 ± 2 °C was measured with a CC180 model (ELICO) direct reading conductivity bridge. The electronic spectra were recorded in DMSO with a Shimadzu UV-160A spectrophotometer. FT-IR spectra were recorded in the range 4000–270 cm 1 in KBr discs on a Nicolet protégé 460 IR Spectrometer. The cyclic voltammetric measurements were performed on a Bio Analytical System (BAS) CV-27 assembly equipped with an X–Y recorder. Measurements were made on degassed (N2 bubbling for 5 min) ligand/complex solutions (10 3 M) in DMF and ethanol containing tetrabutyl ammonium perchlorate (0.1 M) as a supporting electrolyte. The three-electrode system consisted of a glassy carbon (working), platinum wire (auxiliary) and Ag/AgCl (reference). The 1H and 13C{1H} NMR spectra were recorded on a Bruker Spectrospin DPX-300 NMR spectrometer at 300.13 and 75.47 MHz, respectively. 2.2. Synthesis of CETH (I) and [Cu(CETH)2Cl](Ia) 2.2.1. Synthesis and structural characterisation of CETH (I) The ligand (CH3)2CH(C6H4)CH@NNHC(S)NH(C2H5) (I) (Fig. 1) was synthesised by reactions given in the Scheme 1. The synthe-
H N
H N
H N
Slow recrystallization of Ia from chloroform gave a single crystal of size 0.348 0.218 0.109 mm3 and was mounted on a glass fibre. The cell parameters and the intensity data were obtained using a Bruker SMART APEX CCD diffractometer to give a complete asymmetric unit using radiations of wave length
CH3 Fig. 1. Structure of CETH.
H
H O H3C CH 3
2.2.2. Synthesis and structure of [Cu(CETH)2Cl] (Ia) Boiled methanolic solution of CETH (1.00 g, 0.004 mol) was mixed with CuCl22H2O (0.29 g, 0.002 mol) and refluxed for 30 min and allowed to stand over night, the solid complex that separated out was collected by filtration and dried in vacuo. Yield: 51%, M.P. 175–176 °C Anal. Calc. for C26H38N6S2ClCu: C, 52.24; H, 6.40; N, 14.05; S, 10.72. Found: C, 52.34; H, 6.64; N, 14.03; S, 10.75%. IR (KBr, m, cm 1): 3299(br), 3147(br). 1526 (s), 1332(m), 800 (m), 387 (m), 265 (s), and cyclic voltammetry (E1/2, vs. Ag/ AgCl): 0.410 V. 2.3. X-ray crystallography
CH2 - CH3
S
H3C
sis of I was based on the general procedure described in the literature [36]. To a hot ethanolic solution (175 ml) of cuminaldehyde (6.22 g, 0.042 mol) in 500 ml round bottom flask, 150 ml of 5% CH3COOH–H2O solution of 4-ethyl-3-thiosemicarbazide (5.00 g, 0.042 mol) was mixed and heated the reaction mixture under reflux on a steam bath for 30–45 min. The crystalline product which formed was collected by filtration, washed several times with hot water and dried in vacuo and was recrystallized in methanol. Yield 45%, M.P.140-142 °C, Anal. Calc. for C13H19N3S: C, 62.6; H, 7.7; N, 16.8; S, 14.1. Found: C, 62.9; H, 7.6; N, 16.9; S, 14.1%. IR (KBr, m, cm 1): 3300(br), 3143(br). 1607 (s), 1379 (m), 1092 (m), 833 (m). 1H NMR (DMSO-d6, 25 °C): d (vs TMS) 1.14 (t, 1H, H13), 1.19–1.21 (d, J = 7 Hz, 6H, ipr-Me), 2.82–2.90 (m, 1H, ipr-CH), 3.52–3.54 (quintet, 1H, H12), 7.27–7.30 (d, J = 8 Hz, m, 2H, H3, 5), 7.69–7.72 (d, J = 8 Hz, 2H, H2, 6), 8.02 (s, 1H, H9), 8.48 (t, 1H, H11), 11.31 (s, 1H, H7).13C NMR (75.4 MHz, CDCl3): d 14.62 (C13), 23.65 (ipr-CH3), 33.33 (ipr-CH), 38.66 (C12), 126.56 (C2, 6), 127.28 (C3, 5), 131.90 (C1), 141.83 (C7), 150.34 (C4) and 176.58 (C10). ESI-Mass (m/z, E+) 249. The pka values of CETH are: 5.4 and 8.1.
+
H N H2N
N
EtOH C S
N H (C 2 H 5 )
H N
NH (C 2 H 5 )
+
S
H3C
5% CH 3 COOH -H 2 O
I
CH 3
MeoH
NH(C 2 H 5)
(C 2 H 5)HN
H
HN N
S
Cu
S
NH N
H
Cl
H 3C
CH 3
H 3C
Ia Scheme 1.
CH 3
CuCl 2
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0.71073 Å. Frames were collected at T = 298 by x, U, and 2h rotation at 10 s per frames with SMART [37]. The measured intensities were reduced to F2 and corrected for absorptions with SADABS [38]. Structural solution, refinement, and data output were carried out with the SHELXTL program [39]. Non-hydrogen atoms were refined anisotropically. C–H hydrogen atoms were placed in geometrically calculated positions using a rigid model. The program PARST [40,41] was employed for geometrical description. Images were created with the ORTEP 3 program [42] and PLATON [43,44]. Cell dimensions and other experimental parameters are given in Table 1.
3. Results and discussion 3.1. Characterisation of synthesised complex The reaction of CuCl22H2O with cuminaldehyde-4-ethyl-3-thiosemicarbazone led to the formation of complex Ia (Scheme 1). The new compound is pale yellow coloured solid and insoluble in most of the organic solvents except chloroform, dimethyl formamide
2.4. Assay of nuclease activity The extent of cleavage of DNA by the copper(I) complex was monitored by agarose gel electrophoresis using pUC18 DNA. The samples after incubation for 30 min at 37 °C were added to the loading buffer containing 0.25% bromophenol blue + 0.25% Xylene cyanol + 30% glycerol and solutions were loaded on 0.8% agarose gel containing 100 lg of ethidium bromide. Electrophoresis was performed at 100 V in TBE buffer until the bromophenol blue reached to the 3/4th of the gel. Bands were visualised by UV transilluminator and photographed. The efficiency of DNA cleavage was measured by determining the ability of the complex to form open circular (OC) or nicked circular (NC) DNA from its super coiled (SC) form. The reactions were carried out under oxidative and/or hydrolytic conditions. Control experiments were done in the presence of hydroxyl radical scavenger DMSO (4 lL). Fig. 2. Cyclic voltammetric profile of [Cu(CETH)2Cl] at scan rates (1) 25 (2) 50, (3) 75 and (4) 100 mVS 1. Table 1 Crystal data and structure refinement for [Cu(CET)2Cl]. Empirical formula Formula weight Colour Crystal size Crystal system Space group Unit cell dimensions a, Å b (Å) c (Å) a=b=c Volume (Å3) Z Density (calculated), g/cm3 Absorption coefficient, (mm F(0 0 0) Index ranges
1
)
Data collection Diffractometer used Radiation Temperature (K) Monochromator 2h range (°) Scan speed(°/min in w) Std. reflections Number of reflections Number of independent reflections Number of observed reflections Solution and refinement: System used Space group Refinement method Hydrogen atoms Final R indices R indices (all data) Maximum and minimum transmission Data/restraints/parameters Goodness-of-fit on F2 Largest difference in peak and hole, e.Å 3
C26H38ClCuN6S2 597.76 brown 0.348 0.218 0.109 orthorhombic Pna2 (1) 9.8598(14) 15.411(2) 0.817(3) 90 3163(8) 4 1.225 0.931 1256 11 6 h 6 11, 18 6 k 6 18, 24 6 l 6 24 Bruker Smart Apex CCD Mo Ka 298 graphite 2.45–25 10 27915 27915 5566 3996 orthorhombic Pna2(1) Full-matrix least-squares on F2 Constrained [F2 > 4r(F2)] R1 = 0.0405, wR2 = 0.0816 R1 = 0.0643, wR2 = 0.0879 0.906 and 0.782 5566/1/331 0.953 0.231 0.163
Fig. 3. ORTEP plot of the [Cu(CETH)2Cl] with numbering scheme. Ellipsoids drawn at 50% probability level.
Table 2 Selected bond distances [Å] and bond angles [°]. Bond lengths (Ao)
Bond angles (°)
Cu(1)–S(1) 2.2110 (11) Cu(1)–S(2) 2.2211 (12) Cu(1)–Cl(1) 2.3034 (12) S(1)–C(1) 1.7015 (37) S(2)–C(3) 1.7057 (40) N(3)–C(1) 1.3353 (55) C(3)–N(5) 1.3177 (52) N(2)–N(3) 1.3755 (44) N(4)–N(5) 1.3802 (42) C(2)–N(2) 1.2545 (51) N(4)–C(7) 1.2537 (54) N(1)–C(6) 1.4566 (55) N(6)–C(9) 1.4619
Cl(1)–Cu(1)–S(1) 120.01 Cl(1)–Cu(1)–S(2) 118.33 S(1)–Cu(1)–S(2) 121.61 Cu(1)–S(1)–C(1) 110.81 Cu(1)–S(2)–C(3) 108.09 N(1)–C(1)–N(3) 117.20 N(5)–C(3)–N(6) 117.52 N(2)–N(3)–C(1) 122.06 N(4)–N(5)–C(3) 121.49 N(2)–C(2)–C(11) 123.33 N(4)–C(7)–C(4) 123.00
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Table 3 Hydrogen-bond geometry (Å, °). D—H A
D–H
H A
D A
N1—H1 N2 N3—H3 Cl1 N5—H5A Cl1 N6—H6 N4 C6—H6B S3 C9—H9B S4 N1—H1 S3i C12–H12–Cg1ii C22–H22C–Cg2iii
0.86 0.86 0.86 0.86 0.97 0.97 0.86 0.97 0.97
2.28 2.33 2.37 2.26 2.70 2.66 2.72 2.93 2.91
2.666 3.187 3.156 2.643 3.105 3.094 3.402 3.519 3.853
Symmetry codes: (i) 2 Y,Z.
D—H A (4) (3) (3) (5) (5) (5) (3) (5) (9)
x + 1/2, y 1/2, z + 1/2. (ii) 1/2 + X,1/2 Y,Z (iii)
107 178 152 107 105 107 137 123 167 1/2 + X,1/
and dimethyl sulfoxide. The molar conductivity (15 Ohm 1 cm 2 mol 1) value indicates that the complex is non-electrolyte in nature [45]. The pale yellow coloured solution of the complex in DMF shows a metal to ligand charge transfer transition at 351 and398 nm. An infrared spectrum of the complex is compared with ligand spectrum. The IR spectrum of ligand shows one medium band at 3300 cm 1 region and is assigned to t(N–H) stretching vibration of the terminal –NHR group. This band is not effected in complex is suggesting non-participation of terminal –NHR group in coordination. In the spectrum of the ligand the bands in the region 1379 and 833 cm 1 are due to t(C@S) and d(C@S), respectively. The negative shift (Dt = 30–50 cm 1) of these values indicates the neutral thiosemicarbazone ligand coordinates [46] in a thioketonic way in contrast with the usual anionic thioenolic ligand. Two new bands which appear at 387 and 265 cm 1 are assigned to (Cu–S) and (Cu–Cl), respectively. The far infrared spectrum of the complexes provides valuable insights into the nature of the Cl atoms. The Cu–Cl strechining frequencies generally occur in the region 360–220 cm 1, these frequencies change directly with the oxidation number of the copper and inversely with the coordination number. For tri coordinated Cu(I), the expected and reported values are within 320–220 cm 1 [46,47]. These frequen-
cies should be expected to lie near the upper limit for the terminal chlorine, whilst they should be near the lower limit for the bridging chlorine. The intense band around 265 cm 1 for the present complexes can be attributed to the terminal chloride atom. Complex Ia in DMF 0.1 M tetrabutyl ammonium perchlorate (TBAPCl) solution shows quasi irreversible cyclic voltammetric response due to Cu(I)/Cu(II) oxidation peak at 0.41 V vs Ag/AgCl reference electrode with ic/ia = 0.575 V and DEp value 300 mV at a scan rate 50 mV/S (Fig. 2). The DGo (643 Kcal) value of the complex in DMF indicates the stability in solution state. 3.2. Description of the crystal structures The complex Ia has been characterised by single crystal X-ray diffraction method. A perspective view of monomeric entity with
Fig. 4. Intermolecular N–H S bonding network of the complex.
Fig. 5. Packing of the molecules in the unit cell viewed along ‘a’ axis. Dotted lines indicate both intra and intermolecular N–H S hydrogen bonds.
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Fig. 7. Agarose gel (0.8%) showing results of electrophoresis of 4 lL of pUC18 DNA; 2 lL 0.1 M TBE buffer (pH8); 2 lL complex in DMF (10 3 M); 8 lL water, 2 lL H2O2 , 2 lL azide, 4 lL DMSO, 2 lL EDTA, 2 lL DTT (Total volume 20 lL) were added respectively, incubated at 37 °C (30 min): Marker, Lane 1. DNA control, Lane 2. DNA + H2O2, Lane 3. CETH + DNA, Lane 4. CETH + DNA + H2O2, Lane 5. CuCl2 + DNA, Lane 7. [Cu(CETH)2Cl] + DNA, Lane 8. Lane 6. CuCl2 + DNA + H2O2, [Cu(CETH)2Cl] + DNA + H2O2, Lane 9. [Cu(CETH)2Cl] + DNA + azide, Lane 10. [Cu(CETH)2Cl] + DNA + DMSO, Lane 11. [Cu(CETH)2Cl] + DNA + EDTA, Lane 12. [Cu(CETH)2Cl] + DNA + H2O2 + DTT.
Fig. 6. Packing diagram of complex. dotted lines indicates C–H p interactions.
in presence and absence of hydrogen peroxide (Fig. 7). Nuclease activity of complex Ia, was investigated in presence of free radical scavenger (DMSO), chelating agent (EDTA), and reducing agent, DTT with H2O2. From Fig. 7, it is evident that complex Ia, shows marginal nuclease activity in the presence of DTT with hydrogen peroxide (Lane 12).
the atomic number scheme is depicted in Fig. 3. Selected bond distances and angles are given in Table 2. The structure consists of a mono nuclear copper(I) complex, is coordinated via S atom of each thiosemicarbazone ligand and one chlorine atom. The Cu–S bond distances were found Cu(1)–S(2) = 2.2211 Å and Cu(1)– S(1) = 2.2110 Å, whilst and Cu(1)–Cl(1) bond distance was 2.3034 Å. The structural feature in complex is similar to those complexes in the literature [48–53]. The copper atom of the complex is coordinate with two sulphur, S(1) and S(2) and one chlorine, Cl(1) atom (evident by bond length data, Table 2) resulting in three coordinated structure. The Cu–Cl bond distance is much less than the sum of ionic radii of copper and chloride (Cu+ and Cl 2.58 Å) [54,55]. The S–C bond distance found was 1.701–1.705 Å and are close to those (1.66–1.72 Å) observed in [CuX(4-H2-stbc)(PPh3)2] where X = Br, I; [48,50] and shorter than those observed in Hg(II)complex [56]. The bond angles (Table 2) around the copper, Cl(1)–Cu(1)–S(4) = 118.330, S(3)– Cl(1)–Cu(1)–S(3) = 120.010, 0 Cu(1)–S(4) = 121.61 , sum 359.950. The angles of 118.330 is smaller than 1200, is suggest that the complex has unusual stereochemistry for copper(I) and metal site has Y-Shape (geometry) [57].
4. Conclusion
3.3. Hydrogen bonding
Appendix A. Supplementary data
In solid state each –NH group forms an intramolecular hydrogen bond to the S, N and Cl atoms (Table 3) whilst one of the hydrogen forms bifurcated H-bond. The –N1H Cl hydrogen bond (Fig. 4) forming pseudo-six member ring and strong N–H N hydrogen bond also forms a pseudo five member ring, consequently complex locking molecular conformation and thus eliminating conformational flexibility. The intermolecular N–H S bond links the molecules forming dimers in ab plane (Fig. 5). The two isopropyl benzene groups are making dihedral angles of 14.9 and 19.8°, respectively with the plane of three coordinated copper and further the two chains constitute a two fold rotation axis passing through Cu–Cl bond. A weak intermolecular N6–H6 Cl1 contact (x 1/2, y + 1/2, +z) also seen between the molecules. The molecules are also stabilized by C–H p interactions (Fig. 6).
CCDC 12 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ica.2009.06. 022.
3.4. Nuclease activity of complex Ia Nuclease activity of complex Ia has been studied by agarose gel electrophoresis using pUC18 DNA in TBE buffer (pH 8) in the presence/absence of H2O2. CETH shows no significant cleavage activity
We have synthesised copper(I) complex of cuminaldehyde-4ethyl-3-thiosemicarbazone. The complex was characterised including by X-ray crystallography, and its efficiency to cleave DNA was investigated using pUC18 DNA. These studies reveal that the complex has three coordinate ‘Y’ shape structure and it shows marginal cleavage activity in presence DTT with hydrogen peroxide. Acknowledgements The authors thank Department of Science & Technology (DST), New Delhi (India) for giving financial support (Grant No. SR/S1/ IC-37-2007) in the form of major research project. Authors thank Prof. A.K. Gaunguly and Dr. Shailesh Upreti, Indian Institute of Technology-Delhi, New Delhi for providing X-ray crystallography data and for solving the crystal structure and G. N. Anil Kumar, Department of Physics, MSRIT, Bangalore, for providing additional structural information.
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Synthesis, single crystal structure and DNA cleavage studies on first 4N-ethyl substituted three coordinate copper(I) complex of thiosemicarbazone Panchangam Murali Krishna, Katreddi Hussain Reddy * Department of Chemistry, Sri Krishnadevaray University, Anantapur 515 003, AP, India
a r t i c l e
i n f o
Article history: Received 20 December 2008 Received in revised form 7 June 2009 Accepted 16 June 2009 Available online 21 June 2009 Keywords: Copper(I) complex Cuminaldehyde-4-ethyl-3thiosemicarbazone X-ray structure Three coordinate ‘Y-shaped’ complex Nuclease activity
a b s t r a c t A copper(I) complex [Cu(CETH)2Cl] (Ia), where CETH = cuminaldehyde-4-ethyl-3-thiosemicarbazone (I), is prepared and structurally characterised. The complex crystallizes in orthorhombic space group pna2(1) with the unit cell parameters; a = 9.8598(14) Å, b = 15.411(2) Å, c = 0.817(3) Å, V = 2055.9(4) Å3 and Z = 4. The neutral complex has the copper(I) centre bonded to two thioketonic sulphur donor in g1-S bonding mode and one chloride giving ‘Y’ shape geometry. The complex is diamagnetic and exhibits a copper to ligand charge transfer bands at 351 and 398 nm in dimethylformamide. The complex shows quasireversible cyclic voltammetric response at 0.41 V (DEp = 300 mV) at 50 mVS 1 in DMF for the Cu(II)/ Cu(I) oxidation couple. Complex Ia shows marginal nuclease activity with pUC18 DNA in the presence of reducing agent (Dithiotretal) and hydrogen peroxide. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Of the sulphur donor ligands thiosemicarbazones and their derivatives are very interesting molecules ranging from bonding and structural features presented by their transition metal complexes in coordination chemistry. Thiosemicarbazones are biologically active pharmacophores and their activity enhanced on complexation with metal ions [1–10]. They have been used for the analysis of metals, device application in telecommunications, optical communicating, optical storage and optical information process [11,12]. Therefore, the transition metal complexes with these ligands have raised interest amongst many researchers, and they continue to be subjected of many studies, especially in anticancer chemotherapeutic agents [13] and as superoxide dismutase-like radical scavengers [14]. Thiosemicarbazones usually act as chelating ligands for transition metal ions by bonding through the sulphur and azomethine nitrogen atoms, although in some cases they behave as mono dentate ligand where bind through sulphur only. Thiosemicarbazones present in thione – thiol equilibrium that confers the presence of two configurations i.e. E and Z. If the neutral thiosemicarbazone binds to the metal via sulphur in E form and in thiol form binds via enolic sulphur and azomethine nitrogen in Z form. Copper is bio-essential metal for living organisms [15] with two oxidation states +1 and +2. Coordination compounds of copper * Corresponding author. Tel.: +91 8554 224347x255742; fax: +91 8554 229043. E-mail address: [email protected] (K.H. Reddy). 0020-1693/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2009.06.022
have been extensively used in metal ion mediated DNA cleavage through the generation of hydrogen ion abstracting activated oxygen species [16]. The most widely exploited oxidative cleavage agent is the bis (phenonthroline) copper(I) cation [17] and effectively cleaves in presence of oxidative or dioxygen plus reducing agent. Copper(II) complexes of thiosemicarbazones have been widely investigated. However, reports on copper(I) complexes are very limited [18–23]. Copper(I) complexes displays wide diversity in structural chemistry. The coordination number of copper is in the range from two to six. Copper(I) usually forms four-coordinate tetrahedral complexes [24], but three coordination numbers are known but less common [23,25–28]. Copper(I) complexes are diamagnetic, since Cu(I) has d10 electronic configuration and no d–d transitions are possible. Copper(I) complexes plays very important role in coordination chemistry and in catalytic reactions [29]. As a part of our research programme concerning the chelating behaviour of neutral or deprotonated thiosemicarbazones [30–34], herein we reported synthesis, structural characterisation and nuclease activity of three coordinate ‘Y’ shaped [Cu(CETH)2Cl] complex. 2. Experimental 2.1. Materials and techniques 4-Ethyl-3-thiosemicarbazide and cuminaldehyde (p-isopropyl benzaldehyde) were of reagent grade purchased from Sigma–Aldrich. All other chemicals were of AR grade and used as supplied. The solvents were distilled before use. The plasmid pUC18 DNA
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was isolated from E. coli DH5a strains in Lubria Broth (LB) medium supplemented by ampicillin cells from 5 ml culture by Qiagen column following the manufacturer’s protocol [35]. Elemental analysis was carried out on a Perkin–Elmer 2400 CHNS elemental analyser. Magnetic susceptibility measurements were carried out on a magnetic susceptibility balance (Sherwood scientific, Cambridge, England), high purity CuSO45H2O was used as a standard. Molar conductance (10 3 M) in DMF at 30 ± 2 °C was measured with a CC180 model (ELICO) direct reading conductivity bridge. The electronic spectra were recorded in DMSO with a Shimadzu UV-160A spectrophotometer. FT-IR spectra were recorded in the range 4000–270 cm 1 in KBr discs on a Nicolet protégé 460 IR Spectrometer. The cyclic voltammetric measurements were performed on a Bio Analytical System (BAS) CV-27 assembly equipped with an X–Y recorder. Measurements were made on degassed (N2 bubbling for 5 min) ligand/complex solutions (10 3 M) in DMF and ethanol containing tetrabutyl ammonium perchlorate (0.1 M) as a supporting electrolyte. The three-electrode system consisted of a glassy carbon (working), platinum wire (auxiliary) and Ag/AgCl (reference). The 1H and 13C{1H} NMR spectra were recorded on a Bruker Spectrospin DPX-300 NMR spectrometer at 300.13 and 75.47 MHz, respectively. 2.2. Synthesis of CETH (I) and [Cu(CETH)2Cl](Ia) 2.2.1. Synthesis and structural characterisation of CETH (I) The ligand (CH3)2CH(C6H4)CH@NNHC(S)NH(C2H5) (I) (Fig. 1) was synthesised by reactions given in the Scheme 1. The synthe-
H N
H N
H N
Slow recrystallization of Ia from chloroform gave a single crystal of size 0.348 0.218 0.109 mm3 and was mounted on a glass fibre. The cell parameters and the intensity data were obtained using a Bruker SMART APEX CCD diffractometer to give a complete asymmetric unit using radiations of wave length
CH3 Fig. 1. Structure of CETH.
H
H O H3C CH 3
2.2.2. Synthesis and structure of [Cu(CETH)2Cl] (Ia) Boiled methanolic solution of CETH (1.00 g, 0.004 mol) was mixed with CuCl22H2O (0.29 g, 0.002 mol) and refluxed for 30 min and allowed to stand over night, the solid complex that separated out was collected by filtration and dried in vacuo. Yield: 51%, M.P. 175–176 °C Anal. Calc. for C26H38N6S2ClCu: C, 52.24; H, 6.40; N, 14.05; S, 10.72. Found: C, 52.34; H, 6.64; N, 14.03; S, 10.75%. IR (KBr, m, cm 1): 3299(br), 3147(br). 1526 (s), 1332(m), 800 (m), 387 (m), 265 (s), and cyclic voltammetry (E1/2, vs. Ag/ AgCl): 0.410 V. 2.3. X-ray crystallography
CH2 - CH3
S
H3C
sis of I was based on the general procedure described in the literature [36]. To a hot ethanolic solution (175 ml) of cuminaldehyde (6.22 g, 0.042 mol) in 500 ml round bottom flask, 150 ml of 5% CH3COOH–H2O solution of 4-ethyl-3-thiosemicarbazide (5.00 g, 0.042 mol) was mixed and heated the reaction mixture under reflux on a steam bath for 30–45 min. The crystalline product which formed was collected by filtration, washed several times with hot water and dried in vacuo and was recrystallized in methanol. Yield 45%, M.P.140-142 °C, Anal. Calc. for C13H19N3S: C, 62.6; H, 7.7; N, 16.8; S, 14.1. Found: C, 62.9; H, 7.6; N, 16.9; S, 14.1%. IR (KBr, m, cm 1): 3300(br), 3143(br). 1607 (s), 1379 (m), 1092 (m), 833 (m). 1H NMR (DMSO-d6, 25 °C): d (vs TMS) 1.14 (t, 1H, H13), 1.19–1.21 (d, J = 7 Hz, 6H, ipr-Me), 2.82–2.90 (m, 1H, ipr-CH), 3.52–3.54 (quintet, 1H, H12), 7.27–7.30 (d, J = 8 Hz, m, 2H, H3, 5), 7.69–7.72 (d, J = 8 Hz, 2H, H2, 6), 8.02 (s, 1H, H9), 8.48 (t, 1H, H11), 11.31 (s, 1H, H7).13C NMR (75.4 MHz, CDCl3): d 14.62 (C13), 23.65 (ipr-CH3), 33.33 (ipr-CH), 38.66 (C12), 126.56 (C2, 6), 127.28 (C3, 5), 131.90 (C1), 141.83 (C7), 150.34 (C4) and 176.58 (C10). ESI-Mass (m/z, E+) 249. The pka values of CETH are: 5.4 and 8.1.
+
H N H2N
N
EtOH C S
N H (C 2 H 5 )
H N
NH (C 2 H 5 )
+
S
H3C
5% CH 3 COOH -H 2 O
I
CH 3
MeoH
NH(C 2 H 5)
(C 2 H 5)HN
H
HN N
S
Cu
S
NH N
H
Cl
H 3C
CH 3
H 3C
Ia Scheme 1.
CH 3
CuCl 2
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0.71073 Å. Frames were collected at T = 298 by x, U, and 2h rotation at 10 s per frames with SMART [37]. The measured intensities were reduced to F2 and corrected for absorptions with SADABS [38]. Structural solution, refinement, and data output were carried out with the SHELXTL program [39]. Non-hydrogen atoms were refined anisotropically. C–H hydrogen atoms were placed in geometrically calculated positions using a rigid model. The program PARST [40,41] was employed for geometrical description. Images were created with the ORTEP 3 program [42] and PLATON [43,44]. Cell dimensions and other experimental parameters are given in Table 1.
3. Results and discussion 3.1. Characterisation of synthesised complex The reaction of CuCl22H2O with cuminaldehyde-4-ethyl-3-thiosemicarbazone led to the formation of complex Ia (Scheme 1). The new compound is pale yellow coloured solid and insoluble in most of the organic solvents except chloroform, dimethyl formamide
2.4. Assay of nuclease activity The extent of cleavage of DNA by the copper(I) complex was monitored by agarose gel electrophoresis using pUC18 DNA. The samples after incubation for 30 min at 37 °C were added to the loading buffer containing 0.25% bromophenol blue + 0.25% Xylene cyanol + 30% glycerol and solutions were loaded on 0.8% agarose gel containing 100 lg of ethidium bromide. Electrophoresis was performed at 100 V in TBE buffer until the bromophenol blue reached to the 3/4th of the gel. Bands were visualised by UV transilluminator and photographed. The efficiency of DNA cleavage was measured by determining the ability of the complex to form open circular (OC) or nicked circular (NC) DNA from its super coiled (SC) form. The reactions were carried out under oxidative and/or hydrolytic conditions. Control experiments were done in the presence of hydroxyl radical scavenger DMSO (4 lL). Fig. 2. Cyclic voltammetric profile of [Cu(CETH)2Cl] at scan rates (1) 25 (2) 50, (3) 75 and (4) 100 mVS 1. Table 1 Crystal data and structure refinement for [Cu(CET)2Cl]. Empirical formula Formula weight Colour Crystal size Crystal system Space group Unit cell dimensions a, Å b (Å) c (Å) a=b=c Volume (Å3) Z Density (calculated), g/cm3 Absorption coefficient, (mm F(0 0 0) Index ranges
1
)
Data collection Diffractometer used Radiation Temperature (K) Monochromator 2h range (°) Scan speed(°/min in w) Std. reflections Number of reflections Number of independent reflections Number of observed reflections Solution and refinement: System used Space group Refinement method Hydrogen atoms Final R indices R indices (all data) Maximum and minimum transmission Data/restraints/parameters Goodness-of-fit on F2 Largest difference in peak and hole, e.Å 3
C26H38ClCuN6S2 597.76 brown 0.348 0.218 0.109 orthorhombic Pna2 (1) 9.8598(14) 15.411(2) 0.817(3) 90 3163(8) 4 1.225 0.931 1256 11 6 h 6 11, 18 6 k 6 18, 24 6 l 6 24 Bruker Smart Apex CCD Mo Ka 298 graphite 2.45–25 10 27915 27915 5566 3996 orthorhombic Pna2(1) Full-matrix least-squares on F2 Constrained [F2 > 4r(F2)] R1 = 0.0405, wR2 = 0.0816 R1 = 0.0643, wR2 = 0.0879 0.906 and 0.782 5566/1/331 0.953 0.231 0.163
Fig. 3. ORTEP plot of the [Cu(CETH)2Cl] with numbering scheme. Ellipsoids drawn at 50% probability level.
Table 2 Selected bond distances [Å] and bond angles [°]. Bond lengths (Ao)
Bond angles (°)
Cu(1)–S(1) 2.2110 (11) Cu(1)–S(2) 2.2211 (12) Cu(1)–Cl(1) 2.3034 (12) S(1)–C(1) 1.7015 (37) S(2)–C(3) 1.7057 (40) N(3)–C(1) 1.3353 (55) C(3)–N(5) 1.3177 (52) N(2)–N(3) 1.3755 (44) N(4)–N(5) 1.3802 (42) C(2)–N(2) 1.2545 (51) N(4)–C(7) 1.2537 (54) N(1)–C(6) 1.4566 (55) N(6)–C(9) 1.4619
Cl(1)–Cu(1)–S(1) 120.01 Cl(1)–Cu(1)–S(2) 118.33 S(1)–Cu(1)–S(2) 121.61 Cu(1)–S(1)–C(1) 110.81 Cu(1)–S(2)–C(3) 108.09 N(1)–C(1)–N(3) 117.20 N(5)–C(3)–N(6) 117.52 N(2)–N(3)–C(1) 122.06 N(4)–N(5)–C(3) 121.49 N(2)–C(2)–C(11) 123.33 N(4)–C(7)–C(4) 123.00
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Table 3 Hydrogen-bond geometry (Å, °). D—H A
D–H
H A
D A
N1—H1 N2 N3—H3 Cl1 N5—H5A Cl1 N6—H6 N4 C6—H6B S3 C9—H9B S4 N1—H1 S3i C12–H12–Cg1ii C22–H22C–Cg2iii
0.86 0.86 0.86 0.86 0.97 0.97 0.86 0.97 0.97
2.28 2.33 2.37 2.26 2.70 2.66 2.72 2.93 2.91
2.666 3.187 3.156 2.643 3.105 3.094 3.402 3.519 3.853
Symmetry codes: (i) 2 Y,Z.
D—H A (4) (3) (3) (5) (5) (5) (3) (5) (9)
x + 1/2, y 1/2, z + 1/2. (ii) 1/2 + X,1/2 Y,Z (iii)
107 178 152 107 105 107 137 123 167 1/2 + X,1/
and dimethyl sulfoxide. The molar conductivity (15 Ohm 1 cm 2 mol 1) value indicates that the complex is non-electrolyte in nature [45]. The pale yellow coloured solution of the complex in DMF shows a metal to ligand charge transfer transition at 351 and398 nm. An infrared spectrum of the complex is compared with ligand spectrum. The IR spectrum of ligand shows one medium band at 3300 cm 1 region and is assigned to t(N–H) stretching vibration of the terminal –NHR group. This band is not effected in complex is suggesting non-participation of terminal –NHR group in coordination. In the spectrum of the ligand the bands in the region 1379 and 833 cm 1 are due to t(C@S) and d(C@S), respectively. The negative shift (Dt = 30–50 cm 1) of these values indicates the neutral thiosemicarbazone ligand coordinates [46] in a thioketonic way in contrast with the usual anionic thioenolic ligand. Two new bands which appear at 387 and 265 cm 1 are assigned to (Cu–S) and (Cu–Cl), respectively. The far infrared spectrum of the complexes provides valuable insights into the nature of the Cl atoms. The Cu–Cl strechining frequencies generally occur in the region 360–220 cm 1, these frequencies change directly with the oxidation number of the copper and inversely with the coordination number. For tri coordinated Cu(I), the expected and reported values are within 320–220 cm 1 [46,47]. These frequen-
cies should be expected to lie near the upper limit for the terminal chlorine, whilst they should be near the lower limit for the bridging chlorine. The intense band around 265 cm 1 for the present complexes can be attributed to the terminal chloride atom. Complex Ia in DMF 0.1 M tetrabutyl ammonium perchlorate (TBAPCl) solution shows quasi irreversible cyclic voltammetric response due to Cu(I)/Cu(II) oxidation peak at 0.41 V vs Ag/AgCl reference electrode with ic/ia = 0.575 V and DEp value 300 mV at a scan rate 50 mV/S (Fig. 2). The DGo (643 Kcal) value of the complex in DMF indicates the stability in solution state. 3.2. Description of the crystal structures The complex Ia has been characterised by single crystal X-ray diffraction method. A perspective view of monomeric entity with
Fig. 4. Intermolecular N–H S bonding network of the complex.
Fig. 5. Packing of the molecules in the unit cell viewed along ‘a’ axis. Dotted lines indicate both intra and intermolecular N–H S hydrogen bonds.
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Fig. 7. Agarose gel (0.8%) showing results of electrophoresis of 4 lL of pUC18 DNA; 2 lL 0.1 M TBE buffer (pH8); 2 lL complex in DMF (10 3 M); 8 lL water, 2 lL H2O2 , 2 lL azide, 4 lL DMSO, 2 lL EDTA, 2 lL DTT (Total volume 20 lL) were added respectively, incubated at 37 °C (30 min): Marker, Lane 1. DNA control, Lane 2. DNA + H2O2, Lane 3. CETH + DNA, Lane 4. CETH + DNA + H2O2, Lane 5. CuCl2 + DNA, Lane 7. [Cu(CETH)2Cl] + DNA, Lane 8. Lane 6. CuCl2 + DNA + H2O2, [Cu(CETH)2Cl] + DNA + H2O2, Lane 9. [Cu(CETH)2Cl] + DNA + azide, Lane 10. [Cu(CETH)2Cl] + DNA + DMSO, Lane 11. [Cu(CETH)2Cl] + DNA + EDTA, Lane 12. [Cu(CETH)2Cl] + DNA + H2O2 + DTT.
Fig. 6. Packing diagram of complex. dotted lines indicates C–H p interactions.
in presence and absence of hydrogen peroxide (Fig. 7). Nuclease activity of complex Ia, was investigated in presence of free radical scavenger (DMSO), chelating agent (EDTA), and reducing agent, DTT with H2O2. From Fig. 7, it is evident that complex Ia, shows marginal nuclease activity in the presence of DTT with hydrogen peroxide (Lane 12).
the atomic number scheme is depicted in Fig. 3. Selected bond distances and angles are given in Table 2. The structure consists of a mono nuclear copper(I) complex, is coordinated via S atom of each thiosemicarbazone ligand and one chlorine atom. The Cu–S bond distances were found Cu(1)–S(2) = 2.2211 Å and Cu(1)– S(1) = 2.2110 Å, whilst and Cu(1)–Cl(1) bond distance was 2.3034 Å. The structural feature in complex is similar to those complexes in the literature [48–53]. The copper atom of the complex is coordinate with two sulphur, S(1) and S(2) and one chlorine, Cl(1) atom (evident by bond length data, Table 2) resulting in three coordinated structure. The Cu–Cl bond distance is much less than the sum of ionic radii of copper and chloride (Cu+ and Cl 2.58 Å) [54,55]. The S–C bond distance found was 1.701–1.705 Å and are close to those (1.66–1.72 Å) observed in [CuX(4-H2-stbc)(PPh3)2] where X = Br, I; [48,50] and shorter than those observed in Hg(II)complex [56]. The bond angles (Table 2) around the copper, Cl(1)–Cu(1)–S(4) = 118.330, S(3)– Cl(1)–Cu(1)–S(3) = 120.010, 0 Cu(1)–S(4) = 121.61 , sum 359.950. The angles of 118.330 is smaller than 1200, is suggest that the complex has unusual stereochemistry for copper(I) and metal site has Y-Shape (geometry) [57].
4. Conclusion
3.3. Hydrogen bonding
Appendix A. Supplementary data
In solid state each –NH group forms an intramolecular hydrogen bond to the S, N and Cl atoms (Table 3) whilst one of the hydrogen forms bifurcated H-bond. The –N1H Cl hydrogen bond (Fig. 4) forming pseudo-six member ring and strong N–H N hydrogen bond also forms a pseudo five member ring, consequently complex locking molecular conformation and thus eliminating conformational flexibility. The intermolecular N–H S bond links the molecules forming dimers in ab plane (Fig. 5). The two isopropyl benzene groups are making dihedral angles of 14.9 and 19.8°, respectively with the plane of three coordinated copper and further the two chains constitute a two fold rotation axis passing through Cu–Cl bond. A weak intermolecular N6–H6 Cl1 contact (x 1/2, y + 1/2, +z) also seen between the molecules. The molecules are also stabilized by C–H p interactions (Fig. 6).
CCDC 12 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ica.2009.06. 022.
3.4. Nuclease activity of complex Ia Nuclease activity of complex Ia has been studied by agarose gel electrophoresis using pUC18 DNA in TBE buffer (pH 8) in the presence/absence of H2O2. CETH shows no significant cleavage activity
We have synthesised copper(I) complex of cuminaldehyde-4ethyl-3-thiosemicarbazone. The complex was characterised including by X-ray crystallography, and its efficiency to cleave DNA was investigated using pUC18 DNA. These studies reveal that the complex has three coordinate ‘Y’ shape structure and it shows marginal cleavage activity in presence DTT with hydrogen peroxide. Acknowledgements The authors thank Department of Science & Technology (DST), New Delhi (India) for giving financial support (Grant No. SR/S1/ IC-37-2007) in the form of major research project. Authors thank Prof. A.K. Gaunguly and Dr. Shailesh Upreti, Indian Institute of Technology-Delhi, New Delhi for providing X-ray crystallography data and for solving the crystal structure and G. N. Anil Kumar, Department of Physics, MSRIT, Bangalore, for providing additional structural information.
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